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11858995 | DETAILED DESCRIPTION Definitions The term “immunoglobulin” as used herein refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J Mol Biol 1987; 196:90117). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to IMGT rules (Brochet X.,Nucl Acids Res2008; 36:W503-508; Lefranc M P.,Nucl Acids Res1999; 27:209-12; www.imgt.org/). Unless otherwise stated or contradicted by context, reference to amino acid positions in the constant regions is according to the EU-numbering (Edelman et al.,PNAS.1969; 63:78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). For example, SEQ ID NO: 15 sets forth amino acids positions 118-447, according to EU numbering, of the IgG1 heavy chain constant region. The term “amino acid corresponding to position . . . ” as used herein refers to an amino acid position number in a human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that “corresponds to” an amino acid or segment in another sequence is one that aligns with the other amino acid or segment using a standard sequence alignment program such as ALIGN, ClustalW or similar, typically at default settings and has at least 50%, at least 80%, at least 90%, or at least 95% identity to a human IgG1 heavy chain. It is within the ability of one of ordinary skill in the art to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention. The term “antibody” (Ab) as used herein in the context of the present invention refers to an immunoglobulin molecule which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term antibody, unless specified otherwise, also encompasses polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, chimeric antibodies and humanized antibodies. An antibody as generated can possess any isotype. The term “antibody fragment” or “antigen-binding fragment” as used herein refers to a fragment of an immunoglobulin molecule which retains the ability to specifically bind to an antigen, and can be generated by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Examples of antibody fragments include (i) a Fab′ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature1989; 341: 54446), which consists essentially of a VH domain and also called domain antibodies (Holt et al;Trends Biotechnol2003; 21:484-90); (vi) camelid or nanobodies (Revets et al;Expert Opin Biol Ther2005; 5:111-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see, e.g., Bird et al.,Science1988; 242:42326 and Huston et al.,PNAS1988; 85:587983). Such single chain antibodies are encompassed within the term antibody fragment unless otherwise noted or clearly indicated by context. The term “antibody-binding region” or “antigen-binding region” as used herein refers to the region which interacts with the antigen and comprises both the VH and the VL regions. The term antibody when used herein refers not only to monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g., three or more, different antigen-binding regions. The term antigen-binding region, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen. As used herein, the term “isotype” refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is mentioned, the term is not limited to a specific isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgG1, than to other isotypes. Thus, e.g., an IgG1 antibody may be a sequence variant of a naturally-occurring IgG1 antibody, which may include variations in the constant regions. The term “bispecific antibody” or “bs” or “bsAb” as used herein refers to an antibody having two different antigen-binding regions defined by different antibody sequences. A bispecific antibody can be of any format. The terms “half molecule”, “Fab-arm”, and “arm”, as used herein, refer to one heavy chain-light chain pair. When a bispecific antibody is described as comprising a half-molecule antibody “derived from” a first parental antibody, and a half-molecule antibody “derived from” a second parental antibody, the term “derived from” indicates that the bispecific antibody was generated by recombining, by any known method, said half-molecules from each of said first and second parental antibodies into the resulting bispecific antibody. In this context, “recombining” is not intended to be limited by any particular method of recombining and thus includes all of the methods for producing bispecific antibodies described herein, including for example recombining by half-molecule exchange (also known as “controlled Fab-arm exchange”), as well as recombining at nucleic acid level and/or through co-expression of two half-molecules in the same cells. The term “full-length” as used herein in the context of an antibody indicates that the antibody is not a fragment but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g., the VH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1 antibody. A full-length antibody may be engineered. An example of a “full-length” antibody is epcoritamab. The term “Fc region” as used herein refers to an antibody region consisting of the Fc sequences of the two heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain. The term “heterodimeric interaction between the first and second CH3 regions” as used herein refers to the interaction between the first CH3 region and the second CH3 region in a first-CH3/second-CH3 heterodimeric protein. The term “homodimeric interactions of the first and second CH3 regions” as used herein refers to the interaction between a first CH3 region and another first CH3 region in a first-CH3/first-CH3 homodimeric protein and the interaction between a second CH3 region and another second CH3 region in a second-CH3/second-CH3 homodimeric protein. The term “binding” as used herein in the context of the binding of an antibody to a predetermined antigen typically refers to binding with an affinity corresponding to a KDof about 10−6M or less, e.g., 10−7M or less, such as about 10−8M or less, such as about 10−9M or less, about 10−10M or less, or about 10−11M or even less, when determined by, e.g., BioLayer Interferometry (BLI) technology in a Octet HTX instrument using the antibody as the ligand and the antigen as the analyte, and wherein the antibody binds to the predetermined antigen with an affinity corresponding to a KDthat is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KDof binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely related antigen. The amount with which the KDof binding is lower is dependent on the KDof the antibody, so that when the KDof the antibody is very low, then the amount with which the KDof binding to the antigen is lower than the KDof binding to a non-specific antigen may be at least 10,000-fold (i.e., the antibody is highly specific). The term “KD” (M) as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity, as used herein, and KDare inversely related, that is that higher affinity is intended to refer to lower KD, and lower affinity is intended to refer to higher KD. The term “isolated antibody” as used herein refers to an antibody which is substantially free of other antibodies having different antigenic specificities. In a preferred embodiment, an isolated bispecific antibody that specifically binds to CD20 and CD3 is in addition substantially free of monospecific antibodies that specifically bind to CD20 or CD3. The term “CD3” as used herein refers to the human Cluster of Differentiation 3 protein which is part of the T-cell co-receptor protein complex and is composed of four distinct chains. CD3 is also found in other species, and thus, the term “CD3” is not limited to human CD3 unless contradicted by context. In mammals, the complex contains a CD3γ (gamma) chain (human CD3γ chain UniProtKB/Swiss-Prot No P09693, or cynomolgus monkey CD3γ UniProtKB/Swiss-Prot No Q95LI7), a CD3δ (delta) chain (human CD3δ UniProtKB/Swiss-Prot No P04234, or cynomolgus monkey CD3δ UniProtKB/Swiss-Prot No Q95LI8), two CD3ε (epsilon) chains (human CD3ε UniProtKB/Swiss-Prot No P07766, SEQ ID NO: 28); cynomolgus CD3ε UniProtKB/Swiss-Prot No Q95LI5; or rhesus CD3ε UniProtKB/Swiss-Prot No G7NCB9), and a CD3ζ-chain (zeta) chain (human CD3ζ UniProtKB/Swiss-Prot No P20963, cynomolgus monkey CD3ζ UniProtKB/Swiss-Prot No Q09TK0). These chains associate with a molecule known as the T-cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex. The term “CD3 antibody” or “anti-CD3 antibody” as used herein refers to an antibody which binds specifically to the antigen CD3, in particular human CD3ε (epsilon). The term “human CD20” or “CD20” refers to human CD20 (UniProtKB/Swiss-Prot No P11836, SEQ ID NO: 29) and includes any variants, isoforms, and species homologs of CD20 which are naturally expressed by cells, including tumor cells, or are expressed on cells transfected with the CD20 gene or cDNA. Species homologs include rhesus monkey CD20 (Macaca mulatta; UniProtKB/Swiss-Prot No H9YXP1) and cynomolgus monkey CD20 (Macaca fascicularis; UniProtKB No G7PQ03). The term “CD20 antibody” or “anti-CD20 antibody” as used herein refers to an antibody which binds specifically to the antigen CD20, in particular to human CD20. The term “CD3xCD20 antibody”, “anti-CD3xCD20 antibody”, “CD20xCD3 antibody” or “anti-CD20xCD3 antibody” as used herein refers to a bispecific antibody which comprises two different antigen-binding regions, one of which binds specifically to the antigen CD20 and one of which binds specifically to CD3. The term “DuoBody-CD3xCD20” as used herein refers to an IgG1 bispecific CD3xCD20 antibody comprising a first heavy and light chain pair as defined in SEQ ID NO: 24 and SEQ ID NO: 25, respectively, and comprising a second heavy and light chain pair as defined in SEQ ID NO: 26 and SEQ ID NO: 27. The first heavy and light chain pair comprises a region which binds to human CD3ε (epsilon), the second heavy and light chain pair comprises a region which binds to human CD20. The first binding region comprises the VH and VL sequences as defined by SEQ ID NOs: 6 and 7, and the second binding region comprises the VH and VL sequences as defined by SEQ ID NOs: 13 and 14. This bispecific antibody can be prepared as described in WO 2016/110576. Antibodies comprising functional variants of the heavy chain, light chains, VL regions, VH regions, or one or more CDRs of the antibodies of the examples as also provided herein. A functional variant of a heavy chain, a light chain, VL, VH, or CDRs used in the context of an antibody still allows the antibody to retain at least a substantial proportion (at least about 90%, 95% or more) of functional features of the “reference” and/or “parent” antibody, including affinity and/or the specificity/selectivity for particular epitopes of CD20 and/or CD3, Fc inertness and PK parameters such as half-life, Tmax, Cmax. Such functional variants typically retain significant sequence identity to the parent antibody and/or have substantially similar length of heavy and light chains. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm. Exemplary variants include those which differ from heavy and/or light chains, VH and/or VL, and/or CDR regions of the parent antibody sequences mainly by conservative substitutions; e.g., 10, such as 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant may be conservative amino acid residue replacements. Conservative substitutions may be defined by substitutions within the classes of amino acids reflected in the following table: TABLE 1Amino acid residue classes for conservative substitutionsAcidic ResiduesAsp (D) and Glu (E)Basic ResiduesLys (K), Arg (R), and His (H)Hydrophilic Uncharged ResiduesSer (S), Thr (T), Asn (N), andGln (Q)Aliphatic Uncharged ResiduesGly (G), Ala (A), Val (V), Leu (L),and Ile (I)Non-polar Uncharged ResiduesCys (C), Met (M), and Pro (P)Aromatic ResiduesPhe (F), Tyr (Y), and Trp (W) Unless otherwise indicated, the following nomenclature is used to describe a mutation: i) substitution of an amino acid in a given position is written as, e.g., K409R which means a substitution of a Lysine in position 409 with an Arginine; and ii) for specific variants the specific three or one letter codes are used, including the codes Xaa and X to indicate any amino acid residue. Thus, the substitution of Lysine with Arginine in position 409 is designated as: K409R, and the substitution of Lysine with any amino acid residue in position 409 is designated as K409X. In case of deletion of Lysine in position 409 it is indicated by K409*. The term “humanized antibody” as used herein refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody CDRs, which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92/22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e., the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. The VH and VL of the CD3 arm that is used herein in DuoBody-CD3xCD20 represents a humanized antigen-binding region. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties. The term “human antibody” as used herein refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The VH and VL of the CD20 arm that is used in DuoBody-CD3xCD20 represents a human antigen-binding region. Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein,Nature256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well-established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies can thus be generated using, e.g., transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Accordingly, in one embodiment, a human antibody is obtained from a transgenic animal, such as a mouse or a rat, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences. In such embodiments, the antibody originates from human germline immunoglobulin sequences introduced in the animal, but the final antibody sequence is the result of said human germline immunoglobulin sequences being further modified by somatic hypermutations and affinity maturation by the endogenous animal antibody machinery (see, e.g., Mendez et al.Nat Genet1997; 15:146-56). The VH and VL regions of the CD20 arm that is used in DuoBody-CD3xCD20 represents a human antigen-binding region. The term “biosimilar” (e.g., of an approved reference product/biological drug) as used herein refers to a biologic product that is similar to the reference product based on data from (a) analytical studies demonstrating that the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components; (b) animal studies (including the assessment of toxicity); and/or (c) a clinical study or studies (including the assessment of immunogenicity and pharmacokinetics or pharmacodynamics) that are sufficient to demonstrate safety, purity, and potency in one or more appropriate conditions of use for which the reference product is approved and intended to be used and for which approval is sought (e.g., that there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product). In some embodiments, the biosimilar biological product and reference product utilizes the same mechanism or mechanisms of action for the condition or conditions of use prescribed, recommended, or suggested in the proposed labeling, but only to the extent the mechanism or mechanisms of action are known for the reference product. In some embodiments, the condition or conditions of use prescribed, recommended, or suggested in the labeling proposed for the biological product have been previously approved for the reference product. In some embodiments, the route of administration, the dosage form, and/or the strength of the biological product are the same as those of the reference product. A biosimilar can be, e.g., a presently known antibody having the same primary amino acid sequence as a marketed antibody, but may be made in different cell types or by different production, purification, or formulation methods. The term “reducing conditions” or “reducing environment” as used herein refers to a condition or an environment in which a substrate, here a cysteine residue in the hinge region of an antibody, is more likely to become reduced than oxidized. The term “recombinant host cell” (or simply “host cell”) as used herein is intended to refer to a cell into which an expression vector has been introduced, e.g., an expression vector encoding an antibody described herein. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells, and lymphocytic cells. As used herein, “chronic lymphocytic leukemia” or “CLL” refers to a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. CLL can be diagnosed and classified based on WHO classification, which is included herein by reference (Swerdlow S H, Campo E, Harris N L, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised ed. 4th). Lyon, France: IARC Press (2017); Swerdlow S H, Campo E, Harris N L, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (ed. 4th). Lyon, France: IARC Press (2008)). CLL starts from lymphocytes in the bone marrow, and most commonly occurs in those aged >50 years. It is characterized by the clonal proliferation and accumulation of mature B lymphocytes, which ultimately leads to monoclonal B-cell lymphocytosis (reviewed in Uhm,Blood Res2020; 55:S72-82). Monoclonal B cells in CLL express several markers that are characteristic of mature activated B lymphocytes, including CD5, CD19, CD20 and CD23, as well as reduced expression of IgGM, IgGD, and CD79b (Chiorazzi et al.,N Engl J Med2005; 352-804-15). Several prognostic markers have been reported for the disease, including mutations in the IGHV gene, mutations in TP53, del(17p), and del(11q) (Wierda et al.,J Clin Oncol2011; 29:4088-95; Rossi et al.,Blood2013-121:1403-12; CLL-IPI,Lancet Oncol2016; 17:779-90). Treatments for CLL include, for example, chemotherapy, BCL2 inhibitors, BTK inhibitors, PI3Kδ inhibitors, alone or in combination with anti-CD20 antibodies, (Uhm, 2020, supra). The term “treatment” refers to the administration of an effective amount of a therapeutically active antibody described herein for the purpose of easing, ameliorating, arresting or eradicating (curing) symptoms or disease states such as CLL. Treatment may result in a complete response (CR), partial response (PR), or stable disease (SD), for example, as defined by iwCLL response criteria, as shown in Table 2. Treatment may be continued, for example, until disease progression (PD) or unacceptable toxicity. The term “administering” or “administration” as used herein refers to the physical introduction of a composition (or formulation) comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Preferred routes of administration for antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, a therapeutic agent described herein can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. In the methods described herein, the bispecific antibody (e.g., epcoritamab) is administered subcutaneously. Other agents used in combination with the bispecific antibody, such as for cytokine release syndrome prophylaxis or tumor lysis syndrome (TLS) prophylaxis, may be administered via other routes, such as intravenously or orally. The term “effective amount” or “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. For example, dosages as defined herein for the bispecific antibody (e.g., epcoritamab) in the range of 12-60 mg administered subcutaneously can be defined as such an “effective amount” or “therapeutically effective amount”. A therapeutically effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. In some embodiments, patients treated with the methods described herein will show an improvement in ECOG performance status. A therapeutically effective amount or dosage of a drug includes a “prophylactically effective amount” or a “prophylactically effective dosage”, which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or disorder (e.g., cytokine release syndrome) or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The term “inhibits growth” of a tumor as used herein includes any measurable decrease in the growth of a tumor, e.g., the inhibition of growth of a tumor by at least about 10%, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%. The term “subject” as used herein refers to a human patient, for example, a human patient with CLL. The terms “subject” and “patient” are used interchangeably herein. The term “buffer” as used herein denotes a pharmaceutically acceptable buffer. The term “buffer” encompasses those agents which maintain the pH value of a solution, e.g., in an acceptable range and includes, but is not limited to, acetate, histidine, TRIS® (tris (hydroxymethyl) aminomethane), citrate, succinate, glycolate and the like. Generally, the “buffer” as used herein has a pKa and buffering capacity suitable for the pH range of about 5 to about 6, preferably of about 5.5. “Disease progression” or “PD” as used herein refers to a situation in which one or more indices of CLL show that the disease is advancing despite treatment. In some embodiments, disease progression is defined based on iwCLL response criteria, as shown in Table 2. A “surfactant” as used herein is a compound that is typically used in pharmaceutical formulations to prevent drug adsorption to surfaces and or aggregation. Furthermore, surfactants lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. For example, an exemplary surfactant can significantly lower the surface tension when present at very low concentrations (e.g., 5% w/v or less, such as 3% w/v or less, such as 1% w/v or less such as 0.4% w/v or less, such as below 0.1% w/v or less, such as 0.04% w/v). Surfactants are amphiphilic, which means they are usually composed of both hydrophilic and hydrophobic or lipophilic groups, thus being capable of forming micelles or similar self-assembled structures in aqueous solutions. Known surfactants for pharmaceutical use include glycerol monooleate, benzethonium chloride, sodium docusate, phospholipids, polyethylene alkyl ethers, sodium lauryl sulfate and tricaprylin (anionic surfactants); benzalkonium chloride, citrimide, cetylpyridinium chloride and phospholipids (cationic surfactants); and alpha tocopherol, glycerol monooleate, myristyl alcohol, phospholipids, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbintan fatty acid esters, polyoxyethylene sterarates, polyoxyl hydroxystearate, polyoxylglycerides, polysorbates such as polysorbate 20 or polysorbate 80, propylene glycol dilaurate, propylene glycol monolaurate, sorbitan esters sucrose palmitate, sucrose stearate, tricaprylin and TPGS (Nonionic and zwitterionic surfactants). A “diluent” as used herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of dilutions of the pharmaceutical composition or pharmaceutical formulation (the terms “composition” and “formulation” are used interchangeably herein). Preferably, such dilutions of the composition dilute only the antibody concentration but not the buffer and stabilizer. Accordingly, in one embodiment, the diluent contains the same concentrations of the buffer and stabilizer as is present in the pharmaceutical composition of the invention. Further exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution which is preferably an acetate buffer, sterile saline solution, Ringer's solution or dextrose solution. In one embodiment the diluent comprises or consists essentially of acetate buffer and sorbitol. As used herein, the term “about” refers to ±10% of a specified value. CLL Treatment Regimens Provided herein are methods of treating CLL in a human subject using a bispecific antibody which binds to CD3 and CD20 (“anti-CD3xCD20 antibody”), e.g., an isolated anti-CD3xCD20 antibody which binds to human CD3 and human CD20. The methods are also useful for treating recurrent or refractory CLL (R/R CLL). It is understood that the methods of treating CLL with a bispecific antibody which binds to both CD3 and CD20 described herein also encompass corresponding uses of the bispecific antibody for treating CLL in a human subject. Accordingly, in one aspect, provided herein is a method of treating CLL in a human subject, the method comprising administering (e.g., subcutaneously) to the subject an effective amount of a bispecific antibody comprising: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences that are in the VH region sequence of SEQ ID NO: 6, and the VL region comprises the CDR1, CDR2 and CDR3 sequences that are in the VL region sequence of SEQ ID NO: 7; and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region and a VL region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences that are in the VH region sequence of SEQ ID NO: 13, and the VL region comprises the CDR1, CDR2 and CDR3 sequences that are in the VL region sequence of SEQ ID NO: 14; wherein the bispecific antibody is administered at a dose ranging from 12-60 mg in 28-days cycles. In some embodiments, the bispecific antibody is a full length antibody. In some embodiments, the bispecific antibody is an antibody with an inert Fc region. In some embodiments, the bispecific antibody is a full length antibody with an inert Fc region. In some embodiments, the bispecific antibody is administered at a dose of (or a dose of about) 12 mg. In some embodiments, the bispecific antibody is administered at a dose of (or a dose of about) 24 mg. In some embodiments, the bispecific antibody is administered at a dose of (or a dose of about) 48 mg. In some embodiments, the bispecific antibody is administered at a dose of (or a dose of about) 60 mg. With regard to the dose of 12-60 mg of the bispecific antibody that is to be administered, or any other specified dose, it is understood that this amount refers to the amount of a bispecific antibody representing a full-length antibody, such as epcoritamab as defined in the Examples section. Hence, one may refer to administering a dose of a bispecific antibody of 24 mg as administering a dose of a bispecific antibody described herein, wherein the dose corresponds to a dose of 24 mg of epcoritamab. One of ordinary skill in the art can readily determine the amount of antibody to be administered when, for example, the antibody used differs substantially in molecular weight from the molecular weight of a full-length antibody such as epcoritamab. For instance, the amount of antibody can be calculated by dividing the molecular weight of the antibody by the weight of a full-length antibody such as epcoritamab and multiplying the outcome thereof with the specified dose as described herein. As long as the bispecific antibody (e.g., a functional variant of DuoBody-CD3xCD20) has highly similar features as DuoBody-CD3xCD20, with regard to plasma half-life, Fc inertness, and/or binding characteristics for CD3 and CD20, i.e., with regard to CDRs and epitope binding features, such antibodies are suitable for use in the methods provided herein at a dose described for a full-length antibody such as epcoritamab. In one embodiment, the bispecific anti-CD3xCD20 antibody is administered at a dose in the range of between 12 mg and 60 mg. In some embodiments, the bispecific antibody is administered at a dose of 12 mg or about 12 mg. In some embodiments, the bispecific antibody is administered at a dose of 24 mg or about 24 mg. In some embodiments, the bispecific antibody is administered at a dose of 48 mg or about 48 mg. In some embodiments, the bispecific antibody is administered at a dose of 60 mg or about 60 mg. In some embodiments, the dose of bispecific antibody is administered once every week (weekly administration) in 28-day cycles. In some embodiments, the weekly administration is performed for 2.5 28-day cycles (i.e., 10 times). In one embodiment, the dose is administered for 2.5 28-day cycles (i.e., 10 times; on days 15 and 22 of cycle 1, and days 1, 8, 15, and 22 of cycles 2 and 3). In some embodiments, after said weekly administration, one may reduce the interval of administrating the bispecific antibody to an administration once every two weeks (biweekly administration). In some embodiments, such biweekly administration may be performed for six 28-day cycles (i.e., 12 times). In some embodiments, after said biweekly administration, the interval of administrating the bispecific antibody may be reduced further to once every four weeks. In one embodiment, the administration once every four weeks may be performed for an extended period, for example, for at least 1 cycle, at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles, at least 11 cycles, at least 12 cycles, at least 13 cycles, at least 14 cycles, at least 15 cycles, at least 16 cycles, at least 17 cycles, or between 1-20 cycles, 1-19 cycles, 1-18 cycles, 1-17 cycles, 1-16 cycles, 1-15 cycles, 1-14 cycles, 1-13 cycles, 1-12 cycles, 1-10 cycles, 1-5 cycles, 5-20 cycles, 5-15 cycles, or 5-10 cycles of the 28-day cycles. In some embodiments, epcoritamab is administered once every four weeks until disease progression (e.g., as defined by the iwCLL response criteria, as shown in Table 2) or unacceptable toxicity. In one embodiment, the weekly dose is administered on cycles 1-3 (and may include priming and intermediate doses, as described below), the biweekly dose is administered on cycles 4-9, and the dose once every four weeks is administered from cycle 10 onward. It is understood that the doses referred to herein may also be referred to as a full or a flat dose in the scenarios above wherein, e.g., the weekly dose, the biweekly dose, and/or the dose every four weeks is administered is at the same level. Accordingly, when a dose of 48 mg is selected, preferably, at each weekly administration, each biweekly administration, and each administration every four weeks, the same dose of 48 mg is administered. Prior to administering the dose, a priming or a priming and subsequent intermediate (second priming) dose may be administered. This may be advantageous as it may help mitigate cytokine release syndrome (CRS) risk and severity, a side-effect that can occur during treatment with the bispecific anti-CD3xCD20 antibody described herein. Such priming, or priming and intermediate doses, are at a lower dose as compared with the flat or full dose. Accordingly, in some embodiments, prior to administering the weekly dose of 12-60 mg, a priming dose of the bispecific antibody may be administered. In one embodiment, the priming dose is administered two weeks prior to administering the first weekly dose of 12-60 mg in cycle 1. The priming dose may be in the range of 20-2000 μg (0.02 mg-2 mg), for example, in the range of 50-1000 μg (0.05 mg to 1 mg) or in the range of 70-350 μg (0.07 mg to 0.35 mg). The priming dose can be, for example, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 μg, or about 80, about 100, about 120, about 140, about 160, about 180, about 200, about 220, about 240, about 260, about 280, about 300, about 320, about 350, about 400, about 450, about 500, about 600, about 700, about 800, about 900, or about 1000 μg. In a preferred embodiment, the priming dose is in the range of 50 and 350 μg (0.05 and 0.35 mg, respectively). In a more preferred embodiment, the priming dose is 160 μg (0.16 mg) or about 160 μg (about 0.16 mg). In most preferred embodiments, the priming dose is 160 μg (0.16 mg) or about 160 μg (about 0.16 mg) of the full-length bispecific antibody. In some embodiments, after administering the priming dose and prior to administering the first weekly dose of 12-60 mg, an intermediate dose of said bispecific antibody is administered. In one embodiment, the priming dose is administered on day 1 and the intermediate dose is administered on day 8 before the first weekly dose of 12-60 mg on days 15 and 22 of cycle 1 i.e. the priming dose is administered one week before the intermediate dose (i.e., day 1 of cycle 1), and the intermediate dose is administered one week before the first weekly dose of 12-60 mg (day 8 of cycle 1). The intermediate dose is selected from a range in between the priming dose and the flat or full dose. For example, the intermediate dose may be in the range of 200-8000 μg (0.2-8 mg), e.g., in the range of 400-4000 μg (0.4-4 mg) or 600-2000 μg (0.6-2 mg). The intermediate dose can be, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or 1600 μg, or about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, or about 1600 μg. In a preferred embodiment, the intermediate dose is in the range of 600 and 1200 μg (0.6 and 1.2 mg, respectively). In a more preferred embodiment, the intermediate dose is 800 μg (0.8 mg) or about 800 μg (0.8 mg). In a most preferred embodiment, the intermediate dose is 800 μg or about 800 μg (0.8 mg) of the full-length bispecific antibody. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 12 mg or about 12 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12 mg or about 12 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12 mg or about 12 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12 mg or about 12 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 12 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 12 mg or about 12 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12 mg or about 12 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12 mg or about 12 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12 mg or about 12 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 24 mg or about 24 mg on days 15 and 22;b) in cycles 2-3, a full dose of 24 mg or about 24 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 24 mg or about 24 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 24 mg or about 24 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05 and 0.35 is administered on day 1, an intermediate dose in the range of 0.6 and 1.2 mg on day 8, and a full dose of 24 mg on days 15 and 22;b) in cycles 2-3, a full dose of 24 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 24 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 24 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 24 mg or about 24 mg on days 15 and 22;b) in cycles 2-3, a full dose of 24 mg or about 24 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 24 mg or about 24 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 24 mg or about 24 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 48 mg or about 48 mg on days 15 and 22;b) in cycles 2-3, a full dose of 48 mg or about 48 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 48 mg or about 48 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 48 mg or about 48 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 48 mg on days 15 and 22;b) in cycles 2-3, a full dose of 48 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 48 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 48 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 48 mg or about 48 mg on days 15 and 22;b) in cycles 2-3, a full dose of 48 mg or about 48 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 48 mg or about 48 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 48 mg or about 48 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 60 mg or about 60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 60 mg or about 60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 60 mg or about 60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 60 mg or about 60 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 60 mg is administered on day 1. In some embodiments, the bispecific antibody is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 60 mg or about 60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 60 mg or about 60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 60 mg or about 60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 60 mg or about 60 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 12 mg or about 12 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12 mg or about 12 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12 mg or about 12 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12 mg or about 12 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 12 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 12 mg or about 12 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12 mg or about 12 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12 mg or about 12 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12 mg or about 12 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 24 mg or about 24 mg on days 15 and 22;b) in cycles 2-3, a full dose of 24 mg or about 24 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 24 mg or about 24 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 24 mg or about 24 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 24 mg on days 15 and 22;b) in cycles 2-3, a full dose of 24 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 24 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 24 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 24 mg or about 24 mg on days 15 and 22;b) in cycles 2-3, a full dose of 24 mg or about 24 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 24 mg or about 24 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 24 mg or about 24 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 48 mg or about 48 mg on days 15 and 22;b) in cycles 2-3, a full dose of 48 mg or about 48 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 48 mg or about 48 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 48 mg or about 48 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 48 mg on days 15 and 22;b) in cycles 2-3, a full dose of 48 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 48 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 48 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 48 mg or about 48 mg on days 15 and 22;b) in cycles 2-3, a full dose of 48 mg or about 48 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 48 mg or about 48 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 48 mg or about 48 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 60 mg or about 60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 60 mg or about 60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 60 mg or about 60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 60 mg or about 60 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered (e.g., subcutaneously) in 28-day cycles, whereina) in cycle 1, a priming dose in the range of 0.05-0.35 is administered on day 1, an intermediate dose in the range of 0.6-1.2 mg on day 8, and a full dose of 60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 60 mg is administered on day 1. In some embodiments, the bispecific antibody is epcoritamab, which is administered subcutaneously in 28-day cycles, whereina) in cycle 1, a priming dose of 160 μg is administered on day 1, an intermediate dose of 800 μg on day 8, and a full dose of 60 mg or about 60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 60 mg or about 60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 60 mg or about 60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 60 mg or about 60 mg is administered on day 1. In one embodiment, on days 1 and 8 of the first cycle, a priming dose of 80 μg and an intermediate dose of 800 μg, respectively, is selected. In some embodiments, on days 1 and 8 of the first cycle, a priming dose of 80 μg and an intermediate dose of 1200 μg, respectively, is selected. In some embodiments, on days 1 and 8 of the first cycle, a priming dose of 80 μg and an intermediate dose of 1600 μg, respectively, is selected. In some embodiments, on days 1 and 8 of the first cycle, a priming dose of 160 μg and an intermediate dose of 1200 μg, respectively, is selected. In some embodiments, on days 1 and 8 of the first cycle, a priming dose of 160 μg and an intermediate dose of 1600 μg, respectively, is selected. In one embodiment, the human subject has active CLL disease that needs treatment, meeting at least one of the following criteria: (1) evidence of progressive marrow failure as manifested by the development of, or worsening of, anemia and/or thrombocytopenia; (2) Massive (i.e., ≥6 cm below the left costal margin) or progressive or symptomatic splenomegaly; (3) Massive nodes (i.e., ≥10 cm in longest diameter) or progressive or symptomatic lymphadenopathy; (4) Progressive lymphocytosis with an increase of ≥50% over a 2-month period, or lymphocyte doubling time (LDT)<6 months; (5) Autoimmune complications including anemia or thrombocytopenia poorly responsive to corticosteroids; (6) Symptomatic or functional extra nodal involvement (e.g., skin, kidney, lung, spine); and/or (7) Disease-related symptoms as defined by any of the following: unintentional weight loss ≥10% within the previous 6 months, significant fatigue, fevers ≥38.0° C. (100.5° F.) for 2 or more weeks without evidence of infection, and night sweats for ≥1 month without evidence of infection. In some embodiments, the CLL disease is relapsed and/or refractory CLL. In some embodiments, the CLL is refractory to a BTK inhibitor. In some embodiments, the CLL relapsed during treatment with a BTK inhibitor. In some embodiments, the human subject has received at least one line of treatment prior to being treated with the methods described herein. For instance, in one embodiment, the subject has received one prior line of treatment. In some embodiments, the subject has received two prior lines of treatment. In some embodiments, the subject has received two prior lines of systemic antineoplastic therapy. In some embodiments, the subject has received two prior lines of systemic antineoplastic therapy, wherein at least one of the at least two prior antineoplastic therapy comprises treatment with (or intolerance of) a BTK inhibitor (e.g., ibrutinib). In some embodiments, the subject has refractory and/or relapsed CLL after receiving the two prior antineoplastic therapies. Relapse may be defined as evidence of disease progression in a subject who has previously achieved a CR or PR for at least 6 months. Refractory disease may be defined as treatment failure (not achieving a CR or PR) or as progression within 6 months from the last dose of therapy. In some embodiments, the subject has received three prior lines of treatment. In some embodiments, the subject has received more than three prior lines of treatment. In some embodiments, the subject has received one, two, three, or more prior lines of treatment. In some embodiments, the subject has received at least two prior lines of treatment. In one embodiment, a prior line of treatment comprises systemic antineoplastic therapy. In one embodiment, the systemic antineoplastic therapy comprises treatment with a BTK inhibitor, e.g., ibrutinib. In some embodiments, the subject is intolerant to a BTK inhibitor, e.g., ibrutinib. In other embodiments, a prior line of therapy comprises treatment with a BCL2 inhibitor, e.g. venetoclax. In still further embodiments, a prior line of therapy comprises a treatment with a combination of a BTK inhibitor and a BCL2 inhibitor (e.g., ibrutinib and venetoclax). In some embodiments, the human subject has measurable disease meeting at least one of (a) ≥5×109/L (5,000/μL) B lymphocytes in peripheral blood and (b) presence of measurable lymphadenopathy and/or organomegaly. In some embodiments, the human subject has an ECOG performance status score of 0 or 1. Information regarding ECOG performance status scores can be found in, e.g., Oken et al,Am J Clin Oncol1982 December; 5(6):649-55). In some embodiments, the human subject has acceptable laboratory parameters for (1) creatine clearance or serum creatine (>45 mL/min using Cockcroft-Gault formula or serum creatinine ≤1.5 times the upper limit of normal (×ULN)), (2) serum alanine transaminase (≤2.5×ULN), (3) serum aspartate transaminase (≤2.5×ULN), (4) bilirubin (≤1.5×ULN unless due to Gilbert syndrome), (5) hemoglobin (≥9.0 g/dL unless anemia is due to marrow involvement of CLL), (6) absolute neutrophil count (≥1.0×109/L (1000/μL) unless neutropenia is due to bone marrow involvement of CLL), platelet count (≥30×109/L (30,000/μL)), and coagulation status (PT/INR/aPTT≤1.5×ULN). A human subject receiving a treatment described herein may be a patient having one or more of the inclusion criteria set forth in Example 2, or not having one or more of the exclusion criteria set forth in Example 2. Human subjects with CLL are classified as having a CD20-positive cancer. Thus, prior cancer treatments such human subjects may have received include anti-CD20 monoclonal antibodies (e.g., rituximab). During such treatments, or any other treatments, the CLL may be refractory or have relapsed to said treatment. Accordingly, in one embodiment, the subject has received prior to treatment with the bispecific antibody a treatment with an anti-CD20 monoclonal antibody, such as rituximab or obinutuzumab. In some embodiments, during said prior treatment with the anti-CD20 antibody or combinations of anti-CD20 monoclonal antibody with one therapeutic agent, e.g., venetoclax (a Bcl2 inhibitor), the CLL relapsed or was refractory to treatment. The methods described herein are advantageous for treating CLL, such as refractory or recurrent CLL. The treatment is maintained continuously using, e.g., the treatment regimens described above. However, treatment may be terminated when progressive disease develops or unacceptable toxicity occurs. The response of subjects with CLL to the methods described herein may be assessed according to the iwCLL response criteria, as shown in Table 2 (source: Hallek et al.,Lancet2018; 391:1524-1537). TABLE 2International Workshop on Chronic Lymphocytic Leukemia Response CriteriaGroupParameterCRPRPDSDGroup A: Assessment of lymphoid tumor load and constitutional symptomsLymph nodesNone ≥1.5 cmDecrease ≥50%Increase ≥50%Change of −49%(from baseline)1from baseline orto +49%from responseLiver and/orSpleen size <13Decrease ≥50%Increase ≥50%Change of −49%spleen size2cm; liver sizefrom baselinefrom baseline orto +49%normalfrom responseConstitutionalNoneAnyAnyAnysymptomsCirculatingNoneDecrease ≥50%Increase ≥50%Change of −49%lymphocytefrom baselinefrom baseline orto +49%countfrom responseGroup B: Assessment of hematopoietic systemPlatelet count≥100 × 109/L≥100 × 109/LDecrease ≥50%Change of −49%(≥100,000 μL)(≥100,000 μL)from baselineto +49%or increase ≥50%secondary toover baselineCLLHemoglobin≥11.0 g/dL≥11.0 g/dL orDecrease of ≥2.0Increase <11.0(untransfusedincrease of ≥50%g/dL fromg/dL or <50%and withoutover baselinebaseline,over baseline orerythropoietin)secondary todecrease <2.0CLLg/dLBone marrowNormocellular,Presence of CLLIncrease of CLLNo change inno CLL cells, nocells or B-cells by ≥50%marrowB-lymphoidlymphoidon successiveinfiltrationnodulesnodules, or notbiopsiesdoneAbbreviations:CLL = chronic lymphocytic leukemia;CR = complete response;PD = progressive disease;PR = partial response;SD = stable disease.Definitions:CR, complete remission (all the criteria have to be met);PD, progressive disease (at least 1 of the criteria of group A or group B has to be met);PR, partial remission (for a PR, at least 2 of the parameters of group A and 1 parameter of group B need to improve if previously abnormal; if only 1 parameter of both groups A and B is abnormal before therapy, only 1 needs to improve);SD, stable disease (all of the criteria have to be met; constitutional symptoms alone do not define PD).1Sum of the products of 6 or fewer lymph nodes (as evaluated by CT scans and physical examination in clinical trials or by physical examination in general practice).2Spleen size is considered normal if <13 cm. There is not firmly established international consensus on the size of a normal liver; therefore, liver size should be evaluated by imaging and manual palpation and recorded in eCRF. Subjects treated according to the methods described herein preferably experience improvement in at least one sign of CLL. In one embodiment, improvement is measured by a reduction in the number of circulating lymphocytes. In some embodiments, improvement is measured by a reduction in the quantity and/or size of measurable tumor lesions. In some embodiments, lesions can be measured on CT or MRI films. In some embodiments, cytology or histology can be used to evaluate responsiveness to a therapy. In some embodiments, bone marrow aspirate and bone marrow biopsy can be used to evaluate response to therapy. In one embodiment, the subject treated exhibits a complete response (CR), a partial response (PR), or stable disease (SD), as defined by iwCLL response criteria (see, e.g., Table 2). In some embodiments, the methods described herein produce at least one therapeutic effect chosen from prolonged survival, such as progression-free survival or overall survival, optionally compared to another therapy or placebo. In some embodiments, the subjects are treated with the methods described herein until disease progression (PD) or unacceptable toxicity. Cytokine release syndrome (CRS) can occur when methods are used in human subjects that utilize immune cell- and bispecific antibody-based approaches that function by activation of immune effector cell, such as by engaging CD3 (Lee et al.,Biol Blood Marrow Transplant2019; 25:625-38, which is incorporated herein by reference). Hence, in some embodiments, CRS mitigation is performed together with the methods described herein. As part of CRS mitigation, the selection of a priming dose and/or intermediate dose is performed prior to administering the full dose (e.g., 12-60 mg), as described herein. CRS can be classified in accordance with standard practice (e.g. as outlined in Lee et al.,Biol Blood Marrow Transplant.2019 April; 25(4):625-638, which is incorporated herein by reference). CRS may include excessive release of cytokines, for example of proinflammatory cytokines, e.g., IL-6, TNF-alpha or IL-8, that may result in adverse effects like fever, nausea, vomiting and chills. Thus, despite the unique anti-tumor activity of bispecific antibodies such as epcoritamab, their immunological mode of action may trigger unwanted “side” effects, i.e., the induction of unwanted inflammatory reactions. Hence, patients may be further subjected to a concomitant treatment, prophylaxis, and/or premedication with, e.g., analgesics, antipyretics, and/or anti-inflammatory drugs to mitigate possible CRS symptoms. Accordingly, in one embodiment, human subjects in the methods described herein are treated with prophylaxis for CRS. In preferred embodiments, the prophylaxis comprises the administration of a corticosteroid to the subject. In one embodiment, the prophylaxis (e.g. corticosteroid) is administered on the same day as the bispecific antibody. The prophylaxis (e.g. corticosteroid) can also be administered on the subsequent days as well. In some embodiments, the prophylaxix (e.g. corticosteroid) is further administered on subsequent days 2, 3, and 4. It is understood that days 2, 3 and 4 when relating to further medication, such as prophylaxis, is relative to the administration of the bispecific antibody which is administered on day 1. For example, when in a cycle the antibody is administered on day 15, and prophylaxis is also administered, the prophylaxis corresponding to days 2, 3 and 4 are days 16, 17, and 18 of the cycle. In some embodiments, the prophylaxis is administered on the day when the bispecific antibody is administered and on subsequent days 2-4. When said prophylaxis is administered on the same day as the bispecific antibody, the prophylaxis is preferably administered 30-120 minutes prior to said administration of the bispecific antibody. An exemplary corticosteroid suitable for use in the methods and uses described herein is prednisolone. In some embodiments, the corticosteroid is prednisolone. In some embodiments, prednisolone is administered at an intravenous dose of 100 mg, or an equivalent thereof, including an oral dose. Exemplary corticosteroid equivalents of prednisolone, along with dosage equivalents, which can be used for CRS prophylaxis are shown in Table 6. Furthermore, in some embodiments, human subjects in the methods described herein are treated with premedication to reduce reactions to injections. In one embodiment, the premedication includes the administration of antihistamines. In some embodiments, the premedication includes the administration of antipyretics. In a further embodiment, the premedication includes systemic administration of antihistamines and antipyretics. An exemplary antihistamine suitable for use in premedication is diphenhydramine. In some embodiments, the antihistamine is diphenhydramine. In one embodiment, diphenhydramine is administered at an intravenous or oral dose 50 mg, or an equivalent thereof. An exemplary antipyretic suitable for use in premedication is acetaminophen. In some embodiments, the antipyretic is acetaminophen. In one embodiment, acetaminophen is administered at an oral dose of 560-1000 mg, such as 650-1000 mg, or equivalent thereof. In some embodiments, the premedication is administered on the same day as the bispecific antibody. In some embodiments, the premedication is administered on the same day as the bispecific antibody prior to the injection with the bispecific antibody, e.g., 30-120 minutes prior to administration of the bispecific antibody. Premedication and/or prophylaxis can be administered at least in the initial phase of the treatment. In some embodiments, premedication and/or prophylaxis is administered during the first four administrations of the bispecific antibody. For example, the premedication and/or prophylaxis can be administered as described herein, during the first 28 day cycle of the bispecific antibody administration. In some embodiments, the premedication is administered during cycle 1. In some embodiments, the prophylaxis is administered during cycle 1. Usually, risk of reactions during the initial treatment subsides after a few administrations, e.g., after the first four administrations (first cycle). Hence, and when the human subject does not experience CRS, prophylaxis for CRS may be stopped. However, when the human subject experiences a CRS greater than grade 1, CRS prophylaxis may continue. Likewise, premedication may also optionally continue. CRS grading can be performed as described in Tables 7 and 8. In a further embodiment, in the methods described herein, the prophylaxis is administered during the second 28-day cycle i.e cycle 2, when the human subject experiences CRS greater than grade 1 after the fourth i.e. last administration of the bispecific antibody in cycle 1. Furthermore, the prophylaxis can be continued during a subsequent cycle, when in the last administration of the bispecific antibody of the previous cycle, the human subject experiences CRS greater than grade 1. Any premedication may be optionally administered during the second cycle. In some embodiments, the premedication is administered during cycle 2. Further premedication may be optionally administered during subsequent cycles as well. In some embodiments, the premedication is administered during subsequent cycles (after cycle 2). In one embodiment, premedication and prophylaxis for CRS is administered, wherein the premedication comprises an antihistamine such as diphenhydramine (e.g., at an intravenous or oral dose 50 mg, or an equivalent thereof) and the prophylaxis comprises an antipyretic such as acetaminophen (e.g., at an oral dose of 650-1000 mg, or an equivalent thereof), and a corticosteroid such as prednisolone (e.g., at an intravenous dose of 100 mg, or an equivalent thereof). In some embodiments, the premedication and prophylaxis is administered 30-120 minutes prior to administration of the bispecific antibody. On subsequent days 2, 3, and optionally day 4, further prophylaxis is administered comprising the systemic administration of a corticosteroid such as prednisolone (e.g., at an intravenous dose of 100 mg, or an equivalent thereof). In some embodiments, the premedication and prophylaxis schedule preferably is administered during the first four administrations of the bispecific antibody, e.g., during the first 28-day cycle of bispecific antibody administration described herein. Furthermore, subsequent cycles, in case of, e.g., CRS greater than grade 1 occurring during the last administration of the prior cycle, can include the same administration schedule, wherein the premedication as part of the administration schedule is optional. During the treatment of a human subject with CLL using the doses and treatment regimens described herein, CRS can be well managed while at the same time effectively controlling and/or treating CLL. As described in the Examples, subjects treated with the methods described herein may experience manageable CRS. In some cases, subjects receiving the treatment described herein may develop CRS of grade 1 as defined in accordance with standard practice. In other cases, subjects may develop manageable CRS of grade 2 as defined in accordance with standard practice. Hence, subjects receiving the treatments described herein may have manageable CRS of grade 1 or grade 2 during as defined in accordance with standard practice. In accordance with standard classification for CRS, a grade 1 CRS includes a fever to at least 38° C., no hypotension, no hypoxia, and a grade 2 CRS includes a fever to at least 38° C. plus hypotension, not requiring vasopressors and/or hypoxia requiring oxygen by low flow nasal cannula or blow by. Such manageable CRS can occur during cycle 1. Human subjects receiving the treatments described herein may also have CRS greater than grade 2 during the treatments as defined in accordance with standard practice. Hence, human subjects receiving the treatments described herein may also have CRS of grade 3 during said treatments as defined in accordance with standard practice. Such manageable CRS may further occur during cycle 1 and subsequent cycles. Human subjects treated according to the methods described herein may also experience pyrexia, fatigue, and injection site reactions. They may also experience neurotoxicity, partial seizures, agraphia related to CRS, or confusional state related to CRS. As mentioned above, subjects may develop CRS during treatment with the methods described herein, despite having received CRS prophylaxis. CRS grading criteria are described in Tables 7 and 8. In one embodiment, subject is administered antibiotics if the subject develops Grade 1 CRS i.e. subjects who develop Grade 1 CRS are treated with antibiotics if they present with infection. In some embodiments, the antibiotics are continued until neutropenia, if present, resolves. In some embodiments, subjects with Grade 1 CRS who exhibit constitutional symptoms are treated with NSAIDs. In one embodiments, subjects who develop Grade 2 CRS are treated with intravenous fluid boluses and/or supplemental oxygen. In some embodiments, subjects who develop Grade 2 CRS are treated with a vasopressor. In some embodiments, subjects with Grade 2 CRS with comorbidities are treated with tocilizumab (a humanized antibody against IL-6 receptor, commercially available as, e.g., ACTEMRA®) and/or steroids (e.g., dexamethasone or its equivalent of methylprednisolone). In a further embodiment, a subject who presents with concurrent ICANS is administered dexamethasone. In yet a further embodiment, if the subject does not show improvement in CRS symptoms within, e.g., 6 hours, or if the subject starts to deteriorate after initial improvement, then a second dose of tocilizumab is administered together with a dose of corticosteroids. In some embodiments, if the subject is refractory to tocilizumab after three administrations, then additional cytokine therapy, e.g., an anti-IL-6 antibody (e.g., siltuximab) or an IL-1R antagonist (e.g., anakinra) is administered to the subject. In one embodiment, subjects who develop Grade 3 CRS are treated with vasopressor (e.g., norepinephrine) support and/or supplemental oxygen. In some embodiments, subjects with Grade 3 CRS are treated with tocilizumab, or tocilizumab in combination with steroids (e.g., dexamethasone or its equivalent of methylprednisolone). In some embodiments, a subject who presents with concurrent ICANS is administered dexamethasone. In a further embodiment, if the subject is refractory to tocilizumab after three administrations, then additional cytokine therapy, e.g., an anti-IL-6 antibody (e.g., siltuximab) or an IL-1R antagonist (e.g., anakinra) is administered to the subject. In one embodiment, subjects who develop Grade 4 CRS are treated with vasopressor support and/or supplemental oxygen (e.g., via positive pressure ventilation, such as CPAP, BiPAP, intubation, or mechanical ventilation). In some embodiments, the subject is administered at least two vasopressors if the subject develops Grade 4 CRS. In some embodiments, the subject is further administered a steroid i.e. the subject is administered tocilizumab and a steroid. In some embodiments, the steroid is dexamethasone. In some embodiments, the steroid is methylprednisolone. In a further embodiment, a subject who presents with concurrent ICANS is administered dexamethasone. In a further embodiment, if the subject is refractory to tocilizumab after three administrations, then additional cytokine therapy, e.g., an anti-IL-6 antibody (e.g., siltuximab) or an IL-1R antagonist (e.g., anakinra) is administered to the subject. In some embodiments, administration of tocilizumab is switched to administration of an anti-IL-6 antibody (e.g., siltuximab) if the subject is refractory to tocilizumab. In some embodiments, tocilizumab is switched to an IL-1R antagonist (e.g., anakinra) if the subject is refractory to tocilizumab. In some embodiments, the human subject receives prophylactic treatment for tumor lysis syndrome (TLS) i.e. the subject is treated with prophylaxis for tumor lysis syndrome (TLS). Classification and grading of tumor lysis syndrome can be performed using methods known in the art, for example, as described in Howard et al.N Engl J Med2011; 364:1844-54, and Coiffier et al.,J Clin Oncol2008; 26:2767-78. In some embodiments, prophylactic treatment of TLS comprises administering one or more uric acid reducing agents prior to administering the bispecific antibody i.e. the prophylaxis for TLS comprises administering one or more uric acid reducing agents prior to administration of the bispecific antibody. Exemplary uric acid reducing agents include allopurinol and rasburicase. Accordingly, in one embodiment, the prophylactic treatment of TLS comprises administering allopurinol and/or rasburicase. In some embodiments, the prophylactic treatment of TLS comprises administering allopurinol and/or rasburicase prior to administering the bispecific antibody. In one embodiment, allopurinol is administered 72 hours prior to the bispecific antibody. In some embodiments, rasburicase is initiated after administering allopurinol but prior to administering the bispecific antibody. Reassessment of the subject's TLS risk category can be performed prior to subsequent doses of the bispecific antibody. A subject is considered to be at low risk of TLS if all measurable lymph nodes have a largest diameter <5 cm and ALC<25×109/L. A subject is considered to be at medium risk of TLS if any measurable lymph node has a largest diameter ≥5 cm but <10 cm or ALC≥25×109/L. A subject is considered to be at high risk of TLS if (a) any measurable lymph node has a largest diameter ≥10 cm, or (b) ALC≥25×109/L and any measurable lymph node has a largest diameter ≥5 cm but <10 cm. Subjects with a lymphocyte count >100×109/L are considered as high risk. In some embodiments, when the subject shows signs of TLS, supportive therapy, such as rasburicase and/or allopurinol, may be used. In one embodiment, the bispecific antibody used in the methods described herein is administered subcutaneously, and thus is formulated in a pharmaceutical composition such that it is compatible with subcutaneous (s.c.) administration, i.e., having a formulation and/or concentration that allows pharmaceutical acceptable s.c. administration at the doses described herein. In some embodiments, subcutaneous administration is carried out by injection. For example, formulations for DuoBody-CD3xCD20 that are compatible with subcutaneous formulation and can be used in the methods described herein have been described previously (see, e.g., WO2019155008, which is incorporated herein by reference). In some embodiments, the bispecific antibody may be formulated using sodium acetate trihydrate, acetic acid, sodium hydroxide, sorbitol, polysorbate 80, and water for injection, and have a pH of 5.5 or about 5.5. In some embodiments, the bispecific antibody is provided as a 5 mg/mL or 60 mg/mL concentrate. In other embodiments, the desired dose of the bispecific antibody is reconstituted to a volume of about 1 mL for subcutaneous injection. In one embodiment, a suitable pharmaceutical composition for the bispecific antibody can comprise the bispecific antibody, 20-40 mM acetate, 140-160 mM sorbitol, and a surfactant, such as polysorbate 80, and having a pH of 5.3-5.6. In some embodiments, the pharmaceutical formulation may comprise an antibody concentration in the range of 5-100 mg/mL, e.g., 48 or 60 mg/mL of the bispecific antibody, 30 mM acetate, 150 mM sorbitol, 0.04% w/v polysorbate 80, and have a pH of 5.5. Such a formulation may be diluted with, e.g., the formulation buffer to allow proper dosing and subcutaneous administration. The volume of the pharmaceutical composition is appropriately selected to allow for subcutaneous administration of the antibody. For example, the volume to be administered is in the range of about 0.3 mL to about 3 mL, such as from 0.3 mL to 3 mL. The volume to be administered can be 0.5 mL, 0.8 mL, 1 mL, 1.2 mL, 1.5 ml, 1.7 mL, 2 mL, or 2.5 mL, or about 0.5 mL, about 0.8 mL, about 1 mL, about 1.2 mL, about 1.5 ml, about 1.7 mL, about 2 mL, or about 2.5 mL. Accordingly, in some embodiments, the volume to be administered is 0.5 mL or about 0.5 mL. In some embodiments, the volume to be administered is 0.8 mL or about 0.8 mL. In some embodiments, the volume to be administered is 1 mL or about 1 mL. In some embodiments, the volume to be administered is 1.2 mL or about 1.2 mL. In some embodiments, the volume to be administered is 1.5 mL or about 1.5 mL. In some embodiments, the volume to be administered is 1.7 mL or about 1.7 mL. In some embodiments, the volume to be administered is 2 mL or about 2 mL. In some embodiments, the volume to be administered is 2.5 mL or about 2.5 mL. The methods (or uses of CD3xCD20 antibodies) described herein are for the treatment of human patients with CLL. It is understood that the methods described herein may be the first, or part of the first, treatment provided to such patients. However, patients may have been subjected to prior treatments for CLL. Prior treatments may include, but are not limited to, one or more of chemotherapy, immunotherapy, and targeted therapy, or combinations thereof. Most commonly, the standard of care comprises treatments with a combination of cytotoxic chemotherapy and anti-CD20 monoclonal antibodies. It is understood that the methods described herein may also be used in combination with other treatments. In one embodiment, the bispecific antibody used in the methods described herein comprises: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences within the amino acid sequence of SEQ ID NO: 6, and the VL region comprises the CDR1, CDR2 and CDR3 sequences within the amino acid sequence of SEQ ID NO: 7; and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region and a VL region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences within the amino acid sequence of SEQ ID NO: 13, and the VL region comprises the CDR1, CDR2 and CDR3 sequences within the amino acid sequence SEQ ID NO: 14. CDR1, CDR2 and CDR3 regions can be identified from variable heavy and light chain regions using methods known in the art. The CDR regions from said variable heavy and light chain regions can be annotated according to IMGT (see Lefranc et al.,Nucleic Acids Research1999; 27:209-12 and Brochet.Nucl Acids Res2008; 36:W503-8). In some embodiments, the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises VHCDR1, VHCDR2 and VHCDR3 the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 4, the sequence GTN, and SEQ ID NO: 5, respectively; and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 11, the sequence DAS, and SEQ ID NO: 12, respectively. In some embodiments, the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6, and a VL region comprising the amino acid sequence of SEQ ID NO: 7; and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13, and a VL region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments, the bispecific antibody is a full-length antibody. In some embodiments, the bispecific antibody comprises an inert Fc region. In one embodiment, the bispecific antibody is a full-length antibody and have an inert Fc region. In some embodiments, the first binding arm for CD3 is derived from a humanized antibody, e.g., from a full-length IgG1,λ (lambda) antibody such as H1L1 described in WO2015001085, which is incorporated herein by reference, and/or the second binding arm for CD20 is derived from a human antibody, e.g., from a full-length IgG1,κ (kappa) antibody such as clone 7D8 as described in WO2004035607, which is incorporated herein by reference. The bispecific antibody may be produced from two half molecule antibodies, wherein each of the two half molecule antibodies comprises, e.g., the respective first and second binding arms set forth in SEQ ID NOs: 24 and 25, and SEQ ID NOs: 26 and 27. The half-antibodies may be produced in CHO cells and the bispecific antibodies generated by, e.g., Fab-arm exchange. In one embodiment, the bispecific antibody is a functional variant of DuoBody-CD3xCD20. Accordingly, in some embodiments, the bispecific antibody comprises (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a VH region comprising an amino acid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6 or a VH region comprising the amino acid sequence of SEQ ID NO: 6, but with 1, 2, or 3 mutations (e.g., amino acid substitutions), and a VL region comprising an amino acid sequence which is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or a VL region comprising the amino acid sequence of SEQ ID NO: 7, but with 1, 2, or 3 mutations (e.g., amino acid substitutions); and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region comprising an amino acid sequence which is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 13 or a VH region comprising the amino acid sequence of SEQ ID NO: 13, but with 1, 2, or 3 mutations (e.g., amino acid substitutions), and a VL region comprising an amino acid sequence which is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 14 or a VL region comprising the amino acid sequence of SEQ ID NO: 14, but with 1, 2, or 3 mutations (e.g., amino acid substitutions). In one embodiment, the bispecific antibody comprises: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, and a light chain comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region comprising the amino acid sequence of SEQ ID NO: 26, and a VL region comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody comprises (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a heavy chain comprising an amino acid sequence which is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 24 or a heavy chain comprising the amino acid sequence of SEQ ID NO: 24, but with 1, 2, or 3 mutations (e.g., amino acid substitutions), and a light chain comprising an amino acid sequence which is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 25 or a light chain region comprising the amino acid sequence of SEQ ID NO: 25, but with 1, 2, or 3 mutations (e.g., amino acid substitutions); and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a heavy chain comprising an amino acid sequence which is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 26 or a heavy chain comprising the amino acid sequence of SEQ ID NO: 26, but with 1, 2, or 3 mutations (e.g., amino acid substitutions), and a light chain comprising an amino acid sequence which is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 27 or a light chain region comprising the amino acid sequence of SEQ ID NO: 27, but with 1, 2, or 3 mutations (e.g., amino acid substitutions). Various constant regions or variants thereof may be used in the bispecific antibody. In one embodiment, the antibody comprises an IgG constant region, such as a human IgG1 constant region, e.g., a human IgG1 constant region as defined in SEQ ID NO: 15, or any other suitable IgG1 allotype. In some embodiments, the bispecific antibody is a full-length antibody with a human IgG1 constant region. In some embodiments, the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full-length IgG1,λ (lambda) antibody. In one embodiment, the first binding arm of the bispecific antibody is derived from a humanized antibody, e.g., from a full-length IgG1) (lambda) antibody, and thus comprises a κ light chain constant region. In some embodiments, the first binding arm comprises a κ light chain constant region as defined in SEQ ID NO: 22. In some embodiments, the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody. In some embodiments the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1,κ (kappa) antibody, and thus may comprise a κ light chain constant region. In some embodiments, the second binding arm comprises a κ light chain constant region as defined in SEQ ID NO: 23. In a preferred embodiment, the first binding arm comprises a κ light chain constant region as defined in SEQ ID NO: 22 and the second binding arm comprises a κ light chain constant region as defined in SEQ ID NO: 23. It is understood that the constant region portion of the bispecific antibody may comprise modifications that allow for efficient formation/production of bispecific antibodies and/or provide for an inert Fc region. Such modifications are well known in the art. Different formats of bispecific antibodies are known in the art (reviewed by Kontermann,Drug Discov Today2015; 20:838-47; MAbs,2012; 4:182-97). Thus, the bispecific antibody used in the methods and uses described herein are not limited to any particular bispecific format or method of producing it. For example, bispecific antibodies may include, but are not limited to, bispecific antibodies with complementary CH3 domains to force heterodimerization, Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329), or electrostatically-matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304). Preferably, the bispecific antibody comprises an Fc-region comprising a first heavy chain with a first Fc sequence comprising a first CH3 region, and a second heavy chain with a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions. Further details on these interactions and how they can be achieved are provided in e.g. WO2011131746 and WO2013060867 (Genmab), which are hereby incorporated by reference. In one embodiment, the bispecific antibody comprises in the first heavy chain (i) the amino acid L in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15, and comprises in the second heavy chain the amino acid R in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15, or vice versa. Bispecific antibodies may comprise modifications in the Fc region to render the Fc region inert, or non-activating. Thus, in the bispecific antibodies disclosed herein, one or both heavy chains may be modified so that the antibody induces Fc-mediated effector function to a lesser extent relative to the bispecific antibody which does not have the modification. Fc-mediated effector function may be measured by determining Fc-mediated CD69 expression on T cells (i.e. CD69 expression as a result of CD3 antibody-mediated, Fcγ receptor-dependent CD3 crosslinking), by binding to Fcγ receptors, by binding to C1q, or by induction of Fc-mediated cross-linking of FcγRs. In particular, the heavy chain constant region sequence may be modified so that Fc-mediated CD69 expression is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100% when compared to a wild-type (unmodified) antibody, wherein said Fc-mediated CD69 expression is determined in a PBMC-based functional assay, e.g. as described in Example 3 of WO2015001085. Modifications of the heavy and light chain constant region sequences may also result in reduced binding of C1q to said antibody. As compared to an unmodified antibody, the reduction may be by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100%, and C1q binding may be determined, e.g., by ELISA. Further, the Fc region which may be modified so that the antibody mediates reduced Fc-mediated T-cell proliferation compared to an unmodified antibody by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or 100%, wherein said T-cell proliferation is measured in a PBMC-based functional assay. Examples of amino acid positions that may be modified, e.g., in an IgG1 isotype antibody, include positions L234 and L235. Thus, in one embodiment, the bispecific antibody may comprises a first heavy chain and a second heavy chain, and wherein in both the first heavy chain and the second heavy chain, the amino acid residues at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to Eu numbering are F and E, respectively. In addition, a D265A amino acid substitution can decrease binding to all Fcγ receptors and prevent ADCC (Shields et al.,JBC2001; 276:6591-604). Therefore, the bispecific antibody may comprise a first heavy chain and a second heavy chain, wherein in both the first heavy chain and the second heavy chain, the amino acid residue at the position corresponding to position D265 in a human IgG1 heavy chain according to Eu numbering is A. In one embodiment, in the first heavy chain and second heavy chain of the bispecific antibody, the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively. An antibody having these amino acids at these positions is an example of an antibody having an inert Fc region, or a non-activating Fc region. In some embodiments, the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in both the first and second heavy chains, the amino acids in the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 are F, E, and A, respectively. In some embodiments, the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is R, or vice versa. In a preferred embodiment, the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein (i) in both the first and second heavy chains, the amino acids in the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 are F, E, and A, respectively, and (ii) in the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is R, or vice versa. With regard to the bispecific antibodies described herein, those which have the combination of three amino acid substitutions L234F, L235E and D265A and in addition the K409R or the F405L mutation, as described above, may be referred to with the suffix “FEAR” or “FEAL”, respectively. An amino acid sequence of a wild type IgG1 heavy chain constant region may be identified herein as SEQ ID NO: 15. Consistent with the embodiments disclosed above, the bispecific antibody may comprise an IgG1 heavy chain constant region carrying the F405L substitution and may have the amino acid sequence set forth in SEQ ID NO: 17 and/or an IgG1 heavy chain constant region carrying the K409R substitution and may have the amino acid sequence set forth in SEQ ID NO: 18, and have further substitutions that render the Fc region inert or non-activating. Hence, in one embodiment, the bispecific antibody comprises a combination of IgG1 heavy chain constant regions, with the amino acid sequence of one of the IgG1 heavy chain constant regions carrying the L234F, L235E, D265A and F405L substitutions (e.g., as set forth in SEQ ID NO: 19) and the amino acid sequence of the other IgG1 heavy chain constant region carrying the L234F, L235E, D265A and K409R substitutions (e.g., as set forth in SEQ ID NO: 20). Thus, in some embodiments, the bispecific antibody comprises heavy chain constant regions comprising the amino acid sequences of SEQ ID NOs: 19 and 20. In preferred embodiments, the bispecific antibody used in the methods and uses described herein comprises a first binding arm comprising a heavy chain and a light chain as defined in SEQ ID NOs: 24 and 25, respectively, and a second binding arm comprising a heavy chain and a light chain as defined in SEQ ID NOs: 26 and 27, respectively. Such an antibody can also be referred to herein as DuoBody-CD3xCD20. Also, variants of such antibodies are contemplated use in the methods and uses as described herein. In some embodiment, the bispecific antibody comprising a heavy chain and a light chain consisting of the amino acid sequences set forth in SEQ ID NOs: 24 and 25, respectively, and a heavy chain and a light chain consisting of the amino acid sequences set forth in SEQ ID NOs: 26 and 27, respectively. In some embodiments, the bispecific antibody is epcoritamab (CAS 2134641-34-0), or a biosimilar thereof. Kits Also provided herein are kits which include a pharmaceutical composition containing a bispecific antibody which binds to CD3 and CD20 in accordance with the invention, such as DuoBody-CD3xCD20 or epcoritamab, and a pharmaceutically-acceptable carrier, in a therapeutically effective amount adapted for use in the methods described herein. The kits optionally also can include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to administer the composition to a patient with CLL. The kit also can include a syringe. Optionally, the kits include multiple packages of the single-dose (e.g., a dose between 12-60 mg, such as 12 mg, 24 mg, 36 mg, 48 mg, or 60 mg) pharmaceutical compositions each containing an effective amount of the bispecific antibody for a single administration in accordance with the methods described herein. Instruments or devices necessary for administering the pharmaceutical composition(s) also may be included in the kits. For instance, a kit may provide one or more pre-filled syringes containing an amount of the bispecific antibody. Further Embodiments 1. A bispecific antibody comprising: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences that are in the VH region sequence of SEQ ID NO: 6, and the VL region comprises the CDR1, CDR2 and CDR3 sequences that are in the VL region sequence of SEQ ID NO: 7; and (ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region and a VL region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences that are in the VH region sequence of SEQ ID NO: 13, and the VL region comprises the CDR1, CDR2 and CDR3 sequences that are in the VL region sequence of SEQ ID NO: 14; for use in the treatment of chronic lymphocytic leukemia (CLL) in a human subject, wherein the treatment comprises administering the bispecific antibody to the human subject at a dose ranging from 12-60 mg in 28-day cycles. 2. The bispecific antibody according to embodiment 1, wherein the bispecific antibody is administered at a dose of 24 mg. 3. The bispecific antibody according to embodiment 1, wherein the bispecific antibody is administered at a dose of 48 mg. 4. The bispecific antibody according to any one of embodiments 1-3, wherein the bispecific antibody is administered once every week (weekly administration). 5. The bispecific antibody according to embodiment 4, wherein the weekly administration is performed for 2.5 28-day cycles. 6. The bispecific antibody according to embodiment 4 or 5, wherein after the weekly administration, the bispecific antibody is administered once every two weeks (biweekly administration). 7. The bispecific antibody according to embodiment 6, wherein the biweekly administration is performed for six 28-day cycles. 8. The bispecific antibody according to embodiment 6 or 7, wherein after the biweekly administration, the bispecific antibody is administered once every four weeks. 9. The bispecific antibody according to any one of embodiments 4-8, wherein prior to administering the first weekly dose of 12-60 mg, a priming dose of the bispecific antibody is administered in cycle 1 of the 28-day cycles. 10. The bispecific antibody according to embodiment 9, wherein the priming dose is administered two weeks prior to administering the first weekly dose of 12-60 mg. 11. The bispecific antibody according to embodiment 9 or 10, wherein the priming dose is in the range of 0.05-0.35 mg. 12. The bispecific antibody according to any one of embodiments 9-11, wherein said priming dose is 0.16 mg or about 0.16 mg. 13. The bispecific antibody according to any one of embodiments 9-12, wherein after administering the priming dose and prior to administering the first weekly dose of 12-60 mg, an intermediate dose of the bispecific antibody is administered. 14. The bispecific antibody according to embodiment 13, wherein the priming dose is administered on day 1 and the intermediate dose is administered on day 8 before the first weekly dose of 12-60 mg on days 15 and 22 of cycle 1. 15. The bispecific antibody according to embodiment 13 or 14, wherein said intermediate dose is in the range of 0.6-1.2 mg. 16. The bispecific antibody according to any one of embodiments 13-15, wherein said intermediate dose is 0.8 mg or about 0.8 mg. 17. The bispecific antibody according to any one of embodiments 13-16, wherein the bispecific antibody is administered in 28-day cycles, wherein: a) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. 18. The bispecific antibody according to embodiment 17, wherein the full dose is 24 mg or about 24 mg. 19. The bispecific antibody according to embodiment 17, wherein the full dose is 48 mg or about 48 mg. 20. The bispecific antibody according to any one of embodiments 1-19, wherein the bispecific antibody is administered subcutaneously. 21. The bispecific antibody according to any one of embodiments 1-20, wherein the CLL is relapsed and/or refractory CLL. 22. The bispecific antibody according to any one of embodiments 1-21, wherein the subject is intolerant to a BTK inhibitor. 23. The bispecific antibody according to any one of embodiments 1-22, wherein the subject has received at least two prior lines of antineoplastic therapy. 24. The bispecific antibody according to embodiment 23, wherein at least one of the at least two prior antineoplastic therapy comprises treatment with a BTK inhibitor. 25. The bispecific antibody according to any one of embodiments 1-24, wherein the CLL is refractory to a BTK inhibitor. 26. The bispecific antibody according to any one of embodiments 1-25, wherein the CLL relapsed during treatment with a BTK inhibitor. 27. The bispecific antibody according to any one of embodiments 21-26, wherein the subject has refractory and/or relapsed CLL after receiving the two prior antineoplastic therapies. 28. The bispecific antibody according to any one of embodiments 1-27, wherein the subject is treated with prophylaxis for cytokine release syndrome (CRS). 29. The bispecific antibody according to embodiment 28, wherein the prophylaxis comprises administering a corticosteroid to the subject. 30. The bispecific antibody according to any one of embodiment 28 or 29, wherein the corticosteroid is administered on the same day as the bispecific antibody. 31. The bispecific antibody according to embodiment 30, wherein the corticosteroid is further administered on the second, third, and fourth days after administering the bispecific antibody. 32. The bispecific antibody according to any one of embodiments 29-31, wherein the corticosteroid is prednisolone. 33. The bispecific antibody according to embodiment 32, wherein the prednisolone is administered at an intravenous dose of 100 mg, or equivalent thereof, including oral dose. 34. The bispecific antibody according to any one of embodiments 1-33, wherein the subject is administered premedication to reduce reactions to injections. 35. The bispecific antibody according to embodiment 34, wherein the premedication comprises an antihistamine. 36. The bispecific antibody according to embodiment 35, wherein the antihistamine is diphenhydramine. 37. The bispecific antibody according to embodiment 36, wherein the diphenhydramine is administered at an intravenous or oral dose of 50 mg, or equivalent thereof. 38. The bispecific antibody according to any one of embodiments 34-37, wherein the premedication comprises an antipyretic. 39. The bispecific antibody according to embodiment 38, wherein the antipyretic is acetaminophen. 40. The bispecific antibody according to embodiment 39, wherein the acetaminophen is administered at an oral dose of 560 to 1000 mg, or equivalent thereof. 41. The bispecific antibody according to any one of embodiments 34-40, wherein the premedication is administered on the same day as the bispecific antibody. 42. The bispecific antibody according to any one of embodiments 28-41, wherein the prophylaxis is administered during cycle 1. 43. The bispecific antibody according to any one of embodiments 34-42, wherein the premedication is administered during cycle 1. 44. The bispecific antibody according to embodiment 42 or 43, wherein the prophylaxis is administered during cycle 2 when the subject experiences CRS greater than grade 1 after the last administration of the bispecific antibody in cycle 1. 45. The bispecific antibody according to embodiment 44, wherein the prophylaxis is continued in a subsequent cycle, when in the last administration of the bispecific antibody of the previous cycle, the subject experiences CRS greater than grade 1. 46. The bispecific antibody according to any one of embodiments 34-45, wherein the premedication is administered during cycle 2. 47. The bispecific antibody according to embodiment 46, wherein the premedication is administered during subsequent cycles. 48. The bispecific antibody according to any one of embodiments 1-47, wherein the subject is administered antibiotics if the subject develops Grade 1 CRS. 49. The bispecific antibody according to any one of embodiments 1-47, wherein the subject is administered a vasopressor if the subject develops Grade 2 or Grade 3 CRS. 50. The bispecific antibody according to any one of embodiments 1-47, wherein the subject is administered at least two vasopressors if the subject develops Grade 4 CRS. 51. The bispecific antibody according to any one of embodiments 1-50, wherein the subject is administered tocilizumab if the subject develops Grade 2, Grade 3, or Grade 4 CRS. 52. The bispecific antibody according to embodiment 51, wherein the subject is further administered a steroid. 53. The bispecific antibody according to embodiment 52, wherein the steroid is dexamethasone. 54. The bispecific antibody according to embodiment 52, wherein the steroid is methylprednisolone. 55. The bispecific antibody according to any one of embodiments 51-54, wherein tocilizumab is switched to an anti-IL-6 antibody (e.g., siltuximab) if the subject is refractory to tocilizumab. 56. The bispecific antibody according to any one of embodiments 51-54, wherein tocilizumab is switched to an IL-1R antagonist (e.g., anakinra) if the subject is refractory to tocilizumab. 57. The bispecific antibody according to any one of embodiments 1-56, wherein the subject is treated with prophylaxis for tumor lysis syndrome (TLS). 58. The bispecific antibody according to embodiment 57, wherein the prophylaxis for TLS comprises administering one or more uric acid reducing agents prior to administration of the bispecific antibody. 59. The bispecific antibody according to embodiment 58, wherein the one or more uric acid reducing agents comprise rasburicase and/or allopurinol. 60. The bispecific antibody according to any one of embodiments 1-59, wherein the subject achieves a complete response, a partial response, or stable disease. 61. The bispecific antibody according to any one of embodiments 1-60, wherein:(i) the first antigen-binding region comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 4, the sequence GTN, and SEQ ID NO: 5, respectively; and(ii) the second antigen-binding region comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 11, the sequence DAS, and SEQ ID NO: 12, respectively. 62. The bispecific antibody according to any one of embodiments 1-61, wherein:(i) the first antigen-binding region comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6, and the VL region comprising the amino acid sequence of SEQ ID NO: 7; and(ii) the second antigen-binding region comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13, and the VL region comprising the amino acid sequence of SEQ ID NO: 14. 63. The bispecific antibody according to any one of embodiments 1-62, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full-length IgG1,λ (lambda) antibody. 64. The bispecific antibody according to embodiment 63, wherein the first binding arm of the bispecific antibody comprises a λ light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 22. 65. The bispecific antibody according to any one of embodiments 1-64, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length Ig 1κ (kappa) antibody. 66. The bispecific antibody according to embodiment 65, wherein the second binding arm comprises a κ light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 23. 67. The bispecific antibody according to any one of embodiments 1-66, wherein the bispecific antibody is a full-length antibody with a human IgG1 constant region. 68. The bispecific antibody according to any one of embodiments 1-67, wherein the bispecific antibody comprises an inert Fc region. 69. The bispecific antibody according to any one of embodiments 1-68, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in both the first and second heavy chains, the amino acids in the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 are F, E, and A, respectively. 70. The bispecific antibody according to any one of embodiments 1-69, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is R, or vice versa. 71. The bispecific antibody according to any one of embodiments 1-70, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein(i) in both the first and second heavy chains, the amino acids in the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 are F, E, and A, respectively, and(ii) in the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is R, or vice versa. 72. The bispecific antibody according to embodiment 71, wherein the bispecific antibody comprises heavy chain constant regions comprising the amino acid sequences of SEQ ID NOs: 19 and 20. 73. The bispecific antibody according to any one of embodiments 1-72, wherein the bispecific antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 24 and 25, respectively, and a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 26 and 27, respectively. 74. The bispecific antibody according to any one of embodiments 1-73, wherein the bispecific antibody comprises a heavy chain and a light chain consisting of the amino acid sequence of SEQ ID NOs: 24 and 25, respectively, and a heavy chain and a light chain consisting of the amino acid sequence of SEQ ID NOs: 26 and 27, respectively. 75. The bispecific antibody according to any one of embodiments 1-74, wherein the bispecific antibody is epcoritamab, or a biosimilar thereof. The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. 1a. A method of treating chronic lymphocytic leukemia (CLL) in a human subject, the method comprising administering to the subject a bispecific antibody comprising: (i) a first binding arm comprising a first antigen-binding region which binds to human CD3ε (epsilon) and comprises a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences that are in the VH region sequence of SEQ ID NO: 6, and the VL region comprises the CDR1, CDR2 and CDR3 sequences that are in the VL region sequence of SEQ ID NO: 7; and(ii) a second binding arm comprising a second antigen-binding region which binds to human CD20 and comprises a VH region and a VL region, wherein the VH region comprises the CDR1, CDR2 and CDR3 sequences that are in the VH region sequence of SEQ ID NO: 13, and the VL region comprises the CDR1, CDR2 and CDR3 sequences that are in the VL region sequence of SEQ ID NO: 14;wherein the bispecific antibody is administered at a dose ranging from 12-60 mg in 28-day cycles. 2a. The method of embodiment 1a, wherein the bispecific antibody is administered at a dose of 24 mg. 3a. The method of embodiment 1a, wherein the bispecific antibody is administered at a dose of 48 mg. 4a. The method of any one of embodiments 1a-3a, wherein the bispecific antibody is administered once every week (weekly administration). 5a. The method of embodiment 4a, wherein the weekly administration is performed for 2.5 28-day cycles. 6a. The method of embodiment 4a or 5a, wherein after the weekly administration, the bispecific antibody is administered once every two weeks (biweekly administration). 7a. The method of embodiment 6a, wherein the biweekly administration is performed for six 28-day cycles. 8a. The method of embodiment 6a or 7a, wherein after the biweekly administration, the bispecific antibody is administered once every four weeks. 9a. The method of any one of embodiments 4a-8a, wherein prior to administering the first weekly dose of 12-60 mg, a priming dose of the bispecific antibody is administered in cycle 1 of the 28-day cycles. 10a. The method of embodiment 9a, wherein the priming dose is administered two weeks prior to administering the first weekly dose of 12-60 mg. 11a. The method of embodiment 9a or 10a, wherein the priming dose is in the range of 0.05-0.35 mg. 12a. The method of any one of embodiments 9a-11a, wherein said priming dose is 0.16 mg or about 0.16 mg. 13a. The method of any one of embodiments 9a-12a, wherein after administering the priming dose and prior to administering the first weekly dose of 12-60 mg, an intermediate dose of the bispecific antibody is administered. 14a. The method of embodiment 13a, wherein the priming dose is administered on day 1 and the intermediate dose is administered on day 8 before the first weekly dose of 12-60 mg on days and 22 of cycle 1. 15a. The method of embodiment 13a or 14a, wherein said intermediate dose is in the range of 0.6-1.2 mg. 16a. The method of any one of embodiments 13a-15a, wherein said intermediate dose is 0.8 mg or about 0.8 mg. 17a. The method of any one of embodiments 13a-16a, wherein the bispecific antibody is administered in 28-day cycles, wherein:a) in cycle 1, a priming dose is administered on day 1, an intermediate dose on day 8, and a full dose of 12-60 mg on days 15 and 22;b) in cycles 2-3, a full dose of 12-60 mg is administered on days 1, 8, 15, and 22;c) in cycles 4-9, a full dose of 12-60 mg is administered on days 1 and 15; andd) in cycle 10 and subsequent cycles, a full dose of 12-60 mg is administered on day 1. 18a. The method of embodiment 17a, wherein the full dose is 24 mg or about 24 mg. 19a. The method of embodiment 17a, wherein the full dose is 48 mg or about 48 mg. 20a. The method of any one of embodiments 1a-19a, wherein the bispecific antibody is administered subcutaneously. 21a. The method of any one of embodiments 1a-20a, wherein the CLL is relapsed and/or refractory CLL. 22a. The method of any one of embodiments 1-21a, wherein the subject is intolerant to a BTK inhibitor. 23a. The method of any one of embodiments 1a-22a, wherein the subject has received at least two prior lines of antineoplastic therapy. 24a. The method of embodiment 23a, wherein at least one of the at least two prior antineoplastic therapy comprises treatment with a BTK inhibitor. 25a. The method of any one of embodiments 1a-24a, wherein the CLL is refractory to a BTK inhibitor. 26a. The method of any one of embodiments 1a-25a, wherein the CLL relapsed during treatment with a BTK inhibitor. 27a. The method of any one of embodiments 21a-26a, wherein the subject has refractory and/or relapsed CLL after receiving the two prior antineoplastic therapies. 28a. The method of any one of embodiments 1a-27a, wherein the subject is treated with prophylaxis for cytokine release syndrome (CRS). 29a. The method of embodiment 28a, wherein the prophylaxis comprises administering a corticosteroid to the subject. 30a. The method of embodiment 28a or 29a, wherein the corticosteroid is administered on the same day as the bispecific antibody. 31a. The method of embodiment 30a, wherein the corticosteroid is further administered on the second, third, and fourth days after administering the bispecific antibody. 32a. The method of any one of embodiments 29a-31a, wherein the corticosteroid is prednisolone. 33a. The method of embodiment 32a, wherein the prednisolone is administered at an intravenous dose of 100 mg, or equivalent thereof, including oral dose. 34a. The method of any one of embodiments 1a-33a, wherein the subject is administered premedication to reduce reactions to injections. 35a. The method of embodiment 34a, wherein the premedication comprises an antihistamine. 36a. The method of embodiment 35a, wherein the antihistamine is diphenhydramine. 37a. The method of embodiment 36a, wherein the diphenhydramine is administered at an intravenous or oral dose of 50 mg, or equivalent thereof. 38a. The method of any one of embodiments 34a-37a, wherein the premedication comprises an antipyretic. 39a. The method of embodiment 38, wherein the antipyretic is acetaminophen. 40a. The method of embodiment 39, wherein the acetaminophen is administered at an oral dose of 560 to 1000 mg, or equivalent thereof. 41a. The method of any one of embodiments 34a-40a, wherein the premedication is administered on the same day as the bispecific antibody. 42a. The method of any one of embodiments 28a-41a, wherein the prophylaxis is administered during cycle 1. 43a. The method of any one of embodiments 34a-42a, wherein the premedication is administered during cycle 1. 44a. The method of embodiment 42a or 43a, wherein the prophylaxis is administered during cycle 2 when the subject experiences CRS greater than grade 1 after the last administration of the bispecific antibody in cycle 1. 45a. The method of embodiment 44a, wherein the prophylaxis is continued in a subsequent cycle, when in the last administration of the bispecific antibody of the previous cycle, the subject experiences CRS greater than grade 1. 46a. The method of any one of embodiments 34a-45a, wherein the premedication is administered during cycle 2. 47a. The method of embodiment 46a, wherein the premedication is administered during subsequent cycles. 48a. The method of any one of embodiments 1a-47a, wherein the subject is administered antibiotics if the subject develops Grade 1 CRS. 49a. The method of any one of embodiments 1a-47a, wherein the subject is administered a vasopressor if the subject develops Grade 2 or Grade 3 CRS. 50a. The method of any one of embodiments 1a-47a, wherein the subject is administered at least two vasopressors if the subject develops Grade 4 CRS. 51a. The method of any one of embodiments 1a-50a, wherein the subject is administered tocilizumab if the subject develops Grade 2, Grade 3, or Grade 4 CRS. 52a. The method of embodiment 51a, wherein the subject is further administered a steroid. 53a. The method of embodiment 52a, wherein the steroid is dexamethasone. 54a. The method of embodiment 52a, wherein the steroid is methylprednisolone. 55a. The method of any one of embodiments 51a-54a, wherein tocilizumab is switched to an anti-IL-6 antibody (e.g., siltuximab) if the subject is refractory to tocilizumab. 56a. The method of any one of embodiments 51a-54a, wherein tocilizumab is switched to an IL-1R antagonist (e.g., anakinra) if the subject is refractory to tocilizumab. 57a. The method of any one of embodiments 1a-56a, wherein the subject is treated with prophylaxis for tumor lysis syndrome (TLS). 58a. The method of embodiment 57a, wherein the prophylaxis for TLS comprises administering one or more uric acid reducing agents prior to administration of the bispecific antibody. 59a. The method of embodiment 58a, wherein the one or more uric acid reducing agents comprise rasburicase and/or allopurinol. 60a. The method of any one of embodiments 1a-59a, wherein the subject achieves a complete response, a partial response, or stable disease. 61a. The method of any one of embodiments 1a-60a, wherein:(i) the first antigen-binding region comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 4, the sequence GTN, and SEQ ID NO: 5, respectively; and(ii) the second antigen-binding region comprises VHCDR1, VHCDR2, and VHCDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 8, 9, and 10, respectively, and VLCDR1, VLCDR2, and VLCDR3 comprising the amino acid sequences set forth in SEQ ID NO: 11, the sequence DAS, and SEQ ID NO: 12, respectively. 62a. The method of any one of embodiments 1a-61a, wherein:(i) the first antigen-binding region comprises a VH region comprising the amino acid sequence of SEQ ID NO: 6, and the VL region comprising the amino acid sequence of SEQ ID NO: 7; and(ii) the second antigen-binding region comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13, and the VL region comprising the amino acid sequence of SEQ ID NO: 14. 63a. The method of any one of embodiments 1a-62a, wherein the first binding arm of the bispecific antibody is derived from a humanized antibody, preferably from a full-length IgG1,λ (lambda) antibody. 64a. The method of embodiment 63a, wherein the first binding arm of the bispecific antibody comprises a λ light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 22. 65a. The method of any one of embodiments 1a-64a, wherein the second binding arm of the bispecific antibody is derived from a human antibody, preferably from a full-length IgG1κ (kappa) antibody. 66a. The method of embodiment 65a, wherein the second binding arm comprises a κ light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 23. 67a. The method of any one of embodiments 1a-66a, wherein the bispecific antibody is a full-length antibody with a human IgG1 constant region. 68a. The method of any one of embodiments 1a-67a, wherein the bispecific antibody comprises an inert Fc region. 69a. The method of any one of embodiments 1a-68a, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in both the first and second heavy chains, the amino acids in the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 are F, E, and A, respectively. 70a. The method of any one of embodiments 1a-69a, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is R, or vice versa. 71a. The method of any one of embodiments 1a-70a, wherein the bispecific antibody comprises a first heavy chain and a second heavy chain, wherein(i) in both the first and second heavy chains, the amino acids in the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 are F, E, and A, respectively, and(ii) in the first heavy chain, the amino acid in the position corresponding to F405 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is L, and wherein in the second heavy chain, the amino acid in the position corresponding to K409 in the human IgG1 heavy chain constant region of SEQ ID NO: 15 is R, or vice versa. 72a. The method of embodiment 71a, wherein the bispecific antibody comprises heavy chain constant regions comprising the amino acid sequences of SEQ ID NOs: 19 and 20. 73a. The method of any one of embodiments 1a-72a, wherein the bispecific antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 24 and 25, respectively, and a heavy chain and a light chain comprising the amino acid sequences set forth in SEQ ID NOs: 26 and 27, respectively. 74a. The method of any one of embodiments 1a-73a, wherein the bispecific antibody comprises a heavy chain and a light chain consisting of the amino acid sequence of SEQ ID NOs: 24 and 25, respectively, and a heavy chain and a light chain consisting of the amino acid sequence of SEQ ID NOs: 26 and 27, respectively. 75a. The method of any one of embodiments 1a-74a, wherein the bispecific antibody is epcoritamab, or a biosimilar thereof. EXAMPLES DuoBody-CD3xCD20 DuoBody-CD3xCD20 is a bsAb recognizing the T-cell antigen CD3 and the B-cell antigen CD20. DuoBody-CD3xCD20 triggers potent T-cell-mediated killing of CD20-expressing cells. DuoBody-CD3xCD20 has a regular IgG1 structure. Two parental antibodies, IgG1-CD3-FEAL, a humanized IgG1λ, CD3ε-specific antibody having heavy and light chain sequences as listed in SEQ ID NOs: 24 and 25, respectively, and IgG1-CD20-FEAR, derived from human IgG1κ CD20-specific antibody 7D8 having heavy and light chain sequences as listed in SEQ ID NOs: 26 and 27, respectively, were manufactured as separate biological intermediates. Each parental antibody contains one of the complementary mutations in the CH3 domain required for the generation of DuoBody molecules (F405L and K409R, respectively). The parental antibodies comprised three additional mutations in the Fc region (L234F, L235E and D265A; FEA). The parental antibodies were produced in mammalian Chinese hamster ovary (CHO) cell lines using standard suspension cell cultivation and purification technologies. DuoBody-CD3xCD20 was subsequently manufactured by a controlled Fab-arm exchange (cFAE) process (Labrijn et al. 2013, Labrijn et al. 2014, Gramer et al. 2013). The parental antibodies are mixed and subjected to controlled reducing conditions. This leads to separation of the parental antibodies that, under re-oxidation, re-assemble. This way, highly pure preparations of DuoBody-CD3xCD20 (˜93-95%) were obtained. After further polishing/purification, final product was obtained, close to 100% pure. The DuoBody-CD3xCD20 concentration was measured by absorbance at 280 nm, using the theoretical extinction coefficient ε=1.597 mL·mg−1cm−1. The product has received the international proprietary name of epcoritamab. Epcoritamab is prepared (5 mg/mL or 60 mg/mL) as a sterile clear colorless to slightly yellow solution supplied as concentrate for solution for subcutaneous (SC) injection. Epcoritamab contains buffering and tonicifying agents. All excipients and amounts thereof in the formulated product are pharmaceutically acceptable for subcutaneous injection products. Appropriate doses are reconstituted to a volume of about 1 mL for subcutaneous injection. Example 1: Epcoritamab-Induced Activation of CD4+ and CD8+ T Cells and Cytotoxicity of B Cells Obtained from CLL Patients Patients with CLL often have intrinsic T cell immune dysfunction which could potentially impact epcoritamab's anti-tumor activity. This experiment was performed to determine whether epcoritamab can activate T cells from CLL patients and induce cytotoxicity against B cells. Briefly, commercially obtained CLL patient PBMCs were co-cultured with healthy donor (HD) PBMCs (ratio patient:healthy donor cells 1:5) and epcoritamab, or with bispecific antibodies containing either the CD3 arm, or the CD20 arm, and a non-binding control arm (bsIgG1-CD3xctrl, and bsIgG1-ctrlxCD20, respectively, wherein controls have the same format as epcoritamab (i.e. having an inert Fc), for 24 hours. The HD PBMCs were added to improve viability of the CLL patient-derived PBMCs and were labeled with CFSE to be able to distinguish them from the CLL patient-derived PBMCs. CD69 expression was used to evaluate (A) CD4+ and (B) CD8+ T-cell activation. B-cell viability (% CD4−CD8−CD22+ cells left) was analyzed as a measure for cytotoxicity induced by epcoritamab. As shown inFIG.1, epcoritamab induced the activation of both CLL and HD CD4+ and CD8+ T cells (See panels A and B). For CD4+ T cells, HD T cells were more efficiently activated than CLL T cells (see panel A, compare the left graph with the right graph, wherein epcoritamab induced CD4+ T cell activation to a higher % of CD69 in healthy donor derived PBMCs as compared to CLL patient derived PBMCs). Epcoritamab activated both CLL and HDD CD8+ T cells to a similar degree (see panel B, the upper line of both graphs represents epcoritamab). Epcoritamab induced T-cell-mediated cytotoxicity of both CLL and HD B cells, indicating that CLL B cells are susceptible to T-cell-mediated cytotoxicity (see panel C, wherein epcoritamab reduced the viability of B cells following a dose response curve). These data demonstrate that epcoritamab activates both CD4+ and CD8+ T cells, and induces killing of B cells, from CLL patients. Example 2: A Phase 1b/2, Open-Label, Safety and Efficacy Study of Epcoritamab in Relapsed/Refractory Chronic Lymphocytic Leukemia An open-label, 2-part (dose escalation and expansion), multicenter study is conducted to evaluate the safety, tolerability, PK, pharmacodynamics, immunogenicity, and preliminary efficacy of single agent epcoritamab in subjects aged 18 years or older with relapsed and/or refractory (R/R) chronic lymphocytic leukemia (CLL). Summary of Ongoing Clinical Trial with Epcoritamab Epcoritamab is currently in a clinical trial for the treatment of R/R B-NHL (ClinicalTrials.gov Identifier: NCT03625037). Preliminary data suggest that the drug is tolerated at doses up to at least 48 mg, including 60 mg in R/R B-NHL patients, with a favourable safety profile, with no dose-limiting toxicities reported. Objectives Dose Escalation The primary objective of the dose escalation part is to identify the recommended phase 2 dose (RP2D) and maximum tolerated dose (MTD) (endpoint: incidence of dose limiting toxicities (DLTs)), and evaluate the safety and tolerability (endpoints: incidence and severity of adverse events (AEs), serious adverse events (SAEs), CRS, ICANs, and TLS, and incidence of dose interruption, dose delay, and dose intensity), of epcoritamab in subjects with R/R CLL. Secondary objectives of the dose escalation part include characterizing the PK properties of epcoritamab (endpoints: PK parameters, including clearance, volume of distribution and AUC0-last and AUC0-x, Cmax, Tmax, predose values, and half-life), evaluating pharmacodynamic markers linked to efficacy and the mechanism of action of epcoritamab (endpoints: pharmacodynamic markers in blood samples), evaluating the immunogenicity of epcoritamab (endpoint: incidence of anti-drug antibodies (ADAs) to epcoritamab), and assessing the preliminary anti-tumor activity of epcoritamab (endpoints: overall response rate (ORR), duration of response (DOR), time to response (TTR), progression free survival (PFS), and overall survival (OS)). Exploratory objectives of the dose escalation part include evaluating biomarkers predictive of clinical response to epcoritamab (endpoints: CD20 expression, evaluation of immune populations, phenotype and function and blood) and assessing the minimal residual disease (MRD) status in peripheral blood and bone marrow (endpoint: incidence of undetectable MRD). Expansion The primary objective of the expansion part is to assess the preliminary efficacy of epcoritamab (endpoint: ORR). Secondary objectives of the expansion part include evaluating the preliminary efficacy of epcoritamab (endpoints: DOR, TTR, PFS, and OS), assessing the MRD status in peripheral blood and bone marrow (endpoint: incidence of undetectable MRD), evaluating the safety and tolerability of epcoritamab (endpoints: endpoints: incidence and severity of AEs, SAEs, CRS, ICANs, and TLS, and incidence of dose interruption, dose delay, and dose intensity), establishing the PK and pharmacodynamic profiles of epcoritamab (endpoints: PK parameters and pharmacodynamic parameters), and evaluating immunogenicity of epcoritamab (endpoint: incidence of ADAs to epcoritamab). Exploratory objectives of the expansion part include evaluating biomarkers predictive of clinical response to epcoritamab (endpoints: expression of CD20 and evaluation of immune populations, phenotype, and function, and blood). Study Design Overview The trial is conducted in 2 parts: dose escalation (Part 1) and expansion (Part 2). A schematic of the overall trial design is shown inFIG.2. Both parts consist of a screening period (up to 21 days prior to Cycle 1 Day 1), a treatment period (Cycle 1 Day 1 until epcoritamab discontinuation), a safety follow-up (60 days after the last dose of epcoritamab), and a survival status follow-up. In both Part 1 and Part 2, epcoritamab is administered as a subcutaneous (SC) injection in 4-week cycles (i.e., 28 days), as shown below, until one or more of the discontinuation criteria are met:Cycle 1-3: Days 1, 8, 15 and 22 (QW)Cycle 4-9: Days 1 and 15 (Q2W)Cycle 10 and beyond: Day 1 (Q4W) A step-up dosing method is used to mitigate the potential for CRS: priming dose on Cycle 1 Day 1, followed by intermediate dose on Cycle 1 Day 8, then full dose on Cycle 1 Day 15 and Day 22, and full dose in subsequent cycles. In Part 1 (Dose Escalation), epcoritamab is tested in subjects with R/R CLL using the modified 3+3 design. DLTs are evaluated during the first treatment cycle (i.e., 28 days). After identifying the RP2D, the preliminary efficacy of single agent epcoritamab is assessed together with safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and biomarkers in Part 2 (Expansion). Dose Escalation (Part 1) The dose escalation part implements a modified 3+3 design. Epcoritamab is studied at 2 full dose levels: 24 mg and 48 mg. A step-up dosing regimen is applied: 0.16 mg/0.8 mg/24 mg and 0.16 mg/0.8 mg/48 mg (priming/intermediate/full dose). Additional doses including intermediate doses and dosing regimens may be explored based on emerging data. At each dose level, 3 subjects are initially treated. Based on the escalation rules specified following Table 3, 3 additional subjects may be needed at the current dose level or 3 subjects are treated at a different dose level. At least 6 subjects will be enrolled in Part 1. Subjects are monitored for DLTs during the first treatment cycle (i.e., 28 days). TABLE 3Escalation Rules Based on Number of Subjects with DLTsNumber of subjects evaluable forDecision, based on the number ofDLT at the current dose-levelsubjects with DLTs (NDLT)36Escalate, if NDLT≤01Remain on dose- level, if NDLT=12De-escalate, if NDLT≥23Disallow dose-level, if NDLT≥34 Subjects who are not DLT evaluable may be replaced, and at least 6 subjects are needed for a dose level to be identified as RP2D. Additional full dose levels, either lower than 24 mg (e.g., 12 mg) or higher than 48 mg (e.g., 60 mg), may be investigated. After all subjects on a dose level have completed the DLT monitoring period (i.e., Cycle 1; 28 days), all available data will be evaluated (including, but not limited to safety, PK, pharmacodynamic, and immunogenicity data) in order to inform the next dose level. Dose escalation stops when:at the lowest dose-level to be investigated, the decision would be to disallow, de-escalate or remain on the same dose-level according to the escalation rulesat the highest dose-level to be investigated, the decision would be to escalate or remain on the same dose-level according to the escalation rulesat the current dose-level, the decision would be to escalate according to the escalation rules, but a higher dose-level has been evaluated which led to dose de-escalation, or the decision would be to de-escalate according to the escalation rules but a lower dose-level has been evaluated which led to dose escalation The MTD is defined as the highest investigated dose level with DLT observed in at most one-third of subjects. The RP2D for R/R CLL will be set at 48 mg if the dose level is found to be safe and tolerable. The totality of data, including safety (e.g., adverse events (AEs) and safety laboratory values, and observations made after the end of the DLT evaluation period), pharmacokinetics, pharmacodynamics, and preliminary efficacy, will guide further development for expansion. Expansion (Part II) Once the RP2D is established, the expansion part will begin. The expansion part enrolls approximately 20 subjects with R/R CLL previously treated with 2 prior lines of systemic antineoplastic therapy, including a BTK inhibitor (e.g., ibrutinib) or are otherwise intolerant of a BTK inhibitor. R/R CLL subjects are treated at the RP2D identified in Part 1. The primary efficacy endpoint of the expansion part is ORR as assessed using the iwCLL 2018 criteria (Table 2). ORR is a widely accepted response endpoint to evaluate the efficacy for subjects with R/R CLL. Secondary efficacy endpoints include DOR, TTR, PFS, and OS. Incidence of MRD negative status is also evaluated as a secondary efficacy endpoint. MRD assessment indicates how many cancer cells still remain in a subject who is in remission either during or after treatment has been implemented. Safety endpoints in the expansion part include the incidence and severity of AEs/SAEs, incidence and severity of tumor lysis syndrome (TLS), immune effector cell-associated neurotoxicity syndrome (ICANS) and CRS, and incidence of treatment interruption and delay. Inclusion Criteria 1. Subjects must be at least 18 years of age.2. Active CLL disease that needs treatment with at least 1 of the following criteria being met:a. Evidence of progressive marrow failure as manifested by the development of, or worsening of, anemia and/or thrombocytopeniab. Massive (i.e., ≥6 cm below the left costal margin) or progressive or symptomatic splenomegalyc. Massive nodes (i.e., ≥10 cm in longest diameter) or progressive or symptomatic lymphadenopathyd. Progressive lymphocytosis with an increase of ≥50% over a 2-month period, or lymphocyte doubling time (LDT)<6 monthse. Autoimmune complications including anemia or thrombocytopenia poorly responsive to corticosteroidsf. Symptomatic or functional extra nodal involvement (e.g., skin, kidney, lung, spine)g. Disease-related symptoms as defined by any of the following:Unintentional weight loss ≥10% within the previous 6 monthsSignificant fatigueFevers ≥38.0° C. (100.5° F.) for 2 or more weeks without evidence of infection.Night sweats for ≥1 month without evidence of infection3. R/R CLL after receiving at least 2 prior lines of systemic antineoplastic therapy, including treatment with (or intolerance of) a BTK inhibitor (e.g., ibrutinib). Relapse is defined as evidence of disease progression in a subject who has previously achieved a CR or PR for ≥6 months. Refractory disease is defined as treatment failure (not achieving a CR or PR) or as progression within 6 months from the last dose of therapy.4. Measurable Disease with at least one of the following criteria:a. ≥5×109/L (5,000/μL) B lymphocytes in peripheral bloodb. Presence of measurable lymphadenopathy and/or organomegaly5. ECOG performance status score of 0 or 16. Screening flow cytometry evidence of CD20 positivity7. Has acceptable laboratory parameters as follows: TABLE 4ParameterResulta.Creatine clearance>45 mL/min (Cockcroft-Gault) or serumor serum creatinecreatinine ≤1.5 times the upper limit of normal(×ULN)b.Serum alanine≤2.5 × ULNtransaminase (ALT)c.Serum aspartate≤2.5 × ULNtransaminase (AST)d.Bilirubin≤1.5 × ULN unless due to Gilbert syndromeNote: Subjects with Gilbert's syndrome may beincluded if total bilirubin is ≤3 × ULN anddirect bilirubin is ≤1.5 × ULNe.Hemoglobin≥9.0 g/dL unless anemia is due to marrowinvolvement of CLLNote: Blood transfusion may be administeredduring screening to meet this requirementf.Absolute≥1.0 × 109/L (1000/μL) unless neutropenia isneutrophildue to bone marrow involvement of CLL.countNote: Growth factor support is allowed in caseof bone marrow involvement.g.Platelet count≥30 × 109/L (30,000/μL)Note: Transfusion may be administered duringscreening to meet this requirement.h.Coagulation statusPT/INR/aPTT ≤1.5 × ULN8. Received a cumulative dose of corticosteroids less than the equivalent of 250 mg of prednisone within the 2-week period before the first dose of epcoritamab9. Subject must have availability of fresh bone marrow material at screening.10. Must take prophylaxis for CRS/TLS.11. Subject must be willing and able to adhere to the prohibitions and restrictions specified in the protocol. Exclusion Criteria1. Transformation of CLL to aggressive non-Hodgkin lymphoma2. Subject received prior treatment with a CD3xCD20 bispecific antibody3. Subject received any prior allogeneic HSCT or solid organ transplantation4. Subject received treatment with an anti-cancer biologic including anti-CD20 therapy, radio-conjugated or toxin-conjugated antibody or CAR T-cell therapy within 4 weeks or 5 half-lives, whichever is shorter, before the first dose of epcoritamab.5. Subject received chemotherapy or radiation therapy within 2 weeks of the first dose of epcoritamab.6. Subject received treatment with an investigational drug or an invasive investigational medical device within 4 weeks or 5 half-lives, whichever is shorter, prior to the first dose of epcoritamab7. Subject has autoimmune disease or other diseases that require permanent or high-dose immunosuppressive therapy8. Subject has uncontrolled intercurrent illness, including but not limited to:a. Ongoing or active infection requiring intravenous antibiotics treatment at the time of enrollment or within the previous 2 weeks prior to the first dose of epcoritamab.b. Symptomatic congestive heart failure (grade III or IV as classified by the New York Heart Association), unstable angina pectoris or cardiac arrhythmiac. Myocardial infarction, intracranial bleed, or stroke within the past 6 months9. Has a baseline QT interval at screening as corrected by Fridericia's formula (QTcF)>480 msec10. Subject received vaccination with live vaccines within 28 days prior to the first dose of epcoritamab11. Subject has toxicities from previous anti-cancer therapies that have not resolved to baseline levels or to Grade 1 or less except for alopecia and peripheral neuropathy12. Subject has known CNS involvement at screening13. Subject has known past or current malignancy other than inclusion diagnosis, except for:a. Cervical carcinoma of Stage 1B or lessb. Non-invasive basal cell or squamous cell skin carcinomac. Non-invasive, superficial bladder cancerd. Prostate cancer with a current PSA level <0.1 ng/mLe. Any curable cancer with a CR of >2 years duration14. Subject has suspected allergies, hypersensitivity, or intolerance to epcoritamab or its excipients15. Subject is unable to tolerate uric acid reducing medications16. Subject has had major surgery (e.g., requiring general anesthesia) within 3 weeks before screening or will not have fully recovered from surgery, or has major surgery planned during the time the subject is expected to participate in the trial (or within 4 weeks after the last dose of epcoritamab); Note: Subjects with planned surgical procedures to be conducted under local anesthesia may participate.17. Subject has known history/positive serology for hepatitis B (unless immune due to vaccination or resolved natural infection or unless passive immunization due to immunoglobulin therapy):a. Positive test for antibodies to the hepatitis B core antigen (anti-HBc) ANDb. Negative test for antibodies to the hepatitis B surface antigen (anti-HBs).18. Known medical history or ongoing hepatitis C infection that has not been cured.19. Subject tests positive for HIV at screening.20. Subject is a woman who is pregnant or breast-feeding, or who is planning to become pregnant while enrolled in this trial or within 12 months after the last dose of epcoritamab.21. Subject is a man who plans to father a child while enrolled in this trial or within 12 months after the last dose of epcoritamab.22. Subject has any condition for which participation would not be in the best interest of the subject (e.g., compromise the well-being) or that could prevent, limit, or confound the protocol-specified assessments. Premedication and CRS Prophylaxis Administration of corticosteroids, antihistamines, and antipyretics for four days is performed to reduce/prevent the severity of symptoms from potential CRS for each dose of epcoritamab in cycle 1. For administration of epcoritamab in Cycle 2 and beyond, CRS prophylaxis with corticosteroids is optional. Corticosteroid administration can be either intravenous or oral route with recommended dose or equivalent. TABLE 5Prophylactic corticosteroid administration pre- and post-epcoritamab administrationCorticosteroidsAntihistaminesAntipyreticsCycle 11stDayPrednisolone 100 mg IVDiphenhydramineParacetamolepcoritamab01*(or equivalent including50 mg IV or oral(acetaminophen)administrationoral dose)(PO) (or equivalent)650 to 1000 mg PO(priming dose)(or equivalent)DayPrednisolone 100 mg IV02(or equivalent includingoral dose)DayPrednisolone 100 mg IV03(or equivalent includingoral dose)DayPrednisolone 100 mg IV04(or equivalent includingoral dose)2ndDayPrednisolone 100 mg IVDiphenhydramineParacetamolepcoritamab08*(or equivalent including50 mg IV or oral(acetaminophen)administrationoral dose)(PO) (or equivalent)650 to 1000 mg PO(intermediate dose)(or equivalent)DayPrednisolone 100 mg IV09(or equivalent includingoral dose)DayPrednisolone 100 mg IV10(or equivalent includingoral dose)DayPrednisolone 100 mg IV11(or equivalent includingoral dose)3rdDayPrednisolone 100 mg IVDiphenhydramineParacetamolepcoritamab15*(or equivalent including50 mg IV or oral(acetaminophen)administrationoral dose)(PO) (or equivalent)650 to 1000 mg PO(1stfull dose)(or equivalent)DayPrednisolone 100 mg IV16(or equivalent includingoral dose)DayPrednisolone 100 mg IV17(or equivalent includingoral dose)DayPrednisolone 100 mg IV18(or equivalent includingoral dose)4thDayPrednisolone 100 mg IVDiphenhydramineParacetamolepcoritamab22*(or equivalent including50 mg IV or oral(acetaminophen)administrationoral dose)(PO) (or equivalent)650 to 1000 mg PO(2ndfull dose)(or equivalent)DayPrednisolone 100 mg IV23(or equivalent includingoral dose)DayPrednisolone 100 mg IV24(or equivalent includingoral dose)DayPrednisolone 100 mg IV25(or equivalent includingoral dose)Cycle 25thDayIf CRS ≥grade 2 occursOptionalOptionalepcoritamab29*following the 4thadministrationDayepcoritamab(3rdfull dose)30administration, 4-dayDayconsecutive31corticosteroidDayadministration is32continued in Cycle 2until an epcoritamabdose is given that doesnot result in subsequentCRS.*30 minutes - 2 hours prior to administration of epcoritamab. TABLE 6Corticosteroid Dose Equivalents - Conversion TableGlucocorticoidApproximate equivalent dose (mg)Short-actingCortisone (PO)500Hydrocortisone (IV or PO)400Intermediate-actingMethylprednisolone (IV or PO)80Prednisolone (PO)100Prednisone (IV or PO)100Triamcinolone (IV)80Long-actingBetamethasone (IV)15Dexamethasone (IV or PO)15 Supportive Care for Cytokine Release Syndrome CRS is graded according to the ASTCT grading for CRS (Tables 7 and 8), and for treatment of CRS, subjects should receive supportive care. Supportive care can include, but is not limited to,Infusion of salineSystemic glucocorticosteroid, antihistamine, antipyrexiaSupport for blood pressure (vasopressin, vasopressors)Support for low-flow and high-flow oxygen and positive pressure ventilationMonoclonal antibody against IL-6R, e.g., IV administration of tocilizumabMonoclonal antibody against IL-6, e.g., IV siltuximab if not responding to repeated tocilizumab. TABLE 7Grading and Management of Cytokine Release SyndromeHarmonized definitions and grading criteria for CRS, per the AmericanSociety for Transplantation and Cellular Therapy (ASTCT), formerly AmericanSociety for Blood and Marrow Transplantation, (ASBMT), are presented below.Grading of Cytokine Release SyndromeCRSparameterGrade 1Grade 2Grade 3Grade 4Grade 5Fever1≥38.0° C.≥38.0° C.≥38.0° C.≥38.0° C.Death dueWithNoneNot requiringRequiringRequiring ≥2to CRS inhypotensionvasopressors1 vasopressorvasopressorswhich anotherwith or without(excludingcause is notvasopressinvasopressin)the principleAnd/orNoneRequiringRequiringRequiringfactorhypoxia2low-flowhigh-flowpositiveleading to(□6 L/minute)(>6 L/minute)pressurethis outcomenasal cannulanasal cannula,ventilation3or blow-byfacemask,(eg, CPAP,nonrebreatherBiPAP,mask, orintubation andventuri maskmechanicalventilation)Abbreviations:BiPAP, Bilevel positive airway pressure;CPAP, continuous positive airway pressure;CRS, cytokine release syndrome;IV, intravenous.Note:organ toxicities or constitutional symptoms associated with CRS may be graded according to CTCAE but they do not influence CRS grading.1Fever is defined as temperature ≥38.0° C. not attributable to any other cause, with or without constitutional symptoms (eg, myalgia, arthralgia, malaise). In subjects who have CRS receiving antipyretics, anticytokine therapy, and/or corticosteroids, fever is no longer required to grade subsequent CRS severity. In this case, CRS grading is driven by hypotension and/or hypoxia.2CRS grade is determined by the more severe event: hypotension or hypoxia not attributable to any other cause. For example, a subject with temperature of 39.5° C., hypotension requiring 1 vasopressor, and hypoxia requiring low-flow nasal cannula is classified as grade 3 CRS. Both systolic blood pressure and mean arterial pressure are acceptable for blood pressure measurement. No specific limits are required, but hypotension should be determined on a case-by-case basis, accounting for age and the subject's individual baseline, i.e., a blood pressure that is below the normal expected for an individual in a given environment.3Intubation of a subject without hypoxia for the possible neurologic compromise of a patent airway alone or for a procedure is not by definition grade 4 CRS.Source: Adapted from Lee et al.,Biol Blood Marrow Transplant2019; 25: 625-638 TABLE 8Grading and Management of Cytokine Release SyndromeCRS gradeManagement1Fever: Patients with a new fever should be admitted to the hospital if not already. Investigatefor infection and rapidly startup broad-spectrum antibiotics. Continuation of antibiotic therapyis recommended until and potential neutropenia resolve. Constitutional symptoms may behelped by NSAIDs.Tocilizumab: No*.Steroids: No.2Fever: As per grade 1.Hypotension: Immediate clinical evaluation and intervention is warranted. At the firstconfirmed decrease ≥20% from baseline systolic, diastolic or mean arterial pressure orevidence of worsening perfusion, administer an IV fluid bolus (20 mL/kg up to 1 L). Consider avasopressor and administer no later than after the 3rdIV fluid bolus due the vasodilatation andcapillary leak associated with CRS.Hypoxia: Consider X-ray or CT-scan if hypoxic and/or tachypneic. Administer oxygen by low-flow nasal cannula (≤6 L/min) or blow-by.Tocilizumab: No* (yes, if the patient has comorbidities†).Steroids: No (consider, if the patient has comorbidities‡).3Fever: As per grade 1.Hypotension: Immediate clinical evaluation and intervention is warranted. Administer avasopressor (norepinephrine), with or without vasopressin, as most patients with CRS haveperipheral vasodilation.Hypoxia: Administer oxygen by high-flow nasal cannula (>6 L/min), facemask, non-breathermask, or Venturi mask.Tocilizumab: Yes†.Steroids: Consider‡.4Fever: As per grade 1.Hypotension: Immediate clinical evaluation and intervention is warranted. Administer at least 2vasopressors, with or without vasopressin, as most patients with CRS have peripheralvasodilation.Hypoxia: Positive pressure (e.g. CPAP, BiPAP, intubation, and mechanical ventilation).Tocilizumab: Yes†.Steroids: Yes‡.*Consider intervening earlier in specific cases. For example, an elderly patient with prolonged fever (>72 hours) or very high fever (>40.5° C./104.9° F.) may not tolerate the resulting sinus tachycardia as well as a younger patient, so tocilizumab may be indicated.†Tocilizumab (anti-IL-6R) remains the only first-line anticytokine therapy approved for CRS. If there is no improvement in symptoms within 6 hours, or if the patient starts to deteriorate after initial improvement, a second dose of tocilizumab should be administered along with a dose of corticosteroids. For patients being refractory to tocilizumab (3 administrations), additional anticytokine therapy such as siltuximab (anti-IL-6) or anakinra (anti-IL-1R) may be considered. However, such use is entirely anecdotal and, as such, is entirely at the discretion of the treating physician.‡Consider dexamethasone over methylprednisolone due to improved CNS penetration even in absence of neurotoxicity, as high-grade CRS is correlated with risk of concurrent or subsequent ICANS. If concurrent ICANS is observed, dexamethasone should be preferred.Source: (Varadarajan I, Kindwall-Keller T L, Lee D W (2020). Management of cytokine release syndrome. In: Chimeric antigen receptor T-cell therapies for cancer (Chapter 5). Elsevier 2020) Tumor Lysis Syndrome Prevention and Management For prophylactic treatment of tumor lysis syndrome, subjects receive uric acid reducing agents prior to the administration of epcoritamab, with allopurinol given at least 72 hours prior to the first dose of epcoritamab and rasburicase initiated prior to starting epcoritamab. Increased oral hydration should be received prior to the first dose and is maintained during dosings. Reassessment of the subject's TLS risk category is performed prior to subsequent doses. Study Assessments Bone Marrow Assessment A fresh bone marrow aspirate is obtained at screening (i.e., within 21 days prior to Cycle 1 Day 1) and at the time of complete response (CR) or when clinically indicated. A fresh bone marrow biopsy is obtained at screening and at the time of CR or nodular partial response (PR) (nPR) or when clinically indicated. Bone marrow evaluations include morphological examination and either flow cytometry or immunohistochemistry. A bone marrow biopsy with aspirate is obtained (a) to confirm a CR or nPR that is supported by physical examination findings, laboratory evaluations and radiographic evaluations according to iwCLL guidelines (Hallek et al,Lancet2018; 391:1524-37), and (b) if progression is only shown in 1 parameter to confirm cytopenic progression (i.e., neutropenia, anemia, and/or thrombocytopenia and to distinguish from autoimmune and treatment-related cytopenias). Radiographic Assessments Imaging scans of the neck, chest, abdomen and pelvis are performed at screening (i.e., within 3 weeks prior to the first dose of epcoritamab) and subsequent response assessments. A CT scan with contrast is the recommended imaging modality. MRI may be used only if CT with contrast is medically contraindicated or if the frequency of CT scans exceeds local standards. MRI studies do not replace the required neck, chest, abdomen, and pelvic CT scans. Additional imaging assessments may be performed to support the efficacy evaluations for a subject as necessary. Bone Marrow Assessment A fresh bone marrow aspirate is obtained at screening (i.e., within 21 days prior to Cycle 1 Day 1) and at the time of CR or as clinically indicated. A fresh bone marrow biopsy is obtained at screening and at the time of CR or nodular PR (nPR) or as clinically indicated. Bone marrow evaluations include a morphological examination and either flow cytometry or immunohistochemistry. A bone marrow biopsy with aspirate is obtained (a) to confirm a CR or nPR according to iwCLL guidelines (Hallek et al., supra) that is supported by physical examination findings, laboratory evaluations and radiographic evaluations, and (b) if progression is only shown in 1 parameter to confirm cytopenic progression (i.e., neutropenia, anemia, and/or thrombocytopenia and to distinguish from autoimmune and treatment-related cytopenias). Minimal Residual Disease (MRD) Assessment MRD is assessed in the blood by flow cytometry and next generation sequencing. After start of treatment, blood samples are requested at the fixed time points and at time of CR. As an exploratory analysis, when a subject reaches a CR, a portion of the aspirate collected to confirm CR is used to assess MRD. Disease Response and Progressive Disease Assessment Tumor response according to imaging assessment is performed to inform decisions on continuation of treatment. Response assessment is completed according to the revised iwCLL guidelines for diagnosis, indications for treatment, response assessment and supportive management of CLL, as described in Table 2. Endpoint definitions are as follows: Overall response rate (ORR), is defined as the proportion of subjects who achieve a response of PR or CR, prior to initiation of subsequent therapy. Time to response (TTR), is defined among responders, as the time between first dose of epcoritamab and the initial documentation of PR or CR. Duration of response (DOR), is defined among responders, as the time from the initial documentation of PR or CR to the date of disease progression or death, whichever occurs earlier. Progression-free survival (PFS), is defined as the time from the first dosing date of epcoritamab and the date of disease progression or death, whichever occurs earlier. Overall survival (OS), is defined as the time from the first dosing date of epcoritamab and the date of death. MRD negativity rate, is defined as the proportion of subjects with at least 1 undetectable MRD result according to the specific threshold, prior to initiation of subsequent therapy. Clinical Safety Assessments Safety will be assessed by measuring adverse events, laboratory test results, ECGs, vital sign measurements, physical examination findings, and ECOG performance status. Also assessed are immune effector cell-associated neurotoxicity syndrome (e.g., as described by Lee et al.,Biol Blood Marrow Transplant2019; 25:625-638), constitutional symptoms (B symptoms), tumor flare reaction, and survival. Immunophenotyping Analyses Absolute B and T-cell counts are determined in fresh whole blood using flow cytometry to monitor changes associated with epcoritamab treatment. The T-cell activation and exhaustion phenotype is evaluated using flow cytometry and markers in order to evaluate the association of such markers with drug target engagement, treatment efficacy, and/or safety of epcoritamab. Additional immunophenotypes of circulating immune cells (e.g., levels of regulatory T-cells which can suppress T-cell function) are determined in fresh whole blood using flow cytometry to evaluate the association of such markers with T-cell activation/exhaustion phenotype, subject response, and epcoritamab's MOA. Cytokine and Endothelial Activation Marker Analyses Since T-cell activation following initial epcoritamab administrations may lead to cytokine release causing CRS, cytokine levels are closely monitored. The levels of cytokines, such as IL-2, IL15, IL-6, IL-8, IL-10, IFNγ, and/or TNFα, are measured in plasma samples using an array based ligand binding assay. Additional cytokines may also be determined to evaluate the association of such markers with treatment-emergent AEs and outcome to epcoritamab. Preliminary Results: The first patient was enrolled on Nov. 30, 2020. As of Jul. 1, 2021, 7 patients with R/R CLL received epcoritamab subcutaneously administered at 2 full-dose levels: 24 (n=3) and 48 mg (n=4). Six patients completed the dose limiting toxicity (DLT) evaluation period, and 5 patients had a response assessment. Patients had received a median of 4 lines of prior therapy (range, 2-5). Six of 7 patients had poor-risk features of del(17p), TP53 mutations, or both. Three of 7 pts had bulky disease. No DLTs occurred at 24 or 48 mg. The most common treatment-emergent AEs (>30%) were cytokine release syndrome (CRS) (100%), fatigue (71%), injection-site reaction (43%), and nausea (43%). All pts experienced CRS in the first cycle, but no CRS events were higher than grade 2. No cases of immune effector cell-associated neurotoxicity syndrome (ICANS) were observed. Despite presence of circulating tumor cells, tumor lysis syndrome (TLS) was not observed. Antileukemic activity has been observed at both dose levels, with partial responses in 3 of 5 patients. As of Sep. 8, 2021, a total of 11 R/R CLL patients have received epcoritamab at 24 mg (N=3) and 48 mg (N=8). Epcoritamab was well tolerated at both 24 mg and 48 mg with the most common treatment-emergent adverse events being CRS, fatigue, and injection site reaction. There was no immune effector cell-associated neurotoxicity syndrome (ICANS). Preliminary activity of epcoritamab in these heavily pretreated patients who had high-risk cytogenetics have been observed. Among 6 response-evaluable subjects, there were 1 non-confirmed partial response (nPR) at 24 mg and 2 partial responses (PR) at 48 mg. These data are preliminary and non-validated and unclean data and response data were not completely entered by site. CONCLUSION These preliminary data suggest that epcoritamab is well tolerated in patients with R/R CLL at dose levels up to 48 mg and has encouraging clinical activity in pts with high-risk disease. TABLE 9Listing of SequencesSEQIDDescriptionSequence1huCD3 VH CDR1GFTFNTYA2huCD3 VH CDR2IRSKYNNYAT3huCD3 VH CDR3VRHGNFGNSYVSWFAY4huCD3 VL CDR1TGAVTTSNY—huCD3 VL CDR2GTN5huCD3 VL CDR3ALWYSNLWV6huCD3 VH1EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS7huCD3 VL1QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVL8VH CD20-7D8GFTFHDYACDR19VH CD20-7D8ISWNSGTICDR210VH CD20-7D8AKDIQYGNYYYGMDVCDR311VL CD20-7D8QSVSSYCDR1—VL CD20-7D8DASCDR212VL CD20-7D8QQRSNWPITCDR313VH CD20-7D8EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTISWINSGTIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS14VL CD20-7D8EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK15IgG1 heavyASTKGPSVFPLAPSSKSTSGchainGTAALGCLVKDYFPEPVTVSconstantWNSGALTSGVHTFPAVLQSSregion-WTGLYSLSSVVTVPSSSLGTQT(amino acidsYICNVNHKPSNTKVDKRVEPpositionsKSCDKTHTCPPCPAPELLGG118-447PSVFLFPPKPKDTLMISRTPaccording to EUEVTCVVVDVSHEDPEVKFNWnumbering).YVDGVEVHNAKTKPREEQYNCH3 regionSTYRVVSVLTVLHQDWLNGKitalicsEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG16IgG1-LFLEDAASTKGPSVFPLAPSSKSTSGHeavy chainGTAALGCLVKDYFPEPVTVSconstantWNSGALTSGVHTFPAVLQSSregionGLYSLSSVVTVPSSSLGTQT(amino acidsYICNVNHKPSNTKVDKRVEPpositionsKSCDKTHTCPPCPAPEFEGG118-447PSVFLFPPKPKDTLMISRTPaccordingEVTCVVVAVSHEDPEVKFNWto EUYVDGVEVHNAKTKPREEQYNnumbering).STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG17IgG1 F405LASTKGPSVFPLAPSSKSTSG(amino acidsGTAALGCLVKDYFPEPVTVSpositionsWNSGALTSGVHTFPAVLQSS118-447GLYSLSSVVTVPSSSLGTQTaccordingYICNVNHKPSNTKVDKRVEPto EUKSCDKTHTCPPCPAPELLGGnumbering)PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG18IgG1-K409RASTKGPSVFPLAPSSKSTSG(amino acidsGTAALGCLVKDYFPEPVTVSpositionsWNSGALTSGVHTFPAVLQSS118-447GLYSLSSVVTVPSSSLGTQTaccordingYICNVNHKPSNTKVDKRVEPto EUKSCDKTHTCPPCPAPELLGGnumbering)PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG19IgG1-LFLEDA-ASTKGPSVFPLAPSSKSTSGF405LGTAALGCLVKDYFPEPVTVS(FEAL)WNSGALTSGVHTFPAVLQSS(amino acidsGLYSLSSVVTVPSSSLGTQTpositionsYICNVNHKPSNTKVDKRVEP118-447KSCDKTHTCPPCPAPEFEGGaccordingPSVFLFPPKPKDTLMISRTPto EUEVTCVVVAVSHEDPEVKFNWnumbering)YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG20IgG1-LFLEDA-ASTKGPSVFPLAPSSKSTSGK409R (FEAR)GTAALGCLVKDYFPEPVTVS(amino acidsWNSGALTSGVHTFPAVLQSSpositionsGLYSLSSVVTVPSSSLGTQT118-447YICNVNHKPSNTKVDKRVEPaccordingKSCDKTHTCPPCPAPEFEGGto EUPSVFLFPPKPKDTLMISRTPnumbering)EVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG21IgG1 CH3GQPREPQVYTLPPSREEMTKregionNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG22ConstantGQPKAAPSVTLFPPSSEELQregionANKATLVCLISDFYPGAVTVhumanAWKADSSPVKAGVETTTPSKlambda LCQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS23ConstantRTVAAPSVFIFPPSDEQLKSregionGTASVVCLLNNFYPREAKVQhumanWKVDNALQSGNSQESVTEQDkappa LCSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC24huCD3-LFLEDA-EVKLVESGGGLVQPGGSLRLF405L (FEAL)SCAASGFTFNTYAMNWVRQAheavy chainPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG25huCD3 VL +QAVVTQEPSFSVSPGGTVTLCL lightTCRSSTGAVTTSNYANWVQQchainTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS26CD20-7D8-EVQLVESGGGLVQPDRSLRLLFLEDA-SCAASGFTFHDYAMHWVRQAK409R (FEAR)PGKGLEWVSTISWNSGTIGYheavy chainADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG27CD20-7D8EIVLTQSPATLSLSPGERATVL + CLLSCRASQSVSSYLAWYQQKPlight chainGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC28Human CD3MQSGTHWRVLGLCLLSVGVW(epsilon)GQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI29Human CD20MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP Bold and underlined are FE; A; L and R, corresponding to positions 234 and 235; 265; 405 and 409, respectively, said positions being in accordance with EU-numbering. In variable regions, said CDR regions that were annotated in accordance with IMGT definitions are underlined. | 178,280 |
11858996 | DETAILED DESCRIPTION ICOS Antibodies according to the present invention bind the extracellular domain of human ICOS. Thus, the antibodies bind ICOS-expressing T lymphocytes. “ICOS” or “the ICOS receptor” referred to herein may be human ICOS, unless the context dictates otherwise. Sequences of human, cynomolgus and mouse ICOS are shown in the appended sequence listing, and are available from NCBI as human NCBI ID: NP_036224.1, mouse NCBI ID: NP_059508.2 and cynomolgus GenBank ID: EHH55098.1. Cross-Reactivity Antibodies according to the present invention are preferably cross-reactive, and may for example bind the extracellular domain of mouse ICOS as well as human ICOS. The antibodies may bind other non-human ICOS, including ICOS of primates such as cynomolgus. An anti-ICOS antibody intended for therapeutic use in humans must bind human ICOS, whereas binding to ICOS of other species would not have direct therapeutic relevance in the human clinical context. Nevertheless, the data herein indicate that antibodies that bind both human and mouse ICOS have properties that render them particularly suitable as agonist and depleting molecules. This may result from one or more particular epitopes being targeted by the cross-reactive antibodies. Regardless of the underlying theory, however, cross-reactive antibodies are of high value and are excellent candidates as therapeutic molecules for pre-clinical and clinical studies. As explained in the experimental Examples, the STIM antibodies described here were generated using Kymouse™ technology where the mouse had been engineered to lack expression of mouse ICOS (an ICOS knock-out). ICOS knock-out transgenic animals and their use for generating cross-reactive antibodies are further aspects of the present invention. One way to quantify the extent of species cross-reactivity of an antibody is as the fold-difference in its affinity for antigen or one species compared with antigen of another species, e.g., fold difference in affinity for human ICOS vs mouse ICOS. Affinity may be quantified as KD, referring to the equilibrium dissociation constant of the antibody-antigen reaction as determined by SPR with the antibody in Fab format as described elsewhere herein. A species cross-reactive anti-ICOS antibody may have a fold-difference in affinity for binding human and mouse ICOS that is 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less or 5-fold or less. To put it another way, the KDof binding the extracellular domain of human ICOS may be within 30-fold, 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of the KDof binding the extracellular domain of mouse ICOS. Antibodies can also be considered cross-reactive if the KDfor binding antigen of both species meets a threshold value, e.g., if the KDof binding human ICOS and the KDof binding mouse ICOS are both 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. The KDmay be 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. The KDmay be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. An alternative measure of cross-reactivity for binding human ICOS and mouse ICOS is the ability of an antibody to neutralise ICOS ligand binding to ICOS receptor, such as in an HTRF assay (see Example 8). Examples of species cross-reactive antibodies are provided herein, including STIM001, STIM002, STIM002-B, STIM003, STIM005 and STIM006, each of which was confirmed as neutralising binding of human B7-H2 (ICOS ligand) to human ICOS and neutralising binding of mouse B7-H2 to mouse ICOS in an HTRF assay. Any of these antibodies or their variants may be selected when an antibody cross-reactive for human and mouse ICOS is desired. A species cross-reactive anti-ICOS antibody may have an IC50 for inhibiting binding of human ICOS to human ICOS receptor that is within 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of the IC50 for inhibiting mouse ICOS to mouse ICOS receptor as determined in an HTRF assay. Antibodies can also be considered cross-reactive if the IC50 for inhibiting binding of human ICOS to human ICOS receptor and the IC50 for inhibiting binding of mouse ICOS to mouse ICOS receptor are both 1 mM or less, preferably 0.5 mM or less, e.g., 30 nM or less, 20 nM or less, 10 nM or less. The IC50s may be 5 nM or less, 4 nM or less, 3 nM or less or 2 nM or less. In some cases the IC50s will be at least 0.1 nM, at least 0.5 nM or at least 1 nM. Specificity Antibodies according to the present invention are preferably specific for ICOS. That is, the antibody binds its epitope on the target protein, ICOS (human ICOS, and preferably mouse and/or cynomolgus ICOS as noted above), but does not show significant binding to molecules that do not present that epitope, including other molecules in the CD28 gene family. An antibody according to the present invention preferably does not bind human CD28. The antibody preferably also does not bind mouse or cynomolgus CD28. CD28 co-stimulates T cell responses when engaged by its ligands CD80 and CD86 on professional antigen presenting cells in the context of antigen recognition via the TCR. For various in vivo uses of the antibodies described herein, the avoidance of binding to CD28 is considered advantageous. Non-binding of the anti-ICOS antibody to CD28 should allow CD28 to interact with its native ligands and to generate appropriate co-stimulatory signal for T cell activation. Additionally, non-binding of the anti-ICOS antibody to CD28 avoids the risk of superagonism. Over-stimulation of CD28 can induce proliferation in resting T cells without the normal requirement for recognition of a cognate antigen via the TCR, potentially leading to runaway activation of T cells and consequent cytokine-release syndrome, especially in human subjects. The non-recognition of CD28 by antibodies according to the present invention therefore represents an advantage in terms of their safe clinical use in humans. As discussed elsewhere herein, the present invention extends to multispecific antibodies (e.g., bispecifics). A multispecific (e.g., bispecific) antibody may comprise (i) an antibody antigen binding site for ICOS and (ii) a further antigen binding site (optionally an antibody antigen binding site, as described herein) which recognises another antigen (e.g., PD-L1). Specific binding of individual antigen binding sites may be determined. Thus, antibodies that specifically bind ICOS include antibodies comprising an antigen binding site that specifically binds ICOS, wherein optionally the antigen binding site for ICOS is comprised within an antigen-binding molecule that further includes one or more additional binding sites for one or more other antigens, e.g., a bispecific antibody that binds ICOS and PD-L1. Affinity The affinity of binding of an antibody to ICOS may be determined. Affinity of an antibody for its antigen may be quantified in terms of the equilibrium dissociation constant KD, the ratio Ka/Kd of the association or on-rate (Ka) and the dissociation or off-rate (kd) of the antibody-antigen interaction. Kd, Ka and Kd for antibody-antigen binding can be measured using surface plasmon resonance (SPR). An antibody according to the present invention may bind the EC domain of human ICOS with a KDof 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. The KDmay be 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. The KDmay be 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. The KDmay be at least 0.001 nM, for example at least 0.01 nM or at least 0.1 nM. Quantification of affinity may be performed using SPR with the antibody in Fab format. A suitable protocol is as follows:1. Coupling anti-human (or other antibody constant region species-matched) IgG to a biosensor chip (e.g., GLM chip) such as by primary amine coupling;2. Exposing the anti-human IgG (or other matched species antibody) to a test antibody, e.g., in Fab format, to capture test antibody on the chip;3. Passing the test antigen over the chip's capture surface at a range of concentrations, e.g., at 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM and 2 nM, and at 0 nM (i.e., buffer alone); and4. Determining the affinity of binding of test antibody to test antigen using SPR at 25° C. Buffer may be at pH 7.6, 150 mM NaCl, 0.05% detergent (e.g., P20) and 3 mM EDTA. Buffer may optionally contain 10 mM HEPES. HBS-EP can be used as running buffer. HBS-EP is available from Teknova Inc (California; catalogue number H8022). Regeneration of the capture surface can be carried out with 10 mM glycine at pH 1.7. This removes the captured antibody and allows the surface to be used for another interaction. The binding data can be fitted to 1:1 model inherent using standard techniques, e.g., using a model inherent to the ProteOn XPR36™ analysis software. A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc). Worked examples of SPR are found in Example 7. As described, affinity may be determined by SPR with the antibody in Fab format, with the antigen coupled to the chip surface and the test antibody passed over the chip in Fab format in solution, to determine affinity of the monomeric antibody-antigen interaction. Affinity can be determined at any desired pH, e.g., pH 5.5 or pH 7.6, and any desired temperature e.g., 25° C. or 37° C. As reported in Example 7, antibodies according to the present invention bound human ICOS with an apparent affinity of less than 2 nM, as determined by SPR using the antibody in monovalent (Fab) format. Other ways to measure binding of an antibody to ICOS include fluorescence activated cell sorting (FACS), e.g., using cells (e.g., CHO cells) with exogenous surface expression of ICOS or activated primary T cells expressing endogenous levels of ICOS. Antibody binding to ICOS-expressing cells as measured by FACS indicates that the antibody is able to bind the extracellular (EC) domain of ICOS. ICOS Receptor Agonism The ICOS ligand (ICOSL, also known as B7-H2) is a cell surface expressed molecule that binds to the ICOS receptor [17]. This intercellular ligand-receptor interaction promotes multimerisation of ICOS on the T cell surface, activating the receptor and stimulating downstream signalling in the T cell. In effector T cells, this receptor activation stimulates the effector T cell response. Anti-ICOS antibodies may act as agonists of ICOS, mimicking and even surpassing this stimulatory effect of the native ICOS ligand on the receptor. Such agonism may result from ability of the antibody to promote multimerisation of ICOS on the T cell. One mechanism for this is where the antibodies form intercellular bridges between ICOS on the T cell surface and receptors on an adjacent cell (e.g., B cell, antigen-presenting cell, or other immune cell), such as Fc receptors. Another mechanism is where antibodies having multiple (e.g., two) antigen-binding sites (e.g., two VH-VL domain pairs) bridge multiple ICOS receptor molecules and so promote multimerisation. A combination of these mechanisms may occur. Agonism can be tested for in in vitro T cell activation assays, using antibody in soluble form (e.g., in immunoglobulin format or other antibody format comprising two spatially separated antigen-binding sites, e.g., two VH-VL pairs), either including or excluding a cross-linking agent, or using antibody bound to a solid surface to provide a tethered array of antigen-binding sites. Agonism assays may use a human ICOS positive T lymphocyte cell line such as MJ cells (ATCC CRL-8294) as the target T cell for activation in such assays. One or more measures of T cell activation can be determined for a test antibody and compared with a reference molecule or a negative control to determine whether there is a statistically significant (p<0.05) difference in T cell activation effected by the test antibody compared with the reference molecule or the control. One suitable measure of T cell activation is production of cytokines, e.g., IFNγ, TNFα or IL-2. The skilled person will include suitable controls as appropriate, standardising assay conditions between test antibody and control. A suitable negative control is an antibody in the same format (e.g., isotype control) that does not bind ICOS, e.g., an antibody specific for an antigen that is not present in the assay system. A significant difference is observed for test antibody relative to a cognate isotype control within the dynamic range of the assay is indicative that the antibody acts as an agonist of the ICOS receptor in that assay. An agonist antibody may be defined as one which, when tested in a T cell activation assay:has a significantly lower EC50 for induction of IFNγ production compared with control antibody;induces significantly higher maximal IFNγ production compared with control antibody;has a significantly lower EC50 for induction of IFNγ production compared with ICOSL-Fc;induces significantly higher maximal IFNγ production compared with ICOSL-Fc;has a significantly lower EC50 for induction of IFNγ production compared with reference antibody C398.4A; and/orinduces significantly higher maximal IFNγ production compared with reference antibody C398.4A. In vitro T cell assays include the bead-bound assay of Example 13, the plate-bound assay of Example 14 and the soluble form assay of Example 15. A significantly lower or significantly higher value may for example be up to 0.5-fold different, up to 0.75-fold different, up to 2-fold different, up to 3-fold different, up to 4-fold different or up to 5-fold different, compared with the reference or control value. Thus, in one example, an antibody according to the present invention has a significantly lower, e.g., at least 2-fold lower, EC50 for induction of IFNγ in an MJ cell activation assay using the antibody in bead-bound format, compared with control. The bead-bound assay uses the antibody (and, for control or reference experiments, the control antibody, reference antibody or ICOSL-Fc) bound to the surface of beads. Magnetic beads may be used, and various kinds are commercially available, e.g., Tosyl-activated DYNABEADS M-450 (DYNAL Inc, 5 Delaware Drive, Lake Success, N.Y. 11042 Prod No. 140.03, 140.04). Beads may be coated as described in Example 13, or generally by dissolving the coating material in carbonate buffer (pH 9.6, 0.2 M) or other method known in the art. Use of beads conveniently allows the quantity of protein bound to the bead surface to be determined with a good degree of accuracy. Standard Fc-protein quantification methods can be used for coupled protein quantification on beads. Any suitable method can be used, with reference to a relevant standard within the dynamic range of the assay. DELFIA is exemplified in Example 13, but ELISA or other methods could be used. Agonism activity of an antibody can also be measured in primary human T lymphocytes ex vivo. The ability of an antibody to induce expression of IFNγ in such T cells is indicative of ICOS agonism. Described herein are two T cell activation assays using primary cells—see Example 2, T cell activation assay 1 and T cell activation assay 2. Preferably, an antibody will show significant (p<0.05) induction of IFNγ at 5 μg/ml compared with control antibody in T cell activation assay 1 and/or T cell activation assay 2. As noted above, an anti-ICOS antibody may stimulate T cell activation to a greater degree than ICOS-L or C398.4 in such an assay. Thus, the antibody may show significantly (p<0.05) greater induction of IFNγ at 5 μg/ml compared with the control or reference antibody in T cell activation assay 1 or 2. TNFα or IL-2 induction may be measured as an alternative assay readout. Agonism of an anti-ICOS antibody may contribute to its ability to change the balance between populations of TReg and TEff cells in vivo, e.g., in a site of pathology such as a tumour microenvironment, in favour of TEff cells. The ability of an antibody to enhance tumour cell killing by activated ICOS-positive effector T cells may be determined, as discussed elsewhere herein. T Cell Dependent Killing Effector T cell function can be determined in a biologically relevant context using an in vitro co-culture assay where tumour cells are incubated with relevant immune cells to trigger immune cell-dependent killing, in which the effect of an anti-ICOS antibody on tumour cell killing by TEffs is observed. The ability of an antibody to enhance tumour cell killing by activated ICOS-positive effector T cells may be determined. An anti-ICOS antibody may stimulate significantly greater (p<0.05) tumour cell killing compared with a control antibody. An anti-ICOS antibody may stimulate similar or greater tumour cell killing in such an assay as compared with a reference molecule such as the ICOS ligand or the C398.4 antibody. A similar degree of tumour cell killing can be represented as the assay readout for the test antibody being less than two-fold different from that for the reference molecule. ICOS Ligand-Receptor Neutralisation Potency An antibody according to the present invention may be one which inhibits binding of ICOS to its ligand ICOSL. The degree to which an antibody inhibits binding of the ICOS receptor to its ligand is referred to as its ligand-receptor neutralising potency. Potency is normally expressed as an IC50 value, in pM unless otherwise stated. In ligand-binding studies, IC50 is the concentration that reduces receptor binding by 50% of maximal specific binding level. IC50 may be calculated by plotting % specific receptor binding as a function of the log of the antibody concentration, and using a software program such as Prism (GraphPad) to fit a sigmoidal function to the data to generate IC50 values. Neutralising potency may be determined in an HTRF assay. A detailed working example of an HTRF assay for ligand-receptor neutralising potency is set out in Example 8. An IC50 value may represent the mean of a plurality of measurements. Thus, for example, IC50 values may be obtained from the results of triplicate experiments, and a mean IC50 value can then be calculated. An antibody may have an IC50 of 1 mM or less in a ligand-receptor neutralisation assay, e.g., 0.5 mM or less. The IC50 may be, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less or 2 nM or less. The IC50 may be at least 0.1 nM, at least 0.5 nM or at least 1 nM. Antibodies As described in more detail in the Examples, we isolated and characterised antibodies of particular interest, designated STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009. In various aspects of the invention, unless context dictates otherwise, antibodies may be selected from any of these antibodies, or from the sub-set of STIM001, STIM002, STIM003, STIM004 and STIM005. Sequences of each of these antibodies are provided in the appended sequence listing, wherein for each antibody the following sequences are shown: nucleotide sequence encoding VH domain; amino acid sequence of VH domain; VH CDR1 amino acid sequence, VH CDR2 amino acid sequence; VH CDR3 amino acid sequence; nucleotide sequence encoding VL domain; amino acid sequence of VL domain; VL CDR1 amino acid sequence; VL CDR2 amino acid sequence; and VL CDR3 amino acid sequence, respectively. The present invention encompasses anti-ICOS antibodies having the VH and/or VL domain sequences of all antibodies shown in the appended sequence listing and/or in the drawings, as well as antibodies comprising the HCDRs and/or LCDRs of those antibodies, and optionally having the full heavy chain and/or full light chain amino acid sequence. STIM001 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:366, comprising the CDRH1 amino acid sequence of Seq ID No:363, the CDRH2 amino acid sequence of Seq ID No:364, and the CDRH3 amino acid sequence of Seq ID No:365. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:367. STIM001 has a light chain variable region (VL) amino acid sequence of Seq ID No:373, comprising the CDRL1 amino acid sequence of Seq ID No:370, the CDRL2 amino acid sequence of Seq ID No:371, and the CDRL3 amino acid sequence of Seq ID No:372. The light chain nucleic acid sequence of the VLdomain is Seq ID No:374. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:368 (heavy chain nucleic acid sequence Seq ID No:369). A full length light chain amino acid sequence is Seq ID No:375 (light chain nucleic acid sequence Seq ID No:376). STIM002 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:380, comprising the CDRH1 amino acid sequence of Seq ID No:377, the CDRH2 amino acid sequence of Seq ID No:378, and the CDRH3 amino acid sequence of Seq ID No:379. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:381. STIM002 has a light chain variable region (VL) amino acid sequence of Seq ID No:387, comprising the CDRL1 amino acid sequence of Seq ID No:384, the CDRL2 amino acid sequence of Seq ID No:385, and the CDRL3 amino acid sequence of Seq ID No:386. The light chain nucleic acid sequence of the VLdomain is Seq ID No:388 or Seq ID No:519. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:382 (heavy chain nucleic acid sequence Seq ID No:383). A full length light chain amino acid sequence is Seq ID No:389 (light chain nucleic acid sequence Seq ID No:390 or Seq ID NO:520). STIM002-B has a heavy chain variable region (VH) amino acid sequence of Seq ID No:394, comprising the CDRH1 amino acid sequence of Seq ID No:391, the CDRH2 amino acid sequence of Seq ID No:392, and the CDRH3 amino acid sequence of Seq ID No:393. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:395. STIM002-B has a light chain variable region (VL) amino acid sequence of Seq ID No:401, comprising the CDRL1 amino acid sequence of Seq ID No:398, the CDRL2 amino acid sequence of Seq ID No:399, and the CDRL3 amino acid sequence of Seq ID No:400. The light chain nucleic acid sequence of the VLdomain is Seq ID No:402. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:396 (heavy chain nucleic acid sequence Seq ID No:397). A full length light chain amino acid sequence is Seq ID No:403 (light chain nucleic acid sequence Seq ID No:404). STIM003 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:408, comprising the CDRH1 amino acid sequence of Seq ID No:405, the CDRH2 amino acid sequence of Seq ID No:406, and the CDRH3 amino acid sequence of Seq ID No:407. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:409 or Seq ID No:521. STIM003 has a light chain variable region (VL) amino acid sequence of Seq ID No:415, comprising the CDRL1 amino acid sequence of Seq ID No:412, the CDRL2 amino acid sequence of Seq ID No:413, and the CDRL3 amino acid sequence of Seq ID No:414. The light chain nucleic acid sequence of the VLdomain is Seq ID No:4416. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:410 (heavy chain nucleic acid sequence Seq ID No:411 or Seq ID No:522). A full length light chain amino acid sequence is Seq ID No:417 (light chain nucleic acid sequence Seq ID No:418). STIM004 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:422, comprising the CDRH1 amino acid sequence of Seq ID No:419, the CDRH2 amino acid sequence of Seq ID No:420, and the CDRH3 amino acid sequence of Seq ID No:421. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:423. STIM004 has a light chain variable region (VL) amino acid sequence of Seq ID No:429, comprising the CDRL1 amino acid sequence of Seq ID No:426, the CDRL2 amino acid sequence of Seq ID No:427, and the CDRL3 amino acid sequence of Seq ID No:428. The light chain nucleic acid sequence of the VLdomain is Seq ID No:430 or Seq ID No:431. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:424 (heavy chain nucleic acid sequence Seq ID No:425). A full length light chain amino acid sequence is Seq ID No:432 (light chain nucleic acid sequence Seq ID No:433 or Seq ID no: 434). STIM005 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:438, comprising the CDRH1 amino acid sequence of Seq ID No:435, the CDRH2 amino acid sequence of Seq ID No:436, and the CDRH3 amino acid sequence of Seq ID No:437. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:439. STIM005 has a light chain variable region (VL) amino acid sequence of Seq ID No:445, comprising the CDRL1 amino acid sequence of Seq ID No:442, the CDRL2 amino acid sequence of Seq ID No:443, and the CDRL3 amino acid sequence of Seq ID No:444. The light chain nucleic acid sequence of the VLdomain is Seq ID No:446. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:440 (heavy chain nucleic acid sequence Seq ID No:441). A full length light chain amino acid sequence is Seq ID No:447 (light chain nucleic acid sequence Seq ID No:448). STIM006 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:452, comprising the CDRH1 amino acid sequence of Seq ID No:449, the CDRH2 amino acid sequence of Seq ID No:450, and the CDRH3 amino acid sequence of Seq ID No:451. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:453. STIM006 has a light chain variable region (VL) amino acid sequence of Seq ID No:459, comprising the CDRL1 amino acid sequence of Seq ID No:456, the CDRL2 amino acid sequence of Seq ID No:457, and the CDRL3 amino acid sequence of Seq ID No:458. The light chain nucleic acid sequence of the VLdomain is Seq ID No:460. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:454 (heavy chain nucleic acid sequence Seq ID No:455). A full length light chain amino acid sequence is Seq ID No:461 (light chain nucleic acid sequence Seq ID No:462). STIM007 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:466, comprising the CDRH1 amino acid sequence of Seq ID No:463, the CDRH2 amino acid sequence of Seq ID No:464, and the CDRH3 amino acid sequence of Seq ID No:465. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:467. STIM007 has a light chain variable region (VL) amino acid sequence of Seq ID No:473, comprising the CDRL1 amino acid sequence of Seq ID No:470, the CDRL2 amino acid sequence of Seq ID No:471, and the CDRL3 amino acid sequence of Seq ID No:472. The light chain nucleic acid sequence of the VLdomain is Seq ID No:474. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:468 (heavy chain nucleic acid sequence Seq ID No:469). A full length light chain amino acid sequence is Seq ID No:475 (light chain nucleic acid sequence Seq ID No:476). STIM008 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:480, comprising the CDRH1 amino acid sequence of Seq ID No:477, the CDRH2 amino acid sequence of Seq ID No:478, and the CDRH3 amino acid sequence of Seq ID No:479. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:481. STIM008 has a light chain variable region (VL) amino acid sequence of Seq ID No:487, comprising the CDRL1 amino acid sequence of Seq ID No:484, the CDRL2 amino acid sequence of Seq ID No:485, and the CDRL3 amino acid sequence of Seq ID No:486. The light chain nucleic acid sequence of the VLdomain is Seq ID No:488. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:482 (heavy chain nucleic acid sequence Seq ID No:483). A full length light chain amino acid sequence is Seq ID No:489 (light chain nucleic acid sequence Seq ID No:490). STIM009 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:494, comprising the CDRH1 amino acid sequence of Seq ID No:491, the CDRH2 amino acid sequence of Seq ID No:492, and the CDRH3 amino acid sequence of Seq ID No:493. The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:495. STIM009 has a light chain variable region (VL) amino acid sequence of Seq ID No:501, comprising the CDRL1 amino acid sequence of Seq ID No:498, the CDRL2 amino acid sequence of Seq ID No:499, and the CDRL3 amino acid sequence of Seq ID No:500. The light chain nucleic acid sequence of the VLdomain is Seq ID No:502. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:496 (heavy chain nucleic acid sequence Seq ID No:497). A full length light chain amino acid sequence is Seq ID No:503 (light chain nucleic acid sequence Seq ID No:504). Antibodies according to the present invention are immunoglobulins or molecules comprising immunoglobulin domains, whether natural or partly or wholly synthetically produced. Antibodies may be IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab′)2, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanised using routine technology. The term antibody covers any polypeptide or protein comprising an antibody antigen-binding site. An antigen-binding site (paratope) is the part of an antibody that binds to and is complementary to the epitope of its target antigen (ICOS). The term “epitope” refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. The antigen binding site is a polypeptide or domain that comprises one or more CDRs of an antibody and is capable of binding the antigen. For example, the polypeptide comprises a CDR3 (e.g., HCDR3). For example the polypeptide comprises CDRs 1 and 2 (e.g., HCDR1 and 2) or CDRs 1-3 of a variable domain of an antibody (e.g., HCDRs1-3). An antibody antigen-binding site may be provided by one or more antibody variable domains. In an example, the antibody binding site is provided by a single variable domain, e.g., a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH/VL pair or two or more of such pairs. Thus, an antibody antigen-binding site may comprise a VH and a VL. The antibody may be a whole immunoglobulin, including constant regions, or may be an antibody fragment. An antibody fragment is a portion of an intact antibody, for example comprising the antigen binding and/or variable region of the intact antibody. Examples of antibody fragments include:(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region;(iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody,(v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and(vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. Further examples of antibodies are H2 antibodies that comprise a dimer of a heavy chain (5′-VH-(optional hinge)-CH2-CH3-3′) and are devoid of a light chain. Single-chain antibodies (e.g., scFv) are a commonly used fragment. Multispecific antibodies may be formed from antibody fragments. An antibody of the invention may employ any such format, as appropriate. Optionally, the antibody immunoglobulin domains may be fused or conjugated to additional polypeptide sequences and/or to labels, tags, toxins or other molecules. Antibody immunoglobulin domains may be fused or conjugated to one or more different antigen binding regions, providing a molecule that is able to bind a second antigen in addition to ICOS. An antibody of the present invention may be a multispecific antibody, e.g., a bispecific antibody, comprising (i) an antibody antigen binding site for ICOS and (ii) a further antigen binding site (optionally an antibody antigen binding site, as described herein) which recognises another antigen (e.g., PD-L1). An antibody normally comprises an antibody VH and/or VL domain. Isolated VH and VL domains of antibodies are also part of the invention. The antibody variable domains are the portions of the light and heavy chains of antibodies that include amino acid sequences of complementarity determining regions (CDRs; ie., CDR1, CDR2, and CDR3), and framework regions (FRs). Thus, within each of the VH and VL domains are CDRs and FRs. A VH domain comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to the methods used in this invention, the amino acid positions assigned to CDRs and FRs may be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature. An antibody may comprise an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDR1, CDR2 and CDR3 and a framework. Examples of antibody VH and VL domains and CDRs according to the present invention are as listed in the appended sequence listing that forms part of the present disclosure. The CDRs shown in the sequence listing are defined according to the IMGT system [18]. All VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the invention. As described herein, a “set of CDRs” comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a “set of CDRs” includes HCDRs and LCDRs. An antibody the invention may comprise one or more CDRs as described herein, e.g. a CDR3, and optionally also a CDR1 and CDR2 to form a set of CDRs. The CDR or set of CDRs may be a CDR or set of CDRs of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or may be a variant thereof as described herein. The invention provides antibodies comprising an HCDR1, HCDR2 and/or HCDR3 of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 and/or an LCDR1, LCDR2 and/or LCDR3 of any of these antibodies, e.g. a set of CDRs. The antibody may comprise a set of VH CDRs of one of these antibodies. Optionally it may also comprise a set of VL CDRs of one of these antibodies, and the VL CDRs may be from the same or a different antibody as the VH CDRs. A VH domain comprising a disclosed set of HCDRs, and/or a VL domain comprising a disclosed set of LCDRs, are also provided by the invention. Typically, a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although as discussed further below a VH or VL domain alone may be used to bind antigen. The STIM003 VH domain may be paired with the STIM003 VL domain, so that an antibody antigen-binding site is formed comprising both the STIM003 VH and VL domains. Analogous embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, the STIM003 VH is paired with a VL domain other than the STIM003 VL. Light-chain promiscuity is well established in the art. Again, analogous embodiments are provided by the invention for the other VH and VL domains disclosed herein. Thus, the VH of any of antibodies STIM001, STIM002, STIM003, STIM004 and STIM005 may be paired with the VL of any of antibodies STIM001, STIM002, STIM003, STIM004 and STIM005. Further, the VH of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 may be paired with the VL of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. An antibody may comprise one or more CDRs, e.g. a set of CDRs, within an antibody framework. The framework regions may be of human germline gene segment sequences. Thus, the antibody may be a human antibody having a VH domain comprising a set of HCDRs in a human germline framework. Normally the antibody also has a VL domain comprising a set of LCDRs, e.g. in a human germline framework. An antibody “gene segment”, e.g., a VH gene segment, D gene segment, or JH gene segment refers to oligonucleotide having a nucleic acid sequence from which that portion of an antibody is derived, e.g., a VH gene segment is an oligonucleotide comprising a nucleic acid sequence that corresponds to a polypeptide VH domain from FR1 to part of CDR3. Human V, D and J gene segments recombine to generate the VH domain, and human V and J segments recombine to generate the VL domain. The D domain or region refers to the diversity domain or region of an antibody chain. J domain or region refers to the joining domain or region of an antibody chain. Somatic hypermutation may result in an antibody VH or VL domain having framework regions that do not exactly match or align with the corresponding gene segments, but sequence alignment can be used to identify the closest gene segments and thus identify from which particular combination of gene segments a particular VH or VL domain is derived. When aligning antibody sequences with gene segments, the antibody amino acid sequence may be aligned with the amino acid sequence encoded by the gene segment, or the antibody nucleotide sequence may be aligned directly with the nucleotide sequence of the gene segment. Alignments of STIM antibody VH and VL domain sequences against related antibodies and against human germline sequences are shown inFIG.35,FIG.36andFIG.37. An antibody of the invention may be a human antibody or a chimaeric antibody comprising human variable regions and non-human (e.g., mouse) constant regions. The antibody of the invention for example has human variable regions, and optionally also has human constant regions. Thus, antibodies optionally include constant regions or parts thereof, e.g., human antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to antibody light chain kappa or lambda constant domains. Similarly, an antibody VH domain may be attached at its C-terminal end to all or part (e.g. a CH1 domain or Fc region) of an immunoglobulin heavy chain constant region derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, such as IgG1 or IgG4. Examples of human heavy chain constant regions are shown in Table S1. Constant regions of antibodies of the invention may alternatively be non-human constant regions. For example, when antibodies are generated in transgenic animals (examples of which are described elsewhere herein), chimaeric antibodies may be produced comprising human variable regions and non-human (host animal) constant regions. Some transgenic animals generate fully human antibodies. Others have been engineered to generate antibodies comprising chimaeric heavy chains and fully human light chains. Where antibodies comprise one or more non-human constant regions, these may be replaced with human constant regions to provide antibodies more suitable for administration to humans as therapeutic compositions, as their immunogenicity is thereby reduced. Digestion of antibodies with the enzyme papain, results in two identical antigen-binding fragments, known also as “Fab” fragments, and a “Fc” fragment, having no antigen-binding activity but having the ability to crystallize. “Fab” when used herein refers to a fragment of an antibody that includes one constant and one variable domain of each of the heavy and light chains. The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The “Fc fragment” refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognised by Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with the enzyme pepsin, results in the a F(ab′)2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab′)2 fragment has the ability to crosslink antigen. “Fv” when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognise and bind antigen, although at a lower affinity than the entire binding site. Antibodies disclosed herein may be modified to increase or decrease serum half-life. In one embodiment, one or more of the following mutations: T252L, T254S or T256F are introduced to increase biological half-life of the antibody. Biological half-life can also be increased by altering the heavy chain constant region CH1domain or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022, the modifications described therein are incorporated herein by reference. In another embodiment, the Fc hinge region of an antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. One or more amino acid mutations are introduced into the CH2—CH3domain interface region of the Fc-hinge fragment such that the antibody or fragment has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. Other methods of increasing serum half-life are known to those skilled in the art. Thus, in one embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody or fragment is fused to an albumin-biding domain, e.g. an albumin binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PASylated (i.e. genetic fusion of polypeptide sequences composed of PAS (XL-Protein GmbH) which forms uncharged random coil structures with large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated®/rPEGylated (i.e. genetic fusion of non-exact repeat peptide sequence (Amunix, Versartis) to the therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e. genetic fusion to ELP repeat sequence (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, which fusions, e.g. in Tables 2 and 6, are incorporated herein by reference. The antibody may have a modified constant region which increases stability. Thus, in one embodiment, the heavy chain constant region comprises a Ser228Pro mutation. In another embodiment, the antibodies and fragments disclosed herein comprise a heavy chain hinge region that has been modified to alter the number of cysteine residues. This modification can be used to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. Fc Effector Functions, ADCC, ADCP and CDC As discussed above, anti-ICOS antibodies can be provided in various isotypes and with different constant regions. Examples of human IgG antibody heavy chain constant region sequences are shown in Table S1. The Fc region of the antibody primarily determines its effector function in terms of Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement dependent cytotoxicity (CDC) activity and antibody-dependent cell phagocytosis (ADCP) activity. These “cellular effector functions”, as distinct from effector T cell function, involve recruitment of cells bearing Fc receptors to the site of the target cells, resulting in killing of the antibody-bound cell. In addition to ADCC and CDC, the ADCP mechanism [19] represents a means of depleting antibody-bound T cells, and thus targeting high ICOS expressing TRegs for deletion. Cellular effector functions ADCC, ADCP and/or CDC may also be exhibited by antibodies lacking Fc regions. Antibodies may comprise multiple different antigen-binding sites, one directed to ICOS and another directed to a target molecule where engagement of that target molecule induces ADCC, ADCP and/or CDC, e.g., an antibody comprising two scFv regions joined by a linker, where one scFv can engage an effector cell. An antibody according to the present invention may be one that exhibits ADCC, ADCP and/or CDC. Alternatively, an antibody according to the present invention may lack ADCC, ADCP and/or CDC activity. In either case, an antibody according to the present invention may comprise, or may optionally lack, an Fc region that binds to one or more types of Fc receptor. Use of different antibody formats, and the presence or absence of FcR binding and cellular effector functions, allow the antibody to be tailored for use in particular therapeutic purposes as discussed elsewhere herein. A suitable antibody format for some therapeutic applications employs a wild-type human IgG1 constant region. A constant region may be an effector-enabled IgG1 constant region, optionally having ADCC and/or CDC and/or ADCP activity. A suitable wild type human IgG1 constant region sequence is SEQ ID NO: 340 (IGHG1*01). Further examples of human IgG1 constant regions are shown in Table S1. For testing of candidate therapeutic antibodies in mouse models of human disease, an effector positive mouse constant region, such as mouse IgG2a (mIgG2a), may be included instead of an effector positive human constant region. A constant region may be engineered for enhanced ADCC and/or CDC and/or ADCP. The potency of Fc-mediated effects may be enhanced by engineering the Fc domain by various established techniques. Such methods increase the affinity for certain Fc-receptors, thus creating potential diverse profiles of activation enhancement. This can achieved by modification of one or several amino acid residues [20]. Human IgG1 constant regions containing specific mutations or altered glycosylation on residue Asn297 (e.g., N297Q, EU index numbering) have been shown to enhance binding to Fc receptors. Example mutations are one or more of the residues selected from 239, 332 and 330 for human IgG1 constant regions (or the equivalent positions in other IgG isotypes). An antibody may thus comprise a human IgG1 constant region having one or more mutations independently selected from N297Q, S239D, 1332E and A330L (EU index numbering). A triple mutation (M252Y/S254T/T256E) may be used to enhance binding to FcRn, and other mutations affecting FcRn binding are discussed in Table 2 of [21], any of which may be employed in the present invention. Increased affinity for Fc receptors can also be achieved by altering the natural glycosylation profile of the Fc domain by, for example, generating under fucosylated or de-fucosylated variants [22]. Non-fucosylated antibodies harbour a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue. These glycoengineered antibodies that lack core fucose residue from the Fc N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of FcγRIIIa binding capacity. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to Fc-gamma RIII [23]. Thus, an antibody may comprise a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human Fcγ receptors selected from the group consisting of FcγRIIB and FcγRIIA with higher affinity than the wild type human IgG heavy chain constant region binds to the human Fcγ receptors. The antibody may comprise a human IgG heavy chain constant region that is a variant of a wild type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human FcγRIIB with higher affinity than the wild type human IgG heavy chain constant region binds to human FcγRIIB. The variant human IgG heavy chain constant region can be a variant human IgG1, a variant human IgG2, or a variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system). In another embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G, and A330R; P238D, E233D, G237D, H268D, P271G, and A330R; G236D and S267E; S239D and S267E; V262E, S267E, and L328F; and V264E, S267E, and L328F (EU index numbering system). The enhancement of CDC may be achieved by amino acid changes that increase affinity for C1q, the first component of the classic complement activation cascade [24]. Another approach is to create a chimeric Fc domain created from human IgG1 and human IgG3 segments that exploit the higher affinity of IgG3 for C1q [25]. Antibodies of the present invention may comprise mutated amino acids at residues 329, 331 and/or 322 to alter the C1q binding and/or reduced or abolished CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain Fc regions with modifications at residues 231 and 239, whereby the amino acids are replaced to alter the ability of the antibody to fix complement. In one embodiment, the antibody or fragment has a constant region comprising one or more mutations selected from E345K, E430G, R344D and D356R, in particular a double mutation comprising R344D and D356R (EU index numbering system). WO2008/137915 described anti-ICOS antibodies with modified Fc regions having enhanced effector function. The antibodies were reported to mediate enhanced ADCC activity as compared to the level of ADCC activity mediated by a parent antibody comprising the VH and VK domains and a wild type Fc region. Antibodies according to the present invention may employ such variant Fc regions having effector function as described therein. ADCC activity of an antibody may be determined in an assay as described herein. ADCC activity of an anti-ICOS antibody may be determined in vitro using an ICOS positive T cell line as described in Example 10. ADCC activity of an anti-PD-L1 antibody may be determined in vitro in an ADCC assay using PD-L1 expressing cells. For certain applications (such as in the context of vaccination) it may be preferred to use antibodies without Fc effector function. Antibodies may be provided without a constant region, or without an Fc region—examples of such antibody formats are described elsewhere herein. Alternatively, an antibody may have a constant region which is effector null. An antibody may have a heavy chain constant region that does not bind Fcγ receptors, for example the constant region may comprise a Leu235Glu mutation (i.e., where the wild type leucine residue is mutated to a glutamic acid residue). Another optional mutation for a heavy chain constant region is Ser228Pro, which increases stability. A heavy chain constant region may be an IgG4 comprising both the Leu235Glu mutation and the Ser228Pro mutation. This “IgG4-PE” heavy chain constant region is effector null. An alternative effector null human constant region is a disabled IgG1. A disabled IgG1 heavy chain constant region may contain alanine at position 235 and/or 237 (EU index numbering), e.g., it may be a IgG1*01 sequence comprising the L235A and/or G237A mutations (“LAGA”). A variant human IgG heavy chain constant region may comprise one or more amino acid mutations that reduce the affinity of the IgG for human FcγRIIIA, human FcγRIIA, or human FcγRI. In one embodiment, the FcγRIIB is expressed on a cell selected from the group consisting of macrophages, monocytes, B-cells, dendritic cells, endothelial cells, and activated T-cells. In one embodiment, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L, and I332E; S298A, E333A, and K334A; G236A, S239D, and I332E; and F243L, R292P, Y300L, V305I, and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises a S239D, A330L, or I332E amino acid mutations (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises an S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region is a variant human IgG1 heavy chain constant region comprising the S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the antibody or fragment comprises an afucosylated Fc region. In another embodiment, the antibody or fragment thereof is defucosylated. In another embodiment, the antibody or fragment is under fucosylated. An antibody may have a heavy chain constant region that binds one or more types of Fc receptor but does not induce cellular effector functions, i.e., does not mediate ADCC, CDC or ADCP activity. Such a constant region may be unable to bind the particular Fc receptor(s) responsible for triggering ADCC, CDC or ADCP activity. Generating and Modifying Antibodies Methods for identifying and preparing antibodies are well known. Antibodies may be generated using transgenic mice (eg, the Kymouse™, Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse® or MeMo Mouse®), rats (e.g., the Omnirat®), camelids, sharks, rabbits, chickens or other non-human animals immunised with ICOS or a fragment thereof or a synthetic peptide comprising an ICOS sequence motif of interest, followed optionally by humanisation of the constant regions and/or variable regions to produce human or humanised antibodies. In an example, display technologies can be used, such as yeast, phage or ribosome display, as will be apparent to the skilled person. Standard affinity maturation, e.g., using a display technology, can be performed in a further step after isolation of an antibody lead from a transgenic animal, phage display library or other library. Representative examples of suitable technologies are described in US20120093818 (Amgen, Inc), which is incorporated by reference herein in its entirety, eg, the methods set out in paragraphs [0309] to [0346]. Immunisation of an ICOS knock out non-human animal with human ICOS antigen facilitates the generation of antibodies that recognise both human and non-human ICOS. As described herein and illustrated in the Examples, an ICOS knock out mouse can be immunised with cells expressing human ICOS to stimulate production of antibodies to human and mouse ICOS in the mouse, which can be recovered and tested for binding to human ICOS and to mouse ICOS. Cross-reactive antibodies can thus be selected, which may be screened for other desirable properties as described herein. Methods of generating antibodies to an antigen (e.g., a human antigen), through immunisation of animals with the antigen where expression of the endogenous antigen (e.g, endogenous mouse antigen) has been knocked-out in the animal, may be performed in animals capable of generating antibodies comprising human variable domains. The genomes of such animals can be engineered to comprise a human or humanised immunoglobulin locus encoding human variable region gene segments, and optionally an endogenous constant region or a human constant region. Recombination of the human variable region gene segments generates human antibodies, which may have either a non-human or human constant region. Non-human constant regions may subsequently be replaced by human constant regions where the antibody is intended for in vivo use in humans. Such methods and knock-out transgenic animals are described in WO2013/061078. Generally, a Kymouse™, VELOCIMMUNE® or other mouse or rat (optionally an ICOS knock out mouse or rat, as noted) can be challenged with the antigen of interest, and lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The lymphatic cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of the heavy chain and light chain. Such an antibody protein may be produced in a cell, such as a CHO cell. Alternatively, DNA encoding the antigen-specific chimaeric antibodies or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes. Initially, high affinity chimaeric antibodies are isolated having a human variable region and a mouse constant region. The antibodies are characterised and selected for desirable characteristics, including affinity, selectivity, agonism, T-cell dependent killing, neutralising potency, epitope, etc. The mouse constant regions are optionally replaced with a desired human constant region to generate the fully human antibody of the invention, for example wild-type or modified IgG1 or IgG4 (for example, SEQ ID NO: 751, 752, 753 in US2011/0065902 (which is incorporated by reference herein in its entirety). While the constant region selected may vary according to specific use, high affinity antigen-binding and target specificity characteristics reside in the variable region. Thus, in a further aspect, the present invention provides a transgenic non-human mammal having a genome comprising a human or humanised immunoglobulin locus, wherein the mammal does not express ICOS. The mammal may for instance be a knock-out mouse or rat, or other laboratory animal species. Transgenic mice such as the Kymouse™ contain human heavy and light chain immunoglobulin loci inserted at the corresponding endogenous mouse immunoglobulin loci. A transgenic mammal according to the present invention may be one that contains such targeted insertions, or it may contain human heavy and light chain immunoglobulin loci or immunoglobulin genes that are randomly inserted in its genome, inserted at a locus other than the endogenous Ig locus, or provided on an additional chromosome or chromosomal fragment. Further aspects of the invention are the use of such non-human mammals for producing antibodies to ICOS, and methods of producing antibodies or antibody heavy and/or light chain variable domains in such mammals. A method of producing an antibody that binds the extracellular domain of human and non-human ICOS may comprise providing a transgenic non-human mammal having a genome comprising a human or humanised immunoglobulin locus, wherein the mammal does not express ICOS, and (a) immunising the mammal with human ICOS antigen (e.g., with cells expressing human ICOS or with purified recombinant ICOS protein); (b) isolating antibodies generated by the mammal; (c) testing the antibodies for ability to bind human ICOS and non-human ICOS; and (d) selecting one or more antibodies that binds both human and non-human ICOS. Testing for ability to bind human ICOS and non-human ICOS may be done using surface plasmon resonance, HTRF, FACS or any other method described herein. Optionally, binding affinities for human and mouse ICOS are determined. The affinity, or fold-difference in affinity, of binding to human ICOS and mouse ICOS may be determined, and antibodies displaying species cross-reactivity may thus be selected (affinity thresholds and fold-differences that may be used as selection criteria are exemplified elsewhere herein). Neutralising potency, or fold difference in neutralising potency, of the antibody for inhibiting human and mouse ICOS ligand binding to the human and mouse ICOS receptor respectively may also or alternatively be determined as a way to screen for cross-reactive antibodies, e.g., in an HTRF assay. Again, possible thresholds and fold-differences that may be used as selection criteria are exemplified elsewhere herein. The method may comprise testing the antibodies for ability to bind non-human ICOS from the same species or from a different species as the immunised mammal. Thus, where the transgenic mammal is a mouse (e.g., a Kymouse™), antibodies may be tested for ability to bind mouse ICOS. Where the transgenic mammal is a rat, antibodies may be tested for ability to bind rat ICOS. However, it may be equally useful to determine cross-reactivity of an isolated antibody for non-human ICOS of another species. Thus, antibodies generated in goats may be tested for binding to rat or mouse ICOS. Optionally, binding to goat ICOS may be determined instead or additionally. In other embodiments, the transgenic non-human mammal may be immunised with non-human ICOS, optionally ICOS of the same mammalian species (e.g., an ICOS knock-out mouse may be immunised with mouse ICOS) instead of human ICOS. Affinity of isolated antibodies for binding to human ICOS and non-human ICOS is then determined in the same way, and antibodies that bind both human and non-human ICOS are selected. Nucleic acid encoding an antibody heavy chain variable domain and/or an antibody light chain variable domain of a selected antibody may be isolated. Such nucleic acid may encode the full antibody heavy chain and/or light chain, or the variable domain(s) without associated constant region(s). As noted, encoding nucleotide sequences may be obtained directly from antibody-producing cells of a mouse, or B cells may be immortalised or fused to generate hybridomas expressing the antibody, and encoding nucleic acid obtained from such cells. Optionally, nucleic acid encoding the variable domain(s) is then conjugated to a nucleotide sequence encoding a human heavy chain constant region and/or human light chain constant region, to provide nucleic acid encoding a human antibody heavy chain and/or human antibody light chain, e.g., encoding an antibody comprising both the heavy and light chain. As described elsewhere herein, this step is particularly useful where the immunised mammal produces chimaeric antibodies with non-human constant regions, which are preferably replaced with human constant regions to generate an antibody that will be less immunogenic when administered to humans as a medicament. Provision of particular human isotype constant regions is also significant for determining the effector function of the antibody, and a number of suitable heavy chain constant regions are discussed herein. Other alterations to nucleic acid encoding the antibody heavy and/or light chain variable domain may be performed, such as mutation of residues and generation of variants, as described herein. The isolated (optionally mutated) nucleic acid may be introduced into host cells, e.g., CHO cells as discussed. Host cells are then cultured under conditions for expression of the antibody, or of the antibody heavy and/or light chain variable domain, in any desired antibody format. Some possible antibody formats are described herein, e.g., whole immunoglobulins, antigen-binding fragments, and other designs. Variable domain amino acid sequence variants of any of the VH and VL domains or CDRs whose sequences are specifically disclosed herein may be employed in accordance with the present invention, as discussed. There are many reasons why it may be desirable to create variants, which include optimising the antibody sequence for large-scale manufacturing, facilitating purification, enhancing stability or improving suitability for inclusion in a desired pharmaceutical formulation. Protein engineering work can be performed at one or more target residues in the antibody sequence, e.g., to substituting one amino acid with an alternative amino acid (optionally, generating variants containing all naturally occurring amino acids at this position, with the possible exception of Cys and Met), and monitoring the impact on function and expression to determine the best substitution. It is in some instances undesirable to substitute a residue with Cys or Met, or to introduce these residues into a sequence, as to do so may generate difficulties in manufacturing—for instance through the formation of new intramolecular or intermolecular cysteine-cysteine bonds. Where a lead candidate has been selected and is being optimised for manufacturing and clinical development, it will generally be desirable to change its antigen-binding properties as little as possible, or at least to retain the affinity and potency of the parent molecule. However, variants may also be generated in order to modulate key antibody characteristics such as affinity, cross-reactivity or neutralising potency. An antibody may comprise a set of H and/or L CDRs of any of the disclosed antibodies with one or more amino acid mutations within the disclosed set of H and/or L CDRs. The mutation may be an amino acid substitution, deletion or insertion. Thus for example there may be one or more amino acid substitutions within the disclosed set of H and/or L CDRs. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations e.g. substitutions, within the set of H and/or L CDRs. For example, there may be up to 6, 5, 4, 3 or 2 mutations, e.g. substitutions, in HCDR3 and/or there may be up to 6, 5, 4, 3, or 2 mutations, e.g. substitutions, in LCDR3. An antibody may comprise the set of HCDRs, LCDRs or a set of 6 (H and L) CDRs shown for any STIM antibody herein or may comprise that set of CDRs with one or two conservative substitutions. One or more amino acid mutations may optionally be made in framework regions of an antibody VH or VL domain disclosed herein. For example, one or more residues that differ from the corresponding human germline segment sequence may be reverted to germline. Human germline gene segment sequences corresponding to VH and VL domains of example anti-ICOS antibodies are indicated in Table E12-1, Table E12-2 and Table E12-3, and alignments of antibody VH and VL domains to corresponding germline sequences are shown in the drawings. An antibody may comprise a VH domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VH domain of any of the antibodies shown in the appended sequence listing, and/or comprising a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of those antibodies. Algorithms that can be used to calculate % identity of two amino acid sequences include e.g. BLAST, FASTA, or the Smith-Waterman algorithm, e.g. employing default parameters. Particular variants may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue). Alterations may be made in one or more framework regions and/or one or more CDRs. Variants are optionally provided by CDR mutagenesis. The alterations normally do not result in loss of function, so an antibody comprising a thus-altered amino acid sequence may retain an ability to bind ICOS. It may retain the same quantitative binding ability as an antibody in which the alteration is not made, e.g. as measured in an assay described herein. The antibody comprising a thus-altered amino acid sequence may have an improved ability to bind ICOS. Alteration may comprise replacing one or more amino acid residue with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences of the invention are described elsewhere herein. Naturally occurring amino acids include the 20 “standard” L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring. The term “variant” as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, substitutions or additions, yet retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring amino acid residue. Such substitutions may be classified as “conservative”, in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be “non-conservative”, in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties, such as naturally-occurring amino acid from a different group (e.g., substituting a charged or hydrophobic amino; acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid. In some embodiments amino acid substitutions are conservative. Also encompassed within the term variant when used with reference to a polynucleotide or polypeptide, refers to a polynucleotide or polypeptide that can vary in primary, secondary, or tertiary structure, as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide). In some aspects, one can use “synthetic variants”, “recombinant variants”, or “chemically modified” polynucleotide variants or polypeptide variants isolated or generated using methods well known in the art. “Modified variants” can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Some aspects use include insertion variants, deletion variants or substituted variants with substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, for example but not limited to insertion of ornithine which do not normally occur in human proteins. The term “conservative substitution,” when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue that has similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.). Conservative amino acid substitutions include replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids can also be considered “conservative substitutions” if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example if the amino acid is on the exterior of the peptide and expose to solvents, or on the interior and not exposed to solvents. One can select the amino acid that will substitute an existing amino acid based on the location of the existing amino acid, including its exposure to solvents (i.e., if the amino acid is exposed to solvents or is present on the outer surface of the peptide or polypeptide as compared to internally localized amino acids not exposed to solvents). Selection of such conservative amino acid substitutions are well known in the art, for example as disclosed in Dordo et al, J. Mol Biol, 1999, 217, 721-739 and Taylor et al, J. Theor. Biol. 119(1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Accordingly, one can select conservative amino acid substitutions suitable for amino acids on the exterior of a protein or peptide (i.e. amino acids exposed to a solvent), for example, but not limited to, the following substitutions can be used: substitution of Y with F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P. In alternative embodiments, one can also select conservative amino acid substitutions encompassed suitable for amino acids on the interior of a protein or peptide, for example one can use suitable conservative substitutions for amino acids is on the interior of a protein or peptide (i.e. the amino acids are not exposed to a solvent), for example but not limited to, one can use the following conservative substitutions: where Y is substituted with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of variants. The invention includes methods of producing antibodies containing VH and/or VL domain variants of the antibody VH and/or VL domains shown in the appended sequence listing. Such antibodies may be produced by a method comprising (i) providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent antibody VH domain, an antibody VH domain that is an amino acid sequence variant of the parent antibody VH domain, wherein the parent antibody VH domain is the VH domain of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or a VH domain comprising the heavy chain complementarity determining regions of any of those antibodies, (ii) optionally combining the VH domain thus provided with a VL domain, to provide a VH/VL combination, and (iii) testing the VH domain or VH/VL domain combination thus provided to identify an antibody with one or more desired characteristics. Desired characteristics include binding to human ICOS, binding to mouse ICOS, and binding to other non-human ICOS such as cynomolgus ICOS. Antibodies with comparable or higher affinity for human and/or mouse ICOS may be identified. Other desired characteristics include increasing effector T cell function indirectly, via depletion of immunosuppressive TRegs, or directly, via ICOS signalling activation on T effector cells. Identifying an antibody with a desired characteristic may comprise identifying an antibody with a functional attribute described herein, such as its affinity, cross-reactivity, specificity, ICOS receptor agonism, neutralising potency and/or promotion of T cell dependent killing, any of which may be determined in assays as described herein. When VL domains are included in the method, the VL domain may be a VL domain of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or may be a variant provided by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain, wherein the parent VL domain is the VL domain of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or a VL domain comprising the light chain complementarity determining regions of any of those antibodies. Methods of generating variant antibodies may optionally comprise producing copies of the antibody or VH/VL domain combination. Methods may further comprise expressing the resultant antibody. It is possible to produce nucleotide sequences corresponding to a desired antibody VH and/or VL domain, optionally in one or more expression vectors. Suitable methods of expression, including recombinant expression in host cells, are set out in detail herein. Encoding Nucleic Acids and Methods of Expression Isolated nucleic acid may be provided, encoding antibodies according to the present invention. Nucleic acid may be DNA and/or RNA. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode an antibody. The present invention provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. Exemplary nucleotide sequences are included in the sequence listing. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise. The present invention also provides a recombinant host cell that comprises one or more nucleic acids encoding the antibody. Methods of producing the encoded antibody may comprise expression from the nucleic acid, e.g., by culturing recombinant host cells containing the nucleic acid. The antibody may thus be obtained, and may be isolated and/or purified using any suitable technique, then used as appropriate. A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host isE. coli. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others. Vectors may contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Nucleic acid encoding an antibody can be introduced into a host cell. Nucleic acid can be introduced to eukaryotic cells by various methods, including calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may be incorporated into the host cell or into an artificial chromosome. Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduction may be followed by expressing the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene, then optionally isolating or purifying the antibody. Nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences that promote recombination with the genome, in accordance with standard techniques. The present invention also provides a method that comprises using nucleic acid described herein in an expression system in order to express an antibody. Therapeutic Use An antibody described herein may be used in a method of treatment of the human or animal body by therapy. The antibodies find use in increasing effector T cell response, which is of benefit for a range of diseases or conditions, including treating cancers or solid tumours and in the context of vaccination. Increased Teff response may be achieved using an antibody that modulates the balance or ratio between Teffs and Tregs in favour of Teff activity. Anti-ICOS antibodies may be used for depleting regulatory T cells and/or increasing effector T cell response in a patient, and may be administered to a patient to treat a disease or condition amenable to therapy by depleting regulatory T cells and/or increasing effector T cell response. An antibody of the present invention, or a composition comprising such an antibody molecule or its encoding nucleic acid, may be used or provided for use in any such method. Use of the antibody, or of a composition comprising it or its encoding nucleic acid, for the manufacture of a medicament for use in any such method is also envisaged. The method typically comprises administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described elsewhere herein. One envisaged therapeutic use of the antibodies is treatment of cancer. The cancer may be a solid tumour, e.g., renal cell cancer (optionally renal cell carcinoma, e.g., clear cell renal cell carcinoma), head and neck cancer, melanoma (optionally malignant melanoma), non-small cell lung cancer (e.g., adenocarcinoma), bladder cancer, ovarian cancer, cervical cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, testicular germ cell carcinoma, or the metastases of a solid tumour such as those listed, or it may be a liquid haematological tumour e.g., lymphoma (such as Hodgkin's lymphoma or Non-Hodgkin's lymphoma, e.g., diffuse large B-cell lymphoma, DLBCL) or leukaemia (e.g., acute myeloid leukaemia). An anti-ICOS antibody may enhance tumour clearance in melanoma, head and neck cancer and non-small cell lung cancer and other cancers with a moderate to high mutational load [26]. By enhancing patients' immune response to their neoplastic lesions, immunotherapy using an anti-ICOS antibody offers the prospect of durable cures or long-term remissions, potentially even in the context of late stage disease. Cancers are a diverse group of diseases, but anti-ICOS antibodies offer the possibility of treating a range of different cancers by exploiting the patient's own immune system, which has the potential to kill any cancer cell through recognition of mutant or overexpressed epitopes that distinguish cancer cells from normal tissue. By modulating the Teff/Treg balance, anti-ICOS antibodies can enable and/or promote immune recognition and killing of cancer cells. While anti-ICOS antibodies are therefore useful therapeutic agents for a wide variety of cancers, there are particular categories of cancers for which anti-ICOS therapy is especially suited and/or where anti-ICOS therapy can be effective when other therapeutic agents are not. One such group is cancer that is positive for expression of ICOS ligand. Cancer cells may acquire expression of ICOS ligand, as has been described for melanoma [27]. Expression of ICOS ligand may provide the cells with a selective advantage as the surface-expressed ligand binds ICOS on Tregs, promoting the expansion and activation of the Tregs and thereby suppressing the immune response against the cancer. Cancer cells expressing ICOS ligand may depend for their survival on this suppression of the immune system by Tregs, and would thus be vulnerable to treatment with anti-ICOS antibodies that target the Tregs. This applies also to cancers derived from cells that naturally express ICOS ligand. Continued expression of ICOS ligand by these cells again provides a survival advantage through immune suppression. A cancer expressing ICOS ligand may be derived from antigen-presenting cells such as B cells, dendritic cells and monocytes and may be a liquid haematological tumour such as those mentioned herein. Interestingly it has been shown that these types of cancer are also high in ICOS and FOXP3 expression (TCGA data)—see Example 25. Example 20 herein demonstrates efficacy of exemplary anti-ICOS antibodies in treating tumours derived from cancerous B cells (A20 syngeneic cells) that express ICOS ligand. Accordingly, anti-ICOS antibodies can be used in methods of treating cancers that are positive for expression of ICOS ligand. Further, a cancer to be treated with anti-ICOS antibody according to the present invention may be one that is positive for expression of ICOS and/or FOXP3, and optionally also expresses ICOS ligand. Patients may undergo testing to determine whether their cancer is positive for expression of the protein of interest (e.g., ICOS ligand, ICOS and/or FOXP3), for example by taking a test sample (e.g., tumour biopsy) from the patient and determining expression of the protein of interest. Patients whose cancer has been characterised as positive for expression of one, two or all such proteins of interest are selected for treatment with anti-ICOS antibody. As discussed elsewhere herein, anti-ICOS antibody may be used as a monotherapy or in combination with one or more other therapeutic agents. Anti-ICOS antibodies also offer hope to patients whose cancers are refractory to treatment with antibodies or other drugs directed to immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, CD137, GITR or CD73. These immunotherapies are effective against some cancers but in some cases a cancer may not respond, or it may become unresponsive to continued treatment with the antibody. In common with antibodies to immune checkpoint inhibitors, anti-ICOS antibodies modulate the patient's immune system—nevertheless an anti-ICOS antibody may succeed where such other antibodies fail. It is shown herein that animals carrying A20 B cell lymphomas could be treated with anti-ICOS antibodies to reduce growth of the tumour, shrink the tumour and indeed clear the tumour from the body, whereas treatment with an anti-PD-L1 antibody was no better than control. The A20 cell line has also been reported to be resistant to anti-CTLA-4 [28]. Accordingly, anti-ICOS antibodies can be used in methods of treating cancers that are refractory to treatment with one or more immunotherapies, such as (any or all of) an anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody, anti-GITR antibody, or anti-CD73 antibody. A cancer may be characterised as being refractory to treatment with an antibody or other drug if treatment with that antibody or drug does not significantly reduce growth of the cancer, e.g., if a tumour continues to grow or does not reduce in size or if after a response period the tumour re-initiates its growth. Non-response to a therapeutic agent may be determined ex vivo by testing a sample (e.g., tumour biopsy sample) for cancer cell killing or growth inhibition, and/or in the clinical setting by observing (e.g., using an imaging technology, including MRI) that a patient treated with the therapy is not responding to treatment. Patients whose cancer has been characterised as refractory to treatment with such an immunotherapy are selected for treatment with anti-ICOS antibody. Further, anti-ICOS antibodies may be used to treat B-cell derived cancer that is resistant to treatment with an anti-CD20 antibody. Anti-ICOS antibodies represent a treatment for cancers that fail to respond to, or become resistant to, therapy with anti-CD20 antibodies like rituximab. Anti-ICOS antibody may be used as a second-line (or further, or additional) treatment for such cancers. The anti-CD20 antibody resistant cancer may be a B cell cancer, e.g., B cell lymphoma, such as diffuse large B cell lymphoma. Resistance of a cancer to anti-CD20 may be determined ex vivo by testing a sample (e.g., tumour biopsy sample) for cancer cell killing or growth inhibition by anti-CD20 antibody, and/or in the clinical setting by observing that a patient treated with the anti-CD20 antibody is not responding to treatment. Alternatively, or additionally, the cancer (e.g., a tumour biopsy sample) may be tested to assess expression of CD20, where an absence or low level of CD20 expression indicates loss of sensitivity to anti-CD20 antibody. Samples obtained from patients may thus be tested to determine surface expression of a protein of interest, for example ICOS ligand, ICOS, FOXP3 and/or a target receptor to which another therapeutic agent (e.g., anti-receptor antibody) is directed. The target receptor may be CD20 (to which anti-CD20 antibody therapy such as rituximab is directed), or another receptor such as PD1, EGFR, HER2 or HER3. Surface expression of ICOS ligand, ICOS, FOXP3 and/or lack or loss of surface expression of the target receptor is an indication that the cancer is susceptible to anti-ICOS antibody therapy. Anti-ICOS antibodies can be provided for administration to a patient whose cancer is characterised by surface expression of ICOS ligand, ICOS, FOXP3 and/or lack or loss of surface expression of a target receptor, optionally where the patient has been previously treated with anti-CTLA4, anti-PD1, anti-PD-L1 or with an antibody to the target receptor and has not responded or has stopped responding to treatment with that antibody, as measured for example by continued or renewed cancer cell growth, e.g., increase in tumour size. Any suitable method may be employed to determine whether cancer cells test positive for surface expression of a protein such as ICOS ligand, CD20 or other target receptors mentioned herein. A typical method is immunohistochemistry, where a sample of the cells (e.g., a tumour biopsy sample) is contacted with an antibody for the protein of interest, and binding of antibody is detected using a labelled reagent—typically a second antibody that recognises the Fc region of the first antibody and carries a detectable label such as a fluorescent marker. A sample may be declared to test positive where at least 5% of cells are labelled, as visualised by cell staining or other detection of the label. Optionally a higher cut-off such as 10% or 25% may be used. The antibody will generally be used in excess. Reagent antibodies to the molecules of interest are available or may be generated by straightforward methods. To test for ICOS ligand, the antibody MAB1651 is currently available from R&D systems as a mouse IgG that recognises human ICOS ligand. To test for CD20 expression, rituximab may be used. Detection of mRNA levels of the ICOS ligand or target receptor of interest is an alternative technique [27]. A further indication that a tumour will respond to treatment with anti-ICOS antibody is the presence of Tregs in the tumour microenvironment. Activated Tregs are characterised by ICOS-high and Foxp3-high surface expression. The presence of Tregs in a tumour, especially in elevated numbers, provides a further basis on which a patient may be selected for treatment with anti-ICOS antibody. Tregs may be detected in a tumour biopsy sample ex vivo, for example by immunohistochemistry (assaying for co-expression of both Foxp3 and ICOS, using antibodies to the target protein followed by detection of labels, as described above) or by single cell dispersion of the sample for use in FACS with labelled antibodies to ICOS and Foxp3. FACS methods are exemplified in Example 17 and Example 18. The anti-ICOS antibodies may be used for treating cancers associated with infectious agents, such as virally-induced cancers. In this category are head and neck squamous cell carcinoma, cervical cancer, Merkel cell carcinoma and many others. Viruses associated with cancer include HBV, HCV, HPV (cervical cancer, oropharyngeal cancer), and EBV (Burkitts lymphomas, gastric cancer, Hodgkin's lymphoma, other EBV positive B cell lymphomas, nasopharyngeal carcinoma and post transplant lymphoproliferative disease). The International Agency for Research on Cancer (Monograph 100B) identified the following major cancer sites associated with infectious agents:Stomach/Gastric:Heliobacter pyloriLiver: Hepatitis B virus, hepatitis C virus (HCV),Opisthorchis viverrini, Clonorchis sinensisCervix uteri: Human papillomavirus (HPV) with or without HIVAnogenital (penile, vulva, vagina, anus): HPV with or without HIVNasopharynx: Epstein-Barr virus (EBV)Oropharynx: HPV with or without tobacco or alcohol consumptionKaposi's sarcoma: Human herpes virus type 8 with or without HIVNon-Hodgkin lymphoma:H. pylori, EBV with or without HIV, HCV, human T-cell lymphotropic virus type 1Hodgkin's lymphoma: EBV with or without HIVBladder:Schistosoma haematobium. Antibodies according to the present invention may be used for treating cancer associated with or induced by any of these infectious agents, such as the cancers specified above. Stimulation of effector T cell response can also contribute to immunity against infectious disease and/or to recovery from infectious disease in a patient. Thus, an anti-ICOS antibody may be used for treating infectious disease by administering the antibody to a patient. Infectious diseases include those caused by pathogens, e.g., bacterial, fungal, viral or protozoal pathogens, and treatment may be to promote immune response in a patient against the pathogen infection. An example of a bacterial pathogen is tuberculosis. Examples of viral pathogens are hepatitis B and HIV. Examples of protozoal pathogens arePlasmodiumspecies, which cause malaria, such asP. falciparum. The antibody may be used for treating infections, e.g., infection by any pathogen mentioned herein. Infection may be persistent or chronic infection. Infection may be localised or systemic. Extended contact between a pathogen and the immune system may lead to exhaustion of the immune system or development of tolerance (manifested for example through increased levels of Tregs, and tipping of the Treg:Teff balance in favour of Tregs) and/or to immune evasion by the pathogen, through evolution and modification of displayed pathogen antigens. These features reflect similar processes that are believed to occur in cancer. Anti-ICOS antibodies present a therapeutic approach to treating infection by a pathogen, e.g., chronic infection, through modulation of the Treg:Teff ratio in favour of Teff and/or other effects described herein. Treatment may be of patients who have been diagnosed as having an infectious disease or an infection. Alternatively, treatment may be preventative, and administered to a patient to guard against contracting a disease, e.g., as a vaccine, as described elsewhere herein. It has also been proposed that an immune response, particularly an IFNγ-dependent systemic immune response, could be beneficial for treatment of Alzheimer's disease and other CNS pathologies that share a neuroinflammatory component as part [29]. WO2015/136541 proposed treatment of Alzheimer's disease using an anti-PD-1 antibody. Anti-ICOS antibodies may be used in the treatment of Alzheimer's disease or other neurodegenerative diseases, optionally in combination with one or more other immunomodulators (e.g., antibody to PD-1). Combination Therapy Treatment with an immunomodulatory antibody such as anti-CTLA4, anti-PD1 or anti-PDL1, especially one with Fc effector function, may create an environment in which further depletion of ICOS highly expressing immune-suppressive cells is beneficial. It may be advantageous to combine an anti-ICOS antibody with such an immunomodulator to enhance its therapeutic effects. A patient who has been treated with an immunomodulatory antibody (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4) may particularly benefit from treatment with an anti-ICOS antibody. One reason for this is that an immunomodulatory antibody may increase the number of ICOS-positive Tregs (e.g., intratumoural Tregs) in the patient. This effect is also observed with certain other therapeutic agents, such as recombinant IL-2. Anti-ICOS antibody may reduce and/or reverse a surge or rise in ICOS+ Tregs (e.g., intratumoural Tregs) resulting from treatment of the patient with another therapeutic agent. A patient selected for treatment with an anti-ICOS antibody may thus be one who has already received treatment with a first therapeutic agent, the first therapeutic agent being an antibody (e.g., immunomodulator antibody) or other agent (e.g., IL-2) that increases the number of ICOS+ Tregs in the patient. Immunomodulators with which an anti-ICOS antibody may be combined include antibodies to any of: PDL1 (e.g., avelumab), PD-1 (e.g., pembrolizumab or nivolumab) or CTLA-4 (e.g., ipilimumab or tremelimumab). An anti-ICOS antibody may be combined with pidilizumab. In other embodiments, an anti-ICOS antibody is not administered in combination with anti-CTLA-4 antibody, and/or optionally is administered in combination with a therapeutic antibody that is not an anti-CTLA-4 antibody. For example, an anti-ICOS antibody may be used in combination therapy with an anti-PDL1 antibody. Preferably, the anti-ICOS antibody is one that mediates ADCC, ADCP and/or CDC. Preferably, the anti-PDL1 antibody is one that mediates ADCC, ADCP and/or CDC. An example of such combination therapy is administration of an anti-ICOS antibody with an anti-PDL1 antibody wherein both antibodies have effector positive constant regions. Thus, the anti-ICOS antibody and the anti-PDL1 antibody may both be able to mediate ADCC, CDC and/or ADCP. Fc effector function and selection of constant regions is described in detail elsewhere herein, but as one example an anti-ICOS human IgG1 may be combined with an anti-PD-L1 human IgG1. The anti-ICOS antibody and/or the anti-PD-L1 antibody may comprise a wild type human IgG1 constant region. Alternatively, the effector positive constant region of an antibody may be one that is engineered for enhanced effector function, e.g., enhanced CDC, ADCC and/or ADCP. Example antibody constant regions, including wild type human IgG1 sequences and mutations that alter effector function, are discussed in detail elsewhere herein. Anti-PDL1 antibodies with which an anti-ICOS antibody may be combined include:Anti-PDL1 antibody that inhibits binding of PD-1 to PDL1 and/or inhibits PDL1, optionally as effector positive human IgG1;Anti-PD-1 antibody that inhibits binding of PD-1 to PDL1 and/or PDL2;Avelumab, a human IgG1 antibody which inhibits PD-1 binding to PDL-1. See WO2013/079174;Durvalumab (or “MED14736”), a variant human IgG1 antibody having mutations L234A, L235A and 331. See WO2011/066389;Atezolizumab, a variant human IgG1 antibody having mutations N297A, D356E and L358M. See US2010/0203056;BMS-936559, a human IgG4 antibody comprising mutation S228P. See WO2007/005874. Numerous further examples of anti-PD-L1 antibodies are disclosed herein and others are known in the art. Characterisation data for many of the anti-PD-L1 antibodies mentioned here has been published in U.S. Pat. Nos. 9,567,399 and 9,617,338, both incorporated by reference herein. Example anti-PD-L1 antibodies have VH and/or VL domains comprising the HCDRs and/or LCDRs of any of 1D05, 84G09, 1D05 HC mutant 1, 1D05 HC mutant 2, 1D05 HC mutant 3, 1D05 HC mutant 4, 1D05 LC mutant 1, 1D05 LC mutant 2, 1D05 LC mutant 3, 411B08, 411C04, 411D07, 385F01, 386H03, 389A03, 413D08, 413G05, 413F09, 414B06 or 416E01 as set out in U.S. Pat. No. 9,567,399 or 9,617,338. The antibody may comprise the VH and VL domain of any of these antibodies, and may optionally comprise a heavy and/or light chain having the heavy and/or light chain amino acid sequence of any of these antibodies. VH and VL domains of these anti-PD-L1 antibodies are further described elsewhere herein. Further example anti-PD-L1 antibodies have VH and/or VL domains comprising the HCDRs and/or LCDRs of KN-035, CA-170, FAZ-053, M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504, or of an anti-PD-L1 antibody disclosed in any of WO2017/034916, WO2017/020291, WO2017/020858, WO2017/020801, WO2016/111645, WO2016/197367, WO2016/061142, WO2016/149201, WO2016/000619, WO2016/160792, WO2016/022630, WO2016/007235, WO2015/179654, WO2015/173267, WO2015/181342, WO2015/109124, WO2015/112805, WO2015/061668, WO2014/159562, WO2014/165082, WO2014/100079, WO2014/055897, WO2013/181634, WO2013/173223, WO2013/079174, WO2012/145493, WO2011/066389, WO2010/077634, WO2010/036959, WO2010/089411 and WO2007/005874. The antibody may comprise the VH and VL domain of any of these antibodies, and may optionally comprise a heavy and/or light chain having the heavy and/or light chain amino acid sequence of any of these antibodies. The anti-ICOS antibody which is used in combination therapy with anti-PD-L1 may be an antibody of the present invention as disclosed herein. Alternatively, the anti-ICOS antibody may comprise the CDRs of, or a VH and/or VL domain of, an anti-ICOS antibody disclosed in any of the following publications: WO2016154177, US2016304610—for example any of antibodies 7F12, 37A10, 35A9, 36E10, 16G10, 37A10S713, 37A10S714, 37A10S715, 37A10S716, 37A10S717, 37A10S718, 16G10S71, 16G10S72, 16G10S73, 16G10S83, 35A9S79, 35A9S710, or 35A9S89; WO16120789, US2016215059—for example the antibody known as 422.2 and/or H2L5; WO14033327, EP2892928, US2015239978—for example the antibody known as 314-8 and/or produced from hybridoma CNCM I-4180; WO12131004, EP2691419, U.S. Pat. No. 9,376,493, US20160264666—example the antibody Icos145-1 and/or antibody produced by hybridoma CNCM I-4179; WO10056804—for example the antibody JMAb 136 or “136”; WO9915553, EP1017723B1, U.S. Pat. Nos. 7,259,247, 7,132,099, 7,125,551, 7,306,800, 7,722,872, WO05103086, EP1740617, U.S. Pat. Nos. 8,318,905, 8,916,155—for example the antibody MIC-944 or 9F3; WO983821, U.S. Pat. No. 7,932,358B2, US2002156242, EP0984023, EP1502920, U.S. Pat. Nos. 7,030,225, 7,045,615, 7,279,560, 7,226,909, 7,196,175, 7,932,358, 8,389,690, WO02070010, EP1286668, EP1374901, U.S. Pat. Nos. 7,438,905, 7,438,905, WO0187981, EP1158004, U.S. Pat. Nos. 6,803,039, 7,166,283, 7,988,965, WO0115732, EP1125585, U.S. Pat. Nos. 7,465,445, 7,998,478—for example any JMAb antibody, e.g., any of JMAb-124, JMAb-126, JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb-138, JMAb-139, JMAb-140, JMAb-141, e.g., JMAb136; WO2014/089113—for example antibody 17G9; WO12174338; US2016145344; WO11020024, EP2464661, US2016002336, US2016024211, U.S. Pat. No. 8,840,889; U.S. Pat. No. 8,497,244. The anti-ICOS antibody optionally comprises the CDRs of 37A10S713 as disclosed in WO2016154177. It may comprise the VH and VL domains of 37A10S713, and may optionally have the antibody heavy and light chains of 37A10S713. Combination of an anti-ICOS antibody with an immunomodulator may provide an increased therapeutic effect compared with monotherapy, and may allow therapeutic benefit to be achieved with a lower dose of the immunomodulator(s). Thus, for example, an antibody (e.g., anti-PD-L1 antibody, optionally ipilimumab) that is used in combination with anti-ICOS antibody may be dosed at 3 mg/kg rather than a more usual dose of 10 mg/kg. The administration regimen of the anti-PD-L1 or other antibody may involve intravenous administration over a 90 minute period every 3 weeks for a total of 4 doses. An anti-ICOS antibody may be used to increase the sensitivity of a tumour to treatment with an anti-PD-L1 antibody, which may be recognised as a reduction in the dose at which the anti-PD-L1 antibody exerts a therapeutic benefit. Thus, anti-ICOS antibody may be administered to a patient to reduce the dose of anti-PD-L1 antibody effective to treat cancer or a tumour in the patient. Administration of anti-ICOS antibody may reduce the recommended or required dosage of anti-PD-L1 antibody administration to that patient to, for example, 75%, 50%, 25%, 20%, 10% or less, compared with the dosage when anti-PD-L1 antibody is administered without anti-ICOS. The patient may be treated by administration of anti-ICOS antibody and anti-PD-L1 antibody in a combination therapy as described herein. The benefit of combining anti-PD-L1 with anti-ICOS may extend to a reduction in dosage of each agent when compared with its use as a monotherapy. Anti-PD-L1 antibody may be used to reduce the dose at which anti-ICOS antibody exerts a therapeutic benefit, and thus may be administered to a patient to reduce the dose of anti-ICOS antibody effective to treat cancer or a tumour in the patient. Thus, an anti-PD-L1 antibody may reduce the recommended or required dosage of anti-ICOS antibody administration to that patient to, for example, 75%, 50%, 25%, 20%, 10% or less, compared with the dosage when anti-ICOS antibody is administered without anti-PD-L1. The patient may be treated by administration of anti-ICOS antibody and anti-PD-L1 antibody in a combination therapy as described herein. As discussed in Example 22 herein, treatment with anti-PD-L1 antibody, especially antibody with effector positive Fc, appears not to increase the expression of ICOS on Teff cells. This is advantageous when administering such antibodies in combination with effector positive anti-ICOS antibodies, where an increase in ICOS expression on Teffs would undesirably render these cells more sensitive to depletion by the anti-ICOS antibody. In a combination with anti-PD-L1, anti-ICOS therapy may thus exploit a differential expression of ICOS on Teffs compared with Tregs, preferentially targeting the ICOS-high Tregs for depletion. This in turn relieves the suppression of TEffs and has a net effect of promoting the effector T cell response in a patient. The effect of targeting immune checkpoint molecules on expression of ICOS on T cells has also been studied previously—see Figure S6C in ref. [30] (supplementary materials), where treatment with CTLA-4 antibody and/or anti-PD-1 antibody was reported to increase the percentage of CD4+ Tregs expressing ICOS. The effect of a therapeutic agent on ICOS expression in Tregs and Teffs may be a factor in selection of appropriate agents for use in combination with anti-ICOS antibodies, noting that effect of the anti-ICOS antibody may be enhanced under conditions where there is high differential expression of ICOS on Tregs versus Teffs. As described herein, a single dose of anti-ICOS antibody may be sufficient to provide therapeutic effect, especially in combination with other therapeutic agents such as anti-PD-L1 antibody. In tumour therapy, the underlying rationale for this single dose benefit may be that the anti-ICOS antibody mediates its effect, at least in part, by resetting or altering the microenvironment of the tumour sufficiently to render the tumour more sensitive to immune attack and/or to the effects of other immunomodulators such as those mentioned. Tumour microenviroment resetting is triggered through for example depletion of ICOS positive tumour infiltrating T-regs. So, for example, a patient may be treated with a single dose of an anti-ICOS antibody followed by one or multiple doses of anti-PD-L1 antibody. Over a period of treatment, for example six months or a year, the anti-ICOS antibody may be administered in a single dose while other agents, e.g., anti-PD-L1 antibody, are optionally administered multiple times over that treatment period, preferably with at least one such dose being administered subsequent to treatment with the anti-ICOS antibody. Further examples of combination therapy include combination of anti-ICOS antibody with:an antagonist of an adenosine A2A receptor (“A2AR inhibitor”);a CD137 agonist (e.g., agonist antibody);an antagonist of the enzyme indoleamine-2,3 dioxygenase, which catalyses the breakdown of tryptophan (“IDO inhibitor”). IDO is an immune checkpoint, activated in dendritic cells and macrophages, which contributes to immune suppression/tolerance. Anti-ICOS antibodies may be used in combination therapy with IL-2 (e.g., recombinant IL-2 such as aldesleukin). The IL-2 may be administered at high dose (HD). Typical HD IL-2 therapy involves bolus infusion of over 500,000 IU/kg, e.g., bolus infusions of 600,000 or 720,000 IU/kg, per cycle of therapy, where 10-15 such bolus infusions are given at intervals of between 5-10 hours, e.g., up to 15 bolus infusions every 8 hours, and repeating the therapy cycle approximately every 14 to 21 days for up to 6 to 8 cycles. HD IL-2 therapy has been successful in treating tumours, especially melanoma (e.g., metastatic melanoma) and renal cell carcinoma, but its use is limited to the high toxicity of IL-2 which can cause severe adverse effects. Treatment with high dose IL-2 has been shown to increase the population of ICOS-positive Tregs in cancer patients [31]. This increase in ICOS+ TRegs following the first cycle of HD IL-2 therapy was reported to correlate with worse clinical outcome—the higher the number of ICOS+ Tregs, the worse the prognosis. An IL-2 variant F42K has been proposed as an alternative therapy to avoid this undesirable increase in ICOS+ Treg cells [32]. However, another approach would be to exploit the increase in ICOS+ T regs by using an antibody in accordance with the present invention as a second-line therapeutic agent. It may be beneficial to combine IL-2 therapy with anti-ICOS antibodies, capitalising on the ability of anti-ICOS antibodies to target TRegs that highly express ICOS, inhibiting these cells and improving the prognosis for patients undergoing IL-2 therapy. Concomitant administration of IL-2 and anti-ICOS antibody may increase the response rate while avoiding or reducing adverse events in the treated patient population. The combination may permit IL-2 to be used at lower dose compared with IL-2 monotherapy, reducing the risk or level of adverse events arising from the IL-2 therapy, while retaining or enhancing clinical benefit (e.g., reduction of tumour growth, clearance of solid tumour and/or reduction of metastasis). In this way, addition of anti-ICOS can improve treatment of patients who are receiving IL-2, whether high-dose (HD) or low-dose (LD) IL-2. Accordingly, one aspect of the invention provides a method of treating a patient by administering an anti-ICOS antibody to the patient, wherein the patient is also treated with IL-2, e.g., HD IL-2. Another aspect of the invention is an anti-ICOS antibody for use in treating a patient, wherein the patient is also treated with IL-2, e.g., HD IL-2. The anti-ICOS antibody may be used as a second-line therapy. Thus, the patient may be one who has been treated with IL-2, e.g., having received at least one cycle of HD IL-2 therapy, and who has an increased level of ICOS+ Tregs. Assays may be performed on samples of cancer cells, e.g., tumour biopsy samples, using immunohistochemistry or FACS as described elsewhere herein to detect cells positive for ICOS, Foxp3, ICOSL and optionally one or more further markers of interest. Methods may comprise determining that the patient has an increased level of ICOS+ Tregs (e.g., in peripheral blood, or in a tumour biopsy) following IL-2 treatment, where an increased level is indicative that the patient would benefit from treatment with the anti-ICOS antibody. The increase in Tregs may be relative to control (untreated) individuals or to the patient prior to IL-2 therapy. Such patients with elevated Tregs represent a group who may not benefit from continued IL-2 treatment alone, but for whom a combination of anti-ICOS antibody and IL-2 therapy, or treatment with anti-ICOS antibody alone, offers therapeutic benefit. Thus, following a positive determination that the patient has an increased level of ICOS+ Tregs, anti-ICOS antibody and/or further IL-2 therapy may be administered. Treatment with the anti-ICOS antibody may selectively target and deplete the ICOS+ Tregs relative to other T cell populations in such patients. This provides a therapeutic effect by relieving the immunosuppression mediated by these cells and thereby enhancing activity of Teffs against the target cells, e.g., tumour cells or infected cells. Combination therapy with anti-ICOS antibodies and IL-2 may be used for any therapeutic indication described herein, and particularly for treating a tumour, e.g., melanoma such as metastatic melanoma, or renal cell carcinoma. Thus, in one example, the patient treated with an anti-ICOS antibody is one who presents with metastatic melanoma and has been treated with IL-2, e.g., HD IL-2 therapy or LD IL-2 therapy. In general, where an anti-ICOS antibody is administered to a patient who has received treatment with a first therapeutic agent (e.g., immunomodulator antibody) or other agent (e.g., IL-2), the anti-ICOS antibody may be administered after a minimum period of, for example, 24 hours, 48 hours, 72 hours, 1 week or 2 weeks following administration of the first therapeutic agent. The anti-ICOS antibody may be administered within 2, 3, 4 or 5 weeks after administration of the first therapeutic agent. This does not exclude additional administrations of either agent at any time, although it may be desirable to minimise the number of treatments administered, for ease of compliance for patients and to reduce costs. Rather, the relative timing of the administrations will be selected to optimise their combined effect, the first therapeutic agent creating an immunological environment (e.g., elevated ICOS+ Tregs, or antigen release as discussed below) in which the effect of the anti-ICOS antibody is especially advantageous. Thus, sequential administration of the first therapeutic agent and then the anti-ICOS antibody may allow time for the first agent to act, creating in vivo conditions in which the anti-ICOS antibody can exhibit its enhanced effect. Various administration regimens, including simultaneous or sequential combination treatments, are described herein and can be utilised as appropriate. Where the first therapeutic agent is one that increases the number of ICOS+ Tregs in the patient, the treatment regimen for the patient may comprise determining that the patient has an increased number of ICOS+ Tregs, and then administering the anti-ICOS antibody. As noted, use of anti-ICOS antibodies in combination therapy may provide advantages of reducing the effective dose of the therapeutic agents and/or countering adverse effects of therapeutic agents that increase ICOS+ Tregs in patients. Yet further therapeutic benefits may be achieved through selecting a first therapeutic agent that causes release of antigens from target cells through “immunological cell death”, and administering the first therapeutic agent in combination with an anti-ICOS antibody. As noted, administration of the anti-ICOS antibody may sequentially follow administration of the first therapeutic agent, administration of the two agents being separated by a certain time window as discussed above. Immunological cell death is a recognised mode of cell death, contrasting with apoptosis. It is characterised by release of ATP and HMGB1 from the cell and exposure of calreticulin on the plasma membrane [33, 34]. Immunological cell death in a target tissue or in target cells promotes engulfment of the cell by an antigen-presenting cell, resulting in display of antigens from the target cell, which in turn induces antigen-specific Teff cells. Anti-ICOS antibody may increase the magnitude and/or duration of the Teff response by acting as an agonist of ICOS on the Teff cells. In addition, where the anti-ICOS antibody is Fc effector function enabled (e.g., a human IgG1 antibody), the anti-ICOS antibody may cause depletion of antigen-specific Tregs. Thus, through a combination of either or both of these effects, the balance between Teff and Treg cells is modulated in favour of enhancing Teff activity. Combination of an anti-ICOS antibody with a treatment that induces immunological cell death in a target tissue or cell type, such as in a tumour or in cancer cells, thereby promotes an immune response in the patient against the target tissue or cells, representing a form of vaccination in which the vaccine antigen is generated in vivo. Accordingly, one aspect of the invention is a method of treating cancer in a patient by in vivo vaccination of the patient against their cancer cells. Another aspect of the invention is an anti-ICOS antibody for use in such a method. Anti-ICOS antibodies may be used in a method comprising:treating the patient with a therapy that causes immunological cell death of the cancer cells, resulting in presentation of antigen to antigen-specific effector T cells, andadministering an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances the antigen-specific effector T cell response against the cancer cells. Treatments that induce immunological cell death include radiation (e.g., ionising irradiation of cells using UVC light or γ rays), chemotherapeutic agents (e.g., oxaliplatin, anthracyclines such as doxorubicin, idarubicin or mitoxantrone, BK channel agonists such as phloretin or pimaric acid, bortezomib, cardiac glycosides, cyclophosphamide, GADD34/PP1 inhibitors with mitomycin, PDT with hypericin, polyinosinic-polycytidylic acid, 5-fluorouracil, gemcitabine, gefitnib, erlotinib, or thapsigargin with cisplatin) and antibodies to tumour-associated antigens. The tumour-associated antigen can be any antigen that is over-expressed by tumour cells relative to non-tumour cells of the same tissue, e.g., HER2, CD20, EGFR. Suitable antibodies include herceptin (anti-HER2), rituximab (anti-CD20), or cetuximab (anti-EGFR). Thus, it is advantageous to combine an anti-ICOS antibody with one or more such treatments. Optionally, the anti-ICOS antibody is adminstered to a patient who has already received such treatment. The anti-ICOS antibody may be administered after a period of, for example, 24 hours, 48 hours, 72 hours, 1 week or 2 weeks following the treatment that induces immunological cell death, e.g., between 24 to 72 hours after the treatment. The anti-ICOS antibody may be administered within 2, 3, 4 or 5 weeks after the treatment. Other regimens for combination therapy are discussed elsewhere herein. While “in vivo vaccination” has been described above, it is also possible to treat tumour cells to induce immunological cell death ex vivo, after which the cells may be reintroduced to the patient. Rather than administering the agent or treatment that induces immunological cell death directly to the patient, the treated tumour cells are administered to the patient. Treatment of the patient may be in accordance with administration regimens described above. As already noted, a single dose of an anti-ICOS antibody may be sufficient to provide therapeutic benefit. Thus, in the methods of treatment described herein, the anti-ICOS antibody is optionally administered as a single dose. A single dose of anti-ICOS antibody may deplete Tregs in a patient, with consequent beneficial effects in diseases such as cancer. It has previously been reported that transient ablation of Tregs has anti-tumour effects, including reducing tumour progression, treating established tumours and metastases and extending survival, and that it can enhance the therapeutic effect of tumour irradiation [35]. Administration of a single dose of anti-ICOS may provide such Treg depletion, and may be used to enhance the effects of other therapeutic approaches used in combination, such as radiotherapy. Antibodies to PD-L1 An antibody to PD-L1 for use in combination with an anti-ICOS antibody, whether as a separate therapeutic agent or in a multispecific antibody as described herein, may comprise the antigen-binding site of any anti-PD-L1 antibody. Numerous examples of anti-PD-L1 antibodies are disclosed herein and others are known in the art. Characterisation data for many of the anti-PD-L1 antibodies mentioned here has been published in U.S. Pat. Nos. 9,567,399 and 9,617,338, both incorporated by reference herein. 1D05 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:34. 1D05 has a light chain variable region (VL) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:44. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No: 526, Seq ID No:528, Seq ID No: 530, Seq ID No: 532 or Seq ID No: 534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46). 84G09 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:13, comprising the CDRH1 amino acid sequence of Seq ID No:7 (IMGT) or Seq ID No:10 (Kabat), the CDRH2 amino acid sequence of Seq ID No:8 (IMGT) or Seq ID No:11 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:9 (IMGT) or Seq ID No:12 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:14. 84G09 has a light chain variable region (VL) amino acid sequence of Seq ID No:23, comprising the CDRL1 amino acid sequence of Seq ID No:17 (IMGT) or Seq ID No:20 (Kabat), the CDRL2 amino acid sequence of Seq ID No:18 (IMGT) or Seq ID No:21 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:19 (IMGT) or Seq ID No:22 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:24. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:15 (heavy chain nucleic acid sequence Seq ID No:16). A full length light chain amino acid sequence is Seq ID No:25 (light chain nucleic acid sequence Seq ID No:26). 1D05 HC mutant 1 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:47, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 1 has a light chain variable region (VL) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:44. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46). 1D05 HC mutant 2 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:48, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 2 has a light chain variable region (VL) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:44. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46). 1D05 HC mutant 3 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:49, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 3 has a light chain variable region (VL) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:44. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46). 1D05 HC mutant 4 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:342, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 4 has a light chain variable region (VL) amino acid sequence of Seq ID No:43, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:44. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46). 1D05 LC mutant 1 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:34. 1D05 LC mutant 1 has a light chain variable region (VL) amino acid sequence of Seq ID No:50, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2 sequence of 1D05 LC Mutant 1 is as defined by the Kabat or IMGT systems from the VLsequence of Seq ID No:50. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). 1D05 LC mutant 2 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:34. 1D05 LC mutant 2 has a light chain variable region (VL) amino acid sequence of Seq ID No:51, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). 1D05 LC mutant 3 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:33, comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:34. 1D05 LC mutant 3 has a light chain variable region (VL) amino acid sequence of Seq ID No:298, comprising the CDRL1 amino acid sequence of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2 sequence of 1D05 LC Mutant 3 is as defined by the Kabat or IMGT systems from the VLsequence of Seq ID No:298. The light chain nucleic acid sequence of the VLdomain is Seq ID No:44. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46). 411B08 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:58, comprising the CDRH1 amino acid sequence of Seq ID No:52 (IMGT) or Seq ID No:55 (Kabat), the CDRH2 amino acid sequence of Seq ID No:53 (IMGT) or Seq ID No:56 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:54 (IMGT) or Seq ID No:57 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:59. 411B08 has a light chain variable region (VL) amino acid sequence of Seq ID No:68, comprising the CDRL1 amino acid sequence of Seq ID No:62 (IMGT) or Seq ID No:65 (Kabat), the CDRL2 amino acid sequence of Seq ID No:63 (IMGT) or Seq ID No:66 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:64 (IMGT) or Seq ID No:67 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:69. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:60 (heavy chain nucleic acid sequence Seq ID No:61). A full length light chain amino acid sequence is Seq ID No:70 (light chain nucleic acid sequence Seq ID No:71). 411C04 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:78, comprising the CDRH1 amino acid sequence of Seq ID No:72 (IMGT) or Seq ID No:75 (Kabat), the CDRH2 amino acid sequence of Seq ID No:73 (IMGT) or Seq ID No:76 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:74 (IMGT) or Seq ID No:77 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:79. 411C04 has a light chain variable region (VL) amino acid sequence of Seq ID No:88, comprising the CDRL1 amino acid sequence of Seq ID No:82 (IMGT) or Seq ID No:85 (Kabat), the CDRL2 amino acid sequence of Seq ID No:83 (IMGT) or Seq ID No:86 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:84 (IMGT) or Seq ID No:87 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:89. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:80 (heavy chain nucleic acid sequence Seq ID No:81). A full length light chain amino acid sequence is Seq ID No:90 (light chain nucleic acid sequence Seq ID No:91). 411D07 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:98, comprising the CDRH1 amino acid sequence of Seq ID No:92 (IMGT) or Seq ID No:95 (Kabat), the CDRH2 amino acid sequence of Seq ID No:93 (IMGT) or Seq ID No:96 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:94 (IMGT) or Seq ID No:97 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:99. 411D07 has a light chain variable region (VL) amino acid sequence of Seq ID No:108, comprising the CDRL1 amino acid sequence of Seq ID No:102 (IMGT) or Seq ID No:105 (Kabat), the CDRL2 amino acid sequence of Seq ID No:103 (IMGT) or Seq ID No:106 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:104 (IMGT) or Seq ID No:107 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:109. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:100 (heavy chain nucleic acid sequence Seq ID No:101). A full length light chain amino acid sequence is Seq ID No: 110 (light chain nucleic acid sequence Seq ID No:111). 385F01 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:118, comprising the CDRH1 amino acid sequence of Seq ID No:112 (IMGT) or Seq ID No:115 (Kabat), the CDRH2 amino acid sequence of Seq ID No:113 (IMGT) or Seq ID No:116 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:114 (IMGT) or Seq ID No:117 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:119. 385F01 has a light chain variable region (VL) amino acid sequence of Seq ID No:128, comprising the CDRL1 amino acid sequence of Seq ID No:122 (IMGT) or Seq ID No:125 (Kabat), the CDRL2 amino acid sequence of Seq ID No:123 (IMGT) or Seq ID No:126 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:124 (IMGT) or Seq ID No:127 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:129. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:120 (heavy chain nucleic acid sequence Seq ID No:121). A full length light chain amino acid sequence is Seq ID No:130 (light chain nucleic acid sequence Seq ID No:131). 386H03 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:158, comprising the CDRH1 amino acid sequence of Seq ID No:152 (IMGT) or Seq ID No:155 (Kabat), the CDRH2 amino acid sequence of Seq ID No:153 (IMGT) or Seq ID No:156 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:154 (IMGT) or Seq ID No:157 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:159. 386H03 has a light chain variable region (VL) amino acid sequence of Seq ID No:168, comprising the CDRL1 amino acid sequence of Seq ID No:162 (IMGT) or Seq ID No:165 (Kabat), the CDRL2 amino acid sequence of Seq ID No:163 (IMGT) or Seq ID No:166 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:164 (IMGT) or Seq ID No:167 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:169. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:160 (heavy chain nucleic acid sequence Seq ID No:161). A full length light chain amino acid sequence is Seq ID No:170 (light chain nucleic acid sequence Seq ID No:171). 389A03 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:178, comprising the CDRH1 amino acid sequence of Seq ID No:172 (IMGT) or Seq ID No:175 (Kabat), the CDRH2 amino acid sequence of Seq ID No:173 (IMGT) or Seq ID No:176 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:174 (IMGT) or Seq ID No:177 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:179. 389A03 has a light chain variable region (VL) amino acid sequence of Seq ID No:188, comprising the CDRL1 amino acid sequence of Seq ID No:182 (IMGT) or Seq ID No:185 (Kabat), the CDRL2 amino acid sequence of Seq ID No:183 (IMGT) or Seq ID No:186 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:184 (IMGT) or Seq ID No:187 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:189. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:180 (heavy chain nucleic acid sequence Seq ID No:181). A full length light chain amino acid sequence is Seq ID No:190 (light chain nucleic acid sequence Seq ID No:191). 413D08 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:138, comprising the CDRH1 amino acid sequence of Seq ID No:132 (IMGT) or Seq ID No:135 (Kabat), the CDRH2 amino acid sequence of Seq ID No:133 (IMGT) or Seq ID No:136 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:134 (IMGT) or Seq ID No:137 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:139. 413D08 has a light chain variable region (VL) amino acid sequence of Seq ID No:148, comprising the CDRL1 amino acid sequence of Seq ID No:142 (IMGT) or Seq ID No:145 (Kabat), the CDRL2 amino acid sequence of Seq ID No:143 (IMGT) or Seq ID No:146 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:144 (IMGT) or Seq ID No:147 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:149. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No: 140 (heavy chain nucleic acid sequence Seq ID No:141). A full length light chain amino acid sequence is Seq ID No:150 (light chain nucleic acid sequence Seq ID No:151). 413G05 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:244, comprising the CDRH1 amino acid sequence of Seq ID No:238 (IMGT) or Seq ID No:241 (Kabat), the CDRH2 amino acid sequence of Seq ID No:239 (IMGT) or Seq ID No:242 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:240 (IMGT) or Seq ID No:243 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:245. 413G05 has a light chain variable region (VL) amino acid sequence of Seq ID No:254, comprising the CDRL1 amino acid sequence of Seq ID No:248 (IMGT) or Seq ID No:251 (Kabat), the CDRL2 amino acid sequence of Seq ID No:249 (IMGT) or Seq ID No:252 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:250 (IMGT) or Seq ID No:253 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:255. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:246 (heavy chain nucleic acid sequence Seq ID No:247). A full length light chain amino acid sequence is Seq ID No:256 (light chain nucleic acid sequence Seq ID No:257). 413F09 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:264, comprising the CDRH1 amino acid sequence of Seq ID No:258 (IMGT) or Seq ID No:261 (Kabat), the CDRH2 amino acid sequence of Seq ID No:259 (IMGT) or Seq ID No:262 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:260 (IMGT) or Seq ID No:263 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:265. 413F09 has a light chain variable region (VL) amino acid sequence of Seq ID No:274, comprising the CDRL1 amino acid sequence of Seq ID No:268 (IMGT) or Seq ID No:271 (Kabat), the CDRL2 amino acid sequence of Seq ID No:269 (IMGT) or Seq ID No:272 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:270 (IMGT) or Seq ID No:273 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:275. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:266 (heavy chain nucleic acid sequence Seq ID No:267). A full length light chain amino acid sequence is Seq ID No:276 (light chain nucleic acid sequence Seq ID No:277). 414B06 has a heavy chain variable (VH) region amino acid sequence of Seq ID No:284, comprising the CDRH1 amino acid sequence of Seq ID No:278 (IMGT) or Seq ID No:281 (Kabat), the CDRH2 amino acid sequence of Seq ID No:279 (IMGT) or Seq ID No:282 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:280 (IMGT) or Seq ID No:283 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:285. 414B06 has a light chain variable region (VL) amino acid sequence of Seq ID No:294, comprising the CDRL1 amino acid sequence of Seq ID No:288 (IMGT) or Seq ID No:291(Kabat), the CDRL2 amino acid sequence of Seq ID No:289 (IMGT) or Seq ID No:292 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:290 (IMGT) or Seq ID No:293 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:295. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:286 (heavy chain nucleic acid sequence Seq ID No:287). A full length light chain amino acid sequence is Seq ID No:296 (light chain nucleic acid sequence Seq ID No:297). 416E01 has a heavy chain variable region (VH) amino acid sequence of Seq ID No:349, comprising the CDRH1 amino acid sequence of Seq ID No:343 (IMGT) or Seq ID No:346 (Kabat), the CDRH2 amino acid sequence of Seq ID No:344 (IMGT) or Seq ID No:347 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:345 (IMGT) or Seq ID No:348 (Kabat). The heavy chain nucleic acid sequence of the VHdomain is Seq ID No:350. 416E01 has a light chain variable region (VL) amino acid sequence of Seq ID No:359, comprising the CDRL1 amino acid sequence of Seq ID No:353 (IMGT) or Seq ID No:356 (Kabat), the CDRL2 amino acid sequence of Seq ID No:354 (IMGT) or Seq ID No:357 (Kabat), and the CDRL3 amino acid sequence of Seq ID No:355 (IMGT) or Seq ID No:358 (Kabat). The light chain nucleic acid sequence of the VLdomain is Seq ID No:360. The VHdomain may be combined with any of the heavy chain constant region sequences described herein, e.g. Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205, Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VLdomain may be combined with any of the light chain constant region sequences described herein, e.g. Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full length heavy chain amino acid sequence is Seq ID No:351 (heavy chain nucleic acid sequence Seq ID No:352). A full length light chain amino acid sequence is Seq ID No:361 (light chain nucleic acid sequence Seq ID No:362). Antibody-Drug Conjugates Anti-ICOS antibodies can be used as carriers of cytotoxic agents, to target Tregs. As reported in Example 18, Tregs located in the tumour microenvironment (TME) strongly express ICOS. ICOS is more strongly expressed on intratumoural Tregs than on intratumoural Teffs or peripheral Tregs. Thus, anti-ICOS antibodies labelled with a toxic drug or pro-drug may preferentially target Tregs in the TME to deliver the toxic payload, selectively inhibiting those cells. Such targeting of cytotoxic agents provides an additional route to removing the immune suppressive effect of Tregs, thereby altering the Treg:Teff balance in favour of Teff activity and may be used as an alternative to, or in combination with, any one or more of the other therapeutic approaches discussed herein (e.g., Fc effector-mediated inhibition of Tregs, agonism of effector T cells). Accordingly, the invention provides an anti-ICOS antibody that is conjugated to a cytotoxic drug or pro-drug. In the case of a pro-drug, the pro-drug is activatable in the TME or other target site of therapeutic activity to generate the cytotoxic agent. Activation may be in response to a trigger such as photoactivation, e.g., using near-infrared light to activate a photoabsorber conjugate [36]. Spatially-selective activation of a pro-drug further enhances the cytotoxic effect of the antibody-drug conjugate, combining with the high ICOS expression on intratumoural Tregs to provide a cytotoxic effect that is highly selective for these cells. For use in an antibody-drug conjugate, the cytotoxic drug or pro-drug is preferably non-immunogenic and non-toxic (dormant or inactive) during circulation of the antibody-drug conjugate in the blood. Preferably the cytotoxic drug (or the pro-drug, when activated) is potent—e.g., two to four molecules of the drug may be sufficient to kill the target cell. A photoactivatable pro-drug is silicapthalocyanine dye (IRDye 700 DX), which induces lethal damage to the cell membrane after near-infrared light exposure. Cytotoxic drugs include anti-mitotic agents such as monomethyl auristatin E and microtubule inhibitors such as maytansine derivatives, e.g., mertansine, DM1, emtansine. Conjugation of the drug (or pro-drug) to the antibody will usually be via a linker. The linker may be a cleavable linker, e.g., disulphide, hydrazone or peptide link. Cathepsin-cleavable linkers may be used, so that the drug is released by cathepsin in tumour cells. Alternatively, non-cleavable linkers can be used, e.g., thioether linkage. Additional attachment groups and/or spacers may also be included. The antibody in the antibody-drug conjugate may be an antibody fragment, such as Fab′2 or other antigen-binding fragment as described herein, as the small size of such fragments may assist penetration to the tissue site (e.g., solid tumour). An anti-ICOS antibody according to the present invention may be provided as an immunocytokine. Anti-ICOS antibodies may also be administered with immunocytokines in combination therapy. A number of examples of antibodies are described herein for use in combination therapy with anti-ICOS, and any of these (e.g., an anti-PD-L1 antibody) may be provided as immunocytokines for use in the present invention. An immunocytokine comprises an antibody molecule conjugated to a cytokine, such as IL-2. Anti-ICOS:IL-2 conjugates and anti-PD-L1:1 L-2 conjugates are thus further aspects of the present invention. An IL-2 cytokine may have activity at the high (any) affinity IL-2 receptor and/or the intermediate affinity (αβ) IL-2 receptor. IL-2 as used in an immunocytokine may be human wild type IL-2 or a variant IL-2 cytokine having one or more amino acid deletions, substitutions or additions, e.g., IL-2 having a 1 to 10 amino acid deletion at the N-terminus. Other IL-2 variants include mutations R38A or R38Q. An example anti-PD-L1 immunocytokine comprises an immunoglobulin heavy chain and an immunoglobulin light chain, wherein the heavy chain comprises in N- to C-terminal direction:a) A VHdomain comprising CDRH1, CDRH2 and CDRH3; andb) A heavy chain constant region; and wherein the light chain comprises in N- to C-terminal direction:c) A VLdomain comprising CDRL1, CDRL2 and CDRL3;d) A light chain constant region, (CL);e) Optionally, a linker, (L); andf) An IL-2 cytokine; wherein the VHdomain and VLdomain are comprised by an antigen-binding site that specifically binds to human PD-L1; and wherein the immunocytokine comprises a VHdomain which comprises a CDRH3 comprising the motif X1GSGX2YGX3X4FD (SEQ ID NO: 609), wherein X1, X2and X3are independently any amino acid, and X4is either present or absent, and if present, may be any amino acid. The VH and VL domain may be the VH and VL domain of any anti-PD-L1 antibody mentioned herein, e.g., the 1D05 VH and VL domains. The IL-2 may be human wild type or variant IL-2. Vaccination Anti-ICOS antibodies may be provided in vaccine compositions or co-administered with vaccines preparations. ICOS is involved in T follicular helper cell formation and the germinal centre reaction [37]. Agonist ICOS antibodies thus have potential clinical utility as molecular adjuvants to enhance vaccine efficacy. The antibodies may be used to increase protective efficacy of numerous vaccines, such as those against hepatitis B, malaria, HIV. In the context of vaccination, the anti-ICOS antibody will generally be one that lacks Fc effector function, and thus does not mediate ADCC, CDC or ADCP. The antibody may be provided in a format lacking an Fc region, or having an effector null constant region. Optionally, an anti-ICOS antibody may have a heavy chain constant region that binds one or more types of Fc receptor but does not induce ADCC, CDC or ADCP activity, or that exhibits lower ADCC, CDC and ADCP activity compared with wild type human IgG1. Such a constant region may be unable to bind, or may bind with lower affinity, the particular Fc receptor(s) responsible for triggering ADCC, CDC or ADCP activity. Alternatively, where cellular effector functions are acceptable or desirable in the context of the vaccination, the anti-ICOS antibody may comprise a heavy chain constant region that is Fc effector function positive. Any of IgG1, IgG4 and IgG4.PE formats may for instance be used for anti-ICOS antibodies in vaccination regimens, and other examples of suitable isotypes and antibody constant regions are set out in more detail elsewhere herein. Formulations and Administration Antibodies may be monoclonal or polyclonal, but are preferably provided as monoclonal antibodies for therapeutic use. They may be provided as part of a mixture of other antibodies, optionally including antibodies of different binding specificity. Antibodies according to the invention, and encoding nucleic acid, will usually be provided in isolated form. Thus, the antibodies, VH and/or VL domains, and nucleic acids may be provided purified from their natural environment or their production environment. Isolated antibodies and isolated nucleic acid will be free or substantially free of material with which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are prepared (e.g., cell culture) when such preparation is by recombinant DNA technology in vitro. Optionally an isolated antibody or nucleic acid (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Antibodies or nucleic acids may be formulated with diluents or adjuvants and still for practical purposes be isolated—for example they may be mixed with carriers if used to coat microtitre plates for use in immunoassays, and may be mixed with pharmaceutically acceptable carriers or diluents when used in therapy. As described elsewhere herein, other active ingredients may also be included in therapeutic preparations. Antibodies may be glycosylated, either naturally in vivo or by systems of heterologous eukaryotic cells such as CHO cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated. The invention encompasses antibodies having a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase ADCC function [38]. In other applications, modification of galactosylation can be made in order to modify CDC. Typically, an isolated product constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. An antibody may be substantially free from proteins or polypeptides or other contaminants that are found in its natural or production environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. An antibody may have been identified, separated and/or recovered from a component of its production environment (eg, naturally or recombinantly). The isolated antibody may be free of association with all other components from its production environment, eg, so that the antibody has been isolated to an FDA-approvable or approved standard. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody or its encoding nucleic acid will be prepared by at least one purification step. The invention provides therapeutic compositions comprising the antibodies described herein. Therapeutic compositions comprising nucleic acid encoding such antibodies are also provided. Encoding nucleic acids are described in more detail elsewhere herein and include DNA and RNA, e.g., mRNA. In therapeutic methods described herein, use of nucleic acid encoding the antibody, and/or of cells containing such nucleic acid, may be used as alternatives (or in addition) to compositions comprising the antibody itself. Cells containing nucleic acid encoding the antibody, optionally wherein the nucleic acid is stably integrated into the genome, thus represent medicaments for therapeutic use in a patient. Nucleic acid encoding the anti-ICOS antibody may be introduced into human B lymphocytes, optionally B lymphocytes derived from the intended patient and modified ex vivo. Optionally, memory B cells are used. Administration of cells containing the encoding nucleic acid to the patient provides a reservoir of cells capable of expressing the anti-ICOS antibody, which may provide therapeutic benefit over a longer term compared with administration of isolated nucleic acid or isolated antibody. Compositions may contain suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311. Compositions may comprise the antibody or nucleic acid in combination with medical injection buffer and/or with adjuvant. Antibodies, or their encoding nucleic acids, may be formulated for the desired route of administration to a patient, e.g., in liquid (optionally aqueous solution) for injection. Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Formulating antibodies for subcutaneous administration typically requires concentrating them into a smaller volume compared with intravenous preparations. The high potency of antibodies according to the present invention may lend them to use at sufficiently low doses to make subcutaneous formulation practical, representing an advantage compared with less potent anti-ICOS antibodies. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138, 1984). The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared can be filled in an appropriate ampoule. A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. It is envisaged that treatment will not be restricted to use in the clinic. Therefore, subcutaneous injection using a needle-free device is also advantageous. With respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly). Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the aforesaid antibody may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. The antibody, nucleic acid, or composition comprising it, may be contained in a medical container such as a phial, syringe, IV container or an injection device. In an example, the antibody, nucleic acid or composition is in vitro, and may be in a sterile container. In an example, a kit is provided comprising the antibody, packaging and instructions for use in a therapeutic method as described herein. One aspect of the invention is a composition comprising an antibody or nucleic acid of the invention and one or more pharmaceutically acceptable excipients, examples of which are listed above. “Pharmaceutically acceptable” refers to approved or approvable by a regulatory agency of the USA Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans. A pharmaceutically acceptable carrier, excipient, or adjuvant can be administered to a patient, together with an agent, e.g., any antibody or antibody chain described herein, and does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. In some embodiments, an anti-ICOS antibody will be the sole active ingredient in a composition according to the present invention. Thus, a composition may consist of the antibody or it may consist of the antibody with one or more pharmaceutically acceptable excipients. However, compositions according to the present invention optionally include one or more additional active ingredients. Detailed description of agents with which the anti-ICOS antibodies may be combined is provided elsewhere herein. Optionally, compositions contain multiple antibodies (or encoding nucleic acids) in a combined preparation, e.g., a single formulation comprising the anti-ICOS antibody and one or more other antibodies. Other therapeutic agents that it may be desirable to administer with antibodies or nucleic acids according to the present invention include analgaesic agents. Any such agent or combination of agents may be administered in combination with, or provided in compositions with antibodies or nucleic acids according to the present invention, whether as a combined or separate preparation. The antibody or nucleic acid according to the present invention may be administered separately and sequentially, or concurrently and optionally as a combined preparation, with another therapeutic agent or agents such as those mentioned. Anti-ICOS antibodies for use in a particular therapeutic indication may be combined with the accepted standard of care. Thus, for anti-cancer treatment, the antibody therapy may be employed in a treatment regimen that also includes chemotherapy, surgery and/or radiation therapy for example. Radiotherapy may be single dose or in fractionated doses, either delivered to affected tissues directly or to the whole body. Multiple compositions can be administered separately or simultaneously. Separate administration refers to the two compositions being administered at different times, e.g. at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One can also administer compositions at 24 hours apart, or even longer apart. Alternatively, two or more compositions can be administered simultaneously, e.g. less than 10 or less than 5 minutes apart. Compositions administered simultaneously can, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components. Antibodies, and their encoding nucleic acids, can be used as therapeutic agents. Patients herein are generally mammals, typically humans. An antibody or nucleic acid may be administered to a mammal, e.g., by any route of administration mentioned herein. Administration is normally in a “therapeutically effective amount”, this being an amount that produces the desired effect for which it is administered, sufficient to show benefit to a patient. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. A therapeutically effective amount or suitable dose of antibody or nucleic acid can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. As indicated by the in vivo studies described in the Examples herein, anti-ICOS antibody may be effective at a range of doses. Pharmacodynamic studies are reported in Example 24. Anti-ICOS antibodies may be administered in an amount in one of the following ranges per dose:about 10 μg/kg body weight to about 100 mg/kg body weight,about 50 μg/kg body weight to about 5 mg/kg body weight,about 100 μg/kg body weight to about 10 mg/kg body weight,about 100 μg/kg body weight to about 20 mg/kg body weight,about 0.5 mg/kg body weight to about 20 mg/kg body weight, orabout 5 mg/kg body weight or lower, for example less than 4, less than 3, less than 2, or less than 1 mg/kg of the antibody. An optimal therapeutic dose may be between 0.1 and 0.5 mg/kg in a human, for example about 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg or 0.5 mg/kg. For fixed dosing in adult humans, a suitable dose may be between 8 and 50 mg, or between 8 and 25 mg, e.g., 15 mg or 20 mg. In methods of treatment described herein, one or more doses may be administered. In some cases, a single dose may be effective to achieve a long-term benefit. Thus, the method may comprise administering a single dose of the antibody, its encoding nucleic acid, or the composition. Alternatively, multiple doses may be administered, usually sequentially and separated by a period of days, weeks or months. Anti-ICOS antibody may be repeatedly administered to a patient at intervals of 4 to 6 weeks, e.g., every 4 weeks, every 5 weeks, or every 6 weeks. Optionally, the anti-ICOS antibody may be administered to a patient once a month, or less frequently, e.g., every two months or every three months. Accordingly, a method of treating a patient may comprise administering a single dose of the anti-ICOS antibody to the patient, and not repeating the administration for at least one month, at least two months, at least three months, and optionally not repeating the administration for at least 12 months. As discussed in Example 11c, comparable therapeutic effects may be obtained using either one or multiple doses of anti-ICOS antibody, which may be a result of a single dose of antibody being effective to reset the tumour microenvironment. Physicians can tailor the administration regimen of the anti-ICOS antibody to the disease and the patient undergoing therapy, taking into account the disease status and any other therapeutic agents or therapeutic measures (e.g., surgery, radiotherapy etc) with which the anti-ICOS antibody is being combined. In some embodiments, an effective dose of an anti-ICOS antibody is administered more frequently than once a month, such as, for example, once every three weeks, once every two weeks, or once every week. Treatment with anti-ICOS antibody may include multiple doses administered over a period of at least a month, at least six months, or at least a year. As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). For treatment to be effective a complete cure is not contemplated. The method can in certain aspects include cure as well. In the context of the invention, treatment may be preventative treatment. T Cell Therapy WO2011/097477 described use of anti-ICOS antibodies for generating and expanding T cells, by contacting a population of T cells with a first agent that provides a primary activation signal (e.g., an anti-CD3 antibody) and a second agent that activates ICOS (e.g., an anti-ICOS antibody), optionally in the presence of a Th17 polarising agent such as IL-1β, IL-6, neutralising anti-IFNγ and/or anti-IL-4. Anti-ICOS antibodies described herein may be used in such methods to provide T cell populations. Populations of cultured expanded T cells having therapeutic activity (e.g., anti-tumour activity) may be generated. As described in WO2011/097477, such T cells may be used therapeutically in methods of treating patients by immunotherapy. Morphological Assay for Anti-ICOS Antibodies as Therapeutic Candidates It was observed that when candidate therapeutic anti-ICOS antibodies were coupled to a solid surface and brought into contact with ICOS-expressing T cells, they were able to induce morphological change in the cells. On addition of ICOS+ T cells to wells that were internally coated with anti-ICOS antibodies, cells were seen to change from their initial rounded shape, adopting a spindle-shape, spreading and adhering to the antibody-coated surface. This morphological change was not observed with control antibody. Moreover, the effect was found to be dose-dependent, with faster and/or more pronounced shape change occurring as the concentration of antibody on the surface increased. The shape change provides a surrogate indicator of T cell binding to ICOS, and/or of agonism by anti-ICOS antibody. The assay may be used to identify an antibody that promotes multimerisation of ICOS on the T cell surface. Such antibodies represent therapeutic candidate agonist antibodies. Conveniently, the visual indicator provided by this assay is a simple method of screening antibodies or cells, particularly in large numbers. The assay may be automated to run in a high-throughput system. Accordingly, one aspect of the invention is an assay for selecting an antibody that binds ICOS, optionally for selecting an ICOS agonist antibody, the assay comprising: providing an array of antibodies immobilised (attached or adhered) to a substrate in a test well; adding ICOS-expressing cells (e.g., activated primary T cells, or MJ cells) to the test well; observing morphology of the cells; detecting shape change in the cells from rounded to flattened against the substrate within the well; wherein the shape change indicates that the antibody is an antibody that binds ICOS, optionally an ICOS agonist antibody, and selecting the antibody from the test well. The assay may be run with multiple test wells, each containing a different antibody for testing, optionally in parallel, e.g., in a 96 well plate format. The substrate is preferably an inner surface of the well. Thus, a two-dimensional surface is provided against which flattening of the cells may be observed. For example, the bottom and/or wall of a well may be coated with antibody. Tethering of antibody to the substrate may be via a constant region of the antibody. A negative control may be included, such an antibody known not to bind ICOS, preferably an antibody that does not bind an antigen on the surface of the ICOS-expressing cells to be used. The assay may comprise quantifying the degree of morphological change and, where multiple antibodies are tested, selecting an antibody that induces greater morphological change than one or more other test antibodies. Selection of antibody may comprise expressing nucleic acid encoding the antibody present in the test well of interest, or expressing an antibody comprising the CDRs or antigen binding domain of that antibody. The antibody may optionally be reformatted, for example to provide an antibody comprising the antigen binding domain of the selected antibody, e.g., an antibody fragment, or an antibody comprising a different constant region. A selected antibody is preferably provided with a human IgG1 constant region or other constant region as described herein. A selected antibody may further be formulated in a composition comprising one or more additional ingredients—suitable pharmaceutical formations are discussed elsewhere herein. Clauses Embodiments of the invention are set out in the following numbered clauses, which are part of the description. Clause 1. An isolated antibody that binds the extracellular domain of human and/or mouse ICOS, wherein the antibody comprises a VH domain comprising an amino acid sequence having at least 95% sequence identity to the STIM003 VH domain SEQ ID NO: 408 and a VL domain comprising an amino acid sequence having at least 95% sequence identity to the STIM003 VL domain SEQ ID NO: 415. Clause 2. An antibody according to clause 1, wherein the VH domain comprises a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, whereinHCDR1 is the STIM003 HCDR1 having amino acid sequence SEQ ID NO: 405,HCDR2 is the STIM003 HCDR2 having amino acid sequence SEQ ID NO: 406,HCDR3 is the STIM003 HCDR3 having amino acid sequence SEQ ID NO: 407. Clause 3. An antibody according to clause 1 or clause 2, wherein the VL domain comprises a set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, whereinLCDR1 is the STIM003 LCDR1 having amino acid sequence SEQ ID NO: 412,LCDR2 is the STIM003 LCDR2 having amino acid sequence SEQ ID NO: 413,LCDR3 is the STIM003 LCDR3 having amino acid sequence SEQ ID NO: 414. Clause 4. An antibody according to clause 1, wherein the VH domain amino acid sequence is SEQ ID NO: 408 and/or wherein the VL domain amino acid sequence is SEQ ID NO: 415. Clause 5. An isolated antibody that binds the extracellular domain of human and/or mouse ICOS, comprisingan antibody VH domain comprising complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, andan antibody VL domain comprising complementarity determining regions LCDR1, LCDR2 and LCDR3, whereinHCDR1 is the HCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that HCDR1 with 1, 2, 3, 4 or 5 amino acid alterations,HCDR2 is the HCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that HCDR2 with 1, 2, 3, 4 or 5 amino acid alterations, and/orHCDR3 is the HCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that HCDR3 with 1, 2, 3, 4 or 5 amino acid alterations. Clause 6. An antibody according to clause 5, wherein the antibody heavy chain CDRs are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprise the STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 heavy chain CDRs with 1, 2, 3, 4 or 5 amino acid alterations. Clause 7. An antibody according to clause 6, wherein the antibody VH domain has the heavy chain CDRs of STIM003. Clause 8. An isolated antibody that binds the extracellular domain of human and/or mouse ICOS, comprisingan antibody VH domain comprising complementarity determining regions HCDR1, HCDR2 and HCDR3, andan antibody VL domain comprising complementarity determining regions LCDR1, LCDR2 and LCDR3,wherein LCDR1 is the LCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004 STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that LCDR1 with 1, 2, 3, 4 or 5 amino acid alterations,LCDR2 is the LCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises that LCDR2 with 1, 2, 3, 4 or 5 amino acid alterations, and/orLCDR3 is the LCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that LCDR3 with 1, 2, 3, 4 or 5 amino acid alterations. Clause 9. An antibody according to any of clauses 5 to 8, wherein the antibody light chain CDRs are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprise the STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 light chain CDRs with 1, 2, 3, 4 or 5 amino acid alterations. Clause 10. An antibody according to clause 9, wherein the antibody VL domain has the light chain CDRs of STIM003. Clause 11. An antibody according to any of clauses 5 to 10, comprising VH and/or VL domain framework regions of human germline gene segment sequences. Clause 12. An antibody according to any of clauses 5 to 11, comprising a VH domain which(i) is derived from recombination of a human heavy chain V gene segment, a human heavy chain D gene segment and a human heavy chain J gene segment, whereinthe V segment is IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g., V2-5*10);the D gene segment is IGHD6-19 (e.g., IGHD6-19*01), IGHD3-10 (e.g., IGHD3-10*01) or IGHD3-9 (e.g., IGHD3-9*01); and/orthe J gene segment is IGHJ6 (e.g., IGHJ6*02), IGHJ4 (e.g., IGHJ4*02) or IGHJ3 (e.g., IGHJ3*02), or(ii) comprises framework regions FR1, FR2, FR3 and FR4, whereinFR1 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations,FR2 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations,FR3 aligns with human germline V gene segment IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g. V3-20*d01), IGVH3-11 (e.g, V3-11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid alterations, and/orFR4 aligns with human germline J gene segment IGJH6 (e.g., JH6*02), IGJH4 (e.g., JH4*02) or IGJH3 (e.g., JH3*02), optionally with 1, 2, 3, 4 or 5 amino acid alterations. Clause 13. An antibody according to any of clauses 5 to 12, comprising an antibody VL domain which(i) is derived from recombination of a human light chain V gene segment and a human light chain J gene segment, whereinthe V segment is IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), and/orthe J gene segment is IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g., IGKJ2*04), IGLJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g., IGKJ1*01); or(ii) comprises framework regions FR1, FR2, FR3 and FR4, whereinFR1 aligns with human germline V gene segment IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4 or 5 amino acid alterations,FR2 aligns with human germline V gene segment IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4 or 5 amino acid alterations,FR3 aligns with human germline V gene segment IGKV2-28 (e.g., IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4 or 5 amino acid alterations, and/orFR4 aligns with human germline J gene segment IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g., IGKJ2*04), IGKJ3 (e.g., IGKJ3*01) or IGKJ1 (e.g., IGKJ1*01), optionally with 1, 2, 3, 4 or 5 amino acid alterations. Clause 14. An antibody according to any of clauses 5 to 13, comprising an antibody VH domain which is the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence at least 90% identical to the antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. Clause 15. An antibody according to any of clauses 5 to 14, comprising an antibody VL domain which is the VL domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence at least 90% identical to the antibody VL domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. Clause 16. An antibody according to clause 15, comprisingan antibody VH domain which is selected from the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or which has an amino acid sequence at least 90% identical to the antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, andan antibody VL domain which is the VL domain of said selected antibody, or which has an amino acid sequence at least 90% identical to the antibody VL domain sequence of said selected antibody. Clause 17. An antibody according to clause 16, comprising the STIM003 VH domain and the STIM003 VL domain. Clause 18. An antibody according to any of the preceding clauses, comprising an antibody constant region. Clause 19. An antibody according to clause 18, wherein the constant region comprises a human heavy and/or light chain constant region. Clause 20. An antibody according to clause 18 or clause 19, wherein the constant region is Fc effector positive. Clause 21. An antibody according to clause 20, comprising an Fc region that has enhanced ADCC, ADCP and/or CDC function compared with a native human Fc region. Clause 22. An antibody according to any of clauses 18 to 21, wherein the antibody is an IgG1. Clause 23. An antibody according to clause 21 or clause 22, wherein the antibody is afucosylated. Clause 24. An antibody according to any of the preceding clauses which is conjugated to a cytotoxic drug or pro-drug. Clause 25. An antibody according to any of the preceding clauses, which is a multispecific antibody. Clause 26. An isolated antibody that binds the extracellular domain of human and mouse ICOS with an affinity (KD) of less than 50 nM as determined by surface plasmon resonance. Clause 27. An antibody according to clause 26, wherein the antibody binds the extracellular domain of human and mouse ICOS with an affinity (KD) of less than 5 nM as determined by surface plasmon resonance. Clause 28. An antibody according to clause 26 or clause 27, wherein the KDof binding the extracellular domain of human ICOS is within 10-fold of the KDof binding the extracellular domain of mouse ICOS. Clause 29. A composition comprising an isolated antibody according to any of the preceding clauses and a pharmaceutically acceptable excipient. Clause 30. A composition comprising isolated nucleic acid encoding an antibody according to any of clauses 1 to 28 and a pharmaceutically acceptable excipient. Clause 31. A method of modulating the balance of regulatory T cells (Tregs) to effector T cells (Teffs) to increase Teff response in a patient, comprising administering an antibody according to any of clauses 1 to 28 or composition according to clause 29 to the patient. Clause 32. A method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Tregs) and/or increasing effector T cell (Teff) response in a patient, the method comprising administering an antibody according to any of clauses 1 to 28 or a composition according to clause 29 to the patient. Clause 33. An antibody according to any of clauses 1 to 28, or a composition according to clause 29, for use in a method of treatment of the human body by therapy. Clause 34. An antibody or composition for use according to clause 33, for use in modulating the balance of regulatory T cells (Tregs) to effector T cells (Teffs) to increase effector T cell response in a patient. Clause 35. An antibody or composition for use according to clause 33, for use in treating a disease or condition amenable to therapy by depleting regulatory T cells (Tregs) and/or increasing effector T cell (Teff) response in a patient. Clause 36. A method according to clause 32, or an antibody or a composition for use according to clause 35, wherein the disease is a cancer or a solid tumour. Clause 37. An antibody according to any of clauses 1 to 28 or a composition according to clause 29, for use in a method of treating cancer in a human patient. Clause 38. A method of treating cancer in a human patient, comprising administering an antibody according to any of clauses 1 to 28 or a composition according to clause 29 to the patient. Clause 39. A method or an antibody or composition for use according to any of clauses 36 to 38, wherein the cancer is renal cell cancer, head and neck cancer, melanoma, non small cell lung cancer or diffuse large B-cell lymphoma. Clause 40. A method or an antibody or composition for use according to any of clauses 31 to 39, wherein the method comprises administering the antibody and another therapeutic agent and/or radiation therapy to the patient. Clause 41. A method or an antibody or composition for use according to clause 40, wherein the therapeutic agent is an anti-PD-L1 antibody. Clause 42. A method or an antibody or composition for use according to clause 41, wherein the anti-PD-L1 antibody comprises a VH domain having amino acid sequence SEQ ID NO: 299 and a VL domain having amino acid sequence SEQ ID NO: 300. Clause 43. A method or an antibody or composition for use according to clause 41 or clause 42, wherein the therapeutic agent is an anti-PD-L1 IL-2 immunocytokine. Clause 44. A method or an antibody or composition for use according to clause 43, wherein the anti-PD-L1 antibody is an immunocytokine comprising human wild type or variant IL-2. Clause 45. A method or an antibody or composition for use according to clause 44, wherein the anti-ICOS antibody and the anti-PDL1 antibody are each able to mediate ADCC, ADCP and/or CDC. Clause 46. A method or an antibody or composition for use according to any of clauses 41 to 45, wherein the anti-ICOS antibody is a human IgG1 antibody and the anti-PDL1 antibody is a human IgG1 antibody. Clause 47. A method or an antibody or composition for use according to clause 40, wherein the therapeutic agent is an anti-PD-1 antibody. Clause 48. A method or an antibody or composition for use according to clause 40, wherein the other therapeutic agent is IL-2. Clause 49. A method or an antibody or composition for use according to any of clauses 40 to 48, wherein the method comprises administering the anti-ICOS antibody after administering the other therapeutic agent and/or radiation therapy. Clause 50. A method or an antibody or composition for use according to any of clauses 31 to 49, whereinthe anti-ICOS antibody is conjugated to a pro-drug, and whereinthe method or use comprisesadministering the anti-ICOS antibody to a patient andselectively activating the pro-drug at a target tissue site. Clause 51. A method or an antibody or composition for use according to clause 50, wherein the patient has a solid tumour and the method comprises selectively activating the pro-drug in the tumour. Clause 52. A method or an antibody or composition for use according to clause 50 or clause 51, comprising selectively activating the pro-drug through photoactivation. Clause 53. Combination of anti-ICOS human IgG1 antibody and anti-PDL1 human IgG1 antibody for use in a method of treating cancer in a patient. Clause 54. A method of treating cancer in a patient, comprising administering an anti-ICOS human IgG1 antibody and an anti-PD-L1 human IgG1 antibody to the patient. Clause 55. Anti-ICOS antibody for use in a method of treating cancer in a patient, the method comprising administering the anti-ICOS antibody and the anti-PD-L1 antibody to the patient, wherein a single dose of the anti-ICOS antibody is administered. Clause 56. Anti-ICOS antibody for use according to clause 55, wherein the anti-ICOS antibody is a human IgG1 antibody and the anti-PD-L1 antibody is a human IgG1 antibody. Clause 57. Combination according to clause 53, method according to clause 54 or anti-ICOS antibody for use according to clause 55 or clause 56, wherein the cancer is renal cell cancer, head and neck cancer, melanoma, non small cell lung cancer or diffuse large B-cell lymphoma. Clause 58. A method or an antibody, composition or combination for use according to any of clauses 41 to 46 or 53 to 54, the method comprising administering the anti-ICOS antibody and the anti-PD-L1 antibody to the patient, wherein a single dose of the anti-ICOS antibody is administered. Clause 59. A method or an antibody, composition or combination for use according to clause 58, wherein the method comprises administering a single dose of the anti-ICOS antibody followed by multiple doses of the anti-PD-L1 antibody. Clause 60. A method or an antibody, composition or combination for use according to any of clauses 41 to 46 or 53 to 54, wherein the anti-ICOS antibody and the anti-PDL1 antibody are provided in separate compositions for administration. Clause 61. A method or an antibody, composition or combination for use according to any of clauses 41 to 46 or 53 to 60, wherein the anti-ICOS antibody and/or the anti-PD-L1 antibody comprises a human IgG1 constant region comprising amino acid sequence SEQ ID NO: 340. Clause 62. Anti-ICOS antibody for use in a method of treating a patient, the method comprising administering the anti-ICOS antibody to a patient who has an increased level of ICOS-positive regulatory T cells following treatment with another therapeutic agent. Clause 63. A method of treating a patient, the method comprising administering an anti-ICOS antibody to a patient who has an increased level of ICOS-positive regulatory T cells following treatment with another therapeutic agent. Clause 64. An anti-ICOS antibody for use according to clause 62, or a method according to clause 63, wherein the method comprises administering a therapeutic agent to the patient, determining that the patient has an increased level of ICOS-positive regulatory T cells following the treatment with said agent, and administering an anti-ICOS antibody to the patient to reduce the level of regulatory T cells. Clause 65. An anti-ICOS antibody for use or a method according to any of clauses 62 to 64, wherein the therapeutic agent is IL-2 or an immunomodulatory antibody (e.g., anti-PDL-1, anti-PD-1 or anti-CTLA-4). Clause 66. An anti-ICOS antibody for use or a method according to any of clauses 62 to 65, wherein the method comprises treating a tumour, e.g., melanoma, such as metastatic melanoma. Clause 67. Anti-ICOS antibody for use in a method of treating cancer in a patient by in vivo vaccination of the patient against their cancer cells, the method comprisingtreating the patient with a therapy that causes immunological cell death of the cancer cells, resulting in presentation of antigen to antigen-specific effector T cells, andadministering an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances the antigen-specific effector T cell response. Clause 68. A method of treating cancer in a patient by in vivo vaccination of the patient against their cancer cells, the method comprisingtreating the patient with a therapy that causes immunological cell death of the cancer cells, resulting in presentation of antigen to antigen-specific effector T cells, andadministering an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances the antigen-specific effector T cell response. Clause 69. A method of treating cancer in a patient by in vivo vaccination of the patient against their cancer cells, the method comprising administering an anti-ICOS antibody to the patient, whereinthe patient is one who has been previously treated with a therapy that causes immunological cell death of the cancer cells, resulting in presentation of antigen to antigen-specific effector T cells, and whereinthe anti-ICOS antibody enhances the antigen-specific effector T cell response. Clause 70. Anti-ICOS antibody for use or a method according to any of clauses 67 to 69, wherein the therapy that causes immunological cell death is radiation of the cancer cells, administration of a chemotherapeutic agent and/or administration of an antibody directed to a tumour-associated antigen. Clause 71. Anti-ICOS antibody for use or a method according to clause 70, wherein the chemotherapeutic agent is oxaliplatin. Clause 72. Anti-ICOS antibody for use or a method according to clause 70, wherein the tumour-associated antigen is HER2 or CD20. Clause 73. Anti-ICOS antibody for use in a method of treating a cancer in a patient, wherein the cancer is or has been characterised as being positive for expression of ICOS ligand and/or FOXP3. Clause 74. A method of treating a cancer in a patient, wherein the cancer is or has been characterised as being positive for expression of ICOS ligand and/or FOXP3, the method comprising administering an anti-ICOS antibody to the patient. Clause 75. Anti-ICOS antibody for use according to clause 73, or a method according to clause 74, wherein the method comprises:testing a sample from a patient to determine that the cancer expresses ICOS ligand and/or FOXP3;selecting the patient for treatment with the anti-ICOS antibody; andadministering the anti-ICOS antibody to the patient. Clause 76. Anti-ICOS antibody for use according to clause 73, or a method according to clause 74, wherein the method comprises administering an anti-ICOS antibody to a patient from whom a test sample has indicated that the cancer is positive for expression of ICOS ligand and/or FOXP3. Clause 77. Anti-ICOS antibody for use or a method according to clause 75 or clause 76, wherein the sample is biopsy sample of a solid tumour. Clause 78. Anti-ICOS antibody for use in a method of treating a cancer in a patient, wherein the cancer is or has been characterised as being refractory to treatment with an immunooncology drug, e.g., anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody or anti-GITR antibody. Clause 79. A method of treating a cancer in a patient, wherein the cancer is or has been characterised as being refractory to treatment with an immunooncology drug, e.g., anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody or anti-GITR antibody, the method comprising administering an anti-ICOS antibody to the patient. Clause 80. Anti-ICOS antibody for use according to clause 78 or a method according to clause 79, wherein the method comprises:treating the patient with the immunooncology drug;determining that the cancer is not responsive to the drug;selecting the patient for treatment with the anti-ICOS antibody; andadministering the anti-ICOS antibody to the patient. Clause 81. Anti-ICOS antibody for use according to clause 78, or a method according to clause 79, wherein the method comprises administering an anti-ICOS antibody to a patient whose cancer was not responsive to prior treatment with the immunooncology drug. Clause 82. Anti-ICOS antibody for use or a method according to any of clauses 73 to 81, wherein the cancer is a tumour derived from cells that have acquired ability to express ICOS ligand. Clause 83. Anti-ICOS antibody for use or a method according to clause 82, wherein the cancer is melanoma. Clause 84. Anti-ICOS antibody for use or a method according to any of clauses 73 to 81, wherein the cancer is derived from an antigen-presenting cell, such as a B lymphocyte (e.g., B cell lymphoma, such as diffuse large B cell lymphoma) or a T lymphocyte. Clause 85. Anti-ICOS antibody for use or a method according to any of clauses 73 to 81, wherein the cancer is resistant to treatment with an anti-CD20 antibody. Clause 86. Anti-ICOS antibody for use or a method according to clause 85, wherein the cancer is B cell lymphoma. Clause 87. Anti-ICOS antibody for use or a method according to clause 86, wherein the anti-CD20 antibody is rituximab. Clause 88. Anti-ICOS antibody for use or a method according to any of clauses 85 to 87, wherein the method comprises treating the patient with the anti-CD20 antibody;determining that the cancer is not responsive to the anti-CD20 antibody;testing a sample from a patient to determine that the cancer expresses ICOS ligand;selecting the patient for treatment with the anti-ICOS antibody; andadministering the anti-ICOS antibody to the patient. Clause 89. Anti-ICOS antibody for use or a method according to any of clauses 85 to 87, wherein the method comprises administering an anti-ICOS antibody to a patient whose cancer was not responsive to prior treatment with anti-CD20 antibody. Clause 90. Anti-ICOS antibody for use or a method according to any of clauses 67 to 89, wherein the cancer is a solid tumour. Clause 91. Anti-ICOS antibody for use or a method according to any of clauses 67 to 89, wherein the cancer is a haemotological liquid tumour. Clause 92. Anti-ICOS antibody for use or a method according to clause 90 or 91, wherein the tumour is high in regulatory T cells. Clause 93. Anti-ICOS antibody for use or a method according to any of clauses 53 to 92, wherein the anti-ICOS antibody is as defined in any of clauses 1 to 28 or is provided in a composition according to clause 29. Clause 94. A transgenic non-human mammal having a genome comprising a human or humanised immunoglobulin locus encoding human variable region gene segments, wherein the mammal does not express ICOS. Clause 95. A method of producing an antibody that binds the extracellular domain of human and non-human ICOS, comprising(a) immunising a mammal according to clause 94 with human ICOS antigen;(b) isolating antibodies generated by the mammal;(c) testing the antibodies for ability to bind human ICOS and non-human ICOS; and(d) selecting one or more antibodies that binds both human and non-human ICOS. Clause 96. A method according to clause 95, comprising immunising the mammal with cells expressing human ICOS. Clause 97. A method according to clause 95 or clause 96, comprising(c) testing the antibodies for ability to bind human ICOS and non-human ICOS using surface plasmon resonance and determining binding affinities; and(d) selecting one or more antibodies for which the KDof binding to human ICOS is less than 50 nM and the KDof binding to non-human ICOS is less than 500 nM. Clause 98. A method according to clause 97, comprising(d) selecting one or more antibodies for which the KDof binding to human ICOS is less than 10 nM and the KDof binding to non-human ICOS is less than 100 nM. Clause 99. A method according to any of clauses 95 to 98, comprising(c) testing the antibodies for ability to bind human ICOS and non-human ICOS using surface plasmon resonance and determining binding affinities; and(d) selecting one or more antibodies for which the KDof binding to human ICOS is within 10-fold of the KDof binding to non-human ICOS. Clause 100. A method according to clause 99, comprising(d) selecting one or more antibodies for which the KDof binding to human ICOS is within 5-fold of the KDof binding to non-human ICOS. Clause 101. A method according to any of clauses 95 to 100, comprising testing the antibodies for ability to bind non-human ICOS from the same species as the mammal. Clause 102. A method according to any of clauses 95 to 101, comprising testing the antibodies for ability to bind non-human ICOS from a different species as the mammal. Clause 103. A method according to any of clauses 95 to 102, wherein the mammal is a mouse or a rat. Clause 104. A method according to any of clauses 95 to 103, wherein the non-human ICOS is mouse ICOS or rat ICOS. Clause 105. A method according to any of clauses 95 to 104, wherein the human or humanised immunoglobulin locus comprises human variable region gene segments upstream of an endogenous constant region. Clause 106. A method according to clause 105, comprising(a) immunising a mammal according to clause 94 with human ICOS antigen, wherein the mammal is a mouse;(b) isolating antibodies generated by the mouse;(c) testing the antibodies for ability to bind human ICOS and mouse ICOS; and(d) selecting one or more antibodies that binds both human and mouse ICOS. Clause 107. A method according to any of clauses 95 to 106, comprising isolating nucleic acid encoding an antibody heavy chain variable domain and/or an antibody light chain variable domain. Clause 108. A method according to any of clauses 95 to 107, wherein the mammal generates antibodies through recombination of human variable region gene segments and an endogenous constant region. Clause 109. A method according to clause 107 or clause 108, comprising conjugating the nucleic acid encoding the heavy and/or light chain variable domain to a nucleotide sequence encoding a human heavy chain constant region and/or human light chain constant region respectively. Clause 110. A method according to any of clauses 107 to 109, comprising introducing the nucleic acid into a host cell. Clause 111. A method according to clause 110, comprising culturing the host cell under conditions for expression of the antibody, or of the antibody heavy and/or light chain variable domain. Clause 112. An antibody, or antibody heavy and/or light chain variable domain, produced by the method according to any of clauses 95 to 111. Clause 113. A method of selecting an antibody that binds ICOS, optionally for selecting an ICOS agonist antibody, the assay comprising:providing an array of antibodies immobilised (attached or adhered) to a substrate in a test well;adding ICOS-expressing cells (e.g., activated primary T cells, or MJ cells) to the test well;observing morphology of the cells;detecting shape change in the cells from rounded to flattened against the substrate within the well; wherein the shape change indicates that the antibody is an antibody that binds ICOS, optionally an ICOS agonist antibody;selecting the antibody from the test well;expressing nucleic acid encoding the CDRs of the selected antibody; andformulating the antibody into a composition comprising one or more additional components. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification, including published US counterparts of any patents or patent applications referred to, are incorporated herein by reference in their entirety. EXPERIMENTAL EXAMPLES The following Examples describe the generation, characterisation and performance of anti-ICOS antibodies. Antibodies were generated using the Kymouse™, a transgenic mouse platform capable of generating antibodies with human variable domains. Antibodies from the Kymouse™ have human variable domains, generated from human V (D) and J segments, and mouse constant domains. The endogenous mouse variable genes have been silenced and make up a very small portion of the repertoire (less than 0.5% of all heavy chain variable regions are of mouse origin). The Kymouse™ system is described in Lee et al 2014 [39], WO2011/004192, WO2011/158009 and WO2013/061098. This project employed the Kymouse™ HK strain, in which the heavy chain locus and light chain kappa locus are humanised. ICOS knock-out Kymouse™ were immunised with either ICOS protein or a combination of alternating boosts of protein and cells expressing human and mouse ICOS. Hits which bound to human ICOS were identified. The primary selection criteria for the screen was binding to human cell expressed ICOS (CHO cells) and binding to ICOS protein (HTRF). Binding to mouse ICOS protein and mouse cell expressed ICOS (CHO cells) was also assessed and taken into consideration when selecting primary screen hits. Using these criteria hits were progressed to a secondary screen. In the secondary screen hits were confirmed by determining binding to human and mouse ICOS expressed on CHO cells by flow cytometry. From a large number of antibodies screened, a small panel were identified which bind to human/cynomolgus and mouse ICOS as determined by surface plasmon resonance and flow cytometry. These antibodies included STIM001, STIM002 and its variant STIM002-B, STIM003, STIM004 and STIM005. An additional four antibodies STIM006, STIM007, STIM008 and STIM009 were also selected, showing less cross-reactivity with mouse ICOS but demonstrating agonism of the human ICOS receptor. The data presented here indicate the ability of anti-ICOS antibodies to act as agonists of the ICOS receptor in an ICOS positive CD4+ cell line and also in a primary T cell-based assay, show cell-killing ability in an ADCC assay and an ability to promote an anti-tumour immune response in vivo. Example 1: Generation of ICOS Knock-Out Mouse An ICOS knock-out Kymouse™ line was generated by homologous recombination in Kymouse™ HK ES cells. In brief, a 3.5 kb targeting vector encoding a puromycin selection was targeted into ES cells. Successful targeting resulted in the replacement of a small region (72 bp) of the mouse ICOS locus with the puromycin cassette, disrupting the signal peptide/start codon of the gene. Positive ES clones were expanded and microinjected into mouse blastocysts and resulting chimaeras bred in order to ultimately generate animals homozygous for both the humanised heavy and kappa immunoglobulin loci and the modified, functionally-null, ICOS locus. Example 2: Antigen and Cell-Line Preparation Generation of Stably Transfected MEF and CHO-S Cells Expressing Human or Mouse ICOS Full length DNA sequences encoding human and mouse ICOS were codon optimised for mammalian expression, ordered as synthetic string DNA and cloned into an expression vector under the control of the CMV promoter and flanked by 3′ and 5′ piggyBac specific terminal repeat sequences facilitating stable integration into the cell genome (see [40]). The expression vector contained a puromycin selection cassette to facilitate stable cell line generation. For generation of human ICOS expressing and mouse ICOS expressing cell lines respectively, the human or mouse ICOS expression plasmid was co-transfected with a plasmid encoding piggyBac transposase into a mouse embryonic fibroblast (MEF) cell line and CHO-S cells using the FreeStyle Max transfection reagent (Invitrogen) according to manufacturer instructions. MEF cells were generated from embryos obtained from a 129S5 crossed to C57BL6 female mouse. Twenty four hours after transfection, the media was supplemented with puromycin and grown for at least two weeks to select stable cell lines. Cell culture medium was replaced every 3-4 days. Expression of human or mouse ICOS protein was assessed by flow cytometry using anti-human or anti-mouse ICOS-PE conjugated antibodies (eBioscience) respectively. Complete MEF media was made up of Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% v/v fetal bovine serum (Gibco). Complete CHO-S media was made up of CD-CHO media supplemented with 8 mM Glutamax (Gibco). CHO-S cells are the CHO-3E7 cell line included with the pTT5 system available from the National Research Council of Canada, but other CHO cell lines could be employed. Preparation of MEF Cells for Mouse Immunisations Cell culture medium was removed and cells washed once with 1×PBS. Cells were treated for 5 minutes with trypsin to loosen cells from tissue culture surface. Cells were collected and the trypsin neutralized by the addition of complete media containing 10% v/v fetal bovine serum (FCS). Cells were then centrifuged at 300 g for 10 minutes and washed with 25 ml of 1×PBS. Cells were counted and resuspended at the appropriate concentration in 1×PBS. Cloning and Expression of Recombinant Proteins Synthetic DNA encoding the extracellular domains of human ICOS (NCBI ID: NP_036224.1), mouse ICOS (NCBI ID: NP_059508.2) and cynomolgus ICOS (GenBank ID: EHH55098.1) were cloned into either a pREP4 (Invitrogen) or a pTT5 (National Research Council Of Canada) expression plasmid using standard molecular biology techniques. The constructs also contained either a human Fc, a mouse Fc or a FLAG His peptide motif to aid purification and detection. These were added to the DNA constructs by overlap extension. All constructs were sequenced prior to expression to ensure their correct sequence composition. Example 3: Immunisation ICOS knock out HK Kymice™ (see Example 1), Kymouse™ wild type HK strain and Kymouse™ wild type HL strain were immunised according to the regimens shown in Table E3. Kymouse™ wild type HK and HL strains express wild type mouse ICOS. In the HK strain the immunoglobulin heavy chain locus and light chain kappa locus are humanised, and in the HL strain the immunoglobulin heavy chain locus and light chain lambda locus are humanised. TABLE E3Immunisation regimens for Kymouse ™ strainsRegimeMousePrimeBoost 1Boost 2Boost 3Final BoostKM103ICOS KOmICOS FchICOS MEFmICOS FchICOS MEFmICOS FcKM103ICOS KOmICOS FchICOS FcmICOS FchICOS FcN/AKM111ICOS KOmICOS Fc +mICOS MEF +mICOS Fc +mICOS MEF +mICOS Fc +hICOS FchICOS MEFhICOS FchICOS MEFhICOS FcKM111ICOS KOhICOS FchICOS MEFhICOS FchICOS MEFhICOS FcKM111ICOS KOmICOS FcmICOS MEFmICOS FcmICOS MEFmICOS FcKM111HK and HLhICOS FchICOS MEFhICOS FchICOS MEFhICOS FcKM135ICOS KOmICOS Fc 1 prime and 6 boosts (RIMMS)KM135ICOS KOhICOS Fc 1 prime and 6 boosts (RIMMS)Key to table:mICOS Fc = mouse ICOS protein with human FchICOS Fc = human ICOS protein with human FcmICOS MEF = mouse ICOS expressed on MEF cellshICOS MEF = human ICOS expressed on MEF cellsmICOS Fc + hICOS Fc = mouse ICOS protein with human Fc + human ICOS protein with human Fc administered simultaneouslymICOS MEF + hICOS MEF = mouse ICOS expressed on MEF cells + human ICOS expressed on MEF cells administered simultaneouslyICOS KO = ICOS knockout HK KymouseHK and HL = wild type Kymouse HK and HL genotypeRIMMS is a modified sub-cutaneous immunisation procedure (rapid immunisation at multiple sites); modified after Kilpatrick et al. [41]). Immunisation regimens KM103 and KM111 were prime-rest-boost by intraperitoneal (i.p.) administration. Sigma Adjuvant System was used for all immunisations and rest intervals were usually between 2 and 3 weeks. Final boosts were administered by intravenously in absence of adjuvant. Sera from serial or terminal blood samples were analysed for the presence of specific antibodies by flow cytometry and the titre data was used (where possible) to select mice to be used for B cell sorting. Example 4: Comparison of Serum Titres Between ICOS KO and Wild Type Mice Serum titres of immunised ICOS KO and immunised wild type Kymouse were determined using flow cytometry. In ICOS KO mice, immunisation with human ICOS antigen induced a serum immunoglobulin response with Ig binding to both human and mouse ICOS expressed on CHO cells (FIG.1a). Conversely, in the wild type Kymouse (expressing mouse ICOS), immunisation with the same human ICOS antigen produced sera that showed markedly reduced binding to mouse ICOS compared with binding of the same serum to human ICOS (FIG.1b). Method CHO-S cells expressing human ICOS or mouse ICOS (see Example 2) or untransfected CHO-S cells (referred to as wild type (WT)), suspended in FACS buffer (PBS+1% w/v BSA+0.1% w/v sodium azide) were distributed to a 96-well, V-bottom plate (Greiner) at a density of 105cells per well. A titration of mouse serum was prepared, diluting samples in FACS buffer. 50 μL/well of this titration was then added to the cell plate. To determine the change in activity level due to immunisation, serum from each animal prior to immunisation was diluted to 1/100 in FACS buffer and 50 μL/well added to the cells. Cells were incubated at 4° C. for 1 hour. Cells were washed twice with 150 μL PBS, centrifuging after each wash step and aspirating supernatant (centrifuged at 300×g for 3 minutes). To detect antibody binding, APC goat-anti-mouse IgG (Jackson ImmunoResearch) was diluted 1/500 in FACS buffer and 50 μL was added to the cells. In some instances AF647 goat-anti-mouse IgG (Jackson ImmunoResearch) was used. Cells were incubated 1 hour at 4° C. in the dark, then washed twice with 150 μL PBS as above. To fix cells, 100 μL 2% v/v paraformaldehyde was added and cells incubated for 30 minutes at 4° C. Cells were then pelleted by centrifugation at 300×g and the plates resuspended in 50 μL of FACS buffer. Fluorescent signal intensity (geometric mean) was measured by flow cytometry using a BD FACS Array instrument. Example 5: Sorting of Antigen-Specific B Cells by FACS B-cells expressing anti-ICOS antibodies were recovered from immunised mice, using techniques substantially as described in Example 1 of WO2015/040401. In brief, splenocytes and/or lymph node cells isolated from the immunisation regimes were stained with an antibody cocktail containing markers for the selection of cells of interest (CD19), whereas unwanted cells were excluded from the final sorted population (IgM, IgD, 7AAD). CD19+B-cells were further labelled with fluorescently-tagged human ICOS ECD-Fc dimers and fluorescently-tagged mouse ICOS ECD-Fc to detect B-cells producing anti-ICOS antibodies. Fluorescent labelling of human and mouse ICOS was with AlexaFluor647 and AlexaFluor488, respectively—see Example 6. Cells binding human ICOS, or both human and mouse ICOS were selected. These cells were single cell sorted by FACS into lysis buffer. V-region sequences were recovered using RT-PCR and two further rounds of PCR, then bridged to mouse IgG1 constant region and expressed in HEK293 cells. Supernatants from HEK293 cells were screened for the presence of ICOS binding and functional antibodies. This method is hereafter referred to as BCT. Example 6: Screening of Antibodies from BCT HTRF Screening of BCT Supernatants for Binding to Recombinant Human and Mouse ICOS-Fc Supernatants collected from BCT in Example 5 were screened for the ability of secreted antibodies to bind to human ICOS Fc and mouse ICOS Fc expressed as recombinant proteins. Binding of secreted antibodies to recombinant human and mouse ICOS were identified by HTRF® (Homogeneous Time-Resolved Fluorescence, Cisbio) assay format using FluoProbes®647H (Innova Biosciences) labelled ICOS (referred to herein as 647 hICOS or 647 mICOS for human ICOS and mouse ICOS labelled with FluoProbes®647H respectively). 5 μL BCT supernatant was transferred to a white 384-well, low-volume, non-binding surface polystyrene plate (Greiner). 5 μL of 20 nM 647 hICOS or 647 mICOS diluted in HTRF assay buffer was added to all wells. For human ICOS binding assay the reference antibody was diluted in BCT media (Gibco #A14351-01) to 120 nM and 5 μL added to plate. For negative control wells for human ICOS binding assay, 5 μL of mouse IgG1 (Sigma M9269 in some instances referred to as CM7) diluted to 120 nM in BCT media. In the case of mouse ICOS binding assay the reference antibody was diluted in BCT media (Gibco #A14351-01) to 120 nM and 5 μL added to plate. A rat IgG2b isotype control (R&D systems) was added to negative control wells (R&D Systems) diluted in BCT media to 120 nM and 5 μL added to plate. Binding of secreted antibodies to human ICOS was detected by addition of 10 μL of goat anti-mouse IgG (Southern Biotech) directly labelled with Europium cryptate (Cisbio) diluted 1/2000 in HTRF assay buffer. In the case of the mouse ICOS binding assay 5 μL of mouse anti-Rat IgG2B-UBLB (Southern Biotech) was added to positive and negative control wells, and 5 μL of HTRF assay buffer added to all other wells of plate. Then 5 μL of goat anti-mouse IgG (Southern Biotech) directly labelled with Europium cryptate (Cisbio) diluted 1/1000 in HTRF assay buffer was added to detect binding. The plate was left to incubate in the dark for 2 hours prior to reading time resolved fluorescence at 620 nm and 665 nm emission wavelengths, 100 flashes, using an EnVision plate reader (Perkin Elmer). Data were analysed by calculating 665/620 ratio and percent effect for each sample according to equation 2 and equation 1 respectively. For KM103 and KM11-B1, primary hits were selected based on greater than or equal to 5 percent effect for binding to human and mouse ICOS. For KM135 primary hits were selected based on greater than or equal to 10 percent effect for binding to human and mouse ICOS. For KM111-B2 primary hits were defined as greater than or equal to 4 percent effect for binding to human and greater than or equal to 3 percent effect for binding to mouse ICOS. Equation1CalculationofPercentageEffectfromPrimaryScreenEnvisioncellbindingandHTRFUsingwellratiovalue(equation3)or665/620nmratio(seeequation2)(HTRF)Percenteffect=(samplewell-non-specificbinding)×100(totalbinding-non-specificbinding)Non-specificbinding=valuesfromwellscontainingisotypecontrolmouseIgG1TotalBinding=valuesfromwellscontainingreferenceantibodyEquation2Calculationof665/620ratio665/620ratio=(sample665/620nmvalue)×10,000Equation3Calculationof647/FITCratioDatawerefirstnormalisedforcellnumberbydividingmAbchannel(647)byFITC(cellstain)channeltogive“wellratiovalue”:WellRatioValue=647ChannelFITCChannel Screening of BCT Supernatants for Binding to Cell-Expressed Human and Mouse ICOS Supernatants collected from BCT in Example 5 were screened for the ability of secreted antibodies to bind to human or mouse ICOS expressed on the surface of CHO-S cells. To determine CHO-S human and mouse ICOS binding, cells were plated in black-walled, clear-bottom tissue culture treated 384-well plates (Perkin Elmer) at 4×104/well in F12 media (Gibco) supplemented with 10% FBS (Gibco) and cultured overnight. Culture media was removed from 384-well assay plates. At least 50 μL of BCT supernatant or 50 μL reference antibody at 2 μg/mL in BCT media or isotype IgG1 control antibody (referred to in some instances as Cm7, Sigma M9269), at a final concentration of 2 μg/mL) diluted in BCT media were added to each well. Plates were incubated for 1 hour at 4° C. Supernatant was aspirated and 50 μL of goat anti-Mouse 647 (Jackson immunoresearch) at 5 μg/ml with vibrant green DNA stain (Life Technologies) diluted 1 in 500 in secondary antibody buffer (1×PBS+1% BSA+0.1% Sodium Azide) was added to detect antibody binding and visualise cells. Plates were incubated for 1 hr at 4 degrees. Supernatant was aspirated and 25 μL of 4% v/v paraformaldehyde added and plates were incubated for 15 minutes at room temperature. Plates were washed twice with 100 μL PBS and then the wash buffer was completely removed. Fluorescence intensity was measured using Envision plate reader (Perkin Elmer) measuring FITC (excitation 494 nm, emission 520 nm) and alexafluor 647 (excitation 650 nm, emission 668 nm). Assay signal was determined as described in equation 3 and percent effect as in equation 1. Total binding was defined using reference antibody at a final assay concentration of 2 μg/mL. Non-specific binding was defined using mouse IgG1 isotype control (Sigma) at a final assay concentration of 2 μg/mL. Criteria for hit selection were based on assay signal and percent effect. For KM103, KM111-B1 and KM135, primary hits were selected based on greater than or equal to 10 percent effect. For KM111-B2, primary hits were selected based on greater than or equal to 4 percent effect. Summary of Primary Screening Results TABLE E6Summary of number of BCT supernatants screened fromimmunisations, and number of supernatants meeting primary screeningselection criteria for binding to human and mouse ICOS.SupernatantsExperiment IDscreenedPrimary hits selectedKM103123240KM111-B11056198KM111-B21056136KM13570431 FACS Screening for Binding to Cell Expressed Human and Mouse ICOS BCT supernatants and HEK293 expressed antibodies from Example 5 were tested for ability to bind to CHO-S cells expressing human or mouse ICOS. CHO-S cells expressing human or mouse ICOS (see Example 2), were diluted in FACS buffer (PBS 1% BSA 0.1% sodium azide) and were distributed to a 96-well, V-bottom plate (Greiner) at a density of 1×105cells per well. Cells were washed with 150 μL PBS and centrifuged at 300 g for 3 minutes. For supernatant screening, supernatant was aspirated and 150 μL PBS added. This wash step was repeated. 30 μL BCT undiluted supernatant or 50 μL of reference antibody or control antibody diluted to 5 μg/ml in BCT media was added to the washed cells. Cells were incubated at 4° C. for 60 minutes. 150 μL FACS buffer was added and cells washed as described above. To detect antibody binding, 50 μL of goat anti-mouse APC (Jackson ImmunoResearch) diluted to 2 μg/ml in FACS buffer was added to cells. Cells were incubated 4° C. for 60 minutes. Cells were washed twice with 150 μL FACS buffer, centrifuging at 300 g for 3 minutes after each wash step and aspirating supernatant. Cells were fixed by addition of 25 μL 4% paraformaldehyde for 20 minutes at room temperature. Cells were washed once as above and resuspended in FACS buffer for analysis. APC signal intensity (geometric mean) was measured by flow cytometry using a BD FACS Array instrument. Data were plotted as geometric mean values without further calculation. A small sub-set of antibodies were selected as meeting more stringent species cross-reactivity criteria in this further screening compared with the primary screening. In brief: From KM103, 4 antibodies were selected by taking the average geomean of the hybrid control binding to hICOS, mICOS and WT CHO cells and identifying mouse and human binders that were >4 fold above. These 4 antibodies were designated STIM001, STIM002-B, STIM007 and STIM009. From KM111-81, 4 antibodies were selected by taking the average of geomean of the negative control (Armenian hamster: clone HTK888) binding to hICOS, mICOS and WT CHO cells and identifying mouse and human binders that were >10 fold above. From KM111-82, 4 antibodies were selected by taking the average of geomean of the negative control (Armenian hamster: clone HTK888) binding to hICOS, mICOS and WT CHO cells and identifying mouse and human binders that were >4 fold above. These 4 antibodies included STIM003, STIM004 and STIM005. From KM135, no cross-reactive antibodies were identified. Due to a technical failure of the FACS secondary screening method, screening was also carried out using SPR and HTRF, but no antibodies were found to meet the desired cross-reactivity level. In conclusion, from the various multiple immunisation regimens described in Example 3, upward of 4000 BCT supernatants (from the ICOS KO mice only) were screened for binding to human ICOS and mouse ICOS, and a small panel of candidates, including STIM001, STIM002-B, STIM003, STIM004, STIM005, STIM007 and STIM009, were identified as having the most promising characteristics for further development. These were taken forward for more detailed characterisation. Separately, two antibodies STIM006 and STIM008, which did not meet the species cross-reactivity criteria, were also chosen for further characterisation on the basis of their ability to bind human ICOS. Example 7: Affinity Determination by Surface Plasmon Resonance (SPR) Fab affinities of the ICOS leads were generated by SPR using the ProteOn XPR3 6 (BioRad). An anti-human IgG capture surface was created on a GLC biosensor chip by primary amine coupling, immobilising three anti-human IgG antibodies (Jackson Labs 109-005-008, 109-006-008 and 309-006-008). The human Fc tagged human ICOS (hICOS) and mouse ICOS (mICOS) were captured individually on the anti-human IgG surface and the purified Fabs were used as analytes at 5000 nM, 1000 nM, 200 nM, 40 nM and 8 nM, except for STIM003 which was used at 1000 nM, 200 nM, 40 nM, 8 nM and 2 nM. Binding sensorgrams were double referenced using a buffer injection (i.e. 0 nM), and the data was fitted to the 1:1 model inherent to the ProteOn XPR36 analysis software. The assay was run at 25° C. and using HBS-EP as running buffer. TABLE E7-1Affinity and kinetic data for selected antibodies as measured by SPR.Sample AbLigandkakdKD (nM)STIM006hICOS6.67E+059.20E−0313.8STIM003hICOS6.56E+058.62E−041.3STIM001hICOS2.54E+041.12E−0344.0STIM002hICOS3.20E+043.43E−021070.0STIM006mICOS1.57E+035.00E−04318.0STIM003mICOS1.29E+065.03E−040.4STIM001mICOS5.66E+042.30E−02407.0STIM002mICOSweakweakweak In addition, a comparison was performed of antibody:antigen binding affinity at different pH values. As before, the dimeric human ICOS protein, presented as the extracellular domain of ICOS fused to a human Fc region, was captured on the anti-human Fc capture surface created using the 3 antibody cocktail, immobilised on the GLC biosensor chip by primary amine coupling. SPR analysis of recombinantly expressed anti-ICOS Fabs was carried out on the ProteOn XPR36 Array system (Biorad). The Fab fragments were used as analyte to generate binding sensorgrams, which were double referenced with a buffer injection (i.e., 0 nM). The subsequent referenced sensorgrams were fitted to the 1:1 model inherent to the ProteOn analysis software. Table E7-2 presents affinity and kinetic data for the antibodies, all run at 3TC unless stated, using either HBS-EP at pH 7.4/7.6 or pH 5.5 as indicated. Data were fitted to the 1:1 model. Note that data for STIM002 fitted poorly to the 1:1 model at both pH 7.4 and 5.5—the affinity for this antibody may therefore be lower than indicated in the table. TABLE E7-2Relative affinity of STIM001, STIM002, STIM002-B and STIM003Fabs against recombinant human ICOS, at 37° C. except where stated.Antibody (Fab)pHkakdKD (nM)STIM0017.45.08E+043.23E−0363.55.54.90E+043.58E−0373.17.68.29E+043.54E−0342.65.56.77E+045.41E−0380.37.6 (25° C.)2.54E+041.12E−0344STIM0027.43.72E+048.31E−032235.58.79E+043.67E−034.17STIM002-B7.48.28E+043.46E−0341.85.58.64E+042.30E−0326.6STIM0037.41.49E+062.54E−031.715.51.55E+061.58E−031.027.61.87E+063.70E−031.985.51.71E+061.94E−031.157.6 (25° C.)6.65E+060.862E−031.31 Comparison of the affinity data at different pH values indicated that the antibodies retain binding to their target across a physiological pH range. The tumour microenvironment may be relatively acidic compared with blood, thus maintenance of affinity at low pH is a potential advantage in vivo to improve intra-tumoural T-reg depletion. Example 8: Neutralisation of ICOS Ligand Binding to ICOS Receptor Assayed by HTRF Selected anti-ICOS antibodies were further assessed for their ability to neutralise ICOS ligand (B7-H2) binding to ICOS, using homogenous time resolved fluorescence (HTRF). Human IgG1 and human IgG4.PE (null-effector) isotypes of the mAbs were assessed in:HTRF assay for neutralisation of human B7-H2 binding to human ICOS; andHTRF assay for neutralisation of mouse B7-H2 binding to mouse ICOS. Anti-ICOS antibody C398.4A (hamster IgG in each case) was included for comparison. A number of antibodies were found to have high neutralising potency for human and/or mouse ICOS receptor-ligand binding, and the results indicated that some of these antibodies showed good cross-reactivity. The antibody isotype had no significant effect, differences in results between the IgG1 and IgG4.PE assays being within experimental error. IgG1 In the human IgG1 assays, antibody C398.4A produced an IC50 of 1.2±0.30 nM for the neutralisation of human ICOS ligand and an IC50 of 0.14±0.01 nM for the neutralisation of mouse ICOS ligand. IgG1 mAbs STIM001, STIM002, STIM003 and STIM005 produced similar IC50 to C398.4A using the human ICOS ligand neutralisation system and were also cross-reactive, neutralising binding of mouse ICOS ligand to the mouse ICOS receptor. Two additional cross-reactive mAbs, STIM002-B and STIM004, showed weaker human and mouse ICOS ligand neutralisation. STIM006, STIM007, STIM008 and STIM009 showed neutralisation of human ICOS ligand but did not demonstrate significant cross-reactivity in the mouse ICOS ligand neutralisation system. Neutralising IC50 values for mouse B7-H2 ligand could not be calculated for these antibodies. TABLE E8-1IC50 values for human IgG1 isotype mAb for neutralisation ofhuman ICOS Receptor binding to human B7-H2. See also FIG. 2.Mean IC50 (nM)SD (nM) (n = 4)STIM0012.21.3STIM0021.90.8STIM002-B3.63.5STIM0031.30.5STIM004233123STIM0052.50.8STIM0062.21.5STIM0071.10.5STIM0081.61.4STIM00930.553C398.4A1.20.3 TABLE E8-2IC50 values for human IgG1 isotype mAb for neutralisation ofmouse ICOS Receptor binding to mouse B7-H2. See also FIG. 3.Mean IC50 (nM)SD (nM) (n = 3)STIM0016.52.5STIM0026.92.1STIM002-B3011.4STIM0030.10STIM00422.115.4STIM0050.30.2C398.4A0.10 IgG4.PE As expected, IgG4.PE mAbs produced similar results to the IgG1 isotypes. STIM001, STIM003 and STIM005 showed similar IC50 values to C398.4A using the human ICOS ligand neutralisation system. These mAbs were also cross-reactive at neutralising mouse ICOS ligand. STIM002-B and STIM004 produced weaker IC50 values for human ICOS B7-H2 neutralisation and mouse B7-H2 ligand. STIM007, STIM008 and STIM009 showed neutralisation of human ICOS ligand binding to human ICOS receptor but neutralising IC50 values for mouse B7-H2 ligand could not be calculated in these assays. IgG4.PE isotypes of STIM006 and STIM002 were not assayed. TABLE E8-3IC50 values for human IgG4.PE isotype mAb for neutralisation ofhuman ICOS Receptor binding to human B7-H2. See also FIG. 4.SD (nM) (n = 4 unlessMean IC50 (nM)otherwise stated)STIM0011.30.2STIM002-B3.41.8STIM0031.20.3STIM004161152 (n = 3)STIM0051.60.2STIM0060.8(n = 1)STIM0070.80.1STIM0080.80.1STIM0094.62.2C398.4A2.83.8 TABLE E8-4IC50 values for human IgG4.PE isotype mAb for neutralisation ofmouse ICOS Receptor binding to mouse B7-H2. See also FIG. 5.Mean IC50 (nM)SD (nM) (n = 3)STIM0014.72.1STIM002-B43.925.7STIM0030.20.1STIM0043014STIM0050.30.1C398.4A0.20.1 Materials and Methods Test antibodies and isotype controls were diluted in assay buffer (0.53 M Potassium Fluoride (KF), 0.1% Bovine Serum Albumin (BSA) in 1×PBS) from a starting working concentration of up to 4 μM, 1 μM final to 0.002 nM, 5.64e-4 nM final over 11 point titration, 1 in 3 dilutions. Titrations of 5 μl of antibody were added to 384w white walled assay plate (Greiner Bio-One). Positive control wells received 5 μl of assay buffer only. 5 μl of ICOS receptor (human ICOS-mFc, 20 nM, 5 nM final or mouse ICOS-mFc 4 nM, 1 nM final (Chimerigen)) was added to required wells. Plate was incubated for 1 hr at room temperature (RT). After incubation, 5 μl of either mouse or human ICOS ligand, (B7-H2, R&D Systems) conjugated to Alexa 647 (Innova Bioscience) was diluted to either 32 nM (8 nM final) for human B7-H2 or 30 nM, 7.5 nM final for mouse B7-H2 and added to all wells of assay plate except negative control wells which instead received 5 μl of assay buffer. Finally, 5 μl of anti-mouse IgG donor mAb (Southern Biotech) labelled with europium cryptate (Cis Bio), 40 nM, 10 nM final was added to each well and the assay was left in the dark at RT to incubate for a further 2 hours. After incubation, assay was read on Envision plate reader (Perkin Elmer) using a standard HTRF protocol. 620 nm and 665 nm channel values were exported to Microsoft Excel (Microsoft) and % Delta-F and % Neutralisation calculations performed. Titration curves and IC50 values [M] were plotted using Graphpad (Prism). IC50 values were calculated by first transforming the data using equation X=Log(X). The transformed data was then fitted using nonlinear regression, using fitting algorithm, log (inhibitor) vs. response—variable slope (four parameters). %Delta-FCalculation:665/620nmratioforratiometricdatareduction.%DeltaF=(665/620nmWellSignalRatio-SignalNegativeControl)(SignalNegativeControl)*100SignalNegativecontrol=averageofminimumsignalratio.%Neutralisation:%Max(neutralisation)=(%Delta-Fofsamplewell-NegativeControl)(PositiveControl-NegativeControl)*100 Example 9a: T-Cell Activation STIM001 and STIM003 agonistic potential on cytokine production was tested in plate-bound and soluble format in a human primary T-cell activation assay where anti-CD3 and anti-CD28 Abs were added concurrently to the anti-ICOS Ab to induce ICOS expression on effector T-cells. Effect of the ICOS co-stimulation on the level of IFNγ produced by these activated T-cells were assessed using ELISA at 72 hrs post-activation. Materials and Methods T-Cell Activation Assay 1: Isolation of Mononuclear Cells from Human Peripheral Blood (PBMC): Leukocyte cones were collected from healthy donors and their content was diluted up to 50 ml with phosphate buffered saline (PBS, from Gibco) and layered into 2 centrifuge tubes on top of 15 mL Ficoll-Paque (from GE Healthcare). PBMC were separated by density gradient centrifugation (400 g for 40 min without brake), transferred to a clean centrifuge tube and then washed with 50 mL PBS, twice by centrifuging at 300 g for 5 min and twice by centrifuging at 200 g for 5 min. PBMC were then resuspended in R10 media (RPMI+10% heat-inactivated Fetal Bovine Serum, both from Gibco) and their cell count and viability assess with EVE™ Automated Cell Counter (from NanoEnTek). ICOS Antibodies (Abs) Preparation and Dilutions: STIM001 and STIM003 were tested in 3 formats: plate-bound, soluble or soluble plus F(ab′)2Fragments (109-006-170 from Jackson Immuno Research) which crosslink the anti-ICOS Abs. For plate-bound format: the anti-ICOS Abs and their isotype control were serially diluted 1:3 in PBS to give final antibody concentrations ranging from 45 to 0.19 μg/mL. 100 μL of diluted antibodies were coated in duplicate into a 96-well, high-binding, flat-bottom plate (Corning EIA/RIA plate) overnight at 4° C. Plate was then washed with PBS and 125 μl of R10 were added to each well. For soluble format: The anti-ICOS Abs and their isotype control were serially diluted 1:3 in R10 media to give an 2× Ab stock concentrations ranging from 90 to 0.38 μg/mL. 125 μl of diluted Abs were pipetted in duplicate into a 96-well, flat-bottom plate. For crosslinked soluble format: The anti-ICOS Abs and their isotype control were mixed with F(ab′)2Fragments at 1M to 1M ratio. Abs/F(ab′)2Fragments mixes were then 1:3 serially diluted in R10 media to give an 2× Ab concentrations ranging from 90 to 0.38 μg/mL for ICOS and from 60 to 0.24 μg/ml for F(ab′)2Fragments. 125 μl of diluted Abs were pipetted in duplicate into a 96-well, flat-bottom plate. T-Cell Isolation, Cultures and IFN-γ Quantification: T-cell were negatively isolated from PBMC using the EasySep Human T Cell Isolation Kit (from Stemcell Technologies) and resuspended at 2×106/mL in R10 media supplemented with 40 μl/ml of Dynabeads Human T-Activator CD3/CD28 (from Life Technologies). 125 μl of T-cell suspension were added to Ab-containing plates to give a final cell concentration of 1×106cells/ml and cultured for 72 hrs at 37° C. and 5% CO2. Cell free supernatants were then collected and kept at −20° C. until analysis of secreted IFNγ by ELISA (duoset kit from R&D). This experiment was repeated on T-cells isolated from 6 independent donors and 2 technical replicates were included for each assay condition. T-Cell Activation Assay 2 (STIM-REST-STIM Assay): STIM001 and STIM003 agonist potential on cytokine release were also tested plate-bound in a human T-cell assay where T-cells were prestimulated by anti-CD3 and anti-CD28 Abs for 3-days to induce ICOS expression before being rested for 3-days to reduce their activation levels. ICOS expression was confirmed by FACS staining after stimulation (Day 3) and resting (Day 6). These stimulated rested T-cells were then cultured with STIM001 or STIM003 in presence or absence of CD3 Ab to assess the requirement of TCR engagement. Effects of the ICOS co-stimulation were assessed after 72 hrs on the levels of IFNγ, TNFα and IL-2 present in the culture. ICOS Abs Dilutions and Coating: Anti-human CD3 (clone UCHT1 from eBioscience) was diluted in PBS to a 2× Ab concentration of 10 μg/mL. 50 μl of PBS or 50 μl of diluted CD3 Ab were pipetted into a 96-well, high-binding, flat-bottom plate. STIM001, STIM003 and their isotype control were 1:2 serially diluted in PBS to give final 2× antibody concentrations ranging from 20 to 0.62 μg/mL. 50 μL of diluted anti-ICOS Ab were added to wells containing either PBS (no TCR engagement) or diluted CD3 Ab (TCR engagement). Plates were coated overnight at 4° C. T-Cell Isolation, Cultures and IFN-γ Quantification: PBMC from leukocyte cones were obtained as described in T-cell activation assay 1. T-cell were negatively isolated from this PBMC using the EasySep Human T Cell Isolation Kit (from Stemcell Technologies). T-cells were resuspended at 1×106/ml in R10 media supplemented with 20 μl/mL of Dynabeads Human T-Activator CD3/CD28 (from Life Technologies) and cultured for 3-days at 37° C. and 5% CO2(Stimulation). At day 3 dynabeads were removed from the culture. T-cells were then washed (300 g for 5 min), counted and resuspended at 1.5×106/ml in R10 media and culture at 37° C. and 5% CO2for 3-more days (Resting phase). At day 6 stimulated rested T-cells were then washed (300 g for 5 min), counted and resuspended at 1×106/mL in R10 media and 250 μl of T-cell suspension were added to ICOS Ab-coated plates and cultured for 72 hrs at 37° C. and 5% CO2. Cell free supernatants were then collected and kept at −20° C. until analysis of secreted cytokines on the MSD platform. This experiment was repeated with T-cells isolated from 5 independent donors and 3 technical replicates were included for each assay condition. Results Both STIM001 and STIM003 tested positive for inducing IFNγ expression therefore demonstrating agonism in both assays. Example 9b: T Cell Activation Assay 1 Data T cell activation assay 1 was performed as described in Example 9a, using T cells isolated from 8 independent donors, testing each of STIM001 and STIM003 in human IgG1 format. Hamster anti-ICOS antibody C398.4A and a hamster antibody isotype control were included for comparison. 2 technical replicates were included for each assay condition. Results are shown inFIG.16,FIG.17andFIG.18. As noted before, both STIM001 and STIM003 tested positive for inducing IFNγ expression therefore demonstrating an agonistic effect on human primary T cells. Cross-linked antibodies acted as agonists of T cell activation, as indicated by the strong enhancement of IFNγ induction in the presence of the Fc-linking F(ab′)2fragments, compared with either soluble antibody or with control. IFNγ expression in the T cells increased with increasing concentration of cross-linked STIM001 or STIM003 (FIG.16, lower panels). Agonism was also observed for both STIM001 and STIM003 in plate-bound form and, more weakly, for the hamster antibody C398.4A, as indicated by the increase in IFNγ expression observed in the T cells with increasing concentration of antibody (FIG.16, top panels). Magnitude of the IFNγ response varied between T cells obtained from different donors, but STIM001 consistently produced an increase in IFNγ expression in T cells compared with IFNγ expression observed with control antibody (HC IgG1). When considering data from assays with T cells from all 8 donors, it is seen that treatment of T cells with STIM001 significantly increased IFNγ expression compared with treatment with isotype control antibody, in plate-bound form, soluble form and cross-linked form (FIG.17). STIM001 thus behaved as an agonist of T cell activation in all three formats. Similar effects were observed with STIM003 (FIG.18). Levels of IFNγ induced by STIM003 hIgG1 were compared with levels of IFNγ induced by its isotype control (HC IgG1) at a given dose of antibody in the assay, for 8 independent donors. Despite the variability between donors, the mean increase in IFNγ level induced by STIM003 was significant when compared against HC IgG1. It is proposed that STIM001, and the other STIM antibodies described here, have the potential to similarly promote T cell activation in vivo. As discussed previously, agonism of activated ICOS-expressing T cells may be mediated by the anti-ICOS antibody binding to and inducing multimerisation of the ICOS receptor on the T cell surface. Example 9c: T cell activation assay 2 data T cell activation assay 2 was performed as described in Example 9a. In the absence of TCR engagement (no anti-CD3 antibody), levels of cytokines produced from the primary T cells were low and no increase was induced by STIM001 (hIgG1), STIM003 (hIgG1) or antibody C398.4A even at the highest concentration of 10 μg/ml. In contrast, when the anti-ICOS antibodies were added to T cells in combination with the anti-CD3 antibody, each of STIM001 (hIgG1), STIM003 (hIgG1) and C398.4A showed a dose-dependent trend to increase expression of IFNγ, TNFα and, to a lesser degree, IL-2. Data from primary T cells treated with anti-ICOS antibodies under conditions of TCR engagement are shown inFIG.19. Although marked increases in cytokine expression were observed for each of STIM001, STIM003 and C389.4A relative to their respective isotype controls, the difference did not reach statistical significance in this assay. Further replicates of the assay with responsive primary T cells from more donors would be expected to generate statistically significant results. Example 10a: ADCC Assay STIM001 and STIM003 potential to kill via ADCC was tested in the Delfia BATDA cytotoxicity assay (Perkin Elmer) using human primary NK cells as effector and ICOS high MJ cell line (ATCC, CRL-8294) as target cells. MJ cells are human CD4 T-lymphocyte cells that express high levels of ICOS protein. This method is based on loading target cells with an acetoxymethyl ester of fluorescence enhancing ligand (BATDA) which quickly penetrates the cell membrane. Within the cell the ester bonds are hydrolysed to form a hydrophilic ligand (TDA) which no longer passes the membrane. After cytolysis the ligand is released and can be detected by addition of Europium which forms with the BATDA a highly fluorescent and stable chelate (EuTDA). The measured signal correlates directly with the amount of lysed cells. Materials and Methods Target Cell Labelling: According to the manufacturer's instructions, MJ cells were resuspended at 1×106/mL in assay media (RPMI+10% ultra-low IgG FBS, from Gibco) and loaded with 5 μl/mL of Delfia BATDA reagent (Perkin Elmer) for 30 min at 37° C. MJ cells were then washed 3 times with 50 mL PBS (300 g for 5 min) and resuspended at 8×105/ml in assay media supplemented with 2 mM Probenecid (from Life technologies) to reduce BATDA spontaneous release from the cells. ICOS Ab Dilution: STIM001, STIM003 and their isotype control were 1:4 serially diluted in assay media +2 mM Probenecid to give final 4× antibody concentrations across a range down to 80 pg/mL. NK-Cell Isolation and Culture: PBMC from leukocyte cones were obtained as described in T-cell activation assay 1. NK-cell were negatively isolated from this PBMC using the EasySep Human NK Cell Isolation Kit (from Stemcell Technologies) and resuspended at 4×106/ml in R10 media +2 mM Probenecid. NK cell purity was checked to be above 90% by staining for CD3−/CD56+. 50 μl of diluted Ab, 50 μl of BATDA loaded MJ cells, 50 μl of NK cells and 50 μl of assay media+2 mM Probenecid (final volume of 200 μl/well) were added in each well to give a final Ab concentration across a range down to 20 pg/mL and an effector: target ratio of 5:1. Wells containing MJ cells only or MJ cells+delfia lysis buffer (Perkin Elmer) are used to determine spontaneous and 100% BATDA release. The assay was run at 37° C., 5% CO2for 2 hrs before transferring 50 μl of cell-free supernatant into a DELFIA Microtitration Plates (Perkin Elmer). 200 μl of Delfia Europium solution (Perkin Elmer) was added to the supernatants and incubated for 15 min at Room Temperature. Fluorescent signal was then quantified with EnVision Multilabel Reader (PerkinElmer). Specific release induced by STIM001 and STIM003 was calculated according to the kit instructions. This experiment was repeated with NK-cells from independent donors and 3 technical replicates were included for each assay condition. Results Anti-ICOS antibodies STIM001 (hIgG1) and STIM003 (hIgG1) kill ICOS positive human MJ cells in a primary NK dependent ADCC assay (2 hour time point). See alsoFIG.6a. Sub-Nanomolar EC50 were achieved in this assay for both molecules tested. TABLE E10-1EC50 (Molar unit) of STIM001 in the NK primary cellsADCC assay from 2 donors (2 hour time point).EC50Donor 1Donor 2STIM0011.21e−105.29e−10 Example 10b: ADCC Assay with MJ Target Cells The experiment was performed according to the Materials and Methods set out in Example 10a. STIM001, STIM003 and isotype control were 1:4 serially diluted in assay media+2 mM Probenecid to give final 4× antibody concentrations ranging from 40 μg/mL to 80 pg/mL. 50 μl of diluted Ab, 50 μl of BATDA loaded MJ cells, 50 μl of NK cells and 50 μl of assay media+2 mM Probenecid (final volume of 200 μl/well) were added in each well to give a final Ab concentration ranging from 10 μg/mL to 20 pg/mL and an effector: target ratio of 5:1. Results are shown inFIG.6(b-d) and in the table below. STIM001 (hIgG1) and STIM003 (hIgG1) killed ICOS positive human MJ cells in the primary NK dependent ADCC assay, measured at the two hour time point. TABLE E10-2EC50 (Molar unit) of STIM001 and STIM003 in the NK primarycell ADCC assay from 3 donors (2 hour time point).EC50Donor 1Donor 2Donor 3STIM0011.21e−10 (0.121 nM)5.29e−10 (0.529 nM)2.92e−09(2.92 nM)STIM0032.33e−12 (2.33 pM)3.58-e−11 (35.8 pM)1.01e−10(0.101 nM) Example 10c: ADCC Assay with ICOS-Transfected CCRF-CEM Target Cells STIM001 and STIM003 hIgG1 potential to kill via ADCC was further tested in the Delfia BATDA cytotoxicity assay (Perkin Elmer) using human primary NK cells as effector and ICOS-transfected CCRF-CEM cells (ATCC, CRL-119) as target cells. CCRF-CEM is a human T lymphoblast line, originating from peripheral blood from a patient with acute lymphoblastic leukaemia. Antibody-mediated killing of CCRF-CEM cells was confirmed for both STIM001 and STIM003 in this assay. Materials and Methods Materials and Methods were as set out in Example 10a, but using CCRF-CEM cells obtained from ATCC (ATCC CCL-119) rather than MJ cells as the target cells, and using an incubation time of 4 hours. CCRF-CEM cells were transfected with ICOS. A synthetic string DNA encoding full length human ICOS (with signal peptide, as shown in the appended sequence listing), codon-optimised for mammalian expression, was cloned into an expression vector under control of the CMV promoter and flanked by 3′ and 5′ piggyBac specific terminal repeat sequences facilitating stable integration into the cell genome (see [40]). The expression vector contained a puromycin selection cassette to facilitate stable cell line generation. The human ICOS expression plasmid was co-transfected with a plasmid encoding piggyBac transposase into CEM CCRF cells by electroporation. 24 hours after transfection, the media was supplemented with puromycin and grown for at least two weeks to select stable cell lines, with media being exchanged every 3-4 days. The expression of human ICOS was assessed by flow cytometry using an anti-human ICOS-PE conjugated antibody (eBioscience). Complete CEM media was made up of Advanced RPMI Medium containing 10% (v/v) FBS and 2 mM Glutamax. STIM001 (hIgG1), STIM003 (hIgG1) and an isotype control antibody (HC IgG1) were serially diluted in assay media to give final 4× antibody concentrations ranging from 20 μg/mL to 80 pg/mL. 50 μl of diluted Ab, 50 μl of BATDA loaded ICOS-transfected CEM cells, 50 μl of NK cells and 50 μl of assay media (final volume of 200 μl/well) were added in each well to give a final Ab concentration ranging from 5 μg/mL to 20 pg/mL and an effector: target ratio of 5:1. Results STIM001 (hIgG1) and STIM003 (hIgG1) killed ICOS-transfected CCRF-CEM cells in the primary NK dependent ADCC assay, measured at the four hour time point. Results are shown inFIG.6(e-g) and in the table below. TABLE E10-3EC50 (Molar unit) of STIM001 and STIM003 in the NK primarycell ADCC assay from 3 donors (4 hour time point).EC50Donor 4Donor 5Donor 6STIM0013.92e−123.95e−12 (3.95 pM)3.75e−12 (3.75 pM)(3.92 pM)STIM003Approx 3 pM*8.95e−13 (0.895 pM)1.03e−12 (1.03 pM)*Value estimated from incomplete curve. Example 11a: CT26 Syngeneic Model Improved anti-tumour in vivo efficacy was shown in a CT26 syngeneic model by combining anti-ICOS (STIM001 mIgG2a, effector enable) with anti-PDL1 (10F9G2). Materials and Methods Efficacy studies were performed in Balb/c mice using the sub-cutaneous CT26 colon carcinoma model (ATCC, CRL-2638). This model is poorly sensitive to PD1/PDL1 blockade and only tumour growth delay (no stable disease or cure) is usually observed in response to 10F9.G2 (anti-PDL1) and RMT1-14 (anti-PD1) monotherapies. Therefore this model constitutes a relevant model for looking at anti-PD1, anti-PDL1 intrinsic resistance for combination studies. All in vivo experiments were performed in accordance with the UK Animal (Scientific Procedures) Act 1986 and the EU Directive 86/609, under a UK Home Office Project Licence and approved by the Babraham Institute Animal Welfare and Ethical Review Body. Balb/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 1×105CT26 cells (passage number below P20) were subcutaneously injected into the left flanks of mice. Unless stated otherwise, treatment were initiated at day 6 post tumour cells injection. The CT26 cells were passaged in vitro by using Accutase (Sigma), washed twice in PBS and resuspended in RPMI supplemented with 10% fetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection. For in vivo studies STIM001 anti-ICOS agonist (cross reactive to mouse ICOS protein) was reformatted as mouse IgG1 and mouse IgG2a to test the as effector function null and as effector function enable, respectively. The Anti-PDL1 was sourced from Biolegend (Cat. no: 124325). The hybrid controls were generated in Kymab (mIgG2a isotype) or from commercial source (hamster isotype HTK888, Biolegend (Part No92257, Lot B215504)). All antibodies were dosed intraperitoneal (IP) at 10 mg/kg (1 mg/ml in 0.9% saline) three times a week from day 6 (dosing for 2 weeks day 6-18) as monotherapies or by combining anti-PDL1 with anti-ICOS antibodies. Animal weight and tumour volume were measured 3 times per week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3or, in rare case, when incidence of tumour ulceration was observed (welfare). The experiment was stopped at day 50. The human endpoint survival statistics were calculated using the Kaplan-Meier method with Prism. This approach was used to determine if specific treatments were associated with improved survival. TABLE E11-1Treatment groupsNumberGroupsof animalsTreatments (T.I.W, IP from day 6)11010 mg/kg mIgG2a and 10 mg/kg IgG isotypeControl (HTK888)21010 mg/kg Anti-ICOS STIM001 mIgG131010 mg/kg Anti-ICOS STIM001 mIgG2a41010 mg/kg Anti-PD-L1 (10F9.G2)51010 mg/kg anti-PD-L1 plus 10 mg/kg Anti-ICOSSTIM001 mIgG161010 mg/kg anti-PD-L1 plus 10 mg/kg Anti-ICOSSTIM001 mIgG2a Results As shown inFIG.7,FIG.8andFIG.9, ICOS agonists can delay disease progression and cure a proportion of animals from the CT-26 subcutaneous tumours either as a monotherapy or in combination with anti-PDL1. Anti-PDL1 monotherapy induced tumour growth delay but no stable disease or curative potential was observed. The combination was more effective at treating the tumours than the anti-ICOS monotherapies. This study also highlighted that STIM001 in the mouse IgG2a format (effector function enable) was more potent than the mouse IgG1 (effector null) format at triggering an anti-tumour response in this model. Example 11b: Strong Anti-Tumour Efficacy In Vivo in CT26 Syngeneic Model for Combination of Anti-ICOS mIgG2a with Anti-PDL1 mIgG2a An in vivo combination study was performed with STIM001 with a mouse cross reactive anti-human PDL1 antibody designated AbW. For this in vivo work, STIM001 was reformatted as mouse IgG1 and mouse IgG2a to compare its efficacy with low effector function or as effector function enabled molecule, respectively. The anti-PDL1 AbW was generated in the same formats (mouse IgG1 and mouse IgG2A). The efficacy studies were performed in Balb/c mice using the sub-cutaneous CT26 colon carcinoma model (ATCC, CRL-2638). Balb/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 1×10E5 CT26 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. Unless stated otherwise, treatment were initiated at day 6 post tumour cells injection. The CT26 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection. STIM001 and anti-PDL1 antibodies were dosed concurrently in combinations intraperitoneal (IP) at 200 μg each (1 mg/ml in 0.9% saline) three times a week from day 6 (dosing for 2 weeks between day 6-17) post tumour cell implantation. Tumour growth was monitored and compared to tumours of animals treated with a mixture of isotype control antibodies (mIgG1 and mIgG2A). Animal weight and tumour volume were measured 3 times a week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3 or, in rare case, when incidence of tumour ulceration was observed (welfare). The experiment was stopped at day 60. The human endpoint survival statistics were calculated using the Kaplan-Meier method with Prism. This approach was used to determine if specific treatments were associated with improved survival. TABLE E11-2Treatment groups for STIM001 2 × 2 combinationsNumber ofGroupanimalsTreatment regimen (3 time a week for 2 weeks)110mIgG2a + mIgG1 isotypes 200 μg each210Anti-ICOS mIgG1 STIM001 + Anti-PD-L1 mIGg1(AbW) 200 μg each310Anti-ICOS mIgG2a STIM001 + Anti-PD-L1mIGg2a (AbW) 200 μg each410Anti-ICOS mIgG2a STIM001 + Anti-PD-L1 mIGg1(AbW) 200 μg each510Anti-ICOS mIgG1 STIM001 + Anti-PD-L1 mIGg2a(AbW) 200 μg each Results are shown inFIG.10. All antibody combinations delayed tumour growth and extended the survival (time to reach human endpoint) of treated animals when compared to isotype control-treated animals. Interestingly, when combined with anti-PDL1 (independently of its format, mIgG1 or mIgG2a), STIM001 mIgG2a antibody was more effective at inhibiting tumour growth than STIM001 in the mIgG1 format. These data suggest the advantage of an anti-ICOS antibody having effector function to maximize anti-tumour efficacy. Notably, STIM001 mIgG2a in combination with aPD-L1 mIgG2a demonstrated the strongest anti-tumour efficacy and improved survival (90% of animals to show response and 60% were cured from the disease at day 60). Similarly, STIM003 mIgG1 and mIgG2a were tested as monotherapy or in combination with anti-PDL1 (AbW) mIgG2a in the same CT26 tumour models. STIM003 and anti-PDL1 antibodies were dosed in animals as monotherapy or in combination by intraperitoneal injection (IP) of 200 μg of antibodies each (1 mg/ml in 0.9% saline) three times a week from day 6 (dosing for 2 weeks between day 6-17) post tumour cell implantation. In this experiment tumour sizes were monitored for 41 days. The human endpoint survival statistics were calculated using the Kaplan-Meier method with Prism. This approach was used to determine if specific treatments were associated with improved survival. TABLE E11-3Treatment groups for STIM003 combination with anti-PDL1 AbW IgG2aNumberTreatment regimenGroupof animals(3 times a week for 2 weeks from day 6)110mIgG2a + mIgG1 isotypes control 200 μg each210Anti-PD-L1 mIgG2a (AbW) 200 μg310STIM003 mIgG1 200 μg410STIM003 mIgG2a 200 μg510STIM003 mIgG1 + Anti-PD-L1 mIGg2a (AbW)200 μg each610STIM003 mIgG2a + Anti-PD-L1 mIGg2a (AbW)200 μg each Results are shown inFIG.11. Monotherapies using aPDL1 (AbW) and STIM003 mIgG2a demonstrated mild anti-tumour activity (one animal was cured of the disease in each group). Combinations of STIM003 mIgG1 or mIgG2a with aPDL1 (AbW) mIgG2a showed strong anti-tumour efficacy. Interestingly, by day 41, when combined with aPDL1 mIgG2a, STIM003 mIgG2a was more potent at inhibiting tumour growth than STIM003 mIgG1 (60% vs 30% of animals cured of the disease, respectively). The data further highlighted the advantage of an effector format for anti-ICOS antibodies to maximize anti-tumour efficacy. Altogether, these data demonstrate that combination of an anti-ICOS antibody STIM001 or STIM003 with anti-PDL1 results in the strongest anti-tumour response when both antibodies have an effector enabled function. Suitable corresponding human antibody isotypes would include human IgG1, optionally with further enhanced effector function e.g., afucosylated IgG1. Kaplan Meier curves for mice treated with the combination of anti-PDL1 mIgG2a and STIM003 mIgG2a and for each agent individually are shown inFIG.29. Example 11c: Single Dose of STIM003 Antibody Resets the Tumour Microenvironment (TME) and Results in Strong Anti-Tumour Efficacy in Combination with Continuous Anti-PD-L1 Dosing This study compared single vs multiple dosing of STIM003 mIgG2A together with multiple dosing of anti-PDL1 antibody (AbW). The data indicate that a single dose of anti-ICOS antibody could alter the tumour microenvironment so as to allow an anti-PD-L1 antibody to exert a greater effect. This can be envisaged as a “resetting” of the TME by the anti-ICOS antibody. As before, these efficacy studies were performed in Balb/c mice using the sub-cutaneous CT26 colon carcinoma model (ATCC, CRL-2638). Balb/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 1×10E5 CT26 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. Unless stated otherwise, treatments were initiated at day 6 post tumour cells injection. The CT26 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection. Treatment groups are shown in Table E11-4. STIM003 and anti-PDL1 antibodies were dosed intraperitoneal (IP) at 10 mg/kg (1 mg/ml in 0.9% saline). Treatments were initiated from day 6 post tumour cell implantation. Tumour growth was monitored and compared with tumours of animals treated with saline. Animal weight and tumour volume were measured 3 times a week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3or, in rare case, when incidence of tumour ulceration was observed (welfare). The experiment was stopped at day 55. Data are shown inFIG.34. Concurrent dosing of STIM003 and anti-PDL1 for 6 doses from day 6 resulted in a strong anti-tumour efficacy in the CT26 model with 5/8 animals being tumour free at the end of the study (day 55). Interestingly, similar anti-tumour efficacy was achieved with a single dose of STIM003 followed by multiple dose of anti-PDL1 as monotherapy. When combined with anti-PD-L1 mIgG2a, similar overall efficacy was observed between dosing STIM003 once (C) vs dosing 6 times (B). When compared with saline treated group (A) where only one animal had a spontaneous tumour rejection (rare in this model), the groups treated with combined drugs had full tumour rejection in 62.5% of the animals by the end of the experiment (day 55). The data suggest that the STIM003 antibody could be used to reset the tumour microenvironment and that the antibody allows immune-checkpoint resistant tumours to become sensitive to anti-PDL1. As previously shown (Example 11b), the CT26 tumour cell line is not strongly responsive to anti-PDL1 monotherapy. It appears that STIM003 causes changes that facilitate anti-tumour activity of the anti-PDL1 therapy. Example 12: Antibody Sequence Analysis Framework regions of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 were compared with human germline gene segments to identify the closest match. See Table E12-1 and Table E12-2. TABLE E12-1Heavy chain germline gene segments of anti-ICOS AbsHeavy chainVDJSTIM001IGHV1-18*01IGHD6-19*01IGHJ6*02STIM002IGHV1-18*01IGHD3-10*01IGHJ6*02STIM002-BIGHV1-18*01IGHD3-10*01IGHJ6*02STIM003IGHV3-20*d01IGHD3-10*01IGHJ4*02STIM004IGHV3-20*d01IGHD3-10*01IGHJ4*02STIM005IGHV1-18*01IGHD3-9*01IGHJ3*02STIM006IGHV3-11*01IGHD3-10*01IGHJ6*02STIM007IGHV2-5*10IGHD3-10*01IGHJ6*02STIM008IGHV2-5*10IGHD3-10*01IGHJ6*02STIM009IGHV3-11*01IGHD3-9*01IGHJ6*02 TABLE E12-2Kappa light chain germline gene segments of anti-ICOS AbsLight chainVJSTIM001IGKV2-28*01IGKJ4*01STIM002IGKV2-28*01IGKJ2*04STIM002-BIGKV2-28*01IGKJ2*04STIM003IGKV3-20*01IGKJ3*01STIM004IGKV3-20*01IGKJ3*01STIM005IGKV1D-39*01IGKJ1*01STIM006IGKV2-28*01IGKJ2*04STIM007IGKV3-11*01IGKJ4*01STIM008IGKV3-11*01IGKJ4*01STIM009IGKV2-28*01IGKJ1*01 Additional antibody sequences were obtained by next generation sequencing of PCR-amplified antibody DNA from further ICOS-specific cells that were sorted from the immunised mice as described in Example 3. This identified a number of antibodies that could be grouped into clusters with STIM001, STIM002 or STIM003 based their heavy and light chain v and j gene segments and CDR3 length. CL-61091 clustered with STIM001; CL-64536, CL-64837, CL-64841 and CL-64912 clustered with STIM002; and CL-71642 and CL-74570 clustered with STIM003. Sequence alignments of the antibody VH and VL domains are shown inFIGS.35to37. TABLE E12-3Antibodies clustered by sequence.ANTIBODIESVH_V_ GENEVH_J_GENEVH_CDR3_NT_LENGTHSTIM001, CL-610911-18642STIM002, CL-64536,1-18651CL-64837,CL-64841,CL-64912STIM003, CL-71642,3-20451CL-74570STI M 0043-20451STI M 0051-18351STI M 0063-11663STIM007, STIM0082-5648STI M 0093-11660ANTIBODIESVL_V_GENEVL_J_GENEVL_CDR3_NT_LENGTHSTIM001, CL-610912-28427STIM002, CL-64536,2-28227CL-64837,CL-64841,CL-64912STIM003, CL-71642,3-20327CL-74570STI M 0043-20324STI M 0051D-39124STI M 0062-28230STIM007, STIM0083-11427STI M 0092-28127 Example 13: Agonism of ICOS-Expressing MJ Cells by Bead-Bound Antibody Antibodies STIM001, STIM002 and STIM003, the anti-ICOS antibody C398.4A, and ICOS ligand (ICOSL-Fc), were each covalently coupled to beads and assessed for their ability to induce expression of the cytokine IFN-γ from MJ cells grown in culture. Human IgG1 and Clone C398.4A isotype controls coupled to beads were assessed in parallel. Data are shown inFIG.12and Table E13 below. Each of the anti-ICOS antibodies demonstrated agonism in this assay, stimulating MJ cells as determined by IFN-γ quantification significantly above that observed by their cognate isotype controls within the dynamic range of the assay. STIM003 and Clone C398.4A produced lower top asymptote values (95% CI: 3.79 to 5.13 and 3.07 to 4.22, respectively) but more potent Log EC50 values (95% CI: −9.40 to −9.11 and −9.56 to −9.23, respectively) compared with STIM001 (Top 95% CI: 7.21 to 8.88 and Log EC50 95% CI: −8.82 to −8.63) and STIM002 (Top 95% CI: 5.38 to 6.95 and Log EC50 95% CI: −9.00 to −8.74). Because incomplete curves (Top out of dynamic range of assay) were produced for ICOSL-Fc and Clone C398.4A isotype control, the fitted Top and Log EC50 values were not treated as reliable. Human IgG1 Hybrid Control produced a complete curve, however the area under the curve was not significantly different from 0 and it was therefore not deemed to be an agonist. TABLE E13Summary table of bead bound MJ cell in vitro activation assay.Human IgG1Best-fitHybridCloneClonevaluesSTIM001-STIM002-STIM003-Control-C398.4A-C398.4A95% CIbeadsbeadsbeadsbeadsbeadsIC-beadsICOSL-Fc-beadsBottom−0.24 to 0.36−0.38 to 0.56−0.39 to 0.34−0.26 to 0.06−0.37 to 0.42−0.20 to −0.09−0.62 to 0.49Top7.21 to 8.885.38 to 6.953.79 to 5.13−0.04 to 0.203.07 to 4.22NA−45.66 to 65.37LogEC50−8.82 to −8.63−9.00 to −8.74−9.40 to −9.11−10.43 to −8.60−9.56 to −9.23NA−12.80 to −2.37HillSlope0.89 to 1.380.82 to 1.850.69 to 1.56−3.47 to 6.610.64 to 1.90NA−0.38 to 1.88NA—not applicable. MJ Cell Activation Assay Materials and Methods—Bead-Bound Coupling Proteins of Interest to Magnetic Particles Anti-ICOS antibodies, control antibodies, and ICOSL-Fc, were coupled to beads as follows. Dynabeads M-450 Tosylactivated (Invitrogen; approximately 2×10{circumflex over ( )}8 beads/sample) were incubated with 100 μg of each protein sample overnight at room temperature with agitation. Beads were washed three times with DPBS (Gibco) and incubated with 1M Tris-HCl, pH 8.0 (UltraPure™, Gibco) for 1 hr at room temperature with agitation to block the uncoupled reactive sites. Beads were washed again three times with DPBS and finally resuspended in 0.5 ml of DPBS/sample. The quantity of each protein of interest on the beads was then determined as follows. Black flat bottom, high binding ELISA plates (Greiner) were coated with Anti-human IgG (Southern Biotech) or Anti-Armenian Hamster IgG (Jackson ImmunoResearch) capture antibody at 4 μg/ml in DPBS, 50 μl/well, overnight at 4° C. Wells were then washed three times with DPBS+0.1% Tween, 200 μl/well and blocked with 200 μl/well of DPBS+1% BSA for 1 hr at RT. Wells were washed again three times with DPBS+0.1% Tween. Stock protein samples were quantified spectrometrically and beads were counted on a cell counter. Dilution series of protein samples and beads were then incubated in the plates at 50 μl/well for 1 hr at RT before washing again three times with DPBS+0.1% Tween. 50 μl/well of either biotinylated anti-armenian hamster antibody or anti-human IgG—europium in DPBS+0.1% BSA were added and incubated for 1 hr at RT. In the case of addition of biotinylated anti-Armenian hamster antibody C398.4A another incubation step with 50 μl/well of streptavidin-europium (Perkin Elmer) diluted 1:500 in Assay buffer (Perkin Elmer) were added and incubated for 1 hr at RT. The wells were washed three times with 200 μl/well of TBS+0.1% Tween before developing the assay by adding 50 μl/well of Delfia enhancement solution (Perkin Elmer), incubating for 10 mins at RT and measuring the fluorescence emitted at 615 nm on the EnVision Multilabel Plate Reader. The quantity of protein on the beads was determined by extrapolating values from the signals obtained from known concentrations of uncoupled protein samples. MJ Cell In Vitro Activation Assay—Bead Bound MJ [G11] cell line (ATCC CRL-8294) was grown in IMDM (Gibco or ATCC) supplemented with 20% heat inactivated FBS. Cells were counted and 15000 cells/well (50 μl/well) of cell suspension was added to 96-well clear flat bottom polystyrene sterile TC-treated microplates. Beads were counted and serial 1:2 dilutions ranging from 1.5×10{circumflex over ( )}6 beads/well to approximately 5860 beads/well (50 μl/well) were added to the cells in duplicate or in triplicate. To account for background several wells of the plate contained MJ cells only (100 μl/well). The cells and beads were co-cultured in the plates for 3 days at 37° C. and 5% CO2after which supernatants were harvested by centrifugation and collected for IFN-γ content determination. Measuring IFN-γ Levels The IFN-γ content in each well was determined using a modification of the Human IFN-gamma DuoSet ELISA kit (R&D systems). Capture antibody (50 μl/well) was coated overnight at 4 μg/ml in DPBS on black flat bottom, high binding plates (Greiner). The wells were washed three times with 200 μl/well of DPBS+0.1% Tween. The wells were blocked with 200 μl/well of 1% BSA in DPBS (w/v), washed three times with 200 μl/well of DPBS+0.1% Tween and then 50 μl/well of either the IFN-γ standard solutions in RPMI or neat cell supernatant were added to each well and incubated for 1 hr at RT. The wells were washed three times with 200 μl/well of DPBS+0.1% Tween before adding 50 μl/well of biotinylated detection antibody at 200 ng/ml in DPBS+0.1% BSA and incubated for 1 hr at RT. The wells were washed three times with 200 μl/well of DPBS+0.1% Tween before adding 50 μl/well of streptavidin-europium (Perkin Elmer) diluted 1:500 in Assay buffer (Perkin Elmer) and incubated for 1 hr at RT. The wells were washed three times with 200 μl/well of TBS+0.1% Tween before developing the assay by adding 50 μl/well of Delfia enhancement solution (Perkin Elmer) and incubating for 10 mins at RT and measuring the fluorescence emitted at 615 nm on the EnVision Multilabel Plate Reader. Data Analysis IFN-γ values for each well were interpolated from the standard curve and the average background levels from cell-only wells were subtracted. The background corrected values were then used in GraphPad prism to fit a 4-parameter log-logistic concentration response curve. Example 14: Agonism of ICOS-Expressing MJ Cells by Plate-Bound Antibody An alternative assay for agonism of ICOS-expressing T cells uses antibodies in a plate-bound format. MJ Cell Activation Assay Materials and Methods—Plate-Bound Antibody coating: 96-well, sterile, flat, high binding plates (Costar) were coated overnight at 4° C. with 100 μl/well of serial 1:2 dilutions of proteins of interest (anti-ICOS antibodies, control antibodies, and ICOSL-Fc) in DPBS (Gibco) ranging from 10 μg/ml to 0.02 μg/ml of in duplicate or in triplicate. To account for background several wells of the plate were coated with DPBS only. Plates were then washed three times with 200 μl/well of DPBS before the addition of cells. Cell stimulation: MJ [G11] cell line (ATCC CRL-8294) was grown in IMDM (Gibco or ATCC) supplemented with 20% heat inactivated FBS. The cells were counted and 15000 cells/well (100 μl/well) of cell suspension were added to the protein coated plates. Cells were cultured in the plates for 3 days at 37° C. and 5% CO2. Cells were separated from the media by centrifugation and the supernatants collected for IFN-γ content determination. Measurement of IFNγ levels and data analysis was as described in Example 13. Results Results are shown inFIG.13and in Table E14-1 below. In summary, STIM001, STIM002 and STIM003 all showed potent agonism as measured by IFN-γ secretion with similar Log EC50 values (Log EC50 95% CI: −7.76 to −7.64, −7.79 to −7.70 and −7.82 to −7.73, respectively) and Top values (Top 95% CI: 2.06 to 2.54, 2.44 to 2.93 and 2.01 to 2.41, respectively). Clone C398.4A exhibited a similar Log EC50 value (Log EC50 95% CI: −7.78 to −7.60) but lower Top value (Top 95% CI: 1.22 to 1.63) than STIM001 to STIM003. STIM004 also showed agonism in this assay, but was less potent, reaching a moderate Top value (Top 95% CI: 0.16 to 0.82) with a similar Log EC50 value (Log EC50 95% CI: −7.91 to −7.21). STIM001, STIM002 and STIM003 were stronger agonists than ICOSL-Fc (Log EC50 95% CI: −7.85 to −7.31 and Top 95% CI: 0.87 to 2.45). TABLE E14-1Summary of plate-bound MJ cell in vitro activation assay.Best-fitClonevaluesCloneC398.4A95% CISTIM001STIM002STIM003STIM004IgG1C398.4AICOSL-FcICBottom−0.03 to 0.13−0.08 to 0.11−0.10 to 0.07−0.06 to 0.07NA−0.03 to 0.11−0.16 to 0.09−0.07 to 0.04Top2.06 to 2.542.44 to 2.932.01 to 2.410.16 to 0.82NA1.22 to 1.630.87 to 2.450.05 to 0.29LogEC50−7.76 to −7.64−7.79 to −7.70−7.82 to −7.73−7.91 to −7.21NA−7.78 to −7.60−7.85 to −7.31NAHillSlope2.06 to 5.380.16 to 10.881.77 to 6.5−1.46 to 6.77NA1.24 to 8.200.26 to 3.97NAIgG1 = Human IgG1 hybrid control antibody. Example 15: Agonism of ICOS-Expressing MJ Cells by Antibody in Soluble Form In contrast to the assays described in Example 13 and Example 14, which used antibody arrayed on a solid surface, this assay determines whether antibody in soluble form acts as agonist of ICOS-expressing T cells. MJ Cell Activation Assay Materials and Methods—Soluble MJ [G11] cell line (ATCC CRL-8294) was grown in IMDM (Gibco or ATCC) supplemented with 20% heat inactivated FBS. Cells were counted and 15000 cells/well (50 μl/well) of cell suspension was added to 96-well clear flat bottom polystyrene sterile TC-treated microplates. Serial 1:2 dilutions of proteins of interest ranging from 10 μg/ml to 0.01953125 μg/ml either alone or with the addition of a cross-linking reagent (AffiniPure F(ab′)2 Fragment Goat Anti-Human IgG, Fc Fragment Specific; Jackson ImmunoResearch) were added to the cells in duplicate or in triplicate (50 μl/well). To account for background several wells of the plate contained MJ cells only (100 μl/well). The cells and beads were co-cultured in the plates for 3 days at 37° C. and 5% CO2after which supernatants were harvested by centrifugation and collected for IFN-γ content determination. Measurement of IFNγ levels and data analysis was as described in Example 13. Results STIM001 and STIM002 both showed significant soluble agonism as measured by IFN-γ secretion compared to Human IgG4.PE hybrid control. MAb cross-linking via Goat Anti-Human IgG Fc F(ab′)2 Fragment increased secreted IFN-γ levels even more. Example 16: Binding of Antibody to Activated T Cells A. Human ICOS Ability of anti-ICOS antibodies to recognise the ICOS extracellular domain in its native context on the surface of activated primary human T cells is confirmed in this assay. Pan T-cells (CD3 cells) were isolated and cultured for 3 days with CD3/CD28 dynabeads (Thermofisher) to induce ICOS expression on their surface. Surface staining of STIM001, STIM003 and the hIgG1 hybrid control (HC IgG1) was determined by two methods, namely detection following direct binding of pre-labelled antibodies (antibodies directly conjugated with AF647) or indirectly via the use of a secondary AF647-Goat anti-human Fc antibody. Stained cells were ran on the Attune and staining intensity was presented as Mean of fluorescence intensity (MFI). EC50 was determined using GraphPad Prism. Results are shown inFIG.14. Once activated, pan CD3 T cells were clearly stained by both STIM001 and STIM003 hIgG1. Notably, the saturation of STIM003 binding to activated T cells occurred at a lower concentration than that of STIM001, suggesting higher affinity of STIM003 to human ICOS. The EC50 of STIM003 was roughly 100× lower than that of STIM001 (0.148 nM vs 17 nM for the indirect binding assay). B. ICOS from Non-Human Primates Ability of anti-ICOS antibodies to recognise the ICOS extracellular domain in its native context on the surface of activated primary T cells from non-human primates (NHP) is confirmed in this assay. PBMC from whole blood of 2 Mauritian cynomolgus macaques (Wickham Laboratories) were isolated by gradient centrifugation and cultured for 3 days with CD2/CD3/CD28 MACSiBeads (Miltenyi) to induce ICOS expression on their surface. Surface staining of STIM001, STIM003 and the hIgG1 hybrid control (HC IgG1) was determined following direct binding of AF647 pre-labelled antibodies (from 80 μg to 8 pg/ml). Cells were also labelled with V450-CD3 to assess staining on T-cell subsets. Stained cells were run on Attune (Thermofisher) and staining intensity was presented as mean fluorescence intensity (MFI). EC50 was determined using GraphPad Prism. Results are shown inFIG.28. Once activated, T cells were clearly stained by both STIM001 and STIM003 hIgG1. As was observed with binding to human T cells, saturation of STIM003 binding to activated NHP T cells occurred at lower concentration than that of STIM001, indicating that STIM0003 has the higher affinity of these two antibodies ICOS. EC50 values for binding to NHP ICOS were similar to those obtained for binding to human ICOS. TABLE E16EC50 (Molar) calculated for antibody binding to ICOSon activated NHP T cellsPan T-cellsEC50Cynomolgus donor 1Cynomolgus donor 2NHPSTIM0012.224e−7not testedSTIM0034.581e−94.830e−9HumanSTIM0012.209e−71.207e−7STIM0032.293e−98.953e−10 Example 17: Analysis of T Cell Sub-Populations Among Tumour Infiltrating Lymphocytes and Peripheral T Cells A pharmacodynamics study revealed that anti-ICOS antibodies STIM001 and STIM003 in mIgG2a isotype significantly deplete TRegs, increase the percentage of CD4+ effector cells and increase the CD4+ effector/TReg ratio as well as the CD8+/TReg ratio within the tumour microenvironment (TME). The increased CD8+/TReg ratio and increased number of CD4+ effector cells within the TME may collectively contribute to the CT26 tumour clearance observed when these anti-ICOS antibodies were co-injected with anti-PDL1 antibody in the STIM001 & STIM003 efficacy study (Example 11). Method The pharmacodynamics study was performed in female Balb/c mice bearing CT-26 mouse colon carcinoma cells (ATCC, CRL-2638). Balb/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 1×10E5 CT-26 tumour cells (passage number: P8) were subcutaneously injected in the right flank. All CT-26 tumour bearing animals were assigned to 6 groups (Table E17-1) and individual mice were dosed twice (on Day 13 & Day 15 post tumour cell implantation) with 200 μg of antibody or saline. CD3+ T-cells from the CT-26 tumour bearing animals were analysed by FACS on day 16 post tumour cell implantation. TABLE E17-1Treatment groupsNumber ofTreatment regimen (Day 13 and Day 15 post tumourGroupanimalscell implantation)110Saline210Anti-ICOS (STIM001) mIgG1 200 μg each310Anti-ICOS (STIM001) mIgG2a 200 μg each410Anti-ICOS (STIM003) mIgG1 200 μg each510Anti-ICOS (STIM003) mIgG2a 200 μg each610Anti-CTLA-4 (9H10) 200 μg each Results Animals treated with STIM001 & STIM003 in the mIgG2a isotype showed a lower percentage of CD4+ CD3+ CD45+ cells at the tumour site when compared with saline treated group (FIG.15A), whereas STIM001 or STIM003 treatments had very marginal effect of the percentage of CD8+ CD3+ CD45+ cells at the tumour site (FIG.15B). The decrease in CD4+ T cells could be attributed to a profound decrease in the percentages of T-Regulatory cells in all the groups treated with STIM001 and STIM003 antibodies. Notably, animals treated with STIM001 and STIM003 in the mIgG2a isotype showed a dramatic reduction in T-Regs (CD4+ Foxp3+ CD25+) within the TME, whereas STIM001 & STIM003 in the mIgG1 isotype had only a modest effect on T-Reg content in TME. In addition, animals treated with STIM001 & STIM003 in the mIgG2a isotype had reduced T-Reg in the TME when compared with the animals treated with a commercial anti-CTLA-4 (9H10, Biolegend Cat#106208) antibody which is known to deplete T-Reg [42], but this result did not reach statistical significance (FIG.15C). The effect of STIM001 and STIM003 either in mIgG1 or mIgG2a isotypes on T-Reg compartment was more specific with tumour infiltrating lymphocytes (TILs). T-reg depletion was not observed in the periphery (as previously described for anti-CTLA4 [43]) (FIG.15D). The changes in T-Reg contents also resulted in a significant increase in the percentage of intra-tumoural CD4-effector cells (CD4+Foxp3− CD25−) (FIG.15E), similarly the ratio of CD4 effector/T-Reg and CD8/T-Reg ratio in the animals treated with STIM001 & STIM003 in the mIgG2a was also significantly increased within TME (FIG.15F&FIG.15G). Example 18: Effect of Anti-ICOS Antibody on Level of ICOS-Expressing T Cells in CT26 Tumour and Spleen Analysis was performed to quantify the percentage of immune cells within the tumour compared with the spleen, by analysis of total immune cells in the tumour and spleen tissues, following treatment with anti-ICOS antibody STIM001 or STIM003. STIM001 and STIM003 mIgG2a each caused a significant reduction in Treg within the tumour, but not in the spleen, indicating a tumour-selective effect. This depletion was selective for Tregs compared with other T cell subtypes. The results presented here assist in understanding the effects of the STIM antibodies on the immune contexture, and confirm that anti-ICOS antibodies with effector-function-enabled Fc regions can strongly deplete TRegs. Materials and Methods Mice bearing CT26 tumours were dosed twice with STIM001, STIM003, or anti-CTLA4 antibody (9H10). The anti-CTLA4 antibody was included as a positive control for Treg depletion, as anti-CTLA4 antibody had been previously shown to selectively reduce Tregs in tumours [43]. The immune contexture within the tumours and the spleen of treated animals was analysed by FACS following tissue disaggregation. Details of FACS antibodies used in this study are shown in Table E18. All FACS antibodies were used at a concentration recommended by the supplier. FACS data were acquired using Attune NxT flow cytometer and data were analysed using FlowJo software. TABLE E18FACS antibodies.MarkerSupplierCat. numberLot numberFluorophoreLive/deadLifeL-349591784156Fixable YellowtechnologiesCD45E-bioscience45-0451-82E08336-1636PerCp-Cy5.5CD3E-bioscience48-0032-824278794eFlour 450CD4E-bioscience11-0042-86E0084-1633FITCCD8E-bioscience12-0081-85E01039-1635PEFoxp3E-bioscience17-5773-824291991APCCD25E-bioscience47-0251-824277960APC eF 780ICOSE-bioscience25-9942-82E17665-103PE-CY7Fc/BlockE-bioscience16-0161-86E06357-1633— Results ICOS expression was determined in the CT26 tumours and in the spleen of tumour-bearing animals. We observed an increased percentage of tumour infiltrating immune cells expressing ICOS protein (FIG.20), indicating that immune cells in the tumours are more often positive for ICOS expression than immune cells in the periphery. TRegs in the tumour of untreated animals were nearly all (>90%) positive for ICOS expression, whereas CD8+ effector T cells in the tumour were not (approx. 60%). Comparing T cell subpopulations (again in untreated mice) in tumour with those in spleen, a significantly higher (p<0.0001) percentage of intratumoural Tregs were positive for ICOS compared with Tregs in spleen, and a significantly higher (p<0.001) percentage of intratumoural CD4+ Teff cells were positive for ICOS compared with CD4+ Teff cells in spleen. Also in the mice before treatment, the level of ICOS expression was much higher on immune cells in the microenvironment of CT26 tumours, when compared with immune cells in the spleen (FIG.21). ICOS expression was increased on the surface of all immune cell subsets analysed (CD8 T-Effector, CD4 T-Effector and CD4/FoxP3 TReg cells) in the tumour microenvironment. Note that although immune cells in the tumours and the spleen are both expressing ICOS, immune cells in the tumour are expressing significantly more ICOS (indicated by higher MFI,FIG.21) than cells in the spleen (indicated by lower MFI,FIG.21). Importantly, TRegs in the tumour are expressing the highest levels of ICOS, as previously reported [11]. CT26 tumour bearing animals were treated with 2 doses of antibody STIM001 or STIM003 and with an anti-CTLA-4 antibody. The STIM antibodies did not affect the overall percentage of CD45 positive cells (a marker for immune cells) in the tumours, when used in either mIgG1 or mIgG2 format. Nor did treatment with these antibodies significantly affect the percentage of CD8 effector T cells in CT26 tumours (FIG.22). Treatment with STIM001 in mIgG2a isotype led to a significant (p<0.05) depletion of CD4+ effector T cells, but none of STIM001 mIgG1, STIM003 mIgG1 and STIM003 mIgG2a affected the percentage of CD4+ effector T cells. Anti-CTLA-4 treatment produced a notable (albeit not statistically significant) increase in CD45+ cells and CD8+ effector T cells in the TME, but did not affect CD4+ effector T cells (FIG.22). The STIM antibodies significantly affected regulatory T cells in the tumour. As shown inFIG.23, STIM001 mIgG2a and STIM003 mIgG2a significantly and selectively depleted TRegs (which are high for ICOS expression) in the tumour microenvironment. Interestingly, the anti-CTLA4 antibody which, despite being included as a positive control for TReg depletion in this experiment, was less effective than the STIM mIgG2a antibodies at depleting TRegs. This selective depletion of TRegs resulted in an increase in the ratio CD8 effector T cells to TRegs in the tumour, and an increase in the ratio of CD4 effector T cells to TRegs in the tumour, both of which should favour an anti-tumour immune response. Ratio data are shown inFIG.24. In contrast to the depletion of intratumoural Tregs by STIM001 mIgG2a and STIM003 mIgG2a, no such effect was observed on Tregs in spleen (FIG.25,FIG.26,FIG.27). This indicates that the effects of the anti-ICOS antibodies on depletion of Tregs depletion was not systemic in all the tissues. Such selectivity could be advantageous for therapeutic anti-ICOS antibodies in treating tumours in patients, as preferential depletion of Tregs in the tumour microenvironment could selectively relieve suppression of anti-tumour effector T cells, while minimising side effects at other sites in the body. The anti-ICOS antibodies may thus promote an anti-tumour response in the immune system with a low risk of undesirable activation of a wider T cell response that could cause treatment-limiting autoimmune adverse events. Example 19: Antibody Stability STIM003 human IgG1 was tested for stability during storage, freeze/thawing and purification, and was found to maintain its stability under all tested conditions. % aggregation was determined by HPLC. There was no significant change in the percentage of monomer (>99%) after 3 months storage at 4° C. in buffer (10 mM sodium phosphate, 40 mM sodium chloride, pH 7.0). On thermal denaturation testing, all samples (n=15) had the same Tm (no significant difference between aliquots) and had comparable thermal denaturation curves. There was no significant change in Tm (≈70.3° C.), the percentage of monomer or the profile on SDS-PAGE after 3 cycles of freeze and thaw. There was no significant change in Tm (≈70.3° C.), the percentage of monomer or the profile on SDS-PAGE after 7 days' storage at room temperature. There was 90% recovery post protein A purification. Example 20: Monotherapeutic Efficacy of Anti-ICOS Ab Against A20 Tumour Growth in Mouse Anti-ICOS antibodies STIM001 mIgG2a and STIM003 mIgG2a each showed strong anti-tumour efficacy when used as monotherapies in vivo in a mouse A20 syngeneic model. Materials and Methods The efficacy study was performed in BALB/c mice using the sub-cutaneous A20 reticulum cell sarcoma model (ATCC, TIB-208). The A20 cell line is a BALB/c B cell lymphoma line derived from a spontaneous reticulum cell neoplasm found in an old BALB/cAnN mouse. This cell line has been reported to be positive for ICOSL. BALB/c mice were supplied by Charles River UK>18 gram and housed under specific pathogen-free conditions. A total of 5×10e5 A20 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. The A20 cells were passaged in vitro washed twice in PBS and re-suspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 85% at the time of tumour cell injection. Unless stated otherwise, antibody or isotype administration was initiated from day 8 post tumour cells injection. STIM001 and STIM003 anti-ICOS antibodies were generated in mouse IgG2a isotype format. The mouse cross reactive anti-PD-L1 antibody (AbW) was also generated in the same isotype format (mouse IgG2a). STIM001, STIM003 and anti-PD-L1 antibodies were dosed intraperitoneally (IP) at 200 μg of each antibody twice a week starting from day 8 (dosing for 3 weeks between day 8-29) post tumour cell implantation. Animal weights and tumour volume were measured 3 times a week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on study until their tumour reached an average diameter of 12 mm. The experiment was stopped at day 43 post tumour cell implantation. Tumour growth was monitored and compared with tumours of animals treated with isotype control (mIgG2a) antibody. Treatment groups are shown in Table E20 below. TABLE E20Treatment groups for A20 study.NumberTreatmentGroupof animalsregimen (twice per week for 3 weeks 7 doses)18mIgG2a isotype control 200 μg/mouse/each dose28Anti-PD-L1 mIGg2a (AbW) 200 μg/mouse/eachdose38Anti-ICOS mIgG2a STIM001 200 μg/mouse eachdose48Anti-ICOS mIgG2a STIM003 200 μg/mouse/eachdose Results Monotherapy administration of either STIM001 or STIM003 (mIgG2a) in the A20 tumour model produced a complete anti-tumour response (FIG.32,FIG.33). All the animals administered with either STIM001 or STIM003 were cured of the disease. This contrasts with the results in the isotype control and PD-L1 mIgG2a groups (FIG.30,FIG.31). In rare cases, regression of tumours was observed for some animals in the isotype control (spontaneous regression) and anti-PDL-1 groups, but treatment with anti-ICOS antibody produced significantly greater efficacy. At the end of the study, 3 of 8 control animals and 2 of 8 anti-PDL-1 treated animals had no tumour. However, all animals treated with either STIM001 or STIM003 were tumour free at the end of the study (8 of 8 mice in both groups), representing 100% cure using the anti-ICOS antibodies. Example 21: Strong Anti-Tumour Efficacy In Vivo in the J558 Myeloma Syngeneic Model for Combination of Anti-ICOS Antibody and Anti-PD-L1 Antibody Anti-ICOS antibody STIM003 mIgG2a and anti-PD-L1 antibody AbW mIgG2a were administered individually and in combination in the J558 tumour model. This is a syngeneic mouse model of myeloma. The anti-ICOS antibody was found to inhibit tumour growth when dosed as monotherapy or in combination with anti-PD-L1. Materials & Methods Anti-tumour efficacy studies were performed in Balb/c mice using the sub-cutaneous J558 plasmacytoma:myeloma cell line (ATCC, TIB-6). Balb/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 5×106cells (passage number below P15) were subcutaneously injected (in 100 μl) into the right flanks of mice. Unless stated otherwise, on day 11 post tumour cells injection, the animals were randomised based on tumour size and treatments were initiated. The J558 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in DMEM supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 90% at the time of tumour cell injection. Treatment was initiated when the tumours reached an average volume of ˜140 mm{circumflex over ( )}3. Animals were then allocated to 4 groups with similar average tumour size (see Table E-21 for the dosing groups). Both antibodies, which are mouse cross-reactive, were dosed IP from day 11 (post tumour cell implantation) twice a week for 3 weeks (FIG.38) unless the animals had to be removed from study due to welfare (rare) or tumour size. As a control, a group of animals (n=10) was dosed at the same time using a saline solution. For the combination group, both STIM003 and anti-PDL1 antibodies were dosed concurrently IP at 60 μg and 200 μg respectively (in 0.9% saline). Tumour growth was monitored over 37 days and compared to tumours of animals treated with saline. Animal weight and tumour volume were measured 3 time a week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on studies until their tumour reached an average diameter of 12 mm3or, in rare cases, when incidence of tumour ulceration was observed (welfare). TABLE E21Treatment groups for J558 efficacy study.TreatmentGroupsNumber of animalsregimen twice per week from day 11110Saline28Anti-PD-L1 mIgG2a 200 μg (AbW)38Anti-ICOS STIM003 mIgG2a/anti-PD-L1mIgG2a (AbW)combination 60 μg/200 μg (respectively)48Anti-ICOS STIM003 mIgG2a 60 μg Results J558 syngeneic tumours were highly aggressive and all the animals in the saline control group (n=10) had to be removed from studies by day 21 due to tumour size. The anti-STIM003 mIgG2a and the anti-PDL1 mIgG2a both demonstrated good efficacy as monotherapies in this model with 37.5% and 75% of the animals cured of disease, respectively. Importantly, combination of the two antibodies resulted in 100% of the animals having rejected the plasmacytoma tumours by day 37. Data are shown inFIG.38. Example 22: Administration of Anti-PD1 Increases ICOS Expression on TILs Significantly More than Anti-PD-L1 Antibody A pharmacodynamic study was performed in animals harbouring established CT26 tumours to evaluate the effect of treatment with anti-PD-L1 or anti-PD-1 antibodies on ICOS expression on subsets of tumour infiltrating lymphocytes (TILs). The following antibodies were compared:anti-PD-L1 AbW mIgG1 [limited effector function]anti-PD-L1 AbW mIgG2a [with effector function]anti-PD-L1 10F9.G2 rat IgG2b [with effector function]anti-PD1 antibody RMT1-14 rat IgG2a [effector null]. Tumours of treated mice were isolated, dissociated to single cells and stained for CD45, CD3, CD4, CD8, FOXP3 and ICOS. Materials & Methods Rat anti-PD-1 RMP1-14 IgG2a (BioXCell; Catalog number: BE0146), rat anti-PD-L1 10F9.G2 IgG2b (Bio-Legend; Catalog number: 124325) and anti-PD-L1 AbW mIgG1 and mIgG2a were tested in the CT26 tumour model by dosing i.p. with 130 μg on days 13 and 15 post tumour cell implantation. On day 16, animals were culled and the mouse tumours were harvested for FACS analysis. Tumours were dissociated using a mouse tumour dissociation kit (Miltenyi Biotec) and homogenised. The resulting cell suspensions were clarified through 70 μM filters, pelleted and resuspended in FACS buffer at 2 million cells/well in a 96 well plate. The cell suspensions were incubated with anti-16/32 mAb (eBioscience) and stained with FACS antibodies specific for CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7) and ICOS (7E.17G9) all obtained from eBioscience Ltd. Cells were also stained with LiveDead Yellow fixable viability dye (Life technologies). For the Foxp3 intracellular staining, samples were fixed, permeabilised, and stained with antibody specific for Foxp3 (eBioscience, FJK-16s). The samples were resuspended in PBS and data acquired on the Attune flow cytometer (Invitrogen) and analysed using FlowJo V10 software (Treestar). Results Treatment with anti-PD1 and anti-PD-L1 antibodies only resulted in a marginal increase in the percentage on CD8 cells and T Regs expressing ICOS at the measured timepoint. However, in response to anti-PD1 rat IgG2a, a clear and significant (over the saline treated group) increase in ICOS expression (increased dMFI) was observed on the surface of ICOS+ve CD8 cells. ICOS expression was also noted to be upregulated on CD4 effector and CD4 T Reg cells although this did not reach statistical significance. This anti-PD1 antibody induced a marked increase in ICOS expression on CD8 effector cells that was barely seen with the anti-PD-L1 mIgG2a. Similarly, when comparing the different formats of anti-PD-L1 antibodies, in some of the animals treated it was observed that the antibody having the lowest effector function (mIgG1) was associated with higher ICOS expression on effector CD8 and CD4 cells when compared with antibody having effector function (mIgG2a and ratIgG2b), which rarely showed this. SeeFIG.39. An increase in ICOS expression on effector CD8/CD4 T cells may have the effect of rendering these cells more sensitive to depletion by anti-ICOS antibody (e.g., on treatment of mice with STIM003 mIgG2a). An antibody that exhibits lower ICOS induction in effector CD8 and CD4 T cells may be preferable for use in combination with anti-ICOS antibody. The data from this study indicate that anti-PD-L1 effector positive antibody may be especially suitable for combination with anti-ICOS effector positive antibody, reflecting the anti-tumour efficacy observed when combining anti-PDL1 mIgG2a with STIM003 mIgG2a reported in other Examples herein. Example 23: Strong Anti-Tumour Efficacy of Single Dose Anti-ICOS Antibody Monotherapy In Vivo in a B Cell Lymphoma Syngeneic Model This experiment confirms the anti-tumour efficacy of STIM003 mIgG2a as monotherapy. Strong anti-tumour efficacy was demonstrated after short exposure of STIM003 mIgG2a. Materials & Methods Efficacy studies were performed in BALB/c mice using the sub-cutaneous A20 Reticulum Cell Sarcoma model (ATCC number CRL-TIB-208). BALB/c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 5×10E5 A20 cells (passage number below P20) were subcutaneously injected into the right flanks of mice. Treatments were initiated at day 8 post tumour cells injection as shown in the table below. The A20 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice in PBS and resuspended in RPMI supplemented with 10% foetal calf serum. Cell viability was confirmed to be above 85% at the time of tumour cell injection. STIM003 mIgG2a was used either as a single dose (SD) of 60 μg (equivalent to 3 mg/kg for a 20 g animal) or as multiple doses (MD, twice a week for 3 weeks) of 60 μg. Anti-tumour efficacy observed in response to the two schedules was compared to that of animals “treated” with saline (MD, twice a week for 3 weeks). The antibodies were dosed intraperitoneal (IP) as 1 mg/ml in 0.9% saline. Animal weight and tumour volume were measured 3 times a week from the day of tumour cell injection. Tumour volume was calculated by use of the modified ellipsoid formula ½(Length×Width2). Mice were kept on study until their tumour reached an average diameter of 12 mm or, rarely, when incidence of tumour ulceration was observed (welfare). TABLE E23-1Treatment groups.GroupNumber of animalsTreatment regimen (IP injection)110Saline (multiple dose from day 8, twice aweek for 3 weeks)210STIM003 mIgG2 A (multiple dose fromday 8, twice a week for 3 weeks)310STIM003 mIgG2 A (Single dose on day 8) Results Both multiple and single dose of STIM003 mIgG2a resulted in strong and significant monotherapy anti-tumour efficacy as shown by the number of animals with no signs of tumour growth at endpoint (Day 41). SD resulted in 7 our 10 animals cured from the disease whereas the multiple dose cured 9 out of 10 animals injected with A20 B cell lymphoblast. All animals in the saline treated group had to be removed from the study by day 40 due to tumour size. SeeFIG.40. Humane endpoint survival statistics were calculated from the Kaplan-Meier curves (FIG.41) using GraphPad Prism V7.0. This approach was used to determine if the treatments were associated with improved survival. The Hazard Ratio (Mantel-Haenszel) values and their associated P values (Log-Rank Mantel-Cox) are shown in the table below. TABLE E23-1Hazard Ratio (Mantel-Haenszel) values and their associated P values(Log-Rank Mantel-Cox) corresponding to FIG. 41 Kaplan-Meier curves.Hazard Ratio(Mantel-Haenszel)MD vs SalineSD vs SalineMD vs SDRatio (and its0.099950.10760.5314reciprocal)95% CI of ratio0.02604 to 0.38370.02856 to 0.40520.05522 to 5.115P Value0.00080.0010.5842 Example 24: Time and Dose Dependent Effects of Anti-ICOS Antibody in CT-26 Tumour Bearing Animals This Example presents the results of a pharmacodynamic study evaluating the effects of anti-ICOS antibody on immune cells in mice bearing CT-26 tumours. T and B cell subtypes from different tissues were analysed by FACS after a single dose of STIM003 mIgG2a. Methods CT-26 tumour bearing animals were dosed i.p. with either saline or STIM003 at 200 μg, 60 μg or 6 μg on day 12 post tumour cell implantation. Tumour tissues, blood, tumour draining lymph node (TDLN) and spleen were harvested on day 1, 2, 3, 4, and day 8 post treatment. The tumours were dissociated to make single cell suspension using mouse tumour dissociation kit (Miltenyi Biotec). Spleen tissue was dissociated using gentle MACS dissociation, red blood cells were lysed using RBC lysis buffer. Tumour draining lymph nodes were mechanically disaggregated to make single cells suspensions. The resulting cell suspensions were clarified through either 70 μM or 40 μM filters depending on the tissue, cells were then washed twice in RMPI complete media and finally resuspended in ice cold FACS buffer. Total blood was collected into plasma tubes and red blood cells were lysed using RBC lysis buffer, cells were washed twice in RMPI complete media and finally resuspended in ice cold FACS buffer. The single cell suspension from all the tissues were distributed into 96 deep well plates for FACS analysis. Cells were stained with Live Dead Fixable Yellow viability dye (Life technologies). The cell suspensions were incubated with anti-CD16/CD32 mAb (eBioscience) and stained with FACS antibodies specific for CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61.5), ICOSL (HK5.3), B220 (RA3-6B2), Ki-67 (SolA15), CD107a (eBio1D4B), IFN-γ (XMG1.2), TNF-α (MP6-XT22), Foxp3 (FJK-16s) and ICOS (7E.17G9) all obtained from eBioscience Ltd. For cytokine readout by FACS, single cells suspensions from the tumours were plated in 24 well plate for 4 hours in the presence of Brefeldin-A. For the intracellular staining, samples were fixed, permeabilised, and stained with specific antibodies. The samples were finally resuspended in PBS and data acquired on the Attune flow cytometer (Invitrogen) and analysed using FlowJo V10 software (Treestar). Results are presented and discussed below. ICOS Expression is High on Intra-Tumoral T-Regs in the CT26 Model When the percentage of tumour infiltrating lymphocytes (TILs) expressing ICOS was compared to the percentage of immune cells in the spleen, blood, and TDLN, we demonstrated that more immune cells in the microenvironment of CT-26 tumours expressed ICOS vs other tissues. More importantly, the percentage of ICOS positive T-reg cells in all the tissues and at all the time points was higher than the percentage of CD4 or CD8 effector T cells positive for ICOS. Importantly, the dMFI (relative expression) for ICOS also followed the similar ranking in expression with intra-tumoural T-reg being highly positive for ICOS expression vs other TILs subtypes. Interestingly, there was no striking change in the percentage of ICOS+TILs within the time frame of this experiment. Similar results were also seen in spleen and TDLN. On the other hand, in the blood, ICOS expression is relatively stable on T effector cells but increased on T-regs during the course of the experiment. Altogether the data demonstrated that more cells expressed ICOS in the tumour microenvironment and these positive cells also expressed more ICOS molecules on their surface. More importantly, T regs in TILs are highly positive for ICOS. SeeFIG.42. Strong Depletion of Intra-Tumoural T-Reg Cells in Response to ST/M003 Administration In response to the STIM003 mIgG2a antibody, there was strong and rapid depletion of T-reg cells (CD4+CD25+Foxp3) in TME. As T-regs have high ICOS expression compared with the other T cells subsets, it is expected that an anti-ICOS antibody with effector function would preferentially deplete these cells. At the lower dose of STIM003 (6 μg corresponding to a 0.3 mg/kg for a 20 g animal) there was a continuous depletion of T-reg and by day 3 most of the T-reg were depleted from TME. Interestingly, by day 8, T-reg cells repopulate the TME then reach a level slightly above that observed in the saline treated animals. The repopulation of T-reg cells at lower dose can be attributed to the increase in the proliferating CD4 T cells in TME as evidenced by an observed increase in Ki-67+ CD4 T-cells. At a dose higher than 6 μg there was a long-term depletion of T-reg cells in TME as shown by full T Reg depletion until the last time point analysed in this study (day 8). Whereas in the blood there was a transient depletion of T-reg cells at all doses. Importantly, by day 8, all the treated animals had similar (or higher for the 6 μg dose) level of T-reg cells in the blood when compared to the saline treated animals. Data are shown inFIG.43. Notably, and similarly to data previously published for depleting CTLA-4 antibodies, there was no significant change in the percentage of T-reg cells in the spleen or TDLN tissues, suggesting that T-reg cells may be protected from depletion in these organs. In summary, strong depletion of T-reg cells in TME was achieved in CT-26 model at a dose as low as 6 μg per animal. However, a dose of 60 μg resulted in long term depletion up to 8 days post STIM003 mIgG2a injection. This was not improved by using higher dose (200 μg). STIM003 mIgG2a Increased CD8:T Reg and CD4:T Reg Ratios Effects of STIM003 on T-eff:T-reg ratios are shown inFIG.44. STIM003 mIgG2a increased the CD8:T-reg ratio as well as the CD4 eff:T-reg ratio. Although all the treatment doses were associated with an increase in T-eff to T-reg ratio, the intermediate dose of 60 μg (the equivalent of 3 mg/kg for a 20 g animals) was associated with the highest ratio by day 8 post treatment. Interestingly, at the 6 μg dose, the ratios were high until day 4 but by day 8 post treatment they were matching that of the saline treated animals. This can be explained by the repopulation of TRegs observed for this dose by day 8 post treatment. On the other hand, at a dose of 60 or 200 μg, the Teff to T-reg ratios remained high at all time points. This is explained by a long term depeletion of Tregs at these doses. Notably, at higher dose (200 μg), despite the long term Treg depletion there was only a moderate improvement in the ratio by day 8. This can be explained by some depletion of ICOSINTeffector cells at high concentration of STIM003. Altogether, the data demonstrated TReg depletion and increased Effector:T reg ratio at all doses tested. However, an optimal dose of 60 μg (˜3 mg/kg) achieved both a long-term depletion of T-reg, as well as the highest T-eff to T-reg ratios which would be associated with the most favourable immune context to initiate an anti-tumour immune response. Interestingly a similar pattern was observed in the blood, with the intermediate dose of 60 μg associated with the highest T-eff to T-reg ratio. Importantly, in the blood, improvement of the ratio was observed at an earlier time point (between day 3 and day 4). Activation of Effector Cells in Response to STIM003 Surface expression of CD107a on the tumour infiltrating T effector cells was previously identified as a reliable marker for cells that have been activated and exert cytotoxic activity [44]. In the present study employed this marker to confirm that STIM003, in addition to depleting T-regs, can stimulate the cytotoxic activity of effector T cells in the TME. Interestingly, on day 8 post treatment, there was an increase in surface expression of CD107a on both the CD4 and CD8 effector T cell compartments at all doses of STIM003. Furthermore, this upregulation of CD107a expression on the surface on both CD4 and CD8 T cells appeared to plateau when animals were dosed at 60 μg as no improvement was seen at 200 μg dosing. To further demonstrate activation of effector cells in the TME, the cytokine release by CD4 and CD8 TILs was analysed by FACS. As expected and consistent with the in-vitro agonism data presented in earlier Examples herein, STIM003 mIgG2a at all doses promoted pro-inflammatory cytokine IFN-γ and TNF-α production by effector CD4 and CD8 T cells. The induction of pro-inflammatory cytokine production appeared to be high at the optimum dose of 60 μg. Indeed, 60 μg of STIM003 significantly increased cytokine production by CD4 T cells. A similar trend was seen for the proinflammatory cytokine IFN-γ and TNF-α production by effector CD8 T cells in TME. Data are shown inFIG.45. In summary, STIM003 at all the doses resulted in T cells activation in the TME as shown by (1) the presence of the degranulation marker CD107a on their surface and (2) by the production of Th1 cytokines (IFNγ and TNFα) by T cells. This indicates that STIM003 strongly affects the immune context in the TME and plays the dual role of depleting Treg cells and stimulate the killing activity of T effector cells. Human Dose Estimations Based on the pre-clinical efficacy data seen in mice, initial predictions can be made of the clinical dose appropriate for human patients, based on corresponding biological surface area (BSA) [45]. For example, taking the optimal anti-ICOS IgG dose in mouse to be 3 mg/kg (60 μg), and following the methods of ref. [45], the corresponding dose for a human is 0.25 mg/kg. Using the Mosteller formulae, for an individual of 60 kg and 1.70 m the BSA 1.68 m2. Multiplying the dose in mg/kg by a factor of 35.7 (60/1.68) gives a fixed dose of 15 mg. For an individual of 80 kg the corresponding fixed dose would be 20 mg. Doses may be optimised for human therapy in clinical trials to determine safe and effective treatment regimens. Example 25: Bioinformatic Analysis of Data from Tumour Samples One target group of cancers according to the present invention is those cancers that are associated with a relatively high level of ICOS+ immunosuppressive Tregs. To identify cancer types associated with a high content of Tregs, transcriptome data was obtained from The Cancer Genome Atlas (TCGA) public dataset and analysed for ICOS and FOXP3 expression levels. TOGA is a large-scale study that has catalogued genomic and transcriptomic data accumulated for many different types of cancers, and includes mutations, copy number variation, mRNA and miRNA gene expression, and DNA methylation along with substantial sample metadata. Gene Set enrichment analysis (GSEA) was conducted as follows. Gene expression RNA seq data collected as part of the TOGA consortium was downloaded from the UCSC Xena Functional Genomics Browser as log 2(normalized_count+1). Non-tumour tissue samples were removed from the dataset, leaving data for 20530 genes from 9732 samples. An algorithm from [46] and its implementation in [47] that calculates enrichment scores for genes within a specified gene set was used to transpose gene level counts to gene set scores for each sample. The gene set of interest was defined as containing both ICOS and FOXP3. Samples were grouped by primary disease and the ssGSEA scores for each group were compared across the 33 primary disease groups. The disease groups that showed the highest median scores were found to be lymphoid neoplasm diffuse large b-cell lymphoma, thymoma, head and neck squamous cell carcinoma, although diffuse large b-cell lymphoma showed a multimodal distribution of scores with a subset scoring highly and the rest scoring below the group median. In rank order of highest to lowest ssGSEA score for ICOS and FOXP3 expression, the top 15 cancer types were: DLBC (n = 48)lymphoid neoplasm diffuse large b-cell lymphomaTHYM (n = 120)thymomaHNSC (n = 522)head and neck squamous cell carcinomaTGCT (n = 156)testicular germ cell tumourSTAD (n = 415)stomach adenocarcinomaSKCM (n = 473)skin cutaneous melanomaCESC (n = 305)cervical squamous cell carcinoma and endocervicaladenocarcinomaLUAD (n = 517)lung adenocarcinomaLAML (n = 173)acute myeloid leukemiaESCA (n = 185)esophageal carcinomaLUSC (n = 502)lung squamous cell carcinomaREAD (n = 95)rectum adenocarcinomaCOAD (n = 288)colon adenocarcinomaBRCA (n = 1104)breast invasive carcinomaLIHC (n = 373)liver hepatocellular carcinoma In which n is the number of patient samples for that cancer type in TCGA dataset. Anti-ICOS antibodies described herein may be used for treatment of these and other cancers. Cancers that are associated with a relatively high level of ICOS+ immunosuppressive Tregs and which further express PD-L1 may respond especially well to treatment with a combination of anti-ICOS antibody and anti-PD-L1 antibody. Appropriate treatment regiments and antibodies for this purpose have already been detailed in the foregoing description. Using the TCGA dataset as before, enrichment scores for ICOS and FOXP3 were correlated with expression levels of PD-L1 using Spearman's rank correlation and grouped by primary disease indication. P-values were calculated for each group and a p-value of 0.05 (with Bonferroni's multiple comparison correction) was taken as statistically significant. The disease groups with the highest correlations between ICOS/FOXP3 and PD-L1 expression were: TGCT (n = 156)testicular germ cell tumourCOAD (n = 288)colon adenocarcinomaREAD (n = 95)rectum adenocarcinomaBLCA (n = 407)bladder urothelial carcinomaOV (n = 308)ovarian serous cystadenocarcinomaBRCA (n = 1104)breast invasive carcinomaSKCM (n = 473)skin cutaneous melanomaCESC (n = 305)cervical squamous cell carcinoma and endocervicaladenocarcinomaSTAD (n = 415)stomach adenocarcinomaLUAD (n = 517)lung adenocarcinoma Patients may be selected for treatment following an assay determining that their cancer is associated with ICOS+ immunosuppressive Tregs and expression of PD-L1. For cancer types in which, as above, there is a high correlation score, it may suffice to determine that one of ICOS+ immunosuppressive Tregs and expression of PD-L1 is present (e.g., above a threshold value). PD-L1 immunohistochemistry assays may be used in this context. Example 26: Assessment of Further Anti-ICOS Antibodies CL-74570 and CL-61091 antibody sequences identified in Example 12 were synthesised and expressed in IgG1 format in HEK cells. Functional characterisation of these antibodies was performed using an HTRF assay similar to that described in Example 6, with modifications to adapt the assay to use of purified IgG1 rather than BCT supernatant. 5 μL of supernatant containing human IgG1 antibodies expressed from HEK cells was used in place of the BCT supernatant, and the total volume made up to 20 μl per well using HTRF buffer as before. A human IgG1 antibody was used as a negative control. Both antibodies exhibited greater than 5% effect for binding to human and mouse ICOS as calculated using Equation 1 and were therefore confirmed to test positive in this assay. Ability of these antibodies to bind human and mouse ICOS expressed on the surface of CHO-S cells was further confirmed using a Mirrorball assay. In this assay, 5 μl supernatant containing the anti-ICOS IgG1 was transferred to each well of 384 mirrorball black plates (Corning). Binding of anti-ICOS antibodies was detected by adding 10 μl of goat anti-human 488 (Jackson Immunoresearch) diluted in assay buffer (PBS+1% BSA+0.1% Sodium Azide) at a concentration of 0.8 mg/ml to all wells. For positive control wells, 5 μL reference antibody diluted in assay media to 2.2 μg/mL was added to the plates. For negative control wells, 5 μl of Hybrid control IgG1 diluted in assay media to 2.2 μg/mL was added to the plates. 10 μM of DRAQS (Thermoscientific) was added to 0.4×106/ml cells resuspended in assay buffer and 5 μl was added to all wells. Plates were incubated for 2 hr at 4 degrees. Fluorescence intensity was measured using Mirrorball plate reader (TTP Labtech), measuring Alexafluor 488 (excitation 493 nm, emission 519 nm) from a population of 500-700 single cells. Assay signal was measured as Median (FL2) Mean Intensity. Total binding was defined using reference antibody at an assay concentration of 2.2 μg/mL. Non-specific binding was defined using Hybrid control hIgG1 at an assay concentration of 2.22 μg/mL. Both antibodies exhibited greater than 1 percent effect and were therefore confirmed to test positive in this assay. Percenteffect=(samplewell-non-specificbinding)(totalbinding-non-specificbinding)×100 Each of CL-74570 and CL-61091 also demonstrated binding to human and mouse ICOS expressed on CHO-S cells as determined by flow cytometry. FACS screening was performed using a method similar to that described in Example 6, with modifications to adapt the assay to use of purified IgG1 rather than BCT supernatant. Both antibodies exhibited binding>10 fold above the average of geomean of the negative control binding to hICOS, mICOS and WT CHO cells. TABLE E26-1Functional characterisation of CL-74570 and CL-61091.Primary ScreenHTRF (Protein)Mirrorball (ICOS CHO Cell)Secondary screenHumanMouseHumanMouseFACS1:100 dil1:100 dil1:100 dil1:100 dilHuman ICOS CHOMouse ICOS CHOPercentPercentPercentPercent(1:10 dil)(1:10 dil)Effect [%]Effect [%]Effect [%]Effect [%]% Binding-APC% Binding-APCClone ID94.4260.86107.02127.03122.9796.41CL-7457083.4376.6554.14113.1019.0862.94CL-61091 REFERENCES 1 Hutloff A, et al. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature. 1999 Jan. 21; 397(6716):263-6.2 Beier K C, et al. Induction, binding specificity and function of human ICOS. Eur J Immunol. 2000 December; 30(12):3707-17.3 Coyle A J, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity. 2000 July; 13(1):95-105.4 Dong C, et al. ICOS co-stimulatory receptor is essential for T-cell activation and function. 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R. et al., Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. The Journal of experimental medicine, 210(9):1695-710 201343 Selby, M. J. et al., Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer immunology research, 1(1):32-42 2013.44 Rubio V., et al. Ex vivo identification, isolation and analysis of tumor-cytolytic T cells. Nat Med. 2003 November; 9(11):1377-82.45 Nair & Jacob., A simple practice guide for dose conversion between animals and human. J Basic Clin Pharma 2016; 7:27-3146 D. A. Barbie, et al., “Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.,” Nature, vol. 462, no. 7269, pp. 108-12, 200947 S. Hanzelmann, R. Castelo, and J. Guinney, “GSVA: gene set variation analysis for microarray and RNA-Seq data,” BMC Bioinformatics, vol. 14, no. 1, p. 7, 2013 SequencesAntibody STIM001VH domain nucleotide sequence: SEQ ID NO: 367CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 366QVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAVYYCARSSGHYYYYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GYTFSTFG SEQ ID NO: 363VH CDR2 amino acid sequence: ISAYNGDT SEQ ID NO: 364VH CDR3 amino acid sequence: ARSSGHYYYYGMDV SEQ ID NO: 365VL domain nucleotide sequence: SEQ ID NO: 374GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 373DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYCMQSLQTPLTFGGGTKVEIKVL CDR1 amino acid sequence: QSLLHSNEYNY SEQ ID NO: 370VL CDR2 amino acid sequence: LGS SEQ ID NO: 371VL CDR3 amino acid sequence: MQSLQTPLT SEQ ID NO: 372Antibody STIM002VH domain nucleotide sequence: SEQ ID NO: 381CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 380QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO: 377VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO: 378VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO: 379VL domain nucleotide sequence: 388GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACorrected STIM002 VL domain nucleotide sequence: SEQ ID NO: 519GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 387DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLSFGQGTKLEIKVL CDR1 amino acid sequence: QSLLHSDGYNY SEQ ID NO: 384VL CDR2 amino acid sequence: LGS SEQ ID NO: 385VL CDR3 amino acid sequence: MQALQTPLS SEQ ID NO: 386Antibody STIM002-BVH domain nucleotide sequence: SEQ ID NO: 395CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 394QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO: 391VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO: 392VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO: 393VL domain nucleotide sequence: SEQ ID NO: 402GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 401DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPCSFGQGTKLEIKVL CDR1 amino acid sequence: QSLLHSDGYNC SEQ ID NO: 398VL CDR2 amino acid sequence: LGS SEQ ID NO: 399VL CDR3 amino acid sequence: MQALQTPCS SEQ ID NO: 400Antibody STIM003VH domain nucleotide sequence: SEQ ID NO: 409GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCACorrected STIM003 VH domain nucleotide sequence: SEQ ID NO: 521GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 408EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDFYGSGSYYHVPFDYWGQGILVTVSSVH CDR1 amino acid sequence: GVTFDDYG SEQ ID NO: 405VH CDR2 amino acid sequence: INWNGGDT SEQ ID NO: 406VH CDR3 amino acid sequence: ARDFYGSGSYYHVPFDY SEQ ID NO: 407VL domain nucleotide sequence: SEQ ID NO: 416GAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAVL domain amino acid sequence: SEQ ID NO: 415EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYDMSPFTFGPGTKVDIKVL CDR1 amino acid sequence: QSVSRSY SEQ ID NO: 412VL CDR2 amino acid sequence: GAS SEQ ID NO: 413VL CDR3 amino acid sequence: HQYDMSPFT SEQ ID NO: 414Antibody STIM004VH domain nucleotide sequence:GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTGA SEQ ID NO:423VH domain amino acid sequence:EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEWVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDYYGSGSYYNVPFDYWGQGTLVTVSS SEQ ID NO: 422VH CDR1 amino acid sequence: GLTFDDYG SEQ ID NO: 419VH CDR2 amino acid sequence: INWNGDNT SEQ ID NO: 420VH CDR3 amino acid sequence: ARDYYGSGSYYNVPFDY SEQ ID NO: 421VL domain nucleotide sequence: SEQ ID NO: 431GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAAVL domain amino acid sequence as encoded by the above VL domain nucleotidesequence.Corrected VL domain nucleotide sequence: SEQ ID NO: 430GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAACorrected VL domain amino acid sequence: SEQ ID NO: 432EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYGSSPFFGPGTKVDIKVL CDR1 amino acid sequence: QSVSSSY SEQ ID NO: 426VL CDR2 amino acid sequence: GAS SEQ ID NO: 427VL CDR3 amino acid sequence: QQYGSSPF SEQ ID NO: 428Antibody STIM005VH domain nucleotide sequence: SEQ ID NO: 439CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCAVH domain amino acid sequence: SEQ ID NO: 438QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEWMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAVYYCARAGYDILTDFSDAFDIWGHGTMVTVSSVH CDR1 amino acid sequence: GYTFNSYG SEQ ID NO: 435VH CDR2 amino acid sequence: ISVHNGNT SEQ ID NO: 436VH CDR3 amino acid sequence: ARAGYDILTDFSDAFDI SEQ ID NO: 437VL domain nucleotide sequence: SEQ ID NO: 446GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAACTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAAVL domain amino acid sequence: SEQ ID NO: 445DIQMTQPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYGIPWVGQGTKVEIKVL CDR1 amino acid sequence: QNINNF SEQ ID NO: 442VL CDR2 amino acid sequence: ASS SEQ ID NO: 443VL CDR3 amino acid sequence: QQSYGIPW SEQ ID NO: 444Antibody STIM006VH domain nucleotide sequence: SEQ ID NO: 453CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 454QVQLVESGGGLVKPGGSLRLSCAASGFTESDYFMSWIRQAPGKGLEWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFTFSDYF SEQ ID NO: 449VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO: 450VH CDR3 amino acid sequence: ARDHYDGSGIYPLYYYYGLDV SEQ ID NO: 451VL domain nucleotide sequence: SEQ ID NO: 460ATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGCTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 459IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRSFGQGTTLEIKVL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO: 456VL CDR2 amino acid sequence: LGS SEQ ID NO: 457VL CDR3 amino acid sequence: MQALQTPRS SEQ ID NO: 458Antibody STIM007VH domain nucleotide sequence: SEQ ID NO: 467CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 466QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFSLSTTGVG SEQ ID NO: 463VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO: 464VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO: 465VL domain nucleotide sequence: SEQ ID NO: 474GAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACVL domain amino acid sequence: SEQ ID NO: 473EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWPLTFGGGTKVEIKVL CDR1 amino acid sequence: QSVTNY SEQ ID NO: 470VL CDR2 amino acid sequence: DAS SEQ ID NO: 471VL CDR3 amino acid sequence: QHRSNWPLT SEQ ID NO: 472Antibody STIMV08VH domain nucleotide sequence: SEQ ID NO: 481CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 480QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFSLSTSGVG SEQ ID NO: 477VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO: 478VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO: 479VL domain nucleotide sequence: SEQ ID NO: 488GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAVL domain amino acid sequence: SEQ ID NO: 489EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKVL CDR1 amino acid sequence: QSVTNY SEQ ID NO: 484VL CDR2 amino acid sequence: DAS SEQ ID NO: 485VL CDR3 amino acid sequence: QQRSNWPLT SEQ ID NO: 486Antibody STIM009VH domain nucleotide sequence: SEQ ID NO: 495CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAVH domain amino acid sequence: SEQ ID NO: 494QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSSVH CDR1 amino acid sequence: GFTFSDYY SEQ ID NO: 491VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO: 492VH CDR3 amino acid sequence: ARDFYDILTDSPYFYYGVDV SEQ ID NO: 493VL domain nucleotide sequence: SEQ ID NO: 502GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAVL domain amino acid sequence: SEQ ID NO: 501DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIKVL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO: 498VL CDR2 amino acid sequence: LGS SEQ ID NO: 499VL CDR3 amino acid sequence: MQALQTPRT SEQ ID NO: 500 TABLE S1SEQ ID NOS: 1-342SEQIDNO:NameDescriptionSequence1HumanNCBI number:MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLPD-L1NP_054862.1DLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGN(ECD highlighted inAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVBOLD, cytoplasmicDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNdomain underlined)VTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET2Cyno PD-NCBI number:MGWSCIILFLVATATGVHSMFTVTVPKDLYVVEYGSNMTIECKFPVEKL1XP_014973154.1QLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSL(ECD highlighted inGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILBOLD)VVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERT3HumanHuman PD-L1 ECDMRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLPD-L1with C-terminal HisDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNHistagAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHHHHHH4HumanHuman PD-L1 ECDMRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLPD-L1 Fcwith C-term FcDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNfusion (in bold)AALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTIEGREPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVBNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTQLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSWBEALHNHYTQKSLSLSPGK5Cyno PD-Cynomolgus PD-L1MGWSCIILFLVATATGVHSMFTVTVPKDLYVVEYGSNMTIECKFPVEKL1 FLAGECD with N-termQLDLTSLIVYWEMEDKNIIQFVHGEEDLKVQHSNYRQRAQLLKDQLSLFLAG tagGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTANEIFYCIFRRLDPEENHTAELVIPELPLALPPNERTDYKDDDDK6HumanHuman PD-1 fullMGWSCIILFLVATATGVHSLDSPDRPWNPPTFSPALLVVTEGDNATFTPD-1 Fclength sequenceCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPderived from cDNANGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAas human Fc fusionEVPTAHPSPSPRPAGQKLENLYFQGIEGRMDEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDQSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP784G09 -Amino acid sequenceGFTFDDYACDRH1of CDRH1 of 84G09(IMGT)using IMGT884G09 -Amino acid sequenceISWKSNIICDRH2of CDRH2 of 84G09(IMGT)using IMGT984G09 -Amino acid sequenceARDITGSGSYGWFDPCDRH3of CDRH3 of 84G09(IMGT)using IMGT1084G09 -Amino acid sequenceDYAMHCDRH1of CDRH1 of 84G09(Kabat)using Kabat1184G09 -Amino acid sequenceGISWKSNIIGYADSVKGCDRH2of CDRH2 of 84G09(Kabat)using Kabat1284G09 -Amino acid sequenceDITGSGSYGWFDPCDRH3of CDRH3 of 84G09(Kabat)using Kabat1384G09 -Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVHeavyof VHofSGISWKSNIIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCchain84G09 (mutationsARDITGSGSYGWFDPWGQGTLVTVSSvariablefrom germline areregionshown in boldletters)1484G09 -Nucleic acidCAaGAAAAAGCTTGCCGCCACCATGGAGTTTGGGCTGAGCTGGATTTTHeavysequence of VHofCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTACAATTGGTGGAchain84G09GTCCGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGvariableTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGregionACAAACTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATAAGTTGGAAGAGTAATATCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTATTGTGCAAGAGATATAACGGGTTCGGGGAGTTATGGCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGAATCTGCTAAAACTCAGCCTCCG1584G09 -Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVfullof 84G09 heavySGISWKSNIIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCheavychain (mutationsARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTchainfrom germline areAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTsequenceshown in boldVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPletters)SVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK1684G09 -Nucleic acidGAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCAGAfullsequence of 84G09TCCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCGACGACTACheavyheavy chainGCTATGCACTGGGTGCGACAGACCCCTGGCAAGGGCCTGGAATGGGTGchainTCCGGCATCTCCTGGAAGTCCAACATCATCGGCTACGCCGACTCCGTGsequenceAAGGGCCGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCCTGTACTACTGCGCCAGAGACATCACCGGCTCCGGCTCCTACGGATGGTTCGATCCTTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG1784G09 -Amino acid sequenceQSISSYCDRL1of CDRL1 of 84G09(IMGT)using IMGT1884G09 -Amino acid sequenceVASCDRL2of CDRL2 of 84G09(IMGT)using IMGT1984G09 -Amino acid sequenceQQSYSNPITCDRL3of CDRL3 of 84G09(IMGT)using IMGT2084G09 -Amino acid sequenceRASQSISSYLNCDRL1of CDRL1 of 84G09(Kabat)using Kabat2184G09 -Amino acid sequenceVASSLQSCDRL2of CDRL2 of 84G09(Kabat)using Kabat2284G09 -Amino acid sequenceQQSYSNPITCDRL3of CDRL3 of 84G09(Kabat)using Kabat2384G09 -Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKPLILightof VLof 84G09YVASSLQSGVPSSFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPIchainTFGQGTRLEIKvariableregion2484G09 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGALightsequence of VLofGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATchain84G09TTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCCCCTGATCvariableTATGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGTTTCAGTGGCregionAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTAATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAA2584G09 -Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKPLIfullof 84G09 lightYVASSLQSGVPSSFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSNPIlightchainTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC2684G09 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAfullsequence of 84G09GACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATlightlight chainTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCCCCTGATCchainTATGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGTTTCAGTGGCsequenceAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTAATCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT271D05 -Amino acid sequenceGFTFDDYACDRH1of CDRH1 of 1D05(TMGT)using IMGT281D05 -Amino acid sequenceISWIRTGICDRH2of CDRH2 of 1D05(IMGT)using IMGT291D05 -Amino acid sequenceAKDMKGSGTYGGWFDTCDRH3of CDRH3 of 1D05(IMGT)using IMGT301D05 -Amino acid sequenceDYAMHCDRH1of CDRH1 of 1D05(Kabat)using Kabat311D05 -Amino acid sequenceGISWIRTGIGYADSVKGCDRH2of CDRH2 of 1D05(Kabat)using Kabat321D05 -Amino acid sequenceDMKGSGTYGGWFDTCDRH3of CDRH3 of 1D05(Kabat)using Kabat331D05 -Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVHeavyof VHof 1D05SGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCchain(mutations fromAKDMKGSGTYGGWFDTWGQGTLVTVSSvariablegermline are shownregionin bold letters)341D05 -Nucleic acidAAGCTTGCCGCCACCATGGAGTTTGGGCTGAGCTGGATTTTCCTTTTGHeavysequence of VHofGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGchain1D05GGAGGCTTGGTGCAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCvariableTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGTTregionCCAGGGAAGGGCCTGGAATGGGTCTCAGGCATTAGTTGGATTCGTACTGGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATTTTCAGAGACAACGCCAAGAATTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATGAAGGGTTCGGGGACTTATGGGGGGTGGTTCGACACCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGC351D05 -Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVfullof 1D05 heavy chainSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCheavyAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESchainTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVsequenceTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK361D05 -Nucleic acidGAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCAGAfullsequence of 1D05TCCCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCGACGACTACheavyheavy chainGCTATGCACTGGGTGCGACAGGTGCCAGGCAAGGGCCTGGAATGGGTGchainTCCGGCATCTCTTGGATCCGGACCGGCATCGGCTACGCCGACTCTGTGsequenceAAGGGCCGGTTCACCATCTTCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCCTGTACTACTGCGCCAAGGACATGAAGGGCTCCGGCACCTACGGCGGATGGTTCGATACTTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG371D05 -Amino acid sequenceQSISSYCDRL1of CDRL1 of 1D05(IMGT)using IMGT381D05 -Amino acid sequenceVASCDRL2of CDRL2 of 1D05(IMGT)using IMGT391D05 -Amino acid sequenceQQSYSTPITCDRL3of CDRL3 of 1D05(IMGT)using IMGT401D05 -Amino acid sequenceRASQSISSYLNCDRL1of CDRL1 of 1D05(Kabat)using Kabat411D05 -Amino acid sequenceVASSLQSCDRL2of CDRL2 of 1D05(Kabat)using Kabat421D05 -Amino acid sequenceQQSYSTPITCDRL3of CDRL3 of 1D05(Kabat)using Kabat431D05 -Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLILightof VLofYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPIchain1D05(mutations fromTFGQGTRLEIKvariablegermline are shownregionin bold letters)441D05 -Nucleic acidAAAGCTTGCCGCCACCATGAGGCTCCCTGCTCAGCTTCTGGGGCTCCTLightsequence of VLofGCTACTCTGGCTCCGAGGTGCCAGATGTGACATCCAGATGACCCAGTCchain1D05TCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGvariableCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAregionACCAGGGAAAGCCCCTAAACTCCTGATCTATGTTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACTATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCGATCACCTTCGGCCAAGGGACACGTCTGGAGATCAAACGTACGGATGCTGCACCAACT451D05 -Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIfullof 1D05 light chainYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPIlightTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC461D05 -Nucleic acidGACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCTGCTTCCGTGGGCfullsequence of 1D05GACAGAGTGACCATCACCTGTCGGGCCTCCCAGTCCATCTCCTCCTAClightlight chainCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCchainTACGTGGCCAGCTCTCTGCAGTCCGGCGTGCCCTCTAGATTCTCCGGCsequenceTCTGGCTCTGGCACCGACTTTACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTCCTACTCCACCCCTATCACCTTCGGCCAGGGCACCCGGCTGGAAATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT47MutatedAmino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV1D05 -of 1D05 heavy chainSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCHCwith V to A back-AKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESmutant 1mutation inTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVframework region toTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPELAGAgermlinePSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHhighlighted withNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEIgG1 disabledKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW(LAGA) constantESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHregionEALHNHYTQKSLSLSLGK48MutatedAmino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWV1D05 -of 1D05 heavy chainSGISWIRTGIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCHCwith F to S back-AKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESmutant 2mutation inTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVframework region toTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPELAGAgermlinePSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHhighlighted withNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEIgG1 disabledKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW(LAGA) constantESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHregionEALHNHYTQKSLSLSLGK49MutatedAmino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWV1D05 -of 1D05 heavy chainSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCHCwith ELLG to -PVAAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESmutant 3back-mutation inTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVconstant region toTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP-germlinePVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGhighlightedVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK50MutatedAmino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI1D05 -of 1D05 kappa lightYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPILCchain with V to ATFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAmutant 1back-mutation inKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYCDRL2 to germlineACEVTHQGLSSPVTKSFNRGEChighlighted51MutatedAmino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLFI1D05 -of 1D05 kappa lightYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPILCchain with L to FTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAmutant 2back-mutation inKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYframework toACEVTHQGLSSPVTKSFNRGECgermlinehighlighted52411B08 -Amino acid sequenceGFTFSSYWCDRH1of CDRH1 of 411B08(IMGT)using IMGT53411B08 -Amino acid sequenceIKEDGSEKCDRH2of CDRH2 of 411B08(IMGT)using IMGT54411B08 -Amino acid sequenceARNRLYSDFLDNCDRH3of CDRH3 of 411B08(IMGT)using IMGT55411B08 -Amino acid sequenceSYWMSCDRH1of CDRH1 of 411B08(Kabat)using Kabat56411B08 -Amino acid sequenceNIKEDGSEKYYVDSVKGCDRH2of CDRH2 of 411B08(Kabat)using Kabat57411B08 -Amino acid sequenceNRLYSDFLDNCDRH3of CDRH3 of 411B08(Kabat)using Kabat58411B08 -Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVHeavyof VHof 411B08ANIKEDGSEKYYVDSVKGRFTISRDNAENSLYLQMNSLRAEDTSVYYCchainARNRLYSDFLDNWGQGTLVTVSSvariableregion59411B08 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATchain411B08TGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGvariableGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGregionAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG60411B08 -Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVfullof 411B08 heavyANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCheavychainARNRLYSDFLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALchainGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSsequenceSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK61411B08 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGfullsequence of 411B08TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATheavyheavy chainTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGchainGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGsequenceAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG62411B08 -Amino acid sequenceQGVSSWCDRL1of CDRL1 of 411B08(IMGT)using IMGT63411B08 -Amino acid sequenceGASCDRL2of CDRL2 of 411B08(IMGT)using IMGT64411B08 -Amino acid sequenceQQANSIPFTCDRL3of CDRL3 of 411B08(IMGT)using IMGT65411B08 -Amino acid sequenceRASQGVSSWLACDRL1of CDRL1 of 411B08(Kabat)using Kabat66411B08 -Amino acid sequenceGASSLQSCDRL2of CDRL2 of 411B08(Kabat)using Kabat67411B08 -Amino acid sequenceQQANSIPFTCDRL3of CDRL3 of 411B08(Kabat)using Kabat68411B08 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLILightof VLof 411B08YGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFchainTFGPGTKVDIKvariableregion69411B08 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGALightsequence of VLofGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGchain411B08TTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCvariableTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCregionAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC70411B08 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIfullof 411B08 lightYGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFlightchainTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC71411B08 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAfullsequence of 411B08GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGlightlight chainTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCchainTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCsequenceAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT72411C04 -Amino acid sequenceGFTFSSYWCDRH1of CDRH1 of 411C04(IMGT)using IMGT73411C04 -Amino acid sequenceIKEDGSEKCDRH2of CDRH2 of 411C04(IMGT)using IMGT74411C04 -Amino acid sequenceARVRLYSDFLDYCDRH3of CDRH3 of 411C04(IMGT)using IMGT75411C04 -Amino acid sequenceSYWMSCDRH1of CDRH1 of 411C04(Kabat)using Kabat76411C04 -Amino acid sequenceNIKEDGSEKYYVDSLKGCDRH2of CDRH2 of 411C04(Kabat)using Kabat77411C04 -Amino acid sequenceVRLYSDFLDYCDRH3of CDRH3 of 411C04(Kabat)using Kabat78411C04 -Amino acid sequenceEVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVHeavyof VHof 411C04ANIKEDGSEKYYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCchainARVRLYSDFLDYWGQGTLVTVSSvariableregion79411C04 -Nucleic acidGAGGTGCAGCTGGTGGACTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATchain411C04TGGATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTGvariableGCCAACATAAAAGAAGATGGAAGTGAGAAATACTATGTAGACTCTTTGregionAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAGTTCGACTCTACAGTGACTTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG80411C04 -Amino acid sequenceEVQLVDSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVfullof 411C04 heavyANIKEDGSEKYYVDSLKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCheavychainARVRLYSDFLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALchainGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSsequenceSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK81411C04 -Nucleic acidGAGGTGCAGCTGGTGGACTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGfullsequence of 411C04TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATheavyheavy chainTGGATGAGTTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTGchainGCCAACATAAAAGAAGATGGAAGTGAGAAATACTATGTAGACTCTTTGsequenceAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAGTTCGACTCTACAGTGACTTCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAPG82411C04 -Amino acid sequenceQGVSSWCDRL1of CDRL1 of 411C04(IMGT)using IMGT83411C04 -Amino acid sequenceGASCDRL2of CDRL2 of 411C04(IMGT)using IMGT84411C04 -Amino acid sequenceQQANSIPFTCDRL3of CDRL3 of 411C04(IMGT)using IMGT85411C04 -Amino acid sequenceRASQGVSSWLACDRL1of CDRL1 of 411C04(Kabat)using Kabat86411C04 -Amino acid sequenceGASSLQSCDRL2of CDRL2 of 411C04(Kabat)using Kabat87411C04 -Amino acid sequenceQQANSIPFTCDRL3of CDRL3 of 411C04(Kabat)using Kabat88411C04 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLILightof VLof 411C04YGASSLQSGVPSRFSGSGSGTEFILSISSLQPEDFATYYCQQANSIPFchainTFGPGTKVDIKvariableregion89411C04 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGALightsequence of VLofGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGTTGGchain411C04TTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCvariableTATGGTGCCTCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCregionAGTGGATCTGGGACAGAGTTCATTCTCAGCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC90411C04 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIfullof 411C04 lightYGASSLQSGVPSRFSGSGSGTEFILSISSLQPEDFATYYCQQANSIPFlightchainTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC91411C04 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAfullsequence of 411C04GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGTTGGlightlight chainTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCchainTATGGTGCCTCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCsequenceAGTGGATCTGGGACAGAGTTCATTCTCAGCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT92411D07 -Amino acid sequenceGGSIISSDWCDRH1of CDRH1 of 411D07(IMGT)using IMGT93411D07 -Amino acid sequenceIFHSGRTCDRH2of CDRH2 of 411D07(IMGT)using IMGT94411D07 -Amino acid sequenceARDGSGSYCDRH3of CDRH3 of 411D07(IMGT)using IMGT95411D07 -Amino acid sequenceSSDWWNCDRH1of CDRH1 of 411D07(Kabat)using Kabat96411D07 -Amino acid sequenceEIFHSGRTNYNPSLKSCDRH2of CDRH2 of 411D07(Kabat)using Kabat97411D07 -Amino acid sequenceDGSGSYCDRH3of CDRH3 of 411D07(Kabat)using Kabat98411D07 -Amino acid sequenceQVQLQESGPGLVKPSGTLSLTCIVSGGSIISSDWWNWVRQPPGKGLEWHeavyof VHof 411D07IGEIFHSGRTNYNPSLKSRVTISIDKSKNQFSLRLSSVTAADTAVYYCchainARDGSGSYWGQGTLVTVSSvariableregion99411D07 -Nucleic acidCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGHeavysequence of VHofACCCTGTCCCTCACCTGCATTGTCTCTGGTGGCTCCATCATCAGTAGTchain411D07GACTGGTGGAATTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGvariableATTGGAGAAATCTTTCATAGTGGGAGGACCAACTACAACCCGTCCCTCregionAAGAGTCGAGTCACCATATCAATAGACAAGTCCAAGAATCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG100411D07 -Amino acid sequenceQVQLQESGPGLVKPSGTLSLTCIVSGGSIISSDWWNWVRQPPGKGLEWfullof 411D07 heavyIGEIFHSGRTNYNPSLKSRVTISIDKSKNQFSLRLSSVTAADTAVYYCheavychainARDGSGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVchainKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGsequenceTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK101411D07 -Nucleic acidCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGfullsequence of 411D07ACCCTGTCCCTCACCTGCATTGTCTCTGGTGGCTCCATCATCAGTAGTheavyheavy chainGACTGGTGGAATTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGchainATTGGAGAAATCTTTCATAGTGGGAGGACCAACTACAACCCGTCCCTCsequenceAAGAGTCGAGTCACCATATCAATAGACAAGTCCAAGAATCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG102411D07 -Amino acid sequenceQSVLYSSNNKNYCDRL1of CDRL1 of 411D07(IMGT)using IMGT103411D07 -Amino acid sequenceWASCDRL2of CDRL2 of 411D07(IMGT)using IMGT104411D07 -Amino acid sequenceQQYYSNRSCDRL3of CDRL3 of 411D07(IMGT)using IMGT105411D07 -Amino acid sequenceKSSQSVLYSSNNKNYLACDRL1of CDRL1 of 411D07(Kabat)using Kabat106411D07 -Amino acid sequenceWASTRESCDRL2of CDRL2 of 411D07(Kabat)using Kabat107411D07 -Amino acid sequenceQQYYSNRSCDRL3of CDRL3 of 411D07(Kabat)using Kabat108411D07 -Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKSGQLightof VLof 411D07PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQTEDVAVYYCQQchainYYSNRSFGQGTKLEIKvariableregion109411D07 -Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCLightsequence of VLofGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCchain411D07TCCAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAATCAGGACAGvariableCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCregionCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTAATCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC110411D07 -Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKSGQfullof 411D07 lightPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQTEDVAVYYCQQlightchainYYSNRSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFchainYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEsequenceKHKVYACEVTHQGLSSPVTKSFNRGEC111411D07 -Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCfullsequence of 411D07GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGClightlight chainTCCAACAATAAGAATTACTTAGCTTGGTACCAGCAGAAATCAGGACAGchainCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCsequenceCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGACTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTAATCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT112385F01 -Amino acid sequenceGFTFSSYWCDRH1of CDRH1 of 385F01(IMGT)using IMGT113385F01 -Amino acid sequenceIKEDGSEKCDRH2of CDRH2 of 385F01(IMGT)using IMGT114385F01 -Amino acid sequenceARNRLYSDFLDNCDRH3of CDRH3 of 385F01(IMGT)using IMGT115385F01 -Amino acid sequenceSYWMSCDRH1 ofCDRH1 of 385F01(Kabat)using Kabat116385F01 -Amino acid sequenceNIKEDGSEKYYVDSVKGCDRH2of CDRH2 of 385F01(Kabat)using Kabat117385F01 -Amino acid sequenceNRLYSDFLDNCDRH3of CDRH3 of 385F01(Kabat)using Kabat118385F01 -Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVHeavyof VHof 385F01ANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCchainARNRLYSDFLDNWGQGTLVTVSSvariableregion119385F01 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATchain385F01TGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGvariableGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGregionAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG120385F01 -Amino acid sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVfullof 385F01 heavyANIKEDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTSVYYCheavychainARNRLYSDFLDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALchainGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSsequenceSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK121385F01 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGfullsequence of 385F01TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGTAGCTATheavyheavy chainTGGATGAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGchainGCCAACATCAAAGAAGATGGAAGTGAGAAATACTATGTCGACTCTGTGsequenceAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGTCTGTGTATTACTGTGCGAGAAATCGACTCTACAGTGACTTCCTTGACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG122385F01 -Amino acid sequenceQGVSSWCDRL1of CDRL1 of 385F01(IMGT)using IMGT123385F01 -Amino acid sequenceGASCDRL2of CDRL2 of 385F01(IMGT)using IMGT124385F01 -Amino acid sequenceQQANSIPFTCDRL3of CDRL3 of 385F01(IMGT)using IMGT125385F01 -Amino acid sequenceRASQGVSSWLACDRL1of CDRL1 of 385F01(Kabat)using Kabat126385F01 -Amino acid sequenceGASSLQSCDRL2of CDRL2 of 385F01(Kabat)using Kabat127385F01 -Amino acid sequenceQQANSIPFTCDRL3of CDRL3 of 385F01(Kabat)using Kabat128385F01 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLILightof VL of 385F01YGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFchainTFGPGTKVDIKvariableregion129385F01 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGALightsequence of VLofGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGchain385F01TTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCvariableTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCregionAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC130385F01 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGVSSWLAWYQQKSGKAPKLLIfullof 385F01 lightYGASSLQSGVPSRFSGSGSGTEFILTISSLQPEDFATYYCQQANSIPFlightchainTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC131385F01 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTCGGAfullsequence of 385F01GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTGTTAGCAGCTGGlightlight chainTTAGCCTGGTATCAGCAGAAATCAGGGAAAGCCCCTAAGCTCCTGATCchainTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGATTCAGCGGCsequenceAGTGGATCTGGGACAGAGTTCATTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTATCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT132413D08 -Amino acid sequenceGFTFRIYGCDRH1of CDRH1 of 413D08(IMGT)using IMGT133413D08 -Amino acid sequenceIWYDGSNKCDRH2of CDRH2 of 413D08(IMGT)using IMGT134413D08 -Amino acid sequenceARDMDYFGMDVCDRH3of CDRH3 of 413D08(IMGT)using IMGT135413D08 -Amino acid sequenceIYGMHCDRH1of CDRH1 of 413D08(Kabat)using Kabat136413D08 -Amino acid sequenceVIWYDGSNKYYADSVKGCDRH2of CDRH2 of 413D08(Kabat)using Kabat137413D08 -Amino acid sequenceDMDYFGMDVCDRH3of CDRH3 of 413D08(Kabat)using Kabat138413D08 -Amino acid sequenceQVQLVESGGGVVQPGRSLRLSCAASGFTFRIYGMHWVRQAPGKGLEWVHeavyof VHof 413D08AVIWYDGSNKYYADSVKGRFTISRDNSDNTLYLQMNSLRAEDTAVYYCchainARDMDYFGMDVWGQGTTVTVSSvariableregion139413D08 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCCGTATTTATchain413D08GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGvariableGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCTGACTCCGTGregionAAGGGCCGATTCACCATCTCCAGAGACAATTCCGACAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATATGGACTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG140413D08 -Amino acid sequenceQVQLVESGGGVVQPGRSLRLSCAASGFTFRIYGMHWVRQAPGKGLEWVfullof 413D08 heavyAVIWYDGSNKYYADSVKGRFTISRDNSDNTLYLQMNSLRAEDTAVYYCheavychainARDMDYFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGchainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSsequenceSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK141413D08 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGfullsequence of 413D08TCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCCGTATTTATheavyheavy chainGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGchainGCAGTTATATGGTATGATGGAAGTAATAAATACTATGCTGACTCCGTGsequenceAAGGGCCGATTCACCATCTCCAGAGACAATTCCGACAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATATGGACTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG142413D08 -Amino acid sequenceQGIRNDCDRL1of CDRL1 of 413D08(IMGT)using IMGT143413D08 -Amino acid sequenceAASCDRL2of CDRL2 of 413D08(IMGT)using IMGT144413D08 -Amino acid sequenceLQHNSYPRTCDRL3of CDRL3 of 413D08(IMGT)using IMGT145413D08 -Amino acid sequenceRASQGIRNDLGCDRL1of CDRL1 of 413D08(Kabat)using Kabat146413D08 -Amino acid sequenceAASSLQSCDRL2of CDRL2 of 413D08(Kabat)using Kabat147413D08 -Amino acid sequenceLQHNSYPRTCDRL3of CDRL3 of 413D08(Kabat)using Kabat148413D08 -Amino acid sequenceDLQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLILightof VLof 413D08YAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRchainTFGQGTKVEIKvariableregion149413D08 -Nucleic acidGACCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGALightsequence of VLofGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATchain413D08TTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCvariableTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCregionAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC150413D08 -Amino acid sequenceDLQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIfullof 413D08 lightYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRlightchainTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC151413D08 -Nucleic acidGACCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAfullsequence of 413D08GACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATlightlight chainTTAGGCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCchainTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCsequenceAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT152386H03 -Amino acid sequenceGGSISSSDWCDRH1of CDRH1 of 386H03(IMGT)using IMGT153386H03 -Amino acid sequenceIFHSGNTCDRH2of CDRH2 of 386H03(IMGT)using IMGT154386H03 -Amino acid sequenceVRDGSGSYCDRH3of CDRH3 of 386H03(IMGT)using IMGT155386H03 -Amino acid sequenceSSDWWSCDRH1of CDRH1 of 386H03(Kabat)using Kabat156386H03 -Amino acid sequenceEIFHSGNTNYNPSLKSCDRH2of CDRH2 of 386H03(Kabat)using Kabat157386H03 -Amino acid sequenceDGSGSYCDRH3of CDRH3 of 386H03(Kabat)using Kabat158386H03 -Amino acid sequenceQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWHeavyof VHof 386H03IGEIFHSGNTNYNPSLKSRVTISVDKSKNQISLRLNSVTAADTAVYYCchainVRDGSGSYWGQGTLVTVSSvariableregion159386H03 -Nucleic acidCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGHeavysequence of VHofACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTchain386H03GACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGvariableATTGGGGAAATCTTTCATAGTGGGAACACCAACTACAACCCGTCCCTCregionAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGATCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGTGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG160386H03 -Amino acid sequenceQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSDWWSWVRQPPGKGLEWfullof 386H03 heavyIGEIFHSGNTNYNPSLKSRVTISVDKSKNQISLRLNSVTAADTAVYYCheavychainVRDGSGSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVchainKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGsequenceTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK161386H03 -Nucleic acidCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGfullsequence of 386H03ACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTheavyheavy chainGACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGchainATTGGGGAAATCTTTCATAGTGGGAACACCAACTACAACCCGTCCCTCsequenceAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGATCTCCCTGAGGCTGAACTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGTGAGAGATGGTTCGGGGAGTTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG162386H03 -Amino acid sequenceQSVLYSSNNKNYCDRL1of CDRL1 of 386H03(IMGT)using IMGT163386H03 -Amino acid sequenceWASCDRL2of CDRL2 of 386H03(IMGT)using IMGT164386H03 -Amino acid sequenceQQYYSTRSCDRL3of CDRL3 of 386H03(IMGT)using IMGT165386H03 -Amino acid sequenceKSSQSVLYSSNNKNYLACDRL1of CDRL1 of 386H03(Kabat)using Kabat166386H03 -Amino acid sequenceWASTRESCDRL2of CDRL2 of 386H03(Kabat)using Kabat167386H03 -Amino acid sequenceQQYYSTRSCDRL3of CDRL3 of 386H03(Kabat)using Kabat168386H03 -Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQLightof VLof 386H03PPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQchainYYSTRSFGQGTKLEIKvariableregion169386H03 -Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCLightsequence of VLofGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCchain386H03TCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGvariableCCTCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCregionCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC170386H03 -Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQfullof 386H03 lightPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQlightchainYYSTRSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFchainYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEsequenceKHKVYACEVTHQGLSSPVTKSFNRGEC171386H03 -Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCfullsequence of 386H03GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGClightlight chainTCCAACAATAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGchainCCTCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCsequenceCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT172389A03 -Amino acid sequenceGGSISSSSYYCDRH1of CDRH1 of 389A03(IMGT)using IMGT173389A03 -Amino acid sequenceIYSTGYTCDRH2of CDRH2 of 389A03(IMGT)using IMGT174389A03 -Amino acid sequenceAISTAAGPEYFHRCDRH3of CDRH3 of 389A03(IMGT)using IMGT175389A03 -Amino acid sequenceSSSYYCGCDRH1of CDRH1 of 389A03(Kabat)using Kabat176389A03 -Amino acid sequenceSIYSTGYTYYNPSLKSCDRH2of CDRH2 of 389A03(Kabat)using Kabat177389A03 -Amino acid sequenceSTAAGPEYFHRCDRH3of CDRH3 of 389A03(Kabat)using Kabat178389A03 -Amino acid sequenceQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYCGWIRQPPGKGLDWIHeavyof VHof 389A03GSIYSTGYTYYNPSLKSRVTISIDTSKNQFSCLILTSVTAADTAVYYCchainAISTAAGPEYFHRWGQGTLVTVSSvariableregion179389A03 -Nucleic acidCAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCGGAGACCCTGHeavysequence of VHofTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTATchain389A03TACTGCGGCTGGATCCGCCAGCCCCCTGGGAAGGGGCTGGACTGGATTvariableGGGAGTATCTATTCTACTGGGTACACCTACTACAACCCGTCCCTCAAGregionAGTCGAGTCACCATTTCCATAGACACGTCCAAGAACCAGTTCTCATGCCTGATACTGACCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGATAAGTACAGCAGCTGGCCCTGAATACTTCCATCGCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAG180389A03 -Amino acid sequenceQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYCGWIRQPPGKGLDWIfullof 389A03 heavyGSIYSTGYTYYNPSLKSRVTISIDTSKNQFSCLILTSVTAADTAVYYCheavychainAISTAAGPEYFHRWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAchainLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPsequenceSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK181389A03 -Nucleic acidCAGCTGCAGGAGTCGGGCCCAGGCCTGGTGAAGCCTTCGGAGACCCTGfullsequence of 389A03TCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTATheavyheavy chainTACTGCGGCTGGATCCGCCAGCCCCCTGGGAAGGGGCTGGACTGGATTchainGGGAGTATCTATTCTACTGGGTACACCTACTACAACCCGTCCCTCAAGsequenceAGTCGAGTCACCATTTCCATAGACACGTCCAAGAACCAGTTCTCATGCCTGATACTGACCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGATAAGTACAGCAGCTGGCCCTGAATACTTCCATCGCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG182389A03 -Amino acid sequenceQSVLYSSNSKNFCDRL1of CDRL1 of 389A03(IMGT)using IMGT183389A03 -Amino acid sequenceWASCDRL2of CDRL2 of 389A03(IMGT)using IMGT184389A03 -Amino acid sequenceQQYYSTPRTCDRL3of CDRL3 of 389A03(IMGT)using IMGT185389A03 -Amino acid sequenceKSSQSVLYSSNSKNFLACDRL1of CDRL1 of 389A03(Kabat)using Kabat186389A03 -Amino acid sequenceWASTRGSCDRL2of CDRL2 of 389A03(Kabat)using Kabat187389A03 -Amino acid sequenceQQYYSTPRTCDRL3of CDRL3 of 389A03(Kabat)using Kabat188389A03 -Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNFLAWYQQKPGQLightof VLof 389A03PPKLFIYWASTRGSGVPDRISGSGSGTDFNLTISSLQAEDVAVYYCQQchainYYSTPRTFGQGTKVEIKvariableregion189389A03 -Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCLightsequence of VLofGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGCchain389A03TCCAACAGTAAGAACTTCTTAGCTTGGTACCAGCAGAAACCGGGACAGvariableCCTCCTAAGCTGTTCATTTACTGGGCATCTACCCGGGGATCCGGGGTCregionCCTGACCGAATCAGTGGCAGCGGGTCTGGGACAGATTTCAATCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAAC190389A03 -Amino acid sequenceDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNFLAWYQQKPGQfullof 389A03 lightPPKLFIYWASTRGSGVPDRISGSGSGTDFNLTISSLQAEDVAVYYCQQlightchainYYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNchainFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYsequenceEKHKVYACEVTHQGLSSPVTKSFNRGEC191389A03 -Nucleic acidGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCfullsequence of 389A03GAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTATACAGClightlight chainTCCAACAGTAAGAACTTCTTAGCTTGGTACCAGCAGAAACCGGGACAGchainCCTCCTAAGCTGTTCATTTACTGGGCATCTACCCGGGGATCCGGGGTCsequenceCCTGACCGAATCAGTGGCAGCGGGTCTGGGACAGATTTCAATCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAACAATATTATAGTACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT192HumanIGHG*01 &Heavy ChaingcttccaccaagggcccatccgtcttccccctggcgccctgctccaggIgG4IGHG4*04ConstantagcacctccgagagcacagccgccctgggctgcctggtcaaggactacheavyRegionttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcchainNucleotideggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccconstantSequencectcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagaccregiontacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaag#1agagttgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa193Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSConstantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRegionRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAmino AcidDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDSequenceWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK194HumanIGHG*02Heavy ChaingcttccaccaagggcccatccgtcttccccctggcgccctgctccaggIgG4ConstantagcacctccgagagcacagccgccctgggctgcctggtcaaggactacheavyRegionttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcchainNucleotideggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccconstantSequencectcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagaccregiontacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaag#2agagttgagtccaaatatggtcccccgtgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctctgggtaaa195Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSConstantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRegionRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAmino AcidDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDSequenceWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK196HumanIGHG*03Heavy ChaingcttccaccaagggcccatccgtcttccccctggcgccctgctccaggIgG4ConstantagcacctccgagagcacagccgccctgggctgcctggtcaaggactacheavyRegionttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcchainNucleotideggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccconstantSequencectcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagaccregiontacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaag#3agagttgagtccaaatatggtcccccatgcccatcatgcccagcacctgagttcctggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcaggaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctctgggtaaa197Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSConstantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRegionRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAmino AcidDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDSequenceWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK198IgG4-Heavy ChaingcctccaccaagggcccatccgtcttccccctggcgccctgctccaggheavyIgG4-ConstantagcacctccgagagcacggccgccctgggctgcctggtcaaggactacchainPERegionttccccgaaccagtgacggtgtcgtggaactcaggcgccctgaccagcconstantNucleotideggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccregion -Sequence -ctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagaccIgG4-PESynthetictacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagVersion Aagagttgagtccaaatatggtcccccatgcccaccatgcccagcgcctgaatttgaggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggatccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa199IgG4Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSheavyConstantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKchainRegionRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVconstantAmino AcidDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDregion -Sequence -WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKIgG4-PEEncoded byNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSSyntheticRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKVersion A,B & C(Tworesiduesthat differfrom thewild-typesequenceareidentifiedin bold)200IgG4Heavy ChainGcctccaccaagggacctagcgtgttccctctcgccccctgttccaggheavyConstanttccacaagcgagtccaccgctgccctcggctgtctggtgaaagactacchainRegiontttcccgagcccgtgaccgtctcctggaatagcggagccctgacctccconstantNucleotideggcgtgcacacatttcccgccgtgctgcagagcagcggactgtatagcregion -Sequence -ctgagcagcgtggtgaccgtgcccagctccagcctcggcaccaaaaccIgG4-PESynthetictacacctgcaacgtggaccacaagccctccaacaccaaggtggacaagVersion Bcgggtggagagcaagtacggccccccttgccctccttgtcctgcccctgagttcgagggaggaccctccgtgttcctgtttccccccaaacccaaggacaccctgatgatctcccggacacccgaggtgacctgtgtggtcgtggacgtcagccaggaggaccccgaggtgcagttcaactggtatgtggacggcgtggaggtgcacaatgccaaaaccaagcccagggaggagcagttcaattccacctacagggtggtgagcgtgctgaccgtcctgcatcaggattggctgaacggcaaggagtacaagtgcaaggtgtccaacaagggactgcccagctccatcgagaagaccatcagcaaggctaagggccagccgagggagccccaggtgtataccctgcctcctagccaggaagagatgaccaagaaccaagtgtccctgacctgcctggtgaagggattctacccctccgacatcgccgtggagtgggagagcaatggccagcccgagaacaactacaaaacaacccctcccgtgctcgatagcgacggcagcttctttctctacagccggctgacagtggacaagagcaggtggcaggagggcaacgtgttctcctgttccgtgatgcacgaggccctgcacaatcactacacccagaagagcctctccctgtccctgggcaag201IgG4Heavy ChaingccagcaccaagggcccttccgtgttccccctggccccttgcagcaggheavyConstantagcacctccgaatccacagctgccctgggctgtctggtgaaggactacchainRegiontttcccgagcccgtgaccgtgagctggaacagcggcgctctgacatccconstantNucleotideggcgtccacacctttcctgccgtcctgcagtcctccggcctctactccregion -Sequence -ctgtcctccgtggtgaccgtgcctagctcctccctcggcaccaagaccIgG4-PESynthetictacacctgtaacgtggaccacaaaccctccaacaccaaggtggacaaaVersion Ccgggtcgagagcaagtacggccctccctgccctccttgtcctgcccccgagttcgaaggcggacccagcgtgttcctgttccctcctaagcccaaggacaccctcatgatcagccggacacccgaggtgacctgcgtggtggtggatgtgagccaggaggaccctgaggtccagttcaactggtatgtggatggcgtggaggtgcacaacgccaagacaaagccccgggaagagcagttcaactccacctacagggtggtcagcgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtacaagtgcaaggtcagcaataagggactgcccagcagcatcgagaagaccatctccaaggctaaaggccagccccgggaacctcaggtgtacaccctgcctcccagccaggaggagatgaccaagaaccaggtgagcctgacctgcctggtgaagggattctacccttccgacatcgccgtggagtgggagtccaacggccagcccgagaacaattataagaccacccctcccgtcctcgacagcgacggatccttctttctgtactccaggctgaccgtggataagtccaggtggcaggaaggcaacgtgttcagctgctccgtgatgcacgaggccctgcacaatcactacacccagaagtccctgagcctgtccctgggaaag202IgG4Heavy ChaingcctccaccaagggcccatccgtcttccccctggcgccctgctccaggheavyConstantagcacctccgagagcacggccgccctgggctgcctggtcaaggactacchainRegionttccccgaaccagtgacggtgtcgtggaactcaggcgccctgaccagcconstantNucleotideggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccregionSequence -ctcagcagcgtggtgaccgtgccctccagcagcttgggcacgaagaccSynthetictacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagVersion Dagagttgagtccaaatatggtcccccatgcccaccatgcccagcgcctccagttgcggggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccgtcatcgatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggatccttcttcctctacagcaggctaaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctctgggtaaa203Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSConstantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRegionRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAmino AcidDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDSequence -WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKencoded byNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSSyntheticRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKVersion D204DisabledDisabledHeavy ChaingcctccaccaagggcccatcggtcttccccctggcaccctcctccaagHumanIGHG1ConstantagcacctctgggggcacagcggccctgggctgcctggtcaaggactacIgG1RegionttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcheavyNucleotideggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccchainSequencectcagcagcgtggtgaccgtgccctccagcagcttgggcacccagaccconstanttacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagregionaaagtggagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcgcgggggcaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa205Heavy ChainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSConstantGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRegionKVEPKSCDKTHTCPPCPAPELAGAPSVFLEPPKPKDTLMISRTPEVTCAmino AcidVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLSequenceHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE(TwoLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFresiduesLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKthat differfrom thewild-typesequenceareidentifiedin bold)206Human CκIGKC*01Cκ LightcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagconstantChaincagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttcregionConstanttacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagRegiontccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcNucleotideacctactccctgtcctccaccctgaccctgtccaaggccgactacgagSequenceaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt207Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQChainSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSConstantPVTKSFNRGECRegionAmino AcidSequence208Human CκIGKC*02Cκ LightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagconstantChaincagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcregionConstanttatcccagagaggccaaagtacagtggaaggtggataacgccctccaaRegiontcgggtaactcccaggagagtgtcacagagcaggagagcaaggacagcNucleotideacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagSequenceaaacacaaagtctacgccggcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt209Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQChainSGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYAGEVTHQGLSSConstantPVTKSFNRGECRegionAmino AcidSequence210Human CκIGKC*03Cκ LightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagconstantChaincagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcregionConstanttatcccagagaggccaaagtacagcggaaggtggataacgccctccaaRegiontcgggtaactcccaggagagtgtcacagagcaggagagcaaggacagcNucleotideacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagSequenceaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt211Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQRKVDNALQChainSGNSQESVTEQESKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSConstantPVTKSFNRGECRegionAmino AcidSequence212Human CκIGKC*04Cκ LightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagconstantChaincagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcregionConstanttatcccagagaggccaaagtacagtggaaggtggataacgccctccaaRegiontcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcNucleotideacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagSequenceaaacacaaactctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt213Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQChainSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSConstantPVTKSFNRGECRegionAmino AcidSequence214Human CκIGKC*05Cκ LightcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagconstantChaincagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttcregionConstanttatcccagagaggccaaagtacagtggaaggtggataacgccctccaaRegiontcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcNucleotideacctacagcctcagcaacaccctgacgctgagcaaagcagactacgagSequenceaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc215Cκ LightRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQChainSGNSQESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSConstantPVTKSFNRGECRegionAmino AcidSequence216Human CλIGCλ1*01Cλ LightcccaaggccaaccccacggtcactctgttcccgccctcctctgaggagconstantChainctccaagccaacaaggccacactagtgtgtctgatcagtgacttctacregionConstantccgggagctgtgacagtggcttggaaggcagatggcagccccgtcaagRegiongcgggagtggagacgaccaaaccctccaaacagagcaacaacaagtacNucleotidegcggccagcagctacctgagcctgacgcccgagcagtggaagtcccacSequenceagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca217Cλ LightPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKChainAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKConstantTVAPTECSRegionAmino AcidSequence218Human CλIGCλ1*02Cλ LightggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctconstantChaingaggagctccaagccaacaaggccacactagtgtgtctgatcagtgacregionConstantttctacccgggagctgtgacagtggcctggaaggcagatggcagccccRegiongtcaaggcgggagtggagaccaccaaaccctccaaacagagcaacaacNucleotideaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagSequencetcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca219Cλ LightGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPChainVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVConstantEKTVAPTECSRegionAmino AcidSequence220Human CλIGCλ2*01Cλ LightggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctconstantChaingaggagctccaagccaacaaggccacactagtgtgtctgatcagtgacregionConstantttctacccgggagctgtgacagtggcctggaaggcagatggcagccccRegiongtcaaggcgggagtggagaccaccaaaccctccaaacagagcaacaacNucleotideaagtacgcggccagcagctacctgagcctgacgcccgagcagtggaagSequence -tcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtgVersion Agagaagacagtggcccctacagaatgttca221Cλ LightggccagcctaaggccgctccttctgtgaccctgttccccccatcctccChaingaggaactgcaggctaacaaggccaccctcgtgtgcctgatcagcgacConstantttctaccctggcgccgtgaccgtggcctggaaggctgatagctctcctRegiongtgaaggccggcgtggaaaccaccaccccttccaagcagtccaacaacNucleotideaaatacgccgcctcctcctacctgtccctgacccctgagcagtggaagSequence -tcccaccggtcctacagctgccaagtgacccacgagggctccaccgtgVersion Bgaaaagaccgtggctcctaccgagtgctcc222Cλ LightggccagcctaaagctgcccccagcgtcaccctgtttcctccctccagcChaingaggagctccaggccaacaaggccaccctcgtgtgcctgatctccgacConstantttctatcccggcgctgtgaccgtggcttggaaagccgactccagccctRegiongtcaaagccggcgtggagaccaccacaccctccaagcagtccaacaacNucleotideaagtacgccgcctccagctatctctccctgacccctgagcagtggaagSequence -tcccaccggtcctactcctgtcaggtgacccacgagggctccaccgtgVersion Cgaaaagaccgtcgcccccaccgagtgctcc223Cλ LightGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPChainVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVConstantEKTVAPTECSRegionAmino AcidSequence -Encoded byVersion A,B & C224Human CλIGCλ2*02 &Cλ LightggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctconstantIGLC2*03ChaingaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacregionConstantttctacccgggagccgtgacagtggcctggaaggcagatagcagccccRegiongtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacNucleotideaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagSequencetcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca225Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPChainVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVConstantEKTVAPTECSRegionAmino AcidSequence226Human CλIGCλ3*01Cλ LightcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagconstantChaincttcaagccaacaaggccacactggtgtgtctcataagtgacttctacregionConstantccgggagccgtgacagttgcctggaaggcagatagcagccccgtcaagRegiongcgggggtggagaccaccacaccctccaaacaaagcaacaacaagtacNucleotidegcggccagcagctacctgagcctgacgcctgagcagtggaagtcccacSequenceaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagttgcccctacggaatgttca227Cλ LightPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKChainAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKConstantTVAPTECSRegionAmino AcidSequence228Human CλIGCλ3*02Cλ LightggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctconstantChaingaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacregionConstantttctacccggggccagtgacagttgcctggaaggcagatagcagccccRegiongtcaaggcgggggtggagaccaccacaccctccaaacaaagcaacaacNucleotideaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagSequencetcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacggaatgttca229Cλ LightGQPKAAPSVTLEPPSSEELQANKATLVCLISDFYPGPVTVAWKADSSPChainVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVConstantEKTVAPTECSRegionAmino AcidSequence230Human CλIGCλ3*03Cλ LightggtcagcccaaggctgccccctcggtcactctgttcccaccctcctctconstantChaingaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacregionConstantttctacccgggagccgtgacagtggcctggaaggcagatagcagccccRegiongtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacNucleotideaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagSequencetcccacaaaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca231Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPChainVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVConstantEKTVAPTECSRegionAmino AcidSequence232Human CλIGCλ3*04Cλ LightggtcagcccaaggctgccccctcggtcactctgttcccgccctcctctconstantChaingaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacregionConstantttctacccgggagccgtgacagtggcctggaaggcagatagcagccccRegiongtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacNucleotideaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagSequencetcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca233Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPChainVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVConstantEKTVAPTECSRegionAmino AcidSequence234Human CλIGCλ6*01Cλ LightggtcagcccaaggctgccccatcggtcactctgttcccgccctcctctconstantChaingaggagcttcaagccaacaaggccacactggtgtgcctgatcagtgacregionConstantttctacccgggagctgtgaaagtggcctggaaggcagatggcagccccRegiongtcaacacgggagtggagaccaccacaccctccaaacagagcaacaacNucleotideaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagSequencetcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgttca235Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPChainVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVConstantEKTVAPAECSRegionAmino AcidSequence236Human CλIGLC7*01 &Cλ LightggtcagcccaaggctgccccatcggtcactctgttcccaccctcctctconstantIGCλ7*02ChaingaggagcttcaagccaacaaggccacactggtgtgtctcgtaagtgacregionConstantttctacccgggagccgtgacagtggcctggaaggcagatggcagccccRegiongtcaaggtgggagtggagaccaccaaaccctccaaacaaagcaacaacNucleotideaagtatgcggccagcagctacctgagcctgacgcccgagcagtggaagSequencetcccacagaagctacagctgccgggtcacgcatgaagggagcaccgtggagaagacagtggcccctgcagaatgctct237Cλ LightGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPChainVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVConstantEKTVAPAECSRegionAmino AcidSequence238413G05 -Amino acid sequenceGFTFSDYYCDRH1of CDRH1 of 413G05(IMGT)using IMGT239413G05 -Amino acid sequenceISTSGSTICDRH2of CDRH2 of 413G05(IMGT)using IMGT240413G05 -Amino acid sequenceARGITGTNFYHYGLGVCDRH3of CDRH3 of 413G05(IMGT)using IMGT241413G05 -Amino acid sequenceDYYMSCDRH1of CDRH1 of 413G05(Kabat)using Kabat242413G05 -Amino acid sequenceYISTSGSTIYYADSVKGCDRH2of CDRH2 of 413G05(Kabat)using Kabat243413G05 -Amino acid sequenceGITGTNFYHYGLGVCDRH3of CDRH3 of 413G05(Kabat)using Kabat244413G05 -Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQVPGKGLEWVHeavyof VHof 413G05SYISTSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDAAVYHCchainARGITGTNFYHYGLGVWGQGTTVTVSSvariableregion245413G05 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACchain413G05TACATGAGCTGGATCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTvariableTCATACATTAGTACTAGTGGTAGTACCATATACTACGCAGACTCTGTGregionAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACGCGGCCGTGTATCACTGTGCGAGAGGTATAACTGGAACTAACTTCTACCACTACGGTTTGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG246413G05 -Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQVPGKGLEWVfullof 413G05 heavySYISTSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDAAVYHCheavychainARGITGTNFYHYGLGVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGchainTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVsequenceTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK247413G05 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGfullsequence of 413G05TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACheavyheavy chainTACATGAGCTGGATCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTTchainTCATACATTAGTACTAGTGGTAGTACCATATACTACGCAGACTCTGTGsequenceAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTACAAATGAACAGCCTGAGAGCCGAGGACGCGGCCGTGTATCACTGTGCGAGAGGTATAACTGGAACTAACTTCTACCACTACGGTTTGGGCGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG248413G05 -Amino acid sequenceQGINSWCDRL1of CDRL1 of 413G05(IMGT)using IMGT249413G05 -Amino acid sequenceAASCDRL2of CDRL2 of 413G05(IMGT)using IMGT250413G05 -Amino acid sequenceQQVNSFPLTCDRL3of CDRL3 of 413G05(IMGT)using IMGT251413G05 -Amino acid sequenceRASQGINSWLACDRL1of CDRL1 of 413G05(Kabat)using Kabat252413G05 -Amino acid sequenceAASTLQSCDRL2of CDRL2 of 413G05(Kabat)using Kabat253413G05 -Amino acid sequenceQQVNSFPLTCDRL3of CDRL3 of 413G05(Kabat)using Kabat254413G05 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPKLLILightof VLof 413G05YAASTLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQVNSFPLchainTFGGGTKVEIKvariableregion255413G05 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGALightsequence of VLofGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAACAGCTGGchain413G05TTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCvariableTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCregionAGTGGGTCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC256413G05 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGINSWLAWYQQKPGKAPKLLIfullof 413G05 lightYAASTLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQVNSFPLlightchainTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC257413G05 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAfullsequence of 413G05GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAACAGCTGGlightlight chainTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCchainTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCsequenceAGTGGGTCTGGGGCAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGTTAACAGTTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT258413F09 -Amino acid sequenceGFTFSYYACDRH1of CDRH1 of 413F09(IMGT)using IMGT259413F09 -Amino acid sequenceISGGGGNTCDRH2of CDRH2 of 413F09(IMGT)using IMGT260413F09 -Amino acid sequenceAKDRMKQLVRAYYFDYCDRH3of CDRH3 of 413F09(IMGT)using IMGT261413F09 -Amino acid sequenceYYAMSCDRH1of CDRH1 of 413F09(Kabat)using Kabat262413F09 -Amino acid sequenceTISGGGGNTHYADSVKGCDRH2of CDRH2 of 413F09(Kabat)using Kabat263413F09 -Amino acid sequenceDRMKQLVRAYYFDYCDRH3of CDRH3 of 413F09(Kabat)using Kabat264413F09 -Amino acid sequenceEVPLVESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLDWVHeavyof VHof 413F09STISGGGGNTHYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCchainAKDRMKQLVRAYYFDYWGQGTLVTVSSvariableregion265413F09 -Nucleic acidGAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCTACTATchain413F09GCCATGAGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGACTGGGTCvariableTCAACTATTAGTGGTGGTGGTGGTAACACACACTACGCAGACTCCGTGregionAAGGGCCGATTCACTATATCCAGAGACAATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAAGACACGGCCGTCTATTACTGTGCGAAGGATCGGATGAAACAGCTCGTCCGGGCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG266413F09 -Amino acid sequenceEVPLVESGGGLVQPGGSLRLSCAASGFTFSYYAMSWVRQAPGKGLDWVfullof 413F09 heavySTISGGGGNTHYADSVKGRFTISRDNSKNTLYLHMNSLRAEDTAVYYCheavychainAKDRMKQLVRAYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGchainTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVsequenceTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK267413F09 -Nucleic acidGAGGTGCCGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGfullsequence of 413F09TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTTAGCTACTATheavyheavy chainGCCATGAGCTGGGTCCGTCAGGCTCCAGGGAAGGGGCTGGACTGGGTCchainTCAACTATTAGTGGTGGTGGTGGTAACACACACTACGCAGACTCCGTGsequenceAAGGGCCGATTCACTATATCCAGAGACAATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGAGCCGAAGACACGGCCGTCTATTACTGTGCGAAGGATCGGATGAAACAGCTCGTCCGGGCCTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG268413F09 -Amino acid sequenceQDISTYCDRL1of CDRL1 of 413F09(IMGT)using IMGT269413F09 -Amino acid sequenceGTSCDRL2of CDRL2 of 413F09(IMGT)using IMGT270413F09 -Amino acid sequenceQQLHTDPITCDRL3of CDRL3 of 413F09(IMGT)using IMGT271413F09 -Amino acid sequenceWASQDISTYLGCDRL1of CDRL1 of 413F09(Kabat)using Kabat272413F09 -Amino acid sequenceGTSSLQSCDRL2of CDRL2 of 413F09(Kabat)using Kabat273413F09 -Amino acid sequenceQQLHTDPITCDRL3of CDRL3 of 413F09(Kabat)using Kabat274413F09 -Amino acid sequenceDIQLTQSPSFLSASVGDRVTITCWASQDISTYLGWYQQKPGKAPKLLILightof VLof 413F09YGTSSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLHTDPIchainTFGQGTRLEIKvariableregion275413F09 -Nucleic acidGACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGALightsequence of VLofGACAGAGTCACCATCACTTGCTGGGCCAGTCAGGACATTAGCACTTATchain413F09TTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCvariableTATGGTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCregionAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTCATACTGACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC276413F09 -Amino acid sequenceDIQLTQSPSFLSASVGDRVTITCWASQDISTYLGWYQQKPGKAPKLLIfullof 413F09 lightYGTSSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLHTDPIlightchainTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC277413F09 -Nucleic acidGACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAfullsequence of 413F09GACAGAGTCACCATCACTTGCTGGGCCAGTCAGGACATTAGCACTTATlightlight chainTTAGGCTGGTATCAGCAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCchainTATGGTACATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCsequenceAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGCTTCATACTGACCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT278414806 -Amino acid sequenceGFTFSSYWCDRH1of CDRH1 of 414B06(IMGT)using IMGT279414B06 -Amino acid sequenceIKQDGSEKCDRH2of CDRH2 of 414B06(IMGT)using IMGT280414B06 -Amino acid sequenceARVRQWSDYSDYCDRH3of CDRH3 of 414B06(IMGT)using IMGT281414B06 -Amino acid sequenceSYWMNCDRH1of CDRH1 of 414B06(Kabat)using Kabat282414B06 -Amino acid sequenceNIKQDGSEKYYVDSVKGCDRH2of CDRH2 of 414B06(Kabat)using Kabat283414B06 -Amino acid sequenceVRQWSDYSDYCDRH3of CDRH3 of 414B06(Kabat)using Kabat284414B06 -Amino acid sequenceEVHLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVHeavyof VHof 414B06ANIKQDGSEKYYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCchainARVRQWSDYSDYWGQGTPVTVSSvariableregion285414B06 -Nucleic acidGAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGHeavysequence of VHofTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATchain414B06TGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGvariableGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGregionAAGGGCCGCTTCACCGTCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTCGACAATGGTCCGACTACTCTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAG286414B06 -Amino acid sequenceEVHLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVfullof 414B06 heavyANIKQDGSEKYYVDSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCheavychainARVRQWSDYSDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALchainGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSsequenceSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK287414B06 -Nucleic acidGAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGfullsequence of 414B06TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATheavyheavy chainTGGATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGchainGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGsequenceAAGGGCCGCTTCACCGTCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTCGACAATGGTCCGACTACTCTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAG288414B06 -Amino acid sequenceQGISSWCDRL1of CDRL1 of 414B06(IMGT)using IMGT289414B06 -Amino acid sequenceAASCDRL2of CDRL2 of 414B06(IMGT)using IMGT290414306 -Amino acid sequenceQQANSFPFTCDRL3of CDRL3 of 414B06(IMGT)using IMGT291414306 -Amino acid sequenceRASQGISSWLACDRL1of CDRL1 of 414B06(Kabat)using Kabat292414B06 -Amino acid sequenceAASSLQSCDRL2of CDRL2 of 414B06(Kabat)using Kabat293414B06 -Amino acid sequenceQQANSFPFTCDRL3of CDRL3 of 414B06(Kabat)using Kabat294414306 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLILightof VLof 414B06.YAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFchainTFGPGTKVDIKvariableregion295414B06 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGALightsequence of VLofGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGchain414B06TTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCvariableTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCregionAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC296414B06 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIfullof 414B06 lightYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFlightchainTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAchainKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYsequenceACEVTHQGLSSPVTKSFNRGEC297414B06 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAfullsequence of 414B06GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGlightlight chainTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCchainTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCsequenceAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT298MutatedAmino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI1D05 -of 1D05 kappa lightYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPILCchain with V to YTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAmutant 3mutation in CDRL2KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYhighlightedACEVTHQGLSSPVTKSFNRGEC2991D05 -Amino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWVheavyof IgG1 disabledSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCchainvariant of 1D05AKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGdisabledTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIgG1 FcTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK3001D05 -1D05 Light chainDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIlightsequence fused toYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPIchainwild-type human IL-TFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAIL-22 sequence (IL-2KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYfusionamino acid sequenceACEVTHQGLSSPVTKSFNRGECAPTSSSTKKTQLQLEHLLLDLQMILNis underlined andGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQregion to be variedSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWIis shown in bold)TFCQSIISTLT301HumanUniprot number:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKIL-2P60568KATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELFull length aminoKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTacid sequence ofhuman IL-2 (minussignal sequence)302ControlHeavy chain 1D05EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQVPGKGLEWV1D05IgG1 variant fusedSGISWIRTGIGYADSVKGRFTIFRDNAKNSLYLQMNSLRAEDTALYYCimmunocytokineat the N-terminusAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGHC C-to wild-type humanTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVterminalIL2 sequenceTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELfusion(control)AGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT303IL-2 D5-9IL-2 IC45 (Del 5-9)APTSTQLQLELLLDN terminal IL-2sequence304IL-2 D1-9IL-2 IC46 (Del 1-9)TQLQLEHLLLDN terminal IL-2sequence305IL-2 D5-7IL-2 IC64 (Del 5-7)APTSKKTQLQLEHLLLDN terminal IL-2sequence306IL-2 D1IL-2 D1 N terminalPTSSSTKKTQLQLEHLLLDIL-2 sequence307IL-2 D1-2IL-2 D1-2 NTSSSTKKTQLQLEHLLLDterminal IL-2sequence308IL-2 D1-3IL-2 D1-3 NSSSTKKTQLQLEHLLLDterminal IL-2sequence309IL-2 D1-4IL-2 D1-4 NSSTKKTQLQLEHLLLDterminal IL-2sequence310IL-2 D1-5IL-2 D1-5 NSTKKTQLQLEHLLLDterminal IL-2sequence311IL-2 D1-6IL-2 D1-6 NTKKTQLQLEHLLLDterminal IL-2sequence312IL-2 D1-7IL-2 D1-7 NKKTQLQLEHLLLDterminal IL-2sequence313IL-2 D1-8IL-2 D1-8 NKTQLQLEHLLLDterminal IL-2sequence314IL-2 D9IL-2 D9 N terminalAPTSSSTKTQLQLEHLLLDIL-2 sequence315IL-2 D9-8IL-2 D9-8 NAPTSSSTTQLQLEHLLLDterminal IL-2sequence316IL-2 D9-7IL-2 D9-7 NAPTSSSTQLQLEHLLLDterminal IL-2sequence317IL-2 D9-6IL-2 D9-6 NAPTSSTQLQLEHLLLDterminal IL-2sequence318IL-2 D9-4IL-2 D9-4 NAPTTQLQLEHLLLDterminal IL-2sequence319IL-2 D9-3IL-2 D9-3 NAPTQLQLEHLLLDterminal IL-2sequence320IL-2 D9-2IL-2 D9-2 NATQLQLEHLLLDterminal IL-2sequence321IL-2 D2-6IL-2 D2-6 NATKKTQLQLEHLLLDterminal IL-2sequence322IL-2 D3-7IL-2 D3-7 NAPKKTQLQLEHLLLDterminal IL-2sequence323IL-2 D4-8IL-2 D4-8 NAPTKTQLQLEHLLLDterminal IL-2sequence324C-Amino acids 21 toLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEterminal133 of hIL-2VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEaminoFLNRWITFCQSIISTLTacidsequenceof hIL-2325MouseUniprot number:MRIFAGIIFTACCHLLRAFTITAPEDLYVVEYGSNVTMECRFPVERELPD-L1Q9EP73DLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGN(ECD highlighted inAALQITDVKLQDAGVYCCIISYGGADYKRITLEVNAPYRKINQRISVDBOLD, andPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVcytoplasmic domainTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHWunderlined)VLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET326MouseMouse PD-L1FTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQPD-L1extracellularFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCECD Hisdomain with his tagIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHHHHHH327HumanHuman IL-2 receptorELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGIL-2Rαalpha chainNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDchainQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI328HumanHuman IL-2 receptorAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCIL-2Rβbeta chainELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQchainDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV329HumanHuman IL-2 receptorLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMIL-2Rγcommon gamma chainNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQchainKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET330IL-7Human IL-7 aminoDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDacid sequenceANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH331IL-15Human IL-15 aminoGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESacid sequenceDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS332IL-21Human IL-21 aminoQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSacid sequenceCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLFRFKSLLQKMIHQHLSSRTHGSEDS333GM-CSFHuman GM-CSF aminoAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDacid sequenceLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE334IFNαHuman IFN-α aminoCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLacid sequenceQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTELHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGS335TNFαExtracellularGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWportion of humanLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTNF-α amino acidTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFsequenceQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL336IL-12αAlpha chain ofRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDhuman IL-12 aminoHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFacid sequenceMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS337IL-12βBeta chain of humanIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGIL-12 amino acidSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKsequenceDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS338CXCL9Human CXCL-9 aminoTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGacid sequenceVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT339CXCL10Human CXCL-10 aminoVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKacid sequenceGEKRCLNPESKAIKNLLKAVSKERSKRSP340Human WTIGHG1*01 &WT humanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSIgG1IGHG1*02 &IgG1 aminoGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKconstantIGHG1*05acidKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCregion(IgG1)sequenceVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK341WT humanGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGIgG1TCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACnucleicTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCacidGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCsequenceCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA342MutatedAmino acid sequenceEVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV1D05 -of 1D05 heavy chainSGISWIRTGIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCHCwith V to A and FAKDMKGSGTYGGWFDTWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESmutant 2to S back-mutationTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVin framework regionTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPELAGAto germlinePSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHhighlighted withNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEIgG1 disabledKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW(LAGA) constantESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHregionEALHNHYTQKSLSLSLGK TABLE S2SEQ ID NOS: 343-538SEQIDNO:NameDescriptionSequence343416E01 -Amino acid sequenceGFTFSNYACDRH1of CDRH1 of 416E01(IMGT)using IMGT344416E01 -Amino acid sequenceISFSGGTTCDRH2of CDRH2 of 416E01(IMGT)using IMGT345416E01 -Amino acid sequenceAKDEAPAGATFFDSCDRH3of CDRH3 of 416E01(IMGT)using IMGT346416E01 -Amino acid sequenceNYAMSCDRH1of CDRH1 of 416E01(Kabat)using Kabat347416E01 -Amino acid sequenceAISFSGGTTYYADSVKGCDRH2of CDRH2 of 416E01(Kabat)using Kabat348416E01 -Amino acid sequenceDEAPAGATFFDSCDRH3of CDRH3 of 416E01(Kabat)using Kabat349416E01 -Amino acid sequenceEVQLAESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQTPGKGLEHeavyof VHof 416E01WVSAISFSGGTTYYADSVKGRFTISRDNSKNTLYLHMNSLRADDTAchain(mutations fromVYYCAKDEAPAGATFFDSWGQGTLVTVSSvariablegermline are shownregionin bold letters)350416E01 -Nucleic acidGAAGTGCAACTGGCGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGHeavysequence of VHofGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAAchain416E01CTATGCCATGAGTTGGGTCCGCCAGACTCCAGGAAAGGGGCTGGAGvariableTGGGTCTCAGCTATTAGTTTTAGTGGTGGTACTACATACTACGCTGregionACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCACATGAACAGCCTGAGAGCCGATGACACGGCCGTATATTACTGTGCGAAAGATGAGGCACCAGCTGGCGCAACCTTCTTTGACTCCTGGGGCCAGGGAACGCTGGTCACCGTCTCCTCAG351416E01 -Amino acid sequenceEVQLAESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQTPGKGLEfullof 416E01 heavyWVSAISFSGGTTYYADSVKGRFTISRDNSKNTLYLHMNSLRADDTAheavychainVYYCAKDEAPAGATFFDSWGQGTLVTVSSASTKGPSVFPLAPCSRSchainTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYsequenceSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK352416E01 -Nucleic acidGAAGTGCAACTGGCGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGfullsequence of 416E01GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAAheavyheavy chainCTATGCCATGAGTTGGGTCCGCCAGACTCCAGGAAAGGGGCTGGAGchainTGGGTCTCAGCTATTAGTTTTAGTGGTGGTACTACATACTACGCTGsequenceACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATTTGCACATGAACAGCCTGAGAGCCGATGACACGGCCGTATATTACTGTGCGAAAGATGAGGCACCAGCTGGCGCAACCTTCTTTGACTCCTGGGGCCAGGGAACGCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCTTCCGTGTTCCCCCTGGCCCCTTGCAGCAGGAGCACCTCCGAATCCACAGCTGCCCTGGGCTGTCTGGTGAAGGACTACTTTCCCGAGCCCGTGACCGTGAGCTGGAACAGCGGCGCTCTGACATCCGGCGTCCACACCTTTCCTGCCGTCCTGCAGTCCTCCGGCCTCTACTCCCTGTCCTCCGTGGTGACCGTGCCTAGCTCCTCCCTCGGCACCAAGACCTACACCTGTAACGTGGACCACAAACCCTCCAACACCAAGGTGGACAAACGGGTCGAGAGCAAGTACGGCCCTCCCTGCCCTCCTTGTCCTGCCCCCGAGTTCGAAGGCGGACCCAGCGTGTTCCTGTTCCCTCCTAAGCCCAAGGACACCCTCATGATCAGCCGGACACCCGAGGTGACCTGCGTGGTGGTGGATGTGAGCCAGGAGGACCCTGAGGTCCAGTTCAACTGGTATGTGGATGGCGTGGAGGTGCACAACGCCAAGACAAAGCCCCGGGAAGAGCAGTTCAACTCCACCTACAGGGTGGTCAGCGTGCTGACCGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCAGCAATAAGGGACTGCCCAGCAGCATCGAGAAGACCATCTCCAAGGCTAAAGGCCAGCCCCGGGAACCTCAGGTGTACACCCTGCCTCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGATTCTACCCTTCCGACATCGCCGTGGAGTGGGAGTCCAACGGCCAGCCCGAGAACAATTATAAGACCACCCCTCCCGTCCTCGACAGCGACGGATCCTTCTTTCTGTACTCCAGGCTGACCGTGGATAAGTCCAGGTGGCAGGAAGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGAGCCTGTCCCTGGGAAAG353416E01 -Amino acid sequenceQGIRRWCDRL1of CDRL1 of 416E01(IMGT)using IMGT354416E01 -Amino acid sequenceGASCDRL2of CDRL2 of 416E01(IMGT)using IMGT355416E01 -Amino acid sequenceQQANSFPITCDRL3of CDRL3 of 416E01(IMGT)using IMGT356416E01 -Amino acid sequenceRASQGIRRWLACDRL1of CDRL1 of 416E01(Kabat)using Kabat357416E01 -Amino acid sequenceGASSLQSCDRL2of CDRL2 of 416E01(Kabat)using Kabat358416E01 -Amino acid sequenceQQANSFPITCDRL3of CDRL3 of 416E01(Kabat)using Kabat359416E01 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIRRWLAWYQQKPGKAPKLLightof VLofLISGASSLQSGVPSRFSGSGSGTDFTLIITSLQPEDFATYYCQQANchain416E01 (mutationsSFPITFGQGTRLEIKvariablefrom germline areregionshown in boldletters)360416E01 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGLightsequence of VLofGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGGAGchain416E01GTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCvariableCTGATCTCTGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTregionTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCATCATTACCAGTCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAAC361416E01 -Amino acid sequenceDIQMTQSPSSVSASVGDRVTITCRASQGIRRWLAWYQQKPGKAPKLfullof 416E01 lightLISGASSLQSGVPSRFSGSGSGTDFTLIITSLQPEDFATYYCQQANlightchainSFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNchainFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAsequenceDYEKHKVYACEVTHQGLSSPVTKSFNRGEC362416E01 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGfullsequence of 416E01GAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGGAGlightlight chainGTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCchainCTGATCTCTGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTsequenceTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCATCATTACCAGTCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATCAAACGTACGGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGT363STIM001 -Amino acid sequenceGYTFSTFGCDRH1of CDRH1 of STIM001using IMGT364STIM001 -Amino acid sequenceISAYNGDTCDRH2of CDRH2 of STIM001using IMGT365STIM001 -Amino acid sequenceARSSGHYYYYGMDVCDRH3of CDRH3 of STIM001using IMGT366STIM001 -Amino acid sequenceQVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEHeavyof VHof STIM001WMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAchainVYYCARSSGHYYYYGMDVWGQGTTVTVSSvariableregion367STIM001 -Nucleic acidCAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGHeavysequence of VHofCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACchainSTIM001CTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAAvariableTGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACregionAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA368STIM001 -Amino acid sequenceQVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEfullof STIM001 heavyWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAheavychainVYYCARSSGHYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSchainTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYsequenceSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK369STIM001 -Nucleic acidCAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGfullsequence of STIM001CCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACheavyheavy chainCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAAchainTGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACsequenceAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA370STIM001 -Amino acid sequenceQSLLHSNEYNYCDRL1of CDRL1 of STIM001using IMGT371STIM001 -Amino acid sequenceLGSCDRL2of CDRL2 of STIM001using IMGT372STIM001 -Amino acid sequenceMQSLQTPLTCDRL3of CDRL3 of STIM001using IMGT373STIM001 -Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGLightof VLof STIM001QSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYchainCMQSLQTPLTFGGGTKVEIKvariableregion374STIM001 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGLightsequence of VLofGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAchainSTIM001TAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGvariableCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGregionGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA375STIM001 -Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGfullof STIM001 lightQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYlightchainCMQSLQTPLTFGGGTKVEIKchainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAsequenceLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC376STIM001 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGfullsequence of STIM001GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAlightlight chainTAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGchainCAGTCTCCACAGCTCCTGATCTTTTTGGGTTCTAATCGGGCCTCCGsequenceGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt377STIM002 -Amino acid sequenceGYTFTSYGCDRH1of CDRH1 of STIM002using IMGT378STIM002 -Amino acid sequenceISAYNGNTCDRH2of CDRH2 of STIM002using IMGT379STIM002 -Amino acid sequenceARSTYFYGSGTLYGMDVCDRH3of CDRH3 of STIM002using IMGT380STIM002 -Amino acid sequenceQVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEHeavyof VHof STIM002WMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAchainVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSvariableregion381STIM002 -Nucleic acidCAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGHeavysequence of VHofCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGchainSTIM002CTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGvariableTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACregionAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA382STIM002 -Amino acid sequenceQVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEfullof STIM002 heavyWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAheavychainVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK383STIM002 -Nucleic acidCAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGfullsequence of STIM002CCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGheavyheavy chainCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGchainTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACsequenceAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA384STIM002 -Amino acid sequenceQSLLHSDGYNYCDRL1of CDRL1 of STIM002using IMGT385STIM002 -Amino acid sequenceLGSCDRL2of CDRL2 of STIM002using IMGT386STIM002 -Amino acid sequenceMQALQTPLSCDRL3of CDRL3 of STIM002using IMGT387STIM002 -Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGLightof VLof STIM002QSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYchainCMQALQTPLSFGQGTKLEIKvariableregion388STIM002 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGLightsequence of VLofGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAchainSTIM002TAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGvariableCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGregionGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA389STIM002 -Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGfullof STIM002 lightQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYlightchainCMQALQTPLSFGQGTKLEIKchainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAsequenceLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC390STIM002 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGfullsequence of STIM002GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAlightlight chainTAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGchainCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGsequenceGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt391STIM002-Amino acid sequenceGYTFTSYGB -of CDRH1 ofCDRH1STIM002-B usingIMGT392STIM002-Amino acid sequenceISAYNGNTB -of CDRH2 ofCDRH2STIM002-B usingIMGT393STIM002-Amino acid sequenceARSTYFYGSGTLYGMDVB -of CDRH3 ofCDRH3STIM002-B usingIMGT394STIM002-Amino acid sequenceQVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEB -of VHof STIM002-BWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAHeavyVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSchainvariableregion395STIM002-Nucleic acidCAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGB -sequence of VHofCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGHeavySTIM002-BCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGchainTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACvariableAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGregionCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA396STIM002-Amino acid sequenceQVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEB—fullof STIM002-B heavyWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAheavychainVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK397STIM002-Nucleic acidCAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGB—fullsequence ofCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGheavySTIM002-B heavyCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGchainchainTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACsequenceAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA398STIM002-Amino acid sequenceQSLLHSDGYNCB -of CDRL1 ofCDRL1STIM002-B usingIMGT399STIM002-Amino acid sequenceLGSB -of CDRL2 ofCDRL2STIM002-B usingIMGT400STIM002-Amino acid sequenceMQALQTPCSB -of CDRL3 ofCDRL3STIM002-B usingIMGT401STIM002-Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGB -of VLof STIM002-BQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYLightCMQALQTPCSFGQGTKLEIKchainvariableregion402STIM002-Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGB -sequence of VL ofGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCALightSTIM002-BTAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGchainCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGvariableGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACregionACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA403STIM002-Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGB—fullof STIM002-B lightQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYlightchainCMQALQTPCSFGQGTKLEIKchainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNAsequenceLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC404STIM002-Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGB—fullsequence ofGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAlightSTIM002-B lightTAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGchainchainCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGsequenceGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt405STIM003 -Amino acid sequenceGVTFDDYGCDRH1of CDRH1 of STIM003using IMGT406STIM003 -Amino acid sequenceINWNGGDTCDRH2of CDRH2 of STIM003using IMGT407STIM003 -Amino acid sequenceARDFYGSGSYYHVPFDYCDRH3of CDRH3 of STIM003using IMGT408STIM003 -Amino acid sequenceEVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEHeavyof VH of STIM003WVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAchainLYYCARDFYGSGSYYHVPFDYWGQGILVTVSSvariableregion409STIM003 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGHeavysequence of VHofGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGAchainSTIM003TTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARvariableTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGregionACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA410STIM003 -Amino acid sequenceEVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEfullof STIM003 heavyWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAheavychainLYYCARDFYGSGSYYHVPFDYWGQGILVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK411STIM003 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGfullsequence of STIM003GGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGAheavyheavy chainTTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARchainTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGsequenceACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA412STIM003 -Amino acid sequenceQSVSRSYCDRL1of CDRL1 of STIM003using IMGT413STIM003 -Amino acid sequenceGASCDRL2of CDRL2 of STIM003using IMGT414STIM003 -Amino acid sequenceHQYDMSPFTCDRL3of CDRL3 of STIM003using IMGT415STIM003 -Amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRLightof VLof STIM003LLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYchainDMSPFTFGPGTKVDIKvariableregion416STIM003 -Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGLightsequence of VLofGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGchainSTIM003AAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGvariableCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAregionGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA417STIM003 -Amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRfullof STIM003 lightLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYlightchainDMSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNchainNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKsequenceADYEKHKVYACEVTHQGLSSPVTKSFNRGEC418STIM003 -Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGfullsequence of STIM003GGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGlightlight chainAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGchainCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAsequenceGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt419STIM004 -Amino acid sequenceGLTFDDYGCDRH1of CDRH1 of STIM004using IMGT420STIM004 -Amino acid sequenceINWNGDNTCDRH2of CDRH2 of STIM004using IMGT421STIM004 -Amino acid sequenceARDYYGSGSYYNVPFDYCDRH3of CDRH3 of STIM004using IMGT422STIM004 -Amino acid sequenceEVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEHeavyof VHof STIM004WVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAchainLYYCARDYYGSGSYYNVPFDYWGQGTLVTVSSvariableregion423STIM004 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGHeavysequence of VHofGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGAchainSTIM004TTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGvariableTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGregionACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA424STIM004 -Amino acid sequenceEVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEfullof STIM004 heavyWVSGINWNGDNTDYADSVKGRFTISRDNAENSLYLQMNSLRAEDTAheavychainLYYCARDYYGSGSYYNVPFDYWGQGTLVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK425STIM004 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGfullsequence of STIM004GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGAheavyheavy chainTTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGchainTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGsequenceACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA426STIM004 -Amino acid sequenceQSVSSSYCDRL1of CDRL1 of STIM004using IMGT427STIM004 -Amino acid sequenceGASCDRL2of CDRL2 of STIM004using IMGT428STIM004 -Amino acid sequenceQQYGSSPFCDRL3of CDRL3 of STIM004using IMGT429STIM004 -Amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRCorrectedof corrected VLofLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYlightSTIM004GSSPFFGPGTKVDIKchainvariableregion430STIM004 -Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGCorrectedsequence ofGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGlightcorrected VLofCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGchainSTIM004CTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAvariableGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGregionAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAA431STIM004 -Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGLightsequence of VLofGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGchainSTIM004CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGvariableCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAregionGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAA432STIM004 -Amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRfullof STIM004 lightLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYcorrectedchainGSSPFFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNlightFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAchainDYEKHKVYACEVTHQGLSSPVTKSFNRGECsequence433STIM004 -Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGfullsequence ofGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGcorrectedcorrected STIM004CAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGlightlight chainCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAchainGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGsequenceAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt434STIM004 -Nucleic acidGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGfullsequence of STIM004GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGlightlight chainCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGchainCTCCTCATATATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAsequenceGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt435STIM005 -Amino acid sequenceGYTFNSYGCDRH1of CDRH1 of STIM005using IMGT436STIM005 -Amino acid sequenceISVHNGNTCDRH2of CDRH2 of STIM005using IMGT437STIM005 -Amino acid sequenceARAGYDILTDFSDAFDICDRH3of CDRH3 of STIM005using IMGT438STIM005 -Amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEHeavyof VHof STIM005WMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAchainVYYCARAGYDILTDFSDAFDIWGHGTMVTVSSvariableregion439STIM005 -Nucleic acidCAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGHeavysequence of VHofCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGchainSTIM005TTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGvariableTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACregionAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCA440STIM005 -Amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEfullof STIM005 heavyWMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAheavychainVYYCARAGYDILTDFSDAFDIWGHGTMVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK441STIM005 -Nucleic acidCAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGfullsequence of STIM005CCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGheavyheavy chainTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGchainTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACsequenceAGAAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA442STIM005 -Amino acid sequenceQNINNFCDRL1of CDRL1 of STIM005using IMGT443STIM005 -Amino acid sequenceAASCDRL2of CDRL2 of STIM005using IMGT444STIM005 -Amino acid sequenceQQSYGIPWCDRL3of CDRL3 of STIM005using IMGT445STIM005 -Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLLightof VLof STIM005LIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYchainGIPWVGQGTKVEIKvariableregion446STIM005 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGLightsequence of VLofGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAAchainSTIM005CTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCvariableCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTregionTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAA447STIM005 -Amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLfullof STIM005 lightLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYlightchainGIPWVGQGTKVEIKchainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAsequenceLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC448STIM005 -Nucleic acidGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGfullsequence of STIM005GAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAAlightlight chainCTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCchainCTGATCTATGCAGCATCCAGTTTGCAAAGAGGGATACCATCAACGTsequenceTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt449STIM006 -Amino acid sequenceGFTFSDYFCDRH1of CDRH1 of STIM006using IMGT450STIM006 -Amino acid sequenceISSSGSTICDRH2of CDRH2 of STIM006using IMGT451STIM006 -Amino acid sequenceARDHYDGSGIYPLYYYYGLDVCDRH3of CDRH3 of STIM006using IMGT452STIM006 -Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEHeavyof VHof STIM006WISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAchainVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSSvariableregion453STIM006 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGHeavysequence of VHofGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGAchainSTIM006CTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGvariableTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGregionACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA454STIM006 -Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEfullof STIM006 heavyWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAheavychainVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSSASTKGPSVFPchainLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVsequenceLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK455STIM006 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGfullsequence of STIM006GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGAheavyheavy chainCTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGchainTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGsequenceACTCTGTGAGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA456STIM006 -Amino acid sequenceQSLLHSNGYNYCDRL1of CDRL1 of STIM006using IMGT457STIM006 -Amino acid sequenceLGSCDRL2of CDRL2 of STIM006using IMGT458STIM006 -Amino acid sequenceMQALQTPRSCDRL3of CDRL3 of STIM006using IMGT459STIM006 -Amino acid sequenceIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQLightof VLof STIM006SPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCchainMQALQTPRSFGQGTTLEIKvariableregion460STIM006 -Nucleic acidATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGLightsequence of VLofAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGchainSTIM006TAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGvariableTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGregionTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGCTGGAGATCAAA461STIM006 -Amino acid sequenceIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQfullof STIM006 lightSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYClightchainMQALQTPRSFGQGTTLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCchainLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTsequenceLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC462STIM006 -Nucleic acidATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGfullsequence of STIM006AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGlightlight chainTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGchainTCTCCACAGCTCCTGATCTATTTGGGTTCTTATCGGGCCTCCGGGGsequenceTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGACCACGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt463STIM007 -Amino acid sequenceGFSLSTTGVGCDRH1of CDRH1 of STIM007using IMGT464STIM007 -Amino acid sequenceIYWDDDKCDRH2of CDRH2 of STIM007using IMGT465STIM007 -Amino acid sequenceTHGYGSASYYHYGMDVCDRH3of CDRH3 of STIM007using IMGT466STIM007 -Amino acid sequenceQITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKAHeavyof VHof STIM007LEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTchainATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSvariableregion467STIM007 -Nucleic acidCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACHeavysequence of VHofAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACchainSTIM007TACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCvariableCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAregionGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA468STIM007 -Amino acid sequenceQITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKAfullof STIM007 heavyLEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTheavychainATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK469STIM007 -Nucleic acidCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACfullsequence of STIM007AGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACheavyheavy chainTACTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCchainCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAsequenceGCCCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA470STIM007 -Amino acid sequenceQSVTNYCDRL1of CDRL1 of STIM007using IMGT471STIM007 -Amino acid sequenceDASCDRL2of CDRL2 of STIM007using IMGT472STIM007 -Amino acid sequenceQHRSNWPLTCDRL3of CDRL3 of STIM007using IMGT473STIM007 -Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLightof VLof STIM007LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSchainNWPLTFGGGTKVEIKvariableregion474STIM007 -Nucleic acidGAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGLightsequence of VLofGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAAchainSTIM007CTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCvariableCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTregionTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC475STIM007 -Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLfullof STIM007 lightLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSlightchainNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNchainFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAsequenceDYEKHKVYACEVTHQGLSSPVTKSFNRGEC476STIM007 -Nucleic acidGAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGfullsequence of STIM007GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAAlightlight chainCTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCchainCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTsequenceTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt477STIM008 -Amino acid sequenceGFSLSTSGVGCDRH1of CDRH1 of STIM008using IMGT478STIM008 -Amino acid sequenceIYWDDDKCDRH2of CDRH2 of STIM008using IMGT479STIM008 -Amino acid sequenceTHGYGSASYYHYGMDVCDRH3of CDRH3 of STIM008using IMGT480STIM008 -Amino acid sequenceQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKAHeavyof VHof STIM008LEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTchainATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSvariableregion481STIM008 -Nucleic acidCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACHeavysequence of VHofAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACchainSTIM008TAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCvariableCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAregionGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA482STIM008 -Amino acid sequenceQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKAfullof STIM008 heavyLEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTheavychainATYFCTHGYGSASYYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPSchainSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSsequenceGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK483STIM008 -Nucleic acidCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACfullsequence of STIM008AGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACheavyheavy chainTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCchainCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAsequenceGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA484STIM008 -Amino acid sequenceQSVTNYCDRL1of CDRL1 of STIM008using IMGT485STIM008 -Amino acid sequenceDASCDRL2of CDRL2 of STIM008using IMGT486STIM008 -Amino acid sequenceQQRSNWPLTCDRL3of CDRL3 of STIM008using IMGT487STIM008 -Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLightof VLof STIM008LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSchainNWPLTFGGGTKVEIKvariableregion488STIM008 -Nucleic acidGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGLightsequence of VLofGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAAchainSTIM008CTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCvariableCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTregionTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA489STIM008 -Amino acid sequenceEIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLfullof STIM008 lightLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSlightchainNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNchainFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAsequenceDYEKHKVYACEVTHQGLSSPVTKSFNRGEC490STIM008 -Nucleic acidGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGfullsequence of STIM008GGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAAlightlight chainCTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCchainCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTsequenceTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt491STIM009 -Amino acid sequenceGFTFSDYYCDRH1of CDRH1 of STIM009using IMGT492STIM009 -Amino acid sequenceISSSGSTICDRH2of CDRH2 of STIM009using IMGT493STIM009 -Amino acid sequenceARDFYDILTDSPYFYYGVDVCDRH3of CDRH3 of STIM009using IMGT494STIM009 -Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEHeavyof VHof STIM009WVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAchainVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSSvariableregion495STIM009 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGHeavysequence of VHofGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGAchainSTIM009CTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGvariableTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGregionACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA496STIM009 -Amino acid sequenceQVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEfullof STIM009 heavyWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAheavychainVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSSASTKGPSVFPLchainAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLsequenceQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK497STIM009 -Nucleic acidCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGfullsequence of STIM009GGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGAheavyheavy chainCTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGchainTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGsequenceACTCTGTGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA498STIM009 -Amino acid sequenceQSLLHSNGYNYCDRL1of CDRL1 of STIM009using IMGT499STIM009 -Amino acid sequenceLGSCDRL2of CDRL2 of STIM009using IMGT500STIM009 -Amino acid sequenceMQALQTPRTCDRL3of CDRL3 of STIM009using IMGT501STIM009 -Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGLightof VLof STIM009QSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYchainCMQALQTPRTFGQGTKVEIKvariableregion502STIM009 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGLightsequence of VLofGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAchainSTIM009TAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGvariableCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGregionGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA503STIM009 -Amino acid sequenceDIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGfullof STIM009 lightQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYlightchainCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVchainCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLsequenceTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC504STIM009 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGfullsequence of STIM009GAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCAlightlight chainTAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGchainCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGsequenceGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt505HumanAmino acid sequenceFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIPD-L1of KYPROT286 withIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGFlag HisFLAG tag in boldVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQ(KYPROT286)and underlined andAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTThistidine tag inTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTIEGRDYKDDboldDDKHHHHHH506MatureMature amino acidEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLThumansequence of humanKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSICOSICOSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL507HumanAmino acid sequenceEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTICOSof human ICOSKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSextracellularextracellularIFDPPPFKVTLTGGYLHIYESQLCCQLKFdomaindomain508HumanAmino acid sequenceMKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDICOSof human ICOSIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVwith(signal peptide isSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQsignalunderlined)LKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRpeptideAVNTAKKSRLTDVTL509IsoformAmino acid sequenceThe sequence of this isoform differs from theof humanof a human ICOScanonical sequence in its cytoplasmic domainICOSisoformas follows: 168-199:(Q9Y6W8-KYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLM2)510MatureMature amino acidEINGSADHRMFSFHNGGVQISCKYPETVQQLKMRLFREREVLCELTmousesequence of mouseKTKGSGNAVSIKNPMLCLYHLSNNSVSFFLNNPDSSQGSYYFCSLSICOSICOSIFDPPPFQERNLSGGYLHIYESQLCCQLKIVVQVTE511MouseAmino acid sequenceEINGSADHRMFSFHNGGVQISCKYPETVQQLKMRLFREREVLCELTICOSof theKTKGSGNAVSIKNPMLCLYHLSNNSVSFFLNNPDSSQGSYYFCSLSextracellularextracellularIFDPPPFQERNLSGGYLHIYESQLCCQLKdomaindomain of mouseICOS512MouseAmino acid sequenceMGWSCIILFLVATATGVHSEINGSADHRMFSFHNGGVQISCKYPETICOSof mouse ICOSVQQLKMRLFREREVLCELTKTKGSGNAVSIKNPMLCLYHLSNNSVSwith(signal peptide isFFLNNPDSSQGSYYFCSLSIFDPPPFQERNLSGGYLHIYESQLCCQsignalunderlined)LKIVVQVTEpeptide513CynomolgusAmino acid sequenceMKSGLWYFFL FCLHMKVLTGEINGSANYEM FIFHNGGVQIICOSof cynomolgus ICOSLCKYPDIVQQwith(signal peptide isFKMQLLKGGQILCDLTKTKGSGNKVSIKSLKFCHSQLSNNSVSFFLsignalunderlined)YNLDpeptideRSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCATFVVVCIFGCILICWLTKKKYSSTVHDPNGEYMFMRAVNTAKKSRLTGTTP514CynomolgusAmino acid sequenceEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTICOSof cynomolgus ICOSKTKGextracellularextracellularSGNKVSIKSLKFCHSQLSNNSVSFFLYNLDRSHANYYFCNLSIFDPdomaindomainPPFK VTLTGGYLHIYESQLCCQLK515HumanAmino acid sequenceDTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTICOSof human ICOSYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKligandligand comprisingFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFextracellularTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVELNMRGLYDVVSVdomainLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWS516HumanAmino acid sequenceMRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDICOSof human ICOSLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLligandligand includingRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFsignal peptideSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVELNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHSYAGAWAVSPETELTGHVSEQ ID NO: 610 ICOSL-FcDTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSDIEGRMDPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGKLinker is underlined and in bold. Sequence preceding linker is human ICOSL (B7-H2).Sequence following linker is human IgG1 Fc.517C-terminal aminoAmino acids 21 toLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLacid sequence of133 of hIL-2 withEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGhIL-2R38W mutationSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(bold &underlined)518C-terminal aminoAmino acids 21 toLQMILNGINNYKNPKLTQMLTFKFYMPKKATELKHLQCLacid sequence of133 of hIL-2 withEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGhIL-2R38Q mutationSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(bold &underlined)519STIM002 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCCorrected Lightsequence ofACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTchain variablecorrected VLofCAGAGCCTCCTGCATAGTGATGGATACAACTATTTGGATregionSTIM002TGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA520STIM002 -Nucleic acidGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCCorrected fullsequence ofACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTlight chaincorrected STIM002CAGAGCCTCCTGCATAGTGATGGATACAACTATTTGGATsequencelight chainTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAAcgtacggtggccgctccctccgtgttcatcttcccaccttccgacgagcagctgaagtccggcaccgcttctgtcgtgtgcctgctgaacaacttctacccccgcgaggccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaggaatccgtgaccgagcaggactccaaggacagcacctactccctgtcctccaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgacccaccagggcctgtctagccccgtgaccaagtctttcaaccggggcgagtgt521STIM003 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCorrected heavysequence ofCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAchain variablecorrected VHofGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAregionSTIM003GCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA522STIM003 -Nucleic acidGAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCorrected fullsequence ofCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAheavy chaincorrected STIM003GTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAsequenceheavy chainGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCAGCACCAAGGGCCCCTCTGTGTTCCCTCTGGCCCCTTCCAGCAAGTCCACCTCTGGCGGAACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGA523Human IgG1IGHG1*03Human Heavy ChaingcctccaccaagggcccatcggtcttccccctggcacccconstantConstant Regiontcctccaagagcacctctgggggcacagcggccctgggcregion(IGHG1*03)tgcctggtcaaggactacttccccgaaccggtgacggtgNucleotidetcgtggaactcaggcgccctgaccagcggcgtgcacaccSequencettcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaa524Human Heavy ChainA S T K G P S V F P L A P S S K S T S GConstant RegionG T A A L G C L V K D Y F P E P V T V S(IGHG1*03) ProteinW N S G A L T S G V H T F P A V L Q S SSequenceG L Y S L S S V V T V P S S S L G T Q TY I C N V N H K P S N T K V D K R V E PK S C D K T H T C P P C P A P E L L G GP S V F L F P P K P K D T L M I S R T PE V T C V V V D V S H E D P E V K F N WY V D G V E V H N A K T K P R E E Q Y NS T Y R V V S V L T V L H Q D W L N G KE Y K C K V S N K A L P A P I E K T I SK A K G Q P R E P Q V Y T L P P S R E EM T K N Q V S L T C L V K G F Y P S D IA V E W E S N G Q P E N N Y K T T P P VL D S D G S F F L Y S K L T V D K S R WQ Q G N V F S C S V M H E A L H N H Y TQ K S L S L S P G K525Human IgG1IGHG1*04Human Heavy ChaingcctccaccaagggcccatcggtcttccccctggcacccconstantConstant Regiontcctccaagagcacctctgggggcacagcggccctgggcregion(IGHG1*04)tgcctggtcaaggactacttccccgaaccggtgacggtgNucleotidetcgtggaactcaggcgccctgaccagcggcgtgcacaccSequencettcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacatcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa526Human Heavy ChainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVConstant RegionSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT(IGHG1*04) ProteinQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELSequenceLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNHYTQKSLSLSPGK527Human IgG2IGHG2*01 &Human Heavy ChaingcctccaccaagggcccatcggtcttccccctggcgcccconstantIGHG2*03 &Constant RegiontgctccaggagcacctccgagagcacagccgccctgggcregionIGHG2*05(IGHG2*01)tgcctggtcaaggactacttccccgaaccggtgacggtgNucleotidetcgtggaactcaggcgctctgaccagcggcgtgcacaccSequencettcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcaacttcggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa528Human Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVConstant RegionSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNEGT(IGHG2*01) ProteinQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK529Human IgG2IGHG2*02Human Heavy ChainGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCconstantConstant RegionTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCregion(IGHG2*02)TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGNucleotideTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCSequenceTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGACCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCATGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA530Human Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVConstant RegionSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGT(IGHG2*02) ProteinQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK531Human IgG2IGHG2*04Human Heavy ChaingcctccaccaagggcccatcggtcttccccctggcgcccconstantConstant Regiontgctccaggagcacctccgagagcacagcggccctgggcregion(IGHG2*04)tgcctggtcaaggactacttccccgaaccggtgacggtgNucleotidetcgtggaactcaggcgctctgaccagcggcgtgcacaccSequencettcccagctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagacagttgagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacgttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaaccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa532Human Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVConstant RegionSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT(IGHG2*04) ProteinQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK533Human IgG2IGHG2*06Human Heavy ChainGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCconstantConstant RegionTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCregion(IGHG2*06)TGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGNucleotideTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCSequenceTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTG.GACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCTCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA534Human Heavy ChainASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVConstant RegionSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGT(IGHG2*06) ProteinQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSequenceSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK535Human CλIGLC7*03Cλ Light ChainGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCAconstantConstant RegionCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGregion(IGLC7*03)GTGTGTCTCGTAAGTGACTTCAACCCGGGAGCCGTGACANucleotideGTGGCCTGGAAGGCAGATGGCAGCCCCGTCAAGGTGGGASequenceGTGGAGACCACCAAACCCTCCAAACAAAGCAACAACAAGTATGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCT536Cλ Light ChainGQPKAAPSVTLFPPSSEELQANKATLVCLVSDFNPGAVTConstant RegionVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQ(IGLC7*03) AminoWKSHRSYSCRVTHEGSTVEKTVAPAECSAcid Sequence537Human WTIGHG1*01 &WT human IgG1gcctccaccaagggcccatcggtcttccccctggcacccIgG1IGHG1*05nucleotidetcctccaagagcacctctgggggcacagcggccctgggcconstant(IgG1)sequence #2tgcctggtcaaggactacttccccgaaccggtgacggtgregiontcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa538Human CλIGLC2*01Cλ Light ChainGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTconstantConstant RegionVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQregionAmino AcidWKSHRSYSCQVTHEGSTVEKTVAPTECSSequence #2 -Encoded bynucleotidesequence version A& B TABLE S3SEQ ID NOS: 539-562SequencehIgG1 FIT-Ig bispecific 1aAntibody Aanti-ICOSSTIM003Antibody Banti-PD-L184G09FIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #1539LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFIT-IgSEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYConstruct #2540EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3541IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEChIgG1 FIT-Ig bispecific 1bAntibody Aanti-PD-L184G09Antibody Banti-ICOSSTIM003FIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #1542IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFIT-IgSEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSConstruct #2543GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3544LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEChIgG1 FIT-Ig bispecific 2aAntibody Aanti-ICOSSTIM001Antibody Banti-PD-L11D05FIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #1545LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKFIT-IgSEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYConstruct #2546EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIFIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3547IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEChIgG1 FIT-Ig bispecific 2bAntibody Aanti-PD-L11D05Antibody Banti-ICOSSTIM001FIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #1548IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKFIT-IgSEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSConstruct #2549GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIFIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3550LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEChIgG1 FIT-Ig bispecific 3aAntibody Aanti-ICOSSTIM003Antibody Banti-PD-L11D05FIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #1551LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKFIT-IgSEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYConstruct #2552EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3553IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEChIgG1 FIT-Ig bispecific 3bAntibody Aanti-PD-L11D05Antibody Banti-ICOSSTIM003FIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #1554IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKFIT-IgSEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSConstruct #2555GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3556LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEChIgG1 FIT-Ig bispecific 4aAntibody Aanti-ICOSSTIM001Antibody Banti-PD-L184G09FIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #1557LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSGSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKFIT-IgSEQ ID NO:EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYConstruct #2558EGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #3559IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEChIgG1 FIT-Ig bispecific 4bAntibody Aanti-PD-L184G09Antibody Banti-ICOSSTIM001FIT-IgSEQ ID NO:DIVMTQSPSSLAVSPGEKVTMTCKSSQSLYYSGVKENLLAWYQQKPGQSPKLLConstruct #1560IYYASIRFTGVPDRFTGSGSGTDYTLTITSVQAEDMGQYFCQQGINNPLTFGDGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKFIT-IgSEQ ID NO:EVQLVESGGGLTQPGKSLKLSCEASGFTFSSFTMHWVRQSPGKGLEWVAFIRSConstruct #2561GSGIVFYADAVRGRFTISRDNAKNLLFLQMNDLKSEDTAMYYCARRPLGHNTFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVFIT-IgSEQ ID NO:DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSConstruct #3562LQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC TABLE S4Sequences of antibody heavy chain variable regions obtained from additional clonesCDRs are defined according to IMGT.CLONE_IDVH_NUCLEOTIDE_SEQUENCEVH_AMINO_ACID_SEQHCDR1HCDR2HCDR3CL-61091CAGGTTCAACTGATGCAGTCTGGAACTGAGGTGAAGAAGCCTGGGQVQLMQSGTEVKKPGASVGYTFTTYGISAYSGDTARSSGWPHHGCCTCAGTGAAGGTCTCCTGCAAGACTTCTGGTTACACCTTTACCKVSCKTSGYTFTTYGITWSEQ IDSEQ ID NO:YGMDVACCTATGGTATCACTTGGGTGCGACAGGCCCCTGGACAAGGGCTTVRQAPGQGLEWMGWISAYNO: 565566SEQ IDGAGTGGATGGGATGGATCAGCGCTTACAGTGGTGACACAGACTATSGDTDYAQKFQGRVTVTTNO: 567GCACAGAAGTTCCAGGGCAGAGTCACCGTGACAACAGACACATCCDTSTNTAYMELRSLKSDDACGAACACAGCCTACATGGAGTTGAGGAGCCTGAAATCTGACGACTAVYYCARSSGWPHHYGMACGGCCGTGTATTATTGTGCGAGAAGTAGTGGCTGGCCCCACCACDVWGQGTTVTVSSTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCSEQ ID NO: 564TCAGSEQ ID NO: 563CL-64536CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAAAAGCCTGGGQVQLVQSGGEVKKPGASVGYTFTSYGISAYNGNTARSTSYYGSGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCKVSCKASGYTFTSYGFSWSEQ IDSEQ ID NO:TLYGMDVAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAVRQAPGQGLEWMGWISAYNO: 377378SEQ IDGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATNGNTNYAQKLQGRVSMTTNO: 570GCACAGAAGCTCCAGGGCAGAGTCTCCATGACCACAGACACATCCDTSTSTAYMELRSLRSDDACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACTAVYFCARSTSYYGSGTLACGGCCGTGTATTTCTGTGCGCGATCTACGTCTTACTATGGTTCGYGMDVWGQGTTVTVSSGGGACCCTATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCSEQ ID NO: 569ACCGTCTCCTCAGSEQ ID NO: 568CL-64837CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGQVQLVQSGGEVKKPGASVGYTFTSYGISAYNGNTARSTSYYGSGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCKVSCKASGYTFTSYGFSWSEQ IDSEQ ID NO:TLYGMDVAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAVRQAPGQGLEWMGWISAYNO: 377378SEQ IDGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATNGNTNYAQKLQGRVSMTTNO: 570GCACAGAAGCTCCAGGGCAGAGTCTCCATGACCACAGACACATCCDTSTSTAYMELRSLRSDDACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACTAVYYCARSTSYYGSGTLACGGCCGTGTATTACTGTGCGCGATCTACGTCTTACTATGGTTCGYGMDVWGQGTTVTVSSGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCSEQ ID NO: 572ACCGTCTCCTCAGSEQ ID NO: 571CL-64841CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAAAAGCCTGGGQVQLVQSGGEVKKPGASVGYTFTSYGISAYNGNTARSTSYYGSGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCKVSCKASGYTFTSYGFSWSEQ IDSEQ ID NO:TLYGMDVAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAVRQAPGQGLEWMGWISAYNO: 377378SEQ IDGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATNGNTNYAQKLQGRVSMTTNO: 570GCACAGAAGCTCCAGGGCAGAGTCTCCATGACCACAGACACATCCDTSTSTAYMELRSLRSDDACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACTAVYFCARSTSYYGSGTLACGGCCGTGTATTTCTGTGCGCGATCTACGTCTTACTATGGTTCGYGMDVWGQGTTVTVSSGGGACCCTATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCSEQ ID NO: 574ACCGTCTCCTCAGSEQ ID NO: 573CL-64912CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAAAAGCCTCGGQVQLVQSGGEVKKPRASVGYTFTSYVISGYNGNTARSTSYYGAGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCKVSCKASGYTFTSYVFSWSEQ IDSEQ ID NO:TLYGMDVAGCTATGTGTTCAGCTGGGTGCGACATGCCGCTGGACAAGGACTAVRHAAGQGLEWMGWISGYNO: 577578SEQ IDGAGTGGATGGGATGGATCAGCGGTTACAATGGTAACACAAACTATNGNTNYAQKLQCGVSMTANO: 579GCACAGAAGCTCCAGTGCGGAGTCTCGATGACCGCAGACACATCCDTSTSTAYMELRSLRSDDACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACTAVYFCARSTSYYGAGTLACGGCCGTGTATTTCTGTGCGCGATCTACGTCTTACTATGGTGCGYGMDVWGQGTTVTVSSGGGACCCTATACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCSEQ ID NO: 576ACCGTCTCCTCAGSEQ ID NO: 575CL-71642GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGEVQLVESGGGVVRPGGSLGFTFDDYGINWNGGSTAADYYGSGSYGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATRLSCAASGFTFDDYGMSWSEQ IDSEQ ID NO:YNVPFDYGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGVRQAPGKGLEWVSGINWNNO: 582583SEQ IDGAGTGGGTCTCTGGTATTAATTGGAATGGTGGTAGCACAGGTTATGGSTGYADSVKGRFTISRNO: 584GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCDNAKNSLYLQMNSLRAEDAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACTALYYCAADYYGSGSYYNACGGCCTTGTATTACTGTGCGGCCGATTACTATGGTTCGGGGAGTVPFDYWGQGTLVTVSSTATTATAACGTCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCSEQ ID NO: 581ACCGTCTCCTCAGSEQ ID NO: 580CL-74570GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGATACGGCCTGGGEVQLVESGGGVIRPGGSLGFTFDDYGINWIGDNTARDYFGSGSYGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATRLSCAASGFTFDDYGMSWSEQ IDSEQ ID NO:YNVPFDYGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGVRQAPGKGLEWVSGINWINO: 582587SEQ IDGAGTGGGTCTCTGGTATTAATTGGATTGGTGATAACACAGATTATGDNTDYADSVKGRFTISRNO: 588GCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCDNAKNSLYLQMNSLRAEDAAGAACTCCCTATATCTGCAAATGAACAGTCTGAGAGCCGAGGACTALYYCARDYFGSGSYYNACGGCCTTGTATTACTGTGCGAGAGATTACTTTGGTTCGGGGAGTVPFDYWGQGTLVTVSSTATTATAACGTTCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCSEQ ID NO: 586ACCGTCTCCTCAGSEQ ID NO: 585 TABLE S5Sequences of antibody light chain variable regions obtained from additional clonesN terminal E and 5′ nucleotide additions in CL-71642 are shown in bold.These were not recovered in sequencing but were determined to be presentin the sequence by comparison against the related clones as shown in FIG. 36.CDRs are defined according to IMGT.CLONE_IDVL_NUCLEOTIDE_SEQUENCEVL_AMINO_ACID_SEQLCDR1LCDR2LCDR3CL-61091GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTDIVMTQSPLSLPVTPGEPAQSLLHSNGFNYLVSMQALQTPLTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGSISCRSSQSLLHSNGFNYFSEQ ID NO:SEQ IDSEQ IDCATAGTAATGGATTCAACTATTTCGATTGGTACCTGCAGAAGCCADWYLQKPGQSPQLLIFLVS591NO:NO: 593GGACAGTCTCCACAGCTCCTGATCTTTTTGGTTTCTAATCGGGCCNRASGVPDRFSGSGSGTDF592TCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTLKISRVEAEDVGIYYCMQTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGATTALQTPLTFGGGTKVEIKTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTTTCGGCGGASEQ ID NO: 590GGGACCAAGGTGGAGATCAAACSEQ ID NO: 589CL-64536GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTDIVMTQSPLSLPVTPGEPAQSLLHSNGYNCLGSMQALQTPCSGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGSISCRSSQSLLHSNGYNCLSEQ ID NO:SEQ IDSEQ IDCATAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCADWYLQKPGQSPQLLIYLGS596NO:NO: 400GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTRASGFPDRFSGSGSGTDF371TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTLKISRVEAEDVGVYYCMQTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTALQTPCSFGQGTKLEIKTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGSEQ ID NO: 595GGGACCAAGCTGGAGATCAAACSEQ ID NO: 594CL-64837GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTDIVMTQSPLSLPVTPGEPAQSLLHSNGYNCLGSMQALQTPCSGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGSISCRSSQSLLHSNGYNCLSEQ ID NO:SEQ IDSEQ IDCATAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCADWYLQKPGQSPQLLIYLGS596NO:NO: 400GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTRASGFPDRFSGSGSGTDF371TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTLKISRVEAEDVGVYYCMQTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTALQTPCSFGQGTKLEIKTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGSEQ ID NO: 598GGGACCAAGCTGGAGATCAAACSEQ ID NO: 597CL-64841GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTDIVMTQSPLSLPVTPGEPAQSLLHSNGYNCLGSMQALQTPCSGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGSISCRSSQSLLHSNGYNCLSEQ ID NO:SEQ IDSEQ IDCATAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCADWYLQKPGQSPQLLIYLGS596NO:NO: 400GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTRASGFPDRFSGSGSGTDS371TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTLKISRVEAEDVGVYYCMQTCTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTALQTPCSFGQGTKLEIKTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGSEQ ID NO: 600GGGACCAAGCTGGAGATCAAACSEQ ID NO: 599CL-64912GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTDIVMTQSPLSLPVTPGEPAQSLLHSNGYNCLGSMQALQTPCSGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGSISCRSSQSLLHSNGYNCLSEQ ID NO:SEQ IDSEQ IDCATAGTAATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCADWYLQKPGQSPQLLIYLGS596NO:NO: 400GGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTACTCGGGCCTRASGFPDRFSGSGSGTDF371TCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTLKISRVEAEDVGVYYCMQTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTALQTPCSFGQGTKLEIKTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGSEQ ID NO: 602GGGACCAAGCTGGAGATCAAACSEQ ID NO: 601CL-71642GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAEIVLTQSPGTLSLSPGERAQSVSSSYGASQQYGSSPFTGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTLSCRASQSVSSSYLAWYQSEQ ID NO:SEQ IDSEQ IDAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCQKPGQAPRLLIYGASSRAT426NO:NO: 605AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGIPDRFSGSGSGTDFTLTI413GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCSRLEPEDFAVYYCQQYGSSATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGPFTFGPGTKVDIKCAGTATGGTAGCTCACCTTTCACTTTCGGCCCTGGGACCAAAGTGSEQ ID NO: 604GATATCAAACSEQ ID NO: 603CL-74570GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAEIVLTQSPGTLSLSPGERAQSVSSSYGASHQYGNSPFTGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTLSCRASQSVSSSYLAWYQSEQ ID NO:SEQ IDSEQ IDAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCQKPGQAPRLLIYGASSRAT426NO:NO: 608AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGIPDRFSGSGSGTDFTLTI413GACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCSRLEPEDFAVYYCHQYGNSATCAGCAGACTGGAACCTGAAGATTTTGCAGTATATTACTGTCACPFTFGPGTKVDIKCAGTATGGTAATTCACCATTCACTTTCGGCCCTGGGACCAAAGTGSEQ ID NO: 607GATATCAAACSEQ ID NO: 606 | 545,516 |
11858997 | EXAMPLE 1: MATERIALS A fusion protein consisting of the extracellular part of human CD80 and human Fc gamma 1 was obtained from R&D Systems (Minneapolis, MN) as a recombinant protein produced in NS0 cells (Cat #140-B1). A fusion protein consisting of the extracellular part of human CD86 and human Fc gamma 1 was obtained from R&D Systems as a recombinant protein produced in NS0 cells (Cat #140-B2). A fusion protein consisting of the extracellular (173aa) domain of human CD80 fused to murine IgG2a Fc+ hinge (232 aa) was obtained from Ancell (Bayport, MN) as recombinant protein produced in CHO cells (Cat #: 510-820). A biotinylated fusion protein consisting of the extracellular part of human CD80 and mouse Fc gamma 2a was obtained from Ancell (Bayport, MN) as a recombinant protein produced in CHO cells (Cat #510-030). A fusion protein consisting of the extracellular part of human CD86 and mouse Fc gamma 2a was obtained from Ancell (Bayport, MN) as a recombinant protein produced in CHO cells (Cat #509-820). Human IgG1, purified from a human plasma, was obtained from Sigma-Aldrich (St. Louis, MO) (Cat #I-5154) Mouse IgG2a, purified from mouse myeloma UPC-10, was obtained from Sigma-Aldrich (St. Louis, MO) (Cat #M-9144) Anti-Llama IgG (h&l) HRP conjugated antibody, an affinity purified polyclonal antiserum against llama IgG raised in goat and crosslinked to horseradish peroxidase was obtained from Bethyl Labs (Montgomery, TX). CHO-K1 cells were obtained from ATCC (Manassas, VA) (Cat #CCL-61) and maintained according to the provider's instructions. Raji cells were obtained from ECACC (Porton Down, Salisbury, Wiltshire, UK) (Cat #85011429) and maintained according to the provider's instructions. A fusion protein consisting of the extracellular part of human CD152 and human Fc gamma 1 (CTLA4-Ig) was obtained from Chimerigen (Allston, MA) as a recombinant protein produced in NS1 cells (Cat #HF-211A4). A fusion protein consisting of the extracellular part of human CD152 and human Fc gamma 1 (CTLA4-Ig) was obtained from R&D Systems as a recombinant protein produced in Sf21 insect cells (Cat #325-CT/CF). A fusion protein consisting of the extracellular part of human CD28 and human Fc gamma 1 (CD28-Ig) was obtained from R&D Systems as a recombinant protein produced in NS0 cells (Cat #342-CD). A fusion protein consisting of the extracellular part of human PD-1 and mouse Fc gamma 1 was obtained from R&D Systems as a recombinant protein produced in NS0 cells (Cat #1086-PD). A fusion protein consisting of the extracellular part of human PD-L2 and mouse Fc gamma 1 was obtained from R&D Systems as a recombinant protein produced in NS0 cells (Cat #1224-PL). A fusion protein consisting of the extracellular part of human B7-H1 (PD-L1) and mouse Fc gamma 1 was obtained from R&D Systems as a recombinant protein produced in NS0 cells (Cat #156-B7). A fusion protein consisting of the extracellular part of human B7-H2 (ICOSL) and mouse Fc gamma 1 was obtained from R&D Systems as a recombinant protein produced in NS0 cells (Cat #165-B7). Anti-human IgG1 Fc PE conjugated F(ab′)2, a polyclonal antiserum against human IgG1 Fc raised in goat, affinity purified, digested to F(ab′)2 fragments and crosslinked to R-phycoerythrin was obtained from Jackson Immunoresearch Laboratories (West Grove, PA) (Cat #109-116-170). Mouse-anti-human CTLA4 clone BNI3, a monoclonal antibody known to bind human CTLA4 and block CTLA4 interaction with CD80 and CD86, was obtained from Abcam (Cambridge, UK) (Cat #ab19792). A phycoerythrin labeled version of BNI3 was obtained from BD Biosciences (San Jose, CA) (Cat #555853). EXAMPLE 2: IMMUNIZATIONS WITH CD80 AND/OR CD86 Two llamas (No. 089 and No. 090) were immunized with 100 or 50 μg doses of R&D Systems Cat #140-B1, alternated with 20 or 10 μg doses of Ancell Cat #509-820 according to the scheme outlined in Table C-1. Both proteins were formulated in Stimune adjuvants (Cedi Diagnostics B.V., Lelystad, The Netherlands). Blood was collected from these animals as indicated in Table C-1. EXAMPLE 3: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. The characteristics of the constructed libraries are shown in Table C-2. EXAMPLE 4: SELECTIONS OF CD80 AND/OR CD86 BINDING NANOBODIES Phage libraries from llama No. 089 and No. 090 were used for two rounds (R1, R2) of selections on the same plate-immobilized antigen or on alternating antigens. R&D Systems Cat #140-B1 (“HuCD80-HuIgG1”) and Cat #140-B2 (“HuCD86-HuIgG1”) were used as antigens and immobilized directly on Nunc Maxisorp ELISA plates at 2 μg/ml. Table C-3 summarizes the type of selection used in both rounds. Phage populations were pre-incubated with saturating amounts of Sigma-Aldrich I-5154 (human IgG1) and Sigma-Aldrich M-9144 (mouse IgG2a) both prior to the first selection as well as during the phage absorption phase in first and second selection rounds. Plate-immobilized phages were retrieved from both first and second selection rounds using trypsin elution. Output of both R1 and R2 selections were analyzed for enrichment factor (#phage present in eluate relative to control). Based on these parameters the best selections were chosen for further analysis. To this end, the output from each selection was recloned as a pool into the expression vector pAX51. pAX51 is a derivative of pUC19. It contains the LacZ promoter which enables a controlled induction of expression using IPTG. The vector has a resistance gene for Ampicillin or Carbenicillin. The multicloning sites harbours several restriction sites of which SfiI and BstEII are frequently used for cloning Nanobodies®. In frame with the Nanobody coding sequence the vector codes for a C-terminal c-myc tag and a (His)6 tag. The signal peptide is the geneIII leader sequence which translocates the expressed Nanobody to the periplasm. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). pAX51 cloned Nanobodies were expressed as fusion proteins containing C-terminal both the c-myc as well as the 6His tags. The sequences of the clones obtained are depicted in Table B-1. EXAMPLE 5: BINDING OF THE CD80 AND/OR CD86 BINDING NANOBODIES IN ELISA AND FACS BINDING ASSAYS Periplasmic extracts were analyzed first for their ability to bind HuCD80-HuIgG1, HuCD86-HuIgG1 or HuIgG1. To this end, 3 independent ELISA assays were set up. In these ELISAs, either HuCD80 or HuCD86 fusion proteins or human IgG1 were coated on ELISA plates which were washed and then blocked using Marvel skimmed milk powder (Premier Brands UK Ltd., Wirral, Merseyside, UK). One parallel set of ELISA plates was not coated but only blocked. Four aliquots of periplasmic extract of individual clones prepared as described in Example 4 were allowed to bind in all four types of ELISA plates. Binding of Nanobody to immobilized antigen was detected using mouse anti-c-myc tag monoclonal antibody as a secondary, followed by a goat-anti-mouse (human and bovine serum protein pre-absorbed) HRP conjugate for detection (for detailed protocol, see the prior art and prior applications filed by applicant). Individual clones were scored as putative CD80 monoreactive or CD86 monoreactive if the clones yielded high OD's in either the ELISA plate coated with the CD80- or CD86-HuIgG1 fusion protein but not more than background in the other, nor in the plates coated with human IgG1 or uncoated but blocked plate. Clones were scored as putative CD80/CD86 bireactive if they scored high OD's on both ELISA plates but no more than background on both human IgG1 or blocked-only ELISA plates. To verify if putative mono- or bireactive clones could bind to the native form of the antigen, periplasmic extracts of such clones were allowed to bind to Raji cells, a human B-cell lymphoma line known to express high levels of both molecules. Binding of clones was detected using anti-c-myc tag mouse monoclonal antibody, followed by a phycoerythrin conjugated F(ab′)2 derived from goat-anti-mouse IgG (human and bovine crossabsorbed), and read on a BD FACS Calibur instrument. Binding was evaluated in CellQuest software. Dead cells were excluded from the analysis by gating out 7AAD vital dye positive scoring cells. Based on two separate FACS experiments, both cell binding and non-binding clones were identified in both mono- and bireactive clone selections. Table C-4 summarizes the ELISA and FACS data for a number of representative clones, binding in both ELISA (fusion protein) and FACS (native antigen) formats. ELISA data are presented as optical density (OD), FACS data were scored as moderate increase (“+”) in mean fluorescence intensity (MFI) over background fluorescence (secondary antibody only stained cells or cells stained with irrelevant specificity Nanobody plus secondary antibody), or strong increase over background (“++”). EXAMPLE 6: CD80 AND/OR CD86 BINDING NANOBODY EXPRESSION AND PURIFICATION Selected Nanobodies were expressed inE. colias c-myc, His6-tagged proteins in a culture volume of 200 mL. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 h at 37° C. Cells were harvested by centrifugation and periplasmic extracts were prepared by freeze-thawing the pellets. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC). Nanobodies were eluted from the column with 150 mM imidazole and subsequently subjected to gel filtration to PBS. Total yield and yield per liter of cell culture are listed in Table C-5. SDS-PAGE of purified Nanobodies is shown inFIG.2. EXAMPLE 7: INHIBITION OF INTERACTION OF CD80 AND/OR CD86 WITH CD28 OR CTLA4 In order to determine whether mono- or bireactive Nanobodies could inhibit the interaction of CD80 and/or CD86 with CD28 or CTLA4, ELISA plates were coated with either HuCD80-MuIgG2a or HuCD86-MuIgG2a and free binding sites were blocked using 4% Marvell, as per Example 5. Next, dilution series of various confirmed mono- or bireactive clones were allowed to bind to the immobilized antigen (75 microliter volume) before a fixed amount of either HuCD28-HuIgG1 or HuCTLA4-HuIgG1 was added to the wells (25 microliter volume, 2 microgram/ml final concentration), without washing the plates in between. After incubation and a wash step, plate-bound CD80 or CD86 captured CD28- or CTLA4-HuIgG1 was revealed using a HRP conjugated human IgG1 specific secondary reagent. Inhibition was determined based on OD values of controls having received no Nanobody (high control) or no CD28- or CTLA4-HuIgG1 fusion protein (low control). Example OD value profiles of representative inhibitory and non-inhibitory Nanobodies are shown inFIG.3. EXAMPLE 8: INHIBITION OF INTERACTION OF CD80 AND CD86 WITH CD28 OR CTLA4 In order to determine whether Nanobodies could inhibit the interaction of native CD80 and CD86 with CD28-Ig or CTLA4-Ig, Raji cells were incubated with serial dilutions of purified protein from confirmed clones or an irrelevant Nanobody. Next, either HuCD28-HuIgG1 or HuCTLA4-HuIgG1 was added to the cells/Nanobody suspension without washing the cells in between. After a wash step, cell-bound CD28- or CTLA4-HuIg was revealed using a phycoerythrin-conjugated F(ab′)2 derived from affinity purified goat-anti-human IgG1 antiserum (bovine serum protein crossabsorbed). Percentage inhibition was determined based on MFI values of controls having received an irrelevant specificity Nanobody (high control) or no CD28- or CTLA4-Ig fusion protein at all (low control). Example FACS profiles of representative inhibitory and non-inhibitory Nanobodies are shown inFIG.7. Results from both ELISA and FACS based assays are summarized in Table C-6. EXAMPLE 9: AFFINITY DETERMINATION OF THE CD80 AND/OR CD86 BINDING NANOBODIES Affinity constants (Kd) of individual purified Nanobody clones were determined by surface plasmon resonance on a Biacore 3000 instrument. In brief, HuCD80-HuIgG1 or HuCD86-HuIgG1 were amine-coupled to a CM5 sensor chip at densities of 3000-4000 RU. Remaining reactive groups were inactivated using ethanolamine. Nanobody binding was assessed at 1 and 0.1 microM. Each sample was injected for 4 min at a flow rate of 45 μl/min to allow for binding to chip-bound antigen. Next, binding buffer without Nanobody was sent over the chip at the same flow rate to allow for dissociation of bound Nanobody. After 2 min, remaining bound analyte was removed by injecting regeneration solution (50 mM NaOH or Glycine/HCl pH 1.5). Binding curves obtained at different concentrations of Nanobody were used to calculate Kd values. Kd values of selected Nanobody clones are shown in Table C-7. EXAMPLE 10: CONSTRUCTION AND EXPRESSION OF BISPECIFIC CD80 AND/OR CD86 NEUTRALIZING NANOBODIES Several mono- or bi-reactive Nanobodies were expressed as bispecific fusion proteins, consisting of an N-terminal anti-CD80/CD86 Nanobody, fused to a C-terminal human serum albumin binding Nanobody (ALB1) via a 9 amino acid Gly/Ser linker. These constructs were expressed inE. colias c-myc, His6-tagged proteins in shaker cultures as described in Example 6 and subsequently purified from periplasmic extracts by immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC). Examples of bispecific fusion proteins are shown in Table B-2. EXAMPLE 11: IMMUNIZATIONS WITH PD-1 Two llamas (No. 146 and No. 147) were immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #1086-PD, which is the ectodomain of human PD1 (rhPD1-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against PD-1 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhPD1-Fc. After blocking and adding diluted sera samples, the presence of anti-PD-1 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000 for both animals. EXAMPLE 12: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 146 and No. 147 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. EXAMPLE 13: SELECTIONS OF PD-1 BINDING NANOBODIES Phage libraries obtained from llamas No 146 and No. 147 were used for 2 rounds of phage display selection. rhPD1-Fc (R&D Systems, Minneapolis, US, Cat #1086-PD) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/ml. Preincubation of the phages with total human IgG (100 μg/ml) in 2% marvel PBST was followed by incubation with the phage libraries and extensive washing. In a first round, bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with PD-L1 (50 μg/ml) and PD-L2 (50 μg/ml) or with ICOSL (100 μg/ml) as a control. In a second round, bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with PD-L1 (40 μg/ml) and PD-L2 (40 μg/ml) or with ICOSL (80 μg/ml) as a control. After the second round of selection, enrichment was observed. The output from the selection were plated onto LB/amp/2% glu plates. Colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜90 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). The sequences of the clones obtained are depicted in Table B-4. EXAMPLE 14: BINDING OF THE OBTAINED NANOBODIES IN ELISA In order to determine binding specificity to PD-1 by the Nanobodies obtained from the selection described in Example 13, 96 eluted clones were tested in an ELISA binding assay setup. In short, 5 μg/ml PD-1 ectodomain (rhPD1-Fc, R&D Systems, Minneapolis, US) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing Nanobody of the different clones in 100 μl 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). 72 out of the 96 selected clones were able to bind to PD-1 with some specificity. 3 clones were shown to bind to the Fc part of the PD1-Fc-fusion. EXAMPLE 15: INHIBITION OF INTERACTION OF PD-L1 AND/OR PD-L2 WITH PD-1 In order to determine B7-H1 (PD-L1) and PD-L2 competition efficiency of PD-1 binding Nanobodies, the positive clones from the binding assay of Example 14 were tested in an ELISA competition assay setup. In short, 2 μg/ml PD-1 ectodomain (rhPD1-Fc, R&D Systems, Minneapolis, US) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.5 μg/ml of PD-L2-biotin or 0.5 μg/ml of B7-H1-biotin was preincubated with 10 μl of periplasmic extract containing Nanobody of the different clones and a control with only the biotinylated protein (high control). The biotinylated protein was allowed to bind to the immobilized receptor with or without Nanobody. After incubation and a wash step, biotinylated protein binding was revealed using a HRP-conjugated streptavidine. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control). OD values obtained are depicted inFIG.7. From these values clones were selected for recloning in production vector pAX51. After expression, the obtained Nanobodies were purified via the His-tag on Talon beads. Purified Nanobodies were tested in ELISA for binding to PD-1 as described in Example 14. Results are shown inFIG.8. EXAMPLE 16: DETERMINING COMPETITION EFFICIENCY OF PD-1 BINDING NANOBODIES BY TITRATION OF PURIFIED NANOBODY In order to determine competition efficiency of PD-1 binding Nanobodies, the positive clones of the previous binding assay were tested in an ELISA competition assay setup. In short, 2 μg/ml PD-1 ectodomain (R&D Systems Cat #1086-PD, Minneapolis, US) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.5 μg/ml of biotinylated PD-L2 or B7-H1 was preincubated with a dilution series of purified Nanobody. An irrelevant Nanobody against FcgR1 (49C5) was used as a negative controle, since this Nanobody does not bind to PD-1. PD-L2 or B7-H1 without biotin (cold PD-L2 or cold B7-H1) was used as a positive controle for competition. The results are shown inFIGS.9and10. 4 Nanobody families show competition with PD-L2-biotin for binding to PD-1 in a dose-dependent matter. The same 4 Nanobody families also show competition with B7-H1-biotin for binding to PD-1 in a dose-dependent manner. EXAMPLE 17: IMMUNIZATIONS WITH B7-H1 (PD-L1) One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #156-B7, which is the ectodomain of human B7-H1 (rh B7H1-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against B7-H1 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rh B7H1-Fc. After blocking and adding diluted sera samples, the presence of anti-B7-H1 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000. EXAMPLE 18: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 149 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. EXAMPLE 19: SELECTIONS OF B7-H1 (PD-L1) BINDING NANOBODIES The phage library obtained from llamas No. 149 was used for 2 rounds of phage display selection. In a first round, rhB7H1-Fc (R&D Systems, Minneapolis, US, Cat #156-B7) or rhPDL2-Fc (R&D Systems, Minneapolis, US, Cat #1224-PL) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/m. Preincubation of the phages with total human IgG (100 μg/ml) in 2% marvel PBST was followed by incubation with the phage libraries and extensive washing. Bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with PD-1 (100 μg/ml) or with BSA (100 μg/ml) as a control. Enrichment was observed over non-coated wells and wells aspecifically coated with rhPDL2-Fc. In a second round, rhB7H1-Fc (R&D Systems, Minneapolis, US, Cat #156-B7) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/m. Bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with PD-1 (100 μg/ml) or with BSA (100 μg/ml) as a control. After this second round of selection, high enrichment was observed. The output from the selection were plated onto LB/amp/2% glu plates. Colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). The sequences of the clones obtained are depicted in Table B-5. EXAMPLE 20: BINDING OF THE OBTAINED NANOBODIES IN ELISA In order to determine binding specificity to B7-H1 by the Nanobodies obtained from the selection described in Example 19, 96 eluted clones were tested in an ELISA binding assay setup. In short, 5 μg/ml B7-H1 ectodomain (rhB7H1-Fc, R&D Systems, Minneapolis, US, Cat #156-B7) or control Fc was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing Nanobody of the different clones in 100 μl 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). 17 out of the 96 selected clones were able to bind to B7-H1 with some specificity. 1 clone was shown to bind to the Fc part of the B7-H1-Fc-fusion as it also yielded high OD values in the parallel Fc control ELISA. Based on these binding data, clones were selected for recloning in production vector pAX51. After expression, the obtained Nanobodies were purified via the His-tag on Talon beads. Purified Nanobodies were again tested for binding B7-H1 in the ELISA binding assay as described above. OD values are shown inFIG.11. EXAMPLE 21: INHIBITION OF INTERACTION OF B7-H1 (PD-L1) WITH PD-1 In order to determine PD-1 competition efficiency of B7-H1 binding Nanobodies, the positive clones of the binding assay were tested in an ELISA competition assay setup. In short, 2 μg/ml B7-H1 ectodomain (rhB7H1-Fc, R&D Systems, Minneapolis, US, Cat #156-B7) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.5 μg/ml of PD-1-biotin was preincubated with 10 μl of periplasmic extract containing Nanobody of the different clones and a control with only PD-1-biotin (high control). The PD-1-biotin was allowed to bind to the immobilized ligand with or without Nanobody. After incubation and a wash step, PD-1 binding was revealed using a HRP-conjugated streptavidine. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control). OD values for the different Nanobody clones are depicted inFIG.12. EXAMPLE 22: DETERMINING COMPETITION EFFICIENCY OF B7-H1 BINDING NANOBODIES BY TITRATION OF PURIFIED NANOBODY In order to determine competition efficiency of B7-H1 binding Nanobodies, the positive clones of the previous binding assay were tested in an ELISA competition assay setup. In short, 2 μg/ml B7-H1 ectodomain (rhB7H1-Fc, R&D Systems, Minneapolis, US, Cat #156-B7) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.5 μg/ml of PD-1 was preincubated with a dilution series of purified Nanobody. An irrelevant Nanobody against FcgR1 (49C5) was used as a negative controle, since this Nanobody does not bind to B7-H1. Unlabelled PD-1 was used as a positive controle for competition of PD1-biotin. The results are shown inFIG.13. 3 Nanobody families show competition with PD-1-biotin for binding to B7-H1 in a dose-dependent manner. EXAMPLE 23: IMMUNIZATIONS WITH PD-L2 One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #1224-PL, which is the ectodomain of human PD-L2 (rhPDL2-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against PD-L2 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhPDL2-Fc. After blocking and adding diluted sera samples, the presence of anti-PD-L2 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000. EXAMPLE 24: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 149 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. EXAMPLE 25: SELECTION OF PD-L2 BINDING NANOBODIES The phage library obtained from llamas No 149 was used for 2 rounds of phage display selection. In a first round, rhB7H1-Fc (R&D Systems, Minneapolis, US, Cat #156-B7) or rhPDL2-Fc (R&D Systems, Minneapolis, US, Cat #1224-PL) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/m. Preincubation of the phages with total human IgG (100 μg/ml) in 2% marvel PBST was followed by incubation with the phage libraries and extensive washing. Bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with PD-1 (100 μg/ml) or with BSA (100 μg/ml) as a control. Enrichment was observed over non-coated wells and control wells coated with rhPDL1-Fc. In a second round, rhB7H2-Fc (R&D Systems, Minneapolis, US, Cat #1224-PL) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/m. Bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS), specifically eluted with PD-1 (100 μg/ml), or with BSA (100 μg/ml) as a control. After this second round of selection, high enrichment was observed. The output from the selection were plated onto LB/amp/2% glu plates. Colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). The sequences of the clones obtained are depicted in Table B-6. EXAMPLE 26: BINDING OF THE OBTAINED NANOBODIES IN ELISA In order to determine binding specificity to PD-L2 by the Nanobodies obtained from the selection described in Example 25, 96 eluted clones were tested in an ELISA binding assay setup. In short, 5 μg/ml PD-L2 ectodomain (R&D Systems, Minneapolis, US, Cat #1224-PL) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing Nanobody of the different clones in 100 μl 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). 32 out of the 96 selected clones were able to bind to PD-L2 with some specificity. EXAMPLE 27: INHIBITION OF INTERACTION OF PD-L2 WITH PD-1 In order to determine PD-1 competition efficiency of PD-L2 binding Nanobodies, the positive clones from the binding assay of Example 26 were tested in an ELISA competition assay setup. In short, 2 μg/ml PD-L2 ectodomain (R&D Systems, Minneapolis, US, Cat #1224-PL) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.5 μg/ml of PD1-biotin was preincubated with 10 μl of periplasmic extract containing Nanobody and a control with only PD-1-biotin (high control). The PD-1-biotin was allowed to bind to the immobilized ligand with or without Nanobody. After incubation and a wash step, PD-1 binding was revealed using a HRP-conjugated streptavidine. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control). OD values obtained are depicted inFIG.14. From these values clones were selected for recloning in production vector pAX51. After expression, the obtained Nanobodies were purified via the His-tag on Talon beads. Purified Nanobodies were tested in ELISA for binding to PD-L2 as described in Example 26. Results are shown inFIG.15. EXAMPLE 28: DETERMINING COMPETITION EFFICIENCY OF PD-L2 BINDING NANOBODIES BY TITRATION OF PURIFIED NANOBODY In order to determine competition efficiency of PD-L2 binding Nanobodies, the positive clones of the previous binding assay were tested in an ELISA competition assay setup. In short, 2 μg/ml PD-L2 ectodomain (R&D Systems, Minneapolis, US, Cat #1224-PL) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.5 μg/ml of PD-1 was preincubated with a dilution series of purified Nanobody. An irrelevant Nanobody against FcgR1 (49C5) was used as a negative controle, since this Nanobody does not bind to PD-L2. PD-1 without biotin (cold PD-1) was used as a positive controle for competition of PD-1-biotin. The results are shown inFIG.16. Four clones showed competition with PD1-biotin for binding to PD-L2 in a dose-dependent manner. EXAMPLE 29: IMMUNIZATIONS WITH B7-H2 (ICOSL) One llama (No. 149) was immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of R&D Systems (Minneapolis, MN, US) Cat #165-B7, which is the ectodomain of human B7-H2 (rhB7-H2-Fc), formulated in Titermax Gold (Titermax USA, Norcross, GA, US), according to standard protocols. At week 4, sera were collected to define antibody titers against B7-H2 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with rhB7-H2-Fc. After blocking and adding diluted sera samples, the presence of anti-B7-H2 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000. EXAMPLE 30: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 149 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. EXAMPLE 31: SELECTIONS OF B7-H2 BINDING NANOBODIES The phage library obtained from llamas No 149 was used for 2 rounds of phage display selection. rhB7-H2-Fc (R&D Systems, Minneapolis, US, Cat #165-B7) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/m. Preincubation of the phages with total human IgG (100 μg/ml) in 2% marvel PBST was followed by incubation with the phage libraries and extensive washing. In the first and second round bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with ICOS (100 μg/ml) or with PD-1 (100 μg/ml) as a control. Enrichment was observed over non-coated wells. The output from the selection were plated onto LB/amp/2% glu plates. Colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). The sequences of the clones obtained are depicted in Table B-7. EXAMPLE 32: BINDING OF THE OBTAINED NANOBODIES IN ELISA In order to determine binding specificity to B7-H2 by the Nanobodies obtained from the selection described in Example 31, 96 eluted clones were tested in an ELISA binding assay setup. In short, 5 μg/ml B7-H2 ectodomain (R&D Systems, Minneapolis, US, Cat #165-B7) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing Nanobody of the different clones in 100 ul 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). 75 out of the 96 selected clones were able to bind to B7-H2 with some specificity. EXAMPLE 33: INHIBITION OF INTERACTION OF B7-H2 (ICOSL) WITH ICOS In order to determine ICOS competition efficiency of B7-H2 binding Nanobodies, the positive clones from the binding assay of Example 32 were tested in an ELISA competition assay setup. In short, 1 μg/ml B7-H2 ectodomain (R&D Systems, Minneapolis, US, Cat #165-B7) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.25 μg/ml of ICOS-biotin was preincubated with 10 ul of periplasmic extract containing Nanobody of the different clones and a control with only ICOS-biotin (high control). The ICOS-biotin was allowed to bind to the immobilized receptor with or without Nanobody. After incubation and a wash step, ICOS binding was revealed using a HRP-conjugated streptavidine. Binding specificity was determined based on OD values compared to controls having received no Nanobody (high control). OD values obtained are depicted inFIG.17. Based on these values, clones were selected for recloning in production vector pAX51. After expression, the Nanobodies were purified via the His-tag on Talon beads. Purified Nanobodies were tested in ELISA for binding to B7-H2 as described in Example 32. Results are shown inFIG.18. EXAMPLE 34: DETERMINING COMPETITION EFFICIENCY OF B7-H2 BINDING NANOBODIES BY TITRATION OF PURIFIED NANOBODY In order to determine ICOS competition efficiency of B7-H2 binding Nanobodies, the positive clones of the binding assay were tested in an ELISA competition assay setup. In short, 1 μg/ml B7-H2 ectodomain (R&D Systems, Minneapolis, US, Cat #165-B7) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.25 μg/ml of ICOS-biotin was preincubated with a dilution series of purified Nanobody. An irrelevant Nanobody against FcgR1 (49C5) was used as a negative controle, since this Nanobody does not bind to B7-H2. ICOS without biotin (cold ICOS) was used as a positive controle for competition of ICOS-biotin. The results are shown inFIG.19. 5 Nanobody families show competition with ICOS-biotin for binding to B7-H2 in a dose-dependent manner. EXAMPLE 35: IMMUNIZATIONS WITH CD28 Two llamas (No. 45 and No. 46) were immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of CD28-Fc fusion (R&D Systems, Minneapolis, MN, US), formulated in adjuvant Stimune (Cedi Diagnostics, the Netherlands), according to standard protocols. At week 4, sera were collected to define antibody titers against CD28 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with hCD28-Fc. After blocking and adding diluted sera samples, the presence of anti-CD28 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000 for both animals. EXAMPLE 36: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 45 and No. 46 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. EXAMPLE 37: SELECTIONS OF CD28 BINDING NANOBODIES Phage libraries obtained from llamas No 45 and No. 46 were used for phage display selection. hCD28-Fc (R&D Systems, Minneapolis, US, Cat #342-CD) was coated onto Maxisorp 96-well plates (Nunc, Wiesbaden, Germany) at 0.5 and 5 μg/ml. The phages were incubated with human IgG (100 μg/ml) in 2% marvel PBST prior to incubation with the immobilized CD28. After extensive washing, plate bound phage was aspecifically eluted with trypsin (1 mg/ml in PBS) or specifically eluted with B7-1 and B7-2 (50 μg/ml). Enrichment above background was observed for all conditions. The output from the selection were plated onto LB/amp/2% glu plates. Colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜90 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). The sequences of the clones obtained are depicted in Table B-8. EXAMPLE 38: BINDING OF THE OBTAINED NANOBODIES IN ELISA In order to determine whether the Nanobodies obtained from the selection described in Example 37 bind CD28, 96 eluted clones were tested in an ELISA binding assay setup. In short, 1 μg/ml CD28 (hCD28-Fc, R&D Systems, Minneapolis, US, Cat #342-CD) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 10 μl of periplasmic extract containing Nanobody of the different clones in 100 μl 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, followed by a wash step and an additional incubation with a HRP-conjugated donkey-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control). 57 out of 96 selected clones were able to bind to CD28 with some specificity. EXAMPLE 39: INHIBITION OF INTERACTION OF B7-1 WITH CD28 In order to determine B7-1 competition efficiency of the CD28 binding Nanobodies, a selection of the CD28 binding clones was made and tested for B7-1 competition in an ELISA competition assay setup. In short, 1 μg/ml B7-1-muFc (Ancell, Bayport, MN, US, Cat #510-820) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. In another plate, 90 μl 2.2 μg/ml CD28-hFc was mixed with 10 μl periplasmic extract of the CD28 binding clones. This mixture was applied on the coated B7-1 and CD28 was allowed to bind to the immobilized B7-1. After incubation and a wash step the CD28-hFc was detected with a HRP-conjugated anti-human Fc (Jackson Immunoresearch Laboratories, West Grove, PA, US, Cat #109-116-170). Degree of binding inhibition was determined based on OD values compared to controls having received no Nanobody (high control) of no CD28-hFc (low control). OD values obtained are depicted inFIG.20. From these values, clones were selected for recloning in production vector pAX51. After expression, the obtained Nanobodies were purified via the His-tag on Talon beads. Purified Nanobodies were tested in ELISA for binding to CD28 as described in Example 38. Results are shown inFIG.21. EXAMPLE 40: DETERMINING COMPETITION EFFICIENCY OF CD28 BINDING NANOBODIES BY TITRATION OF PURIFIED NANOBODY In order to determine B7-1 competition efficiency of CD28 binding Nanobodies, the purified Nanobodies that showed binding in the previous binding assay were tested in an ELISA competition assay setup. In short, 1 μg/ml B7-1-muFc (Ancell, Bayport, MN, US, Cat #510-820) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 2 μg/ml CD28-hFc was mixed with a dilution series of purified Nanobody. An irrelevant Nanobody against FcgR1 (49E4) was used as a negative controle, since this Nanobody does not bind to CD28. After incubation and a wash step the CD28-hFc was detected with a HRP-conjugated anti-human Fc (Jackson Immunoresearch Laboratories, West Grove, PA, US, Cat #109-116-170) 1:1500 in 2% MPBST. The results are shown inFIG.22. All Nanobodies selected showed competition with B7-1 for binding to CD28 in a dose-dependent manner. EXAMPLE 41: BINDING OF CD28-FC BINDING NANOBODIES TO HUMAN CD28 EXPRESSING JURKAT CELLS IN FACS To verify if the CD28-Fc binding clones could also bind to the native form of the CD28 antigen, serial dilutions of purified protein preparations of such clones were allowed to bind to the human Jurkat T-cell line, which expresses human CD28. Binding of putative CD28 reactive Nanobodies clones was detected using unlabeled anti-c-myc tag mouse monoclonal antibody 9E10, followed by a phycoerythrin conjugated F(ab′)2 derived from goat-anti-mouse IgG (human and bovine crossabsorbed), and read on a BD FACSarray instrument. Dead cells were excluded from the analysis by gating out TOPRO3 vital dye positive scoring cells. Binding of the Nanobodies to cells was evaluated in BD FACS array control software as PE channel histograms. Based on these FACS experiments, all CD28-Fc binding Nanobody clones bound cell expressed CD28. Results of a representative experiment are depicted inFIG.23. EXAMPLE 42: INHIBITION BY CD28 BINDING NANOBODIES OF THE INTERACTION OF CD28 WITH CD80-FC OR CD86-FC ANALYSED IN FACS The potency of cell-expressed CD28 binding Nanobodies to inhibit the interaction of CD28 with either CD80 or CD86 was also ranked using FACS based screening method. In brief, serial dilutions of purified Nanobodies were prepared and incubated at 4° C. with Jurkat cells. To this suspension, either HuCD80-Hu IgG1 Fc fusion protein or HuCD86-Hu IgG1 Fc fusion protein was added 30 minutes after Nanobody incubation had started. After an additional 30 minutes incubation, cells were washed and cell-bound HuCD80-Fc or HuCD86-Fc was detected using a phycoerythrin labeled F(ab′)2fragment of goat anti human IgG Fc (Jackson Immunoresearch Laboratories, West Grove, PA, US, Cat #109-116-170). Dead cells were stained by including TOPRO3 vital dye in the final resuspension buffer. All samples were read on a BD FACSarray instrument. Dead cells were excluded from the analysis by gating out TOPRO3 vital dye positive scoring cells. Inhibition of CD80-Fc or CD86-Fc binding to cell-displayed CD28 by these Nanobodies was evaluated in BD FACSarray control software as PE channel histograms. Results were summarized as mean fluorescence values of these histograms as a function of Nanobody concentration. Results are depicted inFIG.24. EXAMPLE 43: IMMUNIZATIONS WITH CTLA4-IG Two llamas (No. 119 and No. 120) were immunized with 100 or 50 μg doses of Chimerigen Cat #HF-210A4, according to the scheme outlined in Table C-8. Both proteins were formulated in Stimune adjuvants (Cedi Diagnostics B.V., Lelystad, The Netherlands). Blood was collected from these animals as indicated in Table C-8. EXAMPLE 44: SERUM TITERS OF CTLA4-IG IMMUNIZED ANIMALS Sera from blood samples of llamas 119 and 120 were obtained prior to immunization, during the immunization protocol and after completion of the immunizations. Chimerigen CTLA4-Ig or an irrelevant specificity human IgG1 isotope monoclonal antibody were coated onto Nunc Maxisorb plates at 2 μg/ml, blocked with 1% casein in PBS and incubated with serial dilutions of pre- and postimmune llama sera. Plate-immobilized llama IgG was detected using HRP conjugated goat-anti-llama IgG (Bethyl Labs, Montgomery, TX) and TMB chromogen according to standard methods. Comparison of optical density values clearly indicated immunization induced a humoral immune response against CTLA4-Ig in both animals, and that the response was higher against CTLA4-Ig than the control protein having the same human IgG1 Fc. EXAMPLE 45: LIBRARY CONSTRUCTION Peripheral blood mononuclear cells were prepared from blood samples obtained from llama No. 119 and No. 120 using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored after filter sterilization at 4° C. for further use. The characteristics of the constructed libraries are shown in Table C-9. EXAMPLE 46: SELECTIONS OF CTLA4 BINDING NANOBODIES Phage libraries from llama No. 119 and No. 120 were used for two rounds (R1, R2) of selections on the same plate-immobilized antigen or on soluble antigen. Chimerigen Cat #HF-210A4 CTLA4-Ig was immobilized at concentrations varying from 5 μg/ml to 0.05 μg/ml on Nunc Maxisorp ELISA plates precoated at 10 μg/ml with anti-human IgG1 Fc Nanobody. Plate-immobilized phages were retrieved using trypsin or BNI3 elution and rescued inE. coli. Rescued phages were incubated with concentrations of biotinylated CTLA4-Ig varying from 60 nM to 1 pM (Chimerigen Cat #HF-210A4, biotinylated at Ablynx according to standard procedures), captured on neutravidin precoated Maxisorb plates and eluted using trypsin or BNI3. Phage populations were pre-incubated with saturating amounts of Sigma-Aldrich #I 4506 human IgG prior to both selection rounds. Output of both R1 and R2 selections were analyzed for enrichment factor (#phage present in eluate relative to control). Based on these parameters, the best selections were chosen for further analysis. Individual colonies were picked and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 μl) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein). Nanobodies were expressed as fusion proteins containing C-terminal both the c-myc as well as the 6His tags. The sequences of the clones obtained are depicted in Table B-9. EXAMPLE 47: SELECTIVE BINDING OF THE NANOBODIES TO CTLA4-FC IN ELISA Periplasmic extracts as prepared in example 46 were analyzed first for their ability to bind HuCTLA4-HuIgG1 or HuCD28-HuIgG1. To this end, 2 independent ELISA assays were set up. In these ELISAs, either HuCTLA4 (Chimerigen) or HuCD28 (R&D Systems) fusion proteins were coated on ELISA plates which were washed and then blocked using 4% Marvel skimmed milk powder (Premier Brands UK Ltd., Wirral, Merseyside, UK) in PBS. 10 μl aliquots of periplasmic extract of individual clones prepared as described in Example 46 were allowed to bind in both ELISAs. Binding of Nanobody to immobilized antigen was detected using mouse anti-c-myc tag monoclonal antibody as a secondary antibody, followed by a goat-anti-mouse (human and bovine serum protein pre-absorbed) HRP conjugate for detection (for detailed protocol, see the prior art and prior applications filed by applicant). Individual clones were scored as putative CTLA4 monoreactive if the clones yielded high OD's in the ELISA plate coated with the HuCTLA4-HuIgG1 fusion protein but not more than background in the other. The clones were scored CD28 and/or human IgG1 Fc crossreactive if they yielded high OD's in the ELISA plate coated with the HuCTLA4-HuIgG1 fusion protein as well as in the other. Clones binding both CD28-Fc and CTLA4-Fc were very rare. From the 192 clones tested, 115 were able to bind to CTLA4-Fc with some specificity. Clones were selected for recloning in production vector pAX51. After expression, the obtained Nanobodies were purified via the His-tag on Talon beads. EXAMPLE 48: INHIBITION OF THE INTERACTION OF CTLA4 WITH B7-1 In order to determine B7-1 competition efficiency of CTLA4 binding Nanobodies, the purified clones were tested in an ELISA competition assay setup. In short, 2 μg/ml B7-1-muFc (Ancell, Bayport, MN, US, Cat #510-820) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 0.33 nM CTLA4-hFc was mixed with a dilution series of purified Nanobody. An irrelevant Nanobody (1A1) was used as a negative controle, since this Nanobody does not bind to CTLA4. As a positive controle for competition with B7-1, the commercial CTLA-4 binding antibody (BNI-3; competing for B7-1 and B7-2) was used. After incubation and a wash step, the CTLA4-hFc was detected with a HRP-conjugated anti-human Fc (Jackson Immunoresearch Laboratories, West Grove, PA, US, Cat #109-116-170) 1:1500 in 2% MPBST. OD values obtained, depicted inFIGS.26and27, show that 2 Nanobodies selected show competition with B7-1 for binding to CTLA4 in a dose-dependent manner. EXAMPLE 49: INHIBITION OF THE INTERACTION OF CTLA4 WITH B7-2 In order to determine B7-2 competition efficiency of CTLA4 binding Nanobodies, the purified clones were tested in an ELISA competition assay setup. In short, 5 μg/ml B7-muFc (Ancell, Bayport, MN, Cat #509-820) was immobilized on maxisorp microtiter plates (Nunc, Wiesbaden, Germany) and free binding sites were blocked using 4% Marvel in PBS. Next, 22 nM CTLA4-hFc was mixed with a dilution series of purified Nanobody. An irrelevant Nanobody (1A1) was used as a negative controle, since this Nanobody does not bind to CTLA4. As a positive controle for competition, the commercial CTLA-4 binding antibody (BNI-3; competing for B7-1 and B7-2) was used. After incubation and a wash step the CTLA4-hFc was detected with a HRP-conjugated anti-human Fc (Jackson Immunoresearch Laboratories, West Grove, PA, US, Cat #109-116-170) 1:1500 in 2% MPBST. OD values obtained, depicted inFIGS.28and29, show that 4 Nanobodies selected show competition with B7-2 for binding to CTLA4 in a dose-dependent manner. EXAMPLE 50: INHIBITION OF THE CTLA4-FC/CD80-FC INTERACTION IN ALPHASCREEN Periplasmic extracts as prepared in example 46 were analyzed for their ability to block the interaction of HuCTLA4-HuIgG1 (Chimerigen) with HuCD80-MuIgG2a (Ancell). To this end, an alphascreen assay (Perkin Elmer, Waltham, MA) was set up and used as a screening assay. In brief, 5 μl of periplasmic extract of individual Nanobody clones were incubated with 0.15 μM HuCTLA4-HuIgG1 Fc, 0.10 μM biotinylated HuCD80-MuIgG2a Fc, streptavidin coated donor beads and anti-human IgG1 Fc Nanobody coupled acceptor alphascreen beads. The mouse monoclonal antibody BNI3, known to inhibit the CTLA4/CD80 interaction, was used as a positive control. Assays were read in an Envision alphascreen option fitted multimode reader (Perkin Elmer). Individual clones were scored as putative CTLA4/CD80 interaction inhibiting if the presence of the periplasmic extract decreased the fluorescent signal of the acceptor beads. EXAMPLE 51: CTLA4-IG BINDING NANOBODY EXPRESSION AND PURIFICATION Selected CTLA4/CD80 interaction inhibiting Nanobodies were expressed in the periplasmic space ofE. colias c-myc, His6-tagged proteins in a culture volume of ˜200 mL. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 h at 37° C. Cells were harvested by centrifugation and periplasmic extracts were prepared. These extracts were used as starting material for immobilized metal affinity chromatography (IMAC). Nanobodies were eluted from the column with 150 mM imidazole and subsequently subjected to gel filtration to PBS. Total yield and yield per liter of cell culture are listed in Table C-10. SDS-PAGE of purified Nanobodies is shown inFIG.30. EXAMPLE 52: RANKING CTLA4-FC/CD80-FC INTERACTION INHIBITION POTENCY OF NANOBODIES USING ALPHASCREEN In order to determine which Nanobodies could inhibit the interaction of CTLA4 with CD80 most efficiently, serial dilutions of purified Nanobodies were prepared and tested in the same alphascreen assay as used for screening periplasmic extracts (as described in Example 50). Table C-11 summarizes the IC50 value of selected Nanobodies in this assay, clearly showing a range of potencies. EXAMPLE 53: GENERATION OF HUMAN AND CYNOMOLGUS CTLA4 OVEREXPRESSING STABLE CELL LINES To verify if CTLA4-Ig binding Nanobody clones could bind to the native form of either the human or cynomolog monkey CTLA4 antigen, transfected cells stably expressing high levels of either human or cynomolgus monkey CTLA4 on the cell membrane were generated. These clones were generated by transfecting CHO-K1 cells with either full-length human CTLA4 cDNA cloned into pCI-Neo (Promega, Madison, WI), where the intracellular position Y201 was mutated to valine in order to ensure retention of the protein on the extracellular membrane (Chuang et al. J. Immunol. 159: 144, 1997), or the same human CTLA4 cDNA where three extracellular domain positions were mutated to the corresponding cynomolgus monkey CTLA4 extracellular domain amino acids (S13N, I17T and L105M; U.S. Pat. No. 6,682,736). Introduction of linearized endotoxin-free plasmid DNA and selection for stable transfected cells was performed according to standard transfection and antibiotic selection methods. Individual high CTLA4 expressing transfectants were cloned from the bulk antibiotic resistant CHO cell population by staining the transfected CHO cells populations using the viability dye TOPRO3, PE labeled BNI3 antibody (BD Biosciences, San Jose, CA, Cat #555853)) and sorting individual live (TOPRO3 negative) highly PE fluorescent cells into microtiter plate wells containing selection medium. Outgrowing clones were expanded and re-screened in FACS for homogeneous and high level CTLA4 expression using PE labeled BNI3. EXAMPLE 54: BINDING OF THE CTLA4-IG BINDING NANOBODIES TO HUMAN AND CYNO CTLA4 IN FACS To verify if CTLA4-Ig binding clones which inhibit the interaction of CTLA4 with CD80-Ig could also bind to the native form of the antigen, serial dilutions of purified protein preparations of such clones were allowed to bind to CHO cells expressing either human or cynomolgus monkey CTLA4 (see Example 53). Binding of putative CTLA4 reactive Nanobodies clones was detected using unlabeled anti-c-myc tag mouse monoclonal antibody 9E10, followed by a phycoerythrin conjugated F(ab′)2 derived from goat-anti-mouse IgG (human and bovine crossabsorbed), and read on a BD FACSarray instrument. Dead cells were excluded from the analysis by gating out TOPRO3 vital dye positive scoring cells. Binding of the Nanobodies to cells was evaluated in BD FACSarray control software as PE channel histograms. Based on these FACS experiments, all CTLA4-Ig binding Nanobody clones found to be CD80 interaction inhibitory in alphascreen, also bound cell expressed CTLA4. Both cynomolgus crossreactive as well as essentially non-crossreactive human CTLA4 transfectant binding clones were identified by comparing the mean fluorescence intensity curve of serial dilutions of Nanobody between human and cynomologus CTLA4 transfected CHO cells. Table C-11 summarizes the IC50 value of selected Nanobodies in this assay. EXAMPLE 55: INHIBITION OF CTLA4/CD80-FC INTERACTION IN FACS The potency of selected Nanobodies to inhibit the interaction of CTLA4 with CD80 was also ranked using FACS based screening method. In brief, serial dilutions of purified Nanobodies were prepared and incubated at 4° C. with either human or cynomologus CTLA4 overexpressing stable transfectants (see Example 53). To this suspension, HuCD80-Hu IgG1 Fc fusion protein was added to a concentration of 10 nM (final concentration) 20 minutes after Nanobody incubation had started. This concentration was previously determined to be the minimal amount required to saturate all CD80 binding sites on both human and cyno CTLA4 expressing CHO cell clones in the absence of any CTLA4 interaction blocking proteins. After an additional 30 minutes incubation, cells were washed and cell-bound HuCD80-Hu Fc was detected using a phycoerythrin labeled F(ab′)2fragment of goat anti human IgG Fc (Jackson Immunoresearch Laboratories, Cat #109-116-170). Dead cells were stained by including TOPRO3 vital dye in the final resuspension buffer. All samples were read on a BD FACSarray instrument. Dead cells were excluded from the analysis by gating out TOPRO3 vital dye positive scoring cells. Inhibition of CD80-Fc binding to cell-displayed CTLA4 by these Nanobodies was evaluated in BD FACSarray control software as PE channel histograms. Results are summarized as mean fluorescence values of these histograms as a function of Nanobody concentration inFIGS.31,32and33. EXAMPLE 56: AFFINITY DETERMINATION OF CTLA4 BINDING NANOBODIES Affinity constants (Kd) of individual purified Nanobody clones were determined by surface plasmon resonance on a Biacore T100 instrument. In brief, HuCTLA4-HuIgG1 or HuCD28-HuIgG1 were amine-coupled to a CM5 sensor chip at densities of 740-1700 RU. Remaining reactive groups were inactivated using ethanolamine. Nanobody binding was assessed at concentrations varying from 500 to 0.33 nM. Each sample was injected for 2 min at a flow rate of 45 μl/min to allow for binding to chip-bound antigen. Next, binding buffer without Nanobody was sent over the chip at the same flow rate to allow for dissociation of bound Nanobody. After 2 min, remaining bound analyte was removed by injecting regeneration solution (10 mM Glycine/HCl pH 1.5). Binding curves obtained at different concentrations of Nanobody were used to calculate KDvalues. For some clones, only a single undetermined concentrations of Nanobody was injected. For these clones, only off-rates could be determined. KD, konand koffvalues of selected Nanobody clones are shown in Table C-11. EXAMPLE 57: CELL-STIMULATION ASSAYS BY CTLA4 BINDING NANOBODIES Purified Nanobodies were tested in human PBMC and whole blood T-cell stimulation assays (as described in U.S. Pat. No. 6,682,736). Briefly, fresh peripheral blood were collected from healthy donors in heparin anticoagulant containing vacutainers. Blood was then diluted with RPMI1640 medium containing penicillin, streptomycin, 100 ng/ml of Staphylococcal enterotoxin A fromStaphyloccocus aureus(SEA, Sigma-Aldrich, St. Louis, MO, Cat #S9399) and aliquotted in 96-well microtiter plates which had been coated overnight with 60 ng/well of mitogenic mouse-anti-human anti-CD3 (clone OKT3, eBioscience, San Diego, CA, Cat #16-0037-85). Alternatively, PBMC were isolated from freshly drawn heparin anticoagulated blood or buffy coats using a standard Ficoll gradient, resuspended in medium containing SEA as described above and aliquotted into a CD3 coated microtiter plate. Serial dilutions of CTLA4 neutralizing antibody BNI3, mouse IgG2a isotype control antibody or endotoxin-free CTLA4 reactive Nanobody preparations were added to the wells of either whole blood or PBMC assay. Plates were incubated for 48, 72 or 96 hours at 37° C. under 5% CO2/100% humidity atmosphere. Cell-free supernatant from all wells were collected at each timepoint and frozen at −80° C. until the last timepoint was harvested and frozen. IL-2 concentration in these conditioned supernatants was then analyzed after simultaneous thawing of all timepoint samples of any given assay at fixed 1/10 or ½dilutions, using a standard IL-2 sandwich ELISA (Invitrogen, Carlsbad, CA, Cat #CHC 1244). CTLA4 neutralization in these assay gave rise to increased levels of IL-2 production. Relative potency of Nanobody clones and reference antibody could be scored and ranked according to the IC50 values of the titration curves obtained.FIG.34shows representative IL-2 ELISA results, expressed as optical density. EXAMPLE 58: FORMATTING OF CTLA4 NEUTRALIZING NANOBODIES Next, Nanobodies binding CTLA4 and neutralizing its activity in bioassays were formatted such that they gain binding affinity (avidity) and potency in bioassays. For example, CTLA4 neutralizing monomeric Nanobodies were reformatted into bivalent CTLA4 neutralizing constructs by standard PCR-based DNA manipulation, resulting in anE. coliexpression plasmid encoding a fusion protein comprising two identical copies of the same Nanobody clone, linked in tandem via a gly/ser linker. Alternatively, two identical CTLA4 binding Nanobody clones were reformatted similarly into a trivalent fusion proteins, starting with the bivalent format as described above, but fusing this further to another C-terminal Nanobody clone which binds human serum albumin. These two bi- or trivalent formats Nanobodies were expressed inE. coliand subsequently purified using methods identical to those described in Example 51. Finally, some CTLA4 neutralizing Nanobody clones were reformatted into a bivalent format essentially identical to that described above, but further fused C-terminally to full-length human serum albumin. These fusion protein encoding cassettes were cloned into aPichia pastorisexpression vector which allows for inducible protein expression, secreted into the culture medium. Such fusion proteins were produced according to standard methods and purified from conditioned medium using protein A (GE Healthcare Biosciences, Uppsala, Sweden) chromatography for Nanobody clones known to bind protein A, or Blue Sepharose (GE Healthcare Biosciences, Uppsala, Sweden) for Nanobody clones that do not bind protein A, all according to the manufacturer's instructions. Table B-10 lists the sequences of such bivalent, trivalent and bivalent-albumin fusion proteins. EXAMPLE 59: POTENCY OF MULTIVALENT CTLA4 NEUTRALIZING NANOBODIES AS DETERMINED IN ALPHASCREEN Nanobodies neutralizing CTLA4 and formatted into various multivalent formats as described in Example 58 were titered in a CTLA4-Ig/CD80-Ig interaction alphascreen as described in Example 52.FIG.35shows the results of a representative assay where the potency of a monovalent Nanobody clone is compared to that of the multivalently formatted same Nanobody clone. Table C-12 summarizes the IC50 values of selected multivalent Nanobodies. EXAMPLE 60: POTENCY OF MULTIVALENT CTLA4 NEUTRALIZING NANOBODIES AS DETECTED IN FACS Nanobodies neutralizing CTLA4 and formatted into various multivalent formats as described in example 58 were titered in a CTLA4 transfected CHO cell line/CD80-Ig interaction FACS assay as described in Example 55.FIG.36shows the results of a representative assay where the potency of a monovalent Nanobody clone is compared to that of the multivalently formatted same Nanobody clone. Table C-13 summarizes the IC50 values of selected multivalent Nanobodies. EXAMPLE 61: BINDING AFFINITY OF FORMATTED CTLA4 NEUTRALIZING NANOBODIES AS MEASURED IN BIACORE The CTLA4-Ig binding affinity/avidity of multivalent Nanobodies as described in example 58 was analyzed in BIAcore as described in Example 56.FIG.37shows the results of a representative assay where the association and dissociation of monovalent Nanobody clones was compared to those of the corresponding multivalently formatted Nanobodies. Table C-14 summarizes the off-rates values measured for selected multivalent Nanobodies. To calculate apparent gain of affinity (avidity), the ratio of the formatted clone's off-rate versus the monovalent clone's off-rate was determined. For 11F1, no direct comparison could be made for the 300-375 s dissociation period as most monovalent material was dissociated at that time, so the 300-375 s dissociation phase kinetics were compared to the 60 s dissociation phase of the monomer. EXAMPLE 62: POTENCY OF MULTIVALENT CTLA4 NEUTRALIZING NANOBODIES AS DETECTED IN BIOASSAY The potency gain of multivalent formatted Nanobodies was compared versus the original monovalent Nanobody in a T-cell stimulation bioassay, executed as described in Example 57.FIG.38shows the results of a representative assay where the potency of a monovalent Nanobody clone is compared to that of the multivalently formatted same Nanobody clone. As can readily be observed, formatting of a neutralizing anti-CTLA4 Nanobody such as 11F1 results in an increase in potency in bioassay. EXAMPLE 63: CTLA4 NEUTRALIZING NANOBODIES CAN BE HUMANIZED WITHOUT SIGNIFICANT LOSS OF FUNCTIONALITY FIGS.39and40show the amino acid sequences of respectively Nanobody clones 11F1 and 11E3 aligned with multiple human immunoglobulin germline sequences. Multiple amino acid differences between the Nanobody clones and human germline are thus made evident. In order to reduce the potential immunogenicity of Nanobodies administered to human recipients, Nanobody sequences can be modified to resemble the human germline more than the initial “wild type” sequence, a process termed humanization. However, substitution of certain critical amino acids in Nanobody sequences can lead to reduced or completely abrogated antigen binding. Multiple partially humanized Nanobody variant sequences were generated from each clone, as depicted inFIGS.41and42. Variant sequences were prepared by standard site-directed mutagenensis methods, well known to those skilled in the art. Next, protein was produced and purified from these variants using the same methods as described in the pervious examples, and tested for their potential to inhibit the interaction between CTLA4-Ig and CD80-Ig in alphascreen, as described in Example 52. Results are shown in Table C-15. A “loss factor” was calculated by taking the ratio of the wild type molecule IC50 value and the variant's IC50 value. As can readily be observed, humanization of clone 11F1 does not result in significant loss of potency. Therefore, a variant uniting all mutations in one humanized variant can be expected to retain full potency, while also being very unlikely to induce an immune response in man. A mutation present in all humanized variants of 11E3 results in a 10-20 fold range potency loss, but only one variant showed loss of all inhibition potency. Additional variants containing fewer mutations than the “basic” set contained within all variants listed here (essentially single amino acid reversals to the wild type Nanobody clone sequence) can therefore be designed and tested as described here to determine which mutation(s) is (are) responsible for the overall 10× drop in potency. Then, either all mutations listed and tested here can be united into a single humanized variant, or all individual mutations present in variants 1, 2 and 4 can be used in combination with the mutations found not to result in 10× potency drops in the less humanized “basic” set of mutations not resulting in 10× potency drops. EXAMPLE 64: HUMANIZED CTLA4 NEUTRALIZING NANOBODIES CAN BE FORMATTED Humanized Nanobody sequences found not to result in large losses of affinity and/or potency, as described in Example 63, can be formatted into bivalent, trivalent or higher valency multivalent and/or multispecific Nanobodies, as described in Example 58. These can be produced, purified and tested for gain of affinity/potency using the methods described in Examples 58 to 62. One can, for example, combine the gain of affinity/potency of formatted Nanobodies and the benefit of reduced potential to induce an anti-drug immune response in man in a single molecule. This type of molecule is preferred over molecules with lesser potency and/or shorter half-lives and/or higher anticipated immunogenicity. EXAMPLE 65: IN VIVO EFFICACY TESTING OF CTLA4 BINDING NANOBODIES In vivo neutralization of CTLA4 gives rise to increased levels of T-cell activity. One indirect method of measuring this increase is via determination of humoral (B-cell) immune responses following an antigenic challenge, as this reflects increased T-cell help (Keler et al., J. Immunol. 171: 6251, 2003). Alternatively, one can compare the relative frequency of IL-2 producing T-cells between treated and control animals. Therefore, the therapeutic efficacy of CTLA4 neutralizing Nanobodies having crossreactivity with non-human primate CTLA4 is determined by administering the Nanobodies to primates undergoing an immunization scheme. The antigen used for the purpose may be either a vaccine known to be efficacious by itself (i.e. hepatitis vaccine, tetanus toxoid vaccine) or a vaccine which is less than fully protective when used by itself (i.e. certain cancer vaccines) (Keler et al.). Anti-vaccine serum titers can then be determined in ELISA and compared with primates that did not receive CTLA4 neutralizing Nanobodies. Alternatively, increased T-cell reactivity towards the vaccine antigen can be determined by in vitro restimulating PBMC with antigen and comparing the number of IL-2 producing T-cells in ELIspot. Both the required purified vaccine antigen as well as monkey IL-2 ELISpot assays are commercially available (Rollier et al. 2007, Hepatology 45: 602). TABLE B-1Preferred Nanobodies against B7-1 and/or B7-2<Name, SEQ ID #; PRT (protein); →Sequence>CD8086PMP1A1, SEQ ID NO: 266; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGNSATNVDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAGYYCAAATRRAYLPIRIRDYIYWGQGTQVTVSS>CD8086PMP1A3, SEQ ID NO: 267; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGPTSSSYSMGWFRQAPGKEREFVAAINWSHGVTYYADSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYYCAANEYGLGSSIYAYKHWGQGTQVTVSS>CD8086PMP1B2, SEQ ID NO: 268; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRSFSSYVMGWFRQAPGKEREFVAAIIGRDIGTYYADSVKGRFTISRDNAKTTVYLQMNALKPEDTAVYYCAADSRSRLSGIRSAYDYWGQGTVTVSS>CD8086PMP1C5, SEQ ID NO: 269; PRT; →EVQLVESGGGSVQAGGSLRLSCAATGRTFSSYGMGWFRQAPGKEREFVAAIHWNSGITYYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYICAASSKGLTGTIRAYDDWGQGTQVTVSS>CD8086PMP1C7, SEQ ID NO: 270; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAAGWFRQAPGKERDFVAAINWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASGWGRTTVLADTVXYWGQGTQVTVSS>CD8086PMP1C9, SEQ ID NO: 271; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFXXGIDAMGWFRQAPGKEREFVASIXRSGGXATXADSVKGRFTISRDNAKNTMYLQMNXLKPEDTAGYYCAAATRRPYLPIRISRLYLXGPGXHXVTVSS>CD8086PMP1D1, SEQ ID NO: 272; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSS>CD8086PMP1D4, SEQ ID NO: 273; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGGSATNADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAGYYCAAATRRPYLPIRIRDYIYWGQGTQVTVSS>CD8086PMP1E11, SEQ ID NO: 274; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYSAIGWFRQAPGKEREGVSYISSSDGSTYYADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAGGPFTVSTMPWLANYWGQGTQVTVSS>CD8086PMP1F12, SEQ ID NO: 275; PRT; →EVQLVESGGGLVQAGGSLRLACAASGLSFSFYTMGWFRQAPGEERDFVAAINWSGGSTLYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVRSVGRTYWTRALEYNYWGQGTQVTVSS>CD8086PMP2A7, SEQ ID NO: 276; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSS>CD8086PMP2B10, SEQ ID NO: 277; PRT; →EVQLVESGGGLVQAGGSLRLSCTGSQISFSDNTMNWYRQVPGKQRELVASLSIFGATGYADSVKGRFTISRDIAGNTVYLQMNDLKIEDTAVYYCKLGPVRRSRLEYWGQGTQVTVSS>CD8086PMP2B4, SEQ ID NO: 278; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGEQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS>CD8086PMP2C9, SEQ ID NO: 279; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVMGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSS>CD8086PMP2E6, SEQ ID NO: 280; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS>CD8086PMP2F5, SEQ ID NO: 281; PRT; →EVQLVKSGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAYEEGVYRWDFWGQGTQVTVSS>CD8086PMP2G4, SEQ ID NO: 282; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS>CD8086PMP2G8, SEQ ID NO: 283; PRT; →EVQLVKSGGGLVQPGGSLRLSCAASGFIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSS>CD8086PMP2H11, SEQ ID NO: 284; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSS>CD8086PMP2H9, SEQ ID NO: 285; PRT; →EVQLVESGGGLVQAGGSLRLSCTASGSIFSIDAMGWYRQAPGKQRELVAHISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTVPRETGWDGDYWGQGTQVTVSS TABLE B-2Preferred Nanobodies against B7-1 and/orB7-2 and human serum albumin<Name, SEQ ID #; PRT (protein); →Sequence>CD8086PMP1A1-ALB1, SEQ ID NO: 286; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGNSATNVDSVKGRFTISRDNAKNTMYLQMNSLKPEDTAGYYCAAATRRAYLPIRIRDYIYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1A3-ALB1, SEQ ID NO: 287; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGPTSSSYSMGWFRQAPGKEREFVAAINWSHGVTYYADSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYYCAANEYGLGSSIYAYKHWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1B2-ALB1, SEQ ID NO: 288; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRSFSSYVMGWFRQAPGKEREFVAAIIGRDIGTYYADSVKGRFTISRDNAKTTVYLQMNALKPEDTAVYYCAADSRSRLSGIRSAYDYWGQGTVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1C5-ALB1, SEQ ID NO: 289; PRT; →EVQLVESGGGSVQAGGSLRLSCAATGRTFSSYGMGWFRQAPGKEREFVAAIHWNSGITYYADSVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYICAASSKGLTGTIRAYDDWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1C7-ALB1, SEQ ID NO: 290; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYAAGWFRQAPGKERDFVAAINWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASGWGRTTVLADTVXYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1C9-ALB1, SEQ ID NO: 291; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFXXGIDAMGWFRQAPGKEREFVASIXRSGGXATXADSVKGRFTISRDNAKNTMYLQMNXLKPEDTAGYYCAAATRRPYLPIRISRLYLXGPGXHXVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1D1-ALB1, SEQ ID NO: 292; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1D4-ALB1, SEQ ID NO: 293; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFTDGIDAMGWFRQAPGKEREFVASIGRSGGSATNADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAGYYCAAATRRPYLPIRIRDYIYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1E11-ALB1, SEQ ID NO: 294; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYSAIGWFRQAPGKEREGVSYISSSDGSTYYADSVEGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAGGPFTVSTMPWLANYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP1F12-ALB1, SEQ ID NO: 295; PRT; →EVQLVESGGGLVQAGGSLRLACAASGLSFSFYTMGWFRQAPGEERDFVAAINWSGGSTLYAESVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVRSVGRTYWTRALEYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2A7-ALB1, SEQ ID NO: 296; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSSKAMGWFRQAPGKERDFVAAITWSGGSTYYADHVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATNPYGLGQVGYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2B10-ALB1, SEQ ID NO: 297; PRT; →EVQLVESGGGLVQAGGSLRLSCTGSQISFSDNTMNWYRQVPGKQRELVASLSIFGATGYADSVKGRFTISRDIAGNTVYLQMNDLKIEDTAVYYCKLGPVRRSRLEYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2B4-ALB1, SEQ ID NO: 298; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGEQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2C9-ALB1, SEQ ID NO: 299; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVMGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2E6-ALB1, SEQ ID NO: 300; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRDDAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2F5-ALB1, SEQ ID NO: 301; PRT; →EVQLVKSGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAYEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2G4-ALB1, SEQ ID NO: 302; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYDMGWYRQAPGKQRVLVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2G8-ALB1, SEQ ID NO: 303; PRT; →EVQLVKSGGGLVQPGGSLRLSCAASGFIFSIYAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNANAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2H11-ALB1, SEQ ID NO: 304; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIFSIYTMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIAHEEGVYRWDFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>CD8086PMP2H9-ALB1, SEQ ID NO: 305; PRT; →EVQLVESGGGLVQAGGSLRLSCTASGSIFSIDAMGWYRQAPGKQRELVAHISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTVPRETGWDGDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS TABLE B-3Leader sequences and N-terminal sequences<Name, SEQ ID #; PRT (protein); →Sequence>llama leader 1, SEQ ID NO: 306; PRT; →VKKLLFAIPLVVPFYAAQPAMA<llama leader 2, SEQ ID NO: 307; PRT; →VKKLLFAIPLVVPFYAAQPAIA<llama leader 3, SEQ ID NO: 308; PRT; →FELASVAQA<leader sequence, SEQ ID NO: 309; PRT; →MKKTAIAIAVALAGLATVAQA<leader sequence, SEQ ID NO: 310; PRT; →MKKTAIAFAVALAGLATVAQA<N-terminal sequence, SEQ ID NO: 311; PRT; →AAAEQKLISEEDLNGAAHHHHHH TABLE B-4Preferred Nanobodies against PD-1>102C3, SEQ ID NO: 347; PRT; →EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFVAAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADRAESSWYDYWGQGTQVTVSS>102C12, SEQ ID NO: 348; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDKHQSSWYDYWGQGTQVTVSS>102E2, SEQ ID NO: 349; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSISSIHAMGWFRQAPGKEREFVAAITWSGGITYYADSLKGRFTISRDNAKNTGYLQMNSLKPEDTAIYYCAADRAQSSWYDYWGQGTQVTVSS>102E8, SEQ ID NO: 350; PRT; →EVQLVESGGGLVQAGGSLGLSCAASGSIFSINAMAWFRQAPGKEREFVALISWSGGSTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADRVDSNWYDYWGQGTQVTVSS>102H12, SEQ ID NO: 351; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRRAPGKEREFVASIPWSGGRIYYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCAVKERSTGWDFASWGQGTQVTVSS TABLE B-5Preferred Nanobodies against PD-L1 (B7-H1)>104D2, SEQ ID NO: 394; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREWASSISSSDGSTYYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVYSCAASQAPITIATMMKPFYDYWGQGTQVTVSS>104F5, SEQ ID NO: 395; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREWVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAARHGGPLTVEYFFDYWGQGTQVTVSS>104E12, SEQ ID NO: 396; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAIGWFRQAPGKAREGVSCISGGDNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATGGWKYCSGYDPEYIYWGQGTQVTVSS>104B10, SEQ ID NO: 397; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSTFSQYDVGWYRQAPGKQRELVAFSSSGGRTIYPDSVKGRFTFSRDNTKNTVYLQMTSLKPEDTAVYYCKIDWYLNSYWGQGTQVTVSS>104F10, SEQ ID NO: 398; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGVDASNSAMGWYRQAPGKQREWVARITGGGLIAYTDSVKGRFTISRDNAKSTVYLQMNSLEPEDTAVYYCNTINSRDGWGQGTQVTVSS>104D7, SEQ ID NO: 399; PRT; →EVQLVESGGGLVQAGGSLTISCAASGITFSDSIVSWYRRARGKQREWVAGISNGGTTKYAESVLGRFTISRDNAKNMVYLQMNGLNPEDTAVYLCKVRQYWGQGTQVTVSS TABLE B-6Preferred Nanobodies against PD-L2>103A9, SEQ ID NO: 449; PRT; →EVQLVESGGGLVQAGGSLRLSCAASESTVLINAMGWYRQAPGKQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADVYPQDYGLGYVEGKVYYGMDYWGTGTLVTVSS>103E2, SEQ ID NO: 450; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYVSNYAMGWGRQAPGTQRELVASISNGDTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCFEHQVAGLTWGQGTQVTVSS>103G12, SEQ ID NO: 451; PRT; →EVQLVESGGGLVQAGGSLRLSCVASGXALKIXVMGWYRQAPGKQRELVAAITSGGRTNYSDSVKGRFTISGDNAXNTVYLQMNSLKSEDTAVYYCREWNSGYPPVDYWGQGTQVTVSS>103F10, SEQ ID NO: 452; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSSGTMGWFRRAPGKEREFVASIPWSGGRTYYADSVKDRFTISRDNAQNTVFLQMNSLKPEDTAVYYCAFKERSTGWDFASWGQGIQVTVSS>103E3, SEQ ID NO: 453; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGFTLDYYGIGWFRQAPGKEREGVSFISGSDGSTYYAESVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCAADPWGPPSIATMTSYEYKHWGQGTQVTVSS>103F6, SEQ ID NO: 454; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMIWLRRAPGKGFEWVSTIDKDGNTNYVDSVKGRFAVSRDNTKNTLYLQMNSLKPEDTAMYYCTKHGSSARGQGTRVTVSS>103D3, SEQ ID NO: 455; PRT; →EVQLVESGGGLVEPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLEWVSTINSGGGITYRGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCENGGSSYRRGQGTQVTVSS TABLE B-7Preferred Nanobodies against B7-H2 (ICOSL)>95A6, SEQ ID NO: 505; PRT; →EVQLVESGGGLVQAGGSLRLSCALSGRAVSIAATAMGWYRQAPGKQRELVAARWSGGSIQYLDSVKGRFTISRDNAKNTVYLQMNSLTPEDTAVYYCNTLPWRANYSGQGTQVTVSS>95B11, SEQ ID NO: 506; PRT; →EVQLVESGGGLVQPGGSLRLSCAASRSISSFNLLGWYRQAPGKQRELVAHLLSGGSTVYPDSVKGRFTVSRDNTKNTVYLQMNSLKPEDTAVYYCNAIAPALGSSWGQGTQVTVSS>95F8, SEQ ID NO: 507; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGIAFSIDIMDWYRQAPGKERELVATISGGGSTNYADSVKGRFIVSRDNAKNILYLQMNSLKPDDTAVYYCNARRLIYGRTVYWGQGTQVTVSS>95H8, SEQ ID NO: 508; PRT; →EVQLVESGGGLVQTGGSLRLSCAASSSTSTSSIDVMGWYRQSPGKQRELVASISSFGSTYYADSVKGRFIISRDNAKNTVNLQMNNLKLEDTAVHFCNLRRLSPPPLLDYWGQGTQVTVSS>95G5, SEQ ID NO: 509; PRT; →EVQLVESGGGLVQAGGSLRLSCASSGSTFSIDVMGWYRQAPGKVRERVAIIGTGGFPVYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAARLVALGSWGQGTQVTVSS>95E6, SEQ ID NO: 510; PRT; →EVQLVESGGALVQPGGSLRLSCAASGFTLGDYVIGWFRQAPGKEREWVSGISSRDDTTYYANSVKGRFTISRDNAKNTMYLQMNSLKPEDSAVYYCALRSGIAVARAPTNYDYWGQGTQVTVSS>95G6, SEQ ID NO: 511; PRT; →EVQLVESGGALVQPGGSLRLSCAASGFTLGDYVIGWFRQAPGKEREWVSGISSRDGTTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCALRSGIAVARAPSNYDYWGQGTQVTVSS TABLE B-8Preferred Nanobodies against CD28>65C2, SEQ ID NO: 554; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGLTFSNYVMGWFRQAPGKEREFVGTISWDGSDTYYTHSVKGRFTISRDNAKNVVNLQMNSLKPEDTAVYYCAADYRPGGLLSLGKNEYDYWGQGTQVTVSS>70F9, SEQ ID NO: 555; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAHSWYADYADSVKGRFSISRDNDKNTVYLQMNSLKPEDTAVYYCAASRSQGRRYANSYESWGQGTQVTVSS>65B2, SEQ ID NO: 556; PRT; →EVQLVESGGGLVQAGGSLRLSCATSGRTFSSDVMGWFRQAPGKEREFVAAINRSGHSTSYTGSVKGRFAISRDNTKNTVYLQMNSLKPEDTAVYYCALRLWSDYLAQKSGEYNYWGQGTQVTVSS>65C4, SEQ ID NO: 557; PRT; →EVQLVESGGGLVQAGGSLRLSCKAAGRTFSSYAMGWFRQAPGKEREFVASIEWDGGGAYYEEAVKGRFTISRDNTKNTVYLQMDSLRPEDTAVYYCAASRWRTALTNYYDVADWGQGTQVTVSS>65G2, SEQ ID NO: 558; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDAYAIHWFRQAPGKEREGVSCISSSDGSTYYANSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATAKRCWGLSYEYDYWGQGTQVTVSS>70F10, SEQ ID NO: 559; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVACVSNSDGSTYYANSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAADSRCWGWGMLHMRHGDRGQGTQVTVSS TABLE B-9Preferred Nanobodies against CTLA4>65H7, SEQ ID NO: 1288; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGSILSIAVAGWYRRQPGKERELVATISPGNNTHYVDSVKGRFTISRDNAKNTVYLQMTTLKPDDTAAYYCNAKGSILLNAFDYWGKGTQVTVSS>65D10, SEQ ID NO: 1289; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTSSTATVGWFRQAPGKEREFVAVINWRSGFTYYADSVKGRFTISREYAKNTVYLQMDSLKPEDTAVYSCAADLGGRTLYGGIHYSPEEYAYWGQGTQVTVSS>69A4, SEQ ID NO: 1290; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAISPSGLTSYKDSVVGRFTISRDNAKNTVYLQMNSLKPEDTAVHYCAAGQWTWSPLRVSRLAEYNYWGQGTQVTVSS>66B5, SEQ ID NO: 1291; PRT; →EVQLVESGGGLVQPGESLRLSCAASKSIFSISVMAWYRQAPGKQRELVARITPGGNTNYVDSVQGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAQGSLLLAKYDYYGQGTQVTVSS>66B6, SEQ ID NO: 1292; PRT; →EVQLVESGGGLVQAGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS>66G2, SEQ ID NO: 1293; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIDSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS>69D9, SEQ ID NO: 1294; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREFVAAISRSGSLTSYADSVKGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLLTYDSWGQGTQVTVSS>65F9, SEQ ID NO: 1295; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTLTTYIMGWFRQAPGKEREFVAATSPSGTLTSYADSVKGRFSMSRDNAKKMVDLQMNSLKPEDTAVYYCAAKGGRWGPRNDDRYDYWGQGTQVTVSS>4CTLAPMP11E3, SEQ ID NO: 1296; PRT; →EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQGTQVTVSS>4CTLAPMP12H2, SEQ ID NO: 1297; PRT; →EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQRTQVTVSS>4CTLAPMP33H10, SEQ ID NO: 1298; PRT; →EVQLVESGGGLVEPGGSLRLSCAASGSISSFNVMGWYRQAPGQQRDLVAHIASNGEIMYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQGTQVTVSS>4CTLAPMP29A4, SEQ ID NO: 1299; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGSISSFNVMGWYRQAPGKQRDLVAHIASGGEIMYTDSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQGTQVTVSS>4CTLAPMP17C6, SEQ ID NO: 1300; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIVGSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS>4CTLAPMP22D1OCL7, SEQ ID NO: 1301; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIDSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS>4CTLAPMP32E2, SEQ ID NO: 1302; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISLSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS>4CTLAPMP20F4CL8, SEQ ID NO: 1303; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCIVSSDGSTYYADSVKSRFTISRDNAKNTVYLHMNSLKPEDTAVYYCAAVHGLKLPTLRGLGGSYYYLQARSYDYWGQGTQVTVSS>4CTLAPMP29F7, SEQ ID NO: 1304; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITISDGDTYYADSVKGRFTISRDNANNTVNLQMNSLKPEDTAVYYCAAVHGLKLPSQRGLGGSYYYLLPRSYDYWGQGTQVTVSS>4CTLAPMP10C5, SEQ ID NO: 1305; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCITISDGDTYYADSVKGRFTIARDYAKNTVYLQMNSLKPEDTAVYYCAAVHGLKLPSQRGLGGSYYYLLARSYDYWGQGTQVTVSS>4CTLAPMP11F1, SEQ ID NO: 1306; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS>4CTLAPMP29F2, SEQ ID NO: 1307; PRT; →EMQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS>4CTLAPMPO3C4, SEQ ID NO: 1308; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEREFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSS>4CTLAPMP32F8, SEQ ID NO: 1309; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSSYGMGWFRQAPGKEREFVADIRSSAGRTYYAGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMTGITGWDYWGQGTQVTVSS>4CTLAPMPO7F11, SEQ ID NO: 1310; PRT; →KVQLVESGGGLVQAGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS>4CTLAPMPO2C7, SEQ ID NO: 1311; PRT; →EVQLVESGGGLVQAGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS>4CTLAPMPO3A6, SEQ ID NO: 1312; PRT; →EVQLVESGGGLVQPGGSLRLSCAAPGRTFSNYAMGWFRQAPGKGREFVADIRWSDGRTYYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAAQGGVLSGWDYWGQGTQVTVSS>4CTLAPMP13B2, SEQ ID NO: 1313; PRT; →EVQLVESGGGLVQPGGSLRLSCVASGIHFAISTINWYRQAPGKQRESVAAITGTSVTGYADSVKGRFTLSRDNAKNTVYLQMDNLKPEDTAVYYCNVWSGRDYWGQGTQVTVSS>4CTLAPMPO3G3, SEQ ID NO: 1314; PRT; →EVQLVESGGGLVQPAGSLRLSCADSGSIFSINTMGWYRQAPGKQRELVATITSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADYRDFGLSMERFIDFGSWGQGTQVTVSS>4CTLAPMP16D7, SEQ ID NO: 1315; PRT; →EVQLVESGGGLVQPGGSLRLSCADAGSIFSINTMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADYRDFGLSMERFTDFGSWGQGTQVTVSS>4CTLAPMP27D8, SEQ ID NO: 1316; PRT; →KVQLVESGGGLVQPGGSLRLSCAASGSDFSLNAMGWYRQAPGKQRELVAAITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADYRDFGLSMERFVDFGSWGQGTQVTVSS>4CTLAPMPO4B10, SEQ ID NO: 1317; PRT; →EMQLVESGGGLVQPGGSLRLSCAASGNIFSRYIMGWYRQAPGKQRELVADITPGGNTNYADSVKGRFTISRDGAKNTVGLQMNSLRPEDTAVYSCYARGSDKLLMRTYWGQGTQVTVSS>4CTLAPMPO4B12, SEQ ID NO: 1318; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFSRYIMGWYRQAPGKERELVADITPGGNTNYANSVKGRFTISRDGAKNTVGLQMNSLRPDDTAVYSCYARGSDKLLMRTYWGQGTQVTVSS>4CTLAPMPO6D2, SEQ ID NO: 1319; PRT; →EVQLVESGGGLVQPGGSLRLSCTASGNIFSRYIMGWYRQAPGKQRELVADITPGGNTNYADSVKGRFSISRDGAKNTVDLQMNSLRPEDTAVYYCNALGSDKLLIRTYWGQGTQVTVSS>4CTLAPMPO3B1, SEQ ID NO: 1320; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFSRYIMGWYRQAPGKQRESVATITPGGNTDYADSVKGRFTISRDGAKNTVDLQMNSLKPEDTAVYYCNARGSSGLSMSTYWGQGTQVTVSS>4CTLAPMPO3A7, SEQ ID NO: 1321; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWYRQAPGKQRDLVASITPGGNIYYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCNARGSILLDPINYWGQGTQVTVSS>4CTLAPMPO4A3, SEQ ID NO: 1322; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNIMGWYRQAPGNQRDLVASITPGGNMYYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCNARGSILLDPSNYWGQGTQVTVSS>4CTLAPMPO2A1, SEQ ID NO: 1323; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWYRQAPGNQRDLVASITPGGNIYYADSVKGRFTISRDGAKSTVILQMNSLKPEDTAVYYCNARGSILLDRVNYWGQGTQVTVSS>4CTLAPMPO8E5, SEQ ID NO: 1324; PRT; →EVQLVESGGGLVQPGGSLRLSCAASRDIFTRNIMGWYRQAPGKQRDLVASITPGGNMYYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCNAHGSILLDRSNYWGQGTQVTVSS>4CTLAPMPO3F7, SEQ ID NO: 1325; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNIMGWYRQAPGKQRDLVASITPGGNINYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCNAHGSILLNRSNYWGQGTQVTVSS>4CTLAPMPO2C11, SEQ ID NO: 1326; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFTRHIMGWYRQAPGKQRELVASITPGDNINYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCNAHGSILLDRTNYWGQGTQVTVSS>4CTLAPMPO3B11, SEQ ID NO: 1327; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGNIFTRNVMGWYRQAPGKQRDLVASITPGGNINYADSVKGRFTISRDGAKNTVYLQMNSLKPEDTAVYYCNAHGSILLDRIEYWGQGTQVTVSS>4CTLAPMPO2H3, SEQ ID NO: 1328; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGRTSSTATVGWFRQAPGKEREFVAVINWRSGFTYYADSVKGRFTISREYAKNTVYLQMDSLKPEDTAVYSCAADLGGRTLFGGIHYSPEEYAYWGQGTQVTVSS>4CTLAPMP17E3, SEQ ID NO: 1329; PRT; →EVQLMESGGGLVTAGGSLRLSCAASGGTFGHYAMAWFRRPPGNEREFVAGIGWTYTTFYADSVKGRFAISRDNAENTVYLQMNNLKPDDTAVYYCAAAELKGRNLRVPDYEHWGQGTQVTVSS>4CTLAPMP10G5, SEQ ID NO: 1330; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSRYIMAWFRQAPGKEREFVAVIDGSGYSTDYAGSVKGRFTIARDNTKNTAYLQMNSLKPEDTALYFCGAGRQYSTGPYWYDYWGQGTQVTVSS>4CTLAPMPO2G3, SEQ ID NO: 1331; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVKGRFTISRDNAKKMAYLQMLFLKLEDSAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS>4CTLAPMP25H11, SEQ ID NO: 1332; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGNLKSYADSVKGRFTISRDNAKKMAYLQMNSLKLEDTAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS>4CTLAPMP10A11, SEQ ID NO: 1333; PRT; →EVQLMESGGGLVQTGGSLRLSCVASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVKGRFTISRDNAKKMAYLQMLFLKLEDSAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS>4CTLAPMPO2F6, SEQ ID NO: 1334; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGGLKSYADSVKGRFTISRDNAKKMAYLQMNSLKLEDTAVYYCAAAPVPWGTRPSTFPYDSWGQGTQVTVSS>4CTLAPMPO2F4, SEQ ID NO: 1335; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGALKAYADSVKGRFTPSRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFPYDSWGQGTQVTVSS>4CTLAPMP17C1, SEQ ID NO: 1336; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKAYADSVKGRFTPSRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFPYDSWGQGTQVTVSS>4CTLAPMPO5E7, SEQ ID NO: 1337; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREFVTAISRSGTLTSYADSVKGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAVAPVPWGTRPSLFPYDSWGQGTQVTVSS>4CTLAPMPO2F2, SEQ ID NO: 1338; PRT; →EVQLMESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKAYADSVKGRFTPSRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFPYDSWGQGTQVTVSS>4CTLAPMP10F8, SEQ ID NO: 1339; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVNGRFTISRDNAKKMAYLQMNSLKPEDTASYYCAAAPVPWGTRPSFLTYDSWGQGTQVTVSS>4CTLAPMPO2F8, SEQ ID NO: 1340; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGNLKSYADSVNGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFLTYDSWGQGTQVTVSS>4CTLAPMPO2E2, SEQ ID NO: 1341; PRT; →AVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREYVAAISRSGSLKGYADSVKGRFTISRDNAKNMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLLTYDSWGQGTQVTVSS>4CTLAPMP33D9, SEQ ID NO: 1342; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREYVAAISRSGSLKGYADSVKGRFTISRDNAKNMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLLTYDSWGQGTQVTVSS>4CTLAPMP27C8, SEQ ID NO: 1343; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGTLKAYADSVKGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS>4CTLAPMP17D5, SEQ ID NO: 1344; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTTGWFRQAPGKDREFVAAISRSGSLTSYADSVKGRFTISRDNAKKMAYLQMNSLKPEDAAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS>4CTLAPMPO2H7, SEQ ID NO: 1345; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKAYADSVKGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS>4CTLAPMPO2G2, SEQ ID NO: 1346; PRT; →EVQLVESRGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGSLKSYADSVKGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFTYDSWGQGTQVTVSS>4CTLAPMP10D5, SEQ ID NO: 1347; PRT; →EVQLVESGGGVVQTGGSLRLSCAASGRTFSMYTMGWFRRAPGKDREFVAAISRSGGLKAYADSVLGRFTISRDNANEMAYLQMNSLNPEDTAVYYCAAAPVPWGTRPSHFTYDSWGQGTQVTVSS>4CTLAPMP10G9, SEQ ID NO: 1348; PRT; →EVQLVESGGGVVQTGGSLRLSCAASGRTFSMYTMGWFRQAPGEDREFVAAISRSGGLKAYADSVLGRFTISRDNANEMAYLQMNSLNPEDTAVYYCAAAPVPWGTRPSHFTYDSWGQGTQVTVSS>4CTLAPMPO5G9, SEQ ID NO: 1349; PRT; →EVQLVESGGGVVQTGGSLRLSCAASGRTFSMYTMGWFRQAPGKDREFVAAISRSGGLKAYADSVLGRFTISRDNANEMAYLQMNSLNPEDTAVYYCAAAPVPWGTRPSHFTYDSWGQGTQVTVSS>4CTLAPMP10B7, SEQ ID NO: 1350; PRT; →EVQLVESRGGLVQPGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTISRDNAKKMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS>4CTLAPMP29B10, SEQ ID NO: 1351; PRT; →EVQPVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVKGRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFTYDSWGQGTQVTVSS>4CTLAPMP24E3, SEQ ID NO: 1352; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTISRDNAKEMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS>4CTLAPMP10F4, SEQ ID NO: 1353; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTTSRDNAKKMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS>4CTLAPMP10F11, SEQ ID NO: 1354; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSSYTMGWFRQAPGKDREFVAAISRSGGLTSYADSVKGRFTISRDNGKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSLFTYDSWGQGTQVTVSS>4CTLAPMP32B8, SEQ ID NO: 1355; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRAFNNYTMGWFRQAPGKDREFVAAISRSGNLKAYADSVNGRFTISRDNAKKMAYLQMNSLKPEDTSVYYCTAAPVPWGTRPSLFTYDSWGQGTQVTVSS>4CTLAPMP10G11, SEQ ID NO: 1356; PRT; →EVQLVESGGDLVQPGGSLRLSCAASGRTFSNYTVGWFRQAPGKDREFVTAISRSGSLKAYADSVKDRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAGAPVPWGARPSLFTYDSWGQGTQVTVSS>4CTLAPMP10B9, SEQ ID NO: 1357; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTVGWFRQAPGKDREFVTAISRSGSLKAYADSVKDRFTISRDNAKKMAYLQMNSLKPEDTAVYYCAGAPVPWGARPSLFTYDSWGQGTQVTVSS>4CTLAPMPO5G2, SEQ ID NO: 1358; PRT; →EVQLVESGGELVQAGDSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISPSGALTSYADSVKGRFTISRDNAEKMVYLQMSSLKPEDTDVYYCAAARVPWSPRPSLSPYDYWGQGTQVTVSS>4CTLAPMP17H5, SEQ ID NO: 1359; PRT; →EVQLVESGGELVQAGDSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISPSGALTSYADSVKGRFTISRDNAEKMVYLQMSSLKPEDTDAYYCAAARVPWSPRPSLSPYDYWGQGTQVTVSS>4CTLAPMPO5E10, SEQ ID NO: 1360; PRT; →EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYIMGWFRQAPGKEREFVAAISSSGALTSYADSVKGRFTISRDNAEKMVYLQMSSLKPEDTDVYYCAAARVPWSPRPSLSTYDYWGQGTQVTVSS>4CTLAPMPO5E11, SEQ ID NO: 1361; PRT; →EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYIMGWFRRAPGKEREFVAAISSSGALTSYADSVVGRFTISRDNAKKMVYLQMRSLKPEDTDVYYCAAARVPWSPRPSLSTYDYWGQGTQVTVSS>4CTLAPMPO5E4, SEQ ID NO: 1362; PRT; →EVQLVESGGGLVQAGDSLTLSCAASGGTFSTYVMGWFRQASGKEREFVAAISPSGTLTSYADSVKGRFGISRDNAKKMVYLQVSSLKPEDTDVYYCAAARGPWTPRPSLLTYDYWGQGTQVTVSS>4CTLAPMP17F6, SEQ ID NO: 1363; PRT; →EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISSSGALTSYADSVYGRFTISRDNAKKMVYLQMSSLKPEDTDVYYCAAGRGPWSPRPSLLTYDYWGQGTQVTVSS>4CTLAPMP10E11, SEQ ID NO: 1364; PRT; →EVQLVESGGGLVQAGDSLRLSCAASGRTFSNYVMGWFRQAPGKEREFVSAISPSGTLTSYTDSVKGRFAISRDNAKKMLYLQMSSLKPEDTDVYYCAAARGPWSARPSLLTYDYWGQGTQVTVSS>4CTLAPMP17C5, SEQ ID NO: 1365; PRT; →EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVAAISPSGSLTSYADSVKGRFAISRDNAKVMVYLQMSSLKPDDTDVYYCAAARGPWNARPSLLTYDYWGQGTQVTVSS>4CTLAPMP11D1, SEQ ID NO: 1366; PRT; →EVQLVESGGGLVQAGGSLSLSCAASGRTFSSITMAWFRQTPGKEREFVAAISRSGSLTSYADSLKGRFTISRDNAKNTVSLQMNNLKPEDTAVYYCAADTNGRWRPAIRPSDFEIWGQGTQVTVSS>4CTLAPMP17C3, SEQ ID NO: 1367; PRT; →EVQLVESGGGLVQAGGSLGLSCAASGRSFSMYAMGWFRTAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDNAKNTVYLRMNNLNAEDTAVYYCAARSGWGAAMRSADFRSWGQGTQVTVSS>4CTLAPMP10A1, SEQ ID NO: 1368; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMP31A8, SEQ ID NO: 1369; PRT; →EVQLVESGGQLVQAGDSLRLSCVATGRTYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPDDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMPO2H5, SEQ ID NO: 1370; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMP10G3, SEQ ID NO: 1371; PRT; →EVQLVESGGQLVQAGGSLRLSCTATGHTYNTYPLGWFRQAPGKEREFVAAISPSGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCARHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMPO5F10, SEQ ID NO: 1372; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGRMYNSYSLGWSRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCGRHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMP10B8, SEQ ID NO: 1373; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGHTYNTYPLGWFRQAPGKEREFVAAISPSGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCARHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMPO5H11, SEQ ID NO: 1374; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDNAKNTVCLQMNNLKPEDTAVYYCAQHRSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMP17H9, SEQ ID NO: 1375; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYSLGWFRQAPGKEHEFVAAISASGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCARHHSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMP2G9, SEQ ID NO: 1376; PRT; →EVQLVKSGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDSAKNTVYLQMNNLKPEDTAVYYCARARSVGWRASHHLSDYDNWGQGTQVTVSS>4CTLAPMP1OH5, SEQ ID NO: 1377; PRT; →EVQLVESGGQLVQAGGSLRLSCTATGHTFNTYPLAWFRQAPWKEREFVAAISPSGTLRAYADSVKGRFTISRGNAKNTVYLQMNNLKPEDTAVYYCARDRSVGWRASHHLSDYGNWGQGTQVTVSS>4CTLAPMP10B5, SEQ ID NO: 1378; PRT; →EVQLVESGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCARDRSVGWRASHHLSDFDTWGQGTQVTVSS>4CTLAPMPO2A2, SEQ ID NO: 1379; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSNTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDNANDTVYLQMNSLKPEDTAIYYCAAGLTGWAVIPSRTLTTWGQGTQVTVSS>4CTLAPMPO2B8, SEQ ID NO: 1380; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSNTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRBNANDTVYLQMNSLKPEDTAIYYCAAGLTGWAVIPSRTLTTWGQGTQVTVSS>4CTLAPMPO2A5, SEQ ID NO: 1381; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTNSTTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAIYYCAAGLTSWALIPSRTLTTWGQGTQVTVSS>4CTLAPMPO2B11, SEQ ID NO: 1382; PRT; →EVQLVESGGGLVQAGGSLRLSCAAPGRTNSTTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAIYYCAAGLTSWALIPSRTLTTWGQGTQVTVSS>4CTLAPMPO9C1, SEQ ID NO: 1383; PRT; →EVQLVESGGGLVQPGGSLRLSCAASGRTNSTTLMGWSRRAPGKEREFVAAISGSGTLTSYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAIYYCAAGLTSWALIPSRTLTTWGQGTQVTVSS>4CTLAPMPO5C5, SEQ ID NO: 1384; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRMFSSRSIGWFRQVPGKEREFVAAISPSRSLKAYADSVKGRFTISGDNAKNTVDLQMNSLNVEDMAVYYCAADVISGRWYGGAFTPSRFDYWGQGTQVTVSS>4CTLAPMP12B2, SEQ ID NO: 1385; PRT; →EVQLVESGGGLVQAGGSLALSCAASGRMFSSRSIGWFRQAPGKDREFVAAISPSGSLKAYADSVKGRFTISRDNAKNTVDLQMNSLNTEDMAVYYCAADVISGRWYAGAFTPSRFDYWGQGTQVTVSS>4CTLAPMP17B5, SEQ ID NO: 1386; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTLTTYIMGWFRQAPGKEREFVAATSPSGTLTSYADSVKGRFSMSRDNAKKMVDLQMNSLKPEDTAVYYCAAKGGRWGPRNDDRYDYWGQGTQVTVSS>4CTLAPMPO2B10, SEQ ID NO: 1387; PRT; →EVQLVESEGGLVQPGGSLRLSCSASGRTFANNAMGWFRQAPGKEREFVASISASGTLTSSADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCARNRRAWSLSVHTTREYDDWGQGTQVTVSS>4CTLAPMPO2C9, SEQ ID NO: 1388; PRT; →KVQLVESGGGLVQAGGSLRLSCSASGRTFANNAMGWFRQAPGKEREFVASLSASGSLTSYADSVNGRFTISRDNAKNTVYLQMNSLKPVDTALYYCARNRRAWSLSVHTTREYDDWGQGTQVTVSS>4CTLAPMPO4G10, SEQ ID NO: 1389; PRT; →EVQLVESGGGLVKAGDSLRLSCSASGRTFANNAMGWFRQAPGKEREFVASISASGTLTSSADSVRGRFTISRDNAKNTVYLQMNSLKPEDTALYYCARNRRAWSLSVHTTREYDDWGQGTQVTVSS>4CTLAPMP17B6, SEQ ID NO: 1390; PRT; →EVQLVESGGGLVQAGGSLRLSCVASAEGSFSTYVMAWFRQAPGKEREFAAAISGRSGLTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDAARYYCAADRRAWSARPDMGNYYWGQGTQVTVSS>4CTLAPMPO6C10, SEQ ID NO: 1391; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYTIAYFRQAPGREREFAAAISPHGTLRSFADSVKDRFTISRDNAKNTVWLQMNSLKLEDTAVYYCAADPSGWGLRQHSENEYPYWGLGTQVTVSS TABLE B-10Multivalent CTLA4 binding Nanobodies>11F1-9GS-11F1-9GS-ALB1, SEQ ID NO: 1392; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGGITSFYGMGWERQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>12H2-9GS-12H2-9GS-ALB1, SEQ ID NO: 1393; PRT; →EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>2F4-9GS-2F4-9GS-ALB1, SEQ ID NO: 1394; PRT; →EVQLVESGGGLVQTGGSLRLSCAASGRTFSNYTMGWFRQAPGKDREFVAAISRSGALKAYADSVKGRFTPSRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFPYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQTGGSLRLSCAASGRITSNYTMGWERQAPGKDREFVAAISRSGALKAYADSVKGRFTPSRDNAKKMAYLQMNSLKPEDTAVYYCAAAPVPWGTRPSFFPYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>2G9-9G5-2G9-9G5-ALB1, SEQ ID NO: 1395; PRT; →EVQLVKSGGQLVQAGGSLRLSCAATGRTYNSYPLGWFRQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDSAKNTVYLQMNNLKPEDTAVYYCARARSVGWRASHHLSDYDNWGQGTQVTVSSGGGGSGGGSEVQLVKSGGQLVQAGGSLRLSCAATGRTYNSYPLGWERQAPGKEREFVAAISASGTLRAYADSVKGRFTISRDSAKNTVYLQMNNLKPEDTAVYYCARARSVGWRASHHLSDYDNWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFRSEGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS>11F1-9G5-11F1-GGGC, SEQ ID NO: 1396; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGGITSFYGMGWERQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGC>12H2-9G5-12H2-GGGC, SEQ ID NO: 1397; PRT; →EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGC>11F1-9GS-11F1-HSA, SEQ ID NO: 1398; PRT; →EVQLVESGGGLVQAGGSLRLSCAASGGTFSFYGMGWFRQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAASGGITSFYGMGWERQAPGKEQEFVADIRTSAGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEMSGISGWDYWGQGTQVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEENAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL>12H2-9GS-12H2-HSA, SEQ ID NO: 1399; PRT; →EVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSGGGGSGGGSEVQLVESGGGLVEPGGSLRLSCAASGSISSYNVMGWYRQAPGQQRDLVAHIASNGEIMYADSAKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCKLWVLGNDYWGQETQVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL TABLE C-1Immunization protocol with CD80-Fc and CD86-FcDayLlama 089Llama 090Tissue collection0100 μg100 μg10 ml pre-immune blood720 μg20 μg—1450 μg50 μg—2010 μg10 μg—2850 μg50 μg3510 μg10 μg—39150 ml immune blood (PBL1)lymph node bow biopsy43150 ml immune blood (PBL2) TABLE C-2Size and percentages of inserts of constructed librariesLibrary size% insertLlama No. 0896E796%Llama No. 0906E796% TABLE C-3Experimental conditions used in different selection strategiesMethod 1Antigen 1Elution 1Method 2Antigen 2Elution 2Passive plateHuCD80-HuIgG1TrypsinPassive plateHuCD80-HuIgG1Trypsinimmobilizationat 2 μg/mlimmobilizationat 2 μg/mlPassive plateHuCD86-HuIgG1TrypsinPassive plateHuCD86-HuIgG1Trypsinimmobilizationat 2 μg/mlimmobilizationat 2 μg/mlPassive plateHuCD80-HuIgG1TrypsinPassive plateHuCD86-HuIgG1Trypsinimmobilizationat 2 μg/mlimmobilizationat 2 μg/mlPassive plateHuCD86-HuIgG1TrypsinPassive plateHuCD80-HuIgG1Trypsinimmobilizationat 2 μg/mlimmobilizationat 2 μg/ml TABLE C-4ELISA and FACS data of representativeCD80 and/CD86 binding clonesLibsNo. 089/090CD80-IgG1CD86-IgG1IgG1BLANKFACSPMP1A31.9160.0530.0520.052++PMP1B21.9850.0380.0460.041++PMP1D11.8920.0430.0460.044++PMP2A71.7140.0420.0430.039++PMP1H50.0611.4580.0540.054+PMP2B100.0421.7250.0440.042+PMP2D20.0551.5110.0390.041+PMP2H70.0491.5210.0450.050+PMP2E61.5190.5380.0490.049+PMP2F51.6520.7740.0560.051+ TABLE C-5Expression yields of anti-CD80/CD86 mono-and bireactive Nanobodies inE. coliCloneVolume (l)yield (mg)yield (mg/l)PMP1B20.2000.5542.72PMP1C70.2001.4507.25PMP1E110.2001.7008.50PMP2B40.2000.4642.32 TABLE C-6Screening for Nanobodies that inhibit the CD80and/or CD86 interaction with CD28 or CTLA4HuCD28-HuIgG1HuCTLA4-HuIgG1CD80/CD80/CD80CD86CD86CD80CD86CD86CloneELISAELISAFACSELISAELISAFACSPMP1B2−−−−−−PMP2B10+−−+−−PMP1C7+++−+++++−++PMP1E11+++++++++PMP2B4++−−−− TABLE C-7Affinity constants of Nanobodiesthat bind CD80-Ig and/or CD86-IgCD80-IgCD86-IgkonkoffKdkonkoffKdClone(1/Ms)(1/s)(nM)(1/Ms)(1/s)(nM)PMP1C73.5E53.7E−41.13.3E32.7E−3825PMP1E111.8E44.0E−4233.6E32.0E−3553PMP2B10No reactivity2.5E59.7E−44PMP1B24.4E55.9E−50.13No reactivity TABLE C-8Immunization protocol with CTLA4-Ig as antigenDayLlama 119Llama 120Tissue collection0100 μg100 μg10 ml pre-immune blood7100 μg100 μg—1450 μg50 μg—2150 μg50 μg10 ml immune blood2850 μg50 μg—3550 μg50 μg—39150 ml immune bloodlymph node biopsy43150 ml immune blood4950 μg50 μg—56100 ml immune blood TABLE C-9Size and percentages of inserts of constructed librariesLibrary size% insertLlama No. 1191.3 × 10E896Llama No. 1201.6 × 10E891 TABLE C-10Protein production yields#CloneYield (mg)Production volume (ml)Yield (mg/l)12A51.782008.9122C110.812004.0532F20.662003.2942F40.632003.1552G20.852004.2762G94.8220024.0873A60.882004.3883C40.532002.6696C100.382501.50108E5.41.712506.851110G50.472501.861211E30.202500.781311F10.822503.281412H20.142500.541513B20.812503.241617E32.8125011.24 TABLE C-11Performance characteristics of selected CTLA4 binding NanobodiesAlphascreenBIAcoreBIAcoreBIAcoreIC50 human CTLA4IC50 cyno CTLA4#CloneIC50kon(M−1s−1)koff(s−1)KD(M)binding in FACS (nM)binding in FACS (nM)12A54.56E−95.30E−035.727.7422C112.52E−80.0235Not saturated at 500Not saturated at 50032F21.55E−96.13E+063.91E−036.38E−102.562.4242F41.14E−95.69E+062.66E−034.66E−102.382.3652G21.58E−97.42E+063.55E−034.78E−102.282.0862G94.80E−94.35E+067.32E−031.69E−092.472.6673A63.33E−81.80E−03Not saturated at 500Not saturated at 50083C42.95E−90.02058.066.7396C102.206E−90.0027905.735.47108E5.47.724E−90.003861Not saturated at 500No crossreactivity1110G53.313E−90.0061824.65No crossreactivity1210E31.652E−97.42E+054.96E−031.00E−084.914.501311F11.21E−91.86E+061.87E−021.00E−084.555.511412H21.337E−93.49E+052.58E−037.52E−095.825.651513B21.909E−90.0067013.60No crossreactivity1617E31.655E−80.00489111.22No crossreactivity TABLE C-12IC50 values of monovalent and multivalent CTLA4binding Nanobodies as determined in alphascreenCloneIC50 (M)FormatIC50 (M)Gain vs. monovalent2F48.27E−102F4-2F4-ALB11.89E−104.42G92.01E−092G9-2G9-ALB12.58E−107.811F11.04E−0911F1-11F1-ALB11.44E−107.2 TABLE C-13IC50 values of monovalent and multivalent CTLA4binding Nanobodies as determined in FACSCloneIC50 (nM)FormatIC50 (nM)Gain vs. monovalent2F411.42F4-2F4-ALB13.63.22G9—2G9-2G9-ALB1——11F136.411F1-11F1-ALB13.99.3 TABLE C-14Off rate of monovalent and multivalent CTLA4 binding Nanobodies as determined in BIAcoreOff-rateOff-rateOff-rateOff-rateGainGainClone(60 s)(300-375 s)Format(60 s)(300-375 s)(60 s)(300-375 s)2F42.63E−031.63E−032F4-2F4-ALB11.77E−034.25E−041.493.842G95.66E−033.51E−032G9-2G9-ALB12.28E−036.96E−042.485.0411F11.50E−02—11F1-11F1-ALB15.82E−031.03E−052.581456.31 TABLE C-15Potency of humanized variants of CTLA4 binding Nanobodies 11F1and 11E3 determined in alphascreen as described in Example 63CloneIC50Loss factorComment11F1 WT1.46E−09Reference11F1 basic1.61E−091.1011F1 hum19.90E−100.6811F1 hum24.65E−100.3211F1 hum36.20E−100.4311F1 hum41.29E−090.8811F1 hum58.78E−100.6011E3 WT1.74E−09Reference11E3 basic2.06E−0811.8611E3 hum13.27E−0818.8111E3 hum22.64E−0815.1911E3 hum3NANAVariant no longerinhibits interaction11E3 hum41.71E−089.86 | 112,912 |
11858998 | BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: 1 is a forward primer sequence of human GCNT2 contemplated for use according to the subject invention. SEQ ID NO: 2 is a reverse primer sequence of human GCNT2 contemplated for use according to the subject invention. SEQ ID NOs: 3-4 are shRNA target sequences of human GCNT2 shRNA contemplated for use according to the subject invention. SEQ ID NOs: 5-6 are shRNA target sequences of human LGALS8 shRNA contemplated for use according to the subject invention. SEQ ID NO: 7 is a non-target sequence of human LGALS8 scr control contemplated for use according to the subject invention. SEQ ID NO: 8 is the nucleic acid sequence of GCNT2 contemplated for use according to the subject invention. SEQ ID NO: 9 is the amino acid sequence of GCNT2 contemplated for use according to the subject invention. DETAILED DESCRIPTION The present invention provides methods and compositions for diagnosis, prognosis, prevention and/or treatment of cancers. The subject invention provides biomarkers and methods for assessing the severity of a cancer/tumor and for monitoring the progression of a cancer/tumor. The subject invention also provides compositions for treating a cancer/tumor, and for preventing or reducing the progression of a cancer/tumor. In one embodiment, the cancers exhibit significant transcriptional changes in glycosylation-related genes. In a specific embodiment, the cancer is a skin cancer such as melanoma, preferably, metastatic melanoma (MM). Melanoma is one of the most aggressive forms of cancer, typically beginning in the skin and often metastasizing to vital organs and other tissues. Melanomas include, but are not limited to, superficial spreading melanoma (SSM), nodular melanoma (NM), Lentigo maligna, lentigo maligna melanoma (LMM), mucosal melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, uveal melanoma and acral lentiginous melanoma (ALM). The subject invention further provides methods and compositions for inhibiting the growth of primary melanomas, inhibiting metastasis, inhibiting the growth of metastases, killing circulating melanoma cells, inducing remission, extending remission, and/or inhibiting recurrence. In one embodiment, the subject invention pertains to the identification i-linear poly-LacNAc and/or Galectin 8 (Gal-8) as being involved in the pathogenesis of melanomas, e.g., MM. The methods according to the subject invention use i-linear poly-LacNAc and/or Gal-8 as a biomarker for cancer diagnosis, progression and/or metastasis, for example: (1) the diagnosis of cancer; (2) the prognosis of cancer (e.g., monitoring cancer progression or regression from one biological state to another); (3) the susceptibility or prediction of response to treatment for a cancer; (4) the metastasis of cancer; and/or (5) the evaluation of the efficacy to a treatment for a cancer. For the diagnosis of a cancer, the level of the specific biomarker in a subject or a sample of the subject can be compared to a baseline or control level. If the level is below or above the control level, a certain cancer is implicated. The prognosis of a cancer can be assessed by comparing the level of the specific biomarker at a first time point to the level of the biomarker at a second time point that occurs at a given interval. The prediction of response to treatment for a cancer can be determined by obtaining the level of a specific biomarker and correlating this level to a standard curve. The evaluation of the efficacy of the treatment for a cancer can be assessed by comparing the level of the specific biomarker before administration of the treatment to the level of the biomarker after the administration of the treatment. Expression of genes of the present invention can be measured by many methods known in the art. In general, expression of a nucleic acid molecule (e.g., RNA or DNA) can be detected by any suitable method or technique of measuring or detecting gene or polynucleotide sequence or expression. Such methods include, but are not limited to, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), in situ PCR, quantitative PCR (q-PCR), in situ hybridization, flow cytometry, Western blot, Southern blot, Northern blot, immunohistochemistry, sequence analysis, microarray analysis, mass spectrometry analysis, detection of a reporter gene, or any other DNA/RNA hybridization platforms. The subject invention pertains, in part, to the influence of hypoxia on global glycome alterations in MM cells, including the signature MM glycome featuring loss of GCNT2/I-branching, and whether hypoxia-dependent glycome events altered TIC development. In MM cells subjected to hypoxia, global transcriptional and N-glycomic profiling revealed several dysregulated glycome-related genes and enhanced i-linear poly-LacNAc expression. Of these alterations, including downregulation of GCNT2 and I-branched poly-LacNAcs, there was significant upregulation of Gal-8 directly corresponding to expression of a key TIC factor, nerve growth factor receptor (NGFR)/CD271, which enhances MM progression and therapy resistance. GCNT2 expression on patient melanomas was predictive of patient survival and Gal-8 levels were elevated in melanoma patient sera compared with healthy controls. Using GCNT2-enforced and -silenced MM cell variants, the results show that low GCNT2 expression increased TIC marker levels and in vivo tumor-initiating potential. Importantly, MM cell NGFR expression inversely correlated with GCNT2 expression. Gal-8 incubation with MM cells elevated NGFR, whereas Gal-8 silencing dampened NGFR expression, even under hypoxia, and reduced tumor-forming activity in vivo. Also, Gal-8 bound preferentially to MM cells with depressed GCNT2/I-branching and high i-linear-poly-LacNAcs levels. Gal-8 affinity chromatography and proteomics analysis identified pro-metastatic and TIC marker CD44 as a major cell surface Gal-8 ligand, which was dependent on i-linear poly-LacNAc N-glycans for Gal-8-binding. Extracellular Gal-8 binding to i-linear poly-LacNAchiMM cells increased AKT phosphorylation, which promotes tumor cell survival and is a downstream target of various activating cell surface receptors, including CD44. Interestingly, NGFR loss in Gal-8-silenced MM cells was not rescued by exogenous Gal-8-binding, suggesting that extracellular and intracellular Gal-8 expression may both have key roles in promoting MM cell-TIC potential. The present invention demonstrates the importance of hypoxia in governing the MM glycome to promote TIC formation and provides evidence for GCNT2/I-branching loss and elevated Gal-8 as biomarkers of MM. In one embodiment, the subject invention provides methods for treating a cancer, e.g., MM, in a subject. In a specific embodiment, the method comprises: (i) assessing the expression level of i-linear poly-LacNAc and/or Gal-8 in a sample obtained from the subject; (ii) comparing the expression level of i-linear poly-LacNAc and/or Gal-8 in the sample to a reference derived from the expression level of i-linear poly-LacNAc and/or Gal-8 in samples obtained from healthy subjects; (iii) identifying the cancer, e.g., MM, in the subject based on the increased level of i-linear poly-LacNAc and/or Gal-8 in the test sample; and (iv) administering a treatment to the subject. In a further embodiment, the treatment is systemic and comprises administering Immune Checkpoint Inhibitors (ICIs), e.g., anti-PD1, anti-PDL1 and/or anti-CTLA4 treatments. Immune checkpoints are known in the art and the term is well understood in the context of cancer therapy. Immune checkpoints include, but are not limited to, cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1) and its ligand PDL-1, T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG-3), V-domain immunoglobulin suppressor of T cell activation (VISTA), and B and T lymphocyte attenuator (BTLA). Inhibitors of immune checkpoints inhibit their normal immunosuppressive function, for example, by down regulating the expression of checkpoint molecules or by binding thereto and blocking normal receptor/ligand interactions. As a result, inhibitors of immune checkpoints enhance the immune response to an antigen, in particular, from a tumor cell. Inhibitors of immune checkpoints are known in the art and preferred inhibitors include anti-immune checkpoint antibodies, such as anti-CTLA-4 antibodies (e.g., ipilimumab and tremelimumab), anti-PD-1 antibodies (e.g., nivolumab, lambrolozumab, pidilizumab and RG7446 (Roche)) and anti-PDL-1 antibodies (e.g., BMS-936559 (Bristol-Myers Squibb), MPDL3280A (Genentech), MSB0010718C (EMD-Serono) and MED14736 (AstraZeneca)). With knowledge of an immune checkpoint target, a skilled artisan is able to develop an inhibitor thereof. Inhibitors may be selected from proteins, peptides, peptidomimetics, peptoids, antibodies, antibody fragments, small inorganic molecules, small non-nucleic acid organic molecules or nucleic acids such as anti-sense nucleic acids, small interfering RNA (siRNA) molecules or oligonucleotides. The inhibitor may for example be a modified version of the natural ligand (e.g., for CTLA-4, CD80 (B7-1) and CD86 (B7-2)), such as a truncated version of one of the ligands. They may be naturally occurring, recombinant or synthetic. In one embodiment, the subject invention provides a method of identifying a cancer, e.g., MM, in a subject, the method comprising: (a) determining the level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in: i) a test sample obtained from the subject, and ii) optionally, a control sample; (b) optionally, obtaining at least one reference value corresponding to the level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8); and (c) identifying the cancer, e.g., MM, in the subject based on the increased level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the test sample and optionally, administering a therapy to the subject to treat and/or manage the cancer, e.g., MM. In one embodiment, the control sample is obtained from: i) an individual belonging to the same species as the subject and not having, for example, MM, or ii) the subject at a prior time known to be free from MM. In one embodiment, the subject invention provides methods for treating a cancer, e.g., melanoma, preferably, MM, involving the inhibition of Gal-8 expression and/or function. The method comprises administering to the subject a pharmaceutically effective amount of an inhibitor of Gal-8 expression (e.g., gene silencing, such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). Methods to inhibit Gal-8 function or expression would reduce melanoma metastasis progression and/or enhance therapeutic response to conventional immunotherapies used to treat metastatic melanoma. In specific embodiments, the shRNA targeting Gal-8 is placed in a construct, e.g., a viral plasmid, wherein the construct comprises a DNA sequence that is transcribed into an shRNA targeting Gal-8, the DNA sequence for the shRNA targeting Gal-8 comprising the sequence of SEQ ID NO: 5 or 6. In specific embodiments, the shRNA targeting GCNT2 is placed in a construct, e.g., a viral plasmid, wherein the construct comprises a DNA sequence that is transcribed into an shRNA targeting GCNT2, the DNA sequence for the shRNA targeting GCNT2 comprising the sequence of SEQ ID NO: 3 or 4. In certain embodiments, the shRNA comprises a sequence fully complementary to a sequence in a target gene. In some embodiments, the shRNA targeting Gal-8 comprises a sequence fully complementary to a target sequence of Gal-8 and the shRNA targeting GCNT2 comprises a sequence fully complementary to a target sequence of GCNT2. In a specific embodiment, the shRNA targeting Gal-8 comprises a sequence fully complementary to SEQ ID NO: 5 or 6. In a specific embodiment, the shRNA targeting GCNT2 comprises a sequence fully complementary to SEQ ID NO: 3 or 4. In certain embodiments, the step of administering to the subject a pharmaceutically effective amount of an shRNA targeting a gene, e.g., Gal-8, may comprise administering an expression construct comprising a sequence encoding an shRNA targeting the gene, e.g., Gal-8, wherein administration of the expression construct can attenuate target gene expression. In certain embodiments, the method for treating cancer, e.g., melanoma, preferably, MM, further comprises administering to the subject a pharmaceutically effective amount of 1) a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 2) an amino acid sequence of GCNT2 protein, as well as biologically-active fragments, and variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2, 3) a vector comprising a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 4) a cell that overexpresses a nucleic acid sequence of GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, and/or 5) a cell that overexpresses an amino acid sequence of GCNT2 protein, as well as biologically-active fragments, and variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2. In one embodiment, the nucleic acid sequence of GCNT2 comprises, or consists of, a sequence of Accession No. NM_145649 (SEQ ID NO: 8), or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with Accession No. NM 145649. In one embodiment, the amino acid sequence of GCNT2 comprises, or consists of, a sequence of Accession No. NP_663624 (SEQ ID NO: 9) or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with Accession No. NM 663624. In one embodiment, the subject invention provides methods for treating a cancer, e.g., melanoma, preferably, MM, which involve the reduction of i-linear poly-LacNAc level in MM cells, and/or the reduction of the interaction between i-linear poly-LacNAc and Gal-8. In one embodiment, the subject invention provides methods for treating a cancer, e.g., melanoma, preferably, MM, which involve the reduction/inhibition of CD44 on MM cells, and/or the reduction/inhibition of the interaction between CD44 and Gal-8. In one embodiment, the subject invention provides methods for increasing or improving survival of a MM patient, which involve the inhibition of Gal-8 expression and/or function. The method comprises administering to the subject a pharmaceutically effective amount of an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). In one embodiment, the subject invention provides methods for increasing or improving survival of a MM patient, which involve the reduction of i-linear poly-LacNAc level in MM cells, and/or the reduction of the interaction between i-linear poly-LacNAc and Gal-8. In one embodiment, the subject invention provides methods for reducing the expression of tumor-initiating cell markers, e.g., KLF4, and/or NGFR/CD271, in MM cells, which involve the overexpression of GCNT2, the method comprising administering to the subject a pharmaceutically effective amount of 1) a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 2) an amino acid sequence of GCNT2 protein, as well as biologically-active fragments, and variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2, 3) a vector comprising a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 4) a cell that overexpresses a nucleic acid sequence of GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, and/or 5) a cell that overexpresses an amino acid sequence of GCNT2 protein, biologically-active fragments, variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2. In one embodiment, the subject invention provides methods for reducing the expression of tumor-initiating cell markers, e.g., KLF4, and/or NGFR/CD271, in MM cells, the method comprising contacting the MM cells with 1) a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 2) an amino acid sequence of GCNT2 protein, as well as biologically-active fragments, and variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2, and/or 3) a vector comprising a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2; or a composition comprising 1), 2), and/or 3) above. Contacting the MM cells with the above 1), 2), and/or 3) or compositions results in, for example, the transfection or transduction of the GCNT2 gene into the melanoma cells or the direct delivery of the protein, which leads to overexpression of GCNT2 in these cells. There are various transfection methods, including physical treatment (e.g., electroporation microinjection, cell squeezing, impalefection, hydrostatic pressure, continuous infusion, sonication, nanoparticles, and magnetofection), chemical materials (e.g., lipofection, and polyplexes) or biological particles (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus) that are used as carriers. As used herein, “variants” of a protein refer to sequences that have one or more amino acid substitutions, deletions, additions, or insertions. In preferred embodiments, these substitutions, deletions, additions or insertions do not materially adversely affect the protein activity. Variants that retain one or more biological activities are within the scope of the present invention. “Fragments” and its variants are also within the scope of proteins of the subject invention, so long as the fragment retains one or more biological properties. Preferably, the fragment is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the full length protein, e.g., GCNT2. In one embodiment, the subject invention provides methods for reducing the expression of tumor-initiating cell markers, e.g., KLF4, and/or NGFR/CD271, in MM cells, which involves the inhibition of Gal-8 expression and/or function. The method comprises administering to the subject a pharmaceutically effective amount of an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). The method may comprise administering to the subject a pharmaceutical composition comprising an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). The pharmaceutical composition may also comprise a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” refers to a diluent, adjuvant or excipient with which the antigen disclosed herein can be formulated. Typically, a “pharmaceutically acceptable carrier” is a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a diluent, adjuvant or excipient to facilitate administration of the composition disclosed herein and that is compatible therewith. Examples of carriers suitable for use in the pharmaceutical compositions are known in the art and such embodiments are within the purview of the invention. In one embodiment, the method for reducing the expression of tumor-initiating cell markers, e.g., KLF4, and/or NGFR/CD271, in MM cells comprises contacting the MM cells with an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). In certain embodiments, the step of contacting the MM cells with an inhibitor of Gal-8 may comprise introducing an expression construct comprising a sequence encoding an shRNA targeting Gal-8 into the MM cells (e.g., in vitro or in vivo), wherein the shRNA is expressed in an amount sufficient to attenuate Gal-8 expression and the shRNA is stably expressed in the MM cells. In specific embodiments, the expression construct comprises a sequence encoding an shRNA targeting Gal-8, wherein the sequence encoding an shRNA targeting Gal-8 comprises the sequence of SEQ ID NO: 5 or 6. In certain embodiments, the shRNA comprises a sequence fully complementary or substantially complementary (e.g., at least 80%, 85%, 90%, 95% or 100% complementary) to a sequence in a target gene. In some embodiments, the shRNA targeting Gal-8 comprises a sequence fully complementary or substantially complementary (e.g., at least 80%, 85%, 90%, 95% or 100% complementary) to a target sequence of Gal-8. In a specific embodiment, the shRNA targeting Gal-8 comprises a sequence fully complementary or substantially complementary (e.g., at least 80%, 85%, 90%, 95% or 100% complementary) to SEQ ID NO: 5 or 6. As used herein, the term “fully complementary” with regard to a sequence refers to a complement of the sequence by Watson-Crick base pairing, whereby guanine (G) pairs with cytosine (C), and adenine (A) pairs with either uracil (U) or thymine (T). A sequence may be fully complementary to the entire length of another sequence, or it may be fully complementary to a specified portion or length of another sequence. One of skill in the art will recognize that U may be present in RNA, and that T may be present in DNA. Therefore, an A within either of a RNA or DNA sequence may pair with a U in a RNA sequence or T in a DNA sequence. As used herein, the term “substantially complementary” refers to sequences of nucleotides where a majority (e.g., at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) or all of the bases in the sequence are complementary, or one or more (e.g., no more than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%) bases are non-complementary, or mismatched. A complementary sequence can be a reverse complement of the sequence allowing for Watson-Crick base pairing, wobble base pairing, or both, whereby G pairs with either C or U, and A pairs with either U or T. A sequence may be complementary to the entire length of another sequence or it may be complementary to a specified portion or length of another sequence. One skilled in the art will recognize that the U may be present in RNA, and that T may be present in DNA. Therefore, a U within an RNA sequence may pair with A or G in either an RNA sequence or a DNA sequence, while an A within either of an RNA or DNA sequence may pair with a U in a RNA sequence or T in a DNA sequence. Two sequences that are substantially complementary may hybridize to each other, e.g., under low stringency, medium stringency, high stringency, or very high stringency conditions. As used herein, the term “construct,” “expressing construct” or “expression construct” is a generic term that includes nucleic acid preparations designed to achieve an effect of interest. An expressing construct comprises an RNAi molecule that can be cleaved in vivo to form an siRNA or a mature shRNA. For example, an RNAi construct is an expression vector capable of giving rise to an siRNA or a mature shRNA in vivo. The term “vector” refers to a vehicle for introducing a nucleic acid into a cell, which includes, but is not limited to, plasmid, phagemid, virus, bacterium, and vehicle derived from viral or bacterial sources. A “plasmid” is a circular, double-stranded DNA molecule. A useful type of vector for use in the present invention is a viral vector, wherein heterologous DNA sequences are inserted into a viral genome that can be modified to delete one or more viral genes or parts thereof. Certain vectors are capable of autonomous replication in a host cell (e.g., vectors having an origin of replication that functions in the host cell). Other vectors can be stably integrated into the genome of a host cell, and are thereby replicated along with the host genome. In certain embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors. The use of viral vector-based RNAi delivery not only allows for stable single-copy genomic integrations but also avoids the non-sequence specific response via cell-surface toll-like receptor 3 (TLR3), which has raised many concerns for the specificity of siRNA mediated effects. In some embodiments, the shRNA of the invention can be introduced into the cell directly, or can be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to the cell. In certain embodiments, the shRNA can be a synthetic shRNA, including shRNAs incorporating modified nucleotides, such as those with chemical modifications to the 2′-OH group in the ribose sugar backbone, such as 2′-O-methyl (2′OMe), 2′-fluoro (2′F) substitutions, and those containing 2′OMe, or 2′F, or 2′-deoxy, or “locked nucleic acid” (LNA) modifications. In some embodiments, an shRNA of the invention contains modified nucleotides that increase the stability or half-life of the shRNA molecule in vivo and/or in vitro. In one embodiment, the subject invention provides methods for reducing/slowing down the growth/potential of tumor-initiating cells in a subject, the method comprising administering to the subject a pharmaceutically effective amount of 1) a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 2) an amino acid sequence of GCNT2 protein, biologically-active fragments, variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2, 3) a vector comprising a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 4) a cell that overexpresses a nucleic acid sequence of GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, and/or 5) a cell that overexpresses an amino acid sequence of GCNT2 protein, biologically-active fragments, variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2. In one embodiment, the method of treating/preventing/reducing the progression of MM may further comprises administering to the subject one or more therapeutic agents. The therapeutic agent may comprise a chemotherapeutic agent, immunotherapeutic agent, gene therapy or radio therapeutic agent. The administration routes include, but are not limited to, the local, oral, ophthalmic, nasal, topical, intratumoural, transdermal, intra-articular, parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous or intramuscular), intracranial, intracerebral, intraspinal, intravaginal, intrauterine, or rectal route. Additionally, the composition or therapeutic agents may be administered directly into the tumor of MM. A further embodiment of the invention provides a method for monitoring the effect of a treatment for a cancer, such as MM, in a subject. A method for monitoring the effect of a treatment for a cancer, such as MM, in a subject may comprise:(a) determining the level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in:i) a pre-treatment test sample obtained from the subject before the treatment,ii) a post-treatment test sample obtained from the subject after the treatment, andii) optionally, a control sample;(b) optionally obtaining at least one reference values corresponding to levels of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8); and(c) identifying the treatment for the cancer, e.g., MM, in the subject as effective based on a reduced levels of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the post-treatment test sample compared to the levels of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the pre-treatment test sample and optionally, continuing the treatment in the subject, or(d) identifying the treatment for the cancer, e.g., MM, in the subject as ineffective if the levels of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the post-treatment test sample remains the same or increases compared to the levels of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the pre-treatment test sample and optionally, modifying the treatment in the subject. In one embodiment, the subject invention provides a method for diagnosing and/or assessing the progression of MM in a subject, the method comprising:(i) assessing the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in a sample obtained from the subject;(ii) comparing the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the sample to a reference derived from the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in samples obtained from healthy subjects; and(iii) determining the progression of MM in the subject based on whether the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the subject is up-regulated or down-regulated. In further embodiments, the biomarker is Gal-8 and a down-regulation in the expression level of i-linear poly-LacNAc and/or Gal-8 in the sample is indicative of an improvement in the subject's condition. In one embodiment, the subject invention provides a method for stratifying a tumor stage (e.g., of MM) in a subject, the method comprising:(i) assessing the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in a tumor sample obtained from the subject;(ii) comparing the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the tumor sample to a reference derived from the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in healthy samples obtained from healthy subjects; and(iii) determining the tumor stage in the subject based on whether the expression level of one or more biomarkers (e.g., i-linear poly-LacNAc and/or Gal-8) in the subject is up-regulated or down-regulated. In one embodiment, the subject invention provides a method for predicting an outcome of an anti-cancer therapy, in a subject, the method comprising:(i) assessing the expression level of i-linear poly-LacNAc and/or Gal-8 in a sample (e.g., tumor specimen) obtained from the subject;(ii) comparing the expression level of i-linear poly-LacNAc and/or Gal-8 in the sample to a reference derived from the expression level of i-linear poly-LacNAc and/or Gal-8 in samples obtained from healthy subjects; and(iii) determining/predicting the outcome of the anti-cancer therapy by assessing whether the level of i-linear poly-LacNAc and/or Gal-8 in the subject is up-regulated or down-regulated, wherein a down-regulation in the expression of i-linear poly-LacNAc and/or Gal-8 indicates that the cancer, e.g., MM, will be responsive and/or sensitive to the anti-cancer therapy. In one embodiment, the subject invention provides a method for assessing the response of a melanoma subject to an anti-melanoma therapy, the method comprising:assessing an expression level of Gal-8 in a sample obtained from the melanoma subject before and after the anti-melanoma therapy;comparing the expression level of Gal-8 in the sample before and after the anti-melanoma therapy; anddetermining the melanoma subject being responsive to the anti-melanoma therapy by a decreased expression level of Gal-8 in the sample, or determining the melanoma subject being non-responsive to the anti-melanoma therapy if the expression level of Gal-8 remains the same or increases. The term “sample” as used herein refers to any physical sample that includes a cell or a cell extract from a cell, a tissue, a biofluid or an organ including a biopsy sample. The sample can be from a biological source such as a subject, or a portion thereof, or can be from a cell culture. Samples from a biological source can be from a normal or an abnormal organism, such as an organism known to be suffering from a condition or a disease state, or any portion thereof. Samples can also be from any fluid, e.g., blood and serum, tissue or organ including normal and abnormal (diseased) fluid, tissue or organ. Samples from a subject can be used, processed or cultured such that cells from the sample can be sustained in vitro as a primary or continuous cell culture or cell line. In a specific embodiment, the sample is a skin sample, for example, skin cells, skin extract, and/or skin tissue. Preferably, the skin sample may comprise melanocytes. The term “subject” or “patient,” as used herein, describes an organism, including mammals such as primates, to which diagnosis, prevention, assessment, and/or treatment according to the present invention can be provided. Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes, chimpanzees, orangutans, humans, monkeys; domesticated animals such as dogs, cats; live-stocks such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In a specific embodiment, the subject may have hypoxia. In a specific embodiment, the subject may not have hypoxia. In a specific embodiment, the MM cells are under hypoxia. In a specific embodiment, the MM cells are not under hypoxia. The terms “treatment” or any grammatical variation thereof (e.g., treat, treating, etc.), as used herein, includes but is not limited to, the application or administration to a subject (or application or administration to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term “treating” refers to any indication of success in the treatment or amelioration of a pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the pathology or condition more tolerable to the subject; or improving a subject's physical or mental well-being. In a further embodiment, the melanoma or MM may be a drug-resistant melanoma or MM. In a preferred embodiment, the melanoma is an ICI therapy-resistant melanoma. In one embodiment, the subject in need of the treatment for melanoma, preferably, ICI therapy-resistant melanoma, has been treated by ICI or IC therapy. In one embodiment, the subject invention provides a method for treating an IC therapy-resistant melanoma in a subject, the method comprising administering to the subject an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists) or a composition comprising an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). In some embodiments, the method further comprises administering to the subject a pharmaceutical composition comprising 1) a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2, 2) an amino acid sequence of GCNT2 protein, biologically-active fragments, variants thereof, or an amino acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with GCNT2, and/or 3) a vector comprising a nucleic acid sequence that encodes GCNT2 or a nucleic acid sequence sharing at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the nucleic acid sequence that encodes GCNT2. In one embodiment, the subject invention further provides a method for increasing/enhancing/improving the sensitivity of a subject having melanoma to an IC therapy, the method comprising administering to the subject an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists) or a pharmaceutical composition comprising an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). In a further embodiment, the pharmaceutical composition may be administered prior to the administration of the IC therapy, simultaneously with the IC therapy, or after the administration of the IC therapy. In a preferred embodiment, the IC therapy is an anti-PD-1 therapy. In one embodiment, the subject invention provides a method for increasing/enhancing/improving the sensitivity of melanoma cells to an ICI, the method comprising contacting the melanoma cells with an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists) or a pharmaceutical composition comprising an inhibitor of Gal-8 expression (e.g., gene silencing such as siRNAs and shRNAs targeting Gal-8) and/or function (e.g., anti-Gal-8 antibodies or Gal-8 binding antagonists). In a specific embodiment, the shRNA targeting Gal-8 is encoded by a sequence comprising SEQ ID NO: 5 or 6. In one embodiment, melanoma may be a stage 0, I, II, III or IV melanoma. Stage 0 melanoma is a very early-stage disease known as melanoma in situ. The tumor is limited to the epidermis with no invasion of surrounding tissues, lymph nodes, or distant sites. Stage 0 melanoma is considered to be very low risk for disease recurrence or spread to lymph nodes or distant sites. Stage I melanoma is characterized by tumor thickness, presence and number of mitoses, and ulceration status. Stage I melanomas are considered to be low-risk for recurrence and metastasis. Sentinel lymph node biopsy is recommended for Stage I tumors thicker than 1.0 mm and for any ulcerated tumors of any thickness. Surgery is a common treatment for Stage I melanoma. Stage II melanomas also are localized tumors characterized by tumor thickness and ulceration status. Stage II melanoma is considered to be intermediate-risk for local recurrence or distant metastasis. In addition to biopsy and surgery as described for Stage I, Stage II treatment may include adjuvant therapy, which is a treatment given in addition to a primary cancer treatment, following surgery. Treatments may include interferons therapies (e.g., interferon alfa-2a, and/or alfa-2b), and vaccines therapy. Stage III melanomas are tumors that have spread to regional lymph nodes, or have developed in transit metastasis or satellites. Stage III disease is considered to be intermediate-to high-risk for local recurrence or distant metastasis. In addition to surgery and adjuvant therapy as described above, Stage III melanoma treatment often includes therapeutic lymph node dissection (TLND) to remove regional lymph nodes from the area where cancerous lymph nodes were found. The goal of the surgery is to prevent further spread of the disease through the lymphatic system. Stage IV melanomas often are associated with metastasis beyond the regional lymph nodes to distant sites in the body. Common sites of metastasis are vital organs (lungs, abdominal organs, brain, and bone) and soft tissues (skin, subcutaneous tissues, and distant lymph nodes). Stage IV melanoma may be characterized by the location of the distant metastases; the number and size of tumors; and the serum lactate dehydrogenase (LDH) level. Elevated LDH levels usually indicate that the tumor has spread to internal organs. Treatments may include surgery to remove cancerous tumors or lymph nodes that have metastasized to other areas of the body, systemic therapies and radiation therapy. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The transitional terms/phrases (and any grammatical variations thereof) “comprising,” “comprises,” and “comprise” can be used interchangeably. Use of the term “comprising” contemplates other embodiments that “consist” or “consisting essentially of” the recited component(s). When ranges are used herein, such as for dose ranges, combinations and subcombinations of ranges (e.g., subranges within the disclosed range), specific embodiments therein are intended to be explicitly included. The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 0-20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of concentrations of ingredients where the term “about” is used, these values include a variation (error range) of 0-10% around the value (X±10%). Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and/or as otherwise defined herein. EXAMPLES Methods Cells Human A375, A2058 and SK-MEL-5 MM cell lines were obtained from ATCC and grown in DMEM media with 10% FBS (Atlanta) and 1% Antibiotic-Anti-mitotic (Gibco). GCNT2 overexpressing (OE) and silenced (KD) MM cells were generated by lentiviral transduction. For Gal-8 KD cell lines commercial lentiviral particles with shRNA directed against Gal-8 and scrambled control (Scr) were purchased (GeneCopoeia). A375 and 2058 cells were transduced and selected in 1 mg/ml Hygromycin (Corning) containing media. Reagent details and oligonucleotide details are available in Tables 1 and 2. TABLE 1Antibodies and reagentsAntibodies/ReagentsS. NoNameSourceIdentifierConcentrations1Rabbit polyclonal anti-Sigma-AldrichCat# HPA0267761:500 (IHC)GCNT22Leica Bond PolymerLeicaCat# DS9800N/ADetection Kit3OSK-14 (antiDI) human IgMDr. Yoshihiko TaniN/A1:50-1:100Japanese Red Cross(Batch specific)Kinki Block Blood(FlowCentercytometry)4OSK-28 (antiDI) human IgMDr. Yoshihiko TaniN/A1:20-1:50Japanese Red Cross(Batch specific)Kinki Block Blood(FlowCentercytometry)5IRDye ® 800CW Goat anti-LI-COR BiosciencesCat# 926-322111:10000Rabbit IgG (H + L)(Western)6IRDye ® 800CW Goat anti-LI-COR BiosciencesCat# 926-322101:10000Mouse IgG (H + L)(Western)7IRDye ® 680RD Goat anti-LI-COR BiosciencesCat# 926-680701:10000Mouse IgG (H + L)(Western)8IRDye ® 680LT Donkey anti-LI-COR BiosciencesCat# 926-680231:10000Rabbit IgG (H + L)(Western)9HIF-1α (D2U3T) RabbitCell SignalingCat# 14179S1:1000mAb(Western)10Phospho-Akt (Ser473) (D9E)Cell SignalingCat# 4060S1:1000XP ® Rabbit mAb #4060(Western)11Akt (pan) (40D4) MouseCell SignalingCat# 2920S1:1000mAb(Western)12Rabbit Beta actin (D6A8)Cell SignalingCat# 8457S1:1000(Western)13Anti-beta Actin, Clone:Abcam ™Cat# ab82261:1000mAbcam 8226(Western)14Recombinant Anti-p75 NGFAbcam ™Cat# ab529871:1000Receptor antibody(Western)[EP1039Y]15APC anti-human CD271BiolegendCat# 3451071 μg/ml(NGFR) Antibody(Flowcytometry)16APC Mouse IgG1, κ IsotypeBiolegendCat# 4001201 μg/mlCtrl Antibody(Flowcytometry)17PE anti-human CD56BiolegendCat# 3625081 μg/ml(NCAM) Antibody(Flowcytometry)18PE Mouse IgG1, κ IsotypeBiolegendCat# 4001141 μg/mlCtrl Anitbody(Flowcytometry)19Human Galectin-8 Antibody,R&D BiosystemsCat#AF13051 μg/mlGoat Polyclonal, R&D(FlowSystems ™cytometry)20Human Galectin-8R&D Systems ™Cat# 1305GA05010 μg/ml-40Recombinant Proteinμg/ml21Donkey Anti-Goat IgG H&LAbcam ™Cat# ab1501291:2000(Alexa Fluor ® 488)(Flowcytometry)22Foxp3/Transcription FactoreBioscience ™Cat# 50-112-8857N/AStaining Buffer Set23GCNT2 TaqMan ® primerThermo ScientificAssayID:N/AHs00377334_m124KLF4 TaqMan ® primerThermo ScientificAssayID:N/AHs00358836_m125NGFR TaqMan ® primerThermo ScientificAssayID:N/AHs00609976_m126MITF TaqMan ® primerThermo ScientificAssayID:N/AHs01117294_m12718S TaqMan ® primerThermo ScientificAssayID:N/AHs01117294_m128LGALS8 TaqMan ® primerThermo ScientificHs01057135_m1N/A31TaqMan ® Fast AdvancedThermo ScientificCat# 4444557N/AMaster Mix32SuperScript ™ VILO ™Invitrogen ™Cat# 11-754-050N/AcDNA Synthesis Kit33RIPA Lysis and ExtractionThermo ScientificCat# 89900N/ABuffer34Halt ™ Protease andThermo ScientificCat#78440N/APhosphatase Inhibitor Single-Use Cocktail (100X)35BCA protein assay kitThermo ScientificCat# PI23227N/A36Laemmli SDS sample bufferAlfa AesarCat#1XAAJ61337AC37Immobilon PVDFMilliporeCat# IPFL00010N/A38Criterion 4-12% Bis-TrisBioRadCat# 3450123N/APrecast Gels-12 wells + 239Intercept ® (TBS) blockingLI-COR BiosciencesCat# NC1660550N/Abuffer40anti-human IgM-APCBiolegendCat# 3145101:400 (Flowcytometry)41LIVE/DEAD ™ FixableFisherCat# 50-112-15251:1000 (FlowAqua Dead Cell Stain Kit,cytometry)for 600 nm excitation42Accutase-Enzyme CellFisherCat# 50-112-9055N/ADetachment Medium43NeuraminidaseRocheCat# 102696110010.125 U/ml44RNeasy Plus Mini Kit (250)QIAGENCat# 74136N/A45Sep-Pak C18 6 cc VacWaters CorporationCat#N/ACartridge, 500 mg SorbentSKU186006325per Cartridge, 55-105 μm,300/pk46N-Glycosidase FMilliporeSigma-Cat# 11365169001Roche47AdvanceBio Sialidase SAgilentCat# GK8002148Human Galectin-8 ELISASigmaMilliporeCat# RABI050-N/AKit1KT49Trypsin Singles, ProteomicsSigma AldrichCat# T7575-1KT1:20Grade(enzyme:protein)50Pierce ™ C18 Tips, 100 μLThermo ScientificCat# PI87784N/Abed51New England BiolabsNew England BiolabsCat# 50-811-8321000-2500 UPNGase F (Glycerol-free)-15000 units52Sino Biological HumanSino BiologicalCat# 50-161-456510 μg/mlGalectin-8/LGALS8 Protein(affinity(GST Tag)chromatography)53Pierce ™ GlutathioneThermo ScientificCat# PI78601N/AMagnetic Agarose Beads54CD44 (156-3C11) MouseCell SignalingCat# 3570S1:2000mAb(Western) TABLE 2Oligonucleotide detailsOligonucleotide detailsGeneForward SequenceReverse SequenceComments1HumanCGACAGATCTGCCACCATGAATGTCAAGCTTTCAAAAATACloningGCNT2CTTTTGGAGGTACTGCTTTCCAGCTGGGTTGTA (SEQprimers(SEQ ID NO: 1)ID NO: 2)2HumanGCTAACAAGTTTGAGCTTAAT—shRNAGCNT2(SEQ ID NO: 3)targetshRNA #1sequence3HumanGCTCACCTCTATATTAGTTTA—shRNAGCNT2(SEQ ID NO: 4)targetshRNA #2sequence4HumanCCTACAGAATATCATCTATAA—shRNALGALS8(SEQ ID NO: 5)targetshRNA #1sequence5HumanGCTCGGACTTACAAAGTACCC—shRNALGALS8(SEQ ID NO: 6)targetshRNA #2sequence6HumanGCTTCGCGCCGTAGTCTTA—Non-targetLGALS8(SEQ ID NO: 7)sequencescr ctrl Murine Model NOD-SCID IL-2Rγ-deficient (NSG) mice were used for in vivo tumorigenicity experiments. Age-matched mice of at least 6-weeks were used for experiments. Both male and female mice were equally used for the studies to account for any potential gender-related variability. All experiments were conducted as per FIU IACUC protocol. For limiting dilution analysis, mice were inoculated with A375 EV and GCNT2 OE cell variants and A2058 Src and KD variants subcutaneously from 1×103to 1×105into the flank of NSG mice. Tumor volume was calculated using the formula: [tumor volume (mm3)=(length×(width)2×0.5]. Tumor growth was assessed every 2-4 days by calipers. Immunohistochemistry of GCNT2 Archival FFPE-human normal skin, nevi or melanoma tissue microarray (TMA) sections were kindly provided by Dr. Richard Scolyer (Melanoma Institute of Australia). Sections were deparaffinized in xylene; dehydrated with 100%, 95% and 75% ethanol and deionized water; placed in antigen retrieval solution; and boiled at 100° C. for 20 mins. Sections were then stained with 1:500 dilution of GCNT2 primary antibody (Sigma-Aldrich) for 30 mins at 37° C. Leica Bond Polymer Detection Kit (Leica) was used for GCNT2 antibody detection. The polymer-HRP secondary antibody was incubated for 15 mins at room temperature. Hematoxylin was used as counterstain and images were acquired using Nikon eclipse Ti microscope and Nikon FDX-35 digital camera. TMA “grade” scoring was performed as follows. Individual cores of GCNT2-stained TMAs were excluded if they were absent of skin/melanoma tissue or tissue quality deemed unsuitable for review. Melanoma cells and random fields in nevi/melanoma were identified, confirmed, and graded by a pathologist in blinded manner. Greater than 100 cells/specimen were analyzed and semi-quantitatively graded as 0 (no stained cells); 1 (1-25% cells positive); 2 (25-50% cells positive); 3 (50-75% cells positive); or 4 (75-100% cells positive). Similarly, for TMA “intensity” scoring, random fields in nevi/melanoma were analyzed, where over 100 cells/specimen were assessed and semi-quantitatively graded as 0 (No staining); 1 (Faint staining); 2 (Moderate staining) and 3 (Dark staining). Matched clinical data was then assigned to stained samples—Alive No Recurrence, Alive with Melanoma, and Dead with Melanoma. Statistical significance was analyzed via Cochran-Armitage Trend Test (p value<0.05). ELISA of Gal-8 in Melanoma Patient Serum Melanoma patient samples were obtained from the Biospecimen Repository Facility at Miami Cancer Institute Baptist Health-South Florida and used for Gal-8 expression analysis by ELISA. Peripheral blood samples were collected from patients with melanoma at the following stages of disease: 0 (n=1), I (n=5), II (n=1), III (n=2), IV (n=4). Peripheral blood collected in non-EDTA coated tubes were allowed to coagulate at room temperature for 1 hour. Post incubation, samples were centrifuged at 300 g for 5 min at 4° C. for serum collection. Gal-8 levels were measured in patient sera using commercially available Gal-8 ELISA Kit (Sigma) per manufactures protocol. Briefly, serum dilution of 1:1 and 1:2 was performed using assay buffer. Standards were prepared according to manufacturer's protocol. Samples and standards were loaded in anti-Gal-8 antibody coated plates and incubated overnight with gentle shaking followed by detection antibody incubation. Next, wells were incubated in TMB (3,3′,5,5′-Tetramethylbenzidine) substrate for 30 mins followed by addition of ELISA stop solution and read at 450 nm. Glycome Gene Expression Analysis RNA was isolated for sequencing from A375, A2058 and SkMel5 cells grown under chronic hypoxia (1% oxygen) and normoxia conditions and distributed to the Genomics Core Facility at University of Miami Miller School of Medicine for RNAseq analysis. A375, A2058 and SkMel5 cells were grown under hypoxia (1% oxygen) until cells were able to proliferate with no visible signs of cell death. On the day of RNA isolation cells were washed in PBS and RNA isolation performed using RNeasy® plus mini kit (Qiagen) per manufacturers protocol. RNA sequencing was performed in the John P. Hussman Institute for Human Genomics, Center for Genome Technology Sequencing Core. Extracted total RNA was quantified via Qubit fluorometric assay (ThermoFisher) and qualified on the 2100 Bioanalyzer (Agilent). For RNA samples with RNA integrity scores (RIN)>6, 600 ng of total RNA was used as input for the NuGEN Universal Plus mRNA-Seq kit (Tecan Genomics) per the manufacturer's instructions to create poly-A selected RNA and globin depleted sequencing libraries. Following quantification of libraries via qPCR they were combined into equimolar pools and sequenced to more than 30 million raw single end 100 bp reads on the Illumina NovaSeq 6000. Resulting FASTQ files were processed with a bioinformatics pipeline including quality control, alignment to the GRCh38 human reference genome with STAR aligner v2.5.2a2, and gene quantification performed with the Gene Counts STAR function against the GENCODE v35 annotation gene set. Count data were input into edgeR software for differential expression analysis. Counts were normalized using the trimmed mean of M-values (TMM) method to account for compositional difference between the libraries. Differential expression analysis between groups was performed for paired samples adjusting for differences between individuals using an additive linear model with individual as the blocking factor. Specifically, analysis was focused on glycome-associated genes consisting of glycosyltransferases, glycoproteins, glycan-binding proteins, and other proteins necessary for the process of glycosylation, such as chaperones and nucleotide-sugar transporters. For this, we used the generalized linear model likelihood ratio test (glmLRT) implemented in edgeR. Protein coding genes with a nominal p-value (FDR)<0.05 and the average log counts per million across the samples of at least 0 were considered differentially expressed. Raw FASTQ and gene count matrix are available in the GEO GSE188986. RT-qPCR Analysis RNA samples from MM cells grown under normoxia or hypoxia were used to assess gene expression of TIC markers and candidate glycome factors identified in RNAseq analysis. A375 and A2058 cell lines were cultured for 24 h in normoxic and hypoxic conditions. Cell media was aspirated, and RNA was isolated using RNeasy Plus kit (mini) (Qiagen) per manufacturer protocol. Isolated RNA was converted to cDNA using SuperScript™ VILO™ cDNA synthesis kit (Invitrogen). Real-time quantitative PCR was then performed with TaqMan™ fast advanced master mix (Applied Biosystems) and TaqMan primers to amplify genes (GCNT2, KLF4, NGFR, MITF, and internal control 18S). Assays included Taqman master mix per manufacturer's protocol. Alterations in Gal-8, NCAM, and FUT11 were also assessed utilizing RT-qPCR as detailed above (LGALS8, NCAM1, FUT11, and 18S internal control). Immunoblotting Protein expression was assessed in lysates from MM cells grown under normoxia or hypoxia by Western blot analysis. Cells were lysed in Pierce™ RIPA buffer (Thermo Scientific) with protease and phosphatase inhibitor cocktail (Thermo Scientific). After a 30 min incubation on ice, cell lysates were centrifuged for 10 mins at 10,000 RPM in 4° C. Protein concentrations were calculated using Pierce™ BCA protein assay kit (Thermo Scientific) per manufacturer protocol, and equal protein amounts from each sample were prepared with Laemmli SDS sample buffer (Alfa Aesar). Samples were boiled for 5 mins and subsequently loaded on a 4-12% gradient SDS PAGE gel (BioRad) for electrophoresis. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), blocked for 1 h at room temperature with Intercept® (TBS) blocking buffer (LI-COR), and incubated overnight at 4° C. with primary antibodies. Membranes were later washed and incubated with IRDye® anti-rabbit secondary antibody (LI-COR) for 1 h at room temperature. A LI-COR imager (LI-COR Biosciences, Lincoln, NE) was used to analyze blots. Anti-HIF-1α ant-body (Cell Signaling) was used to confirm for hypoxia induction in lysates of A375 and A2058 cells. A375 and A2058 cells were also incubated with Gal-8 (20 μg/ml) and levels of NGFR (Abeam) were assessed by immunoblotting via the above protocol. β-actin (Abcam) was used as control. A375 and A2058 cells with Gal-8 KD were similarly grown under hypoxia and NGFR (Abcam) levels were assessed by immunoblotting. A2058 cells with GCNT2 KD and control Scr cells were first serum starved overnight and incubated with Gal-8 (10 μg/ml) for 15 minutes and 30 minutes in serum free media. Cells were harvested for immunoblotting for AKT pS473 and total AKT (Cell Signaling) Flow Cytometry To analyze surface and intracellular expression of TIC markers and glycome structures, flow cytometry was performed using validated antibodies and methods as we routinely describe. Cells were harvested using Accutase (Fisher), washed with PBS and resuspended in anti-human CD271 (NGFR) (Biolegend) or NCAM (Biolegend) and Aqua Live/Dead stain for 30 minutes on ice. Single-color, isotype, and unstained controls were also prepared for validation. Cells were washed and subsequently resuspended in 2000 of PBS for analysis. For assessing i-linear glycan expression on cell surface, A375 and A2058 cells were harvested using Accutase, washed in PBS and subsequently treated with 125 mU/ml neuraminidase (sialidase) (Roche) for 2 h at 37° C. Cells were then stained for 45 minutes on ice with primary OSK28 antibody followed by secondary anti-human IgM-APC (Biolegend) and Aqua Live/Dead (Fisher). To analyze total Gal-8 levels, cells were lifted and stained with Aqua Live/Dead stain followed by fixation and permeabilization (kit) and stained with Gal-8 primary ab for 45 minutes. Cells were then washed and incubated with secondary rabbit IgG ab (AF647) for 30 mins, washed and analyzed by flow cytometry. For assessing external rhGal-8 binding cells were lifted with Accutase and incubated with rhGal-8 (R&D) for 30 mins, 10 ug/ml. Cells were then washed and stained with anti-Gal-8 ab as described and analyzed by flow cytometry. Flow cytometry was performed with BD FACSCelesta™ (BD Biosciences). Gal-8 Affinity Chromatography A375, A2058, A375 EV, A375 GCNT2 OE, A2058 Scr, and A2058 GCNT2 KD cells were used for Gal-8 affinity chromatography assay. Glutathione S-transferase (GST)—tagged Gal-8 (SinoBiologicals, Beijing, China) was used at 10 μg/ml concentration with reduced glutathione magnetic beads (Pierce) to isolate Gal-8 ligands per manufacturers protocol. Briefly, cells were plated 24 hrs prior to collection, with at least 10 million cells plated per cell type. Cells were first lifted using 1 mM EDTA (Invitrogen), washed in PBS and lysed using IP lysis buffer (Pierce). Lysates were quantified using BCA and 1 mg of protein was incubated with 10 μg/ml of Gal-8-GST and Glutathione magnetic beads at room temperature for 2 hours. After 2 washed in wash buffer (125 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH7.4), elution buffer (50 mM glutathione, reduced (Product No. 78259) in Equilibration/Wash pH 7.4-pH 9 resulted in higher yield) was used to collect Gal-8 ligands and analyzed by mass-spectrometry and immunoblotting. Anti-CD44 antibody (Cell Signaling) was used to confirm CD44 as a Gal-8 ligand using immunoblotting as described above. Lactose (200 mM) was used as control for the affinity chromatography. For assessing the role of N-glycans in CD44's interaction with Gal-8, peptide N-glycanase F (PNGase F) (New England Biolabs) was used under non-denaturing conditions to treat eluates prior to Gal-8 affinity chromatography as per manufacturer's protocol. N-glycosylated CD44 was then confirmed as a ligand by immunoblotting. Proteolysis and NanoLC-MS/MS Analyses. Following Gal-8 affinity chromatography, proteins in the eluate were reduced in 5 mM DTT in 50 mM ammonium bicarbonate for 30 mins at 37° C. and alkylated with iodoacetamide (IAA) for 1 hour at room temperature in the dark. IAA was quenched by adding DTT to a final concentration of 20 mM. Next, trypsin (Proteomics Grade) was added to the sample a ratio of 1:20 (enzyme: protein), and proteins were incubated at 37 C for 12 hours. Samples were subsequently dried under vacuum and resuspended in 100 μL 1% acetonitrile and 0.1% formic acid in water and desalted using a Pierce C18 tip (Thermo Fisher Scientific™) and dried again. Nano liquid chromatography (nLC) tandem mass spectrometry analyses were performed on an Orbitrap Eclipse Tribrid Mass Spectrometer with an online EASY nLC 1200 system (Thermo Fisher Scientific™). An Acclaim PepMap 100 (75 μm, 2 cm) trapping column and a PepMap RSLC C18 analytical column (2 μm, 100 Å, 75 μm×15 cm) were employed for chromatographic separation. The following gradient was utilized for peptide separation: starting conditions 2% B, 2-6% B from 0-5 mins, 6-35% B from 5-75 mins, 35-60% B from 75-80 mins, 60-95% B for 30 sec, and 95% B for 9.5 min (solvents A and B consisted of 1% acetonitrile/99% water+0.1% formic acid and 80% acetonitrile/20% water+0.1% formic acid, respectively). All MS analyses were performed in positive mode and spectra were acquired using the Orbitrap. For proteomic analyses, MS1 scans were acquired using the following parameters: RF lens 30%; resolution 120,000; m/z range 375-2000; cycle time 3 sec; 50 msec injection time; AGC target 4×105; 1 μscan. For MS2 scans, peptides with charge states 2-6 were selected; min. intensity 2×104; and dynamic exclusion of 1 min. An isolation window of 1.2 was used. Higher-energy collisional dissociation (HCD) at 30% collision energy, and a maximum injection time of 45 msec, and first mass at m/z 130 were used. MS spectra were recorded as profile spectra, and MS2 as centroided spectra. Peptides and proteins were assigned using Proteome Discoverer (Thermo Scientific™) and all searches were performed against theHomo sapiensUniProt Reviewed (Swiss-Prot) protein database and Sequest search algorithm, with trypsin selected as the protease, up to 2 missed cleavages considered, and carbamidomethylation (C) set as a fixed modification. Search results were filtered to a 1% false discovery rate (FDR). Statistical Analysis Prism 8.0 software (GraphPad) was used for statistical analysis. For tests involving two groups, unpaired two-tailed Student's t-test was used. For patient samples, appropriate tests were chosen for assumptions of normality. Throughout, error bars depict Standard Error of Mean (SEM). For analysis of 2 groups with repeated measures, 2-way ANOVA was used followed by Sidak's multiple comparison analysis (in vivo assay). P value of <0.05 was considered significant. Example 1—Loss of GCNT2 Correlates with Reduced Patient Survival in MM Patients and Promotes Expression of TIC Marker in MM Cells To assess consequences of GCNT2 expression in MM patients and disease outcome, 64 samples from MM patients that died from melanoma were stained for GCNT2 by immunohistochemistry. Stained slides were grouped into low, medium, and high depending on the GCNT2 staining score (0-3: 0—No staining, 1—Light staining, 2—Moderate staining, and 3—Dark/strong staining). Patients with GCNT2 expression from 0-1 staining level presented with significantly decreased survival compared to patients with high GCNT2 expression (p<0.04) (FIGS.1A and1B). To explore the pathobiological consequences of GCNT2 loss in MM, human MM A375 (low GCNT2 expression) and A2058 (moderate GCNT2 expression) cells engineered to express or silence GCNT2 were utilized. Corresponding overexpressed (OE) or knockdown (KD) GCNT2 variants in A375 and A2058 cells, including their empty vector (EV) and Scrambled control (Scr) variants, respectively, were generated by lentiviral transduction; and GCNT2 expression was validated by RT-qPCR (FIGS.1C and1D). The expression of a common molecular feature of TICs, NGFR/CD271 was first analyzed. GCNT2 OE exhibited significantly decreased (p<0.01), (FIGS.1E and1F), while GCNT2 KD1 and KD2 resulted in increased NGFR expression (p<0.01) (FIGS.1G and1H). To investigate GCNT2's role in TIC generation, in vivo limiting dilution assays were performed in NOD-SCID IL-2Rγ-deficient (NSG) mice using GCNT2-engineered MM cells. A375 GCNT2 OE and A2058 GCNT2 KD cell lines were injected subcutaneously in the flank with cell concentrations ranging from 103to 105cells per mouse along with their respective controls (EV and Scr). Lower GCNT2 expression led to increased growth potential even at 103, while increased GCNT2 expression hindered tumor growth even when 105cells were injected (p<0.01) (FIGS.1I and1J). Together, these results suggest that GCNT2 expression can regulate TIC potential in MM cells. Example 2—Hypoxia Reduces GCNT2 Expression, Enhances TIC Markers and Globally Alters MM Glycobiology Tumor microenvironmental hypoxia is a major factor contributing to the establishment of TICs. MM is a highly hypoxic tumor-type with intratumoral oxygen tension of only 1.5%. To study the role of hypoxia on the MM glycome signature, human MM cells were subjected to acute (24 hrs) and chronic (>48 hours) hypoxia to assess alterations in glycosylation-related genes and their role in TIC marker expression. In addition to significant Gal-8 upregulation, acute hypoxia lowered GCNT2 expression in A375 and A2058 MM cells (p<0.001) along with significant elevations in known TIC markers, KLF4 and NGFR by PT-qPCR (p<0.01) (FIGS.2A and2C). Hypoxia induction was confirmed by elevated HIF1α expression by Western blot analysis (FIGS.2B and2D). Significant elevations in surface expression of Gal-8 and MM glycome i-linear poly-LacNAcs were also evidenced by flow cytometry with anti-i-linear poly-LacNAc moAb Osk28 of MM cells grown under hypoxia (p<0.01) (FIGS.2E,2F,2G and2H). To further assess glyco-structural elevations in i-linear poly-LacNAcs under hypoxia, N-glycans from MM cells cultured under normoxic or hypoxic conditions were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Human MM A375, A2058 and SK-MEL-5 cells grown under normoxia or hypoxia consisted of high mannose (data not shown) and complex N-glycan structures with extensive poly-LacNAc repeating units (reaching up to 11 total LacNAc units; m/z 8093) terminated mainly in N-acetylneuraminic acid (NeuAc) residues and minor antennal fucosylation (FIG.3). Partial annotated MALDI-TOF MS spectra of high mass N-glycans from A375 and A2058 cells under normoxia, however, revealed that A375 cells contained mainly i-linear poly-LacNAcs (FIG.2I), whereas A2058 cells displayed principally I-branched poly-LacNAcs (FIG.2J). These assignments were based on N-glycan molecular ion MALDI-TOF/TOF MS/MS, which helps differentiate i-linear poly-LacNAcs from I-branched poly-LacNAcs. The ratio of relative abundance of fragment ions corresponding to loss of 2 and 3 LacNAc repeats was characteristic of i-linear or I-branched poly-LacNAcs (FIG.2U, Peaks b and c, respectively). A ratio of b/c >1 corresponded mainly to i-linear poly-LacNAcs, while a ratio <1 corresponded to I-branched poly-LacNAcs. This relationship is based on the assumption that fragmentation of i-linear poly-LacNAcs, and therefore the relative abundance of either (2) or (3) LacNAc units, is relatively proportionate (FIG.2U, Peaks b and c, upper panel); whereas fragmentation of I-branched poly-LacNAcs did not result in fragments containing (2) LacNAcs and the relative abundance of the b fragment was much less than fragment c (FIG.2U, Peaks b and c, lower panel). Using this interpretative model, representative N-glycans of A375 and A2058 cells grown under normoxia or hypoxia were contrasted. The molecular ion at m/z 6384 from A375 cells exhibited a relative abundance of fragmented ions corresponding to loss of (2) LacNAc repeats at m/z 5110 that was higher than those ions corresponding to (3) LacNAc repeats (m/z 4661), which was indicative of mainly i-linear poly-LacNAcs (FIG.2K). On the contrary, the same molecular ion from A2058 cells displayed a relative abundance of fragment ion at m/z 5110 that was substantially lower than the relative abundance of ions at m/z 4661, suggesting that the ion at m/z 6384 consisted mainly of I-branched poly-LacNAcs (FIG.2L). Under hypoxia, while LacNAc repeats in poly-LacNAcs per N-glycan antenna was not markedly changed (FIG.3), there was a noted increase in the ratio of i-linear to I-branched poly-LacNAcs at m/z 6384 of A375 cells (FIG.2M). That is, the ratio of relative abundance of fragment ions at m/z 5110 to 4661 was elevated compared with cells grown under normoxia. Similarly, on A2058 cells grown under hypoxia, the ratio of relative abundance of fragment ions at m/z 5110 to 4661 was elevated compared with levels on cells grown under normoxia (FIGS.2L and2N). These flattening of the “V” patterns indicative of increased i-linear poly-LacNAcs were also observed for other N-glycan species and were additionally observed on N-glycan spectra from human MM SK-MEL-5 cells (data not shown). Altogether, these results suggested that hypoxia helps orchestrate signature i-linear poly-LacNAc structures on MM cells. To determine whether hypoxia influenced sialylation on N-glycans, MALDI-TOF MS and MALDI-TOF/TOF MS/MS were conducted on N-glycans digested with α2,3 sialidase-S (Sial-S) from A375 and A2058 cells cultured under normoxia and hypoxia. Cumulative data indicated that there were no major differences in the abundance of α2,3-NeuAc residues and that the majority of the NeuAc residues at the termini of poly-LacNAcs were α2,3-linked NeuAcs (FIG.4). Partial annotated high mass N-glycan spectra after Sial-S digestion from A375 and A2058 cells did not demonstrate any alteration in the abundance of i-linear poly-LacNAcs or I-branched poly-LacNAcs, respectively (FIGS.2O and2P). Furthermore, compared with Sial-S-treated N-glycan at m/z 6287 from cells grown in normoxia (FIGS.2Q and2R), Sial-S-treated N-glycans from cells grown under hypoxia also exhibited increases in relative abundance of i-linear LacNAcs (FIGS.2S and2T). These data suggested that α2,3 sialylation of N-glycans was not affected by hypoxia and did not neutralize hypoxia-dependent induction of i-linear poly-LacNAcs. To assess other MM glycome gene alterations, including glycan-binding lectins and glycan synthesis and degradation pathways induced under hypoxic conditions, human A375, A2058, and SkMel-5 MM cells were cultured under normoxia or hypoxia and analyzed by RNA-sequencing (FIG.5A). Upregulated and downregulated glycome genes shared among all (3) cell lines were observed. Among cancer-associated galectins, galectin (Gal)-8 was the most upregulated galectin under hypoxia. Elevation in Gal-8 expression was confirmed by RT-qPCR (p<0.01) and flow cytometry (p<0.05) (FIGS.5B,5E,5H and5J). Among other key deregulated glycoproteins, NCAM, which is implicated in tumor formation, was increased as confirmed by RT-qPCR and flow cytometry (p<0.01) (FIGS.5C,5F,5I and5K). Alpha1,3 fucosyltransferase 11 (FUT11) was upregulated in MM cells (p<0.01) (FIGS.5D and5G). FUT11 is associated with cancer progression and is expressed upon HIF1α induction under hypoxia. Similarly, while Gal-1 and Gal-3 are well described pro-tumorigenic factors in melanoma progression, Gal-8 has never been associated with MM. However, in other tumor models, Gal-8 has been shown to promote growth and participate in tumor microenvironmental immune escape and metastasis formation, both characteristic of TICs. Example 3—Galectin (Gal)-8 Modulates NGFR Expression in MM Cells and is Elevated in MM Patient Serum With concomitant hypoxia-induced loss of GCNT2 and increase in Gal-8 expression, we assessed whether Gal-8 binding to i-linear poly-LacNAcs was favored over binding to I-branched poly-LacNAcs. Among MM A375 and A2058 cells, A375 expressed significantly more i-linear poly-LacNAcs compared with A2058 cells (p<0.01) (FIG.6A) and binding of recombinant human Gal-8 (rhGal-8) to A375 cells was significantly higher than binding to A2058 cells (p<0.05) (FIG.6B). Furthermore, intracellular staining of endogenous Gal-8 expression illustrated increased Gal-8 expression in A375 cells that had higher i-linear poly-LacNAc expression (p<0.05) (FIG.6C). To investigate whether i-linear poly-LacNAcs encouraged Gal-8 interactions, rhGal-8 was incubated with A375 GCNT2 OE and EV cells as well as A2058 GCNT2 KD and Scr cells. MM cells with low GCNT2 and high i-linear poly-LacNAcs bound Gal-8 to a greater degree compared with binding to cells with high GCNT2 expression (p<0.01 and p<0.05) (FIGS.6D and6E). To elucidate the clinical significance of Gal-8 in MM, patient serum samples (n=13) were analyzed for Gal-8 levels compared with nominal controls by ELISA. There was a significant increase in Gal-8 levels in patient sera (p<0.05) (FIG.6F). To explore the relationship of elevated Gal-8 levels and MM TIC generation, A375 and A2058 MM cell lines were incubated with rhGal-8, and NGFR expression was analyzed by immunoblotting. There was a significant elevation in NGFR in Gal-8-treated cells (FIGS.6G and6H). Furthermore, to determine whether intrinsic Gal-8 could alter NGFR expression, A375 and A2058 cells silenced for Gal-8 expression (KD) by shRNA technology were analyzed by RT-qPCR. Compared with shRNA control cells (Scr), silencing of Gal-8 in Gal-8 KD cells was confirmed by RT-qPCR (FIGS.6I and6J). A375 and A2058 Scr and Gal-8 KD cells were cultured under normoxia and hypoxia and then assessed for NGFR induction. Under hypoxia, while NGFR was elevated in Scr control cells as assayed by RT-qPCR (FIGS.6I and6J) and Western blotting (FIGS.6K,6L,6M and6N), Gal-8 KD cells were unable to upregulate NGFR. These data implicate both hypoxia-dependent and -independent roles of Gal-8 in NGFR regulation. In accordance with downregulation of NGFR by silencing Gal-8, Gal-8 KD cells formed tumors at a significantly less rate than Scr cells in vivo (FIG.6O) (p<0.01). Further, while exogenous Gal-8 significantly upregulated NGFR in Scr cells, incubating Gal-8 with Gal-8 KD MM cells was not able to rescue NGFR expression (FIG.6P), indicating a dual role of Gal-8 as an extracellular (outside-in) and intracellular modulator of NGFR expression. In pan-galectin inhibitor lactose controls, incomplete diminution is often observed in signaling assessments. Hence, while it is an appropriate control for short-term lectin binding assays, lactose may not be the most ideal galectin neutralizer in signaling analyses. Altogether, the data present a novel mechanistic role of Gal-8 in regulating MM TIC marker, NGFR. Example 4—Galectin (Gal)-8 Preferentially Bound i-Linear Poly-LacNAcs on MM Cells and the MM Cell Surface Receptor CD44, and Removal of CD44—N Glycans Ablated Gal-8 Binding To assess whether Gal-8 binding to i-linear poly-LacNAcs was favored over binding to I-branched poly-LacNAcs on MM cells, Gal-8 was incubated with A375 GCNT2 OE and EV cells as well as A2058 GCNT2 KD and Scr cells. MM cells with low GCNT2 and high i-linear poly-LacNAcs bound Gal-8 at a significantly greater degree than to cells with high GCNT2 expression and low i-linear poly-LacNAcs (p<0.05 and p<0.05) (FIGS.7A and7B). Lactose solution was incubated with Gal-8 as negative control for galectin binding. To identify specific glycoprotein ligands of Gal-8 on MM cell surfaces, Gal-8 affinity chromatography followed by mass spectrometry of eluates was performed using both A375 and A2058 MM cell lines (Tables 3 and 4). TABLE 3Top 10 Gal-8 ligands eluted with rhGal-8 from human A375 MM cell lysatesUniProtExp.Coverage### UniqueAccessionDescriptionq-value[%]PeptidesPSMsPeptidesScoreQ6UVK1Chondroitin sulfate proteoglycan 4 OS =Homo sapiens0.00E+0016283928105.9OX = 9606 GN = CSPG4 PE = 1 SV = 2Q6YHK3CD109 antigen OS =Homo sapiensOX = 9606 GN =0.00E+001317241765.76CD109 PE = 1 SV = 2P15144Aminopeptidase N OS =Homo sapiensOX = 9606 GN =0.00E+001917231761.81ANPEP PE = 1 SV = 4P05023Sodium/potassium-transporting ATPase subunit alpha-10.00E+002016201654.97OS =Homo sapiensOX = 9606 GN = ATP1A1 PE = 1SV = 1Q8NFJ5Retinoic acid-induced protein 3 OS =Homo sapiensOX =0.00E+0011512543.79606 GN = GPRC5A PE = 1 SV = 2Q93050V-type proton ATPase 116 kDa subunit a1 OS =Homo0.00E+0014915942sapiensOX = 9606 GN = ATP6V0A1 PE = 1 SV = 3Q15365Poly(rC)-binding protein 1 OS =Homo sapiensOX = 96060.00E+0032814539.3GN = PCBP1 PE = 1 SV = 2P05556Integrin beta-1 OS =Homo sapiensOX = 9606 GN =0.00E+002113151337.96ITGB1 PE = 1 SV = 2P16070CD44 antigen OS =Homo sapiensOX = 9606 GN = CD440.00E+0012813837.46PE = 1 SV = 3Q15366Poly(rC)-binding protein 2 OS =Homo sapiensOX = 96060.00E+0021612332.11GN = PCBP2 PE = 1 SV = 1 TABLE 4Top 10 Gal-8 ligands eluted with rhGal-8 from human A2058 MM cell lysatesUniProtExp.Coverage### UniqueAccessionDescriptionq-value[%]PeptidesPSMsPeptidesScoreP21741Midkine OS =Homo sapiensOX = 9606 GN = MDK0.00E+0039730788.15PE = 1 SV = 1P09429High mobility group protein B1 OS =Homo sapiensOX =0.00E+00347155459606 GN = HMGB1 PE = 1 SV = 3P37802Transgelin-2 OS =Homo sapiensOX = 9606 GN =0.00E+0027513530.49TAGLN2 PE = 1 SV = 3P02765Alpha-2-HS-glycoprotein OS =Homo sapiensOX = 96060.00E+007424467.68GN = AHSG PE = 1 SV = 2Q99497Parkinson disease protein 7 OS =Homo sapiensOX = 96060.00E+0029418449.41GN = PARK7 PE = 1 SV = 2P16070CD44 antigen OS =Homo sapiensOX = 9606 GN = CD440.00E+00749424.4PE = 1 SV = 3P09382Galectin-1 OS =Homo sapiensOX = 9606 GN = LGALS10.00E+0027317349.01PE = 1 SV = 2P51858Hepatoma-derived growth factor OS =Homo sapiensOX =0.00E+0012311327.319606 GN = HDGF PE = 1 SV = 1P27816Microtubule-associated protein 4 OS =Homo sapiensOX =8.00E−03435311.49606 GN = MAP4 PE = 1 SV = 3Q9H910Jupiter microtubule associated homolog 2 OS =Homo1.10E−02223439.91sapiensOX = 9606 GN = JPT2 PE = 1 SV = 1 After eliminating proteins found in lactose control eluates, CD44 was the top membrane glycoprotein identified in both MM cell lines. CD44 is a metastasis-promoting factor that can potentiate disease progression in multiple tumors, including MM. CD44 was subsequently confirmed as a Gal-8 ligand in Gal-8 eluates from A375 and A2058 cell lines (FIGS.7C and7E). Furthermore, treatment with PNGase, which cleaves N-glycans, ablated the capacity of Gal-8 to bind CD44 (FIGS.7D and7F) (p<0.0001), implicating N-glycan-dependency in Gal-8—CD44 binding interactions. To determine whether Gal-8—Gal-8 ligand interactions were affected by I-branched poly-LacNAc expression, A375 GCNT2 OE and A2058 GCNT2 KD cells and their respective controls were used for Gal-8 affinity chromatography. Gal-8 binding to CD44 was significantly greater on cells with low GCNT2 levels and high i-linear poly-LacNAcs than on cells with high GCNT2 levels and high I-branched poly-LacNAcs (p<0.01) (FIGS.7G and7H). To assess whether Gal-8—Gal-8 ligand interactions triggered canonical intracellular signaling, activation of pro-survival molecule, AKT, was analyzed in MM cells with mainly i-linear poly-LacNAc expression (GCNT2 KD). AKT is a downstream effector of CD44 engagement. Gal-8 incubation with A2058 GCNT2 KD cells expressing i-linear poly-LacNAcs increased AKT activation compared with Scr control cells (FIG.7I) (p<0.05). GCNT2 KD cells even had a higher basal level of AKT activation. These data indicate that Gal-8 modulated MM cell signaling, in part, via cell surface expression of i-linear poly-LacNAcs on the principal identified Gal-8 ligand, CD44. Cell surface Gal-8 ligands include α3, α5, and β1 integrins, IL-2Rβ, TGFβ type I receptor, podoplanin, CD166, and CD44. In MM and other cancers, CD44 can potentiate metastasis. CD44 tumor-promoting activity can be transmitted from several key ligands, including hyaluronic acid (HA), E- and L-selectin (a CD44 glycoform known as “HCELL”), Gal-9, osteopontin, matrix metalloproteases, and now Gal-8 on MM cells. CD44 N- and O-glycans and their terminal sialylation play critical roles in promoting or preventing HA-binding or HCELL activity on hematopoietic cells and human colorectal cancer cells. The putative role of MM cell CD44 N-glycan—Gal-8 interactions via i-linear poly-LacNAcs posits another CD44 glycoform that can convey pro-tumorigenic activity, further broadening the importance of post-translational modifications on CD44 in cancer. CD44 has multiple isoforms (>800) due to alternative exon splicing and related variable amounts of N-glycan, 0-glycan and/or heparan sulfate structures. These glycoforms contain critical binding sites for ligand binding, hyaluronic acid (HA), E-selectin, among other lectins (i.e., galectins) and other heterotypic factors. CD44 is alternatively spliced from 10 variant (v) exons that encode extracellular domains in the stalk region proximal the extracellular head region found on all isoforms. The standard CD44 isoform of CD44 (CD44s), which contains the head region and no variable regions, contains (5) potential N-glycan sites, resolves at 75-95 kDa, and is found on several cell types, including fibroblasts and hematopoietic cells. Some of the more common variant isoforms are CD44v3, CD44v6, CD44v10, CD44v3-10, CD44v6-10, CD44E (v8-10) and are found on endothelial cells, epithelial cells, activated lymphocytes, and tumor cells. Western blot data of Gal-8 affinity chromatography eluates show a ˜75 kDa band from human MM cell extracts using a ubiquitous anti-CD44 moAb (Cell Signaling), so there is, at minimum, the CD44s isoform displaying N-glycans for Gal-8-binding. To reveal CD44 isoforms, FACS stain human and mouse melanoma cell lines are performed using commercially-available moAbs to specific CD44v isoforms (Bio-Rad). Negative control blots are conducted using Gal-8 eluates obtained in presence of Gal-8 inhibitory 50 mM lactose and using 800CW or 680RD-2° Ab alone. To analyze dependency of linear or I-branched poly-LacNAcs on identified CD44 isoform for Gal-8-binding, we will similarly conduct Gal-8-purify CD44 from our validated empty vector control or GCNT2 OE human A375 MM and Scr or GCNT2 KD human A2058 MM models and blot with specific anti-CD44 variant Ab. These experiments are done >3-times and blots will be scanned using Li-Cor Odyssey imaging. To ascertain whether other pro-metastatic N-glycan-dependent CD44 receptor/ligands, endothelial (E)-selectin, and HA, compete for or cooperate with Gal-8-binding, individual and combined incubations of parental human/mouse MM cell lines with rhGal-8 (R&D Systems), FITC-HA (Matexcel) and/or rhE-sel-Ig (R&D systems) are conducted. FACS analysis with Abs to Gal-8, FITC, and Ig is used to assess inhibited, enhanced, or unchanged Gal-8 binding. Controls are to elute any pre-bound Gal-8 by pre-treating cells with 50 mM lactose to avoid interference prior to FITC-HA or rhE-sel-Ig incubations. These data reveal whether Gal-8 partners with other MM-associated CD44 receptor/ligands. To determine whether identified Gal-8 ligand CD44 is a major cell surface Gal-8 ligand and directly correlates with expression of MM TIC factor NGFR and the capacity to fat In tumors in vivo, human A375 and A2058 MM cells and mouse YUMM1.1 cells silenced for CD44 expression are generated. Lentiviral particles with scrambled (Scr) or interfering shRNA against CD44 (KD) (GeneCopoeia) are used. Cells are transduced and selected in 1 mg/ml Hygromycin (Corning), and CD44 levels in Scr and CD44 KD cells are validated by RT-qPCR and Western blotting. To assess the contribution of CD44 as a Gal-8 ligand, rhGal-8 binding to Scr and CD44 KD variants is analyzed by FACS, including 50 mM lactose incubations to control for Gal-8 binding. To assess CD44's role in Gal-8-dependent NGFR expression, Scr and CD44 KD A375 and A2058 cells are incubated with rhGal-8 (20 μg/ml; R&D systems) for 16 hr, and NGFR (Ab from Abcam) is analyzed by Western blotting. Control blots include loading controls for β-actin (Abcam). Scr and CD44 KD A375 and A2058 cells incubated with Gal-8 (20 μg/ml) for 15 min and 30 min in serum free media are then assessed. Cell lysates are blotted for phospho-AKT (pS473) and control total AKT (Cell Signaling). This data help correlate with CD44 dependency for Gal-8-induced NGFR and pro-survival factor pAKT. To assess the impact of Gal-8 on melanoma cell Gal-8 ligand CD44, cellular and mouse tools are used in in vivo TIC capacity/limiting dilution assays. WT or lgals8 (Gal-8) KO mice (bred in house) are inoculated with syngeneic Scr or CD44 KD YUMM1.1 variant melanoma cells from 1×103to 1×105into the flank. Tumor volumes are calculated using the formula: [tumor volume (mm3)=(length×(width)2×0.5] every 3 days. All experiments are done >3-times to meet statistical significance. The N-glycans on MM cell CD44 are the key glycan constituents binding Gal-8. To interrogate the poly-LacNAcs on CD44 N-glycan antennae and ascertain using CD44 immunoprecipitated (IP) from high Gal-8-binding (EV) or low Gal-8-binding (GCNT2 OE) A375 MM cells and from low Gal-8-binding (Scr) or high Gal-8-binding (GCNT2 KD) A2058 MM cells, whether GCNT2 directly adds I-branches to CD44 subunit is determined. Using human A375/UACC62 and mouse D4M melanoma cells, including GCNT2 OE and GCNT2 KD cell variants, MALDI-TOF-TOF MS/MS N-glycomic analysis is conducted on anti-CD44 IP for poly-LacNAc analysis. Precise structure assignments can then be attained by GC-MS linkage analysis and exoglycosidase digestions. In addition, selected glycan molecular ion species can be subjected to MS/MS to produce sequence informative fragment ions. The clinical significance of the loss of GCNT2 with melanoma patient prognosis was analyzed. The results show that reduced levels of GCNT2 in MM corresponded with significantly reduced survival. Hence, the expression level of GCNT2 could serve as a prognostic biomarker for disease progression. Other key data from our analysis of GCNT2 impact on MM cell function revealed that reductions in GCNT2/I-branching expression evokes TIC generation, whereas high levels of GCNT2 abolishes classic MM TIC phenotypes, such as NGFR/CD271 marker expression, and the capability to form tumors in in vivo limiting dilution assays. The data here suggest that elevations in i-linear poly-LacNAcs are promoted by hypoxia, lead to increases in TIC characteristics, and in part, correlate with higher disease relapse and patient mortality rates. MM is one of the most hypoxic tumors having an intratumoral oxygen tension of only 1%. Our study here addressed the role of hypoxia in MM glycosylation signature and pathway derangement. Data illustrated here depict global glycome-gene alterations in MM cells under hypoxia. Among the numerous dysregulated glycome-related genes, Gal-8 was found to be a major hypoxia-induced MM factor. Furthermore, GCNT2 was reduced under hypoxia, along with a corresponding gain in i-linear poly-LacNAc expression and TIC marker expression. The observation of increased i-linear poly-LacNAcs using specialized MS/MS glyco-analytics provides strong structural evidence that hypoxia helps promote i-linear poly-LacNAc expression on MM cells. Altogether, these results portray a concerted action of hypoxia on the MM glycome, and, in part, Gal-8 expression in fostering MM TIC characteristics. Gal-8 has never been linked to MM progression. In the present invention, under hypoxia, Gal-8 was the most upregulated galectin. Gal-8 binding to i-linear poly-LacNAcs was greater than binding to I-branched poly-LacNAcs. Given the profound loss of GCNT2 in MM patient samples, Gal-8 may bind these MM cells preferentially and alter cellular signaling. Exogenous rhGal-8 treatment of MM cells resulted in increased MM TIC marker, NGFR, expression. However, the unexpected observation that loss of MM cell Gal-8 prevented the upregulation of NGFR even under hypoxia, a known driver of NGFR expression, strongly indicates dependence of NGFR induction by both exogenous and melanoma cell-intrinsic Gal-8. Since NGFR has been associated with melanoma progression as well as therapy resistance, neutralization of Gal-8 represents a novel therapeutic approach to prevent disease progression. Of particular clinical importance, significant elevations in Gal-8 from MM patient sera were noted compared with controls, suggesting that Gal-8 may be a biomarker of active melanoma. Therefore, in addition to the loss of GCNT2/I-branching in MM cells, increases in Gal-8 serum levels may also represent a direct correlate with melanoma progression. As presented in the studies herein, the mechanistic data of hypoxia-dependent enforcement of the MM signature glycome and the Gal-8-dependent upregulation of pro-MM TIC marker NGFR represent novel pathways in MM that can be exploited for therapeutic exploitation (Illustrated in the cartoon model;FIG.8). Example 5—the Growth- and Metastasis-Promoting Roles of Gal-8 in Melanoma Gal-8 can drive expression of TIC factor, NGFR, and related MM TIC activity in vivo. Moreover, Gal-8 is elevated in melanoma patients compared with normal healthy volunteers and, when Gal-8 is silenced in human A375 MM cells, they form a xenograft at a significantly less rate. These data support subsequent in vivo studies examining the role of Gal-8 promoting growth and metastatic activity of melanoma cells. Elucidating Gal-8's role provides rationale for therapeutic exploitation to complement the promise of ICI therapies and support strategic therapeutic targeting of the MM glycome. Gal-8 silenced (KD) human and murine melanoma cell lines also transduced with luciferase to allow tracking of metastases formation in distant tissue sites have been developed and validated. Plus, Gal-8 KO (B6) mice were obtained to use in syngeneic tumor growth/metastasis assessments and therapeutic anti-PD1 Ab is used in the syngeneic model to assess Gal-8's role in averting immune boosting, anti-tumor activity. To study the MM cell-intrinsic role of Gal-8 in human MM xenograft formation and metastases formation, 6 to 8-week-old NSG mice are used as hosts for luciferase+Scr control and Gal-8 KD human MM (A375/A2058/SKMEL5) tumor growth and metastasis studies. Cell inocula are injected s.c. into flanks at 1×106viable cells/inoculum for growth studies and i.v. at 1×106viable cells/inoculum for experimental metastasis assays. Tumor growth rates and metastasis formation rates (likely in the lung) are monitored via bioluminescence measurements longitudinally. At necropsy (killed with >10% weight loss or s.c. tumor <2 cm3), Gal-8 WT and KD tumors are also be assessed for vascularity using IHC of CD31 (in house), which may be affected by Gal-8 loss. CD31+ vessel cells are counted by microscopy from >6 fields and analyzed for statistical significance. Tumor burden or the relative optical signal intensity from tumor tissue site is plotted over time. Kaplan-Meier curves assessing Survival and Time to Death are conducted. To identify whether Gal-8 expression corresponds with TIC marker NGFR expression and human melanoma growth in vivo, FFPE-tumors (n=8) are prepared and stained with fluorescent-labelled antibodies against Gal-8 and NGFR (in-house). Sections are counterstained with DAPI and scored and assessed for single and dual-stained cells, which reflect TIC capacity. In all, results reveal the MM cell-intrinsic role of Gal-8 in tumor TIC capacity, growth, and metastasis and reveal whether Gal-8 augments melanoma growth and metastasis coincident with TIC capacity and vessel formation. Example 6—Analyze Role of Gal-8 and its Linear Poly-LacNAc Ligands on Melanoma Growth and Metastasis Using Murine Gal-8+/− or GCNT2+/− Melanoma Models and Gal-8 WT/KO Mice To help distinguish the role of host vs. MM cell-intrinsic Gal-8 on melanoma growth and metastasis, a syngeneic mouse model is used. WT(B6) and Gal-8 KO (B6) mice are used as hosts for inocula of murine luciferase+Scr or Gal-8 KD melanoma YUMM1.1 cells. To examine the role of linear (Gal-8 high binding) vs. I-branched (Gal-8 low binding) poly-LacNAc in Gal-8— ligand axis, luciferase+EV/Scr control or GCNT2 OE/KD YUMM1.1 cell variants are inoculate. Since immunity and angiogenesis are also associated with Gal-8, these experiments critically address how Gal-8 may be lowering tumor growth and metastasis. Cell inocula are injected s.c. into flanks at 106viable cells/inoculum for growth studies and i.v. at 106viable cells/inoculum for experimental metastasis assays (n=3). Tumor growth and metastases rates are assayed by bioluminescence. Tumor burden or relative optical signal intensity from tumor tissue site is plotted over time. Kaplan-Meier curves of Survival and Time to Death are generated. At necropsy, vector control, Gal-8 KD, and GCNT2 OE/KD tumors (n=>6/genotype) grown in Gal-8 WT/KO mice are assessed for vascularity using IHC of CD31 (in-house), which may be affected by Gal-8 loss. CD31+ vessels cells are counted by microscopy from >6 fields and analyzed for statistical significance. To assess whether Gal-8 expression fosters melanoma immune evasion, vector control, Gal-8 KD, and GCNT2 OE/KD tumors (n=6/group) grown in Gal-8 WT/KO mice (n=>6/genotype) are collected and tumor-infiltrating lymphocytes (TIL) are analyzed by FACS. After tumor mincing and straining (20 μm) on ice, cells are FACS analyzed with anti-CD45, CD3, CD4, and CD8 Abs. Anti-tumor or immunosuppressive TIL phenotype is further sorted by using antibodies to immune boosting factors, IFN-γ, IL-12, IL-17 and TNF-α, and to immunoregulators, PD1, TGF-β, IL-10, indolamine 2,3 oxidase (IDO), IL-2RA and FoxP3. Overall, these assays address whether Gal-8-dependent modulation of CD4+ or CD8+ effector or exhausted/regulatory T cell subsets. These TIL assays will decipher whether Gal-8 and Gal-8-binding linear poly-LacNAc expression provide superior immune evading properties. Example 7—Analyze In Vivo Efficacy of Anti-PD1 Ab Using Murine Gal-8+/−MM Models in Gal-8 WT/KO Mice Because ICI therapy is one of the most promising approaches to treat MM, efforts are abound to devise complementary methods or biomarker predictors of outcome to be able to synergize ICI therapeutic response and avoid clinical obstacles. To this end, syngeneic MM models are utilized to assay in vivo efficacy of therapeutic anti-PD1 Ab efficacy in the presence or absence of host or MM cell-intrinsic Gal-8. luciferase+ murine control, Gal-8 WT/KD, and GCNT2 OE/KD YUMM1.1 (B6) are implanted into the s.c. flanks of Gal-8 WT/KO (B6) mice and primary and metastatic tumor growth are assayed via bioluminescent optical imaging. Following implantation into s.c. flank, mice are treated with therapeutic InVivoMAb anti-mouse PD-1 (CD279) (Bxcell) (200 μg/mouse; IP every other day for 4-6 weeks until necropsy (2 cm3). At necropsy, tumors (n=6/group) are FACS analyzed for immune cell infiltration, particularly, the ratio of CD4+ or CD8+ T cells: FOXP3+ Treg cells. These data reveal whether host and MM cell intrinsic Gal-8 can impact the anti-melanoma efficacy of anti-PD1 therapy. Importantly, these results highlight whether Gal-8 expression and/or Gal-8-binding inhibiting glycans synthesized by GCNT2 can boost or compromise anti-tumor activity conferred by anti-PD1 therapy. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. These examples should not be construed as limiting. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated within the scope of the invention without limitation thereto. | 98,806 |
11858999 | DETAILED DESCRIPTION OF THE INVENTION The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification etc. Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the specification. See, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual,3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analyses, pharmaceutical preparation, formulation, and delivery and treatment of patients. IL-17A, IL-17F, and IL-17RA The biologic activities of IL-17A and IL-17F are dependent upon IL-17RA, as shown herein using both cells and mice that are genetically deficient in IL-17RA and with neutralizing mAbs (monoclonal antibodies) directed against IL-17RA (see Examples below). “IL-17 receptor A” or “IL-17RA” (interchangeably used herein, as well as IL-17 receptor and IL-17R to refer to the same receptor) as used herein is meant the cell surface receptor and receptor complexes (such as but not limited to IL-17RA-IL-17RC complex), that bind IL-17A and IL-17F and as a result initiates a signal transduction pathway within the cell. IL-17RA proteins may also include variants. IL-17RA proteins may also include fragments, such as the extracellular domain that don't have all or part of the transmembrane and/or the intracellular domain, as well as fragments of the extracellular domain. The cloning, characterization, and preparation of IL-17RA are described, for example, in U.S. Pat. No. 6,072,033, which is incorporated herein by reference in its entirety. The amino acid sequence of the human IL-17RA is shown in SEQ ID NO:430. Soluble forms of huIL-17RA useful in the methods of the present invention include the extracellular domain or the mature form lacking the signal peptide or a fragment of the extracellular domain that retains the capacity to bind IL-17A and/or IL-17F, or a heteromeric version of IL-17A and/or IL-17F. Other forms of IL-17RA include muteins and variants that are at least between 70% and 99% homologous to the native IL-17RA of SEQ ID NO:430 and as described in U.S. Pat. No. 6,072,033, so long as the IL-17RA retains the capacity to bind IL-17A and/or IL-17F, or a heteromeric version of IL-17A and/or IL-17F. The term “IL-17RA” also includes post-translational modifications of the IL-17RA amino acid sequence. Post-translational modifications include, but is not limited to, N- and O-linked glycosylation. IL-17RA Antigen Binding Proteins The present invention provides antigen binding proteins that specifically bind IL-17RA. Embodiments of antigen binding proteins comprise peptides and/or polypeptides (that optionally include post-translational modifications) that specifically bind IL-17RA. Embodiments of antigen binding proteins comprise antibodies and fragments thereof, as variously defined herein, that specifically bind IL-17RA. Aspects of the invention include antibodies that specifically bind to human IL-17RA and inhibit IL-17A and/or IL-17F from binding and activating IL-17RA, or a heteromeric complex of IL-17RA and IL-17RC. Aspects of the invention include antibodies that specifically bind to human IL-17RA and inhibit an IL-17A/IL-17F heteromer from binding and activating IL-17RA, or a heteromeric complex of IL-17RA and IL-17RC. Throughout the specification, when reference is made to inhibiting IL-17A and/or IL-17F, it is understood that this also includes inhibiting heteromers of IL-17A and IL-17F. Aspects of the invention include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming cither a homomeric or heteromeric functional receptor complex, such as, but not limited to, an IL-17RA-IL-17RC complex. Aspects of the invention include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming either a homomeric or heteromeric functional receptor complex, such as, but not limited to IL-17RA/IL-17RC complex and do not necessarily inhibit IL-17A and/or IL-17F or an IL-17A/IL-17F heteromer from binding to IL-17RA or a IL-17RA heteromeric receptor complex. The antigen binding proteins of the invention specifically bind to IL-17RA. “Specifically binds” as used herein means that the antigen binding protein preferentially binds IL-17RA over other proteins. In some embodiments “specifically binds” means that the IL-17RA antigen binding proteins have a higher affinity for IL-17RA than for other proteins. For example, the equilibrium dissociation constant is <10−7to 10−11M. or <10−8to <10−10M, or <10−9to <10−10M. It is understood that when reference is made to the various embodiments of the IL-17RA antibodies described herein, that it also encompasses IL-17RA-binding fragments thereof. An IL-17RA-binding fragment comprises any of the antibody fragments or domains described herein that retains the ability to specifically bind to IL-17RA. Said IL-17RA-binding fragments may be in any of the scaffolds described herein. Said IL-17RA-binding fragments also have the capacity to inhibit activation of the IL-17RA, as described throughout the specification. In embodiments where the IL-17RA antigen binding protein is used for therapeutic applications, one characteristic of an IL-17RA antigen binding protein is that it can inhibit binding of IL-17A and/or IL-17F to IL-17RA and one or more biological activities of, or mediated by, IL-17RA. Such antibodies are considered neutralizing antibodies because of their capacity to inhibit IL-17A and/or IL-17F from binding and causing IL-17RA signaling and/or biological activity. In this case, an antigen binding protein specifically binds IL-17RA and inhibits binding of IL-17A and/or IL-17F to IL-17RA from anywhere between 10 to 100%, such as by at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more (for example by measuring binding in an in vitro competitive binding assay as described herein). For example, IL-17RA antibodies may be tested for neutralizing ability by testing them for the production of IL-6 in human foreskin fibroblast (HFF) assay (see for example Examples 8 and 9), or any suitable assay known in the art. Examples, for illustrative purposes only, of additional biological activity of IL-17RA (e.g., assay readouts) to test for inhibition of IL-17RA signaling and/or biological activity include in vitro and/or in vivo measurement of one or more of IL-8, CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-1β, TNFα, RANK-L, LIF, PGE2, IL-12, MMPs (such as but not limited to MMP3 and MMP9), GROα, NO, and/or C-telopeptide and the like. Embodiments of antigen binding proteins comprise a scaffold structure, as variously define herein, with one or more complementarity determining regions (CDRs). Embodiments of antigen binding proteins comprise a scaffold structure with one or more variable domains, cither heavy or light. Embodiments include antibodies that comprise a light chain variable region selected from the group consisting of AML1 through AML26 (SEQ ID NO:27-53, respectively, with AML23 having two versions—SEQ ID NOs:49 and 50) and/or a heavy chain variable region selected from the group consisting of AMH1 through AMH26 (SEQ ID NO:1-26, respectively), and fragments, derivatives, muteins, and variants thereof. Additional examples of scaffolds that are envisioned include: fibronectin, neocarzinostatin CBM4-2, lipocalins, T-cell receptor, protein-A domain (protein Z), Im9, TPR proteins, zinc finger domains, pVIII, avian pancreatic polypeptide, GCN4, WW domain, Src homology domain 3, PDZ domains. TEM-1 Beta-lactamase, thioredoxin, staphylococcal nuclease, PHD-finger domains, CL-2, BPTI, APPI, HPSTI, ecotin, LACI-D1, LDTI, MTI-II, scorpion toxins, insect defensin-A peptide, EETI-II, Min-23, CBD, PBP, cytochrome b-562, Ld1 receptor domains, gamma-crystallin, ubiquitin, transferring, and/or C-type lectin-like domains. Aspects of the invention include antibodies comprising the following variable domains: AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1), AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2), AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3), AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4), AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5), AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6), AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7), AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8), AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9), AML10/AMH10 (SEQ ID NO:36/SEQ ID NO: 10), AML11/AMH11 (SEQ ID NO:37/SEQ ID NO:11), AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12), AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13), AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14), AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15), AML6/AMH16 (SEQ ID NO:42/SEQ ID NO:16), AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17). AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18), AML19/AMH19 (SEQ ID NO:45/SEQ ID NO: 19), AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20), AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21), AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22), AML23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23), AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24), AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25), AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26), and combinations thereof, as well as and fragments, derivatives, muteins, and variants thereof. In a further embodiment, a first amino acid sequence comprises CDR3, CDR2, and CDR1, and a second amino acid sequence comprises a CDR3, CDR2, and CDR1 of TABLE 1. In another embodiment, the antigen binding protein comprises: A) a heavy chain amino acid sequence that comprises at least one H-CDR1, H-CDR2, or H-CDR3 of a sequence selected from the group consisting of SEQ ID NO: 1-26; and/or B) a light chain amino acid sequence that comprises at least one L-CDR1, L-CDR2, or L-CDR3 of a sequence selected from the group consisting of SEQ ID NO:27-53. In a further variation, the antigen binding protein comprises A) a heavy chain amino acid sequence that comprises a H-CDR1, a H-CDR2, and a H-CDR3 of any of SEQ ID NO:1-26, and B) a light chain amino acid sequence that comprises a L-CDR1, a L-CDR2, and a L-CDR3 of any of SEQ ID NO:27-53. In another variation, the antigen binding protein comprises an amino acid sequence that is of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1-26 or a light chain amino acid sequence selected from the group consisting of SEQ ID NO:27-53. In certain embodiments, the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions from a H-CDR1 (i.e., CDR1 of the heavy chain, etc.). H-CDR2, H-CDR3, L-CDR1 (i.e., CDR1 of the light chain, etc.). L-CDR2, and L-CDR3, and fragments, derivatives, muteins, and variants thereof. Aspects of the invention include antibodies comprising a heavy chain variable region selected from the group consisting of SEQ ID NO:1-26. Aspects of the invention include antibodies comprising a light chain variable region selected from the group consisting of SEQ ID NO:27-53. Aspects of the invention include antibodies comprising a heavy chain variable region selected from the group consisting of SEQ ID NO:1-26 having no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions. Aspects of the invention include antibodies comprising a light chain variable region selected from the group consisting of SEQ ID NO:27-53 having no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions. Aspects of the invention include antibodies comprising a heavy chain variable region selected from the group consisting of SEQ ID NO:1-26 having no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions and a light chain variable region selected from the group consisting of SEQ ID NO:27-53 having no more than one, two, three, four, five, or six amino acid additions, deletions, or substitutions. In other embodiments, the heavy and light chain variable domains of the antigen binding proteins are defined by having a certain percent identity to a reference heavy and/or light chain variable domain. For example, the antigen binding protein comprises A) a heavy chain variable domain amino acid that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:1-26; and B) a light chain variable domain amino acid that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a light chain amino acid sequence selected from the group consisting of SEQ ID NOs:27-53. Aspects of the invention include a variety of embodiments including, but not limited to, the following exemplary embodiments: Embodiment 1: an isolated antibody, comprising a monoclonal antibody or IL-17 receptor A binding fragment thereof that is not fully murine and that specifically binds IL-17 receptor A and inhibits IL-17A from binding and activating said receptor. Embodiment 2: the antibody of embodiment 1, wherein said antibody further inhibits IL-17F from binding and activating said receptor. Embodiment 3: the antibody of embodiment 1, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 4: the antibody of embodiment 3, wherein said antibody comprises an amino acid sequence selected from the group consisting of:A. a. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML1-26 (SEQ ID NOs:27-53, respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH1-26 (SEQ ID NOs:1-26, respectively); orc. the light chain variable domain of (a) and the heavy chain variable domain of (b); andB. a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, CDR3 that differs by no more than a total of three amino acid additions, substitutions, and/or deletions in each CDR from the following sequences:a. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO: 186), CDR3 (SEQ ID NO:187) and a heavy chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO: 112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO:113), CDR2 (SEQ ID NO:114), CDR3 (SEQ ID NO: 115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO: 196) and a heavy chain CDR1 (SEQ ID NO:116), CDR2 (SEQ ID NO:117), CDR3 (SEQ ID NO: 118) of antibody AM-4;e. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO:127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO: 148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO: 152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO:178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; orz.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO: 183), CDR3 (SEQ ID NO:184) of antibody AM-26; wherein said antibody specifically binds IL-17 receptor A. Embodiment 5: the antibody of embodiment 4, wherein said antibody comprises an amino acid sequence selected from the group consisting of:a. a light chain variable domain and a heavy chain variable domain of AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1);b. a light chain variable domain and a heavy chain variable domain of AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2);c. a light chain variable domain and a heavy chain variable domain of AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3);d. a light chain variable domain and a heavy chain variable domain of AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4);e. a light chain variable domain and a heavy chain variable domain of AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5);f. a light chain variable domain and a heavy chain variable domain of AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6)g. a light chain variable domain and a heavy chain variable domain of AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7);h. a light chain variable domain and a heavy chain variable domain of AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8);i. a light chain variable domain and a heavy chain variable domain of AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9);j. a light chain variable domain and a heavy chain variable domain of AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10);k. a light chain variable domain and a heavy chain variable domain of AML11/AMH11 (SEQ ID NO:37/SEQ ID NO: 11);l. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12);m. a light chain variable domain and a heavy chain variable domain of AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13);n. a light chain variable domain and a heavy chain variable domain of AML14% AMH14 (SEQ ID NO:40/SEQ ID NO:14);o. a light chain variable domain and a heavy chain variable domain of AML5/AMH15 (SEQ ID NO:41/SEQ ID NO:15);p. a light chain variable domain and a heavy chain variable domain of AML16/AMH6 (SEQ ID NO:42/SEQ ID NO:16);q. a light chain variable domain and a heavy chain variable domain of AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17);r. a light chain variable domain and a heavy chain variable domain of AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18);s. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);t. a light chain variable domain and a heavy chain variable domain of AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20);u. a light chain variable domain and a heavy chain variable domain of AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21);v. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22);w. a light chain variable domain and a heavy chain variable domain of AML23/AMH23 (SEQ ID NO: 49 or SEQ ID NO:50/SEQ ID NO:23);x. a light chain variable domain and a heavy chain variable domain of AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24);y. a light chain variable domain and a heavy chain variable domain of AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25); andz. a light chain variable domain and a heavy chain variable domain of AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26); wherein said antibody specifically binds IL-17 receptor A. Embodiment 6: the antibody of embodiment 4, wherein said antibody comprises an amino acid sequence selected from the group consisting of:a. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO:187) and a heavy chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO: 112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO: 113), CDR2 (SEQ ID NO: 114), CDR3 (SEQ ID NO: 115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and a heavy chain CDR1 (SEQ ID NO:116), CDR2 (SEQ ID NO:117), CDR3 (SEQ ID NO: 118) of antibody AM-4;e. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO: 198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO:119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO: 127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO: 154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO: 158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO:178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; orz.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO:183), CDR3 (SEQ ID NO:184) of antibody AM-26; wherein said antibody specifically binds IL-17 receptor A. Embodiment 7: the antibody of embodiment 2, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 8: the antibody of embodiment 7, wherein said antibody comprises an amino acid sequence selected from the group consisting of:A. a. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML14, 18, 19, and 22 (SEQ ID NOs: 40, 44, 45, and 48, respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH14, 18, 19, and 22 (SEQ ID NOs:14, 18, 19, and 22, respectively); orc. the light chain variable domain of (a) and the heavy chain variable domain of (b);B. a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, CDR3 that differs by no more than a total of three amino acid additions, substitutions, and/or deletions in each CDR from the following sequences:a. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;b. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;c. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19; ord. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22; andC. a. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO: 14);b. a light chain variable domain and a heavy chain variable domain of AML8/AMH18 (SEQ ID NO:44/SEQ ID NO:18);c. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19); ord. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22); wherein said antibody specifically binds IL-17 receptor A. Embodiment 9: an isolated antibody, or an IL-17 receptor A binding fragment thereof, comprisinga. a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of:i. X1YGIS (SEQ ID NO:453), wherein X1is selected from the group consisting of R, S and G;b. a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of:i. WISX1YX2GNTX3YAQX4X5QG (SEQ ID NO:456), wherein X1is selected from the group consisting of A, X2is selected from the group consisting of N, S and K, X3is selected from the group consisting of N and K, X4is selected from the group consisting of K and N, and X5is selected from the group consisting of L and F;c. a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of:i. X1QLX2X3DY (SEQ ID NO:459), wherein X1is selected from the group consisting of R and K, X2is selected from the group consisting of Y, V, and A, and X3is selected from the group consisting of F and L;ii. X1QLX2FDY (SEQ ID NO:460), wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V;d. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of:i. RASQSX1X2X3X4LA (SEQ ID NO:462), wherein X1is selected from the group consisting of V and I, X2is selected from the group consisting of I and S, X3is selected from the group consisting of S and T, X4is selected from the group consisting of N and S, and X5is selected from the group consisting of A and N, andii. RASQSX1SSNLA (SEQ ID NO:471), wherein X1is selected from the group consisting of V and I;e. a light chain CDR2 comprising an amino acid sequence selected from the group consisting of:i. X1X2STRAX3(SEQ ID NO:466), wherein X1is selected from the group consisting of G and D, X2is selected from the group consisting of A and T, and X3is selected from the group consisting of T and A, andii. X1ASTRAX2(SEQ ID NO:472), wherein X1is selected from the group consisting of G and D, and X2is selected from the group consisting of A and T; andf. a light chain CDR3 comprising an amino acid sequence selected from the group consisting of:i. QQYDX1WPLT (SEQ ID NO:469) wherein X1is selected from the group consisting of N, T, and I; wherein said antibody specifically binds IL-17 receptor A. Embodiment 10: the antibody of embodiment 9, wherein said antibody comprises:a. a heavy chain CDR1 amino acid sequence comprising X1YGIS, wherein X1is selected from the group consisting of R, S and G;b. a heavy chain CDR2 amino acid sequence comprisingWISX1YX2GNTX3YAQX4X5QG (SEQ ID NO:456) wherein X1is selected from the group consisting of A, X2is selected from the group consisting of N, S and K, X3is selected from the group consisting of N and K, X4is selected from the group consisting of K and N, and X5is selected from the group consisting of L and F;c. a heavy chain CDR3 amino acid sequence comprising X1QLX2FDY (SEQ ID NO:460), wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V;d. a light chain CDR1 amino acid sequence comprising RASQSX1SSNLA (SEQ ID NO:471), wherein X1is selected from the group consisting of V and I;e. a light chain CDR2 amino acid sequence comprising X1ASTRAX2(SEQ ID NO:472), wherein X1is selected from the group consisting of G and D, and X2is selected from the group consisting of A and T; andf. a light chain CDR3 amino acid sequence comprising QQYDX1WPLT (SEQ ID NO:469), wherein X1is selected from the group consisting of N, T, and I; wherein said antibody specifically binds IL-17 receptor A. Embodiment 11: the antibody of embodiment 9, wherein said antibody comprises an amino acid sequence selected from the group consisting of:A. a. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML12, 14, 16, 17, 19, and 22 (SEQ ID NOs:38, 40, 42, 43, 45, and 48 respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH12, 14, 16, 17, 19, and 22 (SEQ ID NOs:12, 14, 16, 17, 19, and 22, respectively); orc. the light chain variable domain of (a) and the heavy chain variable domain of (b);B. a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, CDR3 that differs by no more than a total of three amino acid additions, substitutions, and/or deletions in each CDR from the following sequences:a. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;b. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;c. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;d. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;c. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19; orf. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22; andC. a. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12);b. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);c. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);d. a light chain variable domain and a heavy chain variable domain of AML17 AMH17 (SEQ ID NO:43/SEQ ID NO:17);e. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);c. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22); wherein said antibody specifically binds IL-17 receptor A. Embodiment 12: a pharmaceutical composition, comprising the antibody of embodiment 4. Embodiment 14: the antibody of embodiment 4, wherein said antibody is a derivative of said antibody. Embodiment 15: a polypeptide, comprising an amino acid sequence selected from the group consisting of:A. a. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML1-26 (SEQ ID NOs:27-53, respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH1-26 (SEQ ID NOs:1-26, respectively); orc. the light chain variable domain of (a) and the heavy chain variable domain of (b); andB. a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, CDR3 that differs by no more than a total of three amino acid additions, substitutions, and/or deletions in each CDR from the following sequences:a. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO:187) and a heavy chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO: 112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO: 113), CDR2 (SEQ ID NO: 114), CDR3 (SEQ ID NO: 115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and a heavy chain CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO:118) of antibody AM-4;c. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO:127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO: 137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO: 169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO: 173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO:178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; orz.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO:183), CDR3 (SEQ ID NO:184) of antibody AM-26; wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 16: the polypeptide of embodiment 15, wherein said polypeptide comprises an amino acid is selected from the group consisting of:a. a light chain variable domain and a heavy chain variable domain of AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1);b. a light chain variable domain and a heavy chain variable domain of AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2);c. a light chain variable domain and a heavy chain variable domain of AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3);d. a light chain variable domain and a heavy chain variable domain of AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4);e. a light chain variable domain and a heavy chain variable domain of AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5);f. a light chain variable domain and a heavy chain variable domain of AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6)g. a light chain variable domain and a heavy chain variable domain of AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7);h. a light chain variable domain and a heavy chain variable domain of AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8);i. a light chain variable domain and a heavy chain variable domain of AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9);j. a light chain variable domain and a heavy chain variable domain of AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10);k. a light chain variable domain and a heavy chain variable domain of AML11/AMH11 (SEQ ID NO:37/SEQ ID NO: 11);l. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12);m. a light chain variable domain and a heavy chain variable domain of AML13/AMH3 (SEQ ID NO:39/SEQ ID NO:13);n. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);o. a light chain variable domain and a heavy chain variable domain of AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15);p. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);q. a light chain variable domain and a heavy chain variable domain of AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17);r. a light chain variable domain and a heavy chain variable domain of AML18/AMH18 (SEQ ID NO:44/SEQ ID NO: 18).s. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);t. a light chain variable domain and a heavy chain variable domain of AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20);u. a light chain variable domain and a heavy chain variable domain of AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21);v. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22);w. a light chain variable domain and a heavy chain variable domain of AML23/AMH23 (SEQ ID NO: 49 or SEQ ID NO:50/SEQ ID NO:23);x. a light chain variable domain and a heavy chain variable domain of AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24);y. a light chain variable domain and a heavy chain variable domain of AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25); andz. a light chain variable domain and a heavy chain variable domain of AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26); wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 17: the polypeptide of embodiment 15, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of:a. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO: 187) and a heavy chain CDR1 (SEQ ID NO: 107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO: 110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO:112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO: 113), CDR2 (SEQ ID NO: 114), CDR3 (SEQ ID NO: 115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and a heavy chain CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO:118) of antibody AM-4;c. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO:127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO: 139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO: 143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO: 176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO:178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; orz.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO:183), CDR3 (SEQ ID NO:184) of antibody AM-26; wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 18: the polypeptide of embodiment 15, wherein said polypeptide is a pharmaceutical composition. Embodiment 19: an isolated antibody, selected from the group consisting of:a) an antibody consisting of a heavy chain sequence of SEQ ID NO:427 and a light chain sequence of SEQ ID NO:429;b) an antibody consisting essentially of a heavy chain sequence of SEQ ID NO:427 and a light chain sequence of SEQ ID NO:429;c) an antibody comprising a heavy chain sequence of SEQ ID NO: 427;d) an antibody comprising a light chain sequence of SEQ ID NO:429;e) an antibody comprising a heavy chain sequence of SEQ ID NO: 427 and a light chain sequence of SEQ ID NO:429;f) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain sequence of SEQ ID NO: 427;g) an antibody or an IL-17 receptor A binding fragment thereof comprising a light chain sequence of SEQ ID NO:429;h) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain sequence of SEQ ID NO:427 and a light chain sequence of SEQ ID NO:429;i) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain variable region sequence of SEQ ID NO:14;j) an antibody or an IL-17 receptor A binding fragment thereof comprising a light chain variable region sequence of SEQ ID NO:40;k) an antibody or an IL-17 receptor A binding fragment thereof comprising a light chain variable region sequence of SEQ ID NO:40 and a heavy chain variable region sequence of SEQ ID NO:14;l) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain CDR1 of SEQ ID NO: 146, a heavy chain CDR2 of SEQ ID NO:147, a heavy chain CDR3 of SEQ ID NO:148, a light chain CDR1 of SEQ ID NO:224, a light chain CDR2 of SEQ ID NO:225, and a light chain CDR3 of SEQ ID NO:226; andm) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain CDR3 of SEQ ID NO: 148 and a light chain CDR3 of SEQ ID NO:226. Embodiment 20: the antibody of embodiment 19, wherein said antibody is a pharmaceutical composition. Embodiment 21: the antibody of embodiment 19, wherein said antibody is a derivative of said antibody. Embodiment 22: the antibody of embodiment 7, wherein said antibody comprises an amino acid sequence selected from the group consisting of:A. a. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence SEQ ID NO: 40;b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of SEQ ID NO:14; orc. the light chain variable domain of (a) and the heavy chain variable domain of (b);B. a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, CDR3 that differs by no more than a total of three amino acid additions, substitutions, and/or deletions in each CDR from the following sequences: CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO: 146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148); andC. a light chain variable domain of SEQ ID NO:40 and a heavy chain variable domain SEQ ID NO:14; wherein said antibody specifically binds IL-17 receptor A. Embodiment 23: the polypeptide of embodiment 16, wherein said polypeptide comprises a light chain variable domain of SEQ ID NO:40 and a heavy chain variable domain SEQ ID NO:14, wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 24: the polypeptide of embodiment 16, wherein said polypeptide comprises SEQ ID NO:427 and SEQ ID NO:429, wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 25: the polypeptide of embodiment 24, wherein said polypeptide is a pharmaceutical composition. As a general structure, the antigen binding proteins of the invention comprise (a) a scaffold, and (b) one or a plurality of CDRs. A “complementary determining region” or “CDR,” as used herein, refers to a binding protein region that constitutes the major surface contact points for antigen binding. Embodiments of the invention include one or more CDRs embedded in a scaffold structure of the antigen binding protein. The scaffold structure of the antigen binding proteins may be the framework of an antibody, or fragment or variant thereof, or may be completely synthetic in nature. Examples of various scaffold structures of the antigen binding proteins of the invention are further described hereinbelow. The antigen binding proteins of the invention include scaffold regions and one or more CDRs. An antigen binding protein of the invention may have between one and six CDRs (as typically do naturally occurring antibodies), for example, one heavy chain CDR1 (“H-CDR1”), and/or one heavy chain CDR2 (“H-CDR2”), and/or one heavy chain CDR3 (“H-CDR3”), and/or one light chain CDR1 (“L-CDR1”), and/or one light chain CDR2 (“L-CDR2”), and/or one light chain CDR3 (“L-CDR3”). The term “naturally occurring” as used throughout the specification in connection with biological materials such as peptides, polypeptides, nucleic acids, host cells, and the like, refers to materials which are found in nature. In naturally occurring antibodies, a H-CDR1 typically comprises about five (5) to about seven (7) amino acids, H-CDR2 typically comprises about sixteen (16) to about nineteen (19) amino acids, and H-CDR3 typically comprises about three (3) to about twenty five (25) amino acids. L-CDR1 typically comprises about ten (10) to about seventeen (17) amino acids, L-CDR2 typically comprises about seven (7) amino acids, and L-CDR3 typically comprises about seven (7) to about ten (10) amino acids. Specific CDRs of the various antibodies of the invention are provided in TABLE 1 and the Sequence Listing. TABLE 1CorrespondingPoly-nucleotideSequenceAmino acidSEQ IDNYYWNSEQ ID NO: 266sequence ofNO: 107CDR 1 ofAMH1 VhAmino acidSEQ IDDIYYSGSTNYNPSEQ ID NO: 267sequence ofNO: 108SLKSCDR 2 ofAMH1 VhAmino acidSEQ IDDGELANYYGSGSEQ ID NO: 268sequence ofNO: 109SYQFYYYYCIMCDR 3 ofDVAMH1 VhAmino acidSEQ IDGYYWSSEQ ID NO: 269sequence ofNO: 110CDR 1 ofAMH2 VhAmino acidSEQ IDEINHSGRTNYNPSEQ ID NO: 270sequence ofNO: 111SLKSCDR 2 ofAMH2 VhAmino acidSEQ IDGPYYFDSSGYLSEQ ID NO: 271sequence ofNO: 112YYYYGLDVCDR 3 ofAMH2 VhAmino acidSEQ IDSYGMHSEQ ID NO: 272sequence ofNO: 113CDR ofAMH3 VhAmino acidSEQ IDVIWYDGSNKHYSEQ ID NO: 273sequence ofNO: 114ADSVKGCDR 2 ofAMH3 VhAmino acidSEQ IDDTGVYSEQ ID NO: 274sequence ofNO: 115CDR 3 ofAMH3 VhAmino acidSEQ IDSWATHSEQ ID NO: 275sequence ofNO: 116CDR 1 ofAMH4 VhAmino acidSEQ IDVIWYDGSNKHYSEQ ID NO: 276sequence ofNO: 117ADSVKGCDR 2 ofAMH4 VhAmino acidSEQ IDDTGVYSEQ ID NO: 277sequence ofNO: 118CDR 3 ofAMH4 VhAmino acidSEQ IDSYYWSSEQ ID NO: 278sequence ofNO: 119CDR 1 ofAMH5 VhAmino acidSEQ IDRIYRSGNTIYNPSEQ ID NO: 279sequence ofNO: 120SLKSCDR 2 ofAMH5 VhAmino acidSEQ IDENYSESSGLNYSEQ ID NO: 280sequence ofNO: 121YYGMDVCDR 3 ofAMH5 VhAmino acidSEQ IDRYGISSEQ ID NO: 281sequence ofNO: 122CDR 1 ofAMH6 VhAmino acidSEQ IDWISAYNGNTNYSEQ ID NO: 282sequence ofNO: 123AQKLQGCDR 2 ofAMH6 VhAmino acidSEQ IDRDYDILTGYYNSEQ ID NO: 283sequence ofNO: 124GFDPCDR 3 ofAMH6 VhAmino acidSEQ IDRYGISSEQ ID NO: 284sequence ofNO: 125CDR 1 ofAMH7 VhAmino acidSEQ IDWISAYNGNTNYSEQ ID NO: 285sequence ofNO: 126AQKLQGCDR 2 ofAMH7 VhAmino acidSEQ IDRDYDILTGYYNSEQ ID NO: 286sequence ofNO: 127GFDPCDR 3 ofAMH7 VhAmino acidSEQ IDGYGISSEQ ID NO: 287sequence ofNO: 128CDR 1 ofAMH8 VhAmino acidSEQ IDWISAYNGNTNYSEQ ID NO: 288sequence ofNO: 129AQNLQGCDR 2 ofAMH8 VhAmino acidSEQ IDRDYDILTGYYNSEQ ID NO: 289sequence ofNO: 130GFDPCDR 3 ofAMH8 VhAmino acidSEQ IDRYGISSEQ ID NO: 290sequence ofNO: 131CDR 1 ofAMH9 VhAmino acidSEQ IDWISAYNGNTNYSEQ ID NO: 291sequence ofNO: 132AQKLQGCDR 2 ofAMH9 VhAmino acidSEQ IDRDYDILTGYYNSEQ ID NO: 292sequence ofNO: 133GFDPCDR 3 ofAMH9 VhAmino acidSEQ IDSGGYYWSSEQ ID NO: 293sequence ofNO: 134CDR 1 ofAMH10 VhAmino acidSEQ IDYIYFSGSAYYNPSEQ ID NO: 294sequence ofNO: 135SLKSCDR 2 ofAMH10 VhAmino acidSEQ IDEYYDSSGYPDASEQ ID NO: 295sequence ofNO: 136FDICDR 3 ofAMH10 VhAmino acidSEQ IDSYGMHSEQ ID NO: 296sequence ofNO: 137CDR 1 ofAMH11 VhAmino acidSEQ IDVIWYDGSNKYYSEQ ID NO: 297sequence ofNO: 138ADSVKGCDR 2 ofAMH11 VhAmino acidSEQ IDDTKDYSEQ ID NO: 298sequence ofNO: 139CDR 3 ofAMH11 VhAmino acidSEQ IDSYGISSEQ ID NO: 299sequence ofNO: 140CDR 1 ofAMH12 VhAmino acidSEQ IDWISTYKGNTNYSEQ ID NO: 300sequence ofNO: 141AQKLQGCDR 2 ofAMH12 VhAmino acidSEQ IDKQLVFDYSEQ ID NO: 301sequence ofNO: 142CDR 3 ofAMH12 VhAmino acidSEQ IDSYGMQSEQ ID NO: 302sequence ofNO: 143CDR 1 ofAMH13 VhAmino acidSEQ IDVIWYDGNKKYSEQ ID NO: 303sequence ofNO: 144YADSVKGCDR 2 ofAMH13 VhAmino acidSEQ IDGRVRDYYYGMSEQ ID NO: 304sequence ofNO: 145DVCDR 3 ofAMH13 VhAmino acidSEQ IDRYGISSEQ ID NO: 305sequence ofNO: 146CDR 1 ofAMH14 VhAmino acidSEQ IDWISTYSGNTNYSEQ ID NO: 306sequence ofNO: 147AQKLQGCDR 2 ofAMH14 VhAmino acidSEQ IDRQLYFDYSEQ ID NO: 307sequence ofNO: 148CDR 3 ofAMH14 VhAmino acidSEQ IDSYGMQSEQ ID NO: 308sequence ofNO: 149CDR 1 ofAMH15 VhAmino acidSEQ IDVIWYDGNKKYSEQ ID NO: 309sequence ofNO: 150YADSVKGCDR 2 ofAMH15 VhAmino acidSEQ IDGRVRDYYYGMSEQ ID NO: 310sequence ofNO: 151DVCDR 3 ofAMH15 VhAmino acidSEQ IDSYGISSEQ ID NO: 311sequence ofNO: 152CDR 1 ofAMH16 VhAmino acidSEQ IDWISAYNGNTKYSEQ ID NO: 312sequence ofNO: 153AQKLQGCDR 2 ofAMH16 VhAmino acidSEQ IDKQLVFDYSEQ ID NO: 313sequence ofNO: 154CDR 3 ofAMH16 VhAmino acidSEQ IDSYGISSEQ ID NO: 314sequence ofNO: 155CDR 1 ofAMH17 VhAmino acidSEQ IDWISAYSGNTKYSEQ ID NO: 315sequence ofNO: 156AQKLQGCDR 2 ofAMH17 VhAmino acidSEQ IDKQLVFDYSEQ ID NO: 316sequence ofNO: 157CDR 3 ofAMH17 VhAmino acidSEQ IDDYYMHSEQ ID NO: 317sequence ofNO: 158CDR 1 ofAMH18 VhAmino acidSEQ IDWMHPNSGGTDSEQ ID NO: 318sequence ofNO: 159LAQRFQGCDR 2 ofAMH18 VhAmino acidSEQ IDGGYCSTLSCSFYSEQ ID NO: 319sequence ofNO: 160WYFDLCDR 3 ofAMH18 VhAmino acidSEQ IDSYGISSEQ ID NO: 320sequence ofNO: 161CDR 1 ofAMH19 VhAmino acidSEQ IDWISAYSGNTKYSEQ ID NO: 321sequence ofNO: 162AQKFQGCDR 2 ofAMH19 VhAmino acidSEQ IDRQLALDYSEQ ID NO: 322sequence ofNO: 163CDR 3 ofAMH19 VhAmino acidSEQ IDSYSMNSEQ ID NO: 323sequence ofNO: 164CDR 1 ofAMH20 VhAmino acidSEQ IDFISARSSTIYYASEQ ID NO: 324sequence ofNO: 165DSVKGCDR 2 ofAMH20 VhAmino acidSEQ IDPKVGGGMDVSEQ ID NO: 325sequence ofNO: 166CDR 3 ofAMH20 VhAmino acidSEQ IDSYSMNSEQ ID NO: 326sequence ofNO: 167CDR 1 ofAMH21 VhAmino acidSEQ IDIISSRSSIIHYADSSEQ ID NO: 327sequence ofNO: 168VKGCDR 2 ofAMH21 VhAmino acidSEQ IDPKVGGGMDVSEQ ID NO: 328sequence ofNO: 169CDR 3 ofAMH21 VhAmino acidSEQ IDRYGISSEQ ID NO: 329sequence ofNO: 170CDR 1 ofAMH22 VhAmino acidSEQ IDWISAYSGNTNYSEQ ID NO: 330sequence ofNO: 171AQKLQGCDR 2 ofAMH22 VhAmino acidSEQ IDRQLYFDYSEQ ID NO: 331sequence ofNO: 172CDR 3 ofAMH22 VhAmino acidSEQ IDSYYWSSEQ ID NO: 332sequence ofNO: 173CDR 1 ofAMH23 VhAmino acidSEQ IDRIYPSGRTNYNPSEQ ID NO: 333sequence ofNO: 174SLKSCDR 2 ofAMH23 VhAmino acidSEQ IDEAYELQLGLYYSEQ ID NO: 334sequence ofNO: 175YYGMDVCDR 3 ofAMH23 VhAmino acidSEQ IDSYYWSSEQ ID NO: 335sequence ofNO: 176CDR 1 ofAMH24 VhAmino acidSEQ IDRIYPSGRTNYNPSEQ ID NO: 336sequence ofNO: 177SLKSCDR 2 ofAMH24 VhAmino acidSEQ IDEAYELQLGLYYSEQ ID NO: 337sequence ofNO: 178YYGMDVCDR 3 ofAMH24 VhAmino acidSEQ IDSGGYYWSSEQ ID NO: 338sequence ofNO: 179CDR 1 ofAMH25 VhAmino acidSEQ IDYSGNTYYNPSLSEQ ID NO: 339sequence ofNO: 180RSCDR 2 ofAMH25 VhAmino acidSEQ IDEAGGNSAYYYGSEQ ID NO: 340sequence ofNO: 181MDVCDR 3 ofAMH25 VhAmino acidSEQ IDDYYMSSEQ ID NO: 341sequence ofNO: 182CDR 1 ofAMH26 VhAmino acidSEQ IDYISSSGSTIYYASEQ ID NO: 342sequence ofNO: 183DSVKGCDR 2 ofAMH26 VhAmino acidSEQ IDDRTYYFGSGSYSEQ ID NO: 343sequence ofNO: 184EGMDVCDR 3 ofAMH26 VhAmino acidSEQ IDRASQGIRNDLGSEQ ID NO: 345sequence ofNO: 185CDR 1 ofAML1 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 346sequence ofNO: 186CDR 2 ofAML1 VlAmino acidSEQ IDLQHNSNPFTSEQ ID NO: 347sequence ofNO: 187CDR 3 ofAML1 VlAmino acidSEQ IDRASQSVSRNLVSEQ ID NO: 348sequence ofNO: 188CDR 1 ofAML2 VlAmino acidSEQ IDGASTRANSEQ ID NO: 349sequence ofNO: 189CDR 2 ofAML2 VlAmino acidSEQ IDQQYKSWRTSEQ ID NO: 350sequence ofNO: 190CDR 3 ofAML2 VlAmino acidSEQ IDRASQSISSYLNSEQ ID NO: 351sequence ofNO: 191CDR 1 ofAML3 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 352sequence ofNO: 192CDR 2 ofAML3 VlAmino acidSEQ IDQQSYSTPFTSEQ ID NO: 353sequence ofNO: 193CDR 3 ofAML3 VlAmino acidSEQ IDRASQSVSRNLASEQ ID NO: 354sequence ofNO: 194CDR 1 ofAML4 VlAmino acidSEQ IDGASTRATSEQ ID NO: 355sequence ofNO: 195CDR 2 ofAML4 VlAmino acidSEQ IDQQYNNWPTWTSEQ ID NO: 356sequence ofNO: 196CDR 3 ofAML4 VlAmino acidSEQ IDRASQGIRNDLGSEQ ID NO: 357sequence ofNO: 197CDR 1 ofAML5 VlAmino acidSEQ IDAASSFQSSEQ ID NO: 358sequence ofNO: 198CDR 2 ofAML5 VlAmino acidSEQ IDLQHNSYPPTSEQ ID NO: 359sequence ofNO: 199CDR 3 ofAML5 VlAmino acidSEQ IDRASQGIRNDLGSEQ ID NO: 360sequence ofNO: 200CDR 1 ofAML6 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 361sequence ofNO: 201CDR 2 ofAML6 VlAmino acidSEQ IDLQHKSYPLTSEQ ID NO: 362sequence ofNO: 202CDR 3 ofAML6 VlAmino acidSEQ IDRASQGIRNDLGSEQ ID NO: 363sequence ofNO: 203CDR 1 ofAML7 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 364sequence ofNO: 204CDR 2 ofAML7 VlAmino acidSEQ IDLQHKSYPLTSEQ ID NO: 365sequence ofNO: 205CDR 3 ofAML7 VlAmino acidSEQ IDRASQGIRNDLGSEQ ID NO: 366sequence ofNO: 206CDR 1 ofAML8 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 367sequence ofNO: 207CDR 2 ofAML8 VlAmino acidSEQ IDLQHKSYPLTSEQ ID NO: 368sequence ofNO: 208CDR 3 ofAML8 VlAmino acidSEQ IDRASQGIRNDLGSEQ ID NO: 369sequence ofNO: 209CDR 1 ofAML9 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 370sequence ofNO: 210CDR 2 ofAML9 VlAmino acidSEQ IDLQHKSYPLTSEQ ID NO: 371sequence ofNO: 211CDR 3 ofAML9 VlAmino acidSEQ IDRASQGIRSWLASEQ ID NO: 372sequence ofNO: 212CDR 1 ofAML10 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 373sequence ofNO: 213CDR 2 ofAML10 VlAmino acidSEQ IDQQANNFPRTSEQ ID NO: 374sequence ofNO: 214CDR 3 ofAML10 VlAmino acidSEQ IDRASQSVSSNLASEQ ID NO: 375sequence ofNO: 215CDR 4 ofAML11 VlAmino acidSEQ IDGASTRAASEQ ID NO: 376sequence ofNO: 216CDR 2 ofAML11 VlAmino acidSEQ IDQHYINWPKWTSEQ ID NO: 377sequence ofNO: 217CDR 3 ofAML11 VlAmino acidSEQ IDRASQSISSSLASEQ ID NO: 378sequence ofNO: 218CDR 1 ofAML12 VlAmino acidSEQ IDGASTRATSEQ ID NO: 379sequence ofNO: 219CDR 2 ofAML12 VlAmino acidSEQ IDQQYDNWPLTSEQ ID NO: 380sequence ofNO: 220CDR 3 ofAML12 VlAmino acidSEQ IDKSSQSLLHSDGSEQ ID NO: 381sequence ofNO: 22IKTYLYCDR 1 ofAML13 VlAmino acidSEQ IDEVSTRFSSEQ ID NO: 382sequence ofNO: 222CDR 2 ofAML13 VlAmino acidSEQ IDMQSIQLPLTSEQ ID NO: 383sequence ofNO: 223CDR 3 ofAML13 VlAmino acidSEQ IDRASQSVSSNLASEQ ID NO: 384sequence ofNO: 224CDR 1 ofAML14 VlAmino acidSEQ IDDASTRATSEQ ID NO: 385sequence ofNO: 225CDR 2 ofAML14 VlAmino acidSEQ IDQQYDNWPLTSEQ ID NO: 386sequence ofNO: 226CDR 3 ofAML14 VlAmino acidSEQ IDRASQSVSSNLASEQ ID NO: 387sequence ofNO: 227CDR 1 ofAML15 VlAmino acidSEQ IDDASTRAASEQ ID NO: 388sequence ofNO: 228CDR 2 ofAML15 VlAmino acidSEQ IDQQYDNWPLTSEQ ID NO: 389sequence ofNO: 229CDR 3 ofAML15 VlAmino acidSEQ IDRASQSISTSLASEQ ID NO: 390sequence ofNO: 230CDR 1 ofAML16 VlAmino acidSEQ IDGTSTRATSEQ ID NO: 391sequence ofNO: 231CDR 2 ofAML16 VlAmino acidSEQ IDQQYDIWPLTSEQ ID NO: 392sequence ofNO: 232CDR 3 ofAML16 VlAmino acidSEQ IDRASQSVSSNLASEQ ID NO: 393sequence ofNO: 233CDR 1 ofAML17 VlAmino acidSEQ IDGASTRATSEQ ID NO: 394sequence ofNO: 234CDR 2 ofAML17 VlAmino acidSEQ IDQQYDNWPLTSEQ ID NO: 395sequence ofNO: 235CDR 3 ofAML17 VlAmino acidSEQ IDKTSQSVLYSSKSEQ ID NO: 396sequence ofNO: 236NKNFLACDR 1 ofAML18 VlAmino acidSEQ IDWASTRESSEQ ID NO: 397sequence ofNO: 237CDR 2 ofAML18 VlAmino acidSEQ IDQQYYSTPFTSEQ ID NO: 398sequence ofNO: 238CDR 3 ofAML18 VlAmino acidSEQ IDRASQSISSNLASEQ ID NO: 399sequence ofNO: 239CDR 1 ofAML19 VlAmino acidSEQ IDGASTRATSEQ ID NO:400sequence ofNO: 240CDR 2 ofAML19 VlAmino acidSEQ IDQQYDTWPLTSEQ ID NO: 401sequence ofNO: 241CDR 3 ofAML19 VlAmino acidSEQ IDRASQGISNYLASEQ ID NO: 402sequence ofNO: 242CDR 1 ofAML20 VlAmino acidSEQ IDAASTLQSSEQ ID NO: 403sequence ofNO: 243CDR 2 ofAML20 VlAmino acidSEQ IDQKYNRAPFTSEQ ID NO: 404sequence ofNO: 244CDR 3 ofAML20 VlAmino acidSEQ IDRASQGISNYLASEQ ID NO: 405sequence ofNO: 245CDR 1 ofAML21 VlAmino acidSEQ IDAASTLQSSEQ ID NO: 406sequence ofNO: 246CDR 2 ofAML21 VlAmino acidSEQ IDQKYNRAPFTSEQ ID NO: 407sequence ofNO: 247CDR 3 ofAML21 VlAmino acidSEQ IDRASQSVSSNLASEQ ID NO: 408sequence ofNO: 248CDR 1 ofAML22 VlAmino acidSEQ IDDASTRAASEQ ID NO: 409sequence ofNO: 249CDR 2 ofAML22 VlAmino acidSEQ IDQQYDNWPLTSEQ ID NO: 410sequence ofNO: 250CDR 3 ofAML22 VlAmino acidSEQ IDRASQGIINDLGSEQ ID NO: 411sequence ofNO: 251CDR 1 ofAML23 Vlversion 1Amino acidSEQ IDAASSLQSSEQ ID NO: 412sequence ofNO: 252CDR 2 ofAML23 Vlversion 1Amino acidSEQ IDLQHNSYPPTSEQ ID NO: 413sequence ofNO: 253CDR 3 ofAML23 Vlversion 1Amino acidSEQ IDRSSQSLVYSDGSEQ ID NO: 414sequence ofNO: 254HTCLNCDR 1 ofAML23 Vlversion 2Amino acidSEQ IDKVSNWDSSEQ ID NO: 415sequence ofNO: 255CDR 2 ofAML23 Vlversion 2Amino acidSEQ IDMQGTHWYLCSSEQ ID NO: 416sequence ofNO: 256CDR 3 ofAML23 Vlversion 2Amino acidSEQ IDRSSQSLVYSDGSEQ ID NO: 417sequence ofNO: 257HTCLNCDR 1 ofAML24 VlAmino acidSEQ IDKVSNWDSSEQ ID NO: 418sequence ofNO: 258CDR 2 ofAML24 VlAmino acidSEQ IDMQGTHWPLCSSEQ ID NO: 419sequence ofNO: 259CDR 3 ofAML24 VlAmino acidSEQ IDRASQAISIYLASEQ ID NO: 420sequence ofNO: 260CDR 1 ofAML25 VlAmino acidSEQ IDAASSLQSSEQ ID NO: 421sequence ofNO: 261CDR 2 ofAML25 VlAmino acidSEQ IDQQYSSYPRTSEQ ID NO: 422sequence ofNO: 262CDR 3 ofAML25 VlAmino acidSEQ IDRASQSVYSNLASEQ ID NO: 423sequence ofNO: 263CDR 1 ofAML26 VlAmino acidSEQ IDGASTRATSEQ ID NO: 424sequence ofNO: 264CDR 2 ofAML26 VlAmino acidSEQ IDQQYYNWPWTSEQ ID NO: 425sequence ofNO: 265CDR 3 ofAML26 Vl The general structure and properties of CDRs within naturally occurring antibodies have been described in the art. Briefly, in a traditional antibody scaffold, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions largely responsible for antigen binding and recognition. A variable region comprises at least three heavy or light chain CDRs, see, supra (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al. 1991, supra; see also Chothia and Lesk, 1987, supra). See, infra. The CDRs provided by the present invention, however, may not only be used to define the antigen binding domain of a traditional antibody structure, but may be embedded in a variety of other scaffold structures, as described herein. Antibodies of the invention can comprise any constant region known in the art. The light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region. In another embodiment, the invention provides an antigen binding protein that specifically binds IL-17RA, wherein said antigen binding protein comprises a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, and CDR3 that differs by no more than a total of one, two, three, four, five, or six amino acid additions, substitutions, and/or deletions from the following CDR sequences: CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO:187) and heavy chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1; light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO: 111), CDR3 (SEQ ID NO:112) of antibody AM-2; light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and heavy chain CDR1 (SEQ ID NO:113), CDR2 (SEQ ID NO:114), CDR3 (SEQ ID NO: 115) of antibody AM-3; light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and heavy chain CDR1 (SEQ ID NO:116), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO: 118) of antibody AM-4; light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO: 198), CDR3 (SEQ ID NO:199) and heavy chain CDR1 (SEQ ID NO:119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5; light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and heavy chain CDR1 (SEQ ID NO: 122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6; light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and heavy chain CDR1 (SEQ ID NO: 125), CDR2 (SEQ ID NO: 126), CDR3 (SEQ ID NO:127) of antibody AM-7; light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and heavy chain CDR1 (SEQ ID NO: 128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8; light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9; light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO: 136) of antibody AM-10; light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11; light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and heavy chain CDR1 (SEQ ID NO: 140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12; light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO: 144), CDR3 (SEQ ID NO:145) of antibody AM-13; light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO: 147), CDR3 (SEQ ID NO:148) of antibody AM-14; light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15; light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16; light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17; light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18; light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19; light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20; light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO: 168), CDR3 (SEQ ID NO: 169) of antibody AM-21; light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22; light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23; light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and heavy chain CDR1 (SEQ ID NO: 173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23; light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and heavy chain CDR1 (SEQ ID NO: 176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO:178) of antibody AM-24; light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO: 180), CDR3 (SEQ ID NO:181) of antibody AM-25; or light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO:183), CDR3 (SEQ ID NO:184) of antibody AM-26, and fragments, derivatives, muteins, and variants thereof. The CDRs of the invention also include consensus sequences derived from groups of related monoclonal antibodies. The antibodies may be related by both sequence homology and function, as shown in the Examples. As described herein, a “consensus sequence” refers to amino acid sequences having conserved amino acids common among a number of sequences and variable amino acids that vary within given amino acid sequences. The CDR consensus sequences of the invention include CDRs corresponding to each of H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3. Consensus sequences were determined using standard phylogenic analyses of the CDRs corresponding to the VH (i.e., Variable Heavy, etc.) & VL of anti-IL-17RA antibodies. Two different approaches were employed. In a first approach, the consensus sequences were determined by keeping the CDRs contiguous within the same sequence corresponding to a VH or VL. In a second approach, the consensus sequences were determined by aligning the various types of CDRs, i.e., H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2 and L-CDR3 sequences of the IL-17RA antigen binding proteins disclosed herein independently. In the first approach, briefly, amino acid sequences corresponding to the entire variable domains of either VH or VL were convened to FASTA formatting for ease in processing comparative alignments and inferring phylogenies. Next, framework regions of these sequences were replaced with an artificial linker sequence (GGGAAAGGGAAA, SEQ ID NO:448) so that examination of the CDRs alone could be performed without introducing any amino acid position weighting bias due to coincident events (e.g., such as unrelated antibodies that serendipitously share a common germline framework heritage) whilst still keeping CDRs contiguous within the same sequence corresponding to a VH or VL, VH or VL sequences of this format were then subjected to sequence similarity alignment interrogation using a program that employs a standard ClutalW-like algorithm (see, Thompson et al., 1994, Nucleic Acids Res.22:4673-4680). A gap creation penalty of 8.0 was employed along with a gap extension penalty of 2.0. This program likewise generated phylograms (phylogenic tree illustrations) based on sequence similarity alignments using either UPGMA (unweighted pair group method using arithmetic averages) or Neighbor-Joining methods (see, Saitou and Nei, 1987, Molecular Biology and Evolution4:406-425) to construct & illustrate similarity and distinction of sequence groups via branch length comparison and grouping. Both methods produced similar results but UPGMA-derived trees were ultimately used as the method employs a simpler and more conservative set of assumptions. UPGMA-derived trees are shown inFIG.1where similar groups of sequences were defined as having fewer than 15 substitutions per 100 residues (see legend in tree illustrations for scale) amongst individual sequences within the group and were used to define consensus sequence collections. The original sequence alignments generated were employed to empirically examine and document the occurrence of amino acids tolerated at each position with a consensus group and are shown inFIGS.2and3. Consensus sequences for the groups of similar sequences within each CDR were then prepared. Amino acids that varied within each group were noted with the notation Xnwithin each consensus sequence. The H-CDR1 consensus sequences include amino acid sequences selected from the group consisting of: a) X1YGIS (SEQ ID NO:453), wherein X1is selected from the group consisting of R, S and G; b) X1YX2MX3(SEQ ID NO:454), wherein X1is selected from the group consisting of D and S; X2is selected from the group consisting of Y and S; and X3is selected from the group consisting of S and N; and c) SYGMX1(SEQ ID NO:455), wherein X1is selected from the group consisting of H and Q; The H-CDR2 consensus sequences include amino acid sequence selected from the group consisting of: a) WISX1YX2GNTX3YAQX4X5QG (SEQ ID NO:456), wherein X1is selected from the group consisting of A and T; X2is selected from the group consisting of N. S and K; X3is selected from the group consisting of N and K; X4is selected from the group consisting of K and N; and X5is selected from the group consisting of L and F; b) X1X2SX3X4X5SX6IX7YADSVKG (SEQ ID NO:457), wherein X1is selected from the group consisting of Y, I and F; X2is selected from the group consisting of I and S; X3is selected from the group consisting of S and A; X4is selected from the group consisting of S and R; and X5is selected from the group consisting of G, S and no amino acid; X6is selected from the group consisting of T and I; and X7is selected from the group consisting of Y and H; and c) VIWYDGX1X2KX3YADSVKG (SEQ ID NO:458), wherein X1is selected from the group consisting of S and N; X2is selected from the group consisting of N and K; and X3is selected from the group consisting of H and Y. The H-CDR3 consensus sequences include amino acid sequence selected from the group consisting of: a) X1QLX2X3DY (SEQ ID NO:459), wherein X1is selected from the group consisting of R and K, X2is selected from the group consisting of Y, V, and A, and X3is selected from the group consisting of F and L and b) X1QLX2FDY (SEQ ID NO:460), wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V. The L-CDR1 consensus sequence includes an amino acid sequence selected from the group consisting of: a) RASQX1X2X3X4LX5(SEQ ID NO:461), wherein X1is selected from the group consisting of G, S, and A; X2is selected from the group consisting of R and S; X3is selected from the group consisting of S, I and N; X4is selected from the group consisting of W and Y; and X5is selected from the group consisting of A and N; b) RASQSX1X2X3X4LA (SEQ ID NO:462), wherein X1is selected from the group consisting of V and I; X2is selected from the group consisting of I and S; X3is selected from the group consisting of S and T; X4is selected from the group consisting of N and S; and X5is selected from the group consisting of A and N; and c) RASQSVX1X2NLX3(SEQ ID NO:463), wherein X1is selected from the group consisting of Y and S; X2is selected from the group consisting of S and R; and X3is selected from the group consisting of A and V. The L-CDR2 consensus sequence includes an amino acid sequence selected from the group consisting of: a) AASSX1QS (SEQ ID NO:464), wherein X1is selected from the group consisting of L and F; b) AASX1LQS (SEQ ID NO:465), wherein X1is selected from the group consisting of S and T; c) X1X2STRAX3(SEQ ID NO:466), wherein X1is selected from the group consisting of G and D; X2is selected from the group consisting of A and T; and X3is selected from the group consisting of T and A; and d) GASTRAX1(SEQ ID NO:473), wherein X1is selected from the group consisting of A, T and N. The L-CDR3 consensus sequences include amino acid sequences selected from the group consisting of: a) LQHX1SYX2X3T (SEQ ID NO:467), wherein X1is selected from the group consisting of K and N; X2is selected from the group consisting of P and N; and X3is selected from the group consisting of L, F and P; b) QX1X2X3X4X5PX6T (SEQ ID NO:468), wherein X1is selected from the group consisting of Q and K; X2is selected from the group consisting of A, S and Y; X3is selected from the group consisting of N, Y and S; X4is selected from the group consisting of N, S and R; X5is selected from the group consisting of F, T. Y and A; and X6is selected from the group consisting of R and F; c) QQYDX1WPLT (SEQ ID NO:469), wherein X1is selected from the group consisting of N, T and I; and d) QX1YX2X3WX4X5X6T (SEQ ID NO:470), wherein X1is selected from the group consisting of H and Q; X2is selected from the group consisting of I, Y, N and K; X3is selected from the group consisting of N and S; X4is selected from the group consisting of P and R; X5is selected from the group consisting of K, no amino acid, and T; and X6is selected from the group consisting of W and no amino acid. FIGS.1,2,3,16A,16B,19, and22show that a clear pattern in the data exists between sequence homology in the CDR domains and the antibodies function, as determined by cross-competition binning and the determination of where the antibodies bound to IL-17RA. Thus, a structure/function relation for classes of antibodies has been established for the IL-17RA antibodies provided herein. In a second approach CDR consensus sequences were determined for each separate CDR, independently of their contiguous context within the same sequence corresponding to a VH or VL. In this approach the consensus sequences were determined by aligning each H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 in groups, i.e., by aligning the individual H-CDR1 sequences of the IL-17RA antigen binding proteins disclosed herein to determine a H-CDR1 consensus sequence, by aligning the individual H-CDR2 sequences of the IL-17RA antigen binding proteins disclosed herein to determine a H-CDR2 consensus sequence, by aligning the individual H-CDR3 sequences of the IL-17RA antigen binding proteins disclosed herein to determine a H-CDR3 consensus sequence, by aligning the individual L-CDR1 sequences of the UL-17RA antigen binding proteins disclosed herein to determine a L-CDR1 consensus sequence, by aligning the individual L-CDR2 sequences of the IL-17RA antigen binding proteins disclosed herein to determine a L-CDR2 consensus sequence, and by aligning the individual L-CDR3 sequences of the IL-17RA antigen binding proteins disclosed herein to determine a L-CDR3 consensus sequence. Similarities between sequences within each individual CDR sequences were identified. Consensus sequences for the groups of similar sequences within each CDR were then prepared. Amino acids that varied within each group were noted with the notation Xnwithin each consensus sequence. In another embodiment, the invention provides an antigen binding protein that specifically binds IL-17RA, wherein said antigen binding protein comprises at least one H-CDR region of any of SEQ ID NOs:107-184. Other embodiments include antigen binding proteins that specifically bind to IL-17RA, wherein said antigen binding protein comprises at least one L-CDR region of any of SEQ ID NOs:185-265. Other embodiments include antigen binding proteins that specifically binds IL-17RA, wherein said antigen binding protein comprises at least one H-CDR region of any of SEQ ID NOs:107-184 and at least one L-CDR region of any of SEQ ID NOs:185-265. In another embodiment, the invention provides an antigen binding protein that specifically binds IL-17RA, wherein said antigen binding protein comprises at least two H-CDR regions of any of SEQ ID NOs:107-184. Other embodiments include antigen binding proteins that specifically bind to IL-17RA, wherein said antigen binding protein comprises at least two L-CDR region of any of SEQ ID NOs:185-265. Other embodiments include antigen binding proteins that specifically binds IL-17RA, wherein said antigen binding protein comprises at least two H-CDR region of any of SEQ ID NOs:107-184 and at least two L-CDR region of any of SEQ ID NOs:185-265. In another embodiment, the invention provides an antigen binding protein that specifically binds IL-17RA, wherein said antigen binding protein comprises at least three H-CDR regions of any of SEQ ID NOs:107-184. Other embodiments include antigen binding proteins that specifically bind to IL-17RA, wherein said antigen binding protein comprises at least three L-CDR region of any of SEQ ID NOs:185-265. Other embodiments include antigen binding proteins that specifically binds IL-17RA, wherein said antigen binding protein comprises at least three H-CDR region of any of SEQ ID NOs:107-184 and at least three L-CDR region of any of SEQ ID NOs:185-265. In another embodiment, the invention provides an antigen binding protein that specifically binds IL-17RA, wherein said antigen binding protein comprises at least one, two, or three 1H-CDR regions of any of SEQ ID NOs:107-184, wherein said H-CDR regions are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective H-CDR. Other embodiments include antigen binding proteins that specifically bind to IL-17RA, wherein said antigen binding protein comprises at least one, two, or three L-CDR region of any of SEQ ID NOs:185-265, wherein said L-CDR regions are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective L-CDR. Other embodiments include antigen binding proteins that specifically binds IL-17RA, wherein said antigen binding protein comprises at least one, two, or three H-CDR regions of any of SEQ ID NOs:107-184, wherein said H-CDR regions are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective H-CDR, and comprises at least one, two, or three L-CDR region of any of SEQ ID NOs:185-265, wherein said L-CDR regions are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective L-CDR. In another embodiment, the invention provides an antigen binding protein that binds IL-17RA, wherein said antigen binding protein comprises at least one H-CDR region having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of any of SEQ ID NOs:107-184 and/or at least one L-CDR region having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of any of SEQ ID NOs:185-265. In another embodiment, the invention provides an antigen binding protein that binds IL-17RA, wherein said antigen binding protein comprises one, two, or three H-CDR region having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of any of SEQ ID NOs:107-184 and/or one, two, or three L-CDR region having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of any of SEQ ID NOs:185-265. Additional embodiments utilize antigen binding proteins comprising one CDR having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of the sequence selected from the H-CDR regions of any of SEQ ID NOs:107-184 and a L-CDR region having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of any of SEQ ID NOs:185-265 (e.g., the antigen binding protein has two CDR regions, one H-CDR and one L-CDH. A specific embodiment includes antigen binding proteins comprising both a H-CDR3 and a L-CDR3 region. As will be appreciated by those in the art, for any antigen binding protein comprising more than one CDR from the sequences provided herein, any combination of CDRs independently selected from the CDR in TABLE 1 sequences is useful. Thus, antigen binding proteins comprising one, two, three, four, five, or six independently selected CDRs can be generated. However, as will be appreciated by those in the art, specific embodiments generally utilize combinations of CDRs that are non-repetitive, e.g., antigen binding proteins are generally not made with two H-CDR2 regions, etc. In some embodiments, antigen binding proteins are generated that comprise no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of a H-CDR3 region and a L-CDR3 region, particularly with the H-CDR3 region being selected from a sequence having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of a H-CDR3 region of any of SEQ ID NOs:107-184 and the L-CDR3 region being selected from a L-CDR3 consensus sequence having no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of a L-CDR3 region of any of SEQ ID SEQ ID NOs:185-265. As noted herein, the antigen binding proteins of the present invention comprise a scaffold structure into which the CDR(s) of the invention may be grafted. The genus of IL-17RA antigen binding proteins comprises the subgenus of antibodies, as variously defined herein. Aspects include embodiments wherein the scaffold structure is a traditional, tetrameric antibody structure. Thus, the antigen binding protein combinations described herein include the additional components (framework, J and D regions, constant regions, etc.) that make up a heavy and/or light chain. Embodiments include the use of human scaffold components. An exemplary embodiment of a VH variable region grafted into a traditional antibody scaffold structure is depicted in SEQ ID NO:427 and an exemplary embodiment of a VL variable region grafted into a traditional antibody scaffold structure is depicted in SEQ ID NO:429. Of course it is understood that any antibody scaffold known in the art may be employed. In one aspect, the present invention provides antibodies that comprise a light chain variable region selected from the group consisting of AML1 through AML26 and/or a heavy chain variable region selected from the group consisting of AMH1 through AMH26, and fragments, derivatives, muteins, and variants thereof. Antibodies of the invention include, but are not limited to: antibodies comprising AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1), AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2), AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3), AML4/AML4 (SEQ ID NO:30/SEQ ID NO:4), AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5), AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6), AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7), AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8), AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9), AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10), AML11/AML11 (SEQ ID NO:37/SEQ ID NO: 11), AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12), AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13), AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14), AML15/AMH15 (SEQ ID NO:41/SEQ ID NO: 15), AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16), AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17), AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18), AML19/AMH19 (SEQ ID NO:45/SEQ ID NO: 19), AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20), AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21), AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22), AML23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23), AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24), AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25), AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26), as well as IL-17RA-binding fragments thereof and combinations thereof. In one embodiment, the present invention provides an antibody comprising a light chain variable domain comprising a sequence of amino acids that differs from the sequence of a light chain variable domain selected from the group consisting of AML1 through AML26 only at 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues, wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light-chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 800%, 810%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a light chain variable domain selected from the group consisting of AML1 through AML26. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to a nucleotide sequence that encodes a light chain variable domain selected from the group consisting of AML1 through AML26. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide that encodes a light chain variable domain selected from the group consisting of AML1 through AML26. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide that encodes a light chain variable domain selected from the group consisting of AML1 through AML26. In another embodiment, the light chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complement of a light chain polynucleotide provided in any one of AML1 through AML26 polynucleotide sequences (SEQ ID NOs:80-106). In another embodiment, the present invention provides an antibody comprising a heavy chain variable domain comprising a sequence of amino acids that differs from the sequence of a heavy chain variable domain selected from the group consisting of AMH1 through AMH26 only at 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue(s), wherein each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a heavy chain variable domain selected from the group consisting of AMH1 through AMH26. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence that encodes a heavy chain variable domain selected from the group consisting of AMH1 through AMH26. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent or stringent conditions to the complement of a polynucleotide that encodes a heavy chain variable domain selected from the group consisting of AMH1 through AMH26. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent conditions to the complement of a polynucleotide that encodes a heavy chain variable domain selected from the group consisting of AMH1 through AMH26. In another embodiment, the heavy chain variable domain comprises a sequence of amino acids that is encoded by a polynucleotide that hybridizes under moderately stringent or stringent conditions to a complement of a heavy chain polynucleotide provided in any one of AMH1 through AMH26 polynucleotide sequences (SEQ ID NOs:54-79). Accordingly, in various embodiments, the antigen binding proteins of the invention comprise the scaffolds of traditional antibodies, including human and monoclonal antibodies, bispecific antibodies, diabodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each, respectively. The above described CDRs and combinations of CDRs may be grafted into any of the following scaffolds. As used herein, the term “antibody” refers to the various forms of monomeric or multimeric proteins comprising one or more polypeptide chains that specifically binds to an antigen, as variously described herein. In certain embodiments, antibodies are produced by recombinant DNA techniques. In additional embodiments, antibodies are produced by enzymatic or chemical cleavage of naturally occurring antibodies. In another aspect, the antibody is selected from the group consisting of: a) a human antibody; b) a humanized antibody; c) a chimeric antibody; d) a monoclonal antibody; e) a polyclonal antibody; f) a recombinant antibody; g) an antigen-binding antibody fragment; h) a single chain antibody; i) a diabody; j) a triabody; k) a tetrabody; l) a Fab fragment; m) a F(ab′)2fragment; n) an IgD antibody; o) an IgE antibody; p) an IgM antibody; q) an IgA antibody; r) an IgG1 antibody; s) an IgG2 antibody; t) an IgG3 antibody; and u) an IgG4 antibody. A variable region comprises at least three heavy or light chain CDRs, see, supra (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature342: 877-883), embedded within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see also Chothia and Lesk, 1987, supra). See, infra. Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2. Embodiments of the invention include all such classes of antibodies that incorporate the variable domains or the CDRs of the antigen binding proteins, as described herein. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about twelve (12) or more amino acids, with the heavy chain also including a “D” region of about ten (10) more amino acids. See, generally, Paul, W., ed., 1989, Fundamental Immunology Ch. 7, 2nd ed. Raven Press. N.Y. The variable regions of each light/heavy chain pair form the antibody binding site. Scaffolds of the invention include such regions. Some naturally occurring antibodies, for example found in camels and llamas, are dimers consisting of two heavy chain and include no light chains. Muldermans et al., 2001, J. Biotechnol.74:277-302; Desmyter et al., 2001, J. Biol. Chem.276:26285-26290. Crystallographic studies of a camel antibody have revealed that the CDR3 regions form a surface that interacts with the antigen and thus is critical for antigen binding like in the more typical tetrameric antibodies. The invention encompasses dimeric antibodies consisting of two heavy chains, or fragments thereof, that can bind to and/or inhibit the biological activity of IL-17RA. The variable regions of the heavy and light chains typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, i.e., the complementarity determining regions or CDRs. The CDRs are the hypervariable regions of an antibody (or antigen binding protein, as outlined herein), that are responsible for antigen recognition and binding. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest. Chothia et al., 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature342:878-883. Scaffolds of the invention include such regions. CDRs constitute the major surface contact points for antigen binding. See, e.g., Chothia and Lesk, 1987, J. Mol. Biol.196:901-917. Further, CDR3 of the light chain and, especially, CDR3 of the heavy chain may constitute the most important determinants in antigen binding within the light and heavy chain variable regions. See, e.g., Chothia and Lesk, 1987, supra; Desiderio et al., 2001, J. Mol. Biol.310:603-615; Xu and Davis, 2000, Immunity13:37-45; Desmyter et al., 2001, J. Biol. Chem. 276:26285-26290; and Muyldermans, 2001, J. Biotechnol.74:277-302. In some antibodies, the heavy chain CDR3 appears to constitute the major area of contact between the antigen and the antibody. Desmyter et al., 2001, supra. In vitro selection schemes in which CDR3 alone is varied can be used to vary the binding properties of an antibody. Muyldermans, 2001, supra; Desiderio et al., 2001, supra. Naturally occurring antibodies typically include a signal sequence, which directs the antibody into the cellular pathway for protein secretion and which is not present in the mature antibody. A polynucleotide encoding an antibody of the invention may encode a naturally occurring signal sequence or a heterologous signal sequence as described below. In one embodiment, the antigen binding protein is a monoclonal antibody, comprising from one (1) to six (6) of the depicted CDRs, as outlined herein (see TABLE 1). The antibodies of the invention may be of any type including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD, IgA, or IgE antibody. In specific embodiment, the antigen binding protein is an IgG type antibody. In an even more specific embodiment, the antigen binding protein is an IgG2 type antibody. In some embodiments, for example when the antigen binding protein is an antibody with complete heavy and light chains, the CDRs are all from the same species, e.g., human. Alternatively, for example in embodiments wherein the antigen binding protein contains less than six CDRs from the sequences outlined above, additional CDRs may be either from other species (e.g., murine CDRs), or may be different human CDRs than those depicted in the sequences. For example, human H-CDR3 and L-CDR3 regions from the appropriate sequences identified herein may be used, with H-CDR1, H-CDR2, L-CDR1 and L-CDR2 being optionally selected from alternate species, or different human antibody sequences, or combinations thereof. For example, the CDRs of the invention can replace the CDR regions of commercially relevant chimeric or humanized antibodies. Specific embodiments utilize scaffold components of the antigen binding proteins that are human components. In some embodiments, however, the scaffold components can be a mixture from different species. As such, if the antigen binding protein is an antibody, such antibody may be a chimeric antibody and/or a humanized antibody. In general, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species. For example, “chimeric antibodies” traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human. “Humanized antibodies” generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature321:522-525, Verhoeyen et al., 1988, Science239:1534-1536. Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog.20:639-654. In the present invention, the identified CDRs are human, and thus both humanized and chimeric antibodies in this context include some non-human CDRs; for example, humanized antibodies may be generated that comprise the CDRH3 and CDRL3 regions, with one or more of the other CDR regions being of a different special origin. In one embodiment, the IL-17RA antigen binding protein is a multispecific antibody, and notably a bispecific antibody, also sometimes referred to as “diabodies”. These are antibodies that bind to two (or more) different antigens. Diabodies can be manufactured in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol.4:446-449). e.g., prepared chemically or from hybrid hybridomas. In one embodiment, the IL-17RA antigen binding protein is a minibody. Minibodies are minimized antibody-like proteins comprising a scFv joined to a CH3 domain. Hu et al., 1996, Cancer Res.56:3055-3061. In one embodiment, the IL-17RA antigen binding protein is a domain antibody; see, for example U.S. Pat. No. 6,248,516. Domain antibodies (dAbs) are functional binding domains of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies dABs have a molecular weight of approximately 13 kDa, or less than one-tenth the size of a full antibody, dABs are well expressed in a variety of hosts including bacterial, yeast, and mammalian cell systems. In addition, dAbs are highly stable and retain activity even after being subjected to harsh conditions, such as freeze-drying or heat denaturation. See, for example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; US Serial No. 2004/0110941; European Patent 0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 and WO03/002609. In one embodiment, the IL-17RA antigen binding protein is an antibody fragment, that is a fragment of any of the antibodies outlined herein that retain binding specificity to IL-17RA. In various embodiments, the antibody binding proteins comprise, but are not limited to, a F(ab). F(ab′), F(ab′)2, Fv. or a single chain Fv fragments. At a minimum, an antibody, as meant herein, comprises a polypeptide that can bind specifically to IL-17RA comprising all or part of a light or heavy chain variable region, such as one or more CDRs. Further examples of IL-17RA-binding antibody fragments include, but are not limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii) the Fv fragment consisting of the VLand VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989, Nature341:544-546) which consists of a single variable, (v) isolated CDR regions, (vi) F(ab′)2fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VLdomain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science242:423-426. Huston et al., 1988. Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883), (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies” or “triabodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al., 2000, Methods Enzymol.326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448). The antibody fragments may be modified. For example, the molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245). Aspects of the invention include embodiments wherein the non-CDR components of these fragments are human sequences. In one embodiment, the IL-17RA antigen binding protein is a fully human antibody. In this embodiment, as outlined above, specific structures comprise complete heavy and light chains depicted comprising the CDR regions. Additional embodiments utilize one or more of the CDRs of the invention, with the other CDRs, framework regions, J and D regions, constant regions, etc., coming from other human antibodies. For example, the CDRs of the invention can replace the CDRs of any number of human antibodies, particularly commercially relevant antibodies Single chain antibodies may be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VLand VH). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt el al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). By combining different VL, and VHi-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf et al., 2002, Methods Mol Biol. 178:379-87. Single chain antibodies derived from antibodies provided herein (including but not limited to scFvs comprising the variable domain combinations of AML1/AMH(SEQ ID NO:27/SEQ ID NO:1), AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2), AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3). AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4), AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5), AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6), AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7), AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8), AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9), AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10), AML11/AMH11 (SEQ ID NO:37/SEQ ID NO: 11), AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12), AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13), AML14/AMH14 (SEQ ID NO:40/SEQ ID NO: 14), AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15), AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16), AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17), AML18/AMH18 (SEQ ID NO:44/SEQ ID NO: 18), AML9/AMH19 (SEQ ID NO:45/SEQ ID NO:19), AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20), AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21), AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22), AML23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23), AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24), AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25), AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26), and combinations thereof are encompassed by the present invention. In one embodiment, the IL-17RA antigen binding protein is an antibody fusion protein (sometimes referred to herein as an “antibody conjugate”). The conjugate partner can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antigen binding protein (see the discussion on covalent modifications of the antigen binding proteins) and on the conjugate partner. For example linkers are known in the art; for example, homo- or hetero-bifunctional linkers as are well known (see, 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference). In one embodiment, the IL-17RA antigen binding protein is an antibody analog, sometimes referred to as “synthetic antibodies.” For example, a variety of recent work utilizes cither alternative protein scaffolds or artificial scaffolds with grafted CDRs. Such scaffolds include, but are not limited to, mutations introduced to stabilize the three-dimensional structure of the binding protein as well as wholly synthetic scaffolds consisting for example of biocompatible polymers. See, for example, Korndorfcr et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129. Roque et al., 2004, Biotechnol. Prog.20:639-654. In addition, peptide antibody mimetics (“PAMs”) can be used, as well as work based on antibody mimetics utilizing fibronection components as a scaffold. As it is known in the art, a number of different programs can be used to identify the degree of sequence identity or similarity a protein or nucleic acid has to a known sequence. By “protein,” as used herein, is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. In some embodiments, the two or more covalently attached amino acids are attached by a peptide bond. The protein may be made up of naturally occurring amino acids and peptide bonds, for example when the protein is made recombinantly using expression systems and host cells, as outlined below. Alternatively, the protein may include synthetic amino acids (e.g., homophenylalanine, citrulline, ornithine, and norleucine), or peptidomimetic structures, i.e., “peptide or protein analogs”, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad. Sci. U.S.A.89:9367, incorporated by reference herein), which can be resistant to proteases or other physiological and/or storage conditions. Such synthetic amino acids may be incorporated in particular when the antigen binding protein is synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic and naturally occurring residues/structures can be used. “Amino acid” also includes imino acid residues such as proline and hydroxyproline. The amino acid “R group” or “side chain” may be in either the (L)- or the (S)-configuration. In a specific embodiment, the amino acids are in the (L)- or (S)-configuration. In certain aspects, the invention provides recombinant antigen binding proteins that bind an IL-17RA, in some embodiments a recombinant human IL-17RA or portion thereof. In this context, a “recombinant protein” is a protein made using recombinant techniques using any techniques and methods known in the art, i.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins are well known in the art. Embodiments of the invention include recombinant antigen binding proteins that bind wild-type IL-17RA and variants thereof. “Consisting essentially of” means that the amino acid sequence can vary by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% relative to the recited SEQ ID NO: sequence and still retain biological activity, as described herein. In some embodiments, the antigen binding proteins of the invention are isolated proteins or substantially pure proteins. An “isolated” protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, for example constituting at least about 5%, or at least about 50% by weight of the total protein in a given sample. It is understood that the isolated protein may constitute from 5 to 99.9% by weight of the total protein content depending on the circumstances. For example, the protein may be made at a significantly higher concentration through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. The definition includes the production of an antigen binding protein in a wide variety of organisms and/or host cells that are known in the art. For amino acid sequences, sequence identity and/or similarity is determined by using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A.85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res.12:387-395, preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, “Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis. Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc. An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol.35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS5:151-153. Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al., 1990, J. Mol. Biol.215:403-410; Altschul et al., 1997, Nucleic Acids Res.25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A.90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., 1996, Methods in Enzymology266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span-1, overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. An additional useful algorithm is gapped BLAST as reported by Altschul et al., 1993, Nucl. Acids Res.25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parametcr set to 9; the two-hit method to trigger ungapped extensions, charges gap lcngths of k a cost of 10+k; Xuset to 16, and Xgset to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to about 22 bits. Generally, the amino acid homology, similarity, or identity between individual variant CDRs are at least 80% to the sequences depicted herein, and more typically with preferably increasing homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%. In a similar manner, “percent (%) nucleic acid sequence identity” with respect to the nucleic acid sequence of the binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the antigen binding protein. A specific method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively. Generally, the nucleic acid sequence homology, similarity, or identity between the nucleotide sequences encoding individual variant CDRs and the nucleotide sequences depicted herein are at least 80%, and more typically with preferably increasing homologies or identities of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and almost 100%. Thus, a “variant CDR” is one with the specified homology, similarity, or identity to the parent CDR of the invention, and shares biological function, including, but not limited to, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the specificity and/or activity of the parent CDR. While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed antigen binding protein CDR variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of antigen binding protein activities, such as IL-17RA binding. Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about one (1) to about twenty (20) amino acid residues, although considerably larger insertions may be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, although in some cases deletions may be much larger. Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative or variant. Generally these changes are done on a few amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions are generally made in accordance with the following chart depicted as TABLE 2. TABLE 2Original ResidueExemplary SubstitutionsAlaSerArgLysAsnGln, HisAspGluCysSerGlnAsnGluAspGlyProHisAsn, GlnIleLeu, ValLeuIle, ValLysArg, Gln, GluMetLeu, IlePheMet, Leu, TyrSerThrThrSerTrpTyrTyrTrp, PheValIle, Leu Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in TABLE 2. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine. The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the antigen binding protein proteins as needed. Alternatively, the variant may be designed such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be altered or removed as discussed herein. Such a modification of the IL-17RA antigcn binding proteins, including antibodies, is an example of a dcrivativc. A “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified. e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Other derivatives of IL-17RA antibodies within the scope of this invention include covalent or aggregative conjugates of IL-17RA antibodies, or fragments thereof, with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an IL-17RA antibody polypeptide. For example, the conjugated peptidic may be a heterologous signal (or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag. IL-17RA antibody-containing fusion proteins can comprise peptides added to facilitate purification or identification of the IL-17RA antibody (e.g., poly-His). An IL-17RA antibody polypeptide also can be linked to the FLAG peptide DYKDDDDK (SEQ ID NO:447) as described in Hopp et al., Bio/Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), enabling rapid assay and facile purification of expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO). Oligomers that contain one or more IL-17RA antibody polypeptides may be employed as IL-17RA antagonists. Oligomers may be in the form of covalently-linked or non-covalently-linked dimers, trimers, or higher oligomers. Oligomers comprising two or more IL-17RA antibody polypeptides are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. One embodiment is directed to oligomers comprising multiple IL-17RA antibody polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the IL-17RA antibody polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of IL-17RA antibody polypeptides attached thereto, as described in more detail below. In particular embodiments, the oligomers comprise from two to four IL-17RA antibody polypeptides. The IL-17RA antibody moieties of the oligomer may be in any of the forms described above. e.g., variants or fragments. Preferably, the oligomers comprise IL-17RA antibody polypeptides that have IL-17RA binding activity. In one embodiment, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction of Immunoglobulin Fusion Proteins”, inCurrent Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11. One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing an IL-17RA binding fragment of an IL-17RA antibody to the Fc region of an antibody. The dimer can be made by, for example, inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in host cells transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield the dimer. The term “Fe polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also are included. Fusion proteins comprising Fe moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns. One suitable Fe polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG antibody. Another useful Fe polypeptide is the Fe mutein described in U.S. Pat. No. 5,457,035 and in Baum et al., 1994, EMBO J.13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors. In other embodiments, the variable portion of the heavy and/or light chains of an IL-17RA antibody may be substituted for the variable portion of an antibody heavy and/or light chain. Alternatively, the oligomer is a fusion protein comprising multiple IL-17RA antibody polypeptides, with or without peptidic linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233. Another method for preparing oligomeric IL-17RA antibody derivatives involves use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., 1988, Science240:1759), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, hereby incorporated by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in Fanslow et al., 1994, Semin. Immunol.6:267-78. In one approach, recombinant fusion proteins comprising an IL-17RA antibody fragment or derivative fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomeric IL-17RA antibody fragments or derivatives that form are recovered from the culture supernatant. Covalent modifications are also considered derivatives of the IL-17RA antigen binding proteins and are included within the scope of this invention, and are generally, but not always, done post-translationally. For example, several types of covalent modifications of the antigen binding protein are introduced into the molecule by reacting specific amino acid residues of the antigen binding protein with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromcrcuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butancdionc, 1,2-cyclohcxancdionc, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKaof the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using125I or131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method described above being suitable. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Derivatization with bifunctional agents is useful for crosslinking antigen binding proteins to a water-insoluble support matrix or surface for use in a variety of methods. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton. Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, 1983. pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Another type of covalent modification of the antigen binding protein included within the scope of this invention comprises altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to the antigen binding protein is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For ease, the antigen binding protein amino acid sequence is preferably altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids. Another means of increasing the number of carbohydrate moictics on the antigen binding protein is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N- and O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threoninc, or hydroxyproline, (c) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306. Removal of carbohydrate moieties present on the starting antigen binding protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys.259:52 and by Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol.138:350. Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described by Duskin el al., 1982, J. Biol. Chem.257:3105. Tunicamycin blocks the formation of protein-N-glycoside linkages. Another type of covalent modification of the antigen binding protein comprises linking the antigen binding protein to various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In addition, as is known in the art, amino acid substitutions may be made in various positions within the antigen binding protein to facilitate the addition of polymers such as PEG. In some embodiments, the covalent modification of the antigen binding proteins of the invention comprises the addition of one or more labels. The term “labelling group” means any detectable label. Examples of suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,35N,35S,90Y,99Tc,111In,125I,131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine ripper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labelling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labelling proteins are known in the art and may be used in performing the present invention. In general, labels fall into a variety of classes, depending on the assay in which they are to be detected: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, s-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labelling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labelling proteins are known in the art and may be used in performing the present invention. Specific labels include optical dyes, including, but not limited to, chromophores, phosphors and fluorophores, with the latter being specific in many instances. Fluorophores can be either “small molecule” fluores, or proteinaceous fluores. By “fluorescent label” is meant any molecule that may be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, IAEDANS, EDANS, BODIPY® FL, LC® Red 640, LC® Red 705, Oregon green™, the Alexa Fluor® dyes (Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 660, Alexa Fluor 680), Cascade Blue®, Cascade Yellow (5-[2-[1-[[3-(2,5-dioxopyrrolidin-1-yl)oxycarbonylphenyl]methyl]pyridin-1-ium-4-yl]-1,3-oxaaol-5-yl]-2-me y) and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine, and Texas Red® (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. Suitable proteinaceous fluorescent labels also include, but are not limited to, green fluorescent protein, including aRenilla, Ptilosarcus, orAequoreaspecies of GFP (Chalfie et al., 1994, Science263:802-805), EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607) andRenilla(WO92/15673, WO95/07463, WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, 5,925,558). All of the above-cited references are expressly incorporated herein by reference. Polynucleotides Encoding IL-17RA Antigen Binding Proteins Encompassed within the invention are nucleic acids encoding IL-17RA antigen binding proteins, including antibodies, as defined herein. The polynucleotide sequences for the heavy chain variable regions AMH1-26 are found in SEQ ID NOs:54-79, respectively, and the polynucleotide sequences for the light chain variable regions AML1-26 are found in SEQ ID NOs:80-106, respectively, with AML23 having two versions, as shown in SEQ ID NO:102 and 103. The SEQ ID NOs for the polynucleotide sequences encoding the H-CDR1. H-CDR2, H-CDR3, L-CDR1. L-CDR2, and L-CDR3 are provided in TABLE 1. Aspects of the invention include polynucleotide variants (e.g., due to degeneracy) that encode the amino acid sequences described herein. Aspects of the invention include a variety of embodiments including, but not limited to, the following exemplary embodiments: embodiment 51: an isolated polynucleotide, wherein said polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of:A. a. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML1-26 (SEQ ID NOs:27-53, respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH1-26 (SEQ ID NOs: 1-26, respectively); orc. the light chain variable domain of (a) and the heavy chain variable domain of (b); andB. a light chain CDR1, CDR2, CDR3 and a heavy chain CDR1, CDR2, CDR3 that differs by no more than a total of three amino acid additions, substitutions, and/or deletions in each CDR from the following sequences:a. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO: 187) and a heavy chain CDR1 (SEQ ID NO: 107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO:112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO: 113), CDR2 (SEQ ID NO: 114), CDR3 (SEQ ID NO:115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and a heavy chain CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO:118) of antibody AM-4;e. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO: 123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO:127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO: 148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO: 152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO:178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; orz.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO: 183), CDR3 (SEQ ID NO:184) of antibody AM-26;wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 52: the polynucleotide of embodiment 51, wherein said polynucleotide hybridizes under stringent conditions to the full length complement of a polynucleotide selected from the group consisting of:a. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML1/AMH1 (SEQ ID NO:80/SEQ ID NO:54);b. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML2/AMH2 (SEQ ID NO:81/SEQ ID NO:55);c. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML3/AMH3 (SEQ ID NO:82/SEQ ID NO:56);d. a light chain variable domain-encoding polynucleotide and a heavy chain arabic domain-encoding polynucleotide of AML4/AMH4 (SEQ ID NO:83/SEQ ID NO:57);e. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML5/AMH5 (SEQ ID NO:84/SEQ ID NO:58);f. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML6/AMH6 (SEQ ID NO:85/SEQ ID NO:59)g. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML7/AMH7 (SEQ ID NO:86/SEQ ID NO:60);h. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML8/AMH8 (SEQ ID NO:87/SEQ ID NO:61);i. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML9/AMH9 (SEQ ID NO:88/SEQ ID NO:62);j. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML10/AMH10 (SEQ ID NO:89/SEQ ID NO:63);k. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML11/AMH11 (SEQ ID NO:90/SEQ ID NO:64);l. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML12/AMH12 (SEQ ID NO:91/SEQ ID NO:65);m. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML13/AMH13 (SEQ ID NO:92/SEQ ID NO:66);n. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML14/AMH14 (SEQ ID NO:93/SEQ ID NO:67);o. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML15/AMH15 (SEQ ID NO:94/SEQ ID NO:68);p. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML16/AMH16 (SEQ ID NO:95/SEQ ID NO:69);q. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML17/AMH17 (SEQ ID NO:96/SEQ ID NO:70);r. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML18/AMH18 (SEQ ID NO:97/SEQ ID NO:71);s. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML19/AMH19 (SEQ ID NO:98/SEQ ID NO:72);t. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML20/AMH20 (SEQ ID NO:99/SEQ ID NO:73);u. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML21/AMH21 (SEQ ID NO:100/SEQ ID NO:74);v. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML22/AMH22 (SEQ ID NO:101/SEQ ID NO:75);w. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML23/AMH23 (SEQ ID NO: 102 or SEQ ID NO:103/SEQ ID NO:76);x. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML24/AMH24 (SEQ ID NO:104/SEQ ID NO:77);y. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML25/AMH25 (SEQ ID NO:105/SEQ ID NO:78); andz. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML26/AMH26 (SEQ ID NO:106/SEQ ID NO:79). Embodiment 53: the polynucleotide of embodiment 51, wherein said polynucleotide hybridizes under stringent conditions to the full length complement of a polynucleotide selected from the group consisting of:a. a light chain CDR1-encoding polynucleotide of SEQ ID NO:345, CDR2-encoding polynucleotide of SEQ ID NO:346, CDR3-encoding polynucleotide of SEQ ID NO:347 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:266, CDR2-encoding polynucleotide of SEQ ID NO:267, and CDR3-encoding polynucleotide of SEQ ID NO:268 of antibody AM-1;b. a light chain CDR1-encoding polynucleotide of SEQ ID NO:348, CDR2-encoding polynucleotide of SEQ ID NO:349, CDR3-encoding polynucleotide of SEQ ID NO:350 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:269, CDR2-encoding polynucleotide of SEQ ID NO:270, CDR3-encoding polynucleotide of SEQ ID NO:271 of antibody AM-2;c. a light chain CDR1-encoding polynucleotide of SEQ ID NO:351, CDR2-encoding polynucleotide of SEQ ID NO:352, CDR3-encoding polynucleotide of SEQ ID NO:353 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:272, CDR2-encoding polynucleotide of SEQ ID NO:273, CDR3-encoding polynucleotide of SEQ ID NO:274 of antibody AM-3;d. a light chain CDR1-encoding polynucleotide of SEQ ID NO:354, CDR2-encoding polynucleotide of SEQ ID NO:355, CDR3-encoding polynucleotide of SEQ ID NO:356 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:275, CDR2-encoding polynucleotide of SEQ ID NO:276, CDR3-encoding polynucleotide of SEQ ID NO:277 of antibody AM-4;e. a light chain CDR1-encoding polynucleotide of SEQ ID NO:357. CDR2-encoding polynucleotide of SEQ ID NO:358, CDR3-encoding polynucleotide of SEQ ID NO:359 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:278, CDR2-encoding polynucleotide of SEQ ID NO:279, CDR3-encoding polynucleotide of SEQ ID NO:280 of antibody AM-5;f. a light chain CDR1-encoding polynucleotide of SEQ ID NO:360, CDR2-encoding polynucleotide of SEQ ID NO:361, CDR3-encoding polynucleotide of SEQ ID NO:362 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:281, CDR2-encoding polynucleotide of SEQ ID NO:282, CDR3-encoding polynucleotide of SEQ ID NO:283 of antibody AM-6;g. a light chain CDR1-encoding polynucleotide of SEQ ID NO:363, CDR2-encoding polynucleotide of SEQ ID NO:364, CDR3-encoding polynucleotide of SEQ ID NO:365 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:284, CDR2-encoding polynucleotide of SEQ ID NO:285. CDR3-encoding polynucleotide of SEQ ID NO:286 of antibody AM-7;h. a light chain CDR1-encoding polynucleotide of SEQ ID NO:366, CDR2-encoding polynucleotide of SEQ ID NO:367, CDR3-encoding polynucleotide of SEQ ID NO:368 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:287, CDR2-encoding polynucleotide of SEQ ID NO:288, CDR3-encoding polynucleotide of SEQ ID NO:289 of antibody AM-8;i. a light chain CDR1-encoding polynucleotide of SEQ ID NO:369, CDR2-encoding polynucleotide of SEQ ID NO:370, CDR3-encoding polynucleotide of SEQ ID NO:371 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:290, CDR2-encoding polynucleotide of SEQ ID NO:291, CDR3-encoding polynucleotide of SEQ ID NO:292 of antibody AM-9;j. a light chain CDR1-encoding polynucleotide of SEQ ID NO:372, CDR2-encoding polynucleotide of SEQ ID NO:373, CDR3-encoding polynucleotide of SEQ ID NO:374 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:293, CDR2-encoding polynucleotide of SEQ ID NO:294, CDR3-encoding polynucleotide of SEQ ID NO:295 of antibody AM-10;k. a light chain CDR1-encoding polynucleotide of SEQ ID NO:375, CDR2-encoding polynucleotide of SEQ ID NO:376, CDR3-encoding polynucleotide of SEQ ID NO:377 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:296, CDR2-encoding polynucleotide of SEQ ID NO:297, CDR3-encoding polynucleotide of SEQ ID NO:298 of antibody AM-11;l. a light chain CDR1-encoding polynucleotide of SEQ ID NO:378, CDR2-encoding polynucleotide of SEQ ID NO:379, CDR3-encoding polynucleotide of SEQ ID NO:380 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:299, CDR2-encoding polynucleotide of SEQ ID NO:300, CDR3-encoding polynucleotide of SEQ ID NO:301 of antibody AM-12;m. a light chain CDR1-encoding polynucleotide of SEQ ID NO:381, CDR2-encoding polynucleotide of SEQ ID NO:382, CDR3-encoding polynucleotide of SEQ ID NO:383 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:302, CDR2-encoding polynucleotide of SEQ ID NO:303, CDR3-encoding polynucleotide of SEQ ID NO:304 of antibody AM-13;n. a light chain CDR1-encoding polynucleotide of SEQ ID NO:384, CDR2-encoding polynucleotide of SEQ ID NO:385, CDR3-encoding polynucleotide of SEQ ID NO:386 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:305, CDR2-encoding polynucleotide of SEQ ID NO:306, CDR3-encoding polynucleotide of SEQ ID NO:307 of antibody AM-14;o. a light chain CDR1-encoding polynucleotide of SEQ ID NO:387, CDR2-encoding polynucleotide of SEQ ID NO:388, CDR3-encoding polynucleotide of SEQ ID NO:389 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:308, CDR2-encoding polynucleotide of SEQ ID NO:309, CDR3-encoding polynucleotide of SEQ ID NO:310 of antibody AM-15;p. a light chain CDR1-encoding polynucleotide of SEQ ID NO:390, CDR2-encoding polynucleotide of SEQ ID NO:391, CDR3-encoding polynucleotide of SEQ ID NO:392 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:311, CDR2-encoding polynucleotide of SEQ ID NO:312, CDR3-encoding polynucleotide of SEQ ID NO:313 of antibody AM-16;q. a light chain CDR1-encoding polynucleotide of SEQ ID NO:393, CDR2-encoding polynucleotide of SEQ ID NO:394, CDR3-encoding polynucleotide of SEQ ID NO:395 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:314, CDR2-encoding polynucleotide of SEQ ID NO:315, CDR3-encoding polynucleotide of SEQ ID NO:316 of antibody AM-17;r. a light chain CDR1-encoding polynucleotide of SEQ ID NO:396, CDR2-encoding polynucleotide of SEQ ID NO:397, CDR3-encoding polynucleotide of SEQ ID NO:398 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:317, CDR2-encoding polynucleotide of SEQ ID NO:318, CDR3-encoding polynucleotide of SEQ ID NO:319 of antibody AM-18;s. a light chain CDR1-encoding polynucleotide of SEQ ID NO:399, CDR2-encoding polynucleotide of SEQ ID NO:400, CDR3-encoding polynucleotide of SEQ ID NO:401 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:320, CDR2-encoding polynucleotide of SEQ ID NO:321, CDR3-encoding polynucleotide of SEQ ID NO:322 of antibody AM-19;t. a light chain CDR1-encoding polynucleotide of SEQ ID NO:402, CDR2-encoding polynucleotide of SEQ ID NO:403, CDR3-encoding polynucleotide of SEQ ID NO:404 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:323, CDR2-encoding polynucleotide of SEQ ID NO:324, CDR3-encoding polynucleotide of SEQ ID NO:325 of antibody AM-20;u. a light chain CDR1-encoding polynucleotide of SEQ ID NO:405, CDR2-encoding polynucleotide of SEQ ID NO:406, CDR3-encoding polynucleotide of SEQ ID NO:407 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:326, CDR2-encoding polynucleotide of SEQ ID NO:327, CDR3-encoding polynucleotide of SEQ ID NO:328 of antibody AM-21;v. a light chain CDR1-encoding polynucleotide of SEQ ID NO:408, CDR2-encoding polynucleotide of SEQ ID NO:409, CDR3-encoding polynucleotide of SEQ ID NO:410 and a heavy chain CDR1 SEQ ID NO:329, CDR2-encoding polynucleotide of SEQ ID NO:330, CDR3-encoding polynucleotide of SEQ ID NO:331 of antibody AM-22;w. a light chain CDR1-encoding polynucleotide of SEQ ID NO:411 CDR2-encoding polynucleotide of SEQ ID NO:412, CDR3-encoding polynucleotide of SEQ ID NO:413 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:332, CDR2-encoding polynucleotide of SEQ ID NO:333, CDR3-encoding polynucleotide of SEQ ID NO:334 of antibody AM-23;x. a light chain CDR1-encoding polynucleotide of SEQ ID NO:414, CDR2-encoding polynucleotide of SEQ ID NO:415, CDR3-encoding polynucleotide of SEQ ID NO:416 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:332, CDR2-encoding polynucleotide of SEQ ID NO:333, CDR3-encoding polynucleotide of SEQ ID NO:334 of antibody AM-23;y. a light chain CDR1-encoding polynucleotide of SEQ ID NO:417, CDR2-encoding polynucleotide of SEQ ID NO:418, CDR3-encoding polynucleotide of SEQ ID NO:419 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:335, CDR2-encoding polynucleotide of SEQ ID NO:336, CDR3-encoding polynucleotide of SEQ ID NO:337 of antibody AM-24;z. a light chain CDR1-encoding polynucleotide of SEQ ID NO:420. CDR2-encoding polynucleotide of SEQ ID NO:421, CDR3-encoding polynucleotide of SEQ ID NO:422 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:338, CDR2-encoding polynucleotide of SEQ ID NO:339, CDR3-encoding polynucleotide of SEQ ID NO:340 of antibody AM-25; orz.2. a light chain CDR1-encoding polynucleotide of SEQ ID NO:423, CDR2-encoding polynucleotide of SEQ ID NO:424, CDR3-encoding polynucleotide of SEQ ID NO:425 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:341, CDR2-encoding polynucleotide of SEQ ID NO:342, CDR3-encoding polynucleotide of SEQ ID NO:343 of antibody AM-26. Embodiment 54: the polynucleotide of embodiment 51. wherein said polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of:a. a light chain variable domain and a heavy chain variable domain of AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1);b. a light chain variable domain and a heavy chain variable domain of AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2);c. a light chain variable domain and a heavy chain variable domain of AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3);d. a light chain variable domain and a heavy chain variable domain of AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4);e. a light chain variable domain and a heavy chain variable domain of AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5);f. a light chain variable domain and a heavy chain variable domain of AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6)g. a light chain variable domain and a heavy chain variable domain of AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7);h. a light chain variable domain and a heavy chain variable domain of AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8);i. a light chain variable domain and a heavy chain variable domain of AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9);j. a light chain variable domain and a heavy chain variable domain of AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10);k. a light chain variable domain and a heavy chain variable domain of AML11/AMH11 (SEQ ID NO:37/SEQ ID NO:11);l. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12);m. a light chain variable domain and a heavy chain variable domain of AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13);n. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);o. a light chain variable domain and a heavy chain variable domain of AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15);p. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);q. a light chain variable domain and a heavy chain variable domain of AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17);r. a light chain variable domain and a heavy chain variable domain of AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18);s. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);t. a light chain variable domain and a heavy chain variable domain of AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20);u. a light chain variable domain and a heavy chain variable domain of AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21).v. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22);w. a light chain variable domain and a heavy chain variable domain of AML23/AMH23 (SEQ ID NO: 49 or SEQ ID NO:50/SEQ ID NO:23);x. a light chain variable domain and a heavy chain variable domain of AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24);y. a light chain variable domain and a heavy chain variable domain of AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25); andz. a light chain variable domain and a heavy chain variable domain of AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26). Embodiment 55. The polynucleotide of embodiment 51, wherein said polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of:a. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO:187) and a heavy chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO: 110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO:112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO: 113), CDR2 (SEQ ID NO: 114), CDR3 (SEQ ID NO:115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and a heavy chain CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), CDR3 (SEQ ID NO: 118) of antibody AM-4;e. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO:119), CDR2 (SEQ ID NO: 120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO:127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO: 149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO: 178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; orz.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO: 183), CDR3 (SEQ ID NO:184) of antibody AM-26. Embodiment 6: the polynucleotide of embodiment 2, wherein said polynucleotide is selected from the group consisting of:a. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML1/AMH1 (SEQ ID NO:80/SEQ ID NO:54);b. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML2/AMH2 (SEQ ID NO:81/SEQ ID NO:55);c. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML3/AMH3 (SEQ ID NO:82/SEQ ID NO:56);d. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML4/AMH4 (SEQ ID NO:83/SEQ ID NO:57);e. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML5/AMH5 (SEQ ID NO:84/SEQ ID NO:58);f. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML6/AMH6 (SEQ ID NO:85/SEQ ID NO:59)g. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML7/AMH7 (SEQ ID NO:86/SEQ ID NO:60);h. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML8/AMH8 (SEQ ID NO:87/SEQ ID NO:61);i. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML9/AMH9 (SEQ ID NO:88/SEQ ID NO:62);j. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML10/AMH10 (SEQ ID NO:89/SEQ ID NO:63);k. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML11/AMH11 (SEQ ID NO:90/SEQ ID NO:64);l. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML12/AMH12 (SEQ ID NO:91/SEQ ID NO:65);m. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML13/AMH13 (SEQ ID NO:92/SEQ ID NO:66);n. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML14/AMH14 (SEQ ID NO:93/SEQ ID NO:67);o. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML15/AMH15 (SEQ ID NO:94/SEQ ID NO:68);p. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML16/AMH16 (SEQ ID NO:95/SEQ ID NO:69);q. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML17/AMH7 (SEQ ID NO:96/SEQ ID NO:70);r. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML18/AMH18 (SEQ ID NO:97/SEQ ID NO:71);s. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML19/AMH19 (SEQ ID NO:98/SEQ ID NO:72);t. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML20/AMH20 (SEQ ID NO:99/SEQ ID NO:73);u. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML21/AMH21 (SEQ ID NO:100/SEQ ID NO:74);v. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML22/AMH22 (SEQ ID NO:101/SEQ ID NO:75);w. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML23/AMH23 (SEQ ID NO: 102 or SEQ ID NO:103/SEQ ID NO:76);x. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML24/AMH24 (SEQ ID NO:104/SEQ ID NO:77);y. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML25/AMH25 (SEQ ID NO:105/SEQ ID NO:78); andz. a light chain variable domain-encoding polynucleotide and a heavy chain variable domain-encoding polynucleotide of AML26/AMH26 (SEQ ID NO:106/SEQ ID NO:79). Embodiment 57: the polynucleotide of embodiment 53, wherein said polynucleotide is selected from the group consisting of:a. a light chain CDR1-encoding polynucleoside of SEQ ID NO:345, CDR2-encoding polynucleotide of SEQ ID NO:346, CDR3-encoding polynucleotide of SEQ ID NO:347 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:266, CDR2-encoding polynucleotide of SEQ ID NO:267, and CDR3-encoding polynucleotide of SEQ ID NO:268 of antibody AM-1;b. a light chain CDR1-encoding polynucleotide of SEQ ID NO:348, CDR2-encoding polynucleotide of SEQ ID NO:349, CDR3-encoding polynucleotide of SEQ ID NO:350 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:269, CDR2-encoding polynucleotide of SEQ ID NO:270, CDR3-encoding polynucleotide of SEQ ID NO:271 of antibody AM-2;c. a light chain CDR1-encoding polynucleotide of SEQ ID NO:351, CDR2-encoding polynucleotide of SEQ ID NO:352, CDR3-encoding polynucleotide of SEQ ID NO:353 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:272, CDR2-encoding polynucleotide of SEQ ID NO:273, CDR3-encoding polynucleotide of SEQ ID NO:274 of antibody AM-3;d. a light chain CDR1-encoding polynucleotide of SEQ ID NO:354, CDR2-encoding polynucleotide of SEQ ID NO:355. CDR3-encoding polynucleotide of SEQ ID NO:356 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:275, CDR2-encoding polynucleotide of SEQ ID NO:276, CDR3-encoding polynucleotide of SEQ ID NO:277 of antibody AM-4;e. a light chain CDR1-encoding polynucleotide of SEQ ID NO:357, CDR2-encoding polynucleotide of SEQ ID NO:358, CDR3-encoding polynucleotide of SEQ ID NO:359 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:278, CDR2-encoding polynucleotide of SEQ ID NO:279, CDR3-encoding polynucleotide of SEQ ID NO:280 of antibody AM-5;f. a light chain CDR1-encoding polynucleotide of SEQ ID NO:360. CDR2-encoding polynucleotide of SEQ ID NO:361, CDR3-encoding polynucleotide of SEQ ID NO:362 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:281, CDR2-encoding polynucleotide of SEQ ID NO:282, CDR3-encoding polynucleotide of SEQ ID NO:283 of antibody AM-6;g. a light chain CDR1-encoding polynucleotide of SEQ ID NO:363, CDR2-encoding polynucleotide of SEQ ID NO:364, CDR3-encoding polynucleotide of SEQ ID NO:365 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:284, CDR2-encoding polynucleotide of SEQ ID NO:285, CDR3-encoding polynucleotide of SEQ ID NO:286 of antibody AM-7;h. a light chain CDR1-encoding polynucleotide of SEQ ID NO:366. CDR2-encoding polynucleotide of SEQ ID NO:367, CDR3-encoding polynucleotide of SEQ ID NO:368 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:287, CDR2-encoding polynucleotide of SEQ ID NO:288, CDR3-encoding polynucleotide of SEQ ID NO:289 of antibody AM-8;i. a light chain CDR1-encoding polynucleotide of SEQ ID NO:369, CDR2-encoding polynucleotide of SEQ ID NO:370, CDR3-encoding polynucleotide of SEQ ID NO:371 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:290, CDR2-encoding polynucleotide of SEQ ID NO:291. CDR3-encoding polynucleotide of SEQ ID NO:292 of antibody AM-9;j. a light chain CDR1-encoding polynucleotide of SEQ ID NO:372, CDR2-encoding polynucleotide of SEQ ID NO:373. CDR3-encoding polynucleotide of SEQ ID NO:374 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:293, CDR2-encoding polynucleotide of SEQ ID NO:294, CDR3-encoding polynucleotide of SEQ ID NO:295 of antibody AM-10;k. a light chain CDR1-encoding polynucleotide of SEQ ID NO:375, CDR2-encoding polynucleotide of SEQ ID NO:376, CDR3-encoding polynucleotide of SEQ ID NO:377 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:296, CDR2-encoding polynucleotide of SEQ ID NO:297. CDR3-encoding polynucleotide of SEQ ID NO:298 of antibody AM-11;l. a light chain CDR1-encoding polynucleotide of SEQ ID NO:378, CDR2-encoding polynucleotide of SEQ ID NO:379. CDR3-encoding polynucleotide of SEQ ID NO:380 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:299. CDR2-encoding polynucleotide of SEQ ID NO:300, CDR3-encoding polynucleotide of SEQ ID NO:301 of antibody AM-12;m. a light chain CDR1-encoding polynucleotide of SEQ ID NO:381, CDR2-encoding polynucleotide of SEQ ID NO:382, CDR3-encoding polynucleotide of SEQ ID NO:383 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:302, CDR2-encoding polynucleotide of SEQ ID NO:303, CDR3-encoding polynucleotide of SEQ ID NO:304 of antibody AM-13;n. a light chain CDR1-encoding polynucleotide of SEQ ID NO:384, CDR2-encoding polynucleotide of SEQ ID NO:385, CDR3-encoding polynucleotide of SEQ ID NO:386 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:305, CDR2-encoding polynucleotide of SEQ ID NO:306. CDR3-encoding polynucleotide of SEQ ID NO:307 of antibody AM-14;o. a light chain CDR1-encoding polynucleotide of SEQ ID NO:387, CDR2-encoding polynucleotide of SEQ ID NO:388, CDR3-encoding polynucleotide of SEQ ID NO:389 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:308, CDR2-encoding polynucleotide of SEQ ID NO:309, CDR3-encoding polynucleotide of SEQ ID NO:310 of antibody AM-15;p. a light chain CDR1-encoding polynucleotide of SEQ ID NO:390, CDR2-encoding polynucleotide of SEQ ID NO:391, CDR3-encoding polynucleotide of SEQ ID NO:392 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:311, CDR2-encoding polynucleotide of SEQ ID NO:312, CDR3-encoding polynucleotide of SEQ ID NO:313 of antibody AM-16;q. a light chain CDR1-encoding polynucleotide of SEQ ID NO:393, CDR2-encoding polynucleotide of SEQ ID NO:394, CDR3-encoding polynucleotide of SEQ ID NO:395 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:314, CDR2-encoding polynucleotide of SEQ ID NO:315, CDR3-encoding polynucleotide of SEQ ID NO:316 of antibody AM-17;r. a light chain CDR1-encoding polynucleotide of SEQ ID NO:396, CDR2-encoding polynucleotide of SEQ ID NO:397, CDR3-encoding polynucleotide of SEQ ID NO:398 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:317, CDR2-encoding polynucleotide of SEQ ID NO:318, CDR3-encoding polynucleotide of SEQ ID NO:319 of antibody AM-18;s. a light chain CDR1-encoding polynucleotide of SEQ ID NO:399, CDR2-encoding polynucleotide of SEQ ID NO:400, CDR3-encoding polynucleotide of SEQ ID NO:401 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:320, CDR2-encoding polynucleotide of SEQ ID NO:321, CDR3-encoding polynucleotide of SEQ ID NO:322 of antibody AM-19;t. a light chain CDR1-encoding polynucleotide of SEQ ID NO:402, CDR2-encoding polynucleotide of SEQ ID NO:403, CDR3-encoding polynucleotide of SEQ ID NO:404 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:323, CDR2-encoding polynucleotide of SEQ ID NO:324, CDR3-encoding polynucleotide of SEQ ID NO:325 of antibody AM-20;u. a light chain CDR1-encoding polynucleotide of SEQ ID NO:405, CDR2-encoding polynucleotide of SEQ ID NO:406, CDR3-encoding polynucleotide of SEQ ID NO:407 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:326, CDR2-encoding polynucleotide of SEQ ID NO:327, CDR3-encoding polynucleotide of SEQ ID NO:328 of antibody AM-21;v. a light chain CDR1-encoding polynucleotide of SEQ ID NO:408. CDR2-encoding polynucleotide of SEQ ID NO:409, CDR3-encoding polynucleotide of SEQ ID NO:410 and a heavy chain CDR1 SEQ ID NO:329, CDR2-encoding polynucleotide of SEQ ID NO:330, CDR3-encoding polynucleotide of SEQ ID NO:331 of antibody AM-22;w. a light chain CDR1-encoding polynucleotide of SEQ ID NO:411, CDR2-encoding polynucleotide of SEQ ID NO:412, CDR3-encoding polynucleotide of SEQ ID NO:413 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:332, CDR2-encoding polynucleotide of SEQ ID NO:333, CDR3-encoding polynucleotide of SEQ ID NO:334 of antibody AM-23;x. a light chain CDR1-encoding polynucleotide of SEQ ID NO:414, CDR2-encoding polynucleotide of SEQ ID NO:415, CDR3-encoding polynucleotide of SEQ ID NO:416 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:332, CDR2-encoding polynucleotide of SEQ ID NO:333, CDR3-encoding polynucleotide of SEQ ID NO:334 of antibody AM-23;y. a light chain CDR1-encoding polynucleotide of SEQ ID NO:417, CDR2-encoding polynucleotide of SEQ ID NO:418, CDR3-encoding polynucleotide of SEQ ID NO:419 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:335, CDR2-encoding polynucleotide of SEQ ID NO:336, CDR3-encoding polynucleotide of SEQ ID NO:337 of antibody AM-24;z. a light chain CDR1-encoding polynucleotide of SEQ ID NO:420, CDR2-encoding polynucleotide of SEQ ID NO:421. CDR3-encoding polynucleotide of SEQ ID NO:422 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:338, CDR2-encoding polynucleotide of SEQ ID NO:339, CDR3-encoding polynucleotide of SEQ ID NO:340 of antibody AM-25; orz.2. a light chain CDR1-encoding polynucleotide of SEQ ID NO:423, CDR2-encoding polynucleotide of SEQ ID NO:424, CDR3-encoding polynucleotide of SEQ ID NO:425 and a heavy chain CDR1-encoding polynucleotide of SEQ ID NO:341, CDR2-encoding polynucleotide of SEQ ID NO:342, CDR3-encoding polynucleotide of SEQ ID NO:343 of antibody AM-26. Embodiment 58: an isolated polynucleotide, wherein said polynucleotide encodes a polypeptide comprisinga. a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of:i. X1YGIS, wherein X1is selected from the group consisting of R, S and G;b. a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of:i. WISX1YX2GNTX3YAQX4X5QG, wherein X1is selected from the group consisting of A, X2is selected from the group consisting of N, S and K, X3is selected from the group consisting of N and K, X4is selected from the group consisting of K and N, and X5is selected from the group consisting of L and F;c. a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of:i. X1QLX2X3DY, wherein X1is selected from the group consisting of R and K, X2is selected from the group consisting of Y, V, and A, and X3is selected from the group consisting of F and L;ii. X1QLX2FDY, wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V;d. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of:i. RASQSX1X2X3X4LA, wherein X1is selected from the group consisting of V and I, X2is selected from the group consisting of I and S, X3is selected from the group consisting of S and T, X4is selected from the group consisting of N and S, and X is selected from the group consisting of A and N, andii. RASQSX1SSNLA, wherein X1is selected from the group consisting of V and I;c. a light chain CDR2 comprising an amino acid sequence selected from the group consisting of:i. X1X2STRAX3, wherein X1is selected from the group consisting of G and D, X2is selected from the group consisting of A and T, and X3is selected from the group consisting of T and A, andii. X1ASTRAX2, wherein X1is selected from the group consisting of G and D, and X2is selected from the group consisting of A and T; andf. a light chain CDR3 comprising an amino acid sequence selected from the group consisting of:i. QQYDX1WPLT, wherein X1is selected from the group consisting of N, T, and I; wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 59. The polynucleotide of embodiment 58, wherein said polynucleotide encodes a polypeptide wherein said polypeptide comprises:a. a heavy chain CDR1 amino acid sequence comprising X1YGIS, wherein X1is selected from the group consisting of R, S and G;b. a heavy chain CDR2 amino acid sequence comprising WISX1YX2GNTX3YAQX4X5QG, wherein X1is selected from the group consisting of A, X2is selected from the group consisting of N, S and K, X3is selected from the group consisting of N and K, X4is selected from the group consisting of K and N, and X5is selected from the group consisting of L and F;c. a heavy chain CDR3 amino acid sequence comprising X1QLX2FDY, wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V;d. a light chain CDR1 amino acid sequence comprising RASQSX1SSNLA, wherein X1is selected from the group consisting of V and I;e. a light chain CDR2 amino acid sequence comprising X1ASTRAX2, wherein X1is selected from the group consisting of G and D, and X2is selected from the group consisting of A and T; andf. a light chain CDR3 amino acid sequence comprising QQYDX1WPLT, wherein X1is selected from the group consisting of N, T, and I; wherein said polypeptide specifically binds IL-17 receptor A. Embodiment 60: a plasmid, comprising said polynucleotide of embodiment 51. Embodiment 61: the plasmid of embodiment 60, wherein said plasmid is an expression vector. Embodiment 62: an isolated cell, comprising said plasmid of embodiment 60. Embodiment 63: the isolated cell of embodiment 62, wherein a chromosome of said cell comprises said polynucleotide. Embodiment 64: the isolated cell of embodiment 62, wherein said cell is a hybridoma. Embodiment 65: the isolated cell of embodiment 62, wherein said cell comprises the expression vector of embodiment 61. Embodiment 66: the isolated cell of embodiment 65, wherein said cell is a selected from the group consisting of: a. a prokaryotic cell; b. a eukaryotic cell; c. a mammalian cell; d. an insect cell; and e. a CHO cell. Embodiment 67: a method of making a polypeptide that specifically binds IL-17 receptor A, comprising incubating said isolated cell of embodiment 65 under conditions that allow it to express said polypeptide. Embodiment 68: the polynucleotide of embodiment 51, wherein said polynucleotide encodes said polypeptide and wherein said polypeptide is an antibody that specifically binds IL-17 receptor A, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 69: the polynucleotide of embodiment 68, wherein said polynucleotide encodes said antibody and wherein said antibody is selected from the group consisting of:a) an antibody consisting of a heavy chain sequence of SEQ ID NO:427 and a light chain sequence of SEQ ID NO:429;b) an antibody consisting essentially of a heavy chain sequence of SEQ ID NO:427 and a light chain sequence of SEQ ID NO:429;c) an antibody comprising a heavy chain sequence of SEQ ID NO: 427;d) an antibody comprising a light chain sequence of SEQ ID NO:429;e) an antibody comprising a heavy chain sequence of SEQ ID NO: 427 and a light chain sequence of SEQ ID NO:429;f) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain sequence of SEQ ID NO: 427;g) an antibody or an IL-17 receptor A binding fragment thereof comprising a light chain sequence of SEQ ID NO:429;h) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain sequence of SEQ ID NO:427 and a light chain sequence of SEQ ID NO:429;i) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain variable region sequence of SEQ ID NO:14;j) an antibody or an IL-17 receptor A binding fragment thereof comprising a light chain variable region sequence of SEQ ID NO:40;k) an antibody or an IL-17 receptor A binding fragment thereof comprising a light chain variable region sequence of SEQ ID NO:40 and a heavy chain variable region sequence of SEQ ID NO:14;l) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain CDR1 of SEQ ID NO: 146, a heavy chain CDR2 of SEQ ID NO:147, a heavy chain CDR3 of SEQ ID NO:148, a light chain CDR1 of SEQ ID NO:224, a light chain CDR2 of SEQ ID NO:225, and a light chain CDR3 of SEQ ID NO:226; andm) an antibody or an IL-17 receptor A binding fragment thereof comprising a heavy chain CDR3 of SEQ ID NO: 148 and a light chain CDR3 of SEQ ID NO:226; wherein said antibody specifically binds IL-17 receptor A. Embodiment 70: the polynucleotide of embodiment 69, wherein said antibody comprises a polynucleotide selected from the group consisting of:a) a heavy chain-encoding polynucleotide sequence consisting of SEQ ID NO:426 and a light chain-encoding polynucleotide sequence consisting of SEQ ID NO:428;b) a heavy chain-encoding polynucleotide sequence consisting essentially of SEQ ID NO:426 and a light chain-encoding polynucleotide sequence consisting essentially of SEQ ID NO:428;c) a heavy chain-encoding polynucleotide sequence comprising SEQ ID NO: 426;d) a light chain-encoding polynucleotide sequence comprising SEQ ID NO:428;e) a heavy chain-encoding polynucleotide sequence comprising SEQ ID NO: 426 and a light chain-encoding polynucleotide sequence comprising SEQ ID NO:428;f) a heavy chain or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO: 426;g) a light chain or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO:428;h) a heavy chain or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO: 426 and a light chain or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO:428;i) a heavy chain variable region or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO:67;j) a light chain variable region or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO:93;k) a heavy chain variable region or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO:67 and a light chain variable region or an IL-17 receptor A binding fragment thereof-encoding polynucleotide sequence comprising SEQ ID NO:93;l) a light chain CDR1-encoding polynucleotide comprising SEQ ID NO:384, CDR2-encoding polynucleotide comprising SEQ ID NO:385, CDR3-encoding polynucleotide comprising SEQ ID NO:386 and a heavy chain CDR1-encoding polynucleotide comprising SEQ ID NO:305, CDR2-encoding polynucleotide comprising SEQ ID NO:306, CDR3-encoding polynucleotide comprising SEQ ID NO:307; andm) a heavy chain CDR3-encoding polynucleotide comprising SEQ ID NO:307 and a light chain CDR3-encoding polynucleotide comprising SEQ ID NO:386. Embodiment 71: the plasmid of embodiment 60, wherein the polynucleotide is the polynucleotide of embodiment 69. Embodiment 72: the isolated cell of embodiment 62, wherein the polynucleotide is the polynucleotide of embodiment 69. Embodiment 73: the isolated cell of embodiment 65, wherein said expression vector comprises the polynucleotide of embodiment 69. Embodiment 74: the isolated cell of embodiment 66, wherein the cell is a CHO cell and said CHO cell comprises the polynucleotide of embodiment 69. Embodiment 75: the method according to embodiment 67, wherein the polynucleotide is the polynucleotide of embodiment 69. Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for the isolation of nucleic acids or as query sequences for database searches, can be obtained by “back-translation” from the amino acid sequences, or by identification of regions of amino acid identity with polypeptides for which the coding DNA sequence has been identified. The well-known polymerase chain reaction (PCR) procedure can be employed to isolate and amplify a DNA sequence encoding a IL-17RA antigen binding proteins or a desired combination of IL-17RA antigen binding protein polypeptide fragments. Oligonucleotides that define the desired termini of the combination of DNA fragments are employed as 5′ and 3′ primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al.,Science239:487 (1988);Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; andPCR Protocols: A Guide to Methods and Applications, Innis et. al., eds., Academic Press, Inc. (1990). Nucleic acid molecules of the invention include DNA and RNA in both single-stranded and double-stranded form, as well as the corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. The nucleic acid molecules of the invention include full-length genes or cDNA molecules as well as a combination of fragments thereof. The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well. An “isolated nucleic acid” is a nucleic acid that has been separated from adjacent genetic sequences present in the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally-occurring sources. In the case of nucleic acids synthesized enzymatically from a template or chemically, such as PCR products, cDNA molecules, or oligonucleotides for example, it is understood that the nucleic acids resulting from such processes are isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In one preferred embodiment, the nucleic acids are substantially free from contaminating endogenous material. The nucleic acid molecule has preferably been derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA can be present 5′ or 3′ from an open reading frame, where the same do not interfere with manipulation or expression of the coding region. The present invention also includes nucleic acids that hybridize under moderately stringent conditions, and more preferably highly stringent conditions, to nucleic acids encoding IL-17RA antigen binding proteins as described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the DNA. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of about 55 degrees C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42 degrees C.), and washing conditions of about 60 degrees C., in 0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68 degrees C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH.sub.2 PO.sub.4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 1989). When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10. degrees C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (degrees C.)=2(# of A+T bases)+4(# of #G+C bases). For hybrids above 18 base pairs in length, Tm (degrees C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165M). Preferably, each such hybridizing nucleic acid has a length that is at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides), or at least 25% (more preferably at least 50%, or at least 60%, or at least 70%, and most preferably at least 80%) of the length of the nucleic acid of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and most preferably at least 99.5%) with the nucleic acid of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing nucleic acids when aligned so as to maximize overlap and identity while minimizing sequence gaps as described in more detail above. The variants according to the invention are ordinarily prepared by site specific mutagenesis of nucleotides in the DNA encoding the antigen binding protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the recombinant DNA in cell culture as outlined herein. However, antigen binding protein fragments comprising variant CDRs having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, e.g., binding to IL-17RA and inhibiting signaling, although variants can also be selected which have modified characteristics as will be more fully outlined below. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the CDRs (and heavy and light chains or other components of the antigen binding protein) of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the encoded protein. The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes which comprise at least one polynucleotide as above. In addition, the invention provides host cells comprising such expression systems or constructs. Typically, expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypcptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below. Optionally, the vector may contain a “tag”-encoding sequence, i.e., an oligonucleotide molecule located at the 5′ or 3′ end of the IL-17RA antigen binding protein coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another “tag” such as FLAG, HA (hemaglutinin influenza virus), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification or detection of the IL-17RA antigen binding protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified IL-17RA antigen binding protein by various means such as using certain peptidases for cleavage. Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery. Flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning. Whether all or only a portion of the flanking sequence is known, it may be obtained using polymerase chain reaction (PCR) and/or by screening a genomic library with a suitable probe such as an oligonucleotide and/or flanking sequence fragment from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art. An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly. MA) is suitable for most gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the virus early promoter). A transcription termination sequence is typically located 3′ to the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein. A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene may also be used for selection in both prokaryotic and eukaryotic host cells. Other selectable genes may be used to amplify the gene that will be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby leading to the amplification of both the selectable gene and the DNA that encodes another gene, such as an antigen binding protein antibody that binds to IL-17RA polypeptide. As a result, increased quantities of a polypeptide such as an IL-17RA antigen binding protein are synthesized from the amplified DNA. A ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3′ to the promoter and 5′ to the coding sequence of the polypeptide to be expressed. In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various pre- or prosequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which also may affect glycosylation. The final protein product may have, in the −1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the amino-terminus. Alternatively, use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide. Expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the IL-17RA antigen binding protein. Promoters are untranscribed sequences located upstream (i.e., 5′) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, uniformly transcribe gene to which they are operably linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding heavy chain or light chain comprising an IL-17RA antigen binding protein of the invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector. Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter. Additional promoters which may be of interest include, but are not limited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. U.S.A. 81:659-663); the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell22:787-797); herpes thymidine kinase promoter (Wagner et al., 1981. Proc. Natl. Acad. Sci. U.S.A.78:1444-1445); promoter and regulatory sequences from the metallothionine gene Prinster et al., 1982, Nature296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978. Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:21-25). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the clastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring HarborSymp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology7:425-515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122); the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al. 1987, Mol. Cell. Biol.7:1436-1444); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495); the albumin gene control region that is active in liver (Pinkert et al., 1987, Genes and Devel.1:268-276); the alpha-feto-protein gene control region that is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is active in liver (Kelsey et al., 1987, Genes and Devel.1:161-171); the beta-globin gene control region that is active in mycloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell46:89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, 1985, Nature314:283-286); and the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378). An enhancer sequence may be inserted into the vector to increase transcription of DNA encoding light chain or heavy chain comprising an IL-17RA antigen binding protein of the invention by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5′ and 3′ to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be positioned in the vector either 5′ or 3′ to a coding sequence, it is typically located at a site 5′ from the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector, to promote extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., 1984, Nature312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type 1 interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846. Expression vectors of the invention may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art. After the vector has been constructed and a nucleic acid molecule encoding light chain, a heavy chain, or a light chain and a heavy chain comprising an IL-17RA antigen binding sequence has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector for an IL-17RA antigen binding protein into a selected host cell may be accomplished by well known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods ae well known to the skilled artisan, and are et forth, for example, in Sambrook et al., 2001, supra. A host cell, when cultured under appropriate conditions, synthesizes an IL-17RA antigen binding protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule. A host cell may be eukaryotic or prokaryotic. Mammalian cell lines available as hosts for expression are well known in the art and include, but ae not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC®) and any cell lines used in an expression system known in the art can be used to make the recombinant polypeptides of the invention. In general, host cells ae transformed with a recombinant expression vector that comprises DNA encoding a desired anti-IL-17RA antibody polypeptide. Among the host cells that may be employed am prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram positive organisms, for exampleE. colior bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC® CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC® CCL 163), Chinese hamster ovary (CHO) cells, or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28: 31), HeLa cells, BHK (ATCC® CRL 10) cell lines, and the CVT/EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC® CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, mammalian col lines such as HepG2/3B, KB, NTH 3T3 or S49, for example, can be used for expression of the polypeptide when it is desirable to use the polypeptide in various signal transduction or reporter assays. Alternatively, it is possible to produce the polypeptide in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeasts includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyvcromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is made in yeast or bacteria, it may be desirable to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional polypeptide. Such covalent attachments can be accomplished using known chemical or enzymatic methods. The polypeptide can also be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego. Calif., U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio/Technology 6:47 (1988). Cell-free translation systems could also be employed to produce polypeptides using RNAs derived from nucleic acid constructs disclosed herein. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell that comprises an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a “recombinant host cell”. In certain embodiments, cell lines may be selected through determining which cell lines have high expression levels and constitutively produce antigen binding proteins with IL-17RA binding properties. In another embodiment, a cell line from the B cell lineage that does not make its own antibody but has a capacity to make and secrete a heterologous antibody can be selected. Identification of Domains on Human IL-17RA that Neutralizing Antibodies Bound Examples 14-17 describe various studies elucidating domains on human IL-17RA that neutralizing IL-17RA mAbs bound. These domains are referred to as neutralizing determinants. A neutralizing determinant is a contiguous stretch of IL-17RA, that when mutated, negatively affects the binding of at least one of the neutralizing antibodies disclosed herein. A neutralizing determinant comprises at least one epitope. A neutralizing determinant may have primary, secondary, tertiary, and/or quarternary structural characteristics. A neutralizing antibody is any of the antibodies described herein that specifically binds human IL-17RA and inhibits binding of IL-17A and/or IL-17F and thereby inhibits IL-17RA signaling and/or biological activity. Examples of neutralizing antibodies include antibodies comprising AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1). AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2), AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3), AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4), AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5), AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6), AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7), AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8), AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9), AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10), AML11/AMH11 (SEQ ID NO:37/SEQ ID NO:11), AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12), AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13), AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14). AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15). AML16/AMH6 (SEQ ID NO:42/SEQ ID NO:16), AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17), AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18), AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19), AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20), AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21), AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22), AML23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23), AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24), AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25), AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26), as well as IL-17RA-binding fragments thereof and combinations thereof. Further embodiments of neutralizing antibodies include antibodies that specifically bind to human IL-17RA and inhibit IL-17A and/or IL-17F from binding and activating IL-17RA, or a heteromeric complex of IL-17RA and IL-17RC. Further embodiments include antibodies that specifically bind to human IL-17RA and inhibit an IL-17A/IL-17F heteromer from binding and activating IL-17RA, or a heteromeric complex of IL-17RA and IL-17RC. Further embodiments include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming either a homomeric or heteromeric functional receptor complex, such as, but not limited to IL-17RA-IL-17RC complex. Further embodiments include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming either a homomeric or heteromeric functional receptor complex, such as, but not limited to IL-17RA/IL-17RC complex and do not necessarily inhibit IL-17A and/or IL-17F or an IL-17A/IL-17F heteromer from binding to IL-17RA or a IL-17RA heteromeric receptor complex. Further examples of neutralizing antibodies include antibodies comprising at least one CDR from antibodies comprising AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1), AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2), AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3), AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4), AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5), AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6), AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7), AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8), AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9), AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10), AML11/AMH11 (SEQ ID NO:37/SEQ ID NO:11), AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12), AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13), AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14), AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15), AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16), AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17), AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18). AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19), AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20), AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21), AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22), AML0.23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23), AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24), AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25), AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26), as well as IL-17RA-binding fragments thereof and combinations thereof. See Table 1. FIGS.16A and16Bshow that antibodies A: AMH11/AML11, B: AMH4/AML4, C: AMH8/AML8, D: AMH7/AML7, E: AMH6/AML6, F: AMH10/AML10, and G: AMH18/AML18 competed with one another for binding to human IL-17RA and fell into a defined group (Bin 1). In general, antibodies I: AMH22/AML22, J: AMH23/AML23, K: AMH14/AML14, L: AMH19/AML19, M: AMH12/AML12, N: AMH17/AML17, O: AMH16/AML16 competed with one another for binding to human IL-17RA and as a consequence fell into a different group (Bin 3). Generally speaking, the antibodies of Bin 1 did not compete with the antibodies of Bin 3. Antibody H: AMH1/AML1 was unique in its competition pattern and formed Bin 2, but is most similar to Bin 3. Antibody P: AMH26/AML26 formed Bin 4 and showed little cross-competition with any of the other antibodies, suggesting a neutralizing determinant unique to this antibody. Antibodies Q: AMH21/AML21 and R: AMH20/AML20 showed individually unique competition patterns, but with considerable similarities to Bin 3 antibodies, and formed Bins 5 and 6, respectively. This method identified groups of antibodies binding to different neutralizing determinants and provides evidence of several species within a subgenus of cross-competing antibodies. Example 16 describes the use of human/mouse IL-17RA chimeric proteins to determine neutralizing determinants on human IL-17RA.FIG.19show that at least three neutralizing determinants were identified based on those regions affecting the binding of neutralizing IL-17RA antibodies, namely Domain B spanning amino acids 75-96 of human IL-17RA (SEQ ID NO:431), Domain C spanning amino acids 128-154 of human IL-17RA (SEQ ID NO:431), and Domain D spanning amino acids 176-197 of human IL-17RA (SEQ ID NO:431). Domain B spanning amino acids 75-96 of human IL-17RA (SEQ ID NO:431) negatively affected the binding of neutralizing antibodies AMH1/AML1 and AMH23/AML23. Domain C spanning amino acids 128-154 of human IL-17RA (SEQ ID NO:431) negatively affected the binding of neutralizing antibodies AMH22/AML22 and AMH23/AML23. Domain D spanning amino acids 176-197 of human IL-17RA (SEQ ID NO:431) negatively affected the binding of neutralizing antibodies AMH1/AML1, AMH22/AML22, AMH14/AML14, AMH19/AML19, AMH23/AML23, AMH21/AML21, and AMH20/AML20. Thus, Domains B, C, and D are considered neutralizing determinants. Example 17 describes the use of arginine scan techniques to further elucidate the domains on human IL-17R that the IL-17RA neutralizing antibodies bound. A summary of the arginine scan, binning, and chimera data is presented inFIG.22. The arginine scan methodology identified several neutralizing determinants: AMH18/AML18 bound a domain spanning amino acids 220-284 of human IL-17RA (SEQ ID NO:431); AMH1/AML1 bound a domain focused on amino acid residue 152 of human IL-17RA (SEQ ID NO:431); AMH22/AML22 bound a domain spanning amino acids 152-198 of human IL-17RA (SEQ ID NO:431); AMH14/AML14 bound a domain spanning amino acids 152-297 of human IL-17RA (SEQ ID NO:431); AMH19/AML19 bound a domain spanning amino acids 152-186 of human IL-17RA (SEQ ID NO:431); AMH23/AML23 bound a domain spanning amino acids 97-297 of human IL-17RA (SEQ ID NO:431); AMH26/AML26 bound a domain spanning amino acids 138-270 of human IL-17RA (SEQ ID NO:431); AMH21/AML21 bound a domain spanning amino acids 113-198 of human IL-17RA (SEQ ID NO:431); and AMH20/AML20 bound a domain spanning amino acids 152-270 of human IL-17RA (SEQ ID NO:431). All of the residues shown inFIG.22have been shown to significantly reduce or essentially eliminate binding of a neutralizing human monoclonal antibody that specifically binds to human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds to IL-17RA and competes for binding with any one of antibodies AMH3/AML3, AMH20/AML20, AMH22/AML22, AMH23/AML23, AMH14/AML14, AMH21/AML21, AMH19/AML19, AMH12/AML12, AMH17/AML17, or AMH16/AML16, or any subset therein. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds to IL-17R and competes for binding with any one of antibodies AMH22/AML22, AMH23/AML23, AMH14/AML14, AMH19/AML19, AMH12/AML12, AMH17/AML17, or AMH16/AML16, or any subset therein. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds human IL-17RA of SEQ ID NO:431 but does not specifically bind to a chimeric polypeptide consisting of SEQ ID NO:434. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds human IL-17RA of SEQ ID NO:431 but does not specifically bind to a chimeric polypeptide consisting of SEQ ID NO:435. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds human IL-17RA of SEQ ID NO:431 but does not specifically bind to a chimeric polypeptide consisting of SEQ ID NO:436. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 75-96 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 128-154 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 176-197 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 152-297 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 220-284 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 152-198 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 152-186 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 97-297 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 138-270 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 113-198 of SEQ ID NO:431 of human IL-17RA. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant comprising amino acids 152-270 of SEQ ID NO:431 of human IL-17RA. Further embodiments include an antibody, or IL-17RA-binding fragment thereof, that binds human IL-17RA of SEQ ID NO:431, but does not bind said IL-17RA having an amino acid substituted with arginine at any one of E97R, E113R, S115R, H138R, D152R, D154R, E156R, K166R, Q176R, S177R, D184R, E186R, S198R, H215R, S220R, T228R, T235R, E241R, H243R, L270R, Q284R, H297R of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that binds human IL-17RA of SEQ ID NO:431, but does not bind said IL-17RA having an amino acid substituted with arginine at any one of D152R, D154R, E156R, D184R, E186R, H297R of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that binds human IL-17RA of SEQ ID NO:431, but does not bind said IL-17RA having an amino acid substituted with arginine at D152R of SEQ ID NO:431. Further embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds an epitope defined by any one of amino acids D152, D154, E156, D184, E186, H297 of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds an epitope defined by at least two amino acids selected from the group consisting of: D152, D154, E156, D184, E186, H297 of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds an epitope defined by at least three amino acids selected from the group consisting of: D152, D154, E156, D184, E186, H297 of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds an epitope defined by at least four amino acids selected from the group consisting of: D152, D154, E156, D184, E186, H297 of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds an epitope defined by at least five amino acids selected from the group consisting of: D152, D154, E156, D184, E186, H297 of SEQ ID NO:431. Embodiments include an antibody, or IL-17RA-binding fragment thereof, that specifically binds an epitopic defined by amino acids D152, D154, E156, D184, E186, H297 of SEQ ID NO:431. Aspects of the invention include a variety of embodiments including, but not limited to, the following exemplary embodiments: Embodiment 101: an isolated monoclonal antibody, or IL-17RA-binding fragment thereof, that specifically binds to IL-17RA and competes for binding with an antibody selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML2, 3, 5, 9, 10, 12, 14-17, and 19-25 (SEQ ID NOs:28, 29, 31, 35, 36, 38, 40-43, and 45-53, respectively);b. a heavy chain variable domain sequence that is at least 80°/o identical to a heavy chain variable domain sequence of AMH2, 3, 5, 9, 10, 12, 14-17, and 19-25 (SEQ ID NOs:2, 3, 5, 9, 10, 12, 14-17, and 19-25, respectively);c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO:112) of antibody AM-2;b. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO:192), CDR3 (SEQ ID NO:193) and a heavy chain CDR1 (SEQ ID NO:113), CDR2 (SEQ ID NO:114), CDR3 (SEQ ID NO: 115) of antibody AM-3;c. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;d. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;e. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;f. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;g. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;h. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO:149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;i. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;j. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;k. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;l. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;m. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;n. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;o. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;p. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;q. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO: 178) of antibody AM-24;r. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25; wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain and a heavy chain variable domain of AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2);b. a light chain variable domain and a heavy chain variable domain of AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3);c. a light chain variable domain and a heavy chain variable domain of AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5);d. a light chain variable domain and a heavy chain variable domain of AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9);c. a light chain variable domain and a heavy chain variable domain of AML10/AMH10 (SEQ ID NO:36/SEQ ID NO:10);f. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12);g. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);h. a light chain variable domain and a heavy chain variable domain of AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15);i. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);j. a light chain variable domain and a heavy chain variable domain of AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17);k. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);l. a light chain variable domain and a heavy chain variable domain of AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20);m. a light chain variable domain and a heavy chain variable domain of AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21);n. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22);o. a light chain variable domain and a heavy chain variable domain of AML23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23);p. a light chain variable domain and a heavy chain variable domain of AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24);q. a light chain variable domain and a heavy chain variable domain of AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25); wherein said antibody specifically binds to human IL-17RA. Embodiment 102: the antibody of embodiment 101, wherein said antibody is selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML9, 14, 16, 17, 19-23v2, and 26 (SEQ ID NOs:35, 40, 42, 43, 45-50, and 53, respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH9, 14, 16, 17, 19-23, and 26 (SEQ ID NOs:9, 14, 16, 17, 19-23, and 26, respectively);c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;b. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;c. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;d. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;e. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;f. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;g. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO: 169) of antibody AM-21;h. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;i. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO: 173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;j. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;k. a light chain variable domain and a heavy chain variable domain of AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26); wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain and a heavy chain variable domain of AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9);b. a light chain variable domain and a heavy chain variable domain of AML14/AMH4 (SEQ ID NO:40/SEQ ID NO:14);c. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);d. a light chain variable domain and a heavy chain variable domain of AML7/AMH17 (SEQ ID NO:43/SEQ ID NO: 17);e. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);f. a light chain variable domain and a heavy chain variable domain of AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20);g. a light chain variable domain and a heavy chain variable domain of AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21);h. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22);i. a light chain variable domain and a heavy chain variable domain of AML0.23/AMH23 (SEQ ID NO:49 or SEQ ID NO:50/SEQ ID NO:23);j. a light chain variable domain and a heavy chain variable domain of AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26); wherein said antibody specifically binds to human IL-17RA. Embodiment 103: the antibody of embodiment 101, wherein said antibody selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML12, 14, 16, 17, 19, and 22 (SEQ ID NOs:38, 40, 42, 43, 45, and 48 respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH12, 14, 16, 17, 19, and 22 (SEQ ID NOs:12, 14, 16, 17, 19, and 22, respectively);c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;b. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;c. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;d. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;e. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;f. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22; wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO: 12);b. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);c. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);d. a light chain variable domain and a heavy chain variable domain of AML7/AMH17 (SEQ ID NO:43/SEQ ID NO:17);e. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);c. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22); wherein said antibody specifically binds to human IL-17RA. Embodiment 104: the antibody of embodiment 101, wherein said antibody is selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence SEQ ID NO: 40;b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of SEQ ID NO:14;c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprising a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148); wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprising a light chain variable domain of SEQ ID NO:40 and a heavy chain variable domain SEQ ID NO: 14; wherein said antibody specifically binds to human IL-17RA. Embodiment 105: the antibody of embodiment 101, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 106: the antibody of embodiment 105, wherein said antibody inhibits human IL-17A from binding to human IL-17RA. Embodiment 107: the antibody of embodiment 106, wherein said antibody inhibits human IL-17A and IL-17F from binding to human IL-17RA. Embodiment 108: the antibody of embodiment 106, wherein said antibody inhibits human IL-17A or IL-17F from binding to human IL-17RA. Embodiment 109: an isolated monoclonal antibody, or IL-17RA-binding fragment thereof, selected from the group consisting of:a) a monoclonal antibody that specifically binds human IL-17RA of SEQ ID NO:431 but does not specifically bind to a chimeric polypeptide consisting of SEQ ID NO:434;b) a monoclonal antibody that specifically binds human IL-17RA of SEQ ID NO:431 but does not specifically bind to a chimeric polypeptide consisting of SEQ ID NO:435; andc) a monoclonal antibody that specifically binds human IL-17RA of SEQ ID NO:431 but does not specifically bind to a chimeric polypeptide consisting of SEQ ID NO:436. Embodiment 110: an isolated monoclonal antibody, or IL-17RA-binding fragment thereof, that specifically binds a neutralizing determinant selected from the group consisting of:a) a polypeptide comprising amino acids 75-96 of SEQ ID NO:431 of human IL-17RA;b) a polypeptide comprising amino acids 128-154 of SEQ ID NO:431 of human IL-17RA;c) a polypeptide comprising amino acids 176-197 of SEQ ID NO:431 of human IL-17RA;d) a polypeptide comprising amino acids 152-297 of SEQ ID NO:431 of human IL-17RA;e) a polypeptide comprising amino acids 220-284 of SEQ ID NO:431 of human IL-17RA;f) a polypeptide comprising amino acids 152-198 of SEQ ID NO:431 of human IL-17RA;g) a polypeptide comprising amino acids 152-186 of SEQ ID NO:431 of human IL-17RA;h) a polypeptide comprising amino acids 97-297 of SEQ ID NO:431 of human IL-17RA;i) a polypeptide comprising amino acids 138-270 of SEQ ID NO:431 of human IL-17RA;j) a polypeptide comprising amino acids 113-198 of SEQ ID NO:431 of human IL-17RA; andk) a polypeptide comprising amino acids 152-270 of SEQ ID NO:431 of human IL-17RA. Embodiment 111: an isolated monoclonal antibody, or IL-17RA-binding fragment thereof, that specifically binds human IL-17RA of SEQ ID NO:431, but does not specifically bind said IL-17RA having any one of the following amino acid substitutions E97R, E113R, S115R, H138R, D152R, D154R, E156R, K166R, Q176R, S177R, D184R, E186R, S198R, H215R, S220R, T228R, T235R, E241R, H243R, L270R, Q284R, or H297R of SEQ ID NO:431. Embodiment 112: the antibody of embodiment 111, wherein said antibody specifically binds human IL-17RA of SEQ ID NO:431, but does not specifically bind said IL-17RA having any one of the following amino acid substitutions D152R, D154R, E156R, D184R, E186R, or H297R of SEQ ID NO:431. Embodiment 113: the antibody of embodiment 111, wherein said antibody specifically binds human IL-17RA of SEQ ID NO:431, but does not specifically bind said IL-17RA having the aspartic acid residue at position 152 of SEQ ID NO:431 substituted with an arginine. Embodiment 114: the antibody of embodiment 111, wherein said antibody specifically binds an epitope defined by any one of amino acids D152, D154, E156, D184, E186, or H297 of SEQ ID NO:431. Embodiment 115: the antibody of embodiment 114, wherein said antibody specifically binds an epitope defined by at least two of the following amino acids D152, D154, E156, D184, E186, or H297 of SEQ ID NO:431. Embodiment 116: the antibody of embodiment 114, wherein said antibody specifically binds an epitope defined by at least three of the following amino acids D152, D154, E156, D184, E186, or H297 of SEQ ID NO:431. Embodiment 117: the antibody of embodiment 114, wherein said antibody specifically binds an epitope defined by at least four of the following amino acids D152, D154, E156, D184, E186, or H297 of SEQ ID NO:431. Embodiment 118: the antibody of embodiment 114, wherein said antibody specifically binds an epitope defined by at least five of the following amino acids D152, D154, E156, D184, E186, or H297 of SEQ ID NO:431. Embodiment 119: the antibody of embodiment 114, wherein said antibody specifically binds an epitope defined by amino acids D152, D154. E156, D184, E186, 11297 of SEQ ID NO:431. Embodiment 120: an isolated monoclonal antibody, or IL-17RA-binding fragment thereof, that specifically binds to IL-17RA and competes for binding with an antibody comprising:a. a heavy chain CDR1 comprising an amino acid sequence selected from the group consisting of:i. X1YGIS, wherein X1is selected from the group consisting of R, S and G;b. a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of:i. WISX1YX2GNTX3YAQX4X5QG, wherein X1is selected from the group consisting of A, X2is selected from the group consisting of N, S and K, Xjis selected from the group consisting of N and K, X4is selected from the group consisting of K and N, and X5is selected from the group consisting of L and F;c. a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of:i. X1QLX2X3DY, wherein X1is selected from the group consisting of R and K, X2is selected from the group consisting of Y, V, and A, and X3is selected from the group consisting of F and L;ii. X1QLX2FDY, wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V;d. a light chain CDR1 comprising an amino acid sequence selected from the group consisting of:i. RASQSX1X2X3X4LA, wherein X1is selected from the group consisting of V and I, X2is selected from the group consisting of I and S, X3is selected from the group consisting of S and T, X4is selected from the group consisting of N and S, and X5is selected from the group consisting of A and N;ii. RASQSX1SSNLA, wherein X1is selected from the group consisting of V and I;e. a light chain CDR2 comprising an amino acid sequence selected from the group consisting of:i. X1X2STRAX3, wherein X1is selected from the group consisting of G and D, X2is selected from the group consisting of A and T, and X3is selected from the group consisting of T and A;ii. X1ASTRAX2, wherein X1is selected from the group consisting of G and D, and X2is selected from the group consisting of A and T; andf. a light chain CDR3 comprising an amino acid sequence selected from the group consisting of:i. QQYDX1WPLT, wherein X1is selected from the group consisting of N. T, and I. Embodiment 121: the antibody of embodiment 120, wherein said antibody comprises:a. a heavy chain CDR1 amino acid sequence comprising X1YGIS, wherein X1is selected from the group consisting of R, S and G;b. a heavy chain CDR2 amino acid sequence comprising WISX1YX2GNTX3YAQX4X5QG, wherein X1is selected from the group consisting of A, X2is selected from the group consisting of N, S and K, X3is selected from the group consisting of N and K, X4is selected from the group consisting of K and N, and X5is selected from the group consisting of L and F;c. a heavy chain CDR3 amino acid sequence comprising X1QLX2FDY, wherein X1is selected from the group consisting of R and K, and X2is selected from the group consisting of Y and V;d. a light chain CDR1 amino acid sequence comprising RASQSX1SSNLA, wherein Xtis selected from the group consisting of V and I;e. a light chain CDR2 amino acid sequence comprising X1ASTRAX2, wherein X1is selected from the group consisting of G and D, and X2is selected from the group consisting of A and T; andf. a light chain CDR3 amino acid sequence comprising QQYDX1WPLT, wherein X1is selected from the group consisting of N, T, and I. Embodiment 122: the antibody of embodiment 120, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 123: the antibody of embodiment 122, wherein said antibody inhibits human IL-17A from binding to human IL-17RA. Embodiment 124: the antibody of embodiment 122, wherein said antibody inhibits human IL-17A and IL-17F from binding to human IL-17RA. Embodiment 125: the antibody of embodiment 122, wherein said antibody inhibits human IL-117A or IL-17F from binding to human IL-17R A. Use of IL-17RA Antigen Binding Proteins for Diagnostic and Therapeutic Purposes The IL-17RA antigen binding proteins of the invention can be used in diagnostic assays, e.g., binding assays to detect and/or quantify IL-17RA expressed in a tissue or cell. The IL-17RA antigen binding proteins may be used in research to further investigate the role of IL-17RA in disease. The IL-17RA antigen binding proteins may be used to further investigate the role of IL-17RA in forming homomeric and/or heteromeric receptor complexes and the role of said complexes in disease. The IL-17RA antigen binding proteins may be used to further investigate the role of IL-17RA activation to homomeric and/or heteromeric IL-17 ligand complexes. The IL-17RA antigen binding proteins may be used to further investigate the role of IL-17RA activation to homomeric and/or heteromeric IL-17 ligand complexes and how said homomeric and/or heteromeric IL-17 ligand complexes relate to disease. The IL-17RA antigen binding proteins of the present invention can be used for the prevention or treatment of diseases or conditions associated with the IL-17A and/or IL-17F activity. A disease or condition associated with IL-17A and/or IL-17F means any disease, condition, or pathology whose onset in a patient is caused or exacerbated by the interaction of IL-17A and/or IL-17F with IL-17RA. The severity of the disease, condition, or pathology can also be increased or decreased by the modulating the interaction of IL-17A and/or IL-17F with IL-17RA or a heterologous complex comprising IL-17RA and IL-17RC. Antigen binding proteins of the invention that specifically bind to IL-17RA may be used in treatment of IL-17RA mediated diseases in a patient in need thereof. All aspects of the IL-17RA antigen binding proteins described throughout this specification may be used in the preparation of a medicament for the treatment of the various conditions and diseases described herein. In addition, the IL-17RA antigen binding protein of the invention can be used to inhibit IL-17RA from forming a complex with its ligand, e.g., IL-17A and/or IL-17F or any other IL-17 ligand family member that binds IL-17RA or a heterologous complex comprising IL-17RA and IL-17RC, thereby modulating the biological activity of IL-17RA in a cell or tissue. Antigen binding proteins that bind to IL-17RA thus may modulate and/or inhibit interaction with other binding compounds and as such may have therapeutic use in ameliorating IL-17RA mediated diseases. In specific embodiments, IL-17RA antigen binding proteins may inhibit IL-17A and/or IL-17F from binding IL-17RA, which may result in disruption of the IL-17RA-induced signal transduction cascade. Increased levels of IL-17A and/or involvement of IL-17A mediated signals in disease pathogenesis have been demonstrated in a variety of conditions and diseases. Kolls and Linden, 2004, supra; Miossec, 2003, P. Arthritis Rheum.48:594-601); WO2005/063290; Cannetti el al., 2003, J. Immunol.171:1009-1015; Charles et al., 1999, J. Immunol.163: 1521-1528; Cunnane et al., 2000, Online J. Rheumatol.27:58-63; Yoshimoto, 1998, J. Immunol.161: 3400-3407), (WO2005/063290), (Niederau, 1997, Online NLM), (WO2004/002519), (Tsutsui et al., 2000, supra), (Konishi et al., 2002, Proc. Natl. Acad. Sci. U.S.A.99:11340-11345). Ziolkowska et al., 2000, supra). (Chabaud, 2001, Arth&Rheumatism,44:1293). Thus, IL-17RA is said to influence the pathology of these and other diseases or conditions described herein. As described herein, a surrogate rat anti-mouse IL-17RA antibody inhibits the course of disease and reduces bone and cartilage degradation in both a prophylactic and therapeutic rodent collagen induced arthritis model (see Examples below). As further evidence of the efficacy of interrupting the IL-17A/IL-17RA pathway. IL-17RA knockout mice are resistant to collagen-induced arthritis and IL-17RA antibody treatment is effective in arthritis induced in TNFR knockout mice, showing a TNF independent effect (see Example 6). Inhibiting IL-17RA using the antigen binding proteins disclosed herein represents a novel and effective mechanism to inhibit the symptoms and pathology of inflammatory and autoimmune diseases, and in particular inflammation and joint degradation found in rheumatoid arthritis (RA), Preclinical data and data from RA patient tissues suggest the potential to provide efficacy in those who failed TNF inhibitor therapy and to confer added benefit in combination with TNF inhibitors, IL-6 inhibitors, and IL-1 inhibitors. The antigen binding proteins described herein may be used in combination (pre-treatment, post-treatment, or concurrent treatment) with any of one or more TNF inhibitors for the treatment or prevention of the diseases and disorders recited herein, such as but not limited to, all forms of soluble TNF receptors including Etanercept (such as ENBREL®), as well as all forms of monomeric or multimeric p75 and/or p55 TN F receptor molecules and fragments thereof; anti-human TN F antibodies, such as but not limited to, infliximab (such as REMICADE®), and D2E7 (such as HUMIRA®), and the like. Such TNF inhibitors include compounds and proteins which block in vivo synthesis or extracellular release of TNF. In a specific embodiment, the present invention is directed to the use of an IL-17RA antigen binding protein in combination (pre-treatment, post-treatment, or concurrent treatment) with any of one or more of the following TNF inhibitors: TNF binding proteins (soluble TNF receptor type-I and soluble TNF receptor type-II (“sTNFRs”), as defined herein), anti-TNF antibodies, granulocyte colony stimulating factor; thalidomide; BN 50730; tenidap; E 5531; tiapafant PCA 4248; nimesulide; PANAVIR® (Probucol); rolipram; RP 73401; peptide T; MDL 201,449A; (1R,3S)-Cis-1-[9-(2,6-diaminopurinyl)]-3-hydroxy-4-cyclopentene hydrochloride; (1R,3R)-trans-1-(9-(2,6-diamino)purine]-3-acetoxycyclopentane; (11R,3R)-trans-1-[9-adenyl)-3-azidocyclopentane hydrochloride and (1R,3R)-trans-1-(6-hydroxy-purin-9-yl)-3-azidocyclo-pentane. TNF binding proteins are disclosed in the art (EP 308 378, EP 422 339, GB 2 218 101, EP 393 438, WO 90/13575, EP 398 327, EP 412 486, WO 91/03553. EP 418 014, JP 127,800/1991, EP 433 900, U.S. Pat. No. 5,136,021, GB 2 246 569, EP 464 533, WO 92/01002, WO 92/13095, WO 92/16221, EP 512 528. EP 526 905, WO 93/07863, EP 568 928, WO 93/21946, WO 93/19777, EP 417 563, WO 94/06476, and PCT International Application No. PCT/US97/12244). For example, EP 393 438 and EP 422 339 teach the amino acid and nucleic acid sequences of a soluble TNF receptor type I (also known as “sTNFR-I” or “30 kDa TNF inhibitor”) and a soluble TNF receptor type II (also known as “sTNFR-II” or “40 kDa TNF inhibitor”), collectively termed “sTNFRs”, as well as modified forms thereof (e.g., fragments, functional derivatives and variants). EP 393 438 and EP 422 339 also disclose methods for isolating the genes responsible for coding the inhibitors, cloning the gene in suitable vectors and cell types and expressing the gene to produce the inhibitors. Additionally, polyvalent forms (i.e., molecules comprising more than one active moiety) of sTNFR-I and sTNFR-II have also been disclosed. In one embodiment, the polyvalent form may be constructed by chemically coupling at least one TNF inhibitor and another moiety with any clinically acceptable linker, for example polyethylene glycol (WO 92/16221 and WO 95/34326), by a peptide linker (Neve et al. (1996),Cytokine,8(5):365-370, by chemically coupling to biotin and then binding to avidin (WO 91/03553) and, finally, by combining chimeric antibody molecules (U.S. Pat. No. 5,116,964, WO 89/09622, WO 91/16437 and EP 315062. Anti-TNF antibodies include the MAK 195F Fab antibody (Holler et al. (1993), 1st International Symposium on Cytokines in Bone Marrow Transplantation, 147); CDP 571 anti-TNF monoclonal antibody (Rankin et al. (1995),British Journal of Rheumatology,34:334-342); BAY X 1351 murine anti-tumor necrosis factor monoclonal antibody (Kiefl et al. (1995), 7th European Congress of Clinical Microbiology and Infectious Diseases, page 9); CenTNF cA2 anti-TNF monoclonal antibody (Elliott et al. (1994).Lancet.344:1125-1127 and Elliott et al. (1994),Lancet,344:1105-1110). The antigen binding proteins described herein may be used in combination with all forms of IL-1 inhibitors, such as but not limited to, kineret (for example ANAKINRA®). Interleukin-1 receptor antagonist (IL-1ra) is a human protein that acts as a natural inhibitor of interleukin-1. Interleukin-1 receptor antagonists, as well as the methods of making and methods of using thereof, are described in U.S. Pat. No. 5,075,222; WO 91/08285; WO 91/17184; AU 9173636; WO 92/16221; WO 93/21946; WO 94/06457; WO 94/21275; FR 2706772; WO 94/21235; DE 4219626; WO 94/20517; WO 96/22793 and WO 97/28828. The proteins include glycosylated as well as non-glycosylated IL-1 receptor antagonists. Specifically, three preferred forms of IL-1ra (IL-raα, IL-1raβ and IL-1rax), each being encoded by the same DNA coding sequence and variants thereof, are disclosed and described in U.S. Pat. No. 5,075,222. Methods for producing IL-1 inhibitors, particularly IL-1ras, are also disclosed in the 5,075,222 patent. An additional class of interleukin-1 inhibitors includes compounds capable of specifically preventing activation of cellular receptors to IL-1. Such compounds include IL-1 binding proteins, such as soluble receptors and monoclonal antibodies. Such compounds also include monoclonal antibodies to the receptors. A further class of interleukin-1 inhibitors includes compounds and proteins that block in vivo synthesis and/or extracellular release of IL-1. Such compounds include agents that affect transcription of IL-1 genes or processing of IL-1 preproteins. The antigen binding proteins described herein may be used in combination with all forms of CD28 inhibitors, such as but not limited to, abatacept (for example ORENCIA®). The antigen binding proteins described herein may be used in combination with all forms of IL-6 and/or IL-6 receptor inhibitors, such as but not limited to, tocilizumab (for example ACTEMRA®). The antigen binding proteins may be used in combination with one or more cytokines, lymphokines, hematopoietic factor(s), and/or an anti-inflammatory agent. Treatment of the diseases and disorders recited herein can include the use of first line drugs for control of pain and inflammation in combination (pretreatment, post-treatment, or concurrent treatment) with treatment with one or more of the antigen binding proteins provided herein. These drugs are classified as non-steroidal, anti-inflammatory drugs (NSAIDs). Secondary treatments include corticosteroids, slow acting antirheumatic drugs (SAARDs), or disease modifying (DM) drugs. Information regarding the following compounds can be found in The Merck Manual of Diagnosis and Therapy. Sixteenth Edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (1992) and in Pharmaprojects, PJB Publications Ltd. In a specific embodiment, the present invention is directed to the use of an antigen binding protein and any of one or more NSAIDs for the treatment of the diseases and disorders recited herein. NSAIDs owe their anti-inflammatory action, at least in part, to the inhibition of prostaglandin synthesis (Goodman and Gilman in “The Pharmacological Basis of Therapeutics,” MacMillan 7th Edition (1985)). NSAIDs can be characterized into at least nine groups: (1) salicylic acid derivatives; (2) propionic acid derivatives; (3) acetic acid derivatives; (4) fenamic acid derivatives; (5) carboxylic acid derivatives; (6) butyric acid derivatives; (7) oxicams; (8) pyrazoles and (9) pyrazolones. In another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more salicylic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Such salicylic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: acetaminosalol, aloxiprin, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, choline magnesium trisalicylate, magnesium salicylate, choline salicylate, diflusinal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide O-acetic acid, salsalate, sodium salicylate and sulfasalazine. Structurally related salicylic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In an additional specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more propionic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The propionic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof comprise: alminoprofen, benoxaprofen, bucloxic acid, carprofen, dexindoprofen, fenoprofen, flunoxaprofen, fluprofen, flurbiprofen, fircloprofen, ibuprofen, ibuprofen aluminum, ibuproxam, indoprofen, isoprofen, ketoprofen, loxoprofen, miroprofen, naproxcn, naproxcn sodium, oxaprozin, piketoprofen, pimeprofen, pirprofen, pranoprofen, protizinic acid, pyridoxiprofen, suprofen, tiaprofenic acid and tioxaprofen. Structurally related propionic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In yet another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more acetic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The acetic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof comprise: acemetacin, alclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin, diclofenac potassium, diclofenac sodium, etodolac, felbinac, fenclofenac, fenclorac, fenclozic acid, fentiazac, furofenac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, oxametacin, oxpinac, pimetacin, proglumetacin, sulindac, talmetacin, tiaramide, tiopinac, tolmetin, tolmetin sodium, zidometacin and zomepirac. Structurally related acetic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more fenamic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The fenamic acid derivatives, prodrug esters and pharmaceutically acceptable salts thereof comprise: enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, meclofenamate sodium, medofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid and ufenamate. Structurally related fenamic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In an additional specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more carboxylic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The carboxylic acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof which can be used comprise: clidanac, diflunisal, flufenisal, inoridine, ketorolac and tinoridine. Structurally related carboxylic acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In yet another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more butyric acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. The butyric acid derivatives, prodrug esters, and pharmaceutically acceptable salts thereof comprise: bumadizon, butibufen, fenbufen and xenbucin. Structurally related butyric acid derivatives having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more oxicams, prodrug esters, or pharmaceutically acceptable salts thereof. The oxicams, prodrug esters, and pharmaceutically acceptable salts thereof comprise: droxicam, enolicam, isoxicam, piroxicam, sudoxicam, tenoxicam and 4-hydroxyl-1,2-benzothiazine 1,1-dioxide 4-(N-phenyl)-carboxamide. Structurally related oxicams having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In still another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more pyrazoles, prodrug esters, or pharmaceutically acceptable salts thereof. The pyrazoles, prodrug esters, and pharmaceutically acceptable salts thereof which may be used comprise: difenamizole and epirizole. Structurally related pyrazoles having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In an additional specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment or, concurrent treatment) with any of one or more pyrazolones, prodrug esters, or pharmaceutically acceptable salts thereof. The pyrazolones, prodrug esters and pharmaceutically acceptable salts thereof which may be used comprise: apazone, azapropazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propylphenazone, ramifenazone, suxibuzone and thiazolinobutazone. Structurally related pyrazalones having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more of the following NSAIDs: ε-acetamidocaproic acid, S-adenosyl-methionine, 3-amino-4-hydroxybutyric acid, amixetrine, anitrazafen, antrafenine, bendazac, bendazac lysinate, benzydamine, beprozin, broperamole, bucolome, bufczolac, ciproquazonc, cloximate, dazidaminc, deboxamet, detomidine, difenpiramide, difenpyramide, difisalamine, ditazol, emorfazone, fanetizole mesylate, fenflumizole, floctafenine, flumizole, flunixin, fluproquazone, fopirtoline, fosfosal, guaimesal, guaiazolene, isonixim, lefetamine HCl, leflunomide, lofemizole, lotifazole, lysin clonixinate, meseclazone, nabumetone, nictindole, nimesulide, orgotein, orpanoxin, oxaceprol, oxapadol, paranyline, perisoxal, perisoxal citrate, pifoxime, piproxen, pirazolac, pirfenidone, proquazone, proxazole, thielavin B, tiflamizole, timegadine, tolectin, tolpadol, tryptamid and those designated by company code number such as 480156S, AA861, AD1590, AFP802. AFP860, A177B, AP504, AU8001, BPPC, BW540C, CHINOIN 127, CN100, EB382, EL508, F1044, FK-506, GV3658, ITF182, KCNTEI6090, KMFA, LA2851, MR714, MR897, MY309, ON03144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281, SY6001, TA6W, TA1-901 (4-benzoyl-1-indancarboxylic acid), TVX2706, U60257, UR2301 and WY41770. Structurally related NSAIDs having similar analgesic and anti-inflammatory properties to the NSAIDs are also intended to be encompassed by this group. In still another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment or concurrent treatment) with any of one or more corticosteroids, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders recited herein, including acute and chronic inflammation such as rheumatic diseases, graft versus host disease and multiple sclerosis. Corticosteroids, prodrug esters and pharmaceutically acceptable salts thereof include hydrocortisone and compounds which are derived from hydrocortisone, such as 21-acetoxypregnenolone, alclomerasone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budesonide, chloroprednisone, clobetasol, clobetasol propionate, clobetasone, clobetasone butyrate, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacon, dcsonide, desoximerasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flumethasone pivalate, flucinolone acetonide, flunisolide, fluocinonide, fluorocinolone acetonide, fluocortin butyl, fluocortolone, fluocortolone hexanoate, diflucortolone valerate, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandenolide, formocortal, halcinonide, halometasone, halopredone acetate, hydro-cortamate, hydrocortisone, hydrocortisone acetate, hydro-cortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate, hydrocortisone tebutate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 21-diedryaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21-m-sulfobenzoate, prednisolone sodium 21-stcaroglycolate, prednisolonc tcbutatc, prednisolone 21-trimcthylacctatc, prednisonc, prednival, prednylidene, prednylidene 21-diethylaminoacetate, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide and triamcinolone hexacetonide. Structurally related corticosteroids having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more slow-acting antirheumatic drugs (SAARDs) or disease modifying antirheumatic drugs (DMARDS), prodrug esters, or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders recited herein, including acute and chronic inflammation such as rheumatic diseases, graft versus host disease and multiple sclerosis. SAARDs or DMARDS, prodrug esters and pharmaceutically acceptable salts thereof comprise: allocupreide sodium, auranofin, aurothioglucose, aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium 3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine, clobuzarit, cuproxoline, cyclo-phosphamide, cyclosporin, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquine gold salt, gold sodium thiomalate, gold sodium thiosulfate), hydroxychloroquine, hydroxychloroquine sulfate, hydroxyurea, kcbuzone, lcvamisolc, lobcnzarit, mclittin, 6-mercaptopurinc, mcthotrcxate, mizoribine, mycophenolate mofetil, myoral, nitrogen mustard, D-penicillamine, pyridinol imidazoles such as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine. Structurally related SAARDs or DMARDs having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. In another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more COX2 inhibitors, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders recited herein, including acute and chronic inflammation. Examples of COX2 inhibitors, prodrug esters or pharmaceutically acceptable salts thereof include, for example, celecoxib. Structurally related COX2 inhibitors having similar analgesic and anti-inflammatory properties are also intended to be encompassed by this group. Examples of COX-2 selective inhibitors include but not limited to etoricoxib, valdecoxib, celecoxib, licofelone, lumiracoxib, rofecoxib, and the like. In still another specific embodiment, the present invention is directed to the use of an antigen binding protein in combination (pretreatment, post-treatment, or concurrent treatment) with any of one or more antimicrobials, prodrug esters or pharmaceutically acceptable salts thereof for the treatment of the diseases and disorders recited herein, including acute and chronic inflammation. Antimicrobials include, for example, the broad classes of penicillins, cephalosporins and other beta-lactams, aminoglycosides, azoles, quinolones, macrolides, rifamycins, tetracyclines, sulfonamides, lincosamides and polymyxins. The penicillins include, but are not limited to penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, floxacillin, ampicillin, ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate, hetacillin, cyclacillin, bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin, ticarcillin/clavulanate, azlocillin, mezlocillin, peperacillin, and mecillinam. The cephalosporins and other beta-lactams include, but are not limited to cephalothin, cephapirin, cephalexin, cephradine, cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan, cefoxitin, ceruroxime, cefonicid, ceforadine, cefixime, cefotaxime, moxalactam, ceftizoxime, cetriaxone, cephoperazone, ceftazidime, imipenem and aztreonam. The aminoglycosides include, but are not limited to streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin and neomycin. The azoles include, but are not limited to fluconazole. The quinolones include, but are not limited to nalidixic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin and temafloxacin. The macrolides include, but are not limited to erythomycin, spiramycin and azithromycin. The rifamycins include, but are not limited to rifampin. The tetracyclines include, but are not limited to spicycline, chlortetracycline, clomocycline, demeclocycline, deoxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, penimepicycline, pipacycline, rolitetracycline, sancycline, senociclin and tetracycline. The sulfonamides include, but are not limited to sulfanilamide, sulfamethoxazole, sulfacetamide, sulfadiazinc, sulfisoxazolc and co-trimoxazolc (trimcthoprimisulfamethoxazole). The lincosamides include, but are not limited to clindamycin and lincomycin. The polymyxins (polypeptides) include, but are not limited to polymyxin B and colistin. The most cited activity of IL-17A in vitro is the induction of neutrophil mobilizing cytokines and chemokines by stromal cells (e.g. GM-CSF, IL6, IL8). These activities are potently enhanced in the presence of TNF (Ruddy et al., 2004). Similarly the biologic activities of IL-17F are also enhanced by TNF co-stimulus. Of particular note with respect to a pathogenic role for IL-17A in cartilage destruction and bone erosion associated with rheumatoid arthritis, IL-17A induces the expression of NO, MMPs, PGE2 and RANKL and plays a role in antigen specific T and B cell activation (Kolls and Linden, 2004, supra; Lubberts et al., 2005, Arthritis. Res. Ther.7:29-37). Therefore, the antigen binding proteins may be used to inhibit the IL-17A and/or IL-17F/IL-17RA pathway and subsequent production of NO, MMPs, PGE2 and/or RANKL and treat diseases associated with the IL-17A and/or IL-17F upregulation of NO, MMPs, PGE2 and/or RANKL, as well as other proinflammatory mediators described herein. In addition to the presence of elevated levels of IL-17A in the synovial fluid of rheumatoid arthritis patients, several lines of evidence suggest that IL-17A is a key pathogenic cytokine in arthritis. First, administration of IL-17A to the joints of mice exacerbates the symptoms of collagen-induced arthritis (Lubberts et al., 2003, J. Immunol.170:2655-2662). Second, soluble IL-17RA. Fc inhibits collagen breakdown in human RA synovial and bone explant cultures and attenuates the symptoms in collagen induced arthritis in the mouse (Chabaud and Miossec, 2001, Arthritis Rheum.44:1293-1303) (Lubberts et al., 2001, J. Immunol.167:1004-1013)). As predicted from the low affinity interaction between IL-17F and IL-17R, IL-17R-Fc does not neutralize the activity of IL-17F and so these effects are specific to IL-17A antagonism. Third, mice lacking IL-17A are resistant to IL-1-induced arthritis and have suppressed collagen-induced arthritis (Nakae et al., 2003a,J. Immunol.171:6173-6177; Nakae et al., 2003b, supra). These data indicate that IL-17A signaling through IL-17RA is an important mediator of inflammation and joint damage in arthritis. The antigen binding proteins may be used to inhibit IL-17A and/or IL-17F/IL-17RA activity and thereby reduce the inflammation and joint damage in arthritis. In rheumatoid arthritis, elevated levels of mature IL-17A have been demonstrated in patient sera and synovial fluid. In some studies. IL-17A levels were shown to correlate with disease activity and response to disease modifying treatment. Extremely elevated serum levels of IL-17A have consistently been measured in systemic Juvenile Idiopathic Arthritis and the closely related Adult-Onset Still's Disease. WO2005/063290; Cannetti et al., 2003, J. Immunol.171:1009-1015; Charles et al., 1999, J. Immunol.163: 1521-1528; Cunnane et al., 2000, Online J. Rheumatol.27:58-63; Yoshimoto, 1998, J. Immunol.161: 3400-3407. The antigen binding proteins may be used to inhibit IL-17A and/or IL-17F/IL-17RA activity and thereby treat systemic Juvenile Idiopathic Arthritis and Adult-Onset Still's Disease. Various other autoimmune diseases have been associated with increased levels of IL-17A either in diseased tissue or in the serum. These include Systemic Lupus Erythematosus, atopic dermatitis, myasthenia gravis, type I diabetes, and sarcoidosis. IL-17A may also be involved in asthma and GvHD. The antigen binding proteins taught herein may be used to reduce the effects of the IL-17A and/or IL-17F/IL-17RA pathway in these diseases. The antigen binding proteins may be used to reduce IL-17RA activity, comprising administering an antigen binding protein. The present invention is also directed to methods of inhibiting binding and/or signaling of IL-17A and/or IL-17F to IL-17RA comprising providing the antigen binding protein of the invention to IL-17RA. In certain embodiments, the antigen binding protein inhibits binding and/or signaling of IL-17A and IL-17F to IL-17RA. In additional embodiments, the antigen binding protein inhibits binding and/or signaling of IL-17A but not IL-17F to IL-17RA. In other embodiments, the antigen binding protein inhibits binding and/or signaling of IL-17F and not IL-17A to IL-17RA. The antigen binding proteins may be used in treating the consequences, symptoms, and/or the pathology associated with IL-17RA activity, comprising administering an antigen binding protein. The antigen binding proteins may be used to inhibit the production of one or more of an inflammatory cytokine, chemokine, matrix metalloproteinase, or other molecule associated with IL-17RA activation, comprising administering an antigen binding protein. The antigen binding proteins may be used in methods of inhibiting production of molecules such as but is not limited to: IL-6, IL-8. CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-1β, TNFα, RANK-L, LIF, PGE2, IL-12, MMPs (such as but not limited to MMP3 and MMP9), GROQ, NO, and/or C-telopeptide and the like, comprising administering an antigen binding protein. The antigen binding proteins inhibit proinflammatory and proautoimmune immune responses and may be used to treat diseases associated with activity of the IL-17A and/or IL-17F/IL-17RA pathway. Aspects of the invention include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming either a homomeric or heteromeric functional receptor complex, such as, but not limited to IL-17RA/IL-17RC complex and do not necessarily inhibit IL-17A and/or IL-17F or an IL-17A/IL-17F heteromer from binding to IL-17RA or a IL-17RA heteromeric receptor complex. Thus, disease states associated with IL-17RC are also associated with IL-17RA due to the fact that IL-17RC cannot signal without IL-17RA. For example, see You, Z., et al.,Cancer Res.,2006 Jan. 1;66(1):175-83 and You, Z., et al.,Neoplasia,2007 June;9(6):464-70. The IL-17RA antigen binding proteins may be used in methods of treating IL-17RA associated disease, comprising administering an IL-17RA antigen binding protein. The IL-17RA antigen binding protein may be used to treat diseases including, but are not limited to, inflammation, autoimmune disease, cartilage inflammation, and/or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), dermatomyositis, psoriatic arthritis, sclcroderma, systemic lupus crythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barre disease, Type I diabetes mellitus, Graves' disease, Addison's disease. Raynaud's phenomenon, autoimmune hepatitis, GVHD, and the like. Aspects of the invention include a variety of embodiments including, but not limited to, the following exemplary embodiments: embodiment 151: a method of treating a disease state associated with IL-17RA activation in a patient in need thereof, comprising administering to said patient a composition comprising an antibody that specifically binds human IL-17 Receptor A and inhibits the binding of IL-17A, wherein said antibody is selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML1-26 (SEQ ID NOs:27-53, respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH1-26 (SEQ ID NOs:1-26, respectively);c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain CDR1 (SEQ ID NO:185), CDR2 (SEQ ID NO:186), CDR3 (SEQ ID NO:187) and a heavy chain CDR1 (SEQ ID NO:107), CDR2 (SEQ ID NO:108), CDR3 (SEQ ID NO:109) of antibody AM-1;b. a light chain CDR1 (SEQ ID NO:188), CDR2 (SEQ ID NO:189), CDR3 (SEQ ID NO:190) and a heavy chain CDR1 (SEQ ID NO:110), CDR2 (SEQ ID NO:111), CDR3 (SEQ ID NO: 112) of antibody AM-2;c. a light chain CDR1 (SEQ ID NO:191), CDR2 (SEQ ID NO: 192), CDR3 (SEQ TD NO:193) and a heavy chain CDR1 (SEQ ID NO:113), CDR2 (SEQ ID NO:114), CDR3 (SEQ ID NO: 115) of antibody AM-3;d. a light chain CDR1 (SEQ ID NO:194), CDR2 (SEQ ID NO:195), CDR3 (SEQ ID NO:196) and a heavy chain CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO:117), CDR3 (SEQ ID NO:118) of antibody AM-4;e. a light chain CDR1 (SEQ ID NO:197), CDR2 (SEQ ID NO:198), CDR3 (SEQ ID NO:199) and a heavy chain CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO:120), CDR3 (SEQ ID NO:121) of antibody AM-5;f. a light chain CDR1 (SEQ ID NO:200), CDR2 (SEQ ID NO:201), CDR3 (SEQ ID NO:202) and a heavy chain CDR1 (SEQ ID NO:122), CDR2 (SEQ ID NO:123), CDR3 (SEQ ID NO:124) of antibody AM-6;g. a light chain CDR1 (SEQ ID NO:203), CDR2 (SEQ ID NO:204), CDR3 (SEQ ID NO:205) and a heavy chain CDR1 (SEQ ID NO:125), CDR2 (SEQ ID NO:126), CDR3 (SEQ ID NO:127) of antibody AM-7;h. a light chain CDR1 (SEQ ID NO:206), CDR2 (SEQ ID NO:207), CDR3 (SEQ ID NO:208) and a heavy chain CDR1 (SEQ ID NO:128), CDR2 (SEQ ID NO:129), CDR3 (SEQ ID NO:130) of antibody AM-8;i. a light chain CDR1 (SEQ ID NO:209), CDR2 (SEQ ID NO:210), CDR3 (SEQ ID NO:211) and a heavy chain CDR1 (SEQ ID NO:131), CDR2 (SEQ ID NO:132), CDR3 (SEQ ID NO:133) of antibody AM-9;j. a light chain CDR1 (SEQ ID NO:212), CDR2 (SEQ ID NO:213), CDR3 (SEQ ID NO:214) and a heavy chain CDR1 (SEQ ID NO:134), CDR2 (SEQ ID NO:135), CDR3 (SEQ ID NO:136) of antibody AM-10;k. a light chain CDR1 (SEQ ID NO:215), CDR2 (SEQ ID NO:216), CDR3 (SEQ ID NO:217) and a heavy chain CDR1 (SEQ ID NO:137), CDR2 (SEQ ID NO:138), CDR3 (SEQ ID NO:139) of antibody AM-11;l. a light chain CDR1 (SEQ ID NO:218), CDR2 (SEQ ID NO:219), CDR3 (SEQ ID NO:220) and a heavy chain CDR1 (SEQ ID NO:140), CDR2 (SEQ ID NO:141), CDR3 (SEQ ID NO:142) of antibody AM-12;m. a light chain CDR1 (SEQ ID NO:221), CDR2 (SEQ ID NO:222), CDR3 (SEQ ID NO:223) and a heavy chain CDR1 (SEQ ID NO:143), CDR2 (SEQ ID NO:144), CDR3 (SEQ ID NO:145) of antibody AM-13;n. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;o. a light chain CDR1 (SEQ ID NO:227), CDR2 (SEQ ID NO:228), CDR3 (SEQ ID NO:229) and a heavy chain CDR1 (SEQ ID NO: 149), CDR2 (SEQ ID NO:150), CDR3 (SEQ ID NO:151) of antibody AM-15;p. a light chain CDR1 (SEQ ID NO:230), CDR2 (SEQ ID NO:231), CDR3 (SEQ ID NO:232) and a heavy chain CDR1 (SEQ ID NO:152), CDR2 (SEQ ID NO:153), CDR3 (SEQ ID NO:154) of antibody AM-16;q. a light chain CDR1 (SEQ ID NO:233), CDR2 (SEQ ID NO:234), CDR3 (SEQ ID NO:235) and a heavy chain CDR1 (SEQ ID NO:155), CDR2 (SEQ ID NO:156), CDR3 (SEQ ID NO:157) of antibody AM-17;r. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;s. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;t. a light chain CDR1 (SEQ ID NO:242), CDR2 (SEQ ID NO:243), CDR3 (SEQ ID NO:244) and a heavy chain CDR1 (SEQ ID NO:164), CDR2 (SEQ ID NO:165), CDR3 (SEQ ID NO:166) of antibody AM-20;u. a light chain CDR1 (SEQ ID NO:245), CDR2 (SEQ ID NO:246), CDR3 (SEQ ID NO:247) and a heavy chain CDR1 (SEQ ID NO:167), CDR2 (SEQ ID NO:168), CDR3 (SEQ ID NO:169) of antibody AM-21;v. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22;w. a light chain CDR1 (SEQ ID NO:251), CDR2 (SEQ ID NO:252), CDR3 (SEQ ID NO:253) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;x. a light chain CDR1 (SEQ ID NO:254), CDR2 (SEQ ID NO:255), CDR3 (SEQ ID NO:256) and a heavy chain CDR1 (SEQ ID NO:173), CDR2 (SEQ ID NO:174), CDR3 (SEQ ID NO:175) of antibody AM-23;y. a light chain CDR1 (SEQ ID NO:257), CDR2 (SEQ ID NO:258), CDR3 (SEQ ID NO:259) and a heavy chain CDR1 (SEQ ID NO:176), CDR2 (SEQ ID NO:177), CDR3 (SEQ ID NO: 178) of antibody AM-24;z. a light chain CDR1 (SEQ ID NO:260), CDR2 (SEQ ID NO:261), CDR3 (SEQ ID NO:262) and a heavy chain CDR1 (SEQ ID NO:179), CDR2 (SEQ ID NO:180), CDR3 (SEQ ID NO:181) of antibody AM-25;z.2. a light chain CDR1 (SEQ ID NO:263), CDR2 (SEQ ID NO:264), CDR3 (SEQ ID NO:265) and a heavy chain CDR1 (SEQ ID NO:182), CDR2 (SEQ ID NO: 183), CDR3 (SEQ ID NO:184) of antibody AM-26; wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain and a heavy chain variable domain of AML1/AMH1 (SEQ ID NO:27/SEQ ID NO:1);b. a light chain variable domain and a heavy chain variable domain of AML2/AMH2 (SEQ ID NO:28/SEQ ID NO:2);c. a light chain variable domain and a heavy chain variable domain of AML3/AMH3 (SEQ ID NO:29/SEQ ID NO:3);d. a light chain variable domain and a heavy chain variable domain of AML4/AMH4 (SEQ ID NO:30/SEQ ID NO:4);e. a light chain variable domain and a heavy chain variable domain of AML5/AMH5 (SEQ ID NO:31/SEQ ID NO:5);f. a light chain variable domain and a heavy chain variable domain of AML6/AMH6 (SEQ ID NO:32/SEQ ID NO:6)g. a light chain variable domain and a heavy chain variable domain of AML7/AMH7 (SEQ ID NO:33/SEQ ID NO:7);h. a light chain variable domain and a heavy chain variable domain of AML8/AMH8 (SEQ ID NO:34/SEQ ID NO:8);i. a light chain variable domain and a heavy chain variable domain of AML9/AMH9 (SEQ ID NO:35/SEQ ID NO:9);j. a light chain variable domain and a heavy chain variable domain of AML10/AMH10 (SEQ ID NO:36/SEQ ID NO: 10);k. a light chain variable domain and a heavy chain variable domain of AML11/AMH11 (SEQ ID NO:37/SEQ ID NO: 11);L. a light chain variable domain and a heavy chain variable domain of AML12/AMH12 (SEQ ID NO:38/SEQ ID NO:12);m. a light chain variable domain and a heavy chain variable domain of AML13/AMH13 (SEQ ID NO:39/SEQ ID NO:13);n. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);o. a light chain variable domain and a heavy chain variable domain of AML15/AMH15 (SEQ ID NO:41/SEQ ID NO:15);p. a light chain variable domain and a heavy chain variable domain of AML16/AMH16 (SEQ ID NO:42/SEQ ID NO:16);q. a light chain variable domain and a heavy chain variable domain of AML17/AMH17 (SEQ ID NO:43/SEQ ID NO:17);r. a light chain variable domain and a heavy chain variable domain of AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18);s. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);t. a light chain variable domain and a heavy chain variable domain of AML20/AMH20 (SEQ ID NO:46/SEQ ID NO:20);u. a light chain variable domain and a heavy chain variable domain of AML21/AMH21 (SEQ ID NO:47/SEQ ID NO:21);v. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22);w. a light chain variable domain and a heavy chain variable domain of AML23/AMH23 (SEQ ID NO: 49 or SEQ ID NO:50/SEQ ID NO:23);x. a light chain variable domain and a heavy chain variable domain of AML24/AMH24 (SEQ ID NO:51/SEQ ID NO:24);y. a light chain variable domain and a heavy chain variable domain of AML25/AMH25 (SEQ ID NO:52/SEQ ID NO:25);z. a light chain variable domain and a heavy chain variable domain of AML26/AMH26 (SEQ ID NO:53/SEQ ID NO:26);wherein said antibody specifically binds to human IL-17RA. Embodiment 152: the method of embodiment 151, wherein said disease state selected from the group consisting of: inflammation, autoimmune disease, cartilage inflammation, and/or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis. Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barre disease, Type I diabetes mellitus. Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, and graft versus host disease (GVHD). Embodiment 153: the method of embodiment 151 further comprising administering to said subject a second treatment comprising a pharmaceutical composition. Embodiment 154: the method of embodiment 153, wherein said second pharmaceutical composition is selected from the group consisting of: TNF inhibitors, soluble TNF receptors, Etanercept, ENBREL®, soluble TNF receptor type-I and soluble TNF receptor type-II, monomeric or multimeric p75 and/or p55 TNF receptor molecules and fragments thereof, anti-TNF antibodies, Infliximab, REMICADE®, D2E7, or HUMIRA®, IL-1 inhibitors, IL-1 receptor inhibitors, CD28 inhibitors, non-steroidal anti-inflammatory drugs (NSAID), a slow acting antirheumatic drugs (SAARD), and disease modifying antirheumatic drugs (DMARD). Embodiment 155: a method of inhibiting the production of at least one cytokine, chemokine, matrix metalloproteinase, or other molecule associated with IL-17RA activation, comprising administering the antibody of embodiment 151 to a patient in need thereof. Embodiment 156: the method of embodiment 155, wherein said cytokine, chemokine, matrix metalloproteinase, or other molecule is selected from the group consisting of: IL-6, IL-8, CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-1β, TNFα, RANK-L, LIF, PGE2, IL-12, MMP3, MMP9, GROα, NO, and C-telopeptide. Embodiment 157: a method of treating a disease state associated with IL-17RA activation in a subject in need thereof, comprising administering to said subject a composition comprising an antibody that specifically binds human IL-17 Receptor A and inhibits the binding of IL-17A and IL-17F or inhibits the binding of IL-17A or IL-17F. Embodiment 158: the method of embodiment 157, wherein said antibody is selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML14, 18, 19, and 22 (SEQ ID NOs: 40, 44, 45, and 48 respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH14, 18, 19, and 22 (SEQ ID NOs:14, 18, 19, and 22 respectively);c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;b. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;c. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;d. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22; wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);b. a light chain variable domain and a heavy chain variable domain of AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18);c. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);d. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22); wherein said antibody specifically binds to human IL-17RA. Embodiment 159: the method of embodiment 157, wherein said disease state is the disease state of claim 152. Embodiment 160: a method of inhibiting the production of at least one cytokine, chemokine, matrix metalloproteinase, or other molecule associated with IL-17RA activation, comprising administering the antibody of embodiment 157 to a patient in need thereof. Embodiment 161: the method of embodiment 160, wherein said cytokine, chemokine, matrix metalloproteinase, or other molecule is selected from the group consisting of: IL-6, IL-8, CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-1β, TNFα, RANK-L, LIF, PGE2, IL-12, MMP3, MMP9, GROα, NO, and C-telopeptide. Embodiment 162: a method of treating inflammation and autoimmune disease in a patient in need thereof comprising administering to said patient a composition comprising an antibody selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence of AML14, 18, 19, and 22 (SEQ ID NOs: 40, 44, 45, and 48 respectively);b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of AMH14, 18, 19, and 22 (SEQ ID NOs:14, 18, 19, and 22 respectively);c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148) of antibody AM-14;b. a light chain CDR1 (SEQ ID NO:236), CDR2 (SEQ ID NO:237), CDR3 (SEQ ID NO:238) and a heavy chain CDR1 (SEQ ID NO:158), CDR2 (SEQ ID NO:159), CDR3 (SEQ ID NO:160) of antibody AM-18;c. a light chain CDR1 (SEQ ID NO:239), CDR2 (SEQ ID NO:240), CDR3 (SEQ ID NO:241) and a heavy chain CDR1 (SEQ ID NO:161), CDR2 (SEQ ID NO:162), CDR3 (SEQ ID NO:163) of antibody AM-19;d. a light chain CDR1 (SEQ ID NO:248), CDR2 (SEQ ID NO:249), CDR3 (SEQ ID NO:250) and a heavy chain CDR1 (SEQ ID NO:170), CDR2 (SEQ ID NO:171), CDR3 (SEQ ID NO:172) of antibody AM-22; wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain and a heavy chain variable domain of AML14/AMH14 (SEQ ID NO:40/SEQ ID NO:14);b. a light chain variable domain and a heavy chain variable domain of AML18/AMH18 (SEQ ID NO:44/SEQ ID NO:18);c. a light chain variable domain and a heavy chain variable domain of AML19/AMH19 (SEQ ID NO:45/SEQ ID NO:19);d. a light chain variable domain and a heavy chain variable domain of AML22/AMH22 (SEQ ID NO:48/SEQ ID NO:22); wherein said antibody specifically binds to human IL-17RA. Embodiment 163: the method of embodiment 162, wherein said inflammation and autoimmune disease is selected from the group consisting of: arthritis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis, plaque psoriasis, dermatitis, atopic dermatitis, systemic lupus erythematosus, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis, asthma, and chronic obstructive pulmonary disease. Embodiment 164: the method of claim 151, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 165: the method of embodiment 158, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 166: the method of claim 151, wherein said antibody is selected from the group consisting of:A. an isolated antibody, or IL-17RA-binding fragment thereof, comprisinga. a light chain variable domain sequence that is at least 80% identical to a light chain variable domain sequence SEQ ID NO: 40;b. a heavy chain variable domain sequence that is at least 80% identical to a heavy chain variable domain sequence of SEQ ID NO:14;c. the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein said antibody specifically binds to human IL-17RA;B. an isolated antibody, or IL-17RA-binding fragment thereof, comprising a light chain CDR1 (SEQ ID NO:224), CDR2 (SEQ ID NO:225), CDR3 (SEQ ID NO:226) and a heavy chain CDR1 (SEQ ID NO:146), CDR2 (SEQ ID NO:147), CDR3 (SEQ ID NO:148); wherein said antibody specifically binds to human IL-17RA; andC. an isolated antibody, or IL-17RA-binding fragment thereof, comprising a light chain variable domain of SEQ ID NO:40 and a heavy chain variable domain SEQ ID NO:14; wherein said antibody specifically binds to human IL-17RA. Embodiment 167: the method of embodiment 166, wherein said disease state is rheumatoid arthritis. Embodiment 168: the method of embodiment 166, wherein said disease state is psoriasis. Embodiment 169: the method of embodiment 166, wherein said disease state is inflammatory bowel disease. Embodiment 170: the method of embodiment 166, wherein said disease state is asthma. Embodiment 171: the method of embodiment 166, wherein said antibody comprises a light chain variable domain of SEQ ID NO:40 and a heavy chain variable domain SEQ ID NO: 14; wherein said antibody specifically binds to human IL-17RA. Embodiment 172: the method of embodiment 166, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 173: the method of claim 171, wherein said antibody is selected from the group consisting of: a. a humanized antibody; b. a chimeric antibody; c. a recombinant antibody; d. a single chain antibody; e. a diabody; f. a triabody; g. a tetrabody; h. a Fab fragment; i. a F(ab′)2 fragment; j. an IgD antibody; k. an IgE antibody; l. an IgM antibody; m. an IgG1 antibody; n. an IgG2 antibody; o. an IgG3 antibody; and p. an IgG4 antibody. Embodiment 174: the method of embodiment 167, wherein said antibody comprises a light chain sequence of SEQ ID NO:429 and a heavy chain sequence of SEQ ID NO:427. Embodiment 175: the method of claim 168, wherein said antibody comprises a light chain sequence of SEQ ID NO:429 and a heavy chain sequence of SEQ ID NO:427. It is understood that the above-described methods also encompasses comparable methods for first and second medical uses and claims thereto, as described elsewhere in this specification. Chronic viral hepatitis affects over 500 million people worldwide, including approximately 10 million in the U.S. and Europe with chronic hepatitis C infections. A significant proportion of chronic hepatitis patients develop progressive liver fibrosis and/or hepatocellular carcinoma. While viral hepatitis vaccines are available or in development, current therapy for infected individuals relics on long courses of the combination of antiviral drugs and interferon-alpha (INF-α). INF-α is thought to be beneficial in treating viral hepatitis through its proven antiviral immunological activities and antiproliferative effects on fibroblasts, but the duration and level of its use is limited by severe side effects. Recent data describes how INF-α may be directly apoptotic for Th17 cells (American Association for Immunologists, abstract no. 42.8, May 12-16, 2006, Boston). Th17 cells are a distinct subset of CD4+ T-cells responsible for producing IL-17A and IL-17F in response to IL-23 (Harrington, et al.,Nature Imm,2005 vol. 6, no. 11, 1123-1132 and Park, et al.,Nature Imm,2005 vol. 6, no. 11, 1133-1141). We believe this suggests a new mechanism of action for INF-α in chronic viral hepatitis that does not involve direct action of INF-α on virus or fibroblasts, but indirect actions on Th17 cells. Furthermore, it has recently been discovered that Tumor Growth Factor-Beta (TGF-β) and/or IL-6, (see for example, Kimera, A., et al., PNAS U.S.A., 2007 Jul. 17; 104(29):12099-104), both pro-fibrotic cytokine, also induces the development of TH17 cells by upregulating IL-23 receptor expression and thereby conferring responsiveness to IL-23 ((Mangan, et al.,Nature,2006 vol. 441 no. 11, 231-234). Responsiveness to IL-23 induces the differentiation of naïve CD4+ T-cells into TH17 cells. As mentioned above, the TH17 cells are responsible for releasing IL-17A and IL-17F, and IL-17A is known to have various stimulatory effects on fibroblasts in a number of tissues and organs. Taken together, we believe that inhibition of the IL-17RA-IL-17A/IL-17F pathway may offer a therapeutic benefit in the progressive fibrosis of chronic viral hepatitis. An added benefit of inhibiting the IL-17RA-IL-17A/IL-17F pathway in the treatment of viral hepatitis is that one may reduce the dosage of INF-α given to the patient and consequently limit the deleterious side effects associated with INF-α therapy. A further benefit of inhibiting the IL-17RA-IL-17A/IL-17F pathway in the treatment of viral hepatitis is the possibility of achieving a synergistic therapeutic effect with INF-α therapy in combination with IL-17RA-IL-17A/IL-17F antagonist therapy, or other antagonists as described in more detail below. Therefore, aspects of the invention are drawn to methods of treating the pathology associated with viral hepatitis by inhibiting the interaction between IL-17RA and IL-17A and/or IL-17F. Further aspects of the invention are drawn to methods of inhibiting fibrosis by inhibiting the interaction between IL-17RA and IL-17A and/or IL-17F. Further aspects of the invention are drawn to methods of treating fibrosis associated with viral hepatitis by inhibiting the interaction between IL-17RA and IL-17A and/or IL-17F. Antagonists of the IL-17RA-IL-17A/IL-17F pathway may be used to inhibit the interaction between IL-17RA and IL-17A and/or IL-17F. Antagonists of the IL-17RA-IL-17A pathway include the IL-17RA antigen binding proteins described herein, as well as IL-17RA proteins (as well as biologically active fragments and fusion proteins thereof, such as IL-17RA-Fc fusion proteins), as well as antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to IL-17A and inhibit IL-17A from activating IL-17RA, as well as antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to IL-17F and inhibit IL-17F from activating IL-17RA. Additional aspects are drawn to methods of treating the pathology associated with viral hepatitis by antagonizing the IL-23-IL-23 receptor (IL-23R) pathway. Further aspects of the invention are drawn to methods of inhibiting fibrosis by antagonizing the IL-23-IL-23R pathway. Further aspects of the invention are drawn to methods of treating fibrosis associated with viral hepatitis by antagonizing the IL-23-IL-23R pathway. By antagonizing the IL-23-IL-23R pathway, one prevents the IL-23-induced differentiation of the TH17 cells and thereby ultimately limit the amount of circulating IL-17A and IL-17F, which may reduce the pathology associated with viral hepatitis. Antagonists to the IL-23-IL-23R pathway include antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to IL-23 and block IL-23 from activating IL-23R. Additional antagonists to IL-23-IL-23R pathway include antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to IL-23R and block IL-23 from activating IL-23R. Additional antagonists to IL-23-IL-23R pathway include IL-23R proteins, as well as biologically active fragments and fusion proteins thereof, such as IL-23R-Fc fusion proteins, that bind IL-23 and block IL-23 from activating IL-23R. Additional aspects are drawn to methods of treating the pathology associated with viral hepatitis by antagonizing the TGF-β-TGF-βRI/TGF-βRII pathway. Further aspects of the invention are drawn to methods of inhibiting fibrosis by antagonizing the TGF-β-TGF-βRI/TGF-βRII pathway. Further aspects of the invention are drawn to methods of treating fibrosis associated with viral hepatitis by antagonizing the TGF-β-TGF-βRI/TGF-βRII pathway. By antagonizing the TGF-β-TGF-βRI/TGF-βRII pathway, one prevents the TGF-β-induced development of the TH17 cells and thereby ultimately limit the amount of circulating IL-17A and IL-17F, which may reduce the pathology associated with viral hepatitis. Antagonists to the TGF-β-TGF-βRI/TGF-βRII pathway include antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to TGF-β and block TGF-β from activating TGF-βRI and/or TGF-βRII. Additional antagonists to the TGF-β-TGF-βRI/TGF-βRII pathway include antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to TGF-βRI or TGF-βRII and block TGF-β from activating TGF-βRI or TGF-βRII. Additional aspects are drawn to methods of treating the pathology associated with viral hepatitis by antagonizing the IL-6-IL-6R pathway. Further aspects of the invention are drawn to methods of inhibiting fibrosis by antagonizing the IL-6-IL-6R pathway. Further aspects of the invention are drawn to methods of treating fibrosis associated with viral hepatitis by antagonizing the IL-6-IL-6R pathway. By antagonizing the IL-6-IL-6R pathway, one may reduce the pathology associated with viral hepatitis. Antagonists to the IL-6-IL-6R pathway include antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to IL-6 and block IL-6 from activating IL-6R. Additional antagonists to the IL-6-IL-6R pathway include antigen binding proteins, such as antibodies and biologically active fragments thereof, that bind to IL-6R and block IL-6 from activating IL-6R. Further aspects include combination therapy using the antagonists of the IL-17RA-IL-17A/IL-17F pathway, IL-23-IL-23R pathway, TGF-β-TGF-βRI/TGF-βRII pathway, and/or the IL-6-IL-6R pathway mentioned above in combination with each other, as well as in combination with art-recognized hepatitis therapies, such as but not limited to, interferon, and in particular INF-α. All permutations of these combinations are envisioned. Further aspects include combination therapy using the antagonists of the IL-17RA-IL-17A/IL-17F pathway, IL-23-IL-23R pathway, TGF-β-TGF-βRI/TGF-βRII pathway, and/or the IL-6-IL-6R pathway mentioned above in combination with each other, as well as in combination with art-recognized hepatitis therapies, such as but not limited to, interferon, and in particular INF-α, as well as with antiviral agents, such as but not limited to Adefovir dipivoxil, acyclic analogues of deoxyadenosine monophosphate (Adefovir, Tenofovir disoproxil fumarate), (−) enantiomer of the deoxycytidine analogue 2′-deoxy-3′-thiacytidine (Lamivudine), carbocyclic deoxyguanosine analogues (Entecavir), L-nucleosides (β-L-2′-Deoxythymidine, β-L-2′-deoxycytidine, and β-L-2′-deoxyadenosine), [(−)-β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine] (Emtricitabine), 1-β-2,6-Diaminopurine dioxalane (DAPD, amdoxovir), 2′-Fluoro-5-methyl-β-L-arabinofuranosyluridine (L-FMAU, clevudine), Famciclovir, and/or Penciclovir. All permutations of these combinations are envisioned. Diagnostic Methods The antigen binding proteins of the invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or conditions associated with IL-17A or IL-17RA. The invention provides for the detection of the presence of IL-17RA in a sample using classical immunohistological methods known to those of skill in the art (e.g., Tijssen, 1993. Practice and Theory of Enzyme Immunoassays, vol 15 (Eds R. H. Burdon and P. H. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol.101:976-985; Jalkanen et al., 1987, J. Cell Biol.105:3087-3096). The detection of IL-17RA can be performed in vivo or in vitro. Diagnostic applications provided herein include use of the antigen binding proteins to detect expression of IL-17RA and binding of the ligands to IL-17RA. Examples of methods useful in the detection of the presence of IL-17RA include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For diagnostic applications, the antigen binding protein typically will be labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y,99Tc,111In,125I,131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labelling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labelling proteins are known in the art and may be used in performing the present invention. One aspect of the invention provides for identifying a cell or cells that express IL-17RA. In a specific embodiment, the antigen binding protein is labeled with a labeling group and the binding of the labeled antigen binding protein to IL-17RA is detected. In a further specific embodiment, the binding of the antigen binding protein to IL-17RA detected in vivo. In a further specific embodiment, the antigen binding protein-IL-17RA is isolated and measured using techniques known in the art. See, for example,Harlow and Lane,1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodic supplements); John E. Coligan, ed., 1993, Current Protocols n ImmunologyNew York: John Wiley & Sons. Another aspect of the invention provides for detecting the presence of a test molecule that competes for binding to IL-17RA with the antigen binding proteins of the invention. An example of one such assay would involve detecting the amount of free antigen binding protein in a solution containing an amount of IL-17RA in the presence or absence of the test molecule. An increase in the amount of free antigen binding protein (i.e., the antigen binding protein not bound to IL-17RA) would indicate that the test molecule is capable of competing for IL-17RA binding with the antigen binding protein. In one embodiment, the antigen binding protein is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence and absence of an antigen binding protein. Aspects of the invention include the use of the IL-17RA antigen binding proteins in in vitro assays for research purposes, such as to inhibit production of molecules such as but is not limited to: IL-6, IL-8, CXCL1, CXCL2, GM-CSF, G-CSF, M-CSF, IL-10, TNFα, RANK-L, LIF, PGE2, IL-12, MMPs (such as but not limited to MMP3 and MMP9), GROα, NO, and/or C-telopeptide and the like. Antibodies directed against an IL-17RA can be used, for example, in purifying IL-17RA proteins by immunoaffinity chromatography. Methods of Treatment: Pharmaceutical Formulations, Routes of Administration In some embodiments, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or a plurality of the antigen binding proteins of the invention together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant. In addition, the invention provides methods of treating a patient by administering such pharmaceutical composition. The term “patient” includes human and animal subjects. Pharmaceutical compositions comprising one or more antigen binding proteins may be used to reduce IL-17RA activity. Pharmaceutical compositions comprising one or more antigen binding proteins may be used in treating the consequences, symptoms, and/or the pathology associated with IL-17RA activity. Pharmaceutical compositions comprising one or more antigen binding proteins may be used in methods of inhibiting binding and/or signaling of IL-17A and/or IL-17F to IL-17RA comprising providing the antigen binding protein of the invention to IL-17RA. In certain embodiments, the antigen binding protein inhibits binding and/or signaling of IL-17A and IL-17F to IL-17RA. In additional embodiments, pharmaceutical compositions comprising one or more antigen binding proteins may be used in methods of inhibiting binding and/or signaling of IL-17A but not IL-17F to IL-17RA. In other embodiments, pharmaceutical compositions comprising one or more antigen binding proteins may be used in methods of inhibiting binding and/or signaling of IL-17F and not IL-17A to IL-17RA. Aspects of the invention include antibodies that specifically bind to human IL-17RA and inhibit IL-17A and/or IL-17F from binding and activating IL-17RA, or a heteromeric complex of IL-17RA and IL-17RC. Aspects of the invention include antibodies that specifically bind to human IL-17RA and inhibit an IL-17A/IL-17F heteromer from binding and activating IL-17RA, or a heteromeric complex of IL-17RA and IL-17RC. Throughout the specification, when reference is made to inhibiting IL-17A and/or IL-17F, it is understood that this also includes inhibiting heteromers of IL-17A and IL-17F. Aspects of the invention include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming either a homomeric or heteromeric functional receptor complex, such as, but not limited to IL-17RA-IL-17RC complex. Aspects of the invention include antibodies that specifically bind to human IL-17RA and partially or fully inhibit IL-17RA from forming either a homomeric or heteromeric functional receptor complex, such as, but not limited to IL-17RA/IL-17RC complex and do not necessarily inhibit IL-17A and/or IL-17F or an IL-17A/IL-17F heteromer from binding to IL-17RA or a IL-17RA heteromeric receptor complex. Pharmaceutical compositions comprising one or more antigen binding proteins may be used in methods of treating the consequences, symptoms, and/or the pathology associated with IL-17RA activity. Pharmaceutical compositions comprising one or more antigen binding proteins may be used in methods of inhibiting the production of one or more of an inflammatory cytokine, chemokine, matrix metalloproteinase, or other molecule associated with IL-17RA activation, comprising administering an IL-17RA antigen binding protein. Pharmaceutical compositions comprising one or more antigen binding proteins may be used in methods of inhibiting production of IL-6, IL-8, GM-CSF, NO, MMPs, PGE2 RANKL, and/or C-telopeptide, and the like. Pharmaceutical compositions comprising one or more antigen binding proteins may be used to treat diseases and conditions including, but are not limited to, inflammation, autoimmune disease, cartilage inflammation, and/or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus crythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barre disease, Type I diabetes mellitus, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, and the like Preferably, acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprising a therapeutically effective amount of IL-17RA antigen binding proteins are provided. In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, Triton (octylphenol ethoxylate), tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition, (A. R. Gemmo, ed.), 1990, Mack Publishing Company. In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antigen binding proteins of the invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In specific embodiments, pharmaceutical compositions comprise Tris (Tris(hydroxymethyl) aminomethane) buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute therefor. In certain embodiments of the invention, IL-17RA antigen binding protein compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, the IL-17RA antigen binding protein product may be formulated as a lyophilizate using appropriate excipients such as sucrose. The pharmaceutical compositions of the invention can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally. Preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are present preferably in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. When parenteral administration is contemplated, the therapeutic compositions for use in this invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired IL-17RA antigen binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the IL-17RA antigen binding protein is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which can be delivered via depot injection. In certain embodiments, hyaluronic acid may also be used, having the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired antigen binding protein. Pharmaceutical compositions of the invention can be formulated for inhalation. In these embodiments, IL-17RA antigen binding proteins are advantageously formulated as a dry, inhalable powder. In specific embodiments, IL-17RA antigen binding protein inhalation solutions may also be formulated with a propellant for aerosol delivery. In certain embodiments, solutions may be nebulized. Pulmonary administration and formulation methods therefore are further described in International Patent Application No. PCT/US94/001875, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins. It is also contemplated that formulations can be administered orally. IL-17RA antigen binding proteins that are administered in this fashion can be formulated with or without carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain embodiments, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the IL-17RA antigen binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed. A pharmaceutical composition of the invention is preferably provided to comprise an effective quantity of one or a plurality of IL-17RA antigen binding proteins in a mixture with nontoxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc. Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving IL-17RA antigen binding proteins in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT/US93/00829, which is incorporated by reference and describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated by reference), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res.15:167-277 and Langer, 1982, Chem. Tech.12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A.82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949, incorporated by reference. Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Aspects of the invention includes self-buffering IL-17RA antigen binding protein formulations, which can be used as pharmaceutical compositions, as described in international patent application WO 06138181A2 (PCT/US2006/022599), which is incorporated by reference in its entirety herein. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein in which the total salt concentration is less than 150 mM. One embodiment provides self-buffering IL-17RA antigen binding protein formulations that further comprise an IL-17RA antigen binding protein and one or more polyols and/or one or more surfactants. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein, in which the total salt concentration is less than 150 mM, that further comprise one or more excipients, including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); surfactants, polyols, anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein and one or more other pharmaceutically active agents. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein, wherein the IL-17RA antigen binding protein has a buffer capacity per unit volume per pH unit of at least that of approximately: 2.0 or 3.0 or 4.0 or 5.0 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or 3,000 or 4,000 or 5,000 mM sodium acetate buffer in pure water over the range of pH 5.0 to 4.0 or pH 5.0 to 5.5, or at least 2.0 mM, or at least 3.0 mM, or at least 4.0 mM or at least 5.0 mM, or at least 7.5 mM, or at least 10 mM, or at least 20 mM. One embodiment provides self-buffering IL-17RA antigen binding protein formulations wherein, exclusive of the buffer capacity of the protein, the buffer capacity per unit volume per pH unit of the formulation is equal to or less than that of 1.0 or 1.5 or 2.0 or 3.0 or 4.0 or 5.0 mM sodium acetate buffer in pure water over the range of pH 4.0 to 5.0 or pH 5.0 to 5.5, or optionally less than that of 1.0 mM, optionally less than that of 2.0 mM, optionally less than that of 2.5 mM, optionally less than that of 3.0 mM, and optionally less than that of 5.0 mM. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein over the range of plus or minus 1 pH unit from the pH of the formulation, the buffer capacity of the IL-17RA antigen binding protein is at least approximately: 1.00 or 1.50 or 1.63 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or 3,000 or 4,000 or 5,000 mEq per liter per pH unit, optionally at least approximately 1.00, optionally at least approximately 1.50, optionally at least approximately 1.63, optionally at least approximately 2.00, optionally at least approximately 3.00, optionally at least approximately 5.0, optionally at least approximately 10.0, and optionally at least approximately 20.0. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein over the range of plus or minus 1 pH unit from the pH of the formulation, exclusive of the IL-17RA antigen binding protein, the buffer capacity per unit volume per pH unit of the formulation is equal to or less than that of 0.50 or 1.00 or 1.50 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 20.0 or 25.0 mM sodium acetate buffer in pure water over the range pH 5.0 to 4.0 or pH 5.0 to 5.5. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein over a range of plus or minus 1 pH1 unit from a desired pH, the protein provides at least approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the buffer capacity of the formulation, optionally at least approximately 75%, optionally at least approximately 85%, optionally at least approximately 90%, optionally at least approximately 95%, optionally at least approximately 99% of the buffer capacity of the formulation. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein the concentration of the IL-17RA antigen binding protein is between approximately: 20 and 400, or 20 and 300, or 20 and 250, or 20 and 200, or 20 and 150 mg/ml, optionally between approximately 20 and 400 mg/ml, optionally between approximately 20 and 250, and optionally between approximately 20 and 150 mg/ml. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein the pH maintained by the buffering action of the IL-17RA antigen binding protein is between approximately: 3.5 and 8.0, or 4.0 and 6.0, or 4.0 and 5.5, or 4.0 and 5.0, optionally between approximately 3.5 and 8.0, and optionally between approximately 4.0 and 5.5. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein the salt concentration is less than: 150 mM or 125 mM or 100 mM or 75 mM or 50 mM or 25 mM, optionally 150 mM, optionally 125 mM, optionally 100 mM, optionally 75 mM, optionally 50 mM, and optionally 25 mM. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein and one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein and one or more pharmaceutically acceptable polyols in an amount that is hypotonic, isotonic, or hypertonic, preferably approximately isotonic, particularly preferably isotonic, such as but not limited to any one or more of sorbitol, mannitol, sucrose, trehalose, or glycerol, optionally approximately 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose, or 2.5% glycerol. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein further comprising a surfactant, preferably one or more of polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan, polyethoxylates, and poloxamer 188, preferably polysorbate 20 or polysorbate 80, optionally approximately 0.001 to 0.1% polysorbate 20 or polysorbate 80, optionally approximately 0.002 to 0.02% polysorbate 20 or polysorbate 80, or optionally 0.002 to 0.02% polysorbate 20 or polysorbate 80. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein wherein the formulation is sterile and suitable for treatment of a human or non-human subject. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein and a solvent, the IL-17RA antigen binding protein having a buffer capacity per unit volume per pH unit of at least that of 4.0 mM sodium acetate in water over the range of pH 4.0 to 5.0 or pH 5.0 to 5.5, wherein the buffer capacity per unit volume of the formulation exclusive of the IL-17RA antigen binding protein is equal to or less than that of 2.0 mM sodium acetate in water over the same ranges preferably determined in the same way. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein and a solvent, wherein at the pH of the formulation the buffer capacity of the protein is at least 1.63 mEq per liter for a pH change of the formulation of plus or minus 1 pH unit wherein the buffer capacity of the formulation exclusive of the protein is equal to or less than 0.81 mEq per liter at the pH of the formulation for a pH change of plus or minus 1 pH unit. One embodiment provides self-buffering IL-17RA antigen binding protein formulations comprising an IL-17RA antigen binding protein, wherein the formulation is in the form of a lyophilate which upon reconstitution provides a formulation in accordance with any of the foregoing or following. One embodiment provides self-buffering IL-17RA antigen binding protein formulations in a kit comprising one or more vials containing a self-buffering IL-17RA antigen binding protein formulation or a lyophilate of a self-buffering IL-17RA antigen binding protein formulation in accordance with any of the foregoing or the following, and instructions regarding use thereof. One embodiment provides a process for preparing a self-buffering IL-17RA antigen binding protein formulation or a lyophilate thereof according to any of the foregoing or the following, comprising removing residual buffer using a counter ion. One embodiment provides a process for preparing a self-buffering IL-17RA antigen binding protein formulation or a lyophilate thereof according to any of the foregoing or the following, comprising removing residual buffer using any one or more of the following in the presence of a counter ion: chromatography, dialysis, and/or tangential flow filtration. One embodiment provides a process for preparing a self-buffering IL-17RA antigen binding protein formulation or a lyophilate thereof according to any of the foregoing or the following, comprising removing residual buffer using tangential flow filtration. One embodiment provides a process for preparing a self-buffering IL-17RA antigen binding protein formulation or a lyophilate thereof according to any of the foregoing or the following comprising a step of dialysis against a solution at a pH1 below that of the preparation, and, if necessary, adjusting the pH thereafter by addition of dilute acid or dilute base. As discussed above, certain embodiments provide self-buffering IL-17RA antigen binding proteins protein compositions, particularly pharmaceutical IL-17RA antigen binding protein compositions, that comprise, in addition to the IL-17RA antigen binding protein, one or more excipients such as those illustratively described in this section and elsewhere herein. Excipients can be used in the invention in this regard for a wide variety of purposes, such as adjusting physical, chemical, or biological properties of formulations, such as adjustment of viscosity, and or processes of the invention to improve effectiveness and or to stabilize such formulations and processes against degradation and spoilage due to, for instance, stresses that occur during manufacturing, shipping, storage, pre-use preparation, administration, and thereafter. A variety of expositions are available on protein stabilization and formulation materials and methods useful in this regard, such as Arakawa et al., “Solvent interactions in pharmaceutical formulations,”Pharm Res.8(3): 285-91 (1991); Kendrick et al., “Physical stabilization of proteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology.13: 61-84 (2002), and Randolph et al., “Surfactant-protein interactions,”Pharm Biotechnol.13: 159-75 (2002), each of which is herein incorporated by reference in its entirety, particularly in parts pertinent to excipients and processes of the same for self-buffering protein formulations in accordance with the current invention, especially as to protein pharmaceutical products and processes for veterinary and/or human medical uses. Various excipients useful in the invention are listed in TABLE 3 and further described below. TABLE 3Types of Excipients and Their FunctionsFunctionTypeLiquidsLyophilatesTonicityProvides isotonicity to the formulation suchStabilizers include cryo and lyoprotectantsAgents/that it is suitable for injectionExamples include polyols, sugars andStabilizersExamples include polyols, salts, and aminopolymersacidsCryoprotectants protect proteins fromHelp maintain the protein in a more compactfreezing stressesstate (polyols)Lyoprotectants stabilize proteins in theMinimize electrostatic, solution protein-freeze-dried stateprotein interactions (salts)BulkingNot applicableUsed to enhance product elegance and toAgentsprevent blowoutProvides structural strength to the lyo cakeExamples include mannitol and glycineSurfactantsPrevent/control aggregation, particleEmployed if aggregation during theformation and surface adsorption of druglyophilization process is an issueExamples include polysorbate 20 and 80May serve to reduce reconstitution timesExamples include polysorbate 20 and 80Anti-oxidantsControl protein oxidationUsually not employed, molecular reactions inthe lyophilized cake are greatly retardedMetalA specific metal ion is included in a liquidMay be included if a specific metal ion isIons/formulation only as a co-factorincluded only as a co-factorChelatingDivalent cations such as zinc andChelating agents are generally not needed inAgentsmagnesium are utilized in suspensionlyophilized formulationsformulationsChelating agents are used to inhibit heavymetal ion catalyzed reactionsPreservativesImportant particularly for multi-doseFor multi-dose formulations onlyformulationsProvides protection against microbial growthProtects against microbial growth,in formulationExample: benzyl alcoholIs usually included in the reconstitutiondiluent (e.g. bWFI) Salts may be used in accordance with certain embodiments of the invention to, for example, adjust the ionic strength and/or the isotonicity of a self-buffering formulation and/or to improve the solubility and/or physical stability of a self-buffering protein or other ingredient of a self-buffering protein composition in accordance with the invention. As is well known, ions can stabilize the native state of proteins by binding to charged residues on the protein's surface and by shielding charged and polar groups in the protein and reducing the strength of their electrostatic interactions, attractive, and repulsive interactions. Ions also can stabilize the denatured state of a protein by binding to, in particular, the denatured peptide linkages (—CONH) of the protein. Furthermore, ionic interaction with charged and polar groups in a protein also can reduce intermolecular electrostatic interactions and, thereby, prevent or reduce protein aggregation and insolubility. Ionic species differ significantly in their effects on proteins. A number of categorical rankings of ions and their effects on proteins have been developed that can be used in formulating self-buffering protein compositions in accordance with the invention. One example is the Hofmeister series, which ranks ionic and polar non-ionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are referred to as “kosmotropic.” Destabilizing solutes are referred to as chaotropic. Kosmotropes commonly are used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate proteins from solution (“salting-out”). Chaotropes commonly are used to denture and/or to solubilize proteins (“salting-in”). The relative effectiveness of ions to “salt-in” and “salt-out” defines their position in the Hofmeister series. In addition to their utilities and their drawbacks (as discussed above) salts also are effective for reducing the viscosity of protein formulations and can be used in the invention for that purpose. In order to maintain isotonicity in a parenteral formulation in accordance with preferred embodiments of the invention, improve protein solubility and/or stability, improve viscosity characteristics, avoid deleterious salt effects on protein stability and aggregation, and prevent salt-mediated protein degradation, the salt concentration in self-buffering formulations in accordance with various preferred embodiments of the invention are less than 150 mM (as to monovalent ions) and 150 mEq/liter for multivalent ions. In this regard, in certain particularly preferred embodiments of the invention, the total salt concentration is from about 75 mEq/L to about 140 mEq/L. Free amino acids can be used in self-buffering IL-17RA antigen binding protein formulations in accordance with various embodiments of the invention as bulking agents, stabilizers, and antioxidants, as well as other standard uses. However, amino acids included in self-buffering IL-17RA antigen binding protein formulations do not provide buffering action. For this reason, those with significant buffer capacity either are not employed, are not employed at any pH around which they have significant buffering activity, or are used at low concentration so that, as a result, their buffer capacity in the formulation is not significant. This is particularly the case for histidine and other amino acids that commonly are used as buffers in pharmaceutical formulations. Subject to the foregoing consideration, lysine, proline, serine, and alanine can be used for stabilizing proteins in a formulation. Glycine is useful in lyophilization to ensure correct cake structure and properties. Arginine may be useful to inhibit protein aggregation, in both liquid and lyophilized formulations. Methionine is useful as an antioxidant. Polyols include sugars, e.g., mannitol, sucrose, and sorbitol and polyhydric alcohols such as, for instance, glycerol and propylene glycol, and, for purposes of discussion herein, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. They are useful stabilizing agents in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols also are useful for adjusting the tonicity of formulations. Among polyols useful in select embodiments of the invention is mannitol, commonly used to ensure structural stability of the cake in lyophilized formulations. It ensures structural stability to the cake. It is generally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucrose are among preferred agents for adjusting tonicity and as stabilizers to protect against freeze-thaw stresses during transport or the preparation of bulks during the manufacturing process. Reducing sugars (which contain free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they generally are not among preferred polyols for use in accordance with the invention. In addition, sugars that form such reactive species, such as sucrose, which is hydrolyzed to fructose and glucose under acidic conditions, and consequently engenders glycation, also is not among preferred amino acids of the invention in this regard. PEG is useful to stabilize proteins and as a cryoprotectant and can be used in the invention in this regard, such as it is in Recombinate®. Embodiments of the self-buffering IL-17RA antigen binding protein formulations further comprise surfactants. Protein molecules may be susceptible to adsorption on surfaces and to denaturation and consequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects generally scale inversely with protein concentration. These deleterious interactions generally scale inversely with protein concentration and typically are exacerbated by physical agitation, such as that generated during the shipping and handling of a product. Surfactants routinely are used to prevent, minimize, or reduce surface adsorption. Useful surfactants in the invention in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188. Surfactants also are commonly used to control protein conformational stability. The use of surfactants in this regard is protein-specific since, any given surfactant typically will stabilize some proteins and destabilize others. Polysorbates are susceptible to oxidative degradation and often, as supplied, contain sufficient quantities of peroxides to cause oxidation of protein residue side-chains, especially methionine. Consequently, polysorbates should be used carefully, and when used, should be employed at their lowest effective concentration. In this regard, polysorbates exemplify the general rule that excipients should be used in their lowest effective concentrations. Embodiments of the self-buffering IL-17RA antigen binding protein formulations further comprise one or more antioxidants. To some extent deleterious oxidation of proteins can be prevented in pharmaceutical formulations by maintaining proper levels of ambient oxygen and temperature and by avoiding exposure to light. Antioxidant excipients can be used as well to prevent oxidative degradation of proteins. Among useful antioxidants in this regard are reducing agents, oxygen/free-radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations in accordance with the invention preferably are water-soluble and maintain their activity throughout the shelf life of a product. EDTA is a preferred antioxidant in accordance with the invention in this regard and can be used in the invention in much the same way it has been used in formulations of acidic fibroblast growth factor and in products such as Kineret® and Ontak®. Antioxidants can damage proteins. For instance, reducing agents, such as glutathione in particular, can disrupt intramolecular disulfide linkages. Thus, antioxidants for use in the invention are selected to, among other things, eliminate or sufficiently reduce the possibility of themselves damaging proteins in the formulation. Formulations in accordance with the invention may include metal ions that are protein co-factors and that are necessary to form protein coordination complexes, such as zinc necessary to form certain insulin suspensions. Metal ions also can inhibit some processes that degrade proteins. However, metal ions also catalyze physical and chemical processes that degrade proteins. Magnesium ions (10-120 mM) can be used to inhibit isomerization of aspartic acid to isoaspartic acid. Ca+2ions (up to 100 mM) can increase the stability of human deoxyribonuclease (rhDNase, PulmozymeA). Mg+2, Mn+2, and Zn+2, however, can destabilize rhDNase. Similarly, Ca+2and Sr+2can stabilize Factor VIII, it can be destabilized by Mg+2, Mn+2and Zn+2, Cu+2and Fe+2, and its aggregation can be increased by Al+3ions. Embodiments of the self-buffering IL-17RA antigen binding protein formulations further comprise one or more preservatives. Preservatives are necessary when developing multi-dose parenteral formulations that involve more than one extraction from the same container. Their primary function is to inhibit microbial growth and ensure product sterility throughout the shelf-life or term of use of the drug product. Commonly used preservatives include benzyl alcohol, phenol and m-cresol. Although preservatives have a long history of use with small-molecule parenterals, the development of protein formulations that includes preservatives can be challenging. Preservatives almost always have a destabilizing effect (aggregation) on proteins, and this has become a major factor in limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single-use only. However, when multi-dose formulations are possible, they have the added advantage of enabling patient convenience, and increased marketability. A good example is that of human growth hormone (hGH) where the development of preserved formulations has led to commercialization of more convenient, multi-use injection pen presentations. At least four such pen devices containing preserved formulations of hGH are currently available on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) & Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol while Somatrope® (Eli Lilly) is formulated with m-cresol. Several aspects need to be considered during the formulation and development of preserved dosage forms. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in the dosage form with concentration ranges that confer anti-microbial effectiveness without compromising protein stability. For example, three preservatives were successfully screened in the development of a liquid formulation for interleukin-1 receptor (Type I) using differential scanning calorimetry (DSC). The preservatives were rank ordered based on their impact on stability at concentrations commonly used in marketed products. As might be expected, development of liquid formulations containing preservatives are more challenging than lyophilized formulations. Freeze-dried products can be lyophilized without the preservative and reconstituted with a preservative containing diluent at the time of use. This shortens the time for which a preservative is in contact with the protein, significantly minimizing the associated stability risks. With liquid formulations, preservative effectiveness and stability have to be maintained over the entire product shelf-life (˜18 to 24 months). An important point to note is that preservative effectiveness has to be demonstrated in the final formulation containing the active drug and all excipient components. Self-buffering IL-17RA antigen binding protein formulations generally will be designed for specific routes and methods of administration, for specific administration dosages and frequencies of administration, for specific treatments of specific diseases, with ranges of bio-availability and persistence, among other things. Formulations thus may be designed in accordance with the invention for delivery by any suitable route, including but not limited to orally, aurally, ophthalmically, rectally, and vaginally, and by parenteral routes, including intravenous and intraarterial injection, intramuscular injection, and subcutaneous injection. Compositions in accordance with the invention may be produced using well-known, routine methods for making, formulating, and using proteins, particularly pharmaceutical proteins. In certain of the preferred embodiments of a number of aspects of the invention in this regard, methods for preparing the compositions comprise the use of counter ions to remove residual buffering agents. In this regard the term counter ion is any polar or charged constituent that acts to displace buffer from the composition during its preparation. Counter ions useful in this regard include, for instance, glycine, chloride, sulfate, and phosphate. The term counter ion in this regard is used to mean much the same thing as displacement ion. Residual buffering agents can be removed using the counter ions in this regard, using a variety of well-known methods, including but not limited to, standard methods of dialysis and high performance membrane diffusion-based methods such as tangential flow diafiltration. Methods for residual buffer removal employing a counter ion in this regard can also, in some cases, be carried out using size exclusion chromatography. In certain related preferred embodiments in this regard, compositions in accordance with the invention are prepared by a process that involves dialysis against a bufferless solution at a pH below that of the preparation containing the self-buffering protein. In particularly preferred embodiments of the invention in this regard, the bufferless solution comprises counter ions, particularly those that facilitate removal of residual buffer and do not adversely affect the self-buffering protein or the formulation thereof. In further particularly preferred embodiments of the invention in this regard, following dialysis the pH of the preparation is adjusted to the desired pH using dilute acid or dilute base. In certain related particularly preferred embodiments in this regard, compositions in accordance with the invention are prepared by a process that involves tangential flow diafiltration against a bufferless solution at a pH below that of the preparation containing the self-buffering protein. In particularly preferred embodiments of the invention in this regard, the bufferless solution comprises counter ions, particularly those that facilitate removal of residual buffer and do not adversely affect the self-buffering protein or the formulation thereof. In further particularly preferred embodiments of the invention in this regard, following diafiltration the pH of the preparation is adjusted to the desired pH using dilute acid or dilute base. Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. The invention also provides kits for producing a single-dose administration unit. The kits of the invention may each contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of this invention, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided. The therapeutically effective amount of an IL-17RA antigen binding protein-containing pharmaceutical composition to be employed will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will vary depending, in part, upon the molecule delivered, the indication for which the IL-17RA antigen binding protein is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about 0.1 μg/kg to up to about 30 mg/kg or more, depending on the factors mentioned above. In specific embodiments, the dosage may range from 0.1 μg/kg up to about 30 mg/kg, optionally from 1 μg/kg up to about 30 mg/kg or from 10 μg/kg up to about 5 mg/kg. Dosing frequency will depend upon the pharmacokinetic parameters of the particular IL-17RA antigen binding protein in the formulation used. Typically, a clinician administers the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data. In certain embodiments, the antigen binding proteins of the invention can be administered to patients throughout an extended time period. Chronic administration of an antigen binding protein of the invention minimizes the adverse immune or allergic response commonly associated with antigen binding proteins that are not fully human, for example an antibody raised against a human antigen in a non-human animal, for example, a non-fully human antibody or non-human antibody produced in a non-human species. The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device. The composition also may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration. It also may be desirable to use IL-17RA antigen binding protein pharmaceutical compositions according to the invention ex vivo. In such instances, cells, tissues or organs that have been removed from the patient are exposed to IL-17RA antigen binding protein pharmaceutical compositions after which the cells, tissues and/or organs are subsequently implanted back into the patient. In particular, IL-17RA antigen binding proteins can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. In certain embodiments, such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic. In certain embodiments, the cells may be immortalized. In other embodiments, in order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues. In further embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues. All references cited within the body of the instant specification are hereby expressly incorporated by reference in their entirety. EXAMPLES The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not to be construed as limiting the invention. Example 1 IL-17RA knockout mice were generated as described in Ye et al., 2001, J. Exp. Med. 194:519-527 and tested in a standard collagen induced arthritis (CIA) model. Briefly, Genomic clones encoding murine IL-17R were isolated from a 129 derived lambda library using a murine IL-17R cDNA probe and mapped by a combination of PCR, restriction digest, and sequence analyses using deposited genomic sequences corresponding to IL-17R locus on mouse chromosome 6 (GenBank/EMBL/DDBJ accession no. AC018559). A gene targeting vector was constructed by replacing 5.7 kb of genomic sequence containing exons 4-11 (corresponding to nucleotides 445-1,172 of the murine IL-17R eDNA) with a PGKneo cassette. A thymidine kinase cassette (MC-TK) was inserted into the 5′ end of the vector. 129 derived embryonic stem (ES) cells were electroporated with the targeting vector and selected in the presence of G418 and ganciclovir as described. ES clones carrying a targeted mutation in IL-17R were identified by a combination of PCR and genomic Southern blot analyses and were injected into C57BL/6 blastocysts. The resulting male chimeras were crossed to C57BL/6 females to generate mice heterozygous for the IL-17R mutation (IL-17R+/−), which were subsequently intercrossed to generate IL-17R-deficient mice (IL-17R KO). These mice were moved to a C57BL/6 background by five successive backcrosses to C57BL/6 mice. IL-17RA knockout mice showed reduced mean clinical score in the CIA model, as shown inFIG.4(see also Kolls et al., 2001, J. Ex. Med.194:519-527; Lubberts at al., 2005, supra). In addition, the IL-17RA knockout mice showed only a 5% incidence of disease, whereas the wild-type mice showed a 71% incidence of disease. Example 2 The histopathology of CIA-induced IL-17RA −/− mice and IL-17RA expressing mice was compared to determine the correlation between induced arthritis and the absence of IL-17RA signaling. Mice were prepared as described in Example 1. The animals were sacrificed at fifteen to twenty weeks of age, and the histopathology of joints from the sacrificed animals were then examined. Histopathology of bone and cartilage in IL-17RA −/− knock-out mice and IL-17A/IL-17R expression mice (WT C57/BL6 (No. 2-18)) showed subchondral bone erosion of the talus and marked joint architecture disruption of tarsal-metatarsal joints (subchondral bone and articular cartilage erosion), as well as reactive periosteal bone formation (osteophytosis). Histopathology of ankle joints from mice deficient in IL-17RA −/− in an experimentally induced CIA model showed little joint inflammation and joint cartilage and bone erosion. However, the histopathologic analysis of an ankle joint of the rear paw of IL-17RA expressing mice showed marked chronic active inflammation. The significantly reduced incidence of joint inflammation and joint and bone erosion as compared to WT mice further implicates IL-17RA and IL-17RA signaling in inflammation and erosion. Example 3 A model of MOG (Myelin Oligodendrocyte Glycoprotein)-peptide-induced EAE model mice deficient in IL-17RA showed a delay in the onset of arthritis as well as an overall reduction in clinical scores as compared to WT mice. IL-17RA knockout mice were prepared as described in Example 1.FIG.5shows the incidence and median onset of arthritis as a function of time for both IL-17RA −/− and IL-17RA wild-type mice. 15 out of 15 of the IL-17RA expressing wild-type mice exhibited arthritic symptoms, with a mean onset of 13 days. By contrast, 14 of 15 IL-17RA −/− mice exhibited arthritic symptoms, with a mean onset of 22 days (p<0.0001 versus wild-type). Clinical scores of IL-17RA −/− knockout mice show a lower mean clinical score, with a later onset, than wild-type mice.FIG.6shows reduced clinical scores in IL-17RA −/− knockout mice as compared to wild-type mice in a MOG-induced model. The IL-17RA −/− knockout population showed a significantly later onset of arthritis than the IL-17RA expressing wild-type population. Further, the IL-17RA −/− knockout population had a lower mean clinical score at all time points for onset of arthritis. The longer mean onset of arthritis and lower mean clinical score for arthritis observed in IL-17RA −/− mutants as compared to IL-17RA-expressing wild-type animals further implicates IL-17RA signaling in inflammation and erosion. Example 4 Ovalbumin sensitized and challenged IL-17RA KO mice show a significant reduction of inflammatory cells in BAL (bronchoalveolar lavage) fluid compared to wild-type mice. IL-17RA KO mice were prepared as described in Example 1, and then challenged intra-nasally with ovalbumin. The number of inflammatory cells in the IL-17RA KO population were compared to the IL-17RA expressing wild-type population.FIG.7shows IL-17RA KO mice have reduced total numbers of inflammatory cells in BAL fluid than IL-17RA expressing wild-type mice in an ovalbumin-induced of asthma post-third challenge. The IL-17RA KO mouse population was compared to IL-17RA expressing wild-type mice for the incidence of esoinophils (A), neutrophils (B), lymphocytes (C) and macrophages (D) in BAL fluid in an ovalbumin-induced model of asthma.FIGS.8A-8Dshow that IL-17RA KO mice have reduced numbers of esoinophils (8A), neutrophils (88) and lymphocytes (8C) in BAL fluid in the IL-17RA KO population as compared to the IL-17RA expressing wild-type population. No changes in BAL, fluid macrophage (8D) were noted in either wile-type or IL-17RA KO mice (naïve and OVA-challenged). These data suggest that IL-17RA signaling is important in regulating immune-mediated inflammatory responses. Example 5 IL-17RA antibodies were shown to reduce incidence of arthritis in a CIA (Collagen-Induced Arthritis) mouse model when administered prophylactically and therapeutically. The IL-17RA inhibition reduced clinical arthritis in both a prophylactic and therapeutic manner for several models if CIA. The surrogate neutralizing mouse IL-17RA mAb administered prophylactically reduced mean clinical scores in wild-type CIA model in a dose-dependent manner.FIG.9shows the dose-dependent inhibition by IL-17RA mAb in wild-type CIA model. Mice were treated with either IL-17RA mAb or control Ig on a Monday, Wednesday and Friday schedule for 2.5 weeks post boost. Administration of 100 μg and 300 μg of IL-17RA antibodies resulted in a lower clinical score for 18 days post-boost than compared to isotype control Ig. A reduction in bone loss and cartilage erosion in the joint was associated with the reduction of mean clinical scores at the 300 μg dose of the IL-17RA mAb. Histopathologic analysis and radiographic images analysis were compared to the IgG control. By both means of analysis, the ankle joint of the near paw of CBA/I male mouse treated with an IL-18R mAb (isotype control) showed marked inflammation: subchondrial bone erosion of the talus, marked joint architecture disruption of tarsal-metatarsal joints (subchondrial bone and articular cartilage erosion), and reactive periosteal bone formation (ostcophytosis). In stark contrast, the ankle joint of the rear paw of a DBA/1 mouse treated with 300 μg anti-IL-17RA mAb showed well-defined joint spaces, lack of edema and lack of periosteal reactive bone or lytic lesions indicated reduced bone loss and cartilage erosion. Example 6 IL-17RA inhibition was also shown to be effective in a CIA model when dosing was initiated after the onset of clinical signs (i.e. therapeutic dosing protocol) in a wild-type and TNFR p55/p75 KO model. Treatment was initiated approximately 6-7 days post collagen introduction in both models.FIG.10shows that therapeutic treatment with anti-IL-17RA mAb stabilized mean clinical scores in both wild-type mice.FIG.11shows that therapeutic treatment with anti-IL-17RA mAb stabilized mean clinical scores in TNFR p55/p75 KO models. Mice were treated with either an anti-IL-17RA mAb, anti-IL-1R mAb, or control Ig on a Monday, Wednesday and Friday schedule for 2 weeks post randomization into therapeutic treatment groups. These data are representative of 2 independent experiments performed in both WT and TNFR p55/p75 KO CIA models. Administering anti-IL-17RA mAbs showed a reduced clinical score as compared to control IgG in CIA induced wild-type mice. Surprisingly, the similar efficacy of anti-IL-17RA mAbs in the TNF p55/p75 KO model stabilized CIA independently of TNF signaling. This data suggests anti-IL-17RA antigen binding protein therapy may pick up non-responders to anti-TNF therapies. Combination therapy of an anti-IL-17RA antigen binding protein with anti-TNF therapies may be more beneficial than either alone. Example 7 The development of fully human monoclonal antibodies directed against human IL-17RA was carried out using Abgenix (now Amgen Fremont Inc.) XenoMouse® technology (U.S. Pat. Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244, which are incorporated herein by reference in their entirety; Green et al, 1994, Nature Genetics7:13-21; Mendez et al., 1997, Nature Genetics15:146-156; Green and Jakobovitis, 1998, J. Ex. Med.188:483-495)). TABLE 4 shows the portions of the IL-17RA protein used as an immunogen and cell lines used to generate and screen anti-IL-17RA antibodies. TABLE 4ReagentDescriptionIL-17RA.FcHuman IL-17RA extracellular domain with aC-terminal human Fc domain. Expressed in astable CHO cell line.IL-17RA-FLAG-polyHisHuman IL-17RA extracellular domain with a(SEQ ID NO: 431)C-terminal FLAG-polyHis tag. Expressed bytransient transfection in COS PKB cells.IL-17RA CHO cellsHuman IL-17RA full-length expressed on thesurface of CHO cells. IgG2 XenoMouse® mice were immunized/boosted with IL-17RA-Fc (group 1) and IL-17RA-FLAG-polyHis (group 2). Serum titers were monitored by ELISA and mice with the best titers were fused to generate hybridomas. The resulting polyclonal supernatants were screened for binding to IL-17RA by ELISA, and the positive supernatants were screened for binding to IL-17RA CHO cells by FMAT. Positive supernatants were subjected to additional screening. IgG2 XenoMouse® mice were immunized with the following immunogens: IL-17RA-Fc (group 3) and IL-17RA-FLAG-pHis (group 4) and were tested following additional immunizations. Example 8 The anti-IL-17RA antibodies were characterized. Non-clonal hybridoma supernatants were prepared in volumes of 1-2 mls (the Ig concentrations were not determined for these supernatants). The anti-IL-17RA non-clonal hybridoma supernatants were initially screened by FACS for their ability to inhibit biotinylated human IL-17A binding to CHO cells over-expressing human IL-17RA and another CHO cell line over-expressing cynomolgus IL-17RA. Nonclonal supernatants that were able to completely or nearly completely inhibit binding of human IL-17A to CHO-huIL-17RA and CHO-cynoIL-17RA were subsequently screened at several dilutions in an IL-17A-induced cytokine/chemokine secretion assay using a human foreskin fibroblast (HFF) cell line. Anti-IL-17RA non-clonal supernatants were incubated with HFF cells (5000 cells/well in 96 well plate) for 30 minutes at 36° C. and then stimulated overnight with either IL-17A (5 ng/ml) alone or IL-17F (20 ng/ml) and TNF-alpha (5 ng/ml). Fibroblast culture supernatants were then analyzed by ELISA for the presence of either IL-6 or GRO-alpha. Anti-IL-17RA non-clonal hybridomas were selected for sub-cloning based on their performance in the CHO-IL-17RA FACS assay and HFF bioassay. An example of the selection is shown in TABLES 5, 6, and 7. TABLE 5%%positivepositiveMFINeg. Cntl.1.091.5710Repeat assaysIL-17 biot.HFF1:321:4(500 ng/ml)Bioassay% inhibition ofSupernatant I.D.8.8510.22771:4 dil.IL-6 production1:321:12811.341.78956142 (incl..603.7768072989181AMH15/AML15)31.041.60846−54 (incl.1.720.79109082999284AMH14/AML14)51.591.4311765261.451.9314827971.001.288715881.431.6014693191.342.28185920100.791.961158−2111.931.69117221122.231.69869713 (incl.1.490.4968253AMH21/AML21)141.011.2586323.151.311.4597445161.390.728584170.910.9477338181.372.8513496191.471.1587461201.601.2074246211.301.658474220.931.0285416231.081.1277259 In TABLE 5, anti-IL-17RA non-clonal hybridoma supernatants were screened for binding to IL-17RA. The first half of TABLE 5 shows the % positive and mean fluorescent intensity (MFI) in results from flow cytometry (i.e., FACS). The % positive shows inhibition of biotin-huIL-17A binding to huIL-17RA+CHO cells by the non-clonal hybridoma supernatants. The MFI column shows inhibition of biotinylated huIL-17A binding to cyno IL-17RA+CHO cells by the non-clonal hybridoma supernatants. The second half of TABLE 5 shows the HFF binding intensity for the nonclonal and mAbs as measured by the % intensity of IL-6 production. The first 2 columns show an IL-17A/HFF bioassay with non-clonal hybridoma supernatants and the last 4 columns are repeat IL-17A/HFF bioassay results with non-clonal hybridoma supernatants. TABLE 6FACS results on 293-CynoIL-17R4-expressing Cells%%positivepositiveMFIHFFNeg. Cntl.Bioassay1.091.571.01:4repeatIL-17 biot.dilution1:321:4(500 ng/ml)% inhibition ofSupernatant I.D.8.8510.2277IL-6 production1:321:1281:5121 (incl.1.321.49AMH11/AML11)20.872.92931,04.471641,035.011750.66.53186 (incl.0.734.559AMH5/AML5)70.595.18880.457.25792.342.3666136106.768.35643712110.781.1666124120.611.646745671674535132.985.4822−2−13145.3410.64492223393134150.53.241151−716 (incl.0.542.9318927791737329AMH22/AML22)171.252.217−8−76180.610.997732819 (incl. AMH23)0.691.7210797286766750201.531.94315−31216.669.6366−154226.3310.3271114230.32.5575035240.244.1163415250.810.998−4911260.431.3176748270.71.23115026280.581.329564729 (incl.0.81.8511777690877966AMH1/AML1)300.691.55114016310.561.961212−11320.211.118467331.241.15136843340.740.8111368350.711.3796521360.571.2177832370.591.08713380.651.43863−38390.281.23743−21400.352.48950−39410.641.61849−19420.121.048876896928066430.211.12117934440.321.33868−3450.741.681040−16460.581.7410647 TABLE 6 shows IL-17RA non-clonal hybridoma supernatant screening data. The % positive and MFI columns show results from flow cytometry (FACS). The % positive columns show inhibition of biotin-huIL-17A binding to huIL-17RA+CHO cells by the non-clonal hybridoma supernatants. The MFI column shows inhibition of biotinylated huIL-17A binding to cyno IL-17RA+CHO cells by the non-clonal hybridoma supernatants. The first 2 HFF bioassay columns are IL-17A/HFF bioassay with non-clonal hybridoma supernatants and the last 4 bioassay columns are repeat EL-17A/HFF bioassay results with selected non-clonal hybridoma supernatants. A number of supernatants were selected for sub-cloning. TABLE 7% positiveMFINeg. CntlHFF1.091.5710bioassayIL-17biot.1:41:321:128(500 ng/ml)% inhibition ofSupernatant ID.8.8510.2277IL-6 production11.851.33102992121.081.461690615031.291.39223310441.551.331853665851.690.787646306 (incl.1.520.896737875AMH13/AML13)71.540.98779714581.783.443473633096.348.4553574834101.231.5810827131111.622.128−10−6−10121.151.0416716337132.431.67125823−4141.431.0313421718151.621.5918675931161.792.225615745170.911.851049542318 (incl.11.366758261AMH12/AML12)19 (incl.1.751.233908173AMH17/AML17)202.310.49935203821 (incl.1.840.766869071AMH16/AML16) TABLE 7 shows anti-IL-17RA non-clonal hybridoma supernatant screening data. The first two columns are flow cytometry data (FACS). The % positive columns show inhibition of biotin-huIL-17A binding to huIL-17RA+CHO cells by the non-clonal hybridoma supernatants. The MFI column shows inhibition of biotinylated huIL-17A binding to cynomolgus IL-17RA+CHO cells by the non-clonal hybridoma supernatants. The final three columns show IL-17A/HFF bioassay results with non-clonal hybridoma supernatants. Supernatants 6, 18, 19 and 21 were selected for subcloning. TABLE 8IL-17A/HFFLow resolutionbioassayBIAcoreSub-clone IDIC50(nM)KD(nM)1.Subclone of (AMH14/AML14)0.120.692.Subclone of (AMH14/AML14)20.20ND3.Subclone of (AMH14/AML14)30.075ND4.Subclone of (AMH21/AML21)2.3ND5.Subclone of (AMH21/AML21)3.1ND6.Subclone of (AMH21/AML21)3.316.77.Subclone of (AMH20/AML20)8.1ND8.Subclone of (AMH20/AML20)6.6ND9.Subclone of (AMH20/AML20)6.711.610.Subclone of (AMH19/AML19)0.223.111.Subclone of (AMH19/AML19)1.1ND12.Subclone of (AMH19/AML19)0.50ND13.Subclone of (AMH13/AML13)>107.614.Subclone of (AMH18/AML18)0.44ND15.Subclone of (AMH18/AML18)0.40ND16.Subclone of (AMH18/AML18)0.1714.917.Subclone of (AMH12/AML12)3.5ND18.Subclone of (AMH12/AML12)3.78.220.Subclone of (AMH12/AML12)5.5ND21.Subclone of (AMH17/AML17)2.58.222.Subclone of (AMH17/AML17)5.3ND23.Subclone of (AMH17/AML17)0.57ND24.Subclone of (AMH16/AML16)1.6ND25.Subclone of (AMH16/AML16)2.36.226.Subclone of (AMH16/AML16)1.4ND27.Subclone of (AMH22/AML22)0.0461.528.Subclone of (AMH22/AML22)0.09ND29.Subclone of (AMH22/AML22)0.07NDND = not determined TABLE 8 shows IL-17A/HFF bioassay IC50 values and low resolution BIAcore® KDvalues for subcloned hybridomas. Lower IC50 and KDvalues in the IL-17A/HFF IL-17RA binding assays showed that the IL-17RA mAbs inhibited binding of IL-17A to IL-17 receptor A. Antibodies were selected for further characterization based on low KDvalues for inhibiting IL-17A binding to human IL-17RA. Example 9 IL-17RA human mAb clones having the heavy and light chain sequences (AMH22/AML22), (AMH19/AML19), (AMH18/AML18) and (AMH14/AML14) were selected for further bioassay characterization. TABLE 9 below shows IC50 values for the selected Abs in the HFF bioassay and a primary lung fibroblast bioassay against both IL-17A and IL-17F. TABLE 9IL-17A/lungIL-17A/HFFIL-17F/HFFfibroblastIL-17RA mAbIC50 (nM)IC50(nM)IC50(nM)(AMH14/AML14)0.130.0670.04(AMH22/AML22)0.100.0330.14(AMH19/AML19)0.200.0870.22(AMH18/AML18)0.330.0730.081 The selected human mAbs inhibited IL-17A binding to IL-17RA. In addition to the lower IC50 values observed for IL-17A binding to IL-17RA, the selected human mAbs exhibited reduced IC50 values inhibiting the binding of IL-17F to IL-17RA (second column). Therefore, the selected human mAbs inhibit both IL-17A-IL-17RA binding and IL-17F-IL-17RA binding. Example 10 Exemplary IL-17RA human mAbs were tested in a cynomolgus bioassay utilizing the cynomolgus-derived kidney epithelial cell line JTC-12 stimulated with cynomolgus IL-17A.FIG.12shows IL-17RA mAbs having the heavy and light chain sequences (AMH22/AML22), (AMH19/AML19), (AMH18/AML18) and (AMH14/AML14) in the inhibition of cynomolgus IL-17A-induced IL-6 production from JTC-12 cells. The (----) line depicts the positive control value of cynomolgus IL-17 in combination with TNF-alpha. The (-.-.-) line depicts the positive control value of cynomolgus TNF-alpha. The (....) line depicts the media control value. JTC-12 cells were preincubated for 30 mins with anti-IL-17RA mAbs and then stimulated overnight with cynomolgus IL-17A (5 ng/ml) and human TNF-alpha (5 ng/ml).FIG.12shows that each antibody was able to inhibit cynomolgous IL-17A from binding IL-17RA and inhibit IL-17RA activation, as determined by IL-6 production from JTC-12 cells. The IL-17RA antibody (AMH14/AML14) was able to antagonize cynomolgous IL-17A-induced IL-6 production from JTC-12 cells with an IC50 of approximately 1.2 nM. Example 11 In vitro binding of IL-17RA mAbs was assayed. The binding affinities of IL-17RA antibodies were measured by surface plasmon resonance using a Biacore 3000® instrument by standard methods known in the art. Antibody candidates were captured on CM4 chips derivatized with goat anti-human IgG (H+L) antibody (Jackson immuno Research, Bar Harbor, ME). A CM4 chip coated with goat anti-human IgG (H+L) antibody but without captured antibody was used as a reference. Soluble huIL-17RA-FLAG-polyHis (SEQ ID NO:431) at a concentration range of 0.46-1000 nM was flowed over the chips for 2 minutes (association phase) followed by a 15-30 minute disassociation phase. FLAG peptide. Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:447) as described in Hopp et al.,Bio/Technology6:1204, 1988, and U.S. Pat. No. 5,011,912 enables rapid assay and facile purification of expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO). Experiments were conducted at 25° C. using a 50 uL/min flow rate. Data was fit to a 1:1 Model+Local Rmax using B1Aeval software® (v4.1). TABLE 10Human Antibodyka(1/Ms)KD(1/s)KA(1/M)KD(M)(AMH14/AML14)2.60 × 1056.22 × 10−54.18 × 1092.39 × 10−10(AMH22/AML22)2.35 × 1051.17 × 10−42.01 × 1094.98 × 10−10(AMH19/AML19)1.42 × 1051.14 × 10−41.25 × 1098.02 × 10−10(AMH18/AML18)1.02 × 1051.01 × 10−31.01 × 1089.88 × 10−9 TABLE 10 shows the KDof the human mAb clones was on the order of 10−10to 10−9, with the clone having the heavy and light chain sequences (AMH14/AML14) having the highest affinity. Each of the human monoclonal antibodies' kinetic data was consistent with the equilibrium data. The antibody with the heavy and light chain variable sequences (AMH14/AML14; SEQ ID NO:14 and SEQ ID NO:40, respectively) had the highest affinity for IL-17RA, as well as the slowest off-rate. Example 12 The agonistic potential of IL-17RA human mAb having the heavy and light chain variable sequences (AMH14/AML14) was assessed in vitro. The IL-17RA mAb (AMH14/AML14) was tested for its agonist effects on HFF cells. IL-17RA mAb having the heavy and light chain sequences (AMH14/AML14) was also tested under conditions of cross-linking with goat anti-human F(ab′)2, goat anti-human IgG and mouse anti-human IgG prior to incubation on HFF cells. Recombinant IL-17RA mAb AMH14/AML14 at 0, 0.1, 0.5, 1, 1.5 and 10 μg/ml, alone and pre-cross linked with murine anti-human IgG (Zymed/Invitrogen, San Diego, CA), goat anti-human F(ab′)2 (Goat a-h-Fab) and goat anti-human IgG (Goat a-h IgG) were incubated overnight with HFF cells. GRO-alpha was assessed by ELISA. IL-17A alone served as a positive control for GRO-alpha production in this experiment. These data are representative of 2 independent experiments. IL-17RA mAb (AMH14/AML14) alone had no effect on HFF cells. Pre-crosslinking anti-IL-17RA mAb (AMH14/AML14) had no effect on GRO-alpha production from HFF cells. These data demonstrate that anti-IL-17RA mAb (AMH14/AML14) either alone or pre-cross-linked and incubated with HFF cells was unable to induce a ORO-alpha response and therefore is not an agonistic mAb to IL-17RA. Example 13 The effects of the germline (GL) changes to IL-17RA mAb AMH14/AML14 were tested in the HFF bioassay.FIG.13shows sequence variation in the framework regions of SEQ ID NO:40 (AML14) in relation to germline residues and the effect on IC50 values. SEQ ID NO:40 (AML14) contains four non-germline residues in the framework, two in FR2 and two in FR3. Standard site-directed mutagenesis methods were used to generate germline versions A and B of AMH14/AML14. These variants were tested in the IL-17A and IL-17F HFF bioassay; HFF cells were preincubated for 30 mins with various anti-IL-17RA mAbs and then stimulated overnight with IL-17 (5 ng/ml). FIG.14shows that the two variants that had the residues returned to germline (seeFIG.13) had reduced IL-17A inhibitory activity in relation to AMH14/AML14, indicating that some variation in the framework regions was tolerated but that some residues may influence activity. The (---) line indicates the positive control value of IL-17 stimulation in the absence of antibody (approximately 4062 pg/ml). The media-only control gave a value of approximately 71 pg/ml. FIG.15shows that the two variants that had the residues returned to germline (seeFIG.13) had reduced IL-17F inhibitory activity in relation to AMH14/AML14, indicating that some variation in the framework regions was tolerated but that some residues may influence activity. The positive control value of IL-17F in combination with TNF-alpha stimulation in the absence of antibody was approximately 10994 pg/ml, the value for TNF-alpha only was approximately 1534 pg/ml, and the media-only control gave a value of approximately 55 pg/ml. Example 14 Studies were conducted to determine where the various IL-17RA antigen binding proteins (in the form of human antibodies) bound to human IL-17RA. The ForteBio™ Octet™ System is one of several systems and techniques available for measuring antibody binding. The methods used for screening antibody binding essentially followed the manufacturer's recommendations. For more information see www dot fortebio dot com. In brief, streptavidin sensors (ForteBio™) were presoaked for 10 minutes in PBSAT (1% BSA/PBS+0.05% Tween20® (polyoxyethylene sorbitan monolaurate). Biotinylated AMH14/AML14 at 10 ug/mL in PBSAT was loaded onto the sensors for 900 seconds. A new baseline was run for 600 seconds in PBSAT. Wild-type IL-17RA-FLAG-polyHis (SEC ID NO:431) at 10 ug/mL in PBSAT was then bound to the sensors for 900 seconds. A new baseline was established for 600 seconds in PBSAT. 200 nM of the following mAbs AMH22/AML22, AMH19/AML19, and AMH18/AML18 were associated for 900 seconds, followed by dissociation for 900 seconds in PBSAT. The data showed that AMH18/AML18 did not compete with AMH4/AML4 for binding, showing that AMH14/AML14 and AMH18/AML18 bind to different neutralizing determinants. AMH22/AML22 and AMH19/AML19 did not bind in the presence of AMH14/AML14, suggesting that all three of these antibodies bind to the same or to a similar neutralizing determinant and therefore are considered to bin together. Example 15 Cross-competition studies were performed to determine IL-17RA binding characteristics of exemplary IL-17RA antibodies. A modification of the multiplexed binning method described by Jia, el al. was used (see Jia, et al.,J. Immun. Meth.,2004, 288:91-98). The method employed the Bio-Plex Workstation and software (BioRad, Hercules, CA), as well as reagents from Luminex® Corp. (Austin, TX). The manufacturers' basic protocols were followed except where noted below (see www.bio-rad.com and www.luminexcorp.com for details). Each bead code of streptavidin-coated Luminex® beads (Luminex®, #L100-L1XX-01, where “XX” specifies the bead code) were incubated in 150 ul of 50 ug/ml biotinylated monovalent mouse-anti-human IgG capture antibody (BD Pharmingen. Becton Dickinson, Franklin Lakes, NJ, product #555785) for 1 hour at room temperature in the dark and then washed 3 times with PBSAT. The mouse-anti-human IgG coating was evaluated and the beads quantified by FACS. Each bead code was separately incubated with 10 ul of anti-IL-17RA antibody for 1 hour at room temperature and then washed. The beads were pooled and then dispensed to a 96-well filter plate (Millipore, Billerica, MA, product #MSBVN1250). 80 ul of 2 ug/ml IL-17RA (SEQ ID NO:431) was added to half the wells and buffer to the other half and incubated at room temperature for 1 hour then washed with PBSAT. 10 ul of an anti-IL-17RA antibody was added to one well with IL-17RA (SEQ ID NO:431) and one well without IL-17RA and incubated at room temperature for 1 hour then washed with PBSAT. An irrelevant human-IgG (Jackson Labs., Bar Harbor, ME, product #009-000-003) was included as a negative control. 50 ul PE-conjugated monovalent mouse-anti-human IgG (BD Pharmingen, Becton Dickinson, Franklin Lakes. NJ, #555787) was added to each well and incubated at room temperature for 1 hour and then washed with PBSAT. The PE-tagged monovalent antibody will detect the presence of the second mAb added to the well, but not the first mAb captured by the monovalent mouse-anti-human IgG antibody. Beads were resuspended in 120 ul PBSAT and at least 100 events/bead code were collected on the Bio-Plex workstation as per the manufacturer's recommended protocol. Median Fluorescent Intensity (MFI) of the antibody pair without IL-17RA was subtracted from the MFI signal of the corresponding reaction containing IL-17RA to normalize for background noise. The criteria for determining if two antibodies cross-competed with each other and therefore “binned” together was a matter of determining the degree to which the second antibody was detectable. If the normalized MFI was higher than the highest of any of the following three values, then the anti-IL-17RA antibodies were considered to be simultaneously bound to IL-17RA and were considered to be in different bins (i.e., the antibodies did not cross-compete): the normalized MFI is greater than 3 times the MFI value of the antibody paired with itself, or 3 times the MFI value of the antibody paired with a huIgG control, or a MFI of 300. Generally speaking, antibodies assigned to different bins bind different parts of IL-17RA and antibodies assigned to the same bin(s) bind similar parts of IL-17RA. FIGS.16A and16Bshow the results of multiplexed binning of anti-IL-17RA antibodies. Shaded values indicate antibody pairs that bind to IL-17RA simultaneously, suggesting that these antibodies bind to different neutralizing determinants. Boxed values indicate antibodies paired against themselves and cross-compete. The following monoclonal human antibodies containing the ascribed heavy and light variable domains were tested: A: AMH11/AML11, B: AMH4/AML4, C: AMH8/AML8, D: AMH7/AML7, E: AMH6/AML6, F: AMH10/AML10, G: AMH18/AML18, H: AMH11/AML11, I: AMH22/AML22, J: AMH23/AML23, K: AMH14/AML14, L: AMH19/AML19, M: AMH12/AML12, N: AMH17/AML17, O: AMH16/AML16, P: AMH26/AML26, Q: AMH21/AML21, and R: AMH20/AML20. FIGS.16A and16Balso show that antibodies A: AMH11/AML11, B: AMH4/AML4, C: AMH8/AML8, D: AMH7/AML7, E: AMH6/AML6, F: AMH10/AML10, and G: AMH18/AML18 competed with one another for binding to human IL-17RA and as a consequence fell into a defined group (Bin 1). In general, antibodies I: AMH22/AML22, J: AMH23/AML23, K: AMH14/AML14, L: AMH19/AML19, M: AMH12/AML12, N: AMH17/AML17, O: AMH16/AML16 competed with one another for binding to human IL-17RA and as a consequence fell into a defined group (Bin 3). Generally speaking, the antibodies of Bin 1 did not compete with the antibodies of Bin 3. Antibody H: AMH1/AML1 was unique in its competition pattern and formed Bin 2, but is most similar to Bin 3. Antibody P: AMH26/AML26 formed Bin 4 and showed little cross-competition with any of the other antibodies, suggesting a neutralizing determinant unique to this antibody. Antibodies Q: AMH21/AML21 and R: AMH20/AML20, showed individually unique competition patterns, but with considerable similarities to Bin 3 antibodies, and formed Bins 5 and 6, respectively. This data provides evidence of several species within a subgenus of cross-competing antibodies. Example 16 As described above, antibodies that bind human IL-17RA and inhibit, or neutralize, the binding of IL-17A and/or IL-17F were created and characterized. To determine the neutralizing determinants on human IL-17RA that these various IL-17RA antibodies bound, a number of chimeric human/mouse IL-17RA proteins were constructed. This method takes advantage of the non-cross reactivity of the various IL-17RA antibodies with mouse IL-17RA. For each chimera, one or two regions of human IL-17RA extracellular domain (SEQ ID NO:431) was/were replaced with the corresponding region(s) of mouse IL-17RA (SEQ ID NO:432).FIG.17shows mouse IL-17RA (SEQ ID NO:432) and the 5 domains, A, B, C, D, E, and F that replaced the counterpart domains in the human IL-17RA sequence. Such techniques are known in the art, see for example Stemmer, W. P. C. et al., 1995 Gene 164:49-53. Six single-region and 8 double-region chimeras were constructed in pTT5 vectors. Chimeric constructs A through F (single region chimeras) were made synthetically by PCR annealing of 65-mer sense and antisense oligonucleotides which span the protein from a Sal1 site 5′ of the initiation codon to a Not1 site 3′ of the termination codon. The template used in the first round of PCR was a mix of oligos (sense and antisense) spanning the region from the Sal1 site to the Not1 site. PCR was done in 2 steps as follows: Double chimeric constructs were made by digestion of single chimeras A through D with Sal1 and Sac1 restriction enzymes and a 3-way ligation with Sac1 and Not1 digested chimeras E and F using pTT5 as the expression vector. The chimeras, huIL-17RA-FLAG-polyHis (SEQ ID NO:431), and muIL-17RA-FLAG-polyHis (SEQ ID NO:432) were expressed transiently using 2936-E cells (available from the National Research Council of Canada (NRCC); see NRCC document L-11565 for further information) as host cells in roller bottles. Such transient expression techniques are well known in the art, see for example Durocher, Y. et al., 2002Nucleic Acids Res. January 15;30(2):E9. The supernatants were purified using a HisTrap™ HP column as per the manufacturer's general guidelines (GE Healthcare, Piscataway NJ) and eluted using a standard imidazole gradient (see manufacturer's recommended protocols). Purified protein was desalted into PBS, pH 7.2. The chimeras were aligned using standard analysis tools, such as ClustalW (EMBL-EBI). The resulting chimeric proteins are shown inFIGS.18A-18D. With reference toFIGS.17and18A-18D, Chimera A (SEQ ID NO:433) is human IL-17RA extracellular domain with mouse Domain A; Chimera B (SEQ ID NO:434) is human IL-17RA extracellular domain with mouse Domain B; Chimera C (SEQ ID NO:435) is human IL-17RA extracellular domain with mouse Domain C; Chimera D (SEQ ID NO:436) is human IL-17RA extracellular domain with mouse Domain D; Chimera E (SEQ ID NO:437) is human IL-17RA extracellular domain with mouse Domain E; Chimera F (SEQ ID NO:438) is human IL-17RA extracellular domain with mouse Domain F; Chimera G (SEQ ID NO:439) is human IL-17RA extracellular domain with mouse Domains A and E; Chimera H (SEQ ID NO:440) is human IL-17RA extracellular domain with mouse Domains B and E; Chimera I (SEQ ID NO:441) is human IL-17RA extracellular domain with mouse Domains C and E; Chimera J (SEQ ID NO:442) is human IL-17RA extracellular domain with mouse Domains D and E; Chimera K (SEQ ID NO:443) is human IL-17RA extracellular domain with mouse Domains A and F; Chimera L (SEQ ID NO:444) is human IL-17RA extracellular domain with mouse Domains B and F; Chimera M (SEQ ID NO:445) is human IL-17RA extracellular domain with mouse Domains C and F; and Chimera N (SEQ ID NO:446) is human IL-17RA extracellular domain with mouse Domains D and F. Using methods similar to those described in Example 15, multiplex analysis using the Bio-Plex Workstation and software (BioRad, Hercules, CA) was performed to determine neutralizing determinants on human IL-17RA by analyzing exemplary human IL-17RA mAbs differential binding to chimeric versus wild-type IL-17RA proteins. Twelve bead codes of pentaHis-coated beads (Qiagen, Valencia, CA; see www1.giagen.com) were used to capture histidine-tagged protein. The 12 bead codes allowed the multiplexing of 11 chimeric and the wild type human IL-17RA. To prepare the beads, 100 ul of wild-type IL-17RA supernatant from transient expression culture and 100 ul of 2.5 ug/ml chimeric protein were bound to penta-His-coated beads overnight at 4° C. or 2 hours at room temperature with vigorous shaking. The beads were washed as per the manufacturer's protocol and the 12 bead set was pooled and aliquoted into 2 or 3 columns of a 96-well filter plate (Millipore, Billerica, MA, product #MSBVN1250) for duplicate or triplicate assay points, respectively. 100 ul anti-IL-17RA antibodies in 4-fold dilutions were added to the wells, incubated for 1 hour at room temperature, and washed. 100 ul of a 1:100 dilution of PE-conjugated anti-human IgG Fc (Jackson Labs., Bar Harbor, ME, product #109-116-170) was added to each well, incubated for 1 hour at room temperature and washed. Beads were resuspended in 1% BSA, shaken for 3 minutes, and read on the Bio-Plex workstation. Antibody binding to IL-17RA chimeric protein was compared to antibody binding to the human IL-17RA wild-type from the same pool. A titration of antibody over approximately a 5 log scale was performed. Median Fluorescence Intensity (MFI) of chimeric proteins was graphed as a percent of maximum wild-type human IL-17RA signal. Mutations (i.e., mouse domains) that increase the EC50 (expressed in nM) for the IL-17RA mAb by 3-fold or greater (as calculated by GraphPad Prism®) were considered to have negatively affected IL-17RA mAb binding. Through these methods, neutralizing determinants for various IL-17RA antibodies were elucidated. FIG.19is a table summarizing the IL-17RA mAbs capacity to bind the various chimeric proteins. Shaded values denote where the IL-17RA mAb did not meet the criteria for binding to that particular chimeric protein (“n.d.,” i.e., “not determined” means that the chimera was not assayed). As described above, EC50 values are provided. A zero value indicates that antibody binding was ablated. The underlined value was assigned an EC50 value by the GraphPad Prism® even though the titration curve was essentially flat. TABLE 11 shows the control values in nM for the assay. TABLE 11huWT3x wt2x wtmAbmu WTctrlctrlctrlAMH18/AML180.0000.0610.1820.121AMH1/AML11.8790.1340.4030.269AMH22/AML220.0000.0430.1280.085AMH14/AML143416.0000.0270.0820.055AMH19/AML19770.1000.0620.1870.125AMH23/AML230.0000.0530.1580.106AMH26/AML260.0000.2810.8430.562AMH21/AML210.1960.0180.0550.037AMH20/AML201.3330.0220.0660.044 As can be seen inFIG.19, at least three neutralizing determinants were identified based on those regions affecting the binding of neutralizing IL-17RA antibodies, namely Domain B spanning amino acids 75-96 of human IL-17RA (SEQ ID NO:431). Domain C spanning amino acids 128-154 of human IL-17RA (SEQ ID NO:431), and Domain D spanning amino acids 176-197 of human IL-17RA (SEQ ID NO:431). Domain B spanning amino acids 75-96 of human IL-17RA (SEQ ID NO:431) negatively affected the binding of neutralizing antibodies AMH1/AML1 and AMH23/AML23. Domain C spanning amino acids 128-154 of human IL-17RA (SEQ ID NO:431) negatively affected the binding of neutralizing antibodies AMH22/AML22 and AMH23/AML23. Domain D spanning amino acids 176-197 of human IL-17RA (SEQ ID NO:431) negatively affected the binding of neutralizing antibodies AMH1/AML1, AMH22/AML22, AMH14/AML14. AMH19/AML19, AMH23/AML23, AMH21/AML21, and AMH20/AML20. The binding characteristics of the IL-17RA antibodies in relation to where the antibodies bound on human IL-17RA was confirmed by the double chimeras. Thus, Domain B. C, and D are considered neutralizing determinants. Example 17 As described above, antibodies that bind human IL-17RA and inhibit, or neutralize, the binding of IL-17A and/or IL-17F were created and characterized. To determine the neutralizing determinants on human IL-17RA that these various IL-17RA antibodies bound, a number of mutant IL-17RA proteins were constructed having arginine substitutions at select amino acid residues of human IL-17RA. Arginine scanning is an art-recognized method of evaluating where antibodies, or other proteins, bind to another protein, see for example Nanevicz, T., et al., 1995, J. Biol. Chem., 270:37, 21619-21625 and Zupnick, A., et al., 2006, J. Biol. Chem., 281:29, 20464-20473. In general, the arginine sidechain is positively charged and relatively bulky as compared to other amino acids, which may disrupt antibody binding to a region of the antigen where the mutation is introduced. Arginine scanning is a method that determines if a residue is part of a neutralizing determinant and/or an epitope. 95 amino acids distributed throughout the human IL-17RA extracellular domain were selected for mutation to arginine. The selection was biased towards charged or polar amino acids to maximize the possibility of the residue being on the surface and reduce the likelihood of the mutation resulting in misfolded protein.FIG.20depicts the amino acid residues that were replaced with an arginine residue in SEQ ID NO:431. Using standard techniques known in the art, sense and anti-sense oligonucleotides containing the mutated residues were designed based on criteria provided by Stratagene Quickchange® H protocol kit (Stratagene/Agilent, Santa Clara, CA). Mutagenesis of the wild-type (WT) HuIL-17RA-Flag-pHis was performed using a Quickchange® 11 kit (Stratagene). All chimeric constructs were constructed to encode a FLAG-histidine tag (six histidines) on the carboxy terminus of the extracellular domain to facilitate purification via the poly-His tag. Multiplex analysis using the Bio-Plex Workstation and software (BioRad, Hercules, CA) was performed to determine neutralizing determinants on human IL-17RA by analyzing exemplary human IL-17RA mAbs differential binding to arginine mutants versus wild-type IL-17RA proteins. Twelve bead codes of pentaHis-coated beads (Qiagen, Valencia, CA; see www1.qiagen.com) were used to capture histidine-tagged protein. The 12 bead codes allowed the multiplexing of 11 IL-17RA arginine mutants and wild-type human IL-17RA (SEQ ID NO:431). To prepare the beads, 100 ul of wild-type IL-17RA and IL-17RA arginine mutant supernatants from transient expression culture were bound to penta-His-coated beads overnight at 4° C. or 2 hours at room temperature with vigorous shaking. The beads were washed as per the manufacturer's protocol and the 12 bead set was pooled and aliquoted into 2 or 3 columns of a 96-well filter plate (Millipore, Billerica, MA, product #MSBVN1250) for duplicate or triplicate assay points, respectively. 100 ul anti-IL-17RA antibodies in 4-fold dilutions were added to the wells, incubated for 1 hour at room temperature, and washed. 100 ul of a 1:100 dilution of PE-conjugated anti-human IgG Fc (Jackson Labs., Bar Harbor, ME, product #109-116-170) was added to each well, incubated for 1 hour at room temperature and washed. Beads were resuspended in 1% BSA, shaken for 3 minutes, and read on the Bio-Plex workstation. Antibody binding to IL-17RA arginine mutant protein was compared to antibody binding to the human IL-17RA wild-type from the same pool. A titration of antibody over approximately a 5 log scale was performed. Median Fluorescence Intensity (MFI) of IL-17RA arginine mutant proteins was graphed as a percent of maximum wild-type human IL-17RA signal. Those mutants for which signal from all the antibodies are below 30% of wild-type IL-17RA were deemed to be either of too low a protein concentration on the bead due to poor expression in the transient culture or possibly misfolded and were excluded from analysis: these were T51R, K53R, S55R, H64R, D75R, E110R, Q118R, T121, E123R, S147R, H148R, E158R, T160R, H163R, K191R, T193R, E213R, H251R, T269R, H279R, and D293R. Mutations (i.e., arginine substitutions) that increase the EC50 for the IL-17RA mAb by 3-fold or greater (as calculated by GraphPad Prism®) were considered to have negatively affected IL-17RA mAb binding. Through these methods, neutralizing determinants and epitopes for various IL-17RA antibodies were elucidated. FIG.21illustrates titration curves of various IL-17RA mAbs binding to the D152R IL-17RA mutant (i.e., the aspartic acid at position 152 of SEQ ID NO:431 was mutagenized to be an arginine). Antibodies AMH1/AML1, AMH22/AMH22, AML14/AML14, AMH19/AML0.19, AMH23/AML23, AMH21/AML21, and AMH20/AML20 lost the capacity to bind the D152R IL-17RA mutant. Antibodies AMH18/AML18 and AMH26/AML26 were only marginally affected but did not meet the cutoff criteria. A summary of the arginine scan, binning, and chimera data is presented inFIG.22. The arginine scan methodology identified several neutralizing determinants: AMH18/AML18 bound a domain spanning amino acids 220-284 of human IL-17RA (SEQ ID NO:431); AMH1/AML1 bound a domain focused on amino acid residue 152 of human IL-17RA (SEQ ID NO:431); AMH22/AML22 bound a domain spanning amino acids 152-198 of human IL-17RA (SEQ ID NO:431); AMH14/AML14 bound a domain spanning amino acids 152-297 of human IL-17RA (SEQ ID NO:431); AMH19/AML19 bound a domain spanning amino acids 152-186 of human IL-17RA (SEQ ID NO:431); AMH23/AML23 bound a domain spanning amino acids 97-297 of human IL-17RA (SEQ ID NO:431); AMH26/AML26 bound a domain spanning amino acids 138-270 of human IL-17RA (SEQ ID NO:431); AMH21/AML21 bound a domain spanning amino acids 113-198 of human IL-17RA (SEQ ID NO:431); and AMH20/AML20 bound a domain spanning amino acids 152-270 of human IL-17RA (SEQ ID NO:431). All of the residues shown inFIG.22have been shown to ablate binding of a neutralizing human monoclonal antibody that specifically binds to human IL-17RA. | 430,829 |
11859000 | DETAILED DESCRIPTION OF THE DISCLOSURE Certain aspects of the present disclosure are directed to antibodies or antigen-binding portions thereof the specifically bind CCR8 (“anti-CCR8 antibody”). In certain aspects, the anti-CCR8 antibody specifically binds the N-terminal extracellular domain of human CCR8. Other aspects of the present disclosure are directed to methods of treating a subject in need thereof comprising administering the anti-CCR8 antibodies disclosed herein. I. Terms In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application. It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent, up or down (higher or lower). It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of” are also provided. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleotide sequences are written left to right in 5′ to 3′ orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety. As used herein, the term “amount” or “level” is used in the broadest sense and refers to a quantity, concentration or abundance of a substance (e.g., a metabolite, a small molecule, a protein, an mRNA, a marker). When referring to a metabolite or small molecule (e.g. a drug), the terms “amount”, “level” and “concentration” are generally used interchangeably and generally refer to a detectable amount in a biological sample. “Elevated levels” or “increased levels” refers to an increase in the quantity, concentration or abundance of a substance within a sample relative to a control sample, such as from an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some aspects, the elevated level of a substance (e.g., a drug) in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., HPLC). “Reduced levels” refers to a decrease in the quantity, concentration or abundance of a substance (e.g., a drug) in an individual relative to a control, such as from an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some aspects, a reduced level is little or no detectable quantity, concentration or abundance. In some aspects, the reduced level of a substance (e.g., a drug) in a sample refers to a decrease in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g, HPLC). When referring to a protein, mRNA or a marker, such as those described herein, the terms “level of expression” or “expression level” in general are used interchangeably and generally refer to a detectable amount of a protein, mRNA, or marker in a biological sample. In some aspects, a detectable amount or detectable level of a protein, mRNA or a marker is associated with a likelihood of a response to an agent, such as those described herein. “Expression” generally refers to the process by which information contained within a gene is converted into the structures (e.g., a protein marker, such as PD-L1) present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). “Elevated expression,” “elevated expression levels,” or “elevated levels” refers to an increased expression or increased levels of a substance within a sample relative to a control sample, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some aspects, the elevated expression of a substance (e.g., a protein marker, such as PD-L1) in a sample refers to an increase in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g., FACS). “Reduced expression,” “reduced expression levels,” or “reduced levels” refers to a decrease expression or decreased levels of a substance (e.g., a protein marker) in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., cancer) or an internal control. In some aspects, reduced expression is little or no expression. In some aspects, the reduced expression of a substance (e.g., a protein marker) in a sample refers to a decrease in the amount of the substance of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% relative to the amount of the substance in a control sample, as determined by techniques known in the art (e.g, FACS). As used herein, the term “antagonist” refers to any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides or proteins. In some aspects, inhibition in the presence of the antagonist is observed in a dose-dependent manner. In some aspects, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured with a negative control under comparable conditions. Also disclosed herein, are methods of identifying antagonists suitable for use in the methods of the disclosure. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immuno-absorbent assay (ELISA), ForteBio® systems, radioimmunoassay (RIA), Meso Scale Discovery assay (e.g., Meso Scale Discovery Electrochemiluminescence (MSD-ECL), and bead-based Luminex® assay. These assays determine the ability of an antagonist to bind the polypeptide of interest (e.g., a receptor or ligand) and therefore indicate the ability of the antagonist to inhibit, neutralize or block the activity of the polypeptide. Efficacy of an antagonist can also be determined using functional assays, such as the ability of an antagonist to inhibit the function of the polypeptide or an agonist. For example, a functional assay may comprise contacting a polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The potency of an antagonist is usually defined by its IC50value (concentration required to inhibit 50% of the agonist response). The lower the IC50value the greater the potency of the antagonist and the lower the concentration that is required to inhibit the maximum biological response. As used herein, the term “anti-CCR8 antibody” refers to an antibody that specifically binds to CCR8. In some aspects, the anti-CCR8 antibody inhibits a CCR8 biological activity and/or a downstream pathway(s) mediated by CCR8 signaling or other CCR8-mediated function. An anti-CCR8 antibody includes, but is not limited to, antibodies that block, antagonize, suppress, inhibit or reduce a CCR8 biological activity (e.g., ligand binding, activation of G-protein signaling), including downstream pathways mediated by CCR8 signaling or function, such as receptor binding and/or elicitation of a cellular response to CCR8 or its metabolites (e.g., immune suppression). In some aspects, an anti-CCR8 antibody provided by the disclosure binds to human CCR8 and prevents, blocks, or inhibits binding of human CCR8 to a ligand (e.g., CCL1) or interaction between CCR8 and G-protein. In some aspects, the anti-CCR8 antibody prevents, blocks, or inhibits the binding of human CCR8 to CCL1. In some aspects, the anti-CCR8 antibody prevents, blocks, or inhibits the binding of human CCR8 to the CCL8. In some aspects, the anti-CCR8 antibody prevents, blocks, or inhibits the binding of human CCR8 to the CCL16. In some aspects, the anti-CCR8 antibody prevents, blocks, or inhibits the binding of human CCR8 to the CCL18. As used herein, the term “antibody” refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody. As used herein, the term “antibody fragment,” “antigen-binding fragment,” or similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen (e.g., CCR8) and inhibit the activity of the target antigen. Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab′ fragment, or an F(ab′)2 fragment. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al., (2001)J. Immunol. Methods248(1):47-66; Hudson and Kortt, (1999)J. Immunol. Methods231(1):177-189; Poljak, (1994)Structure2(12):1121-1123; Rondon and Marasco, (1997)Annu. Rev. Microbiol.51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety. As used herein, the term “antibody fragment” also includes, e.g., single domain antibodies such as camelized single domain antibodies. See, e.g., Muyldermans et al., (2001)Trends Biochem. Sci.26:230-235; Nuttall et al., (2000)Curr. Pharm. Biotech.1:253-263; Reichmann et al., (1999)J. Immunol. Meth.231:25-38; PCT application publication nos. WO 94/04678 and WO 94/25591; and U.S. Pat. No. 6,005,079, all of which are incorporated herein by reference in their entireties. In some aspects, the disclosure provides single domain antibodies comprising two VH domains with modifications such that single domain antibodies are formed. In some aspects, an antigen-binding fragment includes the variable region of a heavy chain polypeptide and the variable region of a light chain polypeptide. In some aspects, an antigen-binding fragment described herein comprises the CDRs of the light chain and heavy chain polypeptide of an antibody. As used herein, the term “bispecific” or “bifunctional antibody” refers to an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, (1990)Clin. Exp. Immunol.79:315-321; Kostelny et al., (1992)J. Immunol.148:1547-1553. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chain/light chain pairs have different specificities (Milstein and Cuello, (1983)Nature305:537-539). Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion of the heavy chain variable region is preferably with an immunoglobulin heavy-chain constant domain, including at least part of the hinge, CH2, and CH3 regions. For further details of illustrative currently known methods for generating bispecific antibodies see, e.g., Suresh et al., (1986)Methods Enzymol.121:210; PCT Publication No. WO 96/27011; Brennan et al., (1985)Science229:81; Shalaby et al.,J. Exp. Med. (1992) 175:217-225; Kostelny et al., (1992)J. Immunol.148(5):1547-1553; Hollinger et al., (1993)Proc. Nat. Acad. Sci. USA90:6444-6448; Gruber et al., (1994)J. Immunol.152:5368; and Tutt et al., (1991)J. Immunol.147:60. Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al. (1992) J Immunol 148(5):1547-1553. The leucine zipper peptides from the Fos and Jun proteins may be linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers may be reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al. (1993) Proc Natl Acad Sci USA 90:6444-6448 has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See, e.g., Gruber et al. (1994) J Immunol 152:5368. Alternatively, the antibodies can be “linear antibodies” as described in, e.g., Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (VH—CH1-VH—CH1) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. Antibodies with more than two valencies (e.g., trispecific antibodies) are contemplated and described in, e.g., Tutt et al. (1991) J Immunol 147:60. As used herein, the term “biparatopic” refers to an antibody that is capable of binding two epitopes on a single antigen, e.g., polypeptide, target. In some aspects, the biparatopic antibody comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds a first epitope and the second antigen-binding region binds a second epitope on the same antigen. The disclosure also embraces variant forms of multi-specific antibodies such as the dual variable domain immunoglobulin (DVD-Ig) molecules described in Wu et al. (2007) Nat Biotechnol 25(11): 1290-1297. The DVD-Ig molecules are designed such that two different light chain variable domains (VL) from two different parent antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CH1 and Fc region. Methods for making DVD-Ig molecules from two parent antibodies are further described in, e.g., PCT Publication Nos. WO 08/024188 and WO 07/024715. In some aspects, the bispecific antibody is a Fabs-in-Tandem immunoglobulin, in which the light chain variable region with a second specificity is fused to the heavy chain variable region of a whole antibody. Such antibodies are described in, e.g., International Patent Application Publication No. WO 2015/103072. As used herein, “cancer antigen” or “tumor antigen” refers to (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells that express tumor-specific antigens, (iv) cells that express tumor-associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer. As used herein, the term “cancer-specific immune response” refers to the immune response induced by the presence of tumors, cancer cells, or cancer antigens. In certain aspects, the response includes the proliferation of cancer antigen specific lymphocytes. In certain aspects, the response includes expression and upregulation of antibodies and T-cell receptors and the formation and release of lymphokines, chemokines, and cytokines. Both innate and acquired immune systems interact to initiate antigenic responses against the tumors, cancer cells, or cancer antigens. In certain aspects, the cancer-specific immune response is a T cell response. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The anti-CCR8 antibodies described herein can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma, or any viral disease. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. As used herein, the term “CCR8” or “C—C chemokine receptor type 8” refers to a G-protein coupled receptor. CCR8 is known to have at least four ligands: CCL1, CCL8, CCL16, and CCL18. CCL1 is thought to potentiate human Tregcells by inducing CCR8, FOXp3, CD39, Granzyme B, and IL-10 Expression, in a STAT3-dependent manner. See, e.g., Barsheshet et al.,PNAS114(23):6086-91 (Jun. 6, 2017). CCR8 is expressed primarily on Tregcells and to a lesser extent on small fractions of TH2 cells, monocytic cells, NK cells, and CD8+ cells. CCR8 is a transmembrane receptor having seven transmembrane domains, an extracellular N-terminal domain (SEQ ID NO: 172), and an intracellular C-terminal domain, which interacts with G-protein. The amino acid sequence for human CCR8 (UniProt P51685; SEQ ID NO: 171) is shown in Table 1, below. (SEQ ID NO: 171)MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL As used herein the term “compete,” when used in the context of antigen-binding proteins (e.g., immunoglobulins, antibodies, or antigen-binding fragments thereof) that compete for binding to the same epitope, refers to a interaction between antigen-binding proteins as determined by an assay (e.g., a competitive binding assay; a cross-blocking assay), wherein a test antigen-binding protein (e.g., a test antibody) inhibits (e.g., reduces or blocks) specific binding of a reference antigen-binding protein (e.g., a reference antibody) to a common antigen (e.g., CCR8 or a fragment thereof). A polypeptide or amino acid sequence “derived from” a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence. Polypeptides derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. A polypeptide can comprise an amino acid sequence that is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain aspects, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule. In certain aspects, the antibodies of the disclosure are encoded by a nucleotide sequence. Nucleotide sequences of the invention can be useful for a number of applications, including cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like. It will also be understood by one of ordinary skill in the art that the antibodies suitable for use in the methods disclosed herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. The antibodies suitable for use in the methods disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family. In certain aspects, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side-chain family members. Alternatively, in certain aspects, mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into binding polypeptides of the invention and screened for their ability to bind to the desired target. As used herein, the term “cross-reacts” refers to the ability of an antibody of the disclosure to bind to CCR8 from a different species. For example, an antibody of the present disclosure that binds human CCR8 can also bind another species of CCR8. As used herein, cross-reactivity is measured by detecting a specific reactivity with purified antigen in binding assays (e.g., SPR, ELISA) or binding to, or otherwise functionally interacting with, cells physiologically expressing CCR8. Methods for determining cross-reactivity include standard binding assays as described herein, for example, by BIACORE™ surface plasmon resonance (SPR) analysis using a BIACORE™ 2000 SPR instrument (BIACORE AB, Uppsala, Sweden), or flow cytometric techniques. As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+T cells. As used herein, the term “EC50” refers to the concentration of an antibody or an antigen-binding portion thereof, which induces a response, either in an in vitro or an in vivo assay, which is 50% of the maximal response, i.e., halfway between the maximal response and the baseline. As used herein, the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient's own immune system. As used herein, the term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. The term “epitope mapping” refers to a process or method of identifying the binding site, or epitope, of an antibody, or antigen-binding fragment thereof, on its target protein antigen. Epitope mapping methods and techniques are provided herein. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from CCR8 are tested for reactivity with the given anti-CCR8 antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)). Also encompassed by the present disclosure are antibodies that bind to an epitope on CCR8 which comprises all or a portion of an epitope recognized by the particular antibodies described herein (e.g., the same or an overlapping region or a region between or spanning the region). Also encompassed by the present disclosure are antibodies that bind the same epitope and/or antibodies that compete for binding to human CCR8 with the antibodies described herein. Antibodies that recognize the same epitope or compete for binding can be identified using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the immunoglobulin under test inhibits specific binding of a reference antibody to a common antigen, such as CCR8. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al.,J. Immunol.137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using I-125 label (see Morel et al.,Mol. Immunol.25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al.,Virology176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand.J. Immunol.32:77 (1990)). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more. Other techniques include, for example, epitope mapping methods, such as, x-ray analyses of crystals of antigen:antibody complexes which provides atomic resolution of the epitope and mass spectrometry combined with hydrogen/deuterium (H/D) exchange which studies the conformation and dynamics of antigen:antibody interactions. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes. As used herein, the term “Fc-mediated effector functions” or “Fc effector functions” refer to the biological activities of an antibody other than the antibody's primary function and purpose. For example, the effector functions of a therapeutic agnostic antibody are the biological activities other than the activation of the target protein or pathway. Examples of antibody effect functions include C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); lack of activation of platelets that express Fc receptor; and B cell activation. Many effector functions begin with Fc binding to an Fcγ receptor. In some aspects, the tumor antigen-targeting antibody has effector function, e.g., ADCC activity. In some aspects, a tumor antigen-targeting antibody described herein comprises a variant constant region having increased effector function (e.g. increased ability to mediate ADCC) relative to the unmodified form of the constant region. As used herein, the term “Fc receptor” refers to a polypeptide found on the surface of immune effector cells, which is bound by the Fc region of an antibody. In some aspects, the Fc receptor is an Fcγ receptor. There are three subclasses of Fcγ receptors, FcγRI (CD64), FcγRII (CD32) and FγcRIII (CD16). All four IgG isotypes (IgG1, IgG2, IgG3 and IgG4) bind and activate Fc receptors FcγRI, FcγRIIA and FcγRIIIA. FcγRIIB is an inhibitory receptor, and therefore antibody binding to this receptor does not activate complement and cellular responses. FcγRI is a high affinity receptor that binds to IgG in monomeric form, whereas FcγRIIA and FcγRIIA are low affinity receptors that bind IgG only in multimeric form and have slightly lower affinity. The binding of an antibody to an Fc receptor and/or C1q is governed by specific residues or domains within the Fc regions. Binding also depends on residues located within the hinge region and within the CH2 portion of the antibody. In some aspects, the agonistic and/or therapeutic activity of the antibodies described herein is dependent on binding of the Fc region to the Fc receptor (e.g., FcγR). In some aspects, the agonistic and/or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to the Fc receptor (e.g., FcγR). As used herein, the term “human antibody” includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. Human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (See, e.g., Lonberg et al., (1994)Nature368(6474): 856-859); Lonberg, (1994)Handbook of Experimental Pharmacology113:49-101; Lonberg & Huszar, (1995)Intern. Rev. Immunol.13:65-93, and Harding & Lonberg, (1995) Ann. N.Y. Acad. Sci. 764:536-546). However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e. humanized antibodies). As used herein, the term “humanized” refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In some embodiments of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A “humanized” antibody retains an antigenic specificity similar to that of the original antibody. A “chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody. As used herein, the term a “heterologous antibody” is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal. The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. The terms “induce” as used with respect to inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms. As used herein, the term “immunogenic cell death” (alternatively known as “immunogenic apoptosis” refers to a cell death modality associated with the activation of one or more signaling pathways that induces the pre-mortem expression and emission of damaged-associated molecular pattern (DAMPs) molecules (e.g., adenosine triphosphate, ATP) from the tumor cell, resulting in the increase of immunogenicity of the tumor cell and the death of the tumor cell in an immunogenic manner (e.g., by phagocytosis). As used herein, the term “immunogenic cell death-inducing agent” refers to a chemical, biological, or pharmacological agent that induces an immunogenic cell death process, pathway, or modality. As used herein, the terms “inhibits”, “reduces” or “blocks” (e.g., referring to inhibition or reduction of human CCR8-mediated phosphorylation of STAT1 and/or STAT3 in a cell) are used interchangeably and encompass both partial and complete inhibition/blocking. The inhibition/blocking of CCR8 reduces or alters the normal level or type of activity that occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of CCR8 when in contact with an anti-CCR8 antibody as compared to CCR8 not in contact with an anti-CCR8 antibody, e.g., inhibits binding of CCR8 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. As used herein, the term “inhibits growth” (e.g., referring to a tumor or cells, e.g., tumor cells) is intended to include any measurable decrease in the growth of a tumor or a cell, e.g., the inhibition of growth of a tumor by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. As used herein, a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-CCR8 antibody). The term “in vivo” refers to processes that occur in a living organism. As used herein, the term “isolated antibody” is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human CCR8 is substantially free of antibodies that specifically bind antigens other than CCR8). An isolated antibody that specifically binds to an epitope may, however, have cross-reactivity to other CCR8 proteins from different species. However, the antibody continues to display specific binding to human CCR8 in a specific binding assay as described herein. In addition, an isolated antibody is typically substantially free of other cellular material and/or chemicals. In some aspects, a combination of “isolated” antibodies having different CCR8 specificities is combined in a well-defined composition. As used herein, the term “isolated nucleic acid molecule” refers to nucleic acids encoding antibodies or antibody portions (e.g., VH, VL, CDR3) that bind to CCR8, and is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than CCR8, which other sequences may naturally flank the nucleic acid in human genomic DNA. For example, a sequence selected from a sequence set forth in Table 8 corresponds to the nucleotide sequences comprising the heavy chain (VH) and light chain (VL) variable regions of anti-CCR8 antibody monoclonal antibodies described herein. As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes. In some aspects, a human monoclonal antibody of the disclosure is of the IgG1 isotype. In some aspects, a human monoclonal antibody of the disclosure is of the IgG2 isotype. In some aspects, a human monoclonal antibody of the disclosure is of the IgG3 isotype. In some aspects, a human monoclonal antibody of the disclosure is of the IgG4 isotype. As is apparent to a skilled artisan, identification of antibody isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1 IgA2, IgD, and IgE) is routine in the art and commonly involves a combination of sequence alignments with known antibodies, published Fc variant sequences and conserved sequences. As used herein the term “KD” or “KD” refers to the equilibrium dissociation constant of a binding reaction between an antibody and an antigen. The value of KDis a numeric representation of the ratio of the antibody off-rate constant (kd) to the antibody on-rate constant (ka). The value of KDis inversely related to the binding affinity of an antibody to an antigen. The smaller the KDvalue the greater the affinity of the antibody for its antigen. Affinity is the strength of binding of a single molecule to its ligand and is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. As used herein, the term “kd” or “kd” (alternatively “koff” or “koff”) is intended to refer to the off-rate constant for the dissociation of an antibody from an antibody/antigen complex. The value of kdis a numeric representation of the fraction of complexes that decay or dissociate per second, and is expressed in units sec−1. As used herein, the term “ka” or “ka” (alternatively “kon” or“kon”) is intended to refer to the on-rate constant for the association of an antibody with an antigen. The value of ka is a numeric representation of the number of antibody/antigen complexes formed per second in a 1 molar (1M) solution of antibody and antigen, and is expressed in units M−1sec−1. As used herein, the term “lymphocytes” refers to a type of leukocyte or white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B-cells and T-cells. The term “tumor-infiltrating lymphocyte” (abbreviated “TIL”) or “tumor-infiltrating Treg,” as used herein, refers to a lymphocyte or a Treg, respectively, that is associated with tumor cells, e.g., that is localized within a tumor mass. As used herein, the terms “linked,” “fused,” or “fusion,” are used interchangeably. These terms refer to the joining of two more elements, components, or domains by whatever means including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art. As used herein, the term “monoclonal antibody” refers to an antibody that displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In some aspects, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. As used herein, the term “natural killer (NK) cell” refers to a type of cytotoxic lymphocyte. These are large, usually granular, non-T, non-B lymphocytes, which kill certain tumor cells and play an important role in innate immunity to viruses and other intracellular pathogens, as well as in antibody-dependent cell-mediated cytotoxicity (ADCC). As used herein, the term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. As used herein, the term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Polynucleotides used herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination. As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion. As used herein, the term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. As generally used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. As used herein, a “pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977) J Pharm Sci 66:1-19). As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. As used herein, the term “preventing” when used in relation to a condition, refers to administration of a composition that reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject that does not receive the composition. As used herein, the term “purified” or “isolated” as applied to any of the proteins (antibodies or fragments) described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, or 99%), by weight, of the total protein in a sample. As used herein, the term “rearranged” refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element. As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. As used herein, the term “recombinant antibody” includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies comprise variable and constant regions that utilize particular human germline immunoglobulin sequences are encoded by the germline genes, but include subsequent rearrangements and mutations which occur, for example, during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains the antigen binding domain, which is encoded by various genes that rearrange to form an antibody specific for a foreign antigen. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen. The constant region will change in further response to an antigen (i.e., isotype switch). Therefore, the rearranged and somatically mutated nucleic acid molecules that encode the light chain and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecules, but instead will be substantially identical or similar (i.e., have at least 80% identity). As used herein, the term “reference antibody” (used interchangeably with “reference mAb”) or “reference antigen-binding protein” refers to an antibody, or an antigen-binding fragment thereof, that binds to a specific epitope on CCR8 and is used to establish a relationship between itself and one or more distinct antibodies, wherein the relationship is the binding of the reference antibody and the one or more distinct antibodies to the same epitope on CCR8. As used herein, the term connotes an anti-CCR8 antibody that is useful in a test or assay, such as those described herein, (e.g., a competitive binding assay), as a competitor, wherein the assay is useful for the discovery, identification or development, of one or more distinct antibodies that bind to the same epitope. As used herein, the terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to antibody binding to an epitope on a predetermined antigen. Typically, the antibody binds with an equilibrium dissociation constant (KD) of approximately less than 10−6M, such as approximately less than 10−7, 10−1M, 10−9M or 10−10M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE™ 2000 instrument using recombinant human CCR8 as the analyte and the antibody as the ligand and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. In certain aspects, an antibody that specifically binds to CCR8 binds with an equilibrium dissociation constant (KD) of approximately less than 100 nM (10−7M), optionally approximately less than 50 nM (5×10−8M), optionally approximately less than 15 nM (1.5×10−8M), optionally approximately less than 10 nM (10−8M), optionally approximately less than 5 nM (5×10−9M), optionally approximately less than 1 nM (10−9M), optionally approximately less than 0.1 nM (10−10M), optionally approximately less than 0.01 nM (10−11M), or even lower, when determined by surface plasmon resonance (SPR) technology in a BIACORE™ 2000 instrument using recombinant human CCR8 as the analyte and the antibody as the ligand, where binding to the predetermined antigen occurs with an affinity that is at least two-fold greater than the antibody's affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.” As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below. The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. The nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990)J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997)Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). The nucleic acid compositions of the present disclosure, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof may be mutated, in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where “derived” indicates that a sequence is identical or modified from another sequence). The term “T cell” refers to a type of white blood cell that can be distinguised from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (a.k.a. THcells or CD4+T cells) and subtypes, including TH1, TH2, TH3, TH17, TH9, and TFHcells, cytotoxic T cells (a.k.a TCcells, CD8+T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCMcells), effector memory T cells (TEMand TEMRAcells), and resident memory T cells (TRMcells), regulatory T cells (a.k.a. Tregcells or suppressor T cells) and subtypes, including CD4+FOXP3+Tregcells, CD4+FOXP3−Tregcells, Tr1 cells, Th3 cells, and Treg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention. As used herein, the term “T cell-mediated response” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+cells) and helper T cells (e.g., CD4+cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation. As used herein, the term “regulatory T cell,” “T regulatory cell,” Treg,” or “Treg”, used interchangeably herein, refers to a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs are known to direct effector T cell lysis, support tolerogenic dendritic cell formation, support M2 macrophage formation, produce immunosuppressive metabolites and cytokines, serve as an IL-2 sink, and to promote neovasculature formation. Though there are many types of Tregs, many Tregs express CD4 and FOXP3, with FOXP3 serving as a marker for Tregs in many cases. As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., an anti-CCR8 antibody or an antigen-binding fragment thereof) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a cancer). The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. As used herein, the term “tumor microenvironment” (alternatively “cancer microenvironment;” abbreviated TME) refers to the cellular environment or milieu in which the tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME. The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells. As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Various aspects of the disclosure are described in further detail in the following subsections. II. Compositions of the Disclosure Certain aspects of the present disclosure are directed to antibodies or antigen-binding portions thereof the specifically bind CCR8 (“anti-CCR8 antibody”). In certain aspects, the anti-CCR8 antibody specifically binds the N-terminal extracellular domain of human CCR8. Historically, it has been very difficult to generate therapeutic antibodies to CCR8. CCR8, like other GPCRs, is challenging to raise antibodies against due to their strong membrane association, lack of exposure of sequence on the cell surface, and difficulty expressing the CCR8 full-length protein itself. Numerous previous attempts leveraging multiple antibody generation platforms have failed. (See also Jo and Jung,Experimental&Molecular Medicine48:e207 (2016).) The present disclosure solves this problem by specifically targeting the N-terminal extracellular domain of human CCR8. By raising antibodies specifically targeting to the N-terminal domain, the present disclosure focused on generating antibodies that target the longest extracelluar portion of CCR8. This enabled the achievement of previously unattainable anti-CCR8 antibodies. In doing so, the antibodies described herein are capable of inhibiting CCR8 activity in a way not previously described. In particular, the antibodies disclosed herein are capabale of (a) enhancing an immune response to a tumor; (b) reducing, depleting, or killing tumor infiltrating regulatory T (“Treg”) cells; (c) inducing internalization of CCR8 in tumor infiltrating regulatory T (“Treg”) cells; (d) activating NK cells, (e) inducing NK cell mediated killing of tumor infiltrating regulatory T (“Treg”) cells; (f) binding to cynomolgus monkey (“cyno”) CCR8; (g) binding to human CCR8 with KD of 10 nM or less as measured by BIACORE™; or (h) any combination thereof. In some aspects, the antibody or the antigen-binding portion thereof is further engineered by removing one or more post-translation modification. In some aspects, the antibody or the antigen-binding portion thereof is engineered to remove one or more fucose sugar units. In some aspects, the antibody or the antigen-binding portion thereof is modified to remove one or more fucose sugar units from the (IgG1) Fc region of the antibody. In some aspects, the antibody or the antigen-binding portion thereof is afucosylated. In some aspects, removal of one or more fucose sugar units increases the ADCC of the antibody or antigen-binding fragment thereof. In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units (e.g., afucosylated antibody) is at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, or at least about 5.0-fold higher than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units (e.g., fucosylated antibody). In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units is at least about 3.0-fold higher than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units. In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units is at least about 3.5-fold higher than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units. In some aspects, the ADCC of the anti-CCR8 antibody modified to remove one or more fucose sugar units is at least about 4.0-fold higher than the anti-CCR8 antibody that is not modified to remove one or more fucose sugar units. In certain aspects, the anti-CCR8 antibody is capable of inducing an immune response to a tumor. Treg cells serve to regulate the immune response by down regulating the activity of T cells as a means of keeping the immune system in check. Tumor infiltrating Tregs can act to prevent an immune response targeting the tumor, thereby allowing the tumor to evade destruction by a subject's immune system. The antibodies described herein are capable of inhibiting tumor infiltrating Tregs, thereby reducing this barrier to an anti-tumor immune response. In some aspects, the anti-CCR8 antibody increases an immune response to a tumor in a subject in need thereof by at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% relative to an immune response in the absence of the anti-CCR8 antibody. An anti-tumor immune response can be measured using any indicators known in the art. In some aspects, the anti-tumor immune response is determined by comparing the number of tumor infiltrating T cells (TILs) in a tumor sample obtained by a subject before and after contacting the tumor with the anti-CCR8 antibody. In some aspects, the number of TILs is measured by immunohistochemistry or quantitative polymerase chain reaction (qPCR). In some aspects, the number of TILs in the tumor sample is increase by at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, or at least about 20 fold, relative to the number of TILs in a tumor sample obtained from the subject prior to contacting the tumor with the anti-CCR8 antibody. In certain aspects, the anti-CCR8 antibody is capable of reducing, depleting, or killing tumor infiltrating Treg cells. In some aspects, the anti-CCR8 antibody induces depletion in the number of tumor infiltrating Treg cells in a subject following administration of the antibody or antigen-binding portion thereof, relative to the number of tumor infiltrating Treg cells prior to the administration. In some aspects, the number of tumor infiltrating Treg cells is depleted by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% relative to the number of tumor infiltrating Treg cells prior to the administration. In some aspects, the anti-CCR8 preferentially reduces, depletes, or kills tumor infiltrating Treg cells relative to peripheral Treg cells. In some aspects, the anti-CCR8 antibody induces antibody-dependent cellular cytotoxicity (ADCC) in a subject following administration of the anti-CCR8 antibody. In some aspects, the ADCC comprises an EC50 of about 100 μg/mL or less following the administration of the antibody or antigen-binding portion thereof. In some aspects, the ADCC comprises an EC50 of about 100 μg/mL or less, about 90 μg/mL or less, about 80 μg/mL or less, about 70 μg/mL or less, about 60 μg/mL or less, about 50 μg/mL or less, about 45 μg/mL or less, about 40 μg/mL or less, about 35 μg/mL or less, about 30 μg/mL or less, about 30 μg/mL or less, about 25 μg/mL or less, about 20 μg/mL or less, about 15 μg/mL or less, about 10 μg/mL or less, about 5 μg/mL or less, about 1 μg/mL or less, about 0.5 μg/mL or less, about μg/mL or less, about 0.1 μg/mL or less, or about 0.01 μg/mL or less following the administration of the ant-CCR8 antibody. In some aspects, the ADCC comprises an EC50 of about 1 μg/mL or less following the administration of the antibody or antigen-binding portion thereof. In some aspects, the ADCC comprises an EC50 of about 0.1 μg/mL or less following the administration of the antibody or antigen-binding portion thereof. Without being bound by any theory or particular mechanism, it is hypothesized that inhibition of CCR8 signaling in tumor infiltrating Tregs leads to activation of NK cells in the tumor microenvironment. Activated NK cells are then able to target and kill tumor infiltrating Tregs, reducing their number and enhancing the immune response to the tumor. Accordingly, in some aspects, the anti-CCR8 antibody is capable of activating NK cells. In some aspects, the NK cells are activated in the tumor microenvironment. In some aspects, the NK cells are tumor infiltrating NK cells. NK cell activation can be measured using any techniques known in the art. In some aspects, NK cell activation is determined by measuring the percent of cells that express one or more marker of activated NK cells. In certain aspects, NK cell activation is determined by measuring the percent of cells in the tumor microenvironment that express NKp46, but do not express CD3 (e.g., NKp46+/CD3−cells). In some aspects, NK cell activation is characterized by increased expression of one or more target genes by NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing upregulation of 4-1BB on the surface of NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing upregulation of ICAM-1 on the surface of NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing upregulation of 4-1BB and ICAM-1 on the surface of NK cells. In some aspects, the level of 4-1BB and/or ICAM-1 on the surface of NK cells following the contacting of the anti-CCR8 antibody to a tumor is upregulated by at least about 1.5 fold, 2 fold, 2.5 fold, 3.0 fold, 3.5 fold, 4.0 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold, relative to the level of 4-1BB and/or ICAM-1 on the surface of NK cells in a tumor sample taken prior to the contacting. In some aspects, NK cell activation is characterized by decreased expression of one or more target genes by NK cells. In some aspects, the anti-CCR8 antibody is capable of inducing downregulation of CD16 on the surface of NK cells. In some aspects, the level of CD16 on the surface of NK cells following the contacting of the anti-CCR8 antibody to a tumor is downregulated by at least about 1.5 fold, 2 fold, 2.5 fold, 3.0 fold, 3.5 fold, 4.0 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold, relative to the level of CD16 on the surface of NK cells in a tumor sample taken prior to the contacting. In some aspects, the number of activated NK cells in the tumor microenvironment is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% relative to the percent of activated NK cells in a tumor sample obtained from the subject prior to contacting the tumor with the anti-CCR8 antibody. In some aspects, the percent of activated NK cells in the tumor microenvironment is increased by at least about 1.5 fold, at least about 2 fold, at least about 2.5 fold, at least about 3 fold, at least about 3.5 fold, at least about 4 fold, at least about 4.5 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 15 fold, or at least about 20 fold, relative to the number of activated NK cells in a tumor sample obtained from the subject prior to contacting the tumor with the anti-CCR8 antibody. In some aspects, the anti-CCR8 antibody is capable of inducing NK cell mediated killing of tumor infiltrating Tregs. The anti-CCR8 antibodies described herein are capable of specific binding to human CCR8. However, in some aspects, the anti-CCR8 antibodies are capable of binding CCR8 from non-human animals. In some aspects, the anti-CCR8 antibody are capable of specifically binding human CCR8 and non-human primate CCR8. In some aspects, the anti-CCR8 antibody is capable of binding human CCR8 and cynomolgus (cyno) CCR8. In some aspects, the anti-CCR8 antibody binds human CCR8 with a higher affinity that non-human CC8 (e.g., cyno CCR8). In some aspects, the anti-CCR8 antibody binds human CCR8 but does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody binds human CCR8 with an equilibrium dissociation constant (KD) of about 100 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 50 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 25 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 20 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 15 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 10 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 5 nM or less. In some aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 1 nM or less. In some aspects, KDis measured by BIACORE™ In certain aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 10 nM or less as measured by BIACORE™. In certain aspects, the anti-CCR8 antibody binds human CCR8 with a KDof about 1 nM or less as measured by BIACORE™. Inhibition of CCR8 by the antibodies and antigen-binding portions thereof disclosed herein can occur through any mechanism. Without being bound by any particular mechanism, in some aspects, the anti-CCR8 antibody induces internalization of CCR8 by tumor infiltrating Treg cells. Internalization of the CCR8 receptor from the surface eliminates the ability of the receptor to bind its ligand and potentiate intracellular signaling, effectively inhibiting CCR8 activity in tumor infiltrating Treg cells. In certain aspects, the anti-CCR8 antibody binds CCR8 expressed by tumor infiltrating Treg cells. In some aspects, the anti-CCR8 antibody block the interaction between CCR8 and its ligand, e.g., through steric hindrance, a confirmation change, internalization of the CCR8 receptor, or any combination thereof. In some aspects, binding of the anti-CCR8 antibody to the N-terminal extracellular domain of CCR8 inhibits the ability of the CCR8 receptor to interact with G-protein, e.g., through a conformational change and/or through internalization of the CCR8 receptor. II.A. Epitopes The antibodies described herein specifically bind the N-terminal extracellular domain of CCR8 or a fragment thereof. The N-terminal extracellular domain of human CCR8 is generally defined as consisting of amino acids 1-35 of the full-length CCR8 sequence (e.g., amino acids 1-35 of SEQ ID NO: 171) (see uniprot.org/uniprot/P51685). The amino acid sequence of the N-terminal extracellular domain of human CCR8 comprises the amino acid sequence (SEQ ID NO: 172)MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK In some aspects, the anti-CCR8 antibody binds at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids within the N-terminal extracellular domain of human CCR8, e.g., as set forth in SEQ ID NO: 172. In some aspects, the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids within the N-terminal extracellular domain of human CCR8, e.g., as set forth in SEQ ID NO: 172 are contiguous. In some aspects, the anti-CCR8 antibody binds at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten contiguous amino acids within the N-terminal extracellular domain of human CCR8, e.g., as set forth in SEQ ID NO: 172. In some aspects, the at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids within the N-terminal extracellular domain of human CCR8, e.g., as set forth in SEQ ID NO: 172 are not contiguous. In some aspects, the anti-CCR8 antibody binds at least one amino acid in the N-terminal extracellular domain of human CCR8 and at least one amino acid of human CCR8 that is not within the N-terminal extracellular domain of human CCR8. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-10 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-15 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 1-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-10 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-15 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 5-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-15 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 10-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-20 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 15-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 20-25 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 20-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 20-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 25-30 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 25-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8, comprising one or more amino acids selected from amino acid residues 30-35 of SEQ ID NO: 172. In some aspects, the anti-CCR8 antibody binds an epitope on human CCR8 comprising an amino acid sequence selected from SEQ ID NOs: 180-200. II.B. Antibody Sequences In some aspects, the anti-CCR8 antibody comprises a whole antibody, e.g., an antibody comprising two light chain polypeptides and two heavy chain polypeptides. In some aspects, the anti-CCR8 antibody comprises a fragment of a whole antibody that retains the ability to bind CCR8. In some aspects, the anti-CCR8 antibody is a single chain antibody. In some aspects, the anti-CCR8 antibody is a single chain Fv fragment (scFv). In some aspects, the anti-CCR8 antibody is an Fd fragment. In some aspects, the anti-CCR8 antibody is an Fab fragment. In some aspects, the anti-CCR8 antibody is an Fab′ fragment. In some aspects, the anti-CCR8 antibody is an F(ab′)2 fragment. In some aspects, In some aspects, the anti-CCR8 antibody is selected from an intrabody, a minibody, a triabody, or a diabody. In some aspects, the anti-CCR8 antibody comprises a variable heavy (VH) chain and a variable light (VL) chain. In some aspects, the VH comprises a VH complementarity-determining region (CDR) 1, a VH CDR2, and a VH CDR3; and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3. In some aspects, the VH CDR1 comprises the amino acid sequence set forth in Table 2A. In some aspects, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 201. In some aspects, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 202. In some aspects, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 203. In some aspects, the VH CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204. TABLE 2AVH CDR1 Consensus SequencesSEQ IDNO:VH CDR1 Consensus Sequence201S/D/G/A)Y(Y/A/T)M(H/L/N)202D/G/A)Y(A/T)M(H/L/N)203(G/A)YTM(L/N)204S/D)Y(Y/A)MH Note: amino acid residue listed in parenthesis above (and elsewhere in the present disclosure) designate the amino acid at that particular position in the alternative. For example, Y(Y/A)MH means that the sequence can be either YYMH or YAMH. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in Table 2B. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 205. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 207. In some aspects, the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 208. TABLE 2BVH CDR2 Consensus SequencesSEQ IDNO:VH CDR2 Consensus Sequence205(I/G/A)I(N/S/T)(P/W/A)(S/N)(G/S)G(S/R)(T/I)(S/G/Y)YA(Q/D)(K/S)(F/V)(Q/K)G206AI(T/S)ASGGRTYYADSVKG207(G/A)I(T/S)(W/A)(N/S)(S/G)G(S/R)(I/T)(G/Y)YADSVKG208(I/G)I(N/S)(P/W)(S/N)(G/S)GS(T/I)(S/G)YA(Q/D)(K/S)(F/V)(Q/K)G In some aspects, the VH CDR3 comprises the amino acid sequence set forth in Table 2C. In some aspects, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 210. TABLE 2CVH CDR3 Consensus SequencesSEQ IDNO:VH CDR3 Consensus Sequence209(A/G)V(R/G)N(R/G)FRFDY210GR(K/V/D/E/R)SYR(D/E/K/V)SLRFDY In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 204, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 205, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 204, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 201, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 208, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 209. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 202, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 206, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 210. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 202, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 207, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 210. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 203, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 206, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 210. In some aspects, the anti-CCR8 antibody comprises a VH CDR1 having the amino acid sequence set forth in SEQ ID NO: 203, a VH CDR2 having the amino acid sequence set forth in SEQ ID NO: 207, and a VH CDR3 having the amino acid sequence set forth in SEQ ID NO: 210. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in Table 3A. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 211. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 213. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 214. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 215. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 216. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 217. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 218. In some aspects, the VL CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 219. TABLE 3AVL CDR1 Consensus SequencesSEQ ID NO:VL CDR1 Consensus Sequence211SSY(T/A)G(N/S/P)(I/R/V/S)(N/V/T)(L/-)(P/F/Y/H)VV212SSY(T/A)G(N/S)(I/R/S)(N/V/T)(L/-)(P/F/Y/H)VV213SSYAGSST(F/Y)VV214SSYAGS(R/I)(V/T)(F/H)VV215(A/G)(T/A)WD(Y/S)SL(T/R)(A/M)(V/W)V216(A/G)(T/A)WD(Y/S)SL(T/R/S)(A/M)(V/W)V217(A/G)TWD(Y/S)SL(T/S)A(V/W)V218G(A/T)WDSSL(R/S)(M/A)WV219(S/T)G(S/T)(G/S)SNIG(N/K)N(Y/F)VS In some aspects, the VL CDR2 comprises the amino acid sequence set forth in Table 3B. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 220. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 221. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 222. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 223. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 224. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 225. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 226. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 227. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 228. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 229. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 230. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 231. In some aspects, the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 232. TABLE 3BVL CDR2 Consensus SequencesSEQ IDNO:VL CDR2 Consensus Sequence220E(V/A)(N/T/I/S)KRPS221E(V/A)(N/T/S)KRPS222EV(T/S)KRPS223E(A/V)TKRPS224EV(N/S)KRPS225EV(N/T)KRPS226DN(D/T)(K/R)PS227DN(D/T/N)(K/R)RPS228DN(D/N)KRPS229DN(T/N)(K/R)RPS230D(N/D)(D/T/N)(K/R)RPS231D(N/D)(D/N)KRPS232D(N/D)(T/N)(K/R)RPS In some aspects, the VL CDR3 comprises the amino acid sequence set forth in Table 3C. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 233. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 234. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 235. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 237. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 238. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 239. In some aspects, the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 240. TABLE 3CVL CDR3 Consensus SequencesSEQ ID NO:VL CDR3 Consensus Sequence233SSY(T/A)G(N/S/P)(I/R/V/S)(N/V/T)(L/-)(P/F/Y/H)VV234SSY(T/A)G(N/S)(I/R/S)(N/V/T)(L/-)(P/F/Y/H)VV235SSYAGSST(F/Y)VV236SSYAGS(R/I)(V/T)(F/H)VV237(A/G)(T/A)WD(Y/S)SL(T/R)(A/M)(V/W)V238(A/G)(T/A)WD(Y/S)SL(T/R/S)(A/M)(V/W)V239(A/G)TWD(Y/S)SL(T/S)A(V/W)V240G(A/T)WDSSL(R/S)(M/A)WV In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 211, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 220, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 233. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 212, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 221, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 234. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 213, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 222, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 235. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 213, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 222, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 213, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 223, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 235. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 213, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 223, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 236. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 217, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 227, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 238. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 218, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 231, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 239. In some aspects, the anti-CCR8 antibody comprises a VL CDR1 having the amino acid sequence set forth in SEQ ID NO: 219, a VL CDR2 having the amino acid sequence set forth in SEQ ID NO: 232, and a VL CDR3 having the amino acid sequence set forth in SEQ ID NO: 240. In some aspects, the VH CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, and 167. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, 156, and 166. In some aspects, the VH CDR1 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, and 165. In some aspects, the VH CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 47, 107, 117, 137, and 147. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 46, 106, 116, 136, and 146. In some aspects, the VH CDR1 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 45, 105, 115, 135, and 145. In some aspects, the VH CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157, and 167. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126, 156, and 166. In some aspects, the VH CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65, 75, 85, 95, 125, 155, and 165. In some aspects, the VL CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, and 170. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, 159, and 169. In some aspects, the VL CDR1 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 148, 158, and 168. In some aspects, the VL CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 50, 110, 120, 140, and 150. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 49, 109, 119, 139, and 149. In some aspects, the VL CDR1 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 48, 108, 118, 138, and 148. In some aspects, the VL CDR3 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130, 160, and 170. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129, 159, and 169. In some aspects, the VL CDR2 of the anti-CCR8 antibody comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 58, 68, 78, 88, 98, 128, 158, and 168. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 45, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 105, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 106, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 107, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 115, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 116, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 117, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 135, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 136, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 137, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 145, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 146, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 147, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 17, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 35, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 36, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 37, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 65, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 67, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 95, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 96, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 97, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 125, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 127, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 155, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 156, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 157, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160. In some aspects the anti-CCR8 antibody comprises a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 165, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 166, a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 167, a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170. In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid selected from SEQ ID NOs: 41, 101, 111, 131, and 141. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 41, 101, 111, 131, and 141. In some aspects, the VL chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid selected from SEQ ID NOs: 42, 102, 112, 132, and 142. In some aspects, the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 42, 102, 112, 132, and 142. In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid selected from SEQ ID NOs: 41, 101, 111, 131, and 141; and the VL chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid selected from SEQ ID NOs: 42, 102, 112, 132, and 142; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 41, 101, 111, 131, and 141; and the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 42, 102, 112, 132, and 142; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 42; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 41 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 42; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 101 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 102; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 101 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 102; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 111 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 112; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 111 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 112; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 131 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 132; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 131 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 132; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 141 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 142; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 141 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 142; wherein the anti-CCR8 antibody does not bind cyno CCR8. In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, 51, 61, 71, 81, 91, 121, 151, and 161. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, 51, 61, 71, 81, 91, 121, 151, and 161. In some aspects, the VL chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152, and 162. In some aspects, the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152, and 162. In some aspects, the VH chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, 51, 61, 71, 81, 91, 121, 151, and 161; and the VL chain comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152, and 162; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the VH chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 1, 11, 21, 31, 51, 61, 71, 81, 91, 121, 151, and 161; and the VL chain comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 2, 12, 22, 32, 52, 62, 72, 82, 92, 122, 152, and 162; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 1 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 2; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 11 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 12; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 21 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 22; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 31 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 32; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 31 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 32; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 51 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 52; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 51 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 52; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 62; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 61 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 62; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 71 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 72; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 81 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 82; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 81 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 82; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 91 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 92; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 121 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 122; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 121 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 122; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 151 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 152; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 151 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 152; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 161 and a VL chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 162; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody comprises a VH chain comprising the amino acid sequence set forth in SEQ ID NO: 161 and a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 162; wherein the anti-CCR8 antibody binds human CCR8 and cyno CCR8. In some aspects, the anti-CCR8 antibody is a human antibody. In some aspects, the anti-CCR8 antibody is a humanized antibody. In some aspects, the anti-CCR8 is a chimeric antibody. In some aspects, the anti-CCR8 antibody is selected from the group consisting of an IgG1, an IgG2, an IgG3, an IgG4, an IgM, an IgA1 an IgA2, an IgD, and an IgE antibody. In some aspects, the anti-CCR8 antibody is an IgG1 antibody. In some aspects, the anti-CCR8 antibody is an IgG4 antibody. In some aspects, the anti-CCR8 antibody comprises a wild type IgG1 heavy chain constant region. In some aspects, the anti-CCR8 antibody comprises a wild type IgG4 heavy chain constant region. In some aspects, the anti-CCR8 antibody comprises an Fc domain comprising at least one mutation. In some aspects, the anti-CCR8 antibody comprises a mutant IgG1 heavy chain constant region. In some aspects, the anti-CCR8 antibody comprises a mutant IgG4 heavy chain constant region. In some aspects, the mutant IgG4 heavy chain constant region comprises any one of the substitutions S228P, L235E, L235A, or a combination thereof, according to EU numbering. In some aspects, the disclosure provides an antibody or antigen-binding portion thereof that binds to substantially the same epitope on human CCR8 as the anti-CCR8 antibody according to any one of the aforementioned aspects. In some aspects, the disclosure provides an antibody or antigen-binding portion thereof that cross-competes for binding to human CCR8 as the anti-CCR8 antibody according to any one of the aforementioned aspects. In some aspects, the anti-CCR8 antibody comprises an altered heavy chain constant region that has modified effector function relative to its corresponding unaltered constant region. Effector functions involving the constant region of the anti-CCR8 antibody may be modulated by altering properties of the constant or Fc region. Altered effector functions include, for example, a modulation in one or more of the following activities: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), apoptosis, binding to one or more Fc-receptors, and pro-inflammatory responses. Modulation refers to an increase, decrease, or elimination of an effector function activity exhibited by a subject antibody containing an altered constant region as compared to the activity of the unaltered form of the constant region. In particular aspects, modulation includes situations in which an activity is abolished or completely absent. In one aspect, the anti-CCR8 antibody comprises an IgG4 heavy chain constant region. In one aspect, the IgG4 heavy chain constant region is a wild type IgG4 heavy chain constant region. In another aspect, the IgG4 constant region comprises a mutation, e.g., one or both of S228P and L235E or L235A, e.g., according to EU numbering (Kabat, E. A., et al., supra). In one aspect, the anti-CCR8 antibody described herein comprises an IgG1 constant region. In one aspect, the IgG1 heavy chain constant region is a wild type IgG1 heavy chain constant region. In another aspect, the IgG1 heavy chain constant region comprises a mutation. An altered constant region with altered FcR binding affinity and/or ADCC activity and/or altered CDC activity is a polypeptide that has either an enhanced or diminished FcR binding activity and/or ADCC activity and/or CDC activity compared to the unaltered form of the constant region. An altered constant region that displays increased binding to an FcR binds at least one FcR with greater affinity than the unaltered polypeptide. An altered constant region that displays decreased binding to an FcR binds at least one FcR with lower affinity than the unaltered form of the constant region. Such variants which display decreased binding to an FcR may possess little or no appreciable binding to an FcR, e.g., 0 to 50% (e.g., less than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) of the binding to the FcR as compared to the level of binding of a native sequence immunoglobulin constant or Fc region to the FcR. Similarly, an altered constant region that displays modulated ADCC and/or CDC activity may exhibit either increased or reduced ADCC and/or CDC activity compared to the unaltered constant region. In some aspects, the anti-CCR8 antibody exhibits increased effector function. In some aspects, an anti-CCR8 antibody comprises a hybrid constant region, or a portion thereof, such as a G2/G4 hybrid constant region (see e.g., Burton et al. (1992)Adv Immun51:1-18; Canfield et al. (1991)J Exp Med173:1483-1491; and Mueller et al. (1997)Mol Immunol34(6):441-452). In some aspects, the anti-CCR8 antibody comprises an altered constant region exhibiting enhanced complement dependent cytotoxicity (CDC). Modulated CDC activity may be achieved by introducing one or more amino acid substitutions, insertions, or deletions in an Fc region of the antibody. See, e.g., U.S. Pat. No. 6,194,551. Alternatively, or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing. See, e.g., Caron et al. (1992)J Exp Med176:1191-1195 and Shopes (1992)Immunol148:2918-2922; PCT publication nos. WO 99/51642 and WO 94/29351; Duncan and Winter (1988)Nature322:738-40; and U.S. Pat. Nos. 5,648,260 and 5,624,821. In some aspects, the anti-CCR8 antibody is a bispecific antibody, a bispecific T cell engager (BiTE), a multispecific antibody, a biparatopic antibody, an immunoconjugate, an antibody drug conjugate, or any combination thereof. II.C. Antibody Variants and Immunoconjugates Certain aspects of the present disclosure are directed to antibodies comprising a first antigen-binding region that binds to human CCR8 and a second antigen-binding region that binds a second antigen, wherein the first antigen-binding region comprises an anti-CCR8 antibody described herein. In some aspects, the antibody is a bispecific antibody, e.g., capable of binding only two antigens. In some aspects, the antibody is a multispecific antibody, e.g., capable of binding more than two antigens. In some aspects, the multispecific antibody is capable of binding at least about 3 antigens, at least about 4 antigens, at least about 5 antigens, or at least about 6 antigens. In some aspects, the antibody is a biparatopic antibody. Biparatopic antibodies are capable of binding two epitopes on a single polypeptide target. In some aspects, the biparatopic antibody comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region and/or the second antigen-binding region comprises an anti-CCR8 antibody disclosed herein. In some aspects, the multispecific antibody is a bispecific T cell engager (BiTE). BiTE constructs comprises a first antigen-binding region that binds CD3 receptor on T cell and a second antigen-specific binding region. In some aspects, the BiTE comprises a first antigen-binding region that binds CD3 and a second antigen-binding region that binds human CCR8, wherein the second antigen-binding region comprises an anti-CCR8 antibody disclosed herein. In some aspects, the bispecific antibody, the multispecific antibody, the BiTE, or the biparatopic antibody comprises a first VH CDR1, a first VH CDR2, and a first VH CDR3; a first VL domain, comprising a first VL CDR1, a first VL CDR2, and a first VL CDR3; a second VH domain, comprising a second VH CDR1, a second VH CDR2, and a second VH CDR3; and a second VL domain, comprising a second VL CDR1, a second VL CDR2, and a second VL CDR3; wherein (a) the first VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 45, 105, 115, 135, and 145; (b) the first VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 46, 106, 116, 136, and 146; and (c) the first VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 47, 107, 117, 137, and 147. In some aspects, the first VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 48, 108, 118, 138, and 148; (b) the first VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 49, 109, 119, 139, and 149; and (c) the first VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 50, 110, 120, 140, and 150. In some aspects, (a) the VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65, 75, 85, 95, 125, 155, and 165; (b) the VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126, 156, and 166; and (c) the VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157, and 167. In some aspects, (a) the VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 58, 68, 78, 88, 98, 128, 158, and 168; (b) the VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129, 159, and 169; and (c) the VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130, 160, and 170. In some aspects, the antibody is a biparatopic antibody, and (a) the second VH CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 5, 15, 25, 35, 55, 65, 75, 85, 95, 125, 155, and 165; (b) the second VH CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 6, 16, 26, 36, 56, 66, 76, 86, 96, 126, 156, and 166; and (c) the second VH CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 7, 17, 27, 37, 57, 67, 77, 87, 97, 127, 157, and 167. In some aspects, the antibody is a biparatopic antibody, and (a) the second VL CDR1 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 8, 18, 28, 38, 58, 68, 78, 88, 98, 128, 158, and 168; (b) the second VL CDR2 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 9, 19, 29, 39, 59, 69, 79, 89, 99, 129, 159, and 169; and (c) the second VL CDR3 comprises an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NOs: 10, 20, 30, 40, 60, 70, 80, 90, 100, 130, 160, and 170. Certain aspects of the present disclosure are directed to an immunoconjugate comprising an anti-CCR8 antibody disclosed herein. In some aspects, the immunoconjugate is an antibody-drug conjugate. The immunoconjugate can include any cytotoxic agent known in the art linked to an anti-CCR8 antibody disclosed herein. In some aspects, the antibody-drug conjugate comprises a cytotoxic agent selected from the group consisting of a maytansinoid (e.g., maytansine), a dolastatin, an auristatin drug analogue, cryptophycin, a duocarmycin deriative (e.g., a CC-1065 analog and duocarmycin), an enediyne antibiotic (e.g., esperamicin and calicheamicin), pyrolobenodiazepine (PBD), and any combination thereof. An antibody-drug conjugate comprising an anti-CCR8 antibody and a cytotoxic agent may allow efficacy in tumor indications with low numbers of effector cells, including NK cells and macrophages. II.D. Fc Region Variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions. In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcTR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcTRIII only, whereas monocytes express FcTRI, FcTRII and FcTRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96 non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)). Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).) In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000). Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol.24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (e.g., U.S. Pat. No. 7,371,826). See also Duncan & Winter,Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fc region variants. In some embodiments, an antibody is provided, wherein the isotype is human IgG1. In some embodiments, an antibody is provided, wherein the isotype is human IgG4. In some embodiments, an antibody is provided, wherein the isotype is human IgG4, wherein there is a single mutation at serine 228 to proline (S228P). In some embodiments, an antibody is provided, wherein the isotype is human IgG4, wherein there are two mutations at serine 228 to proline (S228P) and leucine 235 to glutamate (L235E). The S228P mutation occurs at position 228 in the literature, however the exact location of a mutation in an antibody may vary depending on how such antibody is produced. I.E. Chimeric Antigen Receptors (CAR) and T Cell Receptors (TCR) Certain aspects of the present disclosure are directed to a chimeric antigen receptor (CAR) comprising an antigen-binding region that specifically binds the N-terminal extracellular domain of human CCR8. In some aspects, the antigen-binding region comprises an anti-CCR8 antibody disclosed herein or an antibody or antigen-binding fragment thereof that binds the same epitope as an anti-CCR8 antibody disclosed herein. In some aspects, the CAR further comprises a transmembrane domain. In some aspects, the CAR further comprises an intracellular signaling domain. In some aspects, the CAR further comprises a hinge region and/or a spacer region. Certain aspects of the present disclosure are directed to a T cell receptor (TCT) comprising an antigen-binding region that specifically binds the N-terminal extracellular domain of human CCR8. In some aspects, the antigen-binding region comprises an anti-CCR8 antibody disclosed herein or an antibody or antigen-binding fragment thereof that binds the same epitope as an anti-CCR8 antibody disclosed herein. In some aspects, the TCR further comprises a transmembrane domain. In some aspects, the TCR further comprises an intracellular signaling domain. I.F. Nucleic Acid Molecules, Vectors, and Cells Certain aspects of the present disclosure are directed to nucleic acid molecules that encode the anti-CCR8 antibodies disclose herein. The nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., the chromosomal DNA that is linked to the isolated DNA in nature) or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid described herein can be, for example, DNA or RNA and can or cannot contain intronic sequences. In some embodiments, the nucleic acid is a cDNA molecule. Nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), nucleic acid encoding the antibody can be recovered from the library. In some aspects, the nucleic acids can further encode a signal peptide. The nucleic acid molecules described herein may be modified to delete specific sequences, e.g., restriction enzyme recognition sequences, or to optimize codons. A method for making the anti-CCR8 antibody disclosed herein can comprise expressing the heavy chain and the light chains in a cell line comprising the nucleotide sequences encoding the heavy and light chains with a signal peptide. Host cells comprising these nucleotide sequences are encompassed herein. Once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term “operatively linked,” as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame. The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (hinge, CH1, CH2, and/or CH3). The sequences of human heavy chain constant region genes are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG1 region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region. The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554). Also provided herein are nucleic acid molecules encoding VH and VL sequences that are homologous to those of the anti-CCR8 antibodies disclosed herein. Exemplary nucleic acid molecules encode VH and VL sequences that are at least 70% identical, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to nucleic acid molecules encoding the VH and VL sequences disclosed herein. Also provided herein are nucleic acid molecules with conservative substitutions (i.e., substitutions that do not alter the resulting amino acid sequence upon translation of nucleic acid molecule), e.g., for codon optimization. Also provided are nucleic acids encoding the VH and/or VL regions of anti-CCR8 antibodies, such as the anti-CCR8 antibodies described herein, which nucleic acids comprise a nucleotide sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleotide sequences encoding the VH and/or VL regions of anti-CCR8 antibodies described herein. Also provided are nucleic acids encoding the heavy chain and/or the light chain of anti-CCR8 antibodies, such as the anti-CCR8 antibodies described herein, which nucleic acids comprise a nucleotide sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any of the nucleotide sequences encoding the heavy and/or light chains of anti-CCR8 antibodies described herein. Certain aspects of the present disclosure are directed to vectors comprising a nucleic acid molecule disclosed herein. In some aspects, the vector is selected from a viral vector, a mammalian vector, and a bacterial vector. In some aspects, the vector is a viral particle or a virus. In some aspects, the vector is a mammalian vector. In some aspects, the vector is a bacterial vector. In certain aspects, the viral vector is a retroviral vector. In some aspects, the viral vector is selected from the group consisting of an adenoviral vector, a lentivirus, a Sendai virus, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, and an adeno associated virus (AAV) vector. In particular aspects, the vector is an AAV vector. In some aspects, the vector is a lentivirus. In particular aspects, the vector is an AAV vector. In some aspects, the vector is a Sendai virus. In some aspects, the vector is a hybrid vector. Examples of hybrid vectors that can be used in the present disclosure can be found in Huang and Kamihira,Biotechnol. Adv.31(2):208-23 (2103), which is incorporated by reference herein in its entirety. In some aspects, the vector further comprises one or more regulatory elements, including but not limited to one or more enhancers, promoters, miRNA binding sequences, polyA sequences, intronic sequences, splice acceptor sites, and any combination thereof. In some aspects, the vector comprises a tissue specific enhancer. In some aspects, the vector comprises a tissue specific promoter. Certain aspects of the present disclosure are directed to cells, e.g., host cells, comprising an anti-CCR8 antibody disclosed herein, a bispecific antibody disclosed herein, a BiTE disclosed herein, a multispecific antibody disclosed herein, a biparatopic antibody disclosed herein, a CAR disclosed herein, a TCR disclosed herein, a nucleic acid molecule disclosed herein, or a vector disclosed herein. The cell can be any type of cell. In some aspects, the cell is selected from a mammalian cell, a bacterial cell, an insect cell, a plant cell, and a yeast cell. In some aspects, the cell is selected from the group consisting of anE. colicell, a fungi such asSaccharomyces cerevisiaeandPichia pastoris, an insect cell such as SF9, a mammalian cell lines (e.g., human cell lines), and a primary cell line. In some aspects, the cell is an immune cell. In some aspects, the cell is a T cell. As such, certain aspects of the present disclosure are directed to an immune cell, e.g., a T cell, comprising a CAR or a TCR disclosed herein. II.G. Pharmaceutical Compositions In certain aspects, the disclosure provides for a pharmaceutical composition comprising an anti-CCR8 antibody with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In certain aspects, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain aspects, the formulation material(s) are for s.c. and/or I.V. administration. In certain aspects, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain aspects, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In certain aspects, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In certain aspects, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain aspects, such compositions may influence the physical state, stability, rate of in vivo release and/or rate of in vivo clearance of the anti-CCR8 antibody. In certain aspects, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain aspects, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In certain aspects, the saline comprises isotonic phosphate-buffered saline. In certain aspects, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain aspects, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In certain aspects, a composition comprising an anti-CCR8 antibody can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain aspects, a composition comprising an anti-CCR8 antibody can be formulated as a lyophilizate using appropriate excipients such as sucrose. In certain aspects, the pharmaceutical composition can be selected for parenteral delivery. In certain aspects, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. In certain aspects, the formulation components are present in concentrations that are acceptable to the site of administration. In certain aspects, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In certain aspects, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising an anti-CCR8 antibody, in a pharmaceutically acceptable vehicle. In certain aspects, a vehicle for parenteral injection is sterile distilled water in which an anti-CCR8 antibody is formulated as a sterile, isotonic solution, and properly preserved. In certain aspects, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain aspects, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain aspects, implantable drug delivery devices can be used to introduce the desired molecule. In certain aspects, a pharmaceutical composition can be formulated for inhalation. In certain aspects, an anti-CCR8 antibody can be formulated as a dry powder for inhalation. In certain aspects, an inhalation solution comprising an anti-CCR8 antibody can be formulated with a propellant for aerosol delivery. In certain aspects, solutions can be nebulized. Pulmonary administration is further described in PCT application No. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins. In certain aspects, it is contemplated that formulations can be administered orally. In certain aspects, an anti-CCR8 antibody that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain aspects, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain aspects, at least one additional agent can be included to facilitate absorption of an anti-CCR8 antibody. In certain aspects, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed. In certain aspects, a pharmaceutical composition can involve an effective quantity of an anti-CCR8 antibody in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. In certain aspects, by dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. In certain aspects, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc. Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving an anti-CCR8 antibody in sustained- or controlled-delivery formulations. In certain aspects, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain aspects, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain aspects, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949. The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain aspects, this can be accomplished by filtration through sterile filtration membranes. In certain aspects, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain aspects, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain aspects, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. In certain aspects, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain aspects, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. In certain aspects, kits are provided for producing a single-dose administration unit. In certain aspects, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain aspects, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are included. In certain aspects, the effective amount of a pharmaceutical composition comprising an anti-CCR8 antibody to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain aspects, will thus vary depending, in part, upon the molecule delivered, the indication for which an anti-CCR8 antibody is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain aspects, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. In certain aspects, the frequency of dosing will take into account the pharmacokinetic parameters of an anti-CCR8 antibody in the formulation used. In certain aspects, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain aspects, the composition can therefore be administered as a single dose or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain aspects, appropriate dosages can be ascertained through use of appropriate dose-response data. In certain aspects, the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain aspects, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain aspects, individual elements of the combination therapy may be administered by different routes. In certain aspects, the composition can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain aspects, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. In certain aspects, it can be desirable to use a pharmaceutical composition comprising an anti-CCR8 antibody in an ex vivo manner. In such instances, cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising an anti-CCR8 antibody after which the cells, tissues and/or organs are subsequently implanted back into the patient. In certain aspects, an anti-CCR8 antibody can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain aspects, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain aspects, the cells can be immortalized. In certain aspects, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain aspects, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues. III. Methods of the Disclosure Certain aspects of the present disclosure are directed to methods of making and/or using the anti-CCR8 antibodies disclosed herein. III.A. Methods of Use Certain aspects of the present disclosure are directed to methods of reducing, depleting, or killing tumor infiltrating Tregs, comprising administering to the subject an anti-CCR8 antibody disclosed herein. Some aspects of the present disclosure are directed to methods of reducing, depleting, or killing tumor infiltrating Tregs, comprising administering a bispecific antibody, a BiTE, a multispecific antibody, a biparatopic antibody, an immunoconjugate, a CAR, a TCR, a nucleic acid molecule or a set of nucleic acid molecules, a vector or a set of vectors, a cell, or a pharmaceutical composition disclosed herein. Certain aspects of the present disclosure are directed to methods of activating NK cells or inducing NK cell mediated killing of tumor infiltrating regulatory Tregs, comprising administering to the subject an anti-CCR8 antibody disclosed herein. Some aspects of the present disclosure are directed to methods of activating NK cells or inducing NK cell mediated killing of tumor infiltrating regulatory Tregs, comprising administering a bispecific antibody, a BiTE, a multispecific antibody, a biparatopic antibody, an immunoconjugate, a CAR, a TCR, a nucleic acid molecule or a set of nucleic acid molecules, a vector or a set of vectors, a cell, or a pharmaceutical composition disclosed herein. In some aspects, the contacting is in vitro. In some aspects the contacting is in vivo. In some aspects, the contacting treats a disease or condition in a subject in need thereof. In some aspects, the contacting promotes an immune response in a subject. In some aspects, the subject has a tumor, and the contacting enhances an immune response to the tumor. Certain aspects of the present disclosure are directed to methods of treating a tumor in a subject in need thereof, comprising administering to the subject an anti-CCR8 antibody disclosed herein. Some aspects of the present disclosure are directed to methods of treating a tumor in a subject in need thereof comprising administering a bispecific antibody, a BiTE, a multispecific antibody, a biparatopic antibody, an immunoconjugate, a CAR, a TCR, a nucleic acid molecule or a set of nucleic acid molecules, a vector or a set of vectors, a cell, or a pharmaceutical composition disclosed herein. In some aspects, the subject has a tumor. In some aspects, the tumor is selected form the group consisting of Kaposi's sarcoma, leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocytic erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma (HCC), hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancers, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasm, kidney cancer, larynx cancer, liver cancer, lung cancer (small cell, large cell), melanoma, neuroblastoma; oral cavity cancer (for example lip, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of the respiratory system, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and cancer of the urinary system, or any combination thereof. In some aspects, the tumor is refractory to a prior therapy. In some aspects, the tumor is refractory to a prior standard of care therapy. In some aspects, the prior therapy comprises an immunotherapy, a chemotherapy, a surgery, a radiotherapy, or any combination thereof. In some aspects, the tumor is refractory to a prior chemotherapy. In some aspects, the tumor is refractory to a prior immunotherapy. In some aspects, the tumor is relapsed. In some aspects, the tumor is advanced. In some aspects, the tumor is locally advanced. In some aspects, the tumor is metastatic. In some aspects, the anti-CCR8 antibody is administered in combination with an additional anticancer agent. In some aspects, the additional anticancer agent is selected from a small molecule, a polypeptide, a radiation therapy, a surgery, and a combination thereof. In some aspects, the anti-CCR8 antibody is administered prior to the additional anticancer agent. In some aspects, the anti-CCR8 antibody is administered after the additional anticancer agent. In some aspects, the anti-CCR8 antibody is administered at the same time as the additional anticancer agent. In some aspects, the anti-CCR8 antibody and the additional anticancer agent are formulated in a single composition. In some aspects, the additional anticancer agent comprises a chemotherapy. The chemotherapy can be any chemotherapy known in the art. In some aspects, the chemotherapy is a standard of care treatment for the particular cancer type. In some aspects, the chemotherapy is a platinum-based chemotherapy. In some aspects, the chemotherapy is selected from the group consisting of doxorubicin (ADRIAMYCIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®; NEOSAR®), lenalidomide (REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab (RITUXAN®), ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide (THALOMID®), alemtuzumab (CAMPATH®), ofatumumab (ARZERRA®), everolimus (AFINITOR®, ZORTRESS®), carfilzomib (KYPROLIS™), and any combination thereof. In some aspects, the additional anticancer agent comprises an immunotherapy. In some aspects, the immunotherapy is selected from a PD-1 antagonist, a PD-L1 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, a TIGIT inhibitor, a CD112R inhibitor, a TAM inhibitor, a STING agonist, a 4-1BB agonist, a CCL22 inhibitor, an agent that induces NK cell activation, and any combination thereof. In some aspects, the additional anticancer therapy comprises a PD-1 antagonist. Any PD-1 antagonist known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the PD-1 antagonist is is an antibody or antigen-binding portion thereof that specifically binds PD-1. Nonlimiting examples of PD-1 antagonists that can be used in combination with the anti-CCR8 antibodies disclosed herein include PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224. In some aspects, the additional anticancer therapy comprises a PD-L1 inhibitor. Any PD-L1 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the PD-L1 inhibitor is is an antibody or antigen-binding portion thereof that specifically binds PD-L1. Nonlimiting examples of PD-L1 inhibitors that can be used in combination with the anti-CCR8 antibodies disclosed herein include FAZ053, Atezolizumab, Avelumab, Durvalumab, and BMS-936559. In some aspects, the additional anticancer therapy comprises a TIM-3 inhibitor. Any TIM-3 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the TIM-3 inhibitor is is an antibody or antigen-binding portion thereof that specifically binds TIM-3. Nonlimiting examples of TIM-3 inhibitors that can be used in combination with the anti-CCR8 antibodies disclosed herein include MGB453 and TSR-022. In some aspects, the additional anticancer therapy comprises a LAG-3 inhibitor. Any LAG-3 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the LAG-3 inhibitor is is an antibody or antigen-binding portion thereof that specifically binds LAG-3. Nonlimiting examples of LAG-3 inhibitors that can be used in combination with the anti-CCR8 antibodies disclosed herein include LAG525, BMS-986016, and TSR-033. In some aspects, the additional anticancer therapy comprises a TIGIT inhibitor. Any TIGIT inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the TIGIT inhibitor is is an antibody or antigen-binding portion thereof that specifically binds TIGIT. In some aspects, the additional anticancer therapy comprises a CD112R inhibitor. Any CD112R inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the CD112R inhibitor is is an antibody or antigen-binding portion thereof that specifically binds CD112R. In some aspects, the additional anticancer therapy comprises a CCL22 inhibitor. Any CCL22 inhibitor known in the art can be used in combination with the anti-CCR8 antibodies disclosed herein. In some aspects, the CCL22 inhibitor is is an antibody or antigen-binding portion thereof that specifically binds CCL22. In some aspects, the additional anticancer therapy an agent that induces NK cell activation, and therefore enhances ADCC activity of the CCR8 antibody. In some aspects, the agent that induces NK cell activation is an antibody or antigen-binding portion thereof, a small molecule, a cytokine, or a cytokine fusion. In certain aspects, the additional anticancer agent comprises an anticancer agent selected from the group consisting of Sunitinib (SUTENT®), Cabozantinib (CABOMETYX®), Axitinib (INLYTA®), Lenvatinib (LENVIMA®), Everolimus (AFINITOR®), Bevacizumab (AVASTIN®), epacadostat, NKTR-214 (CD-122-biased agonist), Tivozanib (FOTIVDA®), abexinostat, Ipilimumab (YERVOY®), tremelimumab, Pazopanib (VOTRIENT®), Sorafenib (NEXAVAR®), Temsirolimus (TORISEL®), Ramucirumab (CYRAMZA®), niraparib, savolitinib, vorolanib (X-82), Regorafenib (STIVARGO®), Donafenib (multikinase inhibitor), Camrelizumab (SHR-1210), pexastimogene devacirepvec (JX-594), Ramucirumab (CYRAMZA®), apatinib (YN968D1), encapsulated doxorubicin (THERMODOX®), Tivantinib (ARQ197), ADI-PEG 20, binimetinib, apatinib mesylate, nintedanib, lirilumab, Nivolumab (OPDIVO®), Pembrolizumab (KEYTRUDA®), Atezolizumab (TECENTRIQ®), Avelumab (BAVENCIO®), Durvalumab (IMFIMZI®), Cemiplimab-rwlc (LIBTAYO®), tislelizumab, spartalizumab, and any combination thereof. In some aspects, the additional anticancer agent comprises a TAM (Axl, Mer, Tyro) inhibitor. In some aspects, the additional anticancer agent comprises a 4-1BB agonist. In some aspects, the additional anticancer agent comprises a Tyrosine Kinase Inhibitor (TKI). Nonlimiting examples of TKIs that can be used in combination with the anti-CCR8 antibodies disclosed herein include imatinib mesylate, dasatinib, nilotinib, and bosutinib. The anti-CCR8 antibodies of the present disclosure can be administered by any suitable route. In some aspects, the anti-CCR8 antibody is administered intravenously. In some aspects, the anti-CCR8 antibody is subcutaneously. In some aspects, the anti-CCR8 antibody is administered intramuscularly. In some aspects, the anti-CCR8 antibody is administered intraperitoneally. In some aspects, the anti-CCR8 antibody is administered orally. III.B. Methods for Producing Anti-CCR8 Antibodies The disclosure also features methods for producing any of the anti-CCR8 antibodies described herein. In some aspects, methods for preparing an antibody described herein can include immunizing a subject (e.g., a non-human mammal) with an appropriate immunogen. Suitable immunogens for generating any of the antibodies described herein are set forth herein. For example, to generate an antibody that binds to the N-terminal extracellular domain of human CCR8, a skilled artisan can immunize a suitable subject (e.g., a non-human mammal such as a rat, a mouse, a gerbil, a hamster, a dog, a cat, a pig, a goat, a horse, or a non-human primate) with a fragment of human CCR8 comprising the N-terminal extracellular domain. In some aspects, a fragment polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 172 is used as the immunogen. A suitable subject (e.g., a non-human mammal) can be immunized with the appropriate antigen along with subsequent booster immunizations a number of times sufficient to elicit the production of an antibody by the mammal. The immunogen can be administered to a subject (e.g., a non-human mammal) with an adjuvant. Adjuvants useful in producing an antibody in a subject include, but are not limited to, protein adjuvants; bacterial adjuvants, e.g., whole bacteria (BCG,Corynebacterium parvumorSalmonella minnesota) and bacterial components including cell wall skeleton, trehalose dimycolate, monophosphoryl lipid A, methanol extractable residue (MER) of tuberclebacillus, complete or incomplete Freund's adjuvant; viral adjuvants; chemical adjuvants, e.g., aluminum hydroxide, and iodoacetate and cholesteryl hemisuccinate. Other adjuvants that can be used in the methods for inducing an immune response include, e.g., cholera toxin and parapoxvirus proteins. See also Bieg et al. (1999)Autoimmunity31(1):15-24. See also, e.g., Lodmell et al. (2000)Vaccine18:1059-1066; Johnson et al. (1999)J Med Chem42:4640-4649; Baldridge et al. (1999)Methods19:103-107; and Gupta et al. (1995)Vaccine13(14): 1263-1276. In some aspects, the methods include preparing a hybridoma cell line that secretes a monoclonal antibody that binds to the immunogen. For example, a suitable mammal such as a laboratory mouse is immunized with a CCR8 polypeptide as described above. Antibody-producing cells (e.g., B cells of the spleen) of the immunized mammal can be isolated two to four days after at least one booster immunization of the immunogen and then grown briefly in culture before fusion with cells of a suitable myeloma cell line. The cells can be fused in the presence of a fusion promoter such as, e.g., vaccinia virus or polyethylene glycol. The hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells of Balb/c mice immunized with a suitable immunogen can be fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14. After the fusion, the cells are expanded in suitable culture medium, which is supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells. The obtained hybrid cells are then screened for secretion of the desired antibodies, e.g., an antibody that binds to human CCR8 and In some aspects, a skilled artisan can identify an anti-CCR8 antibody from a non-immune biased library as described in, e.g., U.S. Pat. No. 6,300,064 (to Knappik et al.; Morphosys AG) and Schoonbroodt et al. (2005)Nucleic Acids Res33(9):e81. In some aspects, the methods described herein can involve, or be used in conjunction with, e.g., phage display technologies, bacterial display, yeast surface display, eukaryotic viral display, mammalian cell display, and cell-free (e.g., ribosomal display) antibody screening techniques (see, e.g., Etz et al. (2001)J Bacteriol183:6924-6935; Cornelis (2000)Curr Opin Biotechnol11:450-454; Klemm et al. (2000)Microbiology146:3025-3032; Kieke et al. (1997)Protein Eng10:1303-1310; Yeung et al. (2002)Biotechnol Prog18:212-220; Boder et al. (2000)Methods Enzymology328:430-444; Grabherr et al. (2001)Comb Chem High Throughput Screen4:185-192; Michael et al. (1995)Gene Ther2:660-668; Pereboev et al. (2001)J Virol75:7107-7113; Schaffitzel et al. (1999)J Immunol Methods231:119-135; and Hanes et al. (2000)Nat Biotechnol18:1287-1292). Methods for identifying antibodies using various phage display methods are known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains of antibodies, such as Fab, Fv, or disulfide-bond stabilized Fv antibody fragments, expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage used in these methods are typically filamentous phage such as fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to any of the phage coat proteins pIII, pVIII, or pIX. See, e.g., Shi et al. (2010)JMB397:385-396. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, described herein include those disclosed in Brinkman et al. (1995)J Immunol Methods182:41-50; Ames et al. (1995)J Immunol Methods184:177-186; Kettleborough et al. (1994)Eur J Immunol24:952-958; Persic et al. (1997)Gene187:9-18; Burton et al. (1994)Advances in Immunology57:191-280; and PCT publication nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, and WO 95/20401. Suitable methods are also described in, e.g., U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108. In some aspects, the phage display antibody libraries can be generated using mRNA collected from B cells from the immunized mammals. For example, a splenic cell sample comprising B cells can be isolated from mice immunized with a CCR8 polypeptide as described above. mRNA can be isolated from the cells and converted to cDNA using standard molecular biology techniques. See, e.g., Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual, 2ndEdition,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane (1988), supra; Benny K. C. Lo (2004), supra; and Borrebaek (1995), supra. The cDNA coding for the variable regions of the heavy chain and light chain polypeptides of immunoglobulins are used to construct the phage display library. Methods for generating such a library are described in, e.g., Merz et al. (1995)J Neurosci Methods62(1-2):213-9; Di Niro et al. (2005)Biochem J388(Pt 3):889-894; and Engberg et al. (1995)Methods Mol Biol51:355-376. In some aspects, a combination of selection and screening can be employed to identify an antibody of interest from, e.g., a population of hybridoma-derived antibodies or a phage display antibody library. Suitable methods are known in the art and are described in, e.g., Hoogenboom (1997)Trends in Biotechnology15:62-70; Brinkman et al. (1995), supra; Ames et al. (1995), supra; Kettleborough et al. (1994), supra; Persic et al. (1997), supra; and Burton et al. (1994), supra. For example, a plurality of phagemid vectors, each encoding a fusion protein of a bacteriophage coat protein (e.g., pIII, pVIII, or pIX of M13 phage) and a different antigen-combining region are produced using standard molecular biology techniques and then introduced into a population of bacteria (e.g.,E. coli). Expression of the bacteriophage in bacteria can, in some aspects, require use of a helper phage. In some aspects, no helper phage is required (see, e.g., Chasteen et al., (2006)Nucleic Acids Res34(21):e145). Phage produced from the bacteria are recovered and then contacted to, e.g., a target antigen bound to a solid support (immobilized). Phage may also be contacted to antigen in solution, and the complex is subsequently bound to a solid support. A subpopulation of antibodies screened using the above methods can be characterized for their specificity and binding affinity for a particular antigen (e.g., human CCR8) using any immunological or biochemical based method known in the art. For example, specific binding of an antibody to CCR8, may be determined for example using immunological or biochemical based methods such as, but not limited to, an ELISA assay, SPR assays, immunoprecipitation assay, affinity chromatography, and equilibrium dialysis as described above. Immunoassays which can be used to analyze immunospecific binding and cross-reactivity of the antibodies include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blots, RIA, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art. In aspects where the selected CDR amino acid sequences are short sequences (e.g., fewer than 10-15 amino acids in length), nucleic acids encoding the CDRs can be chemically synthesized as described in, e.g., Shiraishi et al. (2007)Nucleic Acids Symposium Series51(1):129-130 and U.S. Pat. No. 6,995,259. For a given nucleic acid sequence encoding an acceptor antibody, the region of the nucleic acid sequence encoding the CDRs can be replaced with the chemically synthesized nucleic acids using standard molecular biology techniques. The 5′ and 3′ ends of the chemically synthesized nucleic acids can be synthesized to comprise sticky end restriction enzyme sites for use in cloning the nucleic acids into the nucleic acid encoding the variable region of the donor antibody. III.C. Recombinant Antibody Expression and Purification The antibodies or antigen-binding fragments thereof described herein can be produced using a variety of techniques known in the art of molecular biology and protein chemistry. For example, a nucleic acid encoding one or both of the heavy and light chain polypeptides of an antibody can be inserted into an expression vector that contains transcriptional and translational regulatory sequences, which include, e.g., promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, transcription terminator signals, polyadenylation signals, and enhancer or activator sequences. The regulatory sequences include a promoter and transcriptional start and stop sequences. In addition, the expression vector can include more than one replication system such that it can be maintained in two different organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Several possible vector systems are available for the expression of cloned heavy chain and light chain polypeptides from nucleic acids in mammalian cells. One class of vectors relies upon the integration of the desired gene sequences into the host cell genome. Cells which have stably integrated DNA can be selected by simultaneously introducing drug resistance genes such asE. coligpt (Mulligan and Berg (1981)Proc Natl Acad Sci USA78:2072) or Tn5 neo (Southern and Berg (1982)Mol Appl Genet1:327). The selectable marker gene can be either linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection (Wigler et al. (1979)Cell16:77). A second class of vectors utilizes DNA elements which confer autonomously replicating capabilities to an extrachromosomal plasmid. These vectors can be derived from animal viruses, such as bovine papillomavirus (Sarver et al. (1982)Proc Natl Acad Sci USA,79:7147), cytomegalovirus, polyoma virus (Deans et al. (1984)Proc Natl Acad Sci USA81:1292), or SV40 virus (Lusky and Botchan (1981)Nature293:79). The expression vectors can be introduced into cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPO4precipitation, liposome fusion, cationic liposomes, electroporation, viral infection, dextran-mediated transfection, polybrene-mediated transfection, protoplast fusion, and direct microinjection. Appropriate host cells for the expression of antibodies or antigen-binding fragments thereof include yeast, bacteria, insect, plant, and mammalian cells. Of particular interest are bacteria such asE. coli, fungi such asSaccharomyces cerevisiaeandPichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines. In some aspects, an antibody or fragment thereof can be expressed in, and purified from, transgenic animals (e.g., transgenic mammals). For example, an antibody can be produced in transgenic non-human mammals (e.g., rodents) and isolated from milk as described in, e.g., Houdebine (2002)Curr Opin Biotechnol13(6):625-629; van Kuik-Romeijn et al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999)J Immunol Methods231(1-2):147-157. The antibodies and fragments thereof can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the antibodies or fragments, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed inE. colican be refolded from inclusion bodies (see, e.g., Hou et al. (1998)Cytokine10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning—A Laboratory Manual—3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells will vary depending on a number of factors, and may be easily optimized as needed. An antibody (or fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000)Protein Expression and Purification18:213-220). Following expression, the antibodies and fragments thereof can be isolated. An antibody or fragment thereof can be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography. For example, an antibody can be purified using a standard anti-antibody column (e.g., a protein-A or protein-G column). Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. See, e.g., Scopes (1994) “Protein Purification, 3rdedition,” Springer-Verlag, New York City, N.Y. The degree of purification necessary will vary depending on the desired use. In some instances, no purification of the expressed antibody or fragments thereof will be necessary. Methods for determining the yield or purity of a purified antibody or fragment thereof are known in the art and include, e.g., Bradford assay, UV spectroscopy, Biuret protein assay, Lowry protein assay, amido black protein assay, high pressure liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoretic methods (e.g., using a protein stain such as Coomassie Blue or colloidal silver stain). III.D. Modification of the Antibodies or Antigen-Binding Fragments Thereof The antibodies or antigen-binding fragments thereof can be modified following their expression and purification. The modifications can be covalent or non-covalent modifications. Such modifications can be introduced into the antibodies or fragments by, e.g., reacting targeted amino acid residues of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Suitable sites for modification can be chosen using any of a variety of criteria including, e.g., structural analysis or amino acid sequence analysis of the antibodies or fragments. In some aspects, the antibodies or antigen-binding fragments thereof can be conjugated to a heterologous moiety. The heterologous moiety can be, e.g., a heterologous polypeptide, a therapeutic agent (e.g., a toxin or a drug), or a detectable label such as, but not limited to, a radioactive label, an enzymatic label, a fluorescent label, a heavy metal label, a luminescent label, or an affinity tag such as biotin or streptavidin. Suitable heterologous polypeptides include, e.g., an antigenic tag (FLAG ( (SEQ ID NO: 241)(FLAG DYKDDDDK), ), polyhistidine (6-His; (SEQ ID NO: 142)(6-His; HHHHHH, hemagglutinin (HA; (SEQ ID NO: 242)(HA; YPYDVPDYA), ), glutathione-S-transferase (GST), or maltose-binding protein (MBP)) for use in purifying the antibodies or fragments. Heterologous polypeptides also include polypeptides (e.g., enzymes) that are useful as diagnostic or detectable markers, for example, luciferase, a fluorescent protein (e.g., green fluorescent protein (GFP)), or chloramphenicol acetyl transferase (CAT). Suitable radioactive labels include, e.g.,32P33P,14C,125I,131I,35S, and3H. Suitable fluorescent labels include, without limitation, fluorescein, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), DyLight™ 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor® 700, Cy5, allophycocyanin, and Cy7. Luminescent labels include, e.g., any of a variety of luminescent lanthanide (e.g., europium or terbium) chelates. For example, suitable europium chelates include the europium chelate of diethylene triamine pentaacetic acid (DTPA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Enzymatic labels include, e.g., alkaline phosphatase, CAT, luciferase, and horseradish peroxidase. Two proteins (e.g., an antibody and a heterologous moiety) can be cross-linked using any of a number of known chemical cross linkers. Examples of such cross linkers are those which link two amino acid residues via a linkage that includes a “hindered” disulfide bond. In these linkages, a disulfide bond within the cross-linking unit is protected (by hindering groups on either side of the disulfide bond) from reduction by the action, for example, of reduced glutathione or the enzyme disulfide reductase. One suitable reagent, 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio) toluene (SMPT), forms such a linkage between two proteins utilizing a terminal lysine on one of the proteins and a terminal cysteine on the other. Heterobifunctional reagents that cross-link by a different coupling moiety on each protein can also be used. Other useful cross-linkers include, without limitation, reagents which link two amino groups (e.g., N-5-azido-2-nitrobenzoyloxysuccinimide), two sulfhydryl groups (e.g., 1,4-bis-maleimidobutane), an amino group and a sulfhydryl group (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), an amino group and a carboxyl group (e.g., 4-[p-azidosalicylamido]butylamine), and an amino group and a guanidinium group that is present in the side chain of arginine (e.g., p-azidophenyl glyoxal monohydrate). In some aspects, a radioactive label can be directly conjugated to the amino acid backbone of the antibody. Alternatively, the radioactive label can be included as part of a larger molecule (e.g.,125I in meta-[125I]iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS) which binds to free amino groups to form meta-iodophenyl (mIP) derivatives of relevant proteins (see, e.g., Rogers et al. (1997)J Nucl Med38:1221-1229) or chelate (e.g., to DOTA or DTPA) which is in turn bound to the protein backbone. Methods of conjugating the radioactive labels or larger molecules/chelates containing them to the antibodies or antigen-binding fragments described herein are known in the art. Such methods involve incubating the proteins with the radioactive label under conditions (e.g., pH, salt concentration, and/or temperature) that facilitate binding of the radioactive label or chelate to the protein (see, e.g., U.S. Pat. No. 6,001,329). Methods for conjugating a fluorescent label (sometimes referred to as a “fluorophore”) to a protein (e.g., an antibody) are known in the art of protein chemistry. For example, fluorophores can be conjugated to free amino groups (e.g., of lysines) or sulfhydryl groups (e.g., cysteines) of proteins using succinimidyl (NHS) ester or tetrafluorophenyl (TFP) ester moieties attached to the fluorophores. In some aspects, the fluorophores can be conjugated to a heterobifunctional cross-linker moiety such as sulfo-SMCC. Suitable conjugation methods involve incubating an antibody protein, or fragment thereof, with the fluorophore under conditions that facilitate binding of the fluorophore to the protein. See, e.g., Welch and Redvanly (2003) “Handbook of Radiopharmaceuticals: Radiochemistry and Applications,” John Wiley and Sons (ISBN 0471495603). In some aspects, the antibodies or fragments can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, the antibody or fragment can be PEGylated as described in, e.g., Lee et al. (1999)Bioconjug Chem10(6): 973-8; Kinstler et al. (2002)Advanced Drug Deliveries Reviews54:477-485; and Roberts et al. (2002)Advanced Drug Delivery Reviews54:459-476 or HESylated (Fresenius Kabi, Germany; see, e.g., Pavisid et al. (2010)Int J Pharm387(1-2):110-119). The stabilization moiety can improve the stability, or retention of, the antibody (or fragment) by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold. In some aspects, the antibodies or antigen-binding fragments thereof described herein can be glycosylated. In some aspects, an antibody or antigen-binding fragment thereof described herein can be subjected to enzymatic or chemical treatment, or produced from a cell, such that the antibody or fragment has reduced or absent glycosylation. Methods for producing antibodies with reduced glycosylation are known in the art and described in, e.g., U.S. Pat. No. 6,933,368; Wright et al. (1991)EMBO J10(10):2717-2723; and Co et al. (1993)Mol Immunol30:1361. EXAMPLES Example 1: Generation of Antibodies Seventeen monoclonal antibodies specific to human CCR8 were generated against an N-terminal fragment of CCR8. Antibody Phage Panning, Cloning and Transfection Recombinant proteins expressing either the N-terminal extracellular domain of human or cynomolgus CCR8 fused to a 6×His tag followed by mouse IgG2a-Fc (CCR8-Fc) were cloned into mammalian expression vectors (SEQ ID NOs: 173 and 174, respectively; Table 4). For the Human protein, the free cysteine as position 25 was mutated to a serine to prevent disulfide bonding. The resulting secreted proteins were expressed by transfection in CHO cells and were purified using Protein A and used as antigen for phage panning using Fab display library. For panning, the purified protein was couple to M280 Tosyl beads or to ELISA plate and panning done using standard methods. Successive rounds of panning were performed on Human CCR8-ECD-Fc or in alternating rounds with cynomolgus CCR8-ECD-Fc, with unbound phage being removed by washing each round. The DNA from the resulting bound phage pool was isolated and the heavy and light chain sequences were cloned into a mammalian expression vector. Individual transformed colonies were grown separately in wells of a 96-well bacterial culture plate and the DNA was purified using Qiagen Turbo Miniprep Kit. The 96-well DNA mini-libraries were used to transfect CHO cells in the same 96 well format and the secreted antibody supernatants were harvested after three days incubation at 37° C., 7% CO2incubator. TABLE 4CCR8-ECD-Fc Sequences (signal peptide; CCR8 extracellular domain;6X His tag and linker; and mouse IgG2a-Fc)HumanMGWSCIILFLVATATGAHSMDYTLDLSVTTVTDYYYPDIFSSPSDAELIQTNGKCCR8-FcHHHHHHSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 173)CynomolgusMGWSCIILFLVATATGAHSMDYTLDPSMTTMTDYYYPDSLSSPSDGELIQRNDKCCR8-FcHHHHHHSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 174) Flow Cytometry of CHO Supernatants 293T cells expressing human CCR8, cynomolgus CCR8, mouse CCR8 and human CCR2 were harvested using Accutase® and 100,000 cells were dispensed to each well of a 96 well v-bottom plate. In some instances, the native human CCR8 expressing cell line Hut78 was also tested. The mini-library CHO supernatants were then added at a 1:2 final dilution to each cell type and allowed to incubate at 4° C. for 1 hour. After pelleting, the cells were incubated with anti-human-Fc-biotin followed by streptavidin-APC or with anti-human-Fc-APC at 4° C. for 30 minutes. After washing and fixation, the cells were run on the FACS Canto II with propidium iodide for live/dead discrimination. The live cells were analyzed and clones that showed specific binding to CCR8 (human and/or cynomologus) but not CCR2 were sent for DNA sequencing and further testing. Development of Anti-CCR8-1 The parental antibody, anti-CCR8-parental-1, was developed by phage panning as outlined above on the human CCR8 N-terminal protein and had an affinity of 1 nM on the 293T-Human CCR8 cell line. To improve affinity, a library was created in Vaccinia Virus where the CDR3 of the heavy chain was randomized. A library of 12,000 CDR3 variants was created in vaccinia. Upon overnight infection with the heavy chain CDR3 library (one clone per cell) and the parental light chain virus construct, full-length human IgG antibody is expressed on the cell surface (reference). Infected A431 cells were incubated with the human CCR8-N-terminal protein at a final concentration of 0.1 μg/ml, washed and stained with anti-Fc-Dylight649 to detect CCR8 protein binding and anti-Hu-Fab-FITC to detect antibody expression on the cell surface. Two thousand cells with high antibody expression and high CCR8 binding were sorted and the virus was amplified. DNA was extracted from the virus pool and the new heavy chain V genes were cloned into the mammalian cell expression vector containing a signal sequence and the human IgG1 constant domain (resulting in full length IgG1) and co-transfected along with the parental light chain in CHO cells for clone evaluation as outlined above. The anti-CCR8-1 antibody (comprising a variable heavy chain having the amino acid sequence set forth in SEQ ID NO: 41 and a variable light chain having the amino acid sequence set forth in SEQ ID NO: 43) was found to have three amino acid mutations in the CDR3 as compared to the parental heavy chain and an affinity of 0.4 nM on the 293T-Human CCR8 cell line. The anti-CCR8-1 antibody was found to bind huCC8-ECD-Fc but not CyCCR8-ECD-Fc (FIG.1A), and the antibody preferentially bound 293T cells expressing HuCCR8 but not cyno CCR8, human CCR2, or mouse CCR8 (FIG.1). Development of Anti-CCR8-1-1, Anti-CCR8-1-2, Anti-CCR8-2-3, Anti-CCR8-1-4, and Anti-CCR8-1-5 The heavy chain of the parental antibody (anti-CCR8-parental-1) was cloned into Vaccinia Virus to facilitate light chain shuffle panning with Vaccinex's Vaccinia Display human IgG libraries. Briefly, 6×108BHK cells were infected with the heavy chain form the anti-CCR8-parental-1 antibody (H23188) and a pool of lambda light chains from naïve sources. After two days incubation at 37° C., 7% CO2, the supernatant was harvested and the vaccinia virus particles expressing a library of human IgG on their surface were pelleted by centrifugation. The pellet was resuspended and incubated with the purified Human CCR8-Fc protein coupled to M280 Tosyl beads. The unbound virus was washed away and the bound virus was amplified for subsequent rounds. After performing two rounds on human CCR8-Fc protein followed by two rounds on cyno CCR8-Fc protein, the DNA from the bound pool was extracted and the new light chains cloned into a mammalian expression vector for CHO transfection along with the parental heavy chain and flow cytometry analysis as detailed above. The anti-CCR8-1-1, anti-CCR8-1-2, anti-CCR8-2-3, anti-CCR8-1-4, and anti-CCR8-1-5 antibodies were found to bind huCC8-ECD-Fc at a higher specificity than CyCCR8-ECD-Fc (FIGS.1C,1E,1G,1H, and1K, respectively), and each antibody preferentially bound 293T cells expressing HuCCR8 over cyno CCR8, human CCR2, and mouse CCR8 (FIGS.1D,1F,1H,1J, and1K, respectively). TABLE 5Anti-CCR8 AntibodiesAffinityBindingBindingHeavyAffinityfor 293T-totoChainfor 293T-CyCCR8HuCCR8-CyCCR8-AntibodyMab(SEQ IDLightHu CCR8cellsFcFcSourceNumberNO)Chaincells (nM)(nM)proteinproteinParentalanti-H23188L10371.06>50++++−CCR8-parental-1HCDR3Anti-41430.41>50+++−randomizationCCR8-1VL ShuffleAnti-1011031.0122.1++++CCR8-1-1VL ShuffleAnti-1111131.06>50++++CCR8-1-2VL ShuffleAnti-1211231.38>50++++CCR8-1-3VL ShuffleAnti-1311330.2835.9++++CCR8-1-4VL ShuffleAnti-1411431.3648.6++++CCR8-1-5 Development of Anti-CCR8-2 and Anti-CCR8-2-1 The parental antibody (anti-CCR8-parental-2) was developed by phage panning as outlined above with two rounds on the Human protein followed by two rounds on the cynomolgus N-terminal protein. It was found to bind to both the human and the cynomolgus CCR8 cell lines, with affinities of 19.9 nM and 11.1 nM respectively. To improve affinity, a library was created in phage where the CDR1 and CDR2 of the heavy chain were randomized. This library was panned one round on the Cyno-CCR8-ECD-Fc protein followed by two consecutive rounds on the Human-CCR8-ECD-Fc protein. Individual phage clones were processed in a 96 well format and analyzed by phage ELISA on both the human and the cynomolgus proteins as well as negative antigens. Clones that were specific for CCR8 were sent for sequencing and cloned into mammalian expression vectors for further characterization. The anti-CCR8-2 antibody had an improved affinity of 0.4 nM on 293T-HuCCR8 cells and 0.9 nM on 293T-Cyno CCR8 cells derived from two amino acid mutations each in CDR1 and CDR2. Both the anti-CCR8-2 and anti-CCR8-2-1 antibodies showed binding to both human and cyno CCR8 protein (FIGS.2C and2E) and cells expressing both human and cyno CCR8 (FIGS.2D and2F). Development of Anti-CCR8-2-2 The heavy chain of the parental antibody, anti-CCR8-parental-2, was cloned into Vaccinia Virus to facilitate light chain shuffle panning with Vaccinia Display human IgG libraries. Briefly, 6×108BHK cells were infected with the heavy chain form the anti-CCR8-parental-2 antibody (H23407) and a pool of lambda light chains from naïve sources. After two days incubation at 37° C., 7% CO2, the supernatant was harvested and the vaccinia virus particles expressing a library of human IgG on their surface were pelleted by centrifugation. The pellet was resuspended and incubated with the purified cynomolgus CCR8-Fc protein coupled to M280 Tosyl beads. The unbound virus was washed away and the bound virus was amplified for subsequent rounds. After performing two rounds on cynomolgus CCR8-Fc protein, the bound pool was sorted for human/cyno cross-binders with 0.1 μg/ml biotin-cynomolgus CCR8-Fc and 0.1 μg/ml human CCR8-Fc protein. The highest binders to both proteins were collected, amplified and the DNA was extracted for cloning into a mammalian expression vector for CHO transfection and flow cytometry analysis as detailed above. The light chains that showed the best binding to 293T-CCR8 cell lines were cross-paired with the heavy chain from the anti-CCR8-2 antibody (H23727) to check for synergistic improvement in affinity. The anti-CCR8-2-2 antibody exhibited improved cross-reactive binding to human CCR8 as compared to the parental (FIGS.2E-2F). TABLE 6Anti-CCR8 AntibodiesAffinityAffinityLightforforBindingBindingHeavyChain293T-293T-totoChain(SEQHuCCR8CyCCR8HuCCR8-CyCCR8-AntibodyMab(SEQIDcellscellsFcFcSourceNumberID NO)NO)(nM)(nM)proteinproteinParentalAnti-H23407L103219.911.1++++++++CCR8-Parental-2HCDR1/HCDR2Anti-11130.410.94++++++randomizationCCR8-2HCDR1/HCDR2Anti-51530.541.09++++++randomizationCCR8-2-1HCDR1/HCDR2Anti-1611630.81.6++++++randomization &CCR8-2-2VL Shuffle Development of Anti-CCR8-2-3 and Anti-CCR8-2-4 The parental antibody, anti-CCR8-parental-3, was developed by phage panning as outlined above with two rounds on the Human protein followed by two rounds on the cynomolgus N-terminal protein. It was found to have binding to the human CCR8 cell lines, with an affinity of 6.8 nM. To improve affinity, a library was created in phage where the CDR3 of the heavy chain was randomized. This library was panned one round on the Cyno-CCR8-ECD-Fc protein followed by one round on the Human-CCR8-ECD-Fc protein. The DNA from the bound pool was extracted and cloned into a mammalian expression vector for CHO transfection and flow cytometry analysis as detailed above. Both the anti-CCR8-2-3 and the anti-CCR8-2-4 antibodies showed cross-reactive binding to both the human and cyno CCR8 cell lines (FIGS.2G-2J). Development of Anti-CCR8-2-5 and Anti-CCR8-2-6 A library of CDR3 variants for the heavy chain of the anti-CCR8-parental-3 antibody was also made in vaccinia virus. The library was sorted for human/cyno cross-binders with 1 μg/ml biotin-cynomolgus CCR8-Fc and 1 μg/ml human CCR8-Fc protein. The highest binders to both proteins were collected, amplified and the DNA was extracted for cloning into a mammalian expression vector for CHO transfection and flow cytometry analysis as detailed above. Both the anti-CCR8-2-5 and the anti-CCR8-2-6 antibodies showed cross-reactive binding to both the human and cyno CCR8 cell lines (FIGS.2K-2N). Development of Anti-CCR8-2-7, Anti-CCR8-2-8, Anti-CCR8-2-9, and Anti-CCR8-2-10 The heavy chain of the anti-CCR8-parental-3 antibody was cloned into Vaccinia Virus to facilitate light chain shuffle panning with Vaccinex's Vaccinia Display human IgG libraries. Briefly, 6×108BHK cells were infected with the heavy chain from the anti-CCR8-parental-3 antibody (H23373) and a pool of lambda light chains from naïve sources. After two days incubation at 37° C., 7% C02, the supernatant was harvested and the vaccinia virus particles expressing a library of human IgG on their surface were pelleted by centrifugation. The pellet was resuspended and incubated with the purified cynomolgus CCR8-Fc protein coupled to M280 Tosyl beads. The unbound virus was washed away and the bound virus was amplified for subsequent rounds. After performing two rounds on cynomologous CCR8-Fc protein, the bound pool was sorted for human/cyno cross-binders with 0.1 μg/ml biotin-cynomolgus CCR8-Fc and 0.1 μg/ml Human CCR8-Fc protein. The highest binders to both proteins were collected, amplified and the DNA was extracted for cloning into a mammalian expression vector for CHO transfection and flow cytometry analysis as detailed above. The light chains that showed the best binding to 293T-CCR8 cell lines were cross-paired with the heavy chain from the anti-CCR8-2-3 antibody (H23499), and other heavy chains developed through the phage heavy chain CDR3 efforts in order to check for synergistic improvement in affinity. The anti-CCR8-2-7, anti-CCR8-2-8, anti-CCR8-2-9, and anti-CCR8-2-10 antibodies all showed enhanced binding to both human and cynomolgus CCR8 cell lines over the anti-CCR8-parental-3 antibody (FIGS.2O-2V). TABLE 7Anti-CCR8 AntibodiesAffinityAffinityLightforforBindingBindingHeavyChain293T-293T-totoChain(SEQHuCCR8CyCCR8HuCCR8-CyCCR8-AntibodyMab(SEQIDcellscellsFcFcSourceNumberID NO)NO)(nM)(nM)proteinproteinParentalAnti-CCR8-H23373L10326.8>50++++++++parental 3HCDR3Anti-CCR8-91932.53.9++++++++randomization2-3HCDR3Anti-CCR8-21230.962.4++++++++randomization2-4HCDR3Anti-CCR8-21232.95.2++++++randomization2-5HCDR3Anti-CCR8-1511531.54.5++++++randomization2-6HCDR3Anti-CCR8-31331.33.5+++++++randomization2-7& VL ShuffleHCDR3Anti-CCR8-71731.52.5+++++++randomization2-8& VL ShuffleHCDR3Anti-CCR8-61631.53.2++++++++randomization2-9& VL ShuffleHCDR3Anti-CCR8-81832.43.9++++++randomization2-10& VL Shuffle Example 2: Antibodies Bind CCR8 Of the 17 antibodies, two were selected for further characterization: anti-CCR8-1, comprising a variable heavy chain having the amino acid sequence set forth in SEQ ID NO. 41 and the variable variable light chain having the amino acid sequence set forth in SEQ ID NO: 43; and anti-CCR8-2, comprising a variable heavy chain having the amino acid sequence set forth in SEQ ID NO: 11 and a variable light chain having the amino acid sequence set forth in SEQ ID NO: 13. To test whether CCR8 antibodies bound to cell-expressed human or cynomolgus monkey CCR8, 293T and Raji cells were infected with lentivirus for either human or cynomolgus monkey CCR8. Celllines expressing CCR8 constructs were incubated with CCR8 antibodies in a dose-dependent manner at 4° C. for 30 min, unbound CCR8 antibodies as removed by washing and bound CCR8 antibodies were detected using a fluorescently-conjugated anti-human secondary antibody at 4° C. for 30 min. The results showed that both anti-CCR8-1 (FIG.3A) and anti-CCR8-2 (FIG.3B) bound to the human CCR8 cell lines, while anti-CCR8-2 (FIG.3B) bound to the cynomolgus monkey CCR8 cell line. Example 3: Antibodies Bind to CCR8+Tumor Tregs To test whether CCR8 antibodies bound to CCR8+tumor Tregs, tumor-infiltrating leukocytes (TILs) were isolated from freshly-resected tumors and plated in 96-well plates. TILs were incubated with a fluorescently-tagged antibody panel (CD3, CD4, FOXP3) to identify tumor Tregs at 4° C. Additionally, CCR8 antibodies were incubated with TILs at a single concentration at 4° C. for 30 min, unbound CCR8 antibodies as removed by washing and bound CCR8 antibodies were detected using a fluorescently-conjugated anti-human secondary antibody at 4° C. for 30 min. The results showed that both anti-CCR8-1 and anti-CCR8-2 bound to the CCR8+tumor Tregs (FIG.4A-4B). Example 4: Antibodies Induce ADCC Signaling in ADCC Reporter Bioassay To test whether CCR8 antibodies induced ADCC signaling, CCR8 antibodies were incubated with 293T cells with forced expression of either human or cynomolgus monkey CCR8 in a dose-dependent manner in 96-well plates for 6 hours at 37° C. according to manufacturer's instructions. CD16 Jurkat antibody-dependent cellular cytotoxicity (ADCC) reporter cells were co-cultured with target cells complexed with CCR8 antibodies. Upon completion of experiment, ADCC signaling was determined by a bioluminescent readout on the CD16 Jurkat ADCC reporter cells. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC signaling using the human CCR8 cell line as targets, while anti-CCR8-2 also induced ADCC signaling using the cynomolgus monkey CCR8 cell line (FIG.5). Example 5: Antibodies Induce ADCC of Cells with Forced Expression of Human and Cynomolgus Monkey CCR8 Using PBMCs as Effector Cells To test whether CCR8 antibodies induced ADCC of CCR8+ cells, CCR8 antibodies were incubated with 293T or Raji target cells with forced expression human CCR8 and labeled with CellTrace Violet in a dose-dependent manner in 96-well plates. PBMCs were co-cultured with target cells complexed with CCR8 antibodies overnight at 37° C. Upon completion of the experiment, the number of CCR8+target cells was assessed by Flow Cytometry. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC of the human CCR8 cell lines (FIG.6A), while anti-CCR8-2 also induced ADCC of the cynomolgus monkey CCR8 cell line (FIG.6B). Example 6: Antibodies Induce ADCC of Cells with Forced Expression of Human CCR8 Using NK Cells as Effector Cells To test whether CCR8 antibodies induced ADCC of CCR8+cells, CCR8 antibodies were incubated with Raji target cells with forced expression human CCR8 and labeled with CellTrace Violet in a dose-dependent manner in 96-well plates. NK cells were co-cultured with target cells complexed with CCR8 antibodies for 4 hours at 37° C. Upon completion of the experiment, the number of CCR8+target cells was assessed by Flow Cytometry. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC of the human CCR8 cell line (FIGS.7A-7B). Example 7: Antibodies Modulate NK Cell Activation Markers in Co-Culture Assay with Cells with Forced Expression of Human CCR8 and Kill Cells Expressing CCR8 To test whether CCR8 antibodies modulated activation markers of NK cells, CCR8 antibodies were incubated with Raji target cells with forced expression human CCR8 and labeled with CellTrace Violet in a dose-dependent manner in 96-well plates. NK cells were co-cultured with target cells complexed with CCR8 antibodies overnight at 37° C. Upon completion of the experiment, the number of CCR8+target cells was assessed by Flow Cytometry measuring 4-1BB, ICAM-1 and CD16 expression on the cell surface of NK cells. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced upregulation of 4-1BB and ICAM-1 on the cell surface while CD16 was down-regulated on the cell surface of NK cells (FIG.8A,FIG.8C). To test whether CCR8 antibodies killed cells expressing CCR8, antibodies were incubated with both Raji cells and Raji target cells with forced expression human CCR8 (“Raji-CCR8 cells”) and labeled with CellTrace Violet in a dose-dependent manner in 96-well plates. Upon completion of the experiment, the number of CCR8+target cells was assessed by Flow Cytometry measuring the number of CellTrace Violet positive cells remaining. The results showed that both anti-CCR8-1 and anti-CCR8-2 killed the Raji-CCR8 cells and did not kill the Raji cells without CCR8 (FIG.8B). Example 8: Antibodies Induce ADCC of Tumor Tregs Using NK Cells as Effector Cells To test whether CCR8 antibodies induced ADCC of CCR8+tumor Tregs, TILs were isolated from freshly-resected human tumors and incubated with CCR8 antibodies. NK cells were co-cultured with TIL: antibody complexes in 96-well plates overnight at 37° C. Upon completion of the experiment, the number of tumor Tregs was assessed by Flow Cytometry. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced killing of the human tumor Tregs (FIG.9A). Example 9: Antibodies Induce Internalization of CCR8 in Cells with Forced Expression of Human CCR8 To test whether CCR8 antibodies induced CCR8-IgG complex internalization, CCR8 antibodies were incubated with pH sensitive FabFluor reagent to generate a CCR8 internalization reporter antibody. This antibody conjugate was used to treat 293T cells with forced expression of human CCR8 for 30 min at 37° C. During antibody incubation on the 293T cells, antibodies bind to CCR8 and induce internalization into an acidic endosome, eliciting a fluorescent signal from the conjugated FabFluor reagent. Upon completion of experiment, fluorescent signal can be analyzed by flow cytometry and compared between antibody conjugates. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced CCR8 internalization using the human CCR8 cell line as targets (FIG.10). Example 10: Retrogenix Antibody Binding Experiment To identify targets of CCR8 antibodies, CCR8 antibodies were incubated with cells forced to express about 4,500 cell surface proteins in a fixed or live state. Anti-CCR8-1 was demonstrated to bind to only CCR8 while anti-CCR8-2 also bound to amyloid precursor-like protein 2 (APLP2), but at a significantly lower concentration (data not shown). Example 11: Characterization of Anti-CCR8 Antibody Epitopes To characterize the binding cite of the CCR8 antibodies, CCR8 antibodies were incubated with Raji-CCR8 cells in a dose-dependent manner in 96-well plates at 4° C. for 30 min. Unbound CCR8 antibodies were washed away and the cells were incubated with a fluorescently-conjugated human monoclonal anti-CCR8 antibody (commercially available) at a single concentration for 30 min at 37° C. Upon completion of the experiment, the binding of the commercially available monoclonal CCR8 antibody was assessed by Flow Cytometry. The results showed that anti-CCR8-1 partially blocked the commercially available monoclonal CCR8 antibody from binding to cells while anti-CCR8-2 did not (FIG.11). Example 12: Afucosylated CCR8 Antibodies Show Enhanced ADCC Activity Anti-CCR8-1 was further optimized to remove fucose sugar units from the (IgG1) Fc region of the antibody. To test induction of ADCC signaling, CCR8 antibodies were incubated with 293T cells with forced expression of human CCR8 in a dose-dependent manner in 96-well plates for 6 h at 37° C. according to manufacturer's instructions. Either CD16VV or CD16FF Jurkat ADCC reporter cells were co-cultured with target cells complexed with CCR8 antibodies. Upon completion of experiment, ADCC signaling was determined by a bioluminescent readout on the CD16 Jurkat ADCC reporter cells. The results showed that both anti-CCR8-1 and anti-CCR8-2 induced ADCC signaling using the human CCR8 cell line as targets, while anti-CCR8-2 also induced ADCC signaling using the cynomolgus monkey CCR8 cell line (FIGS.12A-12B). Increased activity was observed with high and low affinity allelic polymorphisms. Example 12: Antibodies Bind Tumor Tregs and Induce ADCC To test whether CCR8 antibodies bind tumor Tregs and induce ADCC, tumor infiltrating lymphocytes (TILs) were isolated from fresh kidney and breast tumor resections and incubated with the anti-CCR8-1 antibody. Anti-CCR8-1 antibody binding was measured using a PE-conjugated anti-human IgG antibody (FIG.13A), and a commercially purchased purified anti-human CD213a1 (IL-13-Rα1) antibody was used as a positive control (Biolegend catalog number 360404). As shown inFIG.13A, triangles represent TIL from kidney tumors and circles represent TIL from breast tumors. All data were acquired by flow cytometry. In a follow-up experiment, isolated TILs from fresh kidney and breast tumor resections were incubated with allogenic NK cells and the anti-CCR8-1 antibody, mogamulizumab, or isotype control for 24 hours. The percentage of CD3+ cells that were either Tregs (FoxP3+) or other lymphocytes (FoxP3−) was measured using flow cytometry. In the presence of NK cells, the anti-CCR8-1 antibody resulted in a significant loss of Tregs while not effecting the non-Treg population. Collectively, these data show that the anti-CCR8-1 antibody binds tumor Tregs and causes NK cell mediated ADCC. TABLE 8SequencesSEQIDNO:Sequence1VQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRESYRVSLRFDYWGQGTLVTVSS3QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLT5DYAMH6GISWNSGSIGYADSVKG7GRESYRVSLRFDY8SGSSSNIGNNYVS9DNNKRPS10GTWDSSLSAWV11EVQLLESGGGLVQPGGSLRLSCAAGGFTFSAYTMNWVRQAPGKGLEWVSAISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRFARGWFDPWGQGTLVTVSS13QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLT15AYTMN16AISASGGRTYYADSVKG17RFARGWFDP18SGSSSNIGNNYVS19DNNKRPS20GTWDSSLSAWV21EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRKSYRVSLRFDYWGQGTLVTVSS23QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLT25DYAMH26GISWNSGSIGYADSVKG27GRKSYRVSLRFDY28SGSSSNIGNNYVS29DNNKRPS30GTWDSSLSAWV31EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRDSYRKSLRFDYWGQGTLVTVSS33QSVLTQPPSVSGAPGQRVTISCTGSGSNIGNNYVSWYQQLPGTAPKMLIYDNTRRPSGIPDRFSGSKSDTSATLGITGLQTGDEADYYCGAWDSSLRMWVFGGGTKLTVL35DYAMH36GISWNSGSIGYADSVKG37GRDSYRKSLRFDY38TGSGSNIGNNYVS39DNTRRPS40GAWDSSLRMWV41QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARAVRNRFRFDYWGQGTLVTVSS4243QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYAGSSTFVVFGGGTKLTVL45SYYMH46IINPSGGSTSYAQKFQG47AVRNRFRFDY48TGTSSDVGSYNLVS49EVSKRPS50SSYAGSSTFVV51EVQLLESGGGLVQPGGSLRLSCAARGFIFSGYTMLWVRQAPGKGLEWVSAITASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRFARGWFDPWGQGTLVTVSS53QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLT55GYTML56AITASGGRTYYADSVKG57RFARGWFDP58SGSSSNIGNNYVS59DNNKRPS60GTWDSSLSAWV61EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRKSYRDSLRFDYWGQGTLVTVSS63QSVLTQPPSVSGAPGQRVTISCTGSGSNIGNNYVSWYQQLPGTAPKMLIYDNTRRPSGIPDRFSGSKSDTSATLGITGLQTGDEADYYCGAWDSSLRMWVFGGGTKLTVL65DYAMH66GISWNSGSIGYADSVKG67GRKSYRDSLRFDY68TGSGSNIGNNYVS69DNTRRPS70GAWDSSLRMWV71EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRRSYRDSLRFDYWGQGTLVTVSS73QSVLTQPPSVSGAPGQRVTISCTGSGSNIGNNYVSWYQQLPGTAPKMLIYDNTRRPSGIPDRFSGSKSDTSATLGITGLQTGDEADYYCGAWDSSLRMWVFGGGTKLTVL75DYAMH76GISWNSGSIGYADSVKG77GRRSYRDSLRFDY78TGSGSNIGNNYVS79DNTRRPS80GAWDSSLRMWV81EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRKSYRDSLRFDYWGQGTLVTVSS83QSVLTQPPSVSAAPGQKVTISCSGTSSNIGKNFVSWYQQLPGTAPKLLIYDDNKRPSGIPDRFSGSKSATSATLGITGLQTGDGADYYCGTWDSSLSAWVFGGGTKLTVL85DYAMH86GISWNSGSIGYADSVKG87GRKSYRDSLRFDY88SGTSSNIGKNFVS89DDNKRPS90GTWDSSLSAWV91EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRKSYRDSLRFDYWGQGTLVTVSS93QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLT95DYAMH96GISWNSGSIGYADSVKG97GRKSYRDSLRFDY98SGSSSNIGNNYVS99DNNKRPS100GTWDSSLSAWV101QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNGFRFDYWGQGTLVT103QSALTQPPSVSGSPGQSITISCTGTSSDVGTYNLVSWYQQHPGNAPKLMIYEVTKRPSGVSNRFSGSKSGNTATLTISGLQAEDEADYHCSSYAGSITHVVFGGGTKLTVL105SYYMH106IINPSGGSTSYAQKFQG107GVGNGFRFDY108TGTSSDVGTYNLVS109EVTKRPS110SSYAGSITHVV111QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNGFRFDYWGQGTLVT113QSALTQPASVSGSPGQSITISCTGTSGDVGSYSLVSWYQHHPSRAPKLIIYEVNKRPSGVSDRFSGSKSGNTASLTITGLQAEDEAHYFCSSYTGNINLPVVFGGGTKLTVL115SYYMH116IINPSGGSTSYAQKFQG117GVGNGFRFDY118TGTSGDVGSYSLVS119EVNKRPS120SSYTGNINLPVV121QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNGFRFDYWGQGTLVT123QSALTQPPSVSGSPGQSITISCSGTSSDVGIYNLVSWYQQHPGKAPKLIIYEVIKRPSGISNRFSGFKSGNTASLTISGLQAEDEADYYCSSYAGPVTYVVFGGGTKLTVL125SYYMH126IINPSGGSTSYAQKFQG127GVGNGFRFDY128SGTSSDVGIYNLVS129EVIKRPS130SSYAGPVTYVV131QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNGFRFDYWGQGTLVT133QSALTQPASVSGSPGQSITISCSGTSSNIGKYNLVSWYQQHPGEAPTLLIYEATKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYICSSYAGSRVFVVFGGGTKLTVL135SYYMH136IINPSGGSTSYAQKFQG137GVGNGFRFDY138SGTSSNIGKYNLVS139EATKRPS140SSYAGSRVFVV141QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGVGNGFRFDYWGQGTLVT143QSALTQPPSVSGSPGQSITISCSGTSSDVGSYNLVSWYQQEPGKAPKLIIYEVNKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYHCSSYAGSSTYVVFGGGTKLTVL145SYYMH146IINPSGGSTSYAQKFQG147GVGNGFRFDY148SGTSSDVGSYNLVS149EVNKRPS150SSYAGSSTYVV151EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTALYYCARGRVSYRESLRFDYWGQGTLVT153QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAWVFGGGTKLT155DYAMH156GISWNSGSIGYADSVKG157GRVSYRESLRFDY158SGSSSNIGNNYVS159DNNKRPS160GTWDSSLSAWV161EVQLLESGGGLVQPGGSLRLSCAAGGFTFSAYTMNWVRQAPGKGLEWVSAISASGGRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRFARGWFDPWGQGTLVT163QSVLTQPPSVSAAPGQRVTISCSGSTSNIGNHYVSWYQQLPRAVPKLVIYDNDKRPSGISDRFSGSRSGTSATLDISGLQAGDEADYYCATWDYSLTAVVFGGGTKLTVL165AYTMN166AISASGGRTYYADSVKG167RFARGWFDP168SGSTSNIGNHYVS169DNDKRPS170ATWDYSLTAVV171MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVFSLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLLDQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKVRTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTLKWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLVLIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEIISFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCEKSSSCQQHSSRSSSVDYIL172MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGK173MGWSCIILFLVATATGAHSMDYTLDLSVTTVTDYYYPDIFSSPSDAELIQTNGKHHHHHHSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK174MGWSCIILFLVATATGAHSMDYTLDPSMTTMTDYYYPDSLSSPSDGELIQRNDKHHHHHHSGGGGSEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK175MGWSCIILFLVATATGAHS 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11859001 | DETAILED DESCRIPTION OF THE INVENTION Introduction In order for the present disclosure to be more readily understood, certain terms and phrases are defined below as well as throughout the specification. The definitions provided herein are non-limiting and should be read in view of the knowledge of one of skill in the art would know. Before the present methods and compositions are described, it is to be understood that this disclosure is not limited to particular method or composition described, as such may, of course, vary. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It should be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g., polypeptides, known to those skilled in the art, and so forth. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. It will be appreciated that throughout this disclosure reference is made to amino acids according to the single letter or three letter codes. For the reader's convenience, the single and three letter amino acid codes are provided in Table 6 below: TABLE 6Amino Acid AbbreviationsSingle Letter3-letterAbbreviationNameabbreviationGGlycineGlyPProlineProAAlanineAlaVValineValLLeucineLeuIIsoleucineIleMMethionineMetCCysteineCysFPhenylalaninePheYTyrosineTyrWTryptophanTrpHHistidineHisKLysineLysRArginineArgQGlutamineGlnNAsparagineAsnEGlutamic AcidGluDAspartic AcidAspSSerineSerTThreonineThr Standard methods in molecular biology are described in the scientific literature (see, e.g., Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4)). The scientific literature describes methods for protein purification, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, as well as chemical analysis, chemical modification, post-translational modification, production of fusion proteins, and glycosylation of proteins (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wiley and Sons, Inc., NY). Definitions Unless otherwise indicated, the following terms are intended to have the meaning set forth below. Other terms are defined elsewhere throughout the specification. Activate: As used herein the term “activate” is used in reference to a receptor or receptor complex to reflect a biological effect, directly and/or by participation in a multicomponent signaling cascade, arising from the binding of an agonist ligand to a receptor responsive to the binding of the ligand. Activity: As used herein, the term “activity” is used with respect to a molecule to describe a property of the molecule with respect to a test system (e.g., an assay) or biological or chemical property (e.g., the degree of binding of the molecule to another molecule) or of a physical property of a material or cell (e.g., modification of cell membrane potential). Examples of such biological functions include but are not limited to catalytic activity of a biological agent, the ability to stimulate intracellular signaling, gene expression, cell proliferation, the ability to modulate immunological activity such as inflammatory response. “Activity” is typically expressed as a level of a biological activity per unit of agent tested such as [catalytic activity]/[mg protein], [immunological activity]/[mg protein], international units (IU) of activity, [STAT5 phosphorylation]/[mg protein], [proliferation]/[mg protein], plaque forming units (pfu), etc. As used herein, the term proliferative activity refers to an activity that promotes cell proliferation and replication, including dysregulated cell division such as that observed in neoplastic diseases, inflammatory diseases, fibrosis, dysplasia, cell transformation, metastasis, and angiogenesis. Affinity: As used herein the term “affinity” refers to the degree of specific binding of a first molecule (e.g., a ligand) to a second molecule (e.g., a receptor) and is measured by the equilibrium dissociation constant (KD), a ratio of the dissociation rate constant between the molecule and its target (Koff) and the association rate constant between the molecule and its target (Kon). Agonist: As used herein, the term “agonist” refers a first agent that specifically binds a second agent (“target”) and interacts with the target to cause or promote an increase in the activation of the target. In some instances, agonists are activators of receptor proteins that modulate cell activation, enhance activation, sensitize cells to activation by a second agent, or up-regulate the expression of one or more genes, proteins, ligands, receptors, biological pathways, that may result in cell proliferation or pathways that result in cell cycle arrest or cell death such as by apoptosis. In some embodiments, an agonist is an agent that binds to a receptor and alters the receptor state resulting in a biological response that mimics the effect of the endogenous ligand of the receptor. The term “agonist” includes partial agonists, full agonists and superagonists. An agonist may be described as a “full agonist” when such agonist which leads to a substantially full biological response (i.e. the response associated with the naturally occurring ligand/receptor binding interaction) induced by receptor under study, or a partial agonist. A “superagonist” is a type of agonist that can produce a maximal response greater than the endogenous agonist for the target receptor, and thus has an activity of more than 100% of the native ligand. A super agonist is typically a synthetic molecule that exhibits greater than 110%, alternatively greater than 120%, alternatively greater than 130%, alternatively greater than 140%, alternatively greater than 150%, alternatively greater than 160%, or alternatively greater than 170% of the response in an evaluable quantitative or qualitative parameter of the naturally occurring form of the molecule when evaluated at similar concentrations in a comparable assay. It should be noted that the biological effects associated with the full agonist may differ in degree and/or in kind from those biological effects of partial or superagonists. In contrast to agonists, antagonists may specifically bind to a receptor but do not result the signal cascade typically initiated by the receptor and may to modify the actions of an agonist at that receptor. Inverse agonists are agents that produce a pharmacological response that is opposite in direction to that of an agonist. Antagonist: As used herein, the term “antagonist” or “inhibitor” refers a molecule that opposes the action(s) of an agonist. An antagonist prevents, reduces, inhibits, or neutralizes the activity of an agonist, and an antagonist can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even where there is no identified agonist. Inhibitors are molecules that decrease, block, prevent, delay activation, inactivate, desensitize, or down-regulate, e.g., a gene, protein, ligand, receptor, biological pathway including an immune checkpoint pathway, or cell. Antibody: As used herein, the term “antibody” refers collectively to: (a) a glycosylated or non-glycosylated immunoglobulin that specifically binds to target molecule, and (b) immunoglobulin derivatives thereof, including but not limited to antibody fragments such as single domain antibodies. In some embodiments the immunoglobulin derivative competes with the immunoglobulin from which it was derived for binding to the target molecule. The term antibody is not restricted to immunoglobulins derived from any particular species and includes murine, human, equine, camelids, antibodies of cartilaginous fishes including, but not limited to, sharks. The term “antibody” encompasses antibodies isolatable from natural sources or from animals following immunization with an antigen and as well as engineered antibodies including monoclonal antibodies, bispecific antibodies, tri-specific, chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted, veneered, or deimmunized (e.g., to remove T-cell epitopes) antibodies, camelized (in the case of VHHs), or molecules comprising binding domains of antibodies (e.g., CDRs) in non-immunoglobulin scaffolds. The term “antibody” should not be construed as limited to any particular means of synthesis and includes naturally occurring antibodies isolatable from natural sources and as well as engineered antibodies molecules that are prepared by “recombinant” means including antibodies isolated from transgenic animals that are transgenic for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed with a nucleic acid construct that results in expression of an antibody, antibodies isolated from a combinatorial antibody library including phage display libraries. In one embodiment, an “antibody” is a mammalian immunoglobulin of the IgG1, IgG2, IgG3 or IgG4 class. In some embodiments, the antibody is a “full length antibody” comprising variable and constant domains providing binding and effector functions. The term “single domain antibody” (sdAb) as used herein refers an antibody fragment consisting of a monomeric variable antibody domain that is able to bind specifically to an antigen and compete for binding with the parent antibody from which it is derived. The term “single domain antibody” includes scFv and VHH molecules. As used herein, the term “VHH” refers to a single domain antibody derived from camelid antibody typically obtained from immunization of camelids (including camels, llamas and alpacas (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448). VHHs are also referred to as heavy chain antibodies or Nanobodies® as Single domain antibodies may also be derived from non-mammalian sources such as VHHs obtained from IgNAR antibodies immunization of cartilaginous fishes including, but not limited to, sharks. Biological Sample: As used herein, the term “biological sample” or “sample” refers to a sample obtained (or derived) from a subject. By way of example, a biological sample comprises a material selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucus secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), fluids of the eye (e.g., vitreous fluid, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, tumor tissue, including immunoglobulin enriched or cell-type specific enriched fractions derived from one or more of such tissues. IL cell: The terms “IL12RB1 cell”, “IL12RB1-expressing cell”, “IL12RB1-positive cell” and “IL12RB1+” cell are used interchangeably herein to refer to a cell which expresses and displays the IL12RB1 antigen on the extracellular surface of the cell membrane. Similarly, the terms “IL12RB1-negative cell”, “IL12RB1− cells” as are used interchangeably herein to describe cells which do not express or display IL12RB1 antigen on the cell surface. CDR: As used herein, the term “CDR” or “complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain immunoglobulin polypeptides. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, et al., U.S. Dept. of Health and Human Services publication entitled “Sequences of proteins of immunological interest” (1991) (also referred to herein as “Kabat 1991” or “Kabat”); by Chothia, et al. (1987) J. Mol. Biol. 196:901-917 (also referred to herein as “Chothia”); and MacCallum, et al. (1996) J. Mol. Biol. 262:732-745, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the term as defined and used herein. The term “Chothia Numbering” as used herein is recognized in the arts and refers to a system of numbering amino acid residues based on the location of the structural loop regions (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For purposes of the present disclosure, unless otherwise specifically identified, the positioning of CDRs2 and 3 in the variable region of an antibody follows Kabat numbering or simply, “Kabat.” The positioning of CDR1 in the variable region of an antibody follows a hybrid of Kabat and Chothia numbering schemes. Comparable: As used herein, the term “comparable” is used to describe the degree of difference in two measurements of an evaluable quantitative or qualitative parameter. For example, where a first measurement of an evaluable quantitative parameter and a second measurement of the evaluable parameter do not deviate beyond a range that the skilled artisan would recognize as not producing a statistically significant difference in effect between the two results in the circumstances, the two measurements would be considered “comparable.” In some instances, measurements may be considered “comparable” if one measurement deviates from another by less than 35%, alternatively by less than 30%, alternatively by less than 25%, alternatively by less than 20%, alternatively by less than 15%, alternatively by less than 10%, alternatively by less than 7%, alternatively by less than 5%, alternatively by less than 4%, alternatively by less than 3%, alternatively by less than 2%, or by less than 1%. In particular embodiments, one measurement is comparable to a reference standard if it deviates by less than 15%, alternatively by less than 10%, or alternatively by less than 5% from the reference standard. Clonotype: As used herein, a clonotype refers to a collection of binding molecules that originate from the same B-cell progenitor cell. The term “clonotype” is used to refer to a collection of antigen binding molecules that belong to the same germline family, have the same CDR3 lengths, and have 70% or greater homology in CDR3 sequence. Conservative Amino Acid Substitution: As used herein, the term “conservative amino acid substitution” refers to an amino acid replacement that changes a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). For example, the amino acids in each of the following groups can be considered as conservative amino acids of each other: (1) hydrophobic amino acids: alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine; (2) polar amino acids: glutamine, asparagine, histidine, serine, threonine, tyrosine, methionine, and cysteine; (3) basic amino acids: lysine and arginine; and (4) acidic amino acids: aspartic acid and glutamic acid. Derived From: As used herein in the term “derived from”, in the context of an amino acid sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made. By way of example, the term “derived from” includes homologs or variants of reference amino acid or DNA sequences. Effective Concentration (EC): As used herein, the terms “effective concentration” or its abbreviation “EC” are used interchangeably to refer to the concentration of an agent in an amount sufficient to effect a change in a given parameter in a test system. The abbreviation “E” refers to the magnitude of a given biological effect observed in a test system when that test system is exposed to a test agent. When the magnitude of the response is expressed as a factor of the concentration (“C”) of the test agent, the abbreviation “EC” is used. In the context of biological systems, the term Emax refers to the maximal magnitude of a given biological effect observed in response to a saturating concentration of an activating test agent. When the abbreviation EC is provided with a subscript (e.g., EC40, EC50, etc.) the subscript refers to the percentage of the Emax of the biological response observed at that concentration. For example, the concentration of a test agent sufficient to result in the induction of a measurable biological parameter in a test system that is 30% of the maximal level of such measurable biological parameter in response to such test agent, this is referred to as the “EC30” of the test agent with respect to such biological parameter. Similarly, the term “EC100” is used to denote the effective concentration of an agent that results the maximal (100%) response of a measurable parameter in response to such agent. Similarly, the term EC50(which is commonly used in the field of pharmacodynamics) refers to the concentration of an agent sufficient to results in the half-maximal (about 50%) change in the measurable parameter. The term “saturating concentration” refers to the maximum possible quantity of a test agent that can dissolve in a standard volume of a specific solvent (e.g., water) under standard conditions of temperature and pressure. In pharmacodynamics, a saturating concentration of a drug is typically used to denote the concentration sufficient of the drug such that all available receptors are occupied by the drug, and EC50is the drug concentration to give the half-maximal effect. Enriched: As used herein in the term “enriched” refers to a sample that is non-naturally manipulated so that a species (e.g., a molecule or cell) of interest is present in: (a) a greater concentration (e.g., at least 3-fold greater, alternatively at least 5-fold greater, alternatively at least 10-fold greater, alternatively at least 50-fold greater, alternatively at least 100-fold greater, or alternatively at least 1000-fold greater) than the concentration of the species in the starting sample, such as a biological sample (e.g., a sample in which the molecule naturally occurs or in which it is present after administration); or (b) a concentration greater than the environment in which the molecule was made (e.g., a recombinantly modified bacterial or mammalian cell). Extracellular Domain: As used herein the term “extracellular domain” or its abbreviation “ECD” refers to the portion of a cell surface protein (e.g., a cell surface receptor) which is external to of the plasma membrane of a cell. The cell surface protein may be transmembrane protein, a cell surface or membrane associated protein. Identity: The term “identity,” as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J Mol. Biol. 215: 403-410 and Altschul, et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W of the query sequence, which either match or satisfy some positive-valued threshold score “T” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (the reward score for a pair of matching residues; always >0) and “N” (the penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: (a) the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or (b) the end of either sequence is reached. The BLAST algorithm parameters “W”, “T”, and “X” determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) functions similarly but uses as defaults a word size (“W”) of 28, an expectation (“E”) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, (1989) PNAS (USA) 89:10915-10919). In An Amount Sufficient Amount to Cause a Response: As used herein the phrase “in an amount sufficient to cause a response” is used in reference to the amount of a test agent sufficient to provide a detectable change in the level of an indicator measured before (e.g., a baseline level) and after the application of a test agent to a test system. In some embodiments, the test system is a cell, tissue or organism. In some embodiments, the test system is an in vitro test system such as a fluorescent assay. In some embodiments, the test system is an in vivo system which involves the measurement of a change in the level a parameter of a cell, tissue, or organism reflective of a biological function before and after the application of the test agent to the cell, tissue, or organism. In some embodiments, the indicator is reflective of biological function or state of development of a cell evaluated in an assay in response to the administration of a quantity of the test agent. In some embodiments, the test system involves the measurement of a change in the level an indicator of a cell, tissue, or organism reflective of a biological condition before and after the application of one or more test agents to the cell, tissue, or organism. The term “in an amount sufficient to effect a response” may be sufficient to be a therapeutically effective amount but may also be more or less than a therapeutically effective amount. Inhibitor: As used herein the term “inhibitor” refers to a molecule that decreases, blocks, prevents, delays activation of, inactivates, desensitizes, or down-regulates, e.g., a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates a constitutive activity of a cell or organism. Intracellular Domain: As used herein the term “intracellular domain” or its abbreviation “ICD” refers to the portion of a cell surface protein (e.g., a cell surface receptor) which is inside of the plasma membrane of a cell. The ICD may include the entire cytoplasmic portion of a transmembrane protein or membrane associated protein, or intracellular protein. Isolated: As used herein the term “isolated” is used in reference to a polypeptide of interest that, if naturally occurring, is in an environment different from that in which it can naturally occur. “Isolated” is meant to include polypeptides that are within samples that are substantially enriched for the polypeptide of interest and/or in which the polypeptide of interest is partially or substantially purified. Where the polypeptide is not naturally occurring, “isolated” indicates that the polypeptide has been separated from an environment in which it was synthesized, for example isolated from a recombinant cell culture comprising cells engineered to express the polypeptide or by a solution resulting from solid phase synthetic means. Kabat Numbering: The term “Kabat numbering” as used herein is recognized in the art and refers to a system of numbering amino acid residues which are more variable than other amino acid residues (e.g., hypervariable) in the heavy and light chain regions of immunoglobulins (Kabat, et al., (1971)Ann. NY Acad. Sci.190:382-93; Kabat, et al., (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The term “Chothia Numbering” as used herein is recognized in the arts and refers to a system of numbering amino acid residues based on the location of the structural loop regions (Chothia et al. 1986, Science 233:755-758; Chothia & Lesk 1987, JMB 196:901-917; Chothia et al. 1992, JMB 227:799-817). For purposes of the present disclosure, unless otherwise specifically identified, the positioning of CDRs 2 and 3 in the variable region of an antibody follows Kabat numbering or simply, “Kabat.” The positioning of CDR1 in the variable region of an antibody follows a hybrid of Kabat and Chothia numbering schemes. Ligand: As used herein, the term “ligand” refers to a molecule that specifically binds a receptor and causes a change in the receptor so as to effect a change in the activity of the receptor or a response in cell that expresses that receptor. In one embodiment, the term “ligand” refers to a molecule or complex thereof that can act as an agonist or antagonist of a receptor. As used herein, the term “ligand” encompasses natural and synthetic ligands. “Ligand” also encompasses small molecules, peptide mimetics of cytokines and antibodies. The complex of a ligand and receptor is termed a “ligand-receptor complex.” A ligand may comprise one domain of a polyprotein or fusion protein (e.g., either domain of an antibody/ligand fusion protein). Modulate: As used herein, the terms “modulate”, “modulation” and the like refer to the ability of a test agent to cause a response, either positive or negative or directly or indirectly, in a system, including a biological system, or biochemical pathway. The term modulator includes both agonists (including partial agonists, full agonists and superagonists) and antagonists. Nucleic Acid: The terms “nucleic acid”, “nucleic acid molecule”, “polynucleotide” and the like are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), complementary DNA (cDNA), recombinant polynucleotides, vectors, probes, primers and the like. Operably Linked: The term “operably linked” is used herein to refer to the relationship between molecules, typically polypeptides or nucleic acids, which are arranged in a construct such that each of the functions of the component molecules is retained although the operable linkage may result in the modulation of the activity, either positively or negatively, of the individual components of the construct. For example, the operable linkage of a polyethylene glycol (PEG) molecule to a wild-type protein may result in a construct where the biological activity of the protein is diminished relative to the to the wild-type molecule, however the two are nevertheless considered operably linked. When the term “operably linked” is applied to the relationship of multiple nucleic acid sequences encoding differing functions, the multiple nucleic acid sequences when combined into a single nucleic acid molecule that, for example, when introduced into a cell using recombinant technology, provides a nucleic acid which is capable of effecting the transcription and/or translation of a particular nucleic acid sequence in a cell. For example, the nucleic acid sequence encoding a signal sequence may be considered operably linked to DNA encoding a polypeptide if it results in the expression of a preprotein whereby the signal sequence facilitates the secretion of the polypeptide; a promoter or enhancer is considered operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is considered operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, in the context of nucleic acid molecules, the term “operably linked” means that the nucleic acid sequences being linked are contiguous, and, in the case of a secretory leader or associated subdomains of a molecule, contiguous and in reading phase. However, certain genetic elements such as enhancers may function at a distance and need not be contiguous with respect to the sequence to which they provide their effect but nevertheless may be considered operably linked. Parent Polypeptide: As used herein, the terms “parent polypeptide” or “parent protein” are used interchangeably to designate the source of a second polypeptide (e.g., a derivative, mutein or variant) which is modified with respect to a first “parent” polypeptide. In some instances, the parent polypeptide is a wild-type or naturally occurring form of a protein. In some instance, the parent polypeptide may be a modified form a naturally occurring protein that is further modified. The term “parent polypeptide” may refer to the polypeptide itself or compositions that comprise the parent polypeptide (e.g., glycosylated or PEGylated forms and/or fusion proteins comprising the parent polypeptide). Partial Agonist: As used herein, the term “partial agonist” refers to a molecule that specifically binds that bind to and activate a given receptor but possess only partial activation the receptor relative to a full agonist. Partial agonists may display both agonistic and antagonistic effects. For example, when both a full agonist and partial agonist are present, the partial agonist acts as a competitive antagonist by competing with the full agonist for the receptor binding resulting in net decrease in receptor activation relative to the contact of the receptor with the full agonist in the absence of the partial agonist. Partial agonists can be used to activate receptors to give a desired submaximal response in a subject when inadequate amounts of the endogenous ligand are present, or they can reduce the overstimulation of receptors when excess amounts of the endogenous ligand are present. The maximum response (Emax) produced by a partial agonist is called its intrinsic activity and may be expressed on a percentage scale where a full agonist produced a 100% response. An partial agonist may have greater than 10% but less than 100%, alternatively greater than 20% but less than 100%, alternatively greater than 30% but less than 100%, alternatively greater than 40% but less than 100%, alternatively greater than 50% but less than 100%, alternatively greater than 60% but less than 100%, alternatively greater than 70% but less than 100%, alternatively greater than 80% but less than 100%, or alternatively greater than 90% but less than 100%, of the activity of the reference polypeptide when evaluated at similar concentrations in a given assay system. Polypeptide: As used herein the terms “polypeptide,” “peptide,” and “protein”, used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The term polypeptide include fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence; fusion proteins with heterologous and homologous leader sequences; fusion proteins with or without N-terminal methionine residues; fusion proteins with amino acid sequences that facilitate purification such as chelating peptides; fusion proteins with immunologically tagged proteins; fusion proteins comprising a peptide with immunologically active polypeptide fragment (e.g., antigenic diphtheria or tetanus toxin or toxoid fragments) and the like. Prevent: As used herein the terms “prevent”, “preventing”, “prevention” and the like refer to a course of action initiated with respect to a subject prior to the onset of a disease, disorder, condition or symptom thereof so as to prevent, suppress, inhibit or reduce, either temporarily or permanently, a subject's risk of developing a disease, disorder, condition or the like (as determined by, for example, the absence of clinical symptoms) or delaying the onset thereof. A course of action to prevent a disease, disorder or condition in a subject is typically applied in the context of a subject who is predisposed to developing a disease, disorder or condition due to genetic, experiential or environmental factors of developing a particular disease, disorder or condition. In certain instances, the terms “prevent”, “preventing”, “prevention” are also used to refer to the slowing of the progression of a disease, disorder or condition from an existing state to a more deleterious state. Receptor: As used herein, the term “receptor” refers to a polypeptide having a domain that specifically binds a ligand that binding of the ligand results in a change to at least one biological property of the polypeptide. In some embodiments, the receptor is a cell membrane associated protein that comprises and extracellular domain (ECD) and a membrane associated domain which serves to anchor the ECD to the cell surface. In some embodiments of cell surface receptors, the receptor is a membrane spanning polypeptide comprising an intracellular domain (ICD) and extracellular domain (ECD) linked by a membrane spanning domain typically referred to as a transmembrane domain (TM). The binding of a cognate ligand to the receptor results in a conformational change in the receptor resulting in a measurable biological effect. In some instances, where the receptor is a membrane spanning polypeptide comprising an ECD, TM and ICD, the binding of the ligand to the ECD results in a measurable intracellular biological effect mediated by one or more domains of the ICD in response to the binding of the ligand to the ECD. In some embodiments, a receptor is a component of a multi-component complex to facilitate intracellular signaling. For example, the ligand may bind a cell surface receptor that is not associated with any intracellular signaling alone but upon ligand binding facilitates the formation of a heteromultimeric (including heterodimeric, heterotrimeric, etc.) or homomultimeric (including homodimeric, homotrimeric, homotetrameric, etc.) complex that results in a measurable biological effect in the cell such as activation of an intracellular signaling cascade (e.g., the Jak/STAT pathway). In some embodiments, a receptor is a membrane spanning single chain polypeptide comprising ECD, TM and ICD domains wherein the ECD, TM and ICD domains are derived from the same or differing naturally occurring receptor variants or synthetic functional equivalents thereof. Recombinant: As used herein, the term “recombinant” is used as an adjective to refer to the method by which a polypeptide, nucleic acid, or cell was modified using recombinant DNA technology. A “recombinant protein” is a protein produced using recombinant DNA technology and is frequently abbreviated with a lower case “r” preceding the protein name to denote the method by which the protein was produced (e.g., recombinantly produced human growth hormone is commonly abbreviated “rhGH”). Similarly a cell is referred to as a “recombinant cell” if the cell has been modified by the incorporation (e.g., transfection, transduction, infection) of exogenous nucleic acids (e.g., ssDNA, dsDNA, ssRNA, dsRNA, mRNA, viral or non-viral vectors, plasmids, cosmids and the like) using recombinant DNA technology. The techniques and protocols for recombinant DNA technology are well known in the art such as those can be found in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals. Response: The term “response,” for example, of a cell, tissue, organ, or organism, encompasses a quantitative or qualitative change in a evaluable biochemical or physiological parameter, (e.g., concentration, density, adhesion, proliferation, activation, phosphorylation, migration, enzymatic activity, level of gene expression, rate of gene expression, rate of energy consumption, level of or state of differentiation) where the change is correlated with the activation, stimulation, or treatment, with or contact with exogenous agents or internal mechanisms such as genetic programming. In certain contexts, the terms “activation”, “stimulation”, and the like refer to cell activation as regulated by internal mechanisms, as well as by external or environmental factors; whereas the terms “inhibition”, “down-regulation” and the like refer to the opposite effects. A “response” may be evaluated in vitro such as through the use of assay systems, surface plasmon resonance, enzymatic activity, mass spectroscopy, amino acid or protein sequencing technologies. A “response” may be evaluated in vivo quantitatively by evaluation of objective physiological parameters such as body temperature, bodyweight, tumor volume, blood pressure, results of X-ray or other imaging technology or qualitatively through changes in reported subjective feelings of well-being, depression, agitation, or pain. In some embodiments, the level of proliferation of CD3 activated primary human T-cells may be evaluated in a bioluminescent assay that generates a luminescent signal that is proportional to the amount of ATP present which is directly proportional to the number of viable cells present in culture as described in Crouch, et al. (1993) J. Immunol. Methods 160: 81-8 or using commercially available assays such as the CellTiter-Glo® 2.0 Cell Viability Assay or CellTiter-Glo® 3D Cell Viability kits commercially available from Promega Corporation, Madison WI 53711 as catalog numbers G9241 and G9681 in substantial accordance with the instructions provided by the manufacturer. In some embodiments, the level of activation of T cells in response to the administration of a test agent may be determined by flow cytometric methods as described as determined by the level of STAT (e.g., STAT1, STAT3, STAT5) phosphorylation in accordance with methods well known in the art. Significantly Reduced Binding: As used herein, the term “exhibits significantly reduced binding” is used with respect a variant of a first molecule (e.g., a ligand or antibody) which exhibits a significant reduction in the affinity for a second molecule (e.g., receptor or antigen) relative the parent form of the first molecule. With respect to antibody variants, an antibody variant “exhibits significantly reduced binding” if the affinity of the variant antibody for an antigen if the variant binds to the native form of the receptor with and affinity of less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the parent antibody from which the variant was derived. Similarly, with respect to variant ligands, a variant ligand “exhibits significantly reduced binding” if the affinity of the variant ligand binds to a receptor with an affinity of less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the parent ligand from which the variant ligand was derived. Similarly, with respect to variant receptors, a variant ligand “exhibits significantly reduced binding” if the affinity of the variant receptors binds to a with an affinity of less than 20%, alternatively less than about 10%, alternatively less than about 8%, alternatively less than about 6%, alternatively less than about 4%, alternatively less than about 2%, alternatively less than about 1%, or alternatively less than about 0.5% of the parent receptor from which the variant receptor was derived. Small Molecule(s): The term “small molecules” refers to chemical compounds (typically pharmaceutically active compounds) having a molecular weight that is less than about 10 kDa, less than about 2 kDa, or less than about lkDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing an inorganic component, molecules comprising a radioactive atom, and synthetic molecules. The term “small molecule” is a term well understood to those of ordinary skill in the pharmaceutical arts and is typically used to distinguish organic chemical compounds from biologics. Specifically Binds: As used herein the term “specifically binds” refers to the degree of affinity for which a first molecule exhibits with respect to a second molecule. In the context of binding pairs (e.g., ligand/receptor, antibody/antigen) a first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the first molecule of the binding pair does not bind in a significant amount to other components present in the sample. A first molecule of a binding pair is said to specifically bind to a second molecule of a binding pair when the first molecule of the binding pair when the affinity of the first molecule for the second molecule is at least two-fold greater, alternatively at least five times greater, alternatively at least ten times greater, alternatively at least 20-times greater, or alternatively at least 100-times greater than the affinity of the first molecule for other components present in the sample. In a particular embodiment, where the first molecule of the binding pair is an antibody, the antibody specifically binds to the antigen (or antigenic determinant (epitope) of a protein, antigen, ligand, or receptor) if the equilibrium dissociation constant (KD) between antibody and the antigen is lesser than about 10−6M, alternatively lesser than about 10−8M, alternatively lesser than about 10−10M, alternatively lesser than about 1011M, lesser than about 10−12M as determined by, e.g., Scatchard analysis (Munsen, et al. (1980) Analyt. Biochem. 107:220-239). In one embodiment where the ligand is an ILR binding sdAb and the receptor comprises an ILR, the ILR binding sdAb specifically binds if the equilibrium dissociation constant (KD) of the ILR binding sdAb/ILR ECD is lesser than about 10−5M, alternatively lesser than about 10−6M, alternatively lesser than about 10−7M, alternatively lesser than about 10−8M, alternatively lesser than about 10−9M, alternatively lesser than about 1010 M, or alternatively lesser than about 10−11M. Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA assays, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays)) and surface plasmon resonance assays (see, e.g., Drescher et al., (2009) Methods Mol Biol 493:323-343 with commercially available instrumentation such as the Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752). In some embodiments, the present disclosure provides molecules (e.g., ILR binding sdAbs) that specifically bind to the hILR. As used herein, the binding affinity of an ILR binding molecule for the ILR, the binding affinity may be determined and/or quantified by surface plasmon resonance (“SPR”). In evaluating binding affinity of an ILR binding molecule for the ILR, either member of the binding pair may be immobilized, and the other element of the binding pair be provided in the mobile phase. In some embodiments, the sensor chip on which the protein of interest is to be immobilized is conjugated with a substance to facilitate binding of the protein of interest such as nitrilotriacetic acid (NTA) derivatized surface plasmon resonance sensor chips (e.g., Sensor Chip NTA available from Cytiva Global Life Science Solutions USA LLC, Marlborough MA as catalog number BR100407), as anti-His tag antibodies (e.g. anti-histidine CM5 chips commercially available from Cytiva, Marlborough MA), protein A or biotin. Consequently, to evaluate binding, it is frequently necessary to modify the protein to provide for binding to the substance conjugated to the surface of the chip. For example, the one member of the binding pair to be evaluated by incorporation of a chelating peptide comprising poly-histidine sequence (e.g., 6×His (SEQ ID NO: 230) or 8×His (SEQ ID NO: 231)) for retention on a chip conjugated with NTA. In some embodiments, the ILR binding molecule may be immobilized on the chip and ILR (or ECD fragment thereof) be provided in the mobile phase. Alternatively, the ILR (or ECD fragment thereof) may be immobilized on the chip and the ILR binding molecule be provided in the mobile phase. In either circumstance, it should be noted that modifications of some proteins for immobilization on a coated SPR chip may interfere with the binding properties of one or both components of the binding pair to be evaluated by SPR. In such cases, it may be necessary to switch the mobile and bound elements of the binding pair or use a chip with a binding agent that facilitates non-interfering conjugation of the protein to be evaluated. Alternatively, when evaluating the binding affinity of ILR binding molecule for ILR using SPR, the ILR binding molecule may be derivatized by the C-terminal addition of a poly-His sequence (e.g., 6×His (SEQ ID NO: 230) or 8×His (SEQ ID NO: 231)) and immobilized on the NTA derivatized sensor chip and the ILR receptor subunit for which the ILR VHH's binding affinity is being evaluated is provided in the mobile phase. The means for incorporation of a poly-His sequence into the C-terminus of the ILR binding molecule produced by recombinant DNA technology is well known to those of skill in the relevant art of biotechnology. In some embodiments, the binding affinity of ILR binding molecule for an ILR comprises using SPR substantially in accordance with the teaching of the Examples. Substantially Pure: As used herein, the term “substantially pure” indicates that a component of a composition makes up greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%, alternatively greater than about 95% of the total content of the composition. A protein that is “substantially pure” comprises greater than about 50%, alternatively greater than about 60%, alternatively greater than about 70%, alternatively greater than about 80%, alternatively greater than about 90%, alternatively greater than about 95% of the total content of the composition. T-cell: As used herein the term “T-cell” or “T cell” is used in its conventional sense to refer to a lymphocytes that differentiates in the thymus, possess specific cell-surface antigen receptors, and include some that control the initiation or suppression of cell-mediated and humoral immunity and others that lyse antigen-bearing cells. In some embodiments the T cell includes without limitation naïve CD8+T cells, cytotoxic CD8+T cells, naïve CD4+T cells, helper T cells, e.g., TH1, TH2, TH9, TH11, TH22, TFH; regulatory T cells, e.g., TR1, Tregs, inducible Tregs; memory T cells, e.g., central memory T cells, effector memory T cells, NKT cells, tumor infiltrating lymphocytes (TILs) and engineered variants of such T-cells including but not limited to CAR-T cells, recombinantly modified TILs and TCR-engineered cells. In some embodiments the T cell is a T cell expressing the IL12RB1 isoform referred to interchangeably as IL cell, IL12RB1+ cell, IL T cell, or IL12RB1+ T cell). Terminus/Terminal: As used herein in the context of the structure of a polypeptide, “N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxyl terminus”) refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while the terms “N-terminal” and “C-terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C-terminus, respectively. “Immediately N-terminal” refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the N-terminus of the polypeptide. “Immediately C-terminal” refers to the position of a first amino acid residue relative to a second amino acid residue in a contiguous polypeptide sequence, the first amino acid being closer to the C-terminus of the polypeptide. Transmembrane Domain: The term “transmembrane domain” or “TM” refers to a polypeptide domain of a membrane spanning polypeptide (e.g., a transmembrane receptor) which, when the membrane spanning polypeptide is associated with a cell membrane, is which is embedded in the cell membrane and is in peptidyl linkage with the extracellular domain (ECD) and the intracellular domain (ICD) of a membrane spanning polypeptide. A transmembrane domain may be homologous (naturally associated with) or heterologous (not naturally associated with) with either or both of the extracellular and/or intracellular domains. In some embodiments, where the receptor is chimeric receptor comprising the intracellular domain derived from a first parental receptor and a second extracellular domains are derived from a second different parental receptor, the transmembrane domain of the chimeric receptor is the transmembrane domain normally associated with either the ICD or the ECD of the parent receptor from which the chimeric receptor is derived. Treg Cell or Regulatory T Cell. The terms “regulatory T cell”, “Treg cell”, or “Treg” are interchangeably herein to refers to a type of CD4+T cell that can suppress the responses of other T cells including but not limited to effector T cells (Teff). Treg cells are typically characterized by expression of CD4 (CD4+), the CD25 subunit of the IL2 receptor (CD25+), and the transcription factor forkhead box P3 (FOXP3+) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004). In some instances, the term “conventional CD4+T cells” is used to distinguish non-Treg CD4+T cells from CD4+Tregs. Variant: The terms “variant”, “protein variant” or “variant protein” or “variant polypeptide” are used interchangeably herein to refer to a polypeptide that differs from a parent polypeptide by virtue of at least one amino acid modification, substitution, or deletion. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide or may be a modified version of a WT polypeptide. The term variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the nucleic acid sequence that encodes it. In some embodiments, the variant polypeptide comprises from about one to about ten, alternatively about one to about eight, alternatively about one to about seven, alternatively about one to about five, alternatively about one to about four, alternatively from about one to about three alternatively from one to two amino acid modifications, substitutions, or deletions, or alternatively a single amino acid amino acid modification, substitution, or deletion compared to the parent polypeptide. A variant may be at least about 99% identical, alternatively at least about 98% identical, alternatively at least about 97% identical, alternatively at least about 95% identical, or alternatively at least about 90% identical to the parent polypeptide from which the variant is derived. VHH: As used herein, the term “VHH” is a type of sdAb that has a single monomeric heavy chain variable antibody domain. Such antibodies can be found in or produced from Camelid mammals (e.g., camels, llamas) which are naturally devoid of light chainsVHHs can be obtained from immunization of camelids (including camels, llamas, and alpacas (see, e.g., Hamers-Casterman, et al. (1993) Nature 363:446-448) or by screening libraries (e.g., phage libraries) constructed in VHH frameworks. Antibodies having a given specificity may also be derived from non-mammalian sources such as VHHs obtained from immunization of cartilaginous fishes including, but not limited to, sharks. In a particular embodiment, a VHH in a bispecific VHH2binding molecule described herein binds to a receptor (e.g., the first receptor or the second receptor of the natural or non-natural receptor pairs) if the equilibrium dissociation constant (KD) between the VHH and the receptor is lesser than about 10−6M, alternatively lesser than about 10−8M, alternatively lesser than about 1010 M, alternatively lesser than about 10−11M, alternatively lesser than about 1010 M, lesser than about 1012 M as determined by, e.g., Scatchard analysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239). Standardized protocols for the generation of single domain antibodies from camelids are well known in the scientific literature. See, e.g., Vincke, et al (2012) Chapter 8 inMethods in Molecular Biology, Walker, J. editor (Humana Press, Totowa NJ). Specific binding may be assessed using techniques known in the art including but not limited to competition ELISA, BIACORE® assays and/or KINEXA® assays. In some embodiments, a VHH described herein can be humanized to contain human framework regions. Examples of human germlines that could be used to create humanized VHHs include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782). Wild Type: By “wild type” or “WT” or “native” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A wild-type protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been modified by the hand of man. IL12RB1 The IL12RB1 binding molecules of the present disclosure specifically bind to the extracellular domain of the IL12RB1. Human IL12RB1 In one embodiment, specifically bind to the extracellular domain of the human IL receptor subunit (hIL12RB1). hIL12RB1 is expressed as a 662 amino acid precursor comprising a 23 amino acid N-terminal signal sequence which is post-translationally cleaved to provide an 639 amino acid mature protein. The canonical full-length acid hIL12RB1 precursor (including the signal peptide) is a 662 amino acid polypeptide having the amino acid sequence: (SEQ ID NO: 1)MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSLTAPQDPDPAGMATYSWSRESGAMGQEKCYYITIFASAHPEKLTLWSTVLSTYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSDWLIFFASLGSFLSILLVGVLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKM. For purposes of the present disclosure, the numbering of amino acid residues of the human IL12RB1 polypeptides as described herein is made in accordance with the numbering of this canonical sequence (UniProt Reference No P42701, SEQ ID NO:1). Amino acids 1-23 of SEQ ID NO:1 are identified as the signal peptide of hIL12RB1, amino acids 24-545 of SEQ ID NO:1 are identified as the extracellular domain, amino acids 546-570 of SEQ ID NO:1 are identified as the transmembrane domain, and amino acids 571-662 of SEQ ID NO:1 are identified as the intracellular domain. For the purposes of generating antibodies that bind to the ECD of IL12RB1, immunization may be performed with the extracellular domain of the hIL12RB1. The extracellular domain of hIL12RB1 is a 522 amino acid polypeptide of the sequence: (SEQ ID NO: 227)CRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRQLGSQGSSWSKWSSPVCVPPENPPQPQVRFSVEQLGQDGRRRLTLKEQPTQLELPEGCQGLAPGTEVTYRLQLHMLSCPCKAKATRTLHLGKMPYLSGAAYNVAVISSNQFGPGLNQTWHIPADTHTEPVALNISVGTNGTTMYWPARAQSMTYCIEWQPVGQDGGLATCSYHFGGNASAAGTPHHVSVKNHSLDSVSVDWAPSLLSTCPGVLKEYVVRCRDEDSKQVSEHPVQPTETQVTLSGLRAGVAYTVQVRADTAWLRGVWSQPQRFSIEVQVSD. Mouse IL12RB1 In one embodiment, specifically bind to the extracellular domain of the mouse or murine IL12RB1 receptor subunit (mIL12RB1). mIL12RB1 is expressed as a 738 amino acid precursor comprising a 19 amino acid N-terminal signal sequence which is post-translationally cleaved to provide a 719 amino acid mature protein. The canonical full-length acid mIL12RB1 precursor (including the 24 amino acid signal peptide) is a 738 amino acid polypeptide having the amino acid sequence: (SEQ ID NO: 228)MDMMGLAGTSKHITFLLLCQLGASGPGDGCCVEKTSFPEGASGSPLGPRNLSCYRVSKTDYECSWQYDGPEDNVSHVLWCCFVPPNHTHTGQERCRYFSSGPDRTVQFWEQDGIPVLSKVNFWVESRLGNRTMKSQKISQYLYNWTKTTPPLGHIKVSQSHRQLRMDWNVSEEAGAEVQFRRRMPTTNWTLGDCGPQVNSGSGVLGDIRGSMSESCLCPSENMAQEIQIRRRRRLSSGAPGGPWSDWSMPVCVPPEVLPQAKIKFLVEPLNQGGRRRLTMQGQSPQLAVPEGCRGRPGAQVKKHLVLVRMLSCRCQAQTSKTVPLGKKLNLSGATYDLNVLAKTRFGRSTIQKWHLPAQELTETRALNVSVGGNMTSMQWAAQAPGTTYCLEWQPWFQHRNHTHCTLIVPEEEDPAKMVTHSWSSKPTLEQEECYRITVFASKNPKNPMLWATVLSSYYFGGNASRAGTPRHVSVRNQTGDSVSVEWTASQLSTCPGVLTQYVVRCEAEDGAWESEWLVPPTKTQVTLDGLRSRVMYKVQVRADTARLPGAWSHPQRFSFEVQISRLSIIFASLGSFASVLLVGSLGYIGLNRAAWHLCPPLPTPCGSTAVEFPGSQGKQAWQWCNPEDFPEVLYPRDALVVEMPGDRGDGTESPQAAPECALDTRRPLETQRQRQVQALSEARRLGLAREDCPRGDLAHVTLPLLLGGVTQGASVLDDLWRTHKTAEPGPPTLGQEA For purposes of the present disclosure, the numbering of amino acid residues of the mIL12RB1 polypeptides as described herein is made in accordance with the numbering of this canonical sequence (UniProt Reference No. Q60837, SEQ ID NO:228). Amino acids 1-19 of SEQ ID NO:228 are identified as the signal peptide of mIL12RB1, amino acids 20-565 of SEQ ID NO:228 are identified as the extracellular domain, amino acids 566-591 of SEQ ID NO:228 are identified as the transmembrane domain, and amino acids 592-738 of SEQ ID NO:228 are identified as the intracellular domain. For the purposes of generating antibodies that bind to the ECD of IL12RB1, immunization may be performed with the extracellular domain of the mIL12RB1. The extracellular domain of the mIL12RB1 receptor is a 546 amino acid polypeptide of the sequence: (SEQ ID NO: 229)QLGASGPGDGCCVEKTSFPEGASGSPLGPRNLSCYRVSKTDYECSWQYDGPEDNVSHVLWCCFVPPNHTHTGQERCRYFSSGPDRTVQFWEQDGIPVLSKVNFWVESRLGNRTMKSQKISQYLYNWTKTTPPLGHIKVSQSHRQLRMDWNVSEEAGAEVQFRRRMPTTNWTLGDCGPQVNSGSGVLGDIRGSMSESCLCPSENMAQEIQIRRRRRLSSGAPGGPWSDWSMPVCVPPEVLPQAKIKFLVEPLNQGGRRRLTMQGQSPQLAVPEGCRGRPGAQVKKHLVLVRMLSCRCQAQTSKTVPLGKKLNLSGATYDLNVLAKTRFGRSTIQKWHLPAQELTETRALNVSVGGNMTSMQWAAQAPGTTYCLEWQPWFQHRNHTHCTLIVPEEEDPAKMVTHSWSSKPTLEQEECYRITVFASKNPKNPMLWATVLSSYYFGGNASRAGTPRHVSVRNQTGDSVSVEWTASQLSTCPGVLTQYVVRCEAEDGAWESEWLVPPTKTQVTLDGLRSRVMYKVQVRADTARLPGAWSHPQRFSFEVQIS. IL12Rb1 Binding Molecules and Single Domain Antibodies In some embodiments, a IL12Rb1 binding molecule of the present disclosure is a single domain antibody (sdAb). The present disclosure relates to IL12Rb1 binding molecules comprising single domain antibodies (sdAbs) that specifically bind to the extracellular domain of the human IL12Rb1 isoform (hIL12Rb1) which are found on all IL12Rb1-expressing cells. A single-domain antibody (sdAb) is an antibody containing a single monomeric variable antibody domain. Like a full-length antibody, sdAbs are able to bind specifically to an antigenic determinant. hIL12RB1 binding VHH single-domain antibodies can be engineered from heavy chain antibodies isolated from Camelidae mammals (e.g., camels, llamas, dromedary, alpaca, and guanaco) immunized with the extracellular domain of hIL12RB1 or an immunologically active fragment thereof. Descriptions of sdAbs and VHHs can be found in, e.g., De Greve et al., (2019) Curr Opin Biotechnol. 61:96-101; Ciccarese, et al., (2019) Front Genet. 10:997: Chanier and Chames (2019)Antibodies(Basel) 8(1); and De Vlieger, et al. (2018)Antibodies(Basel) 8(1). Alternatively, hIL12RB1 single domain antibodies may be engineered from heavy chain antibodies isolated from the IgNAR heavy chain antibodies isolated from cartilaginous fishes immunized with the extracellular domain of hIL12RB1 or an immunologically active fragment thereof. hIL12RB1 binding sdAbs may also be obtained by splitting the dimeric variable domains from immunoglobulin G (IgG) isotypes from other mammalian species including humans, rats, rabbits immunized with the extracellular domain of hIL12RB1 or an immunologically active fragment thereof. Although most research into sdAbs is currently based on heavy chain variable domains, sdAbs derived from light chains have also been shown to bind specifically to the target proteins comprising the antigenic immunization sequence. Moller et al.,J Biol Chem.285(49):38348-38361, 2010. In some embodiments, the sdAb is a VHH. A VHH is a type of sdAb that has a single monomeric heavy chain variable antibody domain. Similar to a traditional antibody, a VHH is able to bind specifically to a specific antigen. An exemplary VHH has a molecular weight of approximately 12-15 kDa which is much smaller than traditional mammalian antibodies (150-160 kDa) composed of two heavy chains and two light chains. VHHs can be found in or produced from Camelidae mammals (e.g., camels, llamas, dromedary, alpaca, and guanaco) which are naturally devoid of light chains. The present disclosure provides IL12RB1 binding molecules comprising a polypeptide having at least 75%, alternatively 80%, alternatively 90%, alternatively 95%, alternatively 98%, or alternatively 99% or 100% identity to a polypeptide of any one of SEQ ID NOS:2-22. The present disclosure provides IL12RB1 binding molecules comprising a CDR1, a CDR2, and a CDR3 as described in a row of Table 1A provided herein. In some embodiments, the CDR1, CDR2, and CDR3 can each, independently, comprise at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity, or have 0, 1, 2, or 3 amino acid changes, optionally conservative amino acid changes, relative to the sequence described in a row of Table 1 or 4 provided herein. EXPERIMENTAL The single domain antibodies of the present disclosure were obtained from camels by immunization with an extracellular domain of a IL12Rb1 receptor. IL12Rb1 VHH molecules of the present disclosure of the present disclosure were generated in substantial accordance with the teaching of the Examples. Briefly, a camel was sequentially immunized with the ECD of the human IL12Rb1 and mouse IL12Rb1 over a period several weeks of by the subcutaneous an adjuvanted composition containing a recombinantly produced fusion proteins comprising the extracellular domain of the IL12Rb1, the human IgG1 hinge domain and the human IgG1 heavy chain Fc. Following immunization, RNAs extracted from a blood sample of appropriate size VHH-hinge-CH2-CH3 species were transcribed to generate DNA sequences, digested to identify the approximately 400 bp fragment comprising the nucleic acid sequence encoding the VHH domain was isolated. The isolated sequence was digested with restriction endonucleases to facilitate insertion into a phagemid vector for in frame with a sequence encoding a his-tag and transformed intoE. colito generate a phage library. Multiple rounds of biopanning of the phage library were conducted to identify VHHs that bound to the ECD of IL12Rb1 (human or mouse as appropriate). Individual phage clones were isolated for periplasmic extract ELISA (PE-ELISA) in a 96-well plate format and selective binding confirmed by colorimetric determination. The IL12Rb1 binding molecules that demonstrated specific binding to the IL12Rb1 antigen were isolated and sequenced and sequences analyzed to identify VHH sequences, CDRs and identify unique VHH clonotypes. As used herein, the term “clonotypes” refers a collection of binding molecules that originate from the same B-cell progenitor cell, in particular collection of antigen binding molecules that belong to the same germline family, have the same CDR3 lengths, and have 70% or greater homology in CDR3 sequence. The VHH molecules demonstrating specific binding to the hIL12Rb1 ECD antigen (anti-human IL12Rb1 VHHs) and the CDRs isolated from such VHHs are provided in Table 1. The VHH molecules demonstrating specific binding to the mIL12Rb1 ECD antigen (anti-mouse IL12Rb1 VHHs) are provided in Table 4 and the CDRs isolated from such VHHs are provided in Table 3. Nucleic acid sequences encoding the VHHs of Table 1 and 4 are provide in Tables 2 and 5 respectively. In some instances, due to sequence or structural similarities between the extracellular domains of IL12Rb1 receptors from various mammalian species, immunization with an antigen derived from a IL12Rb1 of a first mammalian species (e.g., the hIL12Rb1-ECD) may provide antibodies which specifically bind to IL12Rb1 receptors of one or more additional mammalian species. Such antibodies are termed “cross reactive.” For example, immunization of a camelid with a human derived antigen (e.g., the hIL12Rb1-ECD) may generate antibodies that are cross-reactive the murine and human receptors. Evaluation of cross-reactivity of antibody with respect to the receptors derived from other mammalian species may be readily determined by the skilled artisan, for example using the methods relating to evaluation of binding affinity and/or specific binding described elsewhere herein such as flow cytometry or SPR. Consequently, the use of the term “human IL12Rb1 VHH” or “hIL12Rb1 VHH” merely denotes that the species of the IL12Rb1 antigen used for immunization of the camelid from which the VHH was derived was the human IL12Rb1 (e.g., the hIL12Rb1, ECD, SEQ ID NO:192 but should not be understood as limiting with respect to the specific binding affinity of the VHH for hIL12Rb1 molecules of other mammalian species. Similarly, the use of the term “mouse IL12Rb1 VHH” or “mIL12Rb1” merely denotes that the species of the IL12Rb1 antigen used for immunization of the camelid from which the VHH was derived was the murine IL12Rb1 (e.g., the mIL12Rb1 ECD, SEQ ID NO:194) but should not be understood as limiting with respect to the specific binding affinity of the VHH for IL12Rb1 molecules of other mammalian species. Modified Forms of Single Domain Antibodies CDR Grafted sdAbs In some embodiments, the IL12RB1 binding sdAb of the present disclosure is a CDR grafted IL12RB1 binding sdAb. CDRs obtained from antibodies, heavy chain antibodies, and sdAbs derived therefrom may be grafted onto alternative frameworks as described in Saerens, et al. (2005) J. Mol Biol 352:597-607 to generate CDR-grafted sdAbs. In some embodiments, the present disclosure provides a IL12RB1 binding molecule comprising a CDR grafted IL binding sdAb, said CDR-grafted IL binding sdAb comprising a set of CDRs1, 2, and 3 as shown in a row of the Table 1A above. In some embodiments, the present disclosure provides a IL12RB1 binding molecule comprising a CDR grafted IL12RB1 binding sdAb, said CDR-grafted IL binding sdAb comprising a set of CDRs1, 2, and 3 as shown in a row of the Table 1A above. Elimination of N-Linked Glycosylation Sites In some embodiments, it is possible that an amino acid sequence (particularly a CDR sequence) of the IL binding sdAb may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser (N-X-S) or Asn-X-Thr (N-X-T), wherein X is any amino acid except for proline. In such instances, it is desirable to eliminate such N-linked glycosylation motifs by modifying the sequence of the N-linked glycosylation motif to prevent glycosylation. In some embodiments, the elimination of the Asn-X-Ser (N-X-S) N-linked glycosylation motif may be achieved by the incorporation of conservative amino acid substitution of the Asn (N) residue and/or Ser (S) residue of the Asn-X-Ser (N-X-S) N-linked glycosylation motif. In some embodiments, the elimination of the Asn-X-Thr (N-X-T) N-linked glycosylation motif may be achieved by the incorporation of conservative amino acid substitution of the Asn (N) residue and/or Thr (T) residue of the Asn-X-Thr (N-X-T) N-linked glycosylation motif. In some embodiments, elimination of the As procaryotic host cells do not provide the mechanism for glycosylation of recombinant proteins, when employing a procaryotic expression system to produce a recombinant recombinant IL12RB1 binding sdAb the modification of the sequence to eliminate the N-linked glycosylation sites may be obviated. Chimeric and Humanized sdAbs Any framework region can be used with the CDRs as described herein. In some embodiments, the IL12RB1 binding sdAb is a chimeric sdAb, in which the CDRs are derived from one species (e.g., camel) and the framework and/or constant regions are derived from another species (e.g., human or mouse). In specific embodiments, the framework regions are human or humanized sequences. Thus, humanized IL12RB1 binding sdAbs derived from hIL12RB1 binding VHHs are considered within the scope of the present disclosure. The techniques for humanization of camelid single domain antibodies are well known in the art. See, e.g., Vincke, et al. (2009)General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody ScaffoldJ. Biol. Chem. 284(5)3273-3284. In some embodiments, a VHH described herein can be humanized to contain human framework regions. Examples of human germlines that could be used to create humanized VHHs include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782). IL12Rb1 Binding Molecules Comprising Additional Agents In some embodiments, a IL12Rb1 binding molecule of the present disclosure comprises a IL12Rb1 single domain antibody (sdAb) is operably linked to to one or more additional biologically active agents including but not limited to, therapeutic agents, chemically, optically or radioactively active agents, including combinations thereof. The conjugation of at least one such biologically, chemically, optically or radioactively active agent confer additional biological or chemical properties to IL binding sdAb, the combination providing a IL12Rb1 binding molecule possessing additional or alternative utilities. For example, the additional agent may be a molecule selected from one or more of: immunomodulatory agents (e.g., immunogens); molecules that improve aqueous solubility (e.g., water soluble polymers and hydrophilic molecules such as sugars); carrier molecules that extend in vivo half-life (e.g., PEGylation, Fc fusions or acylation); generation of antibodies for use in detection assays (e.g., epitope tags), enhance ease of purification (e.g., chelating peptides such as poly-His tags); targeting domains that provide selective targeting IL12Rb1 binding molecule to a particular cell or tissue type; therapeutic agents (e.g., therapeutic agents including small molecule or polypeptide agents); agents that visibility to optical or electromagnetic sensors (e.g., radionucleotides or fluorescent agents). In some embodiments, the linker is a cleavable linker or a non-cleavable linker. The use of a cleavable linker in a IL12Rb1 binding molecule as contemplated herein facilitates the release of a therapeutic agent into the intracellular cytoplasm upon internalization of the IL12Rb1 binding molecule. A non-cleavable linker would allow release upon digestion of the IL12Rb1 binding molecule of or it could be used with an agent that does not require release from the antibody (e.g., an imaging agent). In some embodiments, where the IL12Rb1 binding molecule comprises a IL binding sdAb in stable association with an additional agent joined via a linker. A linker is a covalent linkage between two elements of a IL12Rb1 binding molecule (e.g., a hIL12Rb1 binding VHH and PEG polymer). A linker can be a covalent bond, chemical linker or a peptide linker. Suitable linkers include “flexible linkers” which are generally of sufficient length to permit some movement between the IL binding sdAb and the linked agent(s). Examples of chemical linkers include aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. In some embodiments, the linker is a peptide linker. Suitable peptide linkers can be readily selected and can be of any suitable length, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, 30-50 or more than 50 amino acids. Suitable peptide linkers are known in the art, and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. Examples of flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Glycine and glycine-serine polymers are relatively unstructured, and therefore can serve as a neutral tether between components. Further examples of flexible linkers include glycine polymers (G)n, glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers. Glycine and glycine-serine polymers are relatively unstructured, and therefore may serve as a neutral tether between components. A multimer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20, 20-30, or 30-50) of such linker sequences may be linked together to provide flexible linkers that may be used to conjugate a heterologous amino acid sequence to IL12RB1 binding sdAbs disclosed herein. In some embodiments the linkers have the formula (GGGS)n (SEQ ID NO: 232), (GGGSG)n (SEQ ID NO: 233), or (GGSG)n (SEQ ID NO: 234), wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Immunomodulatory Agents In some embodiments, the IL binding molecule is operably linked to n immunomodulatory agent (immunoconjugates). Examples of immunomodulatory agents that may operably linked to the hIL12RB1 binding sdAb of the present disclosure include, but are not limited to, inactivated virus particles, inactivated bacterial toxins such as toxoid from diphtheria, tetanus, cholera, or leukotoxin molecules, inactivated bacteria and dendritic cells. Such immunoconjugates are useful in facilitating an immune response against the IL12RB1 or cells expressing the IL12RB1. Flag Tags In one embodiment, the present disclosure provides a IL12Rb1 binding molecule operably linked to an antigenic tag, such as a FLAG sequence. FLAG sequences are recognized by biotinylated, highly specific, anti-FLAG antibodies, as described herein (see e.g., Blanar et al. (1992) Science 256:1014 and LeClair, et al. (1992) PNAS-USA 89:8145). In some embodiments, the IL12RB1 binding sdAb polypeptide further comprises a C-terminal c-myc epitope tag. Chelating Peptides In one embodiment, the present disclosure provides a IL12RB1 binding molecule is operably linked to one or more transition metal chelating polypeptide sequences. The incorporation of such a transition metal chelating domain facilitates purification immobilized metal affinity chromatography (IMAC) as described in Smith, et al. U.S. Pat. No. 4,569,794 issued Feb. 11, 1986. Examples of transition metal chelating polypeptides useful in the practice of the present IL12RB1 binding molecule are described in Smith, et al. supra and Dobeli, et al. U.S. Pat. No. 5,320,663 issued May 10, 1995, the entire teachings of which are hereby incorporated by reference. Particular transition metal chelating polypeptides useful in the practice of the present IL12RB1 binding molecule are polypeptides comprising 3-6 contiguous histidine residues (SEQ ID NO: 235) such as a six-histidine (His)6(SEQ ID NO: 230) peptide and are frequently referred to in the art as “His-tags.” In addition to providing a purification “handle” for the recombinant proteins or to facilitate immobilization on SPR sensor chips, such the conjugation of the hIL12RB1 binding molecule to a chelating peptide facilitates the targeted delivery to IL12RB1 expressing cells of transition metal ions as kinetically inert or kinetically labile complexes in substantial accordance with the teaching of Anderson, et al., (U.S. Pat. No. 5,439,829 issued Aug. 8, 1995 and Hale, J. E (1996) Analytical Biochemistry 231(1):46-49. The transition metal ion is a reporter molecule such as a fluorescent compound or radioactive agent, including as radiological imaging or therapeutic agents. Carrier Molecules In some embodiments the IL12RB1 binding sdAbs of the present disclosure is operably linked to one or more carrier molecules. Carrier molecules are typically large, slowly metabolized macromolecules which provide for stabilization and/or extended duration of action in vivo. Examples of in vivo carriers that may be incorporated into IL12Rb1 binding molecules, but are not limited to: proteins (including but not limited to human serum albumin); fatty acids (acylation); polysaccharides (including but not limited to (N- and O-linked) sugars, sepharose, agarose, cellulose, or cellulose); polypeptdies amino acid copolymers, acylation, or polysialylation, an polyethylene glycol (PEG) polymers. Water Soluble Polymers In some embodiments, the IL12RB1 binding sdAb is conjugated to one or more water-soluble polymers. Examples of water soluble polymers useful in the practice of the present IL12Rb1 binding molecule include polyethylene glycol (PEG), poly-propylene glycol (PPG), polysaccharides (polyvinylpyrrolidone, copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), polyolefinic alcohol, polysaccharides, poly-alpha-hydroxy acid, polyvinyl alcohol (PVA), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. Polyethylene Glycol In one embodiment, the carrier molecule is a polyethylene glycol (“PEG”) polymer. Conjugation of PEG polymers to proteins (PEGylation) is a well-established method for the extension of serum half-life of biological agents. The PEGylated polypeptide may be further referred to as monopegylated, dipegylated, tripegylated (and so forth) to denote a polypeptide comprising one, two, three (or more) PEG moieties attached to the polypeptide, respectively. In some embodiments, the PEG may be covalently attached directly to the sdAb (e.g., through a lysine side chain, sulfhydryl group of a cysteine or N-terminal amine) or optionally employ a linker between the PEG and the sdAb. In some embodiments, a IL12Rb1 binding molecule comprises more than one PEG molecules each of which is attached to a different amino acid residue. In some embodiments, the sdAb may be modified by the incorporation of non-natural amino acids with non-naturally occurring amino acid side chains to facilitate site specific PEGylation. In other embodiments, cysteine residues may be substituted at one or more positions within the sdAb to facilitate site-specific PEGylation via the cysteine sulfhydryl side chain. In some instances, the IL12RB1 binding molecules of the present disclosure possess an N-terminal glutamine (“1Q”) residue. N-terminal glutamine residues have been observed to spontaneously cyclyize to form pyroglutamate (pE) at or near physiological conditions. (See e.g., Liu, et al (2011) J. Biol. Chem. 286(13): 11211-11217). In some embodiments, the formation of pyroglutamate complicates N-terminal PEG conjugation particularly when aldehyde chemistry is used for N-terminal PEGylation. Consequently, when PEGylating the IL12RB1 binding molecules of the present disclosure, particularly when aldehyde chemistry is to be employed, the IL12RB1 binding molecules possessing an amino acid at position 1 (e.g., 1Q) are substituted at position 1 with an alternative amino acid or are deleted at position 1 (e.g., des-1Q). In some embodiments, the IL12RB1 binding molecules of the present disclosure comprise an amino acid substitution selected from the group Q1E and Q1D. PEGs suitable for conjugation to a polypeptide sequence are generally soluble in water at room temperature, and have the general formula R(O—CH2—CH2)nO—R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. When R is a protective group, it generally has from 1 to 8 carbons. The PEG can be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure. A molecular weight of the PEG used in a IL12Rb1 binding molecule is not restricted to any particular range. The PEG component of a IL12Rb1 binding molecule can have a molecular mass greater than about 5 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In some embodiments, the molecular mass is from about 5 kDa to about 10 kDa, from about 5 kDa to about 15 kDa, from about 5 kDa to about 20 kDa, from about 10 kDa to about 15 kDa, from about 10 kDa to about 20 kDa, from about 10 kDa to about 25 kDa or from about 10 kDa to about 30 kDa. Linear or branched PEG molecules having molecular weights from about 2,000 to about 80,000 daltons, alternatively about 2,000 to about 70,000 daltons, alternatively about 5,000 to about 50,000 daltons, alternatively about 10,000 to about 50,000 daltons, alternatively about 20,000 to about 50,000 daltons, alternatively about 30,000 to about 50,000 daltons, alternatively about 20,000 to about 40,000 daltons, alternatively about 30,000 to about 40,000 daltons. In one embodiment of the IL12Rb1 binding molecule, the PEG is a 40 kD branched PEG comprising two 20 kD arms. The present disclosure also contemplates a IL12Rb1 binding molecule comprising more than one PEG moiety wherein the PEGs have different sizes values, and thus the various different PEGs are present in specific ratios. For example, in the preparation of a PEGylated IL12Rb1 binding molecule, some compositions comprise a mixture of mono-, di-, tri-, and quadra-PEGylated sdAb conjugates. In some compositions, the percentage of mono-PEGylated species is 18-25%, the percentage of di-PEGylated species is 50-66%, the percentage of tri-pegylated species is 12-16%, and the percentage of quadra-pegylated species up to 5%. Such complex compositions can be produced by reaction conditions and purification methods known in the art. Chromatography may be used to resolve conjugate fractions, and a fraction is then identified which contains the conjugate having, for example, the desired number of PEGs attached, purified free from unmodified protein sequences and from conjugates having other numbers of PEGs attached. PEGylation most frequently occurs at the α-amino group at the N-terminus of the polypeptide, the epsilon amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. Since most recombinant polypeptides possess a single alpha and a number of epsilon amino and imidazole groups, numerous positional isomers can be generated depending on the linker chemistry. Two widely used first generation activated monomethoxy PEGs (mPEGs) are succinimdyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotehnol. Appl. Biochem 15:100-114) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al. U.S. Pat. No. 5,650,234), which react preferentially with lysine residues to form a carbamate linkage but are also known to react with histidine and tyrosine residues. Use of a PEG-aldehyde linker targets a single site on the N-terminus of a polypeptide through reductive amination. The PEG can be bound to a IL12Rb1 binding molecule of the present disclosure via a terminal reactive group (a “spacer”) which mediates a bond between the free amino or carboxyl groups of one or more of the polypeptide sequences and polyethylene glycol. The PEG having the spacer which can be bound to the free amino group includes N-hydroxysuccinylimide polyethylene glycol, which can be prepared by activating succinic acid ester of polyethylene glycol with N-hydroxysuccinylimide. In some embodiments, the PEGylation of the sdAb is facilitated by the incorporation of non-natural amino acids bearing unique side chains to facilitate site specific PEGylation. The incorporation of non-natural amino acids into polypeptides to provide functional moieties to achieve site specific PEGylation of such polypeptides is known in the art. See e.g., Ptacin, et al., PCT International Application No. PCT/US2018/045257 filed Aug. 3, 2018 and published Feb. 7, 2019 as International Publication Number WO 2019/028419A1. The PEG moiety of the of a PEGylated IL12Rb1 binding molecule may be linear or branched. Branched PEG derivatives, “star-PEGs” and multi-armed PEGs are contemplated by the present disclosure. Specific embodiments PEGs useful in the practice of the present disclosure include a 10 kDa linear PEG-aldehyde (e.g., Sunbright® ME-100AL, NOF America Corporation, One North Broadway, White Plains, NY 10601 USA), 10 kDa linear PEG-NETS ester (e.g., Sunbright® ME-100CS, Sunbright® ME-100AS, Sunbright® ME-100GS, Sunbright® ME-100HS, NOF), a 20 kDa linear PEG-aldehyde (e.g., Sunbright® ME-200AL, NOF, a 20 kDa linear PEG-NHS ester (e.g., Sunbright® ME-200CS, Sunbright® ME-200AS, Sunbright® ME-200GS, Sunbright® ME-200HS, NOF), a 20 kDa 2-arm branched PEG-aldehyde the 20 kDA PEG-aldehyde comprising two 10 kDA linear PEG molecules (e.g., Sunbright® GL2-200AL3, NOF), a 20 kDa 2-arm branched PEG-NHS ester the 20 kDA PEG-NETS ester comprising two 10 kDA linear PEG molecules (e.g., Sunbright® GL2-200TS, Sunbright® GL200GS2, NOF), a 40 kDa 2-arm branched PEG-aldehyde the 40 kDA PEG-aldehyde comprising two 20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3), a 40 kDa 2-arm branched PEG-NETS ester the 40 kDA PEG-NETS ester comprising two 20 kDA linear PEG molecules (e.g., Sunbright® GL2-400AL3, Sunbright® GL2-400GS2, NOF), a linear 30 kDa PEG-aldehyde (e.g., Sunbright® ME-300AL) and a linear 30 kDa PEG-NETS ester. Fc Fusions In some embodiments, the carrier molecule is a Fc molecule or a monomeric subunit thereof. In some embodiments, the dimeric Fc molecule may be engineered to possess a “knob-into-hole modification.” The knob-into-hole modification is more fully described in Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Pat. No. 5,731,168, issued Mar. 24, 1998, U.S. Pat. No. 7,642,228, issued Jan. 5, 2010, U.S. Pat. No. 7,695,936, issued Apr. 13, 2010, and U.S. Pat. No. 8,216,805, issued Jul. 10, 2012. The knob-into-hole modification refers to a modification at the interface between two immunoglobulin heavy chains in the CH3 domain, wherein: i) in a CH3 domain of a first heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) creating a projection from the surface (“knob”) and ii) in the CH3 domain of a second heavy chain, an amino acid residue is replaced with an amino acid residue having a smaller side chain (e.g., alanine or threonine), thereby generating a cavity (“hole”) within at interface in the second CH3 domain within which the protruding side chain of the first CH3 domain (“knob”) is received by the cavity in the second CH3 domain. In one embodiment, the “knob-into-hole modification” comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other one of the antibody heavy chains. Furthermore, the Fc domains may be modified by the introduction of cysteine residues at positions S354 and Y349 which results in a stabilizing disulfide bridge between the two antibody heavy chains in the Fe region (Carter, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole format is used to facilitate the expression of a first polypeptide (e.g., an IL12RB1 binding sdAb) on a first Fc monomer with a “knob” modification and a second polypeptide on the second Fc monomer possessing a “hole” modification to facilitate the expression of heterodimeric polypeptide conjugates. Targeting Domains In some embodiments, the IL12Rb1 binding molecule is provided as a component of a multivalent (e.g., bivalent) fusion protein with a polypeptide sequence (“targeting domain”) to facilitate selective binding to particular cell type or tissue expressing a cell surface molecule that specifically binds to such targeting domain, optionally incorporating a linker between the IL12RB1 binding sdAb sequence and the sequence of the targeting domain of the fusion protein. In some embodiments of the IL12Rb1 binding molecule is operably linked to a targeting domain As used herein, the term targeting domain refers to a moiety that specifically binds to a molecule expressed on the surface of a target cell. The targeting domain may be any moiety that specifically binds to one or more cell surface molecules (e.g., T cell receptor) expressed on the surface of a target cell. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a IL12Rb1+ T cell. In some embodiments, the targeting domain is a ligand for a receptor. In some embodiments, the targeting domain is a ligand for a receptor expressed on the surface of a T cell. In some embodiments, the ligand is a cytokine. In some embodiments, the cytokine includes but is not limited to the group consisting interleukins, interferons, and functional derivatives thereof. In some embodiments, the cytokine includes but is not limited to the group consisting IL2, IL3, IL4, IL7, IL9, IL12, IL15, IL18, IL21, IL22, IL23, IL27, IL28, IL34, and modified versions or fragments thereof that bind to their cognate ligand expressed on the surface of a T-cell. In some embodiments, the cytokine includes but is not limited to the group consisting of interferon alpha, interferon a2b, interferon gamma, or interferon lambda and modified versions or fragments thereof that bind to their cognate ligand expressed on the surface of a T-cell. In another aspect, the present disclosure provides a multivalent binding molecule, the multivalent binding molecule comprising: (a) a IL12RB1 binding molecule and (b) a second binding molecule that specifically binds to the extracellular domain of a second cell surface molecule, wherein the IL12RB1 binding molecule and second binding molecule are operably linked, optionally through a chemical or polypeptide linker. In some embodiments, the IL12RB1 binding molecules of the present disclosure are useful in the preparation of the multivalent binding molecules described in Gonzalez, et al. PCT/US2018/021301 published as WO 2018/182935 A1 on Oct. 4, 2018. In accordance with the teaching of Gonzalez, the second binding molecule specifically binds to the extracellular domain of: (i) a component of cytokine receptor other than a receptor of which IL12Rb1 forms a signaling complex in response to a natural ligand (e.g. IL12Rb2) that activates the JAK/STAT pathway in the cell; (ii) a receptor tyrosine kinase; or (iii) a TNFR superfamily member. In some embodiments, the second surface molecule is a tyrosine kinase selected from EGFR, ErbB2, ErbB3, ErbB4, InsR, IGF1R, InsRR, PDGFRα, PDGFRβ, CSF1R/Fms, cKit, Flt-3/Flk2, VEGFR1, VEGFR2, VEGFR3, FGFR1, FGFR2, FGFR3, FGFR4, PTK7/CCK4, TrkA, TrkB, TrkC, Ror1, Ror2, MuSK, Met, Ron, Axl, Mer, Tyro3, Tie1, Tie2, EphA1-8, EphA10, EphB1-4, EphB6, Ret, Ryk, DDR1, DDR2, Ros, LMR1, LMR2, LMR3, ALK, LTK, SuRTK106/STYK1. In some embodiments, the second surface molecule is a TNFR superfamily member is selected from TNFR1 (TNFRSF1A), TNFR2 (TNFRSF1B; TNFRSF2), 41-BB (TNFRSF9); AITR (TNFRSF18); BCMA (TNFRSF17), CD27 (TNFRSF7), CD30 (TNFRSF8), CD40 (TNFRSF5), Death Receptor 1 (TNFRSF10C), Death Receptor-3 (TNFRSF25), Death Receptor 4 (TNFRSF10A), Death Receptor 5 (TNFRSF10B), Death Receptor-6 (TNFRSF21), Decoy Receptor-3 (TNFRSF6B), Decoy Receptor 2 (TNFRSF10D), EDAR, Fas (TNFRSF6), HVEM (TNFRSF14), LTBR (TNFRSF3), OX40 (TNFRSF4), RANK (TNFRSF11A), TACI (TNFRSF13B), Troy (TNFRSF19), XEDAR (TNFRSF27), Osteoprotegerin (TNFRSF11B), TWEAK receptor (TNFRSF12A), BAFF Receptor (TNFRSF13C), NGF receptor (TNFRSF16). In some embodiments, the targeting domain of the IL12Rb1 binding molecule is an antibody (as defined hereinabove to include molecules such as VHHs, scFvs, etc.) Examples of antibodies that may incorporated as a targeting domain of a IL12Rb1 binding molecule include but are not limited to the group consisting of: anti-GD2 antibodies, anti-BCMA antibodies, anti-CD19 antibodies, anti-CD33 antibodies, anti-CD38 antibodies, anti-CD70 antibodies, anti-GD2 antibodies and IL3Ra2 antibodies, anti-CD19 antibodies, anti-mesothelin antibodies, anti-Her2 antibodies, anti-EpCam antibodies, anti-Muc1 antibodies, anti-ROR1 antibodies, anti-CD133 antibodies, anti-CEA antibodies, anti-PSMA antibodies, anti-EGRFRVIII antibodies, anti-PSCA antibodies, anti-GPC3 antibodies, anti-Pan-ErbB antibodies, and anti-FAP antibodies. The antibody or antigen-binding fragment thereof can also be linked to another antibody to form, e.g., a bispecific or a multispecific antibody Labels In some embodiments, the IL binding molecule is operably linked to label. In some embodiments, the label is incorporated to facilitate use as imaging agent, diagnostic agent, or for use in cell sorting procedures. The term labels includes but is not limited to fluorescent labels, a biologically active enzyme labels, a radioisotopes (e.g., a radioactive ions), a nuclear magnetic resonance active labels, a luminescent labels, or a magnetic compound. In one embodiment a IL binding sdAb (e.g., a IL binding VHH) molecule in stable association (e.g., covalent, coordinate covalent) with an imaging labels. The term imaging labels is used to describe any of a variety of compounds a signature that facilitates identification, tracing and/or localization of the IL12RB1 binding sdAb (or its metabolites) using diagnostic procedures. Examples of imaging labels include, but are not limited to, fluorescent compounds, radioactive compounds, and compounds opaque to imaging methods (e.g., X-ray, ultrasound). Examples of radioactive compounds useful as imaging label include but are not limited to Technetium-99m (99mTc), Indium-111 (111In), Iodine-131 (131I), Iodine-123 (123I), Iodine-125 (125I) Gallium-67 (67Ga), and Lutetium-177 (177Lu), phosphorus (32P), carbon (14C), tritium (3H), yttrium (90Y), actinium (225Ac), astatine (211At), rhenium (186Re), bismuth (212Bi or213Bi), and rhodium (188Rh). Therapeutic Agents In some embodiments, the IL12RB1 binding molecule is operably linked to a therapeutic agent. Examples of therapeutic agents include therapeutic small molecule (e.g., chemotherapeutic agents) or biologic therapeutic agents including antibodies, cytoxic or cytostatic compounds, a radioisotope, molecules of plant, fungal, or bacterial origin, or biological proteins (e.g., protein toxins) or particles (e.g., nano-particles or recombinant viral particles, e.g., via a viral coat protein), therapeutic antibodies, chemotherapeutic agents, as described more fully herein. In some embodiments, the therapeutic agent which may be incorporated into the IL12Rb1 binding molecules of the present disclosure is short-range radiation emitters, including, for example, short-range, high-energy a-emitters. Examples of such radioisotope include an alpha-emitter, a beta-emitter, a gamma-emitter or a beta/gamma emitter. Radioisotopes useful as therapeutic agents include yttrium 90 (90Y), lutetium-177 (177Lu), actinium-225 (225Ac), astatine-211 (211At), rhenium-186 (186Re), bismuth-212 (212Bi), bismuth-213 (213Bi), and rhodium-188 (188Rh). In some embodiments, the IL12Rb1 binding molecule is operably linked a cytotoxic agent (or derivative thereof), such maytansinol or the DM1 maytansinoid), a taxane, or a calicheamicin, pseudomonas exotoxin A, deBouganin, ricin toxin, diphtheria toxin, an amatoxin, such as a-amanitin, saporin, maytansine, a maytansinoid, an auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, and an indolinobenzodiazepine dimer, or a variant thereof). Synthesis of IL12Rb1 Binding Molecules: In some embodiments, the IL12Rb1 binding molecules of the present disclosure are polypeptides. However, in some embodiments, only a portion of the IL12Rb1 binding molecule is a polypeptide, for example where the IL12Rb1 binding molecule comprises a non-peptidyl domain (e.g., a PEG IL12RB1 binding sdAb conjugate, a radionucleotide IL12RB1 binding sdAb conjugate, or a small molecule IL12RB1 binding sdAb conjugate). The following provides guidance to enable the solid phase and recombinant synthesis of the polypeptide portions (domains) of IL12Rb1 binding molecules of the present disclosure. In those embodiments where only a portion of the IL12Rb1 binding molecule is a polypeptide, it will be understood that the peptidyl domain(s) of the IL12Rb1 binding molecule are an intermediate in the process which may undergo further processing to complete the synthesis of the desired IL12Rb1 binding molecules. The polypeptide domains of IL12Rb1 binding molecules may be produced by conventional methodology for the construction of polypeptides including recombinant or solid phase syntheses as described in more detail below. Chemical Synthesis In addition to generating mutant polypeptides via expression of nucleic acid molecules that have been altered by recombinant molecular biological techniques, polypeptide domains of IL12Rb1 binding molecules can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those of skill in the art. Chemical synthesis includes direct synthesis of a peptide by chemical means of the polypeptide domains of IL12Rb1 binding molecules exhibiting the properties described. This method can incorporate both natural and unnatural amino acids at desired positions that facilitate linkage of particular molecules (e.g., PEG). In some embodiments, the polypeptide domains of IL12Rb1 binding molecules of the present disclosure may be prepared by chemical synthesis. The chemical synthesis of the polypeptide domains of IL12Rb1 binding molecules may proceed via liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS) allows the incorporation of unnatural amino acids and/or peptide/protein backbone modification. Various forms of SPPS are available for synthesizing the polypeptide domains of IL12Rb1 binding molecules of the present disclosure are known in the art (e.g., Ganesan A. (2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. et al., (2005) Protein Pept Lett. 12:723-8). In the course of chemical synthesis, the alpha functions and any reactive side chains may protected with acid-labile or base-labile groups that are stable under the conditions for linking amide bonds but can readily be cleaved without impairing the peptide chain that has formed. In the solid phase synthesis, either the N-terminal or C-terminal amino acid may be coupled to a suitable support material. Suitable support materials are those which are inert towards the reagents and reaction conditions for the stepwise condensation and cleavage reactions of the synthesis process and which do not dissolve in the reaction media being used. Examples of commercially available support materials include styrene/divinylbenzene copolymers which have been modified with reactive groups and/or polyethylene glycol; chloromethylated styrene/divinylbenzene copolymers; hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers; and the like. The successive coupling of the protected amino acids can be carried out according to conventional methods in peptide synthesis, typically in an automated peptide synthesizer. At the end of the solid phase synthesis, the peptide is cleaved from the support material while simultaneously cleaving the side chain protecting groups. The peptide obtained can be purified by various chromatographic methods including but not limited to hydrophobic adsorption chromatography, ion exchange chromatography, distribution chromatography, high pressure liquid chromatography (HPLC) and reversed-phase HPLC. Recombinant Production Alternatively, polypeptide domains of IL12Rb1 binding molecules of the present disclosure may be produced by recombinant DNA technology. In the typical practice of recombinant production of polypeptides, a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression will be accomplish, the nucleic acid sequence being operably linked to one or more expression control sequences encoding by the vector and functional in the target host cell. The recombinant protein may be recovered through disruption of the host cell or from the cell medium if a secretion leader sequence (signal peptide) is incorporated into the polypeptide. The recombinant protein may be purified and concentrated for further use including incorporation. Synthesis of Nucleic Acid Sequences Encoding the IL12Rb1 Binding Molecule In some embodiments, the polypeptide domains of IL12Rb1 binding molecule is produced by recombinant methods using a nucleic acid sequence encoding the polypeptide domains of IL12Rb1 binding molecule (or fusion protein comprising the polypeptide domains of IL12Rb1 binding molecule). The nucleic acid sequence encoding the desired polypeptide domains of IL12Rb1 binding molecule can be synthesized by chemical means using an oligonucleotide synthesizer. The nucleic acid molecules are not limited to sequences that encode polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of the polypeptide domains of IL12Rb1 binding molecule) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription. The nucleic acid molecules encoding the polypeptide domains of IL12Rb1 binding molecule (and fusions thereof) may contain naturally occurring sequences or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (i.e., either a sense or an antisense strand). Nucleic acid sequences encoding the polypeptide domains of the IL12Rb1 binding molecule may be obtained from various commercial sources that provide custom synthesis of nucleic acid sequences. Amino acid sequence variants of the HUMAN IL12Rb1 binding molecules of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code which is well known in the art. Such variants represent insertions, substitutions, and/or specified deletions of, residues as noted. Any combination of insertion, substitution, and/or specified deletion can be made to arrive at the final construct, provided that the final construct possesses the desired biological activity as defined herein. Methods for constructing a DNA sequence encoding the polypeptide domains of IL12Rb1 binding molecule and expressing those sequences in a suitably transformed host include, but are not limited to, using a PCR-assisted mutagenesis technique. Mutations that consist of deletions or additions of amino acid residues to polypeptide domains of IL12Rb1 binding molecule can also be made with standard recombinant techniques. In the event of a deletion or addition, the nucleic acid molecule encoding polypeptide domains of IL12Rb1 binding molecule is optionally digested with an appropriate restriction endonuclease. The resulting fragment can either be expressed directly or manipulated further by, for example, ligating it to a second fragment. The ligation may be facilitated if the two ends of the nucleic acid molecules contain complementary nucleotides that overlap one another, but blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate various mutant sequences. A polypeptide domain of IL12Rb1 binding molecules of the present disclosure may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g., a signal sequence or other polypeptide having a specific cleavage site at the N-terminus or C-terminus of the mature IL12Rb1 binding molecule. In general, the signal sequence may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In some embodiments, the signal sequence is the signal sequence that is natively associated with the IL12Rb1 binding molecule (i.e. the human IL12Rb1 signal sequence). The inclusion of a signal sequence depends on whether it is desired to secrete the IL12Rb1 binding molecule from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be encoded and most preferably that the wild type IL-2 signal sequence be used. Alternatively, heterologous mammalian signal sequences may be suitable, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal. When the recombinant host cell is a yeast cell such asSaccharomyces cerevisiae, the alpha mating factor secretion signal sequence may be employed to achieve extracellular secretion of the IL12Rb1 binding molecule into the culture medium as described in Singh, U.S. Pat. No. 7,198,919 B1. In the event the polypeptide domain of IL12Rb1 binding molecules to be expressed is to be expressed as a chimera (e.g., a fusion protein comprising a IL12Rb1 binding molecule and a heterologous polypeptide sequence), the chimeric protein can be encoded by a hybrid nucleic acid molecule comprising a first sequence that encodes all or part of the polypeptide domains of IL12Rb1 binding molecule and a second sequence that encodes all or part of the heterologous polypeptide. For example, polypeptide domains of IL12Rb1 binding molecules described herein may be fused to a hexa-histidine tag (SEQ ID NO: 230) to facilitate purification of bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells. By first and second, it should not be understood as limiting to the orientation of the elements of the fusion protein and a heterologous polypeptide can be linked at either the N-terminus and/or C-terminus of the polypeptide domains of IL12Rb1 binding molecule. For example, the N-terminus may be linked to a targeting domain and the C-terminus linked to a hexa-histidine tag (SEQ ID NO: 230) purification handle. The complete amino acid sequence of the polypeptide domain of IL12Rb1 binding molecule (or fusion/chimera) to be expressed can be used to construct a back-translated gene. A DNA oligomer containing a nucleotide sequence coding for the polypeptide domain of IL12Rb1 binding molecules can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly. In some embodiments, the nucleic acid sequence encoding the polypeptide domain of the IL12Rb1 binding molecule may be “codon optimized” to facilitate expression in a particular host cell type. Techniques for codon optimization in a wide variety of expression systems, including mammalian, yeast and bacterial host cells, are well known in the and there are online tools to provide for a codon optimized sequences for expression in a variety of host cell types. See e.g., Hawash, et al., (2017) 9:46-53 and Mauro and Chappell inRecombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David Hacker (Human Press New York). Additionally, there are a variety of web based on-line software packages that are freely available to assist in the preparation of codon optimized nucleic acid sequences. Expression Vectors Once assembled (by synthesis, site-directed mutagenesis or another method), the nucleic acid sequence encoding polypeptide domains of IL12Rb1 binding molecule will be inserted into an expression vector. A variety of expression vectors for uses in various host cells are available and are typically selected based on the host cell for expression. An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Plasmids are examples of non-viral vectors. To facilitate efficient expression of the recombinant polypeptide, the nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory control sequences that are functional in the chosen expression host. Expression vectors typically contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media. Expression vectors for polypeptide domain of IL12Rb1 binding molecules of the present disclosure contain a regulatory sequence that is recognized by the host organism and is operably linked to nucleic acid sequence encoding the polypeptide domains of IL12Rb1 binding molecule. The terms “regulatory control sequence,” “regulatory sequence” or “expression control sequence” are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego CA USA Regulatory sequences include those that direct constitute expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. In selecting an expression control sequence, a variety of factors understood by one of skill in the art are to be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject IL12Rb1 binding molecule, particularly as regards potential secondary structures. In some embodiments, the regulatory sequence is a promoter, which is selected based on, for example, the cell type in which expression is sought. Promoters are untranslated sequences located upstream (5′) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known. A T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body. Skilled artisans are well aware of numerous promoters and other regulatory elements which can be used to direct expression of nucleic acids. Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5′ and 3′ to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5′ or 3′ to the coding sequence but is preferably located at a site 5′ from the promoter. Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above-listed components employs standard techniques. In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication, and other genes that encode a selectable marker. For example, the neomycin-resistance (neoR) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells. Additional examples of marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding beta-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context. Proper assembly of the expression vector can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. Host Cells The present disclosure further provides prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a polypeptide domains of IL12Rb1 binding molecule. A cell of the present disclosure is a transfected cell, i.e., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a polypeptide domains of IL12Rb1 binding molecule, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered within the scope of the present disclosure. Host cells are typically selected in accordance with their compatibility with the chosen expression vector, the toxicity of the product coded for by the DNA sequences of this IL12Rb1 binding molecule, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells. In some embodiments the recombinant polypeptide domains of IL12Rb1 binding molecule or biologically active variants thereof can also be made in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., 519 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in yeastS. cerevisiaeinclude pYepSecl (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Examples of useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC #CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. The polypeptide domains of IL12Rb1 binding molecule can be produced in a prokaryotic host, such as the bacteriumE. coli, or in a eukaryotic host, such as an insect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987). In some embodiments, the recombinant polypeptide domains of IL12Rb1 binding molecule may be glycosylated or unglycosylated depending on the host organism used to produce the IL12Rb1 binding molecule. If bacteria are chosen as the host then the polypeptide domains of IL12Rb1 binding molecule produced will be aglycosylated. Eukaryotic cells, on the other hand, will glycosylate the recombinant polypeptide domains of IL12Rb1 binding molecule. For other additional expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Transfection The expression constructs of the can be introduced into host cells to thereby produce the recombinant polypeptide domains of IL12Rb1 binding molecule disclosed herein or to produce biologically active muteins thereof. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals. In order to facilitate transfection of the target cells, the target cell may be exposed directly with the non-viral vector may under conditions that facilitate uptake of the non-viral vector. Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation). Cell Culture Cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Mammalian host cells may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan. Recovery of Recombinant Proteins Recombinantly-produced IL12Rb1 binding polypeptides can be recovered from the culture medium as a secreted polypeptide if a secretion leader sequence is employed. Alternatively, the IL12Rb1 binding polypeptides can also be recovered from host cell lysates. A protease inhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be employed during the recovery phase from cell lysates to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the growth of adventitious contaminants. Purification Various purification steps are known in the art and find use, e.g., affinity chromatography. Affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. Covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural specific binding of one molecular species to separate and purify a second species from a mixture. Antibodies are commonly used in affinity chromatography. Size selection steps may also be used, e.g., gel filtration chromatography (also known as size-exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their size. In gel filtration, a protein solution is passed through a column that is packed with semipermeable porous resin. The semipermeable resin has a range of pore sizes that determines the size of proteins that can be separated with the column. In some embodiments, the His-Tag modified protein is purified by immobilized metal affinity chromatography (IMAC), the His-Tag being optionally removed following purification. The recombinant polypeptide domains of IL12Rb1 binding molecule produced by the transformed host can be purified according to any suitable method. IL12Rb1 binding molecules can be isolated from inclusion bodies generated inE. coli, or from conditioned medium from either mammalian or yeast cultures producing a given IL12Rb1 binding molecule sing cation exchange, gel filtration, and or reverse phase liquid chromatography. The substantially purified forms of the recombinant polypeptides can be used, e.g., as therapeutic agents, as described herein. The biological activity of the recombinant polypeptide domains of IL12Rb1 binding molecule produced in accordance with the foregoing can be confirmed by a IL12Rb1 binding using procedures well known in the art including but not limited to competition ELISA, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs and competition binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET) and surface plasmon resonance assays (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009) with instrumentation commercially available from GE Healthcare Bio-Sciences such as the Biacore 8+, Biacore S200, Biacore T200 (GE Healthcare Bio-Sciences, 100 Results Way, Marlborough MA 01752)); liquid phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid phase ligand binding assays (e.g., multiwell plate assays, on-bead ligand binding assays, on-column ligand binding assays, and filter assays). Methods of Use Inhibition of IL12Rb1 Receptor Activity In one embodiment, the present disclosure provides a method of modulating the activity of cells expressing the IL12Rb1 by the administration of a IL12Rb1 binding molecule to a subject in an amount sufficient to interfere with the activity of receptors comprising the of IL12Rb1. The present disclosure further provides a method of modulating the activity of cells expressing the IL12Rb1 in a mixed population of cells comprising contacting said population of cells, in vivo and/or ex vivo, with a IL12Rb1 binding molecule or complex of the present disclosure to in an amount sufficient to interfere with the activity of receptors comprising the IL12Rb1. Identification Isolation, Enrichment or Depletion of IL12Rb1+ Cells In one embodiment, the present disclosure provides a method of use of the IL12Rb1 binding molecules of the present disclosure useful in a process for in the isolation, enrichment or depletion of IL12Rb1+ cells from a biological sample comprising IL12Rb1+ cells. The biological sample may comprise cells of blood origin such as PBMC, T cells, B cells of cell culture origin or of tissue origin such as brain or bone marrow. Processes suitable for the isolation, enrichment or depletion of IL12Rb1+ cells comprise centrifugation, filtration, magnetic cell sorting and fluorescent cell sorting by techniques well known in the art. The present disclosure further provides a method for the treatment of a subject suffering from a disease, disorder or condition by the administration of a therapeutically effective amount of a cell product enriched or depleted of IL12Rb1+ cells through the use of a IL12Rb1 binding molecule as described herein. In one embodiment, the sorting procedure employs a IL12Rb1 binding molecule comprising a fluorescent label for use in FACS isolation or depletion of IL12Rb1+ cells from a sample. The fluorescent label may be attached to the sdAb of the IL12Rb1 binding molecule directly (e.g., by chemical conjugation optionally employing a linker) or indirectly (e.g., by biotinylation of the sdAb and binding of the biotinylated antibody to a streptavidin fluorochrome conjugate). Such fluorescently labelled IL12Rb1+ cells may be separated from a mixed cell population using conventional FACS technology. In an alternative embodiment, the selection procedure employs IL12Rb1 binding molecules of the present disclosure (e.g., a IL12RB1 binding VHH) conjugated to magnetic particles which provide magnetic labeling of the IL12Rb1+ cells for use in magnetic cell separation procedures. In one embodiment the method comprises: (a) conjugation of one or more IL12Rb1 binding molecule of the present disclosure (e.g., a IL12RB1 binding VHH) to a magnetic particle; (b) creating a mixture by contacting the biological sample with a quantity of the magnetic particles conjugated to IL12Rb1 binding molecule; (c) subjecting to a magnetic field such that the magnetically labelled IL12Rb1+ cells are retained; (d) removing the non-magnetically labelled cells from the mixture; and (e) removal of the magnetic field enabling isolation of the IL12Rb1+ cells. The cell selection procedure (e.g., FACS or magnetic separation) results in two products: (a) a population of cells depleted of IL12Rb1+ cells and (b) a population of cells enriched for IL12Rb1+ cells. Each of these populations may be further processed by convention procedures to identify particular IL12Rb1+ or IL12Rb1− cell subsets which may be useful in research, diagnostic or clinical applications. For example, isolation of specific IL12Rb1+ T cell subsets that also express one or more of CD4, CD8, CD19, CD25, and CD62L, further iterations of the using one or more antibodies that specifically bind to CD4, CD8, CD19, CD25, and CD62L antigens respectively by FACS or magnetic field separation by techniques well known in the art. In one embodiment of the IL12Rb1 binding molecule a humanized antibody or fragment thereof as disclosed herein may be used for depletion of IL12Rb1-expressing cells from a biological sample comprising IL12Rb1-expressing cells such peripheral blood or lymphoid tissue which may optionally be further processed for further isolation of IL12Rb1+ naïve T cell subsets, isolation human IL12Rb1+ memory T cells from a population of CD4+ or CD8+ cells, or isolation of human IL12Rb1RA+naïve T cells from presorted CD4+ or CD8+ cells by depletion of IL12Rb1+ cells. In one embodiment, the IL12Rb1 binding molecule provides a method of generating a population of cells enriched for naïve Tregs from a biological sample, the method comprising depleting IL12Rb1+ cells using a IL12Rb1 binding molecule of the present disclosure as described above, optionally further comprising the steps of depleting CD8+ and/or CD19+ cells. The IL12Rb1+ depleted cell population may optionally be further expanded in vitro for particular cell types to in the preparation of a cell product comprising a therapeutically effective amount of the IL12Rb1+ depleted cell product which may be administered to a subject suffering from a disease, disorder or condition. The IL12Rb1+ enriched cell population may optionally be further expanded in vitro to in the preparation of a cell product comprising a therapeutically effective amount of the IL12Rb1+ cells. Kits The present disclosure also contemplates kits comprising pharmaceutical compositions of IL12Rb1 binding molecules. The kits are generally in the form of a physical structure housing various components, as described below, and can be utilized, for example, in practicing the methods described above. When the IL12Rb1 binding molecule is in a form that requires reconstitution by a user, the kit may also comprise a sterile container providing a reconstitution medium comprising buffers, pharmaceutically acceptable excipients, and the like. A kit of the present disclosure can be designed for conditions necessary to properly maintain the components housed therein (e.g., refrigeration or freezing). A kit may further contain a label or packaging insert including identifying information for the components therein and instructions for their use. Each component of the kit can be enclosed within an individual container, and all of the various containers can be within a single package. Labels or inserts can include manufacturer information such as lot numbers and expiration dates. The label or packaging insert can be, e.g., integrated into the physical structure housing the components, contained separately within the physical structure, or affixed to a component of the kit (e.g., an ampule, syringe or vial). Labels or inserts may be provided in a physical form or a computer readable medium. In some embodiments, the actual instructions are not present in the kit, but rather the kit provides a means for obtaining the instructions from a remote source, e.g., via an internet site, including by secure access by providing a password (or scannable code such as a barcode or QR code on the container of the IL12Rb1 binding molecule or kit comprising) in compliance with governmental regulations (e.g., HIPAA) are provided. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present IL12Rb1 binding molecule and are not intended to limit the scope of what the inventors regard as their IL12Rb1 binding molecule nor are they intended to represent that the experiments below were performed and are all of the experiments that can be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like described therein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Variations of the particularly described procedures employed may become apparent to individuals or skill in the art and it is expected that those skilled artisans may employ such variations as appropriate. Accordingly, it is intended that the IL12Rb1 binding molecule be practiced otherwise than as specifically described herein, and that the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius (° C.), and pressure is at or near atmospheric. Standard abbreviations are used, including the following: bp=base pair(s); kb=kilobase(s); pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s); aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); pg=picogram; ng=nanogram; μg=microgram; mg=milligram; g=gram; kg=kilogram; dl or dL=deciliter; μl or μL=microliter; ml or mL=milliliter; 1 or L=liter; μM=micromolar; mM=millimolar; M=molar; kDa=kilodalton; i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC or SQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; QM=monthly; HPLC=high performance liquid chromatography; BW=body weight; U=unit; ns=not statistically significant; PBS=phosphate-buffered saline; PCR=polymerase chain reaction; NHS=N-hydroxysuccinimide; HSA=human serum albumin; MSA=mouse serum albumin; DMEM=Dulbeco's Modification of Eagle's Medium; GC=genome copy; EDTA=ethylenediaminetetraacetic acid; PBMCs=primary peripheral blood mononuclear cells; FBS=fetal bovine serum; FCS=fetal calf serum; HEPES=4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; LPS=lipopolysaccharide; ATCC=American Type Culture Collection. Example 1. Immunization Protocol The process for isolation of the anti-hIL12Rb2 VHHs was initiated by immunization of a camel with a polypeptide corresponding to amino acids 24-622 of hIL12Rb2, (UNIPROT Reference No. Q99665). The process for isolation of the anti-m IL12Rb2 VHHs was the initiated by immunization of a camel with the with the 614 amino acid extracellular domain of the mIL12Rb2, amino acids 24-637 of the m IL12Rb2 precursor (UNIPROT Reference No. P97378). With respect to each antigen, the following methodology was used to identify and isolate the VHHs. The synthetic DNA sequence encoding the antigen was inserted into the pFUSE_hIgG1_Fc2 vector (Generay Biotechnology) and transfected into the HEK293F mammalian cell host cell for expression. The antigen is expressed as an Fc fusion protein which is purified using Protein A chromatography. The antigen was diluted with 1 xPBS (antigen total about 1 mg). The quality was estimated by SDS-PAGE to ensure the purity was sufficient (>80%) for immunization. The camel was acclimated at the facility for at least 7 days before immunization. The immunization with the antigen was conducted using once weekly administration of the antigen over a period of 7 weeks. For the initial immunization, the immunogen was prepared as follows: 10 mL of complete Freund's Adjuvant (CFA) was added into mortar, then 10 mL antigen in 1 xPBS was slowly added into the mortar with the pestle grinding and sample ground until the antigen was emulsified until milky white and hard to disperse. For the subsequent six immunizations (weeks 2-7) in the immunization protocol, immunogen was prepared as above except that Incomplete Freund's Adjuvant (IFA) was used in place of CFA. At least six sites on the camel were injected subcutaneously with approximately 2 ml of the emulsified antigen for a total of approximately 10 mL per camel. When injecting the antigen, the needle is maintained in the in the subcutaneous space for approximately 10 to 15 seconds after each injection to avoid leakage of the emulsion. Example 2. Phage Library Construction A blood sample was collected from the camel three days following the last injection in the immunization protocol. RNA was extracted from blood and transcribed to cDNA. The approximately 900 bp reverse transcribed sequences encoding the VH-CH1-hinge-CH2-CH3 constructs were isolated from the approximately desired 700 bp fragments encoding the VHH-hinge-CH2-CH3 species. The purified approximately 700 bp fragments were amplified by nested PCR. The amplified sequences were digested using Pst1 and Not1. The approximately 400 bp PST1/Not1 digested fragments were inserted into a Pst1/Not1 digested pMECS phagemid vector such that the sequence encoding the VHH was in frame with a DNA sequence encoding a HA/His sequence. The PCR generated sequences and the vector of pMECS phagemid were digested with PstI and NotI, subsequently, ligated to pMECS/Nb recombinant. After ligation, the products were transformed intoEscherichia coli(E. coli) TG1 cells by electroporation. The transformants were enriched in growth medium, followed by transfer to 2YT+2% glucose agar plates. Example 3: Isolation of Antigen Specific VHHs Bio-panning of the phage library was conducted to identify VHHs that bind IL12Rb1. A 96-well plate was coated with IL12Rb1 and the phage library was incubated in each well to allow phage-expressing IL12Rb1 reactive VHH to bind to the IL12Rb1 on the plate. Non-specifically bound phage were washed off and the specifically bound phage isolated. After the selection, the enriched phage library expressing IL12Rb1 reactive VHH were amplified in TG1 cells. The aforementioned bio-panning process was repeated for 2-3 rounds to enrich the library for VHH selective for IL12Rb1. Example 4: Identification of Antibodies Exhibiting Specific Binding to IL12Rb1 Upon completion of the biopanning of Example 3, three 96-well plates of individual phage clones were isolated in order to perform periplasmic extract ELISA (PE-ELISA) on IL12Rb1 coated plates to identify positive VHH binders that selectively bound IL12Rb1. A 96-well plate was coated with IL12Rb1 and PBS under the same conditions. Next, wells were blocked at 37° C. for 1 h. Then, 100 μl of extracted antibodies was added to each well and incubated for 1 h. Subsequently, 100 μl of anti-tag polyclonal antibody conjugated to HRP was added to each well and incubated at 37° C. for 1 h. Plates were developed with TMB substrate. The reaction was stopped by the addition of H2SO4. Absorbance at 450 nm was read on a microtiter plate reader. Antibodies with absorbance of the antigen-coated well at least threefold greater than PB S-coated control are VHHs that specifically bind to IL12Rb1. Positive clones were sequenced, and sequences analyzed to identify unique clonotypes. | 145,570 |
11859002 | DETAILED DESCRIPTION Antagonistic TNFR2 polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments inhibit the activation of TNFR2 on TNFR2-expressing cells by binding this receptor (e.g., on the exterior surface of a T-reg cell, a cancer cell that expresses TNFR2, or a myeloid-derived suppressor cell (MDSC) and thus prevent the protein from recruiting its cognate ligand, TNFα. TNFα potentiates TNFR2 signaling by nucleating a trimer of TNFR2 proteins. It is this trimerization event that brings individual TNFR2 proteins into close proximity and initiates signaling via the MAPK/NFκB/TRAF2/3 pathway, which ultimately leads to cell growth and escape from apoptosis. TNFR2 antibodies can antagonize this interaction by binding the receptor and preventing TNFα from triggering this structural change. For instance, one mechanism by which this may occur is through the formation of an anti-parallel TNFR2 dimer, which is an inactive structural form of the receptor. The invention is based in part on the discovery of epitopes within TNFR2 that promote receptor antagonism, as well as on the finding that the binding specificity of an antagonistic TNFR2 antibody or antigen-binding fragment thereof is dictated primarily by the CDR-H3 sequence of the antibody or fragment thereof. It has been discovered that binding of distinct residues within TNFR2 promote receptor antagonism, such as residues containing the KCRPG motif (SEQ ID NO: 19) within TNFR2, as well as downstream amino acids (for instance, the LRKCRPGFGVA (SEQ ID NO: 285) and VVCKPCAPGTFSN (SEQ ID NO: 286) epitopes). Additionally, it has been found that replacement of the CDR-H3 sequence of a neutral anti-TNFR2 antibody (i.e., an antibody that is neither antagonistic nor agonistic in function) with the CDR-H3 of an antagonistic TNFR2 antibody converts the phenotype-neutral antibody to an antagonistic TNFR2 antibody, such as a dominant antagonistic TNFR2 antibody. Collectively, these discoveries enable the production of TNFR2-binding polypeptides (e.g., single-chain polypeptides containing a CDR-H3 region optionally bound to one or more additional CDRs, antibodies, and antigen-binding fragments thereof), such as dominant antagonist polypeptides that bind TNFR2 and suppress receptor activation. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs described herein) may exhibit one or more, or all, of the following properties:a. Suppression of T-reg cell proliferation, for instance, by binding and inactivating TNFR2 on the T-reg cell surface;b. Suppression of MDSC proliferation, for instance, by binding and inactivating TNFR2 on the MDSC surface;c. Promotion of the expansion of T effector cells, such as CD8+ T cells; and/ord. Suppression of the proliferation of TNFR2-expressing cancer cells, such as T cell lymphoma cells (e.g., Hodgkin's or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, and renal cell carcinoma cells. In some embodiments, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs described herein) exert one or more, or all, of the above characteristics with a greater potency in the microenvironment of a tumor than in a site that is free of cancer cells. For instance, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs described herein) may exert one or more, or all, of properties (a), (b), and (c) preferentially in a patient (such as a mammalian patient, e.g., a human) suffering from cancer relative to a subject (such as a mammalian subject, e.g., a human) that does not have cancer. The sections that follow provide a description of exemplary characteristics of antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, antigen-binding fragments thereof, and constructs of the invention, and their use in therapeutic methods. Antagonistic TNFR2 Polypeptides Effects on TNFR2/MAPK/TRAF2/3 Signal Transduction Cascades Anti-TFNR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention are capable of interacting with and inhibiting the activity of TNFR2. Thus, the anti-TNFR2 polypeptides of the invention can selectively antagonize the TNFα-TNFR2 interaction rather than promote TNFR2 signaling. This is particularly important for therapeutic applications, such as cancer immunotherapy, as TNFR2 activation upon association with TNFα leads to propagation of the MAPK and TRAF2/3 signal cascade and activation of NFκB-mediated transcription of genes involved in T-reg cell growth and escape from apoptosis (Faustman, et al., Nat. Rev. Drug Disc., 9:482-493, 2010). The TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may bind TNFR2 with high affinity and may sterically sequester the receptor from TNFα rather than allow TNFα binding to TNFR2 initiate TNFR2 signaling, e.g., by binding TNFR2 in an anti-parallel dimer conformation in which TNFα binding sites are sterically inaccessible. This, in turn, prevents TNFα from nucleating an activated trimer of TNFR2, which triggers TNFR2 signal transduction. The antibodies of the invention can therefore be used to suppress T-reg cell growth and proliferation, thereby allowing, for example, the proliferation of T effector cells that can mount an immune response against, e.g., a cancer cell or foreign pathogen. Thus, antagonistic TNFR2 polypeptides described herein can be administered to a mammalian subject, such as a human patient with a cell proliferation disorder or an infectious disease, in order to enhance the effectiveness of an immune response (e.g., an immune response against cancer cells or pathogenic organisms) in the patient. Effects on T-Reg Cell Proliferation Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention can be used to attenuate the activity (e.g., proliferation) of T-reg cells that typically accompanies T cell-mediated cytotoxicity against self cells, such as the attack of a tumor cell by a T-lymphocyte. Antagonistic TNFR2 antibodies can be administered to a mammalian subject, such as a human (e.g., by any of a variety of routes of administration described herein) in order to prolong the duration of an adaptive immune response, such as a response against a cancer cell or a pathogenic organism. In this way, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention may synergize with existing techniques to enhance T-lymphocyte-based therapy for cancer and for infectious diseases. For instance, TNFR2 antagonists of the invention may be administered to suppress T-reg cell activity, thereby enhancing the cytotoxic effect of tumor reactive T cells. TNFR2 antagonists may also synergize with existing strategies to promote tumor-reactive T cell survival, such as lymphodepletion and growth factor therapy, and in turn prolong the duration of anti-tumor reactivity in vivo. Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof can also be used to treat a broad array of infectious diseases in a mammalian subject (e.g., a human), as inhibition of T-reg proliferation promotes the activity of CD8+ T-lymphocytes capable of mounting an attack on pathogenic organisms. Additionally, antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention can be used to treat a wide variety of infectious diseases, such asMycobacterium tuberculosis, in a human or an agricultural farm animal (e.g., a bovine mammal, pig, cow, horse, sheep, goat, cat, dog, rabbit, hamster, guinea pig, or other non-human mammal). Direct Effects on TNFR2+ Cancer Cells Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention may bind and inactivate TNFR2 on the surface of a cancer cell, such as a TNFR2+ tumor cell. For instance, antagonistic TNFR2 antibodies and antigen-binding fragments thereof described herein may bind TNFR2 on the surface a T cell lymphoma cell (e.g., a Hodgkin's or cutaneous non-Hodgkin's lymphoma cell), ovarian cancer cell, colon cancer cell, multiple myeloma cell, or renal cell carcinoma cell, among others. The ability of antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention to bind TNFR2 directly on a cancer cell provides another pathway by which these molecules may attenuate cancer cell survival and proliferation. For instance, an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention, such as an antibody or antigen-binding fragment thereof that contains the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, may bind TNFR2 directly on the surface of a cancer cell (e.g., a cutaneous T cell lymphoma cell, ovarian cancer cell, colon cancer cell, or multiple myeloma cell, such as an ovarian cancer cell) in order to suppress the ability of the cell to proliferate and/or to promote apoptosis of the cell. TNFR2 Antagonist Polypeptides are not Reliant on Additional TNFR2-Binding Agents for Activity Significantly, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention are capable of binding TNFR2 and suppressing TNFR2-mediated signalling without the need for an endogenous TNFR2-binding agent, such as TNFα. Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention do not require TNFα to attenuate T-reg and/or cancer cell proliferation. Without being limited by mechanism, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may exhibit this property due to the ability of these antibodies or antigen-binding fragments thereof to bind TNFR2 and stabilize the anti-parallel dimer conformation of this receptor. This structural configuration is not capable of potentiating NFκB signaling. By maintaining TNFR2 in an inactive structural state, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may prevent TNFR2 agonists from restoring cell growth. For instance, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may bind TNFR2 on the surface of a TNFR2+ cell, such as a T-reg cell, cancer cell, or myeloid-derived suppressor cell (MDSC) and inhibit the proliferation of such cells in the presence or absence of TNFα. For example, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may inhibit the proliferation of such cells by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cells that are not treated with the TNFR2 antagonist polypeptide. The antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-biding fragment thereof) may exhibit an IC50value in such a cell proliferation assay that is largely unchanged by the presence or absence of TNFα (e.g., an IC50value in the presence of TNFα that is changed by less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% relative to the IC50value of the antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) in the same cell proliferation assay in the absence of TNFα). Examples of cell death assays that can be used to measure the antagonistic effects of TNFR2 antibodies are described herein, e.g., in Example 9, below. Similarly, antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention may inhibit TNFR2 signaling as assessed by measuring the expression of one or more genes selected from the group consisting of CHUK, NFKBIE, NFKBIA, MAP3K11, TRAF2, TRAF3, relB, and cIAP2/BIRC3 by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to such cells that are not treated with the TNFR2 antagonist polypeptide. The antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-biding fragment thereof) may exhibit an IC50value in such a gene expression assay that is largely unchanged by the presence or absence of TNFα (e.g., an IC50value in the presence of TNFα that is changed by less than 50%, 45%, 40%, 35%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% relative to the IC50value of the antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) in the same gene expression assay in the absence of TNFα). Examples of gene expression assays that can be used to measure the antagonistic effects of TNFR2 antibodies are described herein, e.g., in Example 12, below. Direct Killing of T-Reg Cells, TNFR2+ Cancer Cells, and MDSCs Antagonistic TNFR2 polypeptides disclosed herein, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof, can not only reduce T-reg cell, TNFR2+ cancer cell, and/or MDSC proliferation, but can also induce the death of T-reg cells, TNFR2+ cancer cells, and/or MDSCs within a sample (e.g., within a patient, such as a human patient). Antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may be capable of reducing the total quantity of T-reg cells, cancer cells (such as cutaneous T cell lymphoma cells, ovarian cancer cells, colon cancer cells, renal cell carcinoma cells or multiple myeloma cells, among others), and/or MDSCs in a sample treated with an antagonist TNFR2 antibody or antigen-binding fragment thereof (such as a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein) by, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, relative to a sample not treated with an antagonist TNFR2 antibody or antigen-binding fragment thereof. The ability of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention to attenuate T-reg, MDSC, and/or cancer cell growth may be due in part to the ability of these antibodies or antigen-binding fragments to diminish the quantity of soluble TNFR2 within a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein). In the absence of this beneficial activity, soluble TNFR2 can be secreted by, e.g., T-reg cells, and could otherwise interfere with the ability of TNFR2 antagonists to localize to TNFR2 at the surface of a T-reg cell, TNFR2+ cancer cell, or MDSC by binding and sequestering such antagonists in the extracellular environment. By reducing TNFR2 secretion, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may render T-reg cells, TNFR2+ cancer cells, and/or MDSCs increasingly susceptible to therapeutic molecules, such as an antagonistic TNFR2 antibody or antigen-binding fragment thereof, and/or additional anti-cancer agents described herein or known in the art that may be used in conjunction with the compositions and methods of the invention. Selective Modulation of Active (CD25Hiand CD45RALow) T-Reg Cells Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may be capable of inhibiting the proliferation or reducing the total quantity of T-reg cells in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein) and may act selectively on T-reg cells in an actively-dividing state. Antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may selectively target active T-reg cells that express CD25Hi and CD45RALow, e.g., over resting T-reg cells that express CD25Medand CD45RAHi. For instance, antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may be capable of reducing the proliferation of T-reg cells expressing CD25Hiand CD45RALowby, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more relative to T-reg cells that do not express the CD25Hiand CD45RALowproteins, such as T-reg cells that express CD25Medand CD45RAHiproteins. Modulation of T-Reg Cells, MDSCs, and T Effector Cells in the Tumor Microenvironment Antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of T-reg cells with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. The antagonist TNFR2 polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of T-reg cells with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. This effect may be determined using, for example, a cell death assay as described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may exhibit an IC50for reducing or inhibiting the proliferation of T-reg cells in the microenvironment of a tumor that is less than the IC50of the polypeptides for reducing or inhibiting the proliferation of T-reg cells in a site that is free of cancer cells by, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more. Examples of cell death assays that can be used to measure the antagonistic effects of anti-TNFR2 polypeptides are described herein, e.g., in Example 9, below. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of T-reg cells with a potency that is greater in the microenvironment of a tumor containing T cell lymphoma cells (e.g., Hodgkin's or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells than in a site that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or a in a subject without cancer. Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of MDSCs with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of MDSCs with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or in a subject without cancer. This effect may be determined using, for example, a cell death assay described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may have an IC50for reducing or inhibiting the proliferation of MDSCs in the microenvironment of a tumor that is less than the IC50of the polypeptides for reducing or inhibiting the proliferation of MDSCs in a site that is free of cancer cells by, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more. Examples of cell death assays that can be used to measure the antagonistic effects of anti-TNFR2 polypeptides are described herein, e.g., in Example 9, below. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may reduce or inhibit the proliferation of MDSCs or may promote the apoptosis of MDSCs with a potency that is greater in the microenvironment of a tumor containing Hodgkin's lymphoma cells, cutaneous non-Hodgkin's lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells than in a site that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in a subject without cancer. Additionally or alternatively, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may expand T effector cells, such as CD8+ cytotoxic T cells, with a greater potency in a patient suffering from cancer relative to a subject that does not have cancer. In some embodiments, the polypeptides of the invention, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, expand T effector cells, such as CD8+ cytotoxic T cells, with a greater potency in the microenvironment of a tumor relative to a site that is free of cancer cells, such as a site distal from a tumor in a patient suffering from cancer or a in a subject without cancer. This effect may be determined using, for example, a cell proliferation assay described herein. For instance, the polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may have an EC50for the expansion of T effector cells in the microenvironment of a tumor that is less than the EC50of the polypeptides for expanding T effector cells in a site that is free of cancer cells by, for example, 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more. Examples of cell proliferation assays that can be used to measure the effects of anti-TNFR2 polypeptides on T effector cells are described herein, e.g., in Example 18, below. The polypeptides described herein, such as single-chain polypeptides, antibodies, and antigen-binding fragments thereof, may directly expand T effector cells, such as CD8+ cytotoxic T cells, with a potency that is greater in the microenvironment of a tumor containing\T cell lymphoma cells (e.g., Hodgkin's or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells than in a site that is free of such cancer cells, such as a site distal from a tumor in a patient suffering from one or more of the foregoing cancers or in a subject without cancer. The T effector cells (e.g., CD8+ cytotoxic T cells) may, for example, specifically react with an antigen present on one or more cancer cells, such as Hodgkin's lymphoma cells, cutaneous non-Hodgkin's lymphoma cells, T cell lymphoma cells, ovarian cancer cells, colon cancer cells, multiple myeloma cells, or renal cell carcinoma cells, among cells of other cancers described herein. Activity of Antigen-Binding Fragments of Full-Length TNFR2 Antagonist Antibodies Antagonistic TNFR2 antibodies of the invention may inhibit, e.g., T-reg, cancer cell, and/or MDSC growth, or promote T effector cell growth, with a similar potency as that exhibited by antigen-binding fragments of such antibodies. For instance, removal of the Fc region of an antagonistic TNFR2 antibody of the invention may not alter the ability of the molecule to attenuate the proliferation or reduce the total quantity of T-reg cells, MDSCs, and/or cancer cells in a sample (e.g., a sample isolated from a human patient undergoing treatment for cancer or an infectious disease as described herein). Antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention may function by a pathway distinct from antibody-dependent cellular cytotoxicity (ADCC), in which a Fc region is required to recruit effector proteins in order to induce cell death. Additionally, antagonistic TNFR2 antibodies or antigen-binding fragments thereof may not be susceptible to a loss of inhibitory capacity in the presence of cross-linking agents. Antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may therefore exhibit therapeutic activity in a variety of isotypes, such as IgG, IgA, IgM, IgD, or IgE, or in a variety of forms, such as a single-chain polypeptide (e.g., a single-chain polypeptide one or more CDRs covalently bound to one another, for instance, by an amide bond, a thioether bond, a carbon-carbon bond, or a disulfide bridge), a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a primatized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a multi-specific antibody or antigen-binding fragment thereof, a dual-variable immunoglobulin domain, a monovalent antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a domain antibody, a Fv fragment, a Fab fragment, a F(ab′)2molecule, and a tandem scFv (taFv). Specific Binding Properties of Antagonistic TNFR2 Polypeptides The specific binding of a polypeptide, such as a single-chain polypeptide, antibody, or antibody fragment of the invention, to human TNFR2 can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody needed to achieve half-maximal inhibition of the TNFα-TNFR2 interaction in vitro (IC50) and the equilibrium constant (KD) of the antibody-TNFR2 complex dissociation. The equilibrium constant, KD, that describes the interaction of TNFR2 with an antibody of the invention is the chemical equilibrium constant for the dissociation reaction of a TNFR2-antibody complex into solvent-separated TNFR2 and antibody molecules that do not interact with one another. Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention include those that specifically bind to TNFR2 with a KDvalue of less than 100 nM (e.g., 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In some embodiments, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are those that specifically bind to TNFR2 with a KDvalue of less than 1 nM (e.g., (e.g., 990 μM, 980 μM, 970 μM, 960 μM, 950 μM, 940 μM, 930 μM, 920 μM, 910 μM, 900 μM, 890 μM, 880 μM, 870 μM, 860 μM, 850 μM, 840 μM, 830 μM, 820 μM, 810 μM, 800 μM, 790 μM, 780 μM, 770 μM, 760 μM, 750 μM, 740 μM, 730 μM, 720 μM, 710 μM, 700 μM, 690 μM, 680 μM, 670 μM, 660 μM, 650 μM, 640 μM, 630 μM, 620 μM, 610 μM, 600 μM, 590 μM, 580 μM, 570 μM, 560 μM, 550 μM, 540 μM, 530 μM, 520 μM, 510 μM, 500 μM, 490 μM, 480 μM, 470 μM, 460 μM, 450 μM, 440 μM, 430 μM, 420 μM, 410 μM, 400 μM, 390 μM, 380 μM, 370 μM, 360 μM, 350 μM, 340 μM, 330 μM, 320 μM, 310 μM, 300 μM, 290 μM, 280 μM, 270 μM, 260 μM, 250 μM, 240 μM, 230 μM, 220 μM, 210 μM, 200 μM, 190 μM, 180 μM, 170 μM, 160 μM, 150 μM, 140 μM, 130 μM, 120 μM, 110 μM, 100 μM, 90 μM, 80 μM, 70 μM, 60 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, or 1 μM). Polypeptides of the invention can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the KDor IC50of an anti-TNFR2 polypeptide include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays, among others. ELISA represents a particularly useful method for analyzing antibody activity, as such assays typically require minimal concentrations of antibodies. A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a TNFR2 antibody of the invention). Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are capable of binding TNFR2 and epitopes derived thereof, such as epitopes containing one or more of residues 142-146 of SEQ ID NO: 7 within human TNFR2 (KCRPG, as shown inFIGS.2A and2B), as well as isolated peptides derived from TNFR2 that structurally pre-organize various residues in a manner that may simulate the conformation of these amino acids in the native protein. For instance, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may bind peptides containing the amino acid sequence of any one of SEQ ID NOs: 11, 19, 20, 34-117, 285, or 286, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2′-azino-di-3-ethylbenzthiazoline sulfonate) with an antigen-antibody complex bound to a HRP-conjugated secondary antibody. For instance, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may induce a luminescence response of about 400 absorbance units or more when incubated with surface-immobilized antigen and a HRP-conjugated secondary antibody in the presence of an HRP substrate (see, e.g., Example 3). In some embodiments, the luminescence observed can be from about 400 to about 900 absorbance units (e.g., 400-900 absorbance units, 500-800 absorbance units, or 600-700 absorbance units). In some embodiments, the luminescence observed can be from about 600 to about 900 absorbance units (e.g., 600-900 absorbance units or 700-800 absorbance units). Kinetic Properties of Antagonistic TNFR2 Polypeptides In addition to the thermodynamic parameters of a TNFR2-polypeptide interaction, it is also possible to quantitatively characterize the kinetic association and dissociation of a polypeptide of the invention with TNFR2. This can be done, e.g., by monitoring the rate of antibody-antigen complex formation according to established procedures. For example, one can use surface plasmon resonance (SPR) to determine the rate constants for the formation (kon) and dissociation (koff) of an antibody-TNFR2 complex. These data also enable calculation of the equilibrium constant of (KD) of antibody-TNFR2 complex dissociation, since the equilibrium constant of this unimolecular dissociation can be expressed as the ratio of the koffto konvalues. SPR is a technique that is particularly advantageous for determining kinetic and thermodynamic parameters of receptor-antibody interactions since the experiment does not require that one component be modified by attachment of a chemical label. Rather, the receptor is typically immobilized on a solid metallic surface which is treated in pulses with solutions of increasing concentrations of antibody. Antibody-receptor binding induces distortion in the angle of reflection of incident light at the metallic surface, and this change in refractive index over time as antibody is introduced to the system can be fit to established regression models in order to calculate the association and dissociation rate constants of an antibody-receptor interaction. Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may exhibit high konand low konvalues upon interaction with TNFR2, consistent with high-affinity receptor binding. For example, antibodies of the invention may exhibit konvalues in the presence of TNFR2 of greater than 104M−1s−1(e.g., 1.0×104M−1s−1, 1.5×104M−1s−1, 2.0×104M−1s−1, 2.5×104M−1s−1, 3.0×104M−1s−1, 3.5×104M−1s−1, 4.0×104M−1s−1, 4.5×104M−1s−1, 5.0×104M−1s−1, 5.5×104M−1s−1, 6.0×104M−1s−1, 6.5×104M−1s−1, 7.0×104M−1s−1, 7.5×104M−1s−1, 8.0×104M−1s−1, 8.5×104M−1s−1, 9.0×104M−1s−1, 9.5×104M−1s−1, 1.0×105M−1s−1, 1.5×105M−1s−1, 2.0×105M−1s−1, 2.5×105M−1s−1, 3.0×105M−1s−1, 3.5×105M−1s−1, 4.0×105M−1s−1, 4.5×105M−1s−1, 5.0×105M−1s−1, 5.5×105M−1s−1, 6.0×105M−1s−1, 6.5×105M−1s−1, 7.0×105M−1s−1, 7.5×105M−1s−1, 8.0×105M−1s−1, 8.5×105M−1s−1, 9.0×105M−1s−1, 9.5×105M−1s−1, or 1.0×106M−1s−1). Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may exhibit low koffvalues when bound to TNFR2, as these polypeptides are capable of interacting with distinct TNFR2 epitopes with a high affinity. Residues within these epitopes form strong intermolecular contacts with TFNR2, which serves to slow the dissociation of the antibody-TNFR2 complex. This high receptor affinity is manifested in low koffvalues. For instance, antibodies of the invention may exhibit koffvalues of less than 10−3s−1when complexed to TNFR2 (e.g., 1.0×10−3s−1, 9.5×10−4s−1, 9.0×10−4s−1, 8.5×10−4s−1, 8.0×10−4s−1, 7.5×10−4s−1, 7.0×10−4s−1, 6.5×10−4s−1, 6.0×10−4s−1, 5.5×10−4s−1, 5.0×10−4s−1, 4.5×10−4s−1, 4.0×10−4s−1, 3.5×10−4s−1, 3.0×10−4s−1, 2.5×10−4s−1, 2.0×10−4s−1, 1.5×10−4s−1, 1.0×10−4s−1, 9.5×10−5s−1, 9.0×10−5s−1, 8.5×10−5s−1, 8.0×10−5s−1, 7.5×10−5s−1, 7.0×10−5s−1, 6.5×10−5s−1, 6.0×10−5s−1, 5.5×10−5s−1, 5.0×10−5s−1, 4.5×10−5s−1, 4.0×10−5s−1, 3.5×10−5s−1, 3.0×10−5s−1, 2.5×10−5s−1, 2.0×10−5s−1, 1.5×10−5s−1, or 1.0×10−5s−1). Epitopes within TNFR2 Bound by Antagonistic TNFR2 Polypeptides Among the difficulties in developing anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that are capable of antagonizing TNFR2 has been the elucidation of epitopes within TNFR2 that participate in antagonistic complex formation rather than epitopes that promote signal transduction. The present invention is based in part on the discovery of epitopes within TNFR2 that, when bound, promote receptor antagonism. Particularly important epitopes that bind antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention and promote receptor antagonism are those that contain one or more residues of the KCRPG motif (SEQ ID NO: 19), located at positions 142-146 of SEQ ID NO: 7 within human TNFR2. One or more of these residues reside within larger epitopes (e.g., residues 142-149 of SEQ ID NO: 7, shown inFIG.2A, and residues 137-144 of SEQ ID NO: 7, shown inFIG.2B) that may interact with antagonistic TNFR2 antibodies of the invention. The knowledge of those residues that selectively bind antagonistic TNFR2 antibodies can be used to identify and design a wide array of antagonistic TNFR2 antibodies and antigen-binding fragments thereof using library screening techniques, e.g., those described herein or known in the art. For instance, peptides containing one or more the residues within the KCRPG sequence (e.g., the LRKCRPGFGVA motif (SEQ ID NO: 285) within human TNFR2) can be used to screen and select for antibodies and antibody-like scaffolds that bind these epitopes with high affinity and selectivity. Importantly, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are capable of selectively binding an epitope of TNFR2 that contains one or more of the residues of the KCRPG motif (SEQ ID NO: 19) and distinctly do not exhibit specific binding to an epitope containing residues 56-60 of SEQ ID NO: 7 within human TNFR2 (KCSPG, SEQ ID NO: 12). Polypeptides that exhibit the ability to bind an epitope containing one or more residues of amino acids 142-146 of SEQ ID NO: 7 within human TNFR2 and an epitope containing residues 56-60 of SEQ ID NO: 7 within human TNFR2 have been shown to lack inhibitory (antagonistic) activity. As such, the ability of a TNFR2 polypeptide to discriminate among these epitopes and specifically interact with an epitope including one or more of residues 142-146 of SEQ ID NO: 7 within human TNFR2 and to not engage in specific binding with an epitope composed of residues 56-60 of SEQ ID NO: 7 within human TNFR2 characterizes antibodies of the invention that antagonize TNFR2 signaling. In addition to one or more residues of amino acids 142-146 of SEQ ID NO: 7, antagonistic TNFR2 polypeptides, such a dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may also bind one or more residues of a larger epitope that includes at least five continuous or discontinuous residues from positions 130-149 of SEQ ID NO: 7 within human TNFR2 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO: 17), or an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. For example, antagonistic TNFR2 antibodies of the invention may specifically bind an epitope containing residues 142-149 of SEQ ID NO: 7 within human TNFR2 (KCRPGFGV, SEQ ID NO: 20), or an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence (so long as one or more residues of the KCRPG sequence is present in the epitope). Additionally or alternatively, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may specifically bind an epitope including residues 137-144 of SEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11), or an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence (so long as one or more residues of the KCRPG sequence is present in the epitope). In addition to the KCRPG motif (SEQ ID NO: 19), it has been discovered that another important epitope present within human TNFR2 that promotes receptor antagonism contains residues 159-171 of SEQ ID NO: 7 (VVCKPCAPGTFSN, SEQ ID NO: 286). Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may therefore bind an epitope downstream of the KCRPG sequence (SEQ ID NO: 19) that contains, for instance, at least five continuous or discontinuous residues from positions 150-190 of SEQ ID NO: 7 within human TNFR2 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO: 22), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. For example, in some embodiments, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof of the invention) may specifically bind an epitope that includes residues from positions 161-169 of SEQ ID NO: 7 within human TNFR2 (CKPCAPGTF, SEQ ID NO: 21), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may specifically bind an epitope that includes residues from positions 140-150 of SEQ ID NO: 7 within human TNFR2 (LRKCRPGFGVA, SEQ ID NO: 285), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Additionally or alternatively, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may specifically bind an epitope that includes residues from positions 159-171 of SEQ ID NO: 7 within human TNFR2 (VVCKPCAPGTFSN, SEQ ID NO: 286), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In addition to the above-described epitopes, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may also specifically bind an epitope within human TNFR2 that includes at least five continuous or discontinuous residues from positions 75-128 of SEQ ID NO: 7 within human TNFR2 (CDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCAL, SEQ ID NO: 13), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Anti-TNFR2 polypeptides of the invention may also specifically bind an epitope within human TNFR2 that includes at least five continuous or discontinuous residues from positions 75-91 of SEQ ID NO: 7 within human TNFR2 (CDSCEDSTYTQLWNWVP, SEQ ID NO: 14), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In some embodiments, anti-TNFR2 polypeptides of the invention may specifically bind an epitope that includes residues at positions 80-86 of SEQ ID NO: 7 within human TNFR2 (DSTYTQL, SEQ ID NO: 8), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. Antagonistic TNFR2 polypeptides of the invention also may specifically bind an epitope that includes at least five continuous or discontinuous residues from positions 86-103 of SEQ ID NO: 7 within human TNFR2 (LWNWVPECLSCGSRCSSD, SEQ ID NO: 15), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In particular cases, antibodies of the invention may specifically bind an epitope that includes residues from positions 91-98 of SEQ ID NO: 7 within human TNFR2 (PECLSCGS, SEQ ID NO: 9), as well as an epitope that exhibits at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. The polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may also specifically bind an epitope that include at least five continuous or discontinuous residues from positions 111-128 of SEQ ID NO: 7 within human TNFR2 (TREQNRICTCRPGWYCAL, SEQ ID NO: 16), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. In some embodiments, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may specifically bind an epitope that includes residues from positions 116-123 of SEQ ID NO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10), as well as epitopes that exhibit at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to this sequence and epitopes that contain conservative amino acid substitutions relative to this sequence. For example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may specifically bind an epitope containing residues 116-123 of SEQ ID NO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10) and residues 137-144 of SEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11). One exemplary procedure that can be used to predict the inhibitory activity of a TNFR2 polypeptide of the invention is to compare the affinity of the antibody or antibody fragment for a peptide containing the KCRPG motif (e.g., a linear peptide having the sequence LRKCRPGFGVA (SEQ ID NO: 285) or VVCKPCAPGTFSN (SEQ ID NO: 286) to the affinity of the same antibody or antibody fragment for a peptide containing the KCSPG sequence (e.g., a linear peptide having the sequence QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). For instance, antagonistic TNFR2 antibody TNFRAB1 specifically binds the peptide fragment defined by residues 130-149 of SEQ ID NO: 7 within human TNFR2 (KQEGCRLCAPLRKCRPGFGV, SEQ ID NO: 17) with a 40-fold greater affinity than the peptide fragment defined by residues 48-67 of SEQ ID NO: 7 within human TNFR2 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). Antagonistic TNFR2 antibodies and antigen-binding fragments of the invention bind an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 19), e.g., with an affinity that is at least 10-fold greater than the affinity of the same antibody or antigen-binding fragment for a peptide that contains the KCSPG sequence of human TNFR2 (SEQ ID NO: 12). For example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may bind an epitope containing one or more residues of the KCRPG sequence (SEQ ID NO: 19) of human TNFR2 with an affinity that is 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, or more than 1000-fold greater than the affinity of the same antibody or antigen-binding fragment for a peptide that contains the KCSPG sequence (SEQ ID NO: 12) of human TNFR2. Antibodies or antibody fragments that bind epitopes containing one or more residues of the KCRPG sequence (amino acids 142-146 of SEQ ID NO: 7 within human TNFR2) and epitopes containing the KCSPG motif (amino acids 56-60 of SEQ ID NO: 7 within human TNFR2) with similar affinity (e.g., less than a 10-fold difference in affinity) are not considered antagonistic TNFR2 antibodies of the invention. Antagonistic TNFR2 Polypeptides that Bind TNFR2 from Non-Human Animals In addition to binding epitopes within human TFNR2 that contain the KCRPG motif (SEQ ID NO: 19), antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention also include those that specifically bind epitopes containing the equivalent motif within TNFR2 derived from non-human animals, such as the KCGPG motif (SEQ ID NO: 287) within TNFR2 of non-human mammals, e.g., in TNFR2 obtained from a cow, bison, mouse, or rat, among others. The location of sequences equivalent to the human KCRPG motif (SEQ ID NO: 19) in TNFR2 derived from exemplary non-human mammals is shown in Table 2, below: TABLE 2Location of sequences equivalent to KCRPG in TNFR2from non-human mammalsAminoSEQGenbankacid positionsID NO. ofAccession No.Sequenceof equivalentfull-lengthof full-lengthSource ofequivalentsequenceTNFR2TNFR2TNFR2to KCRPGwithin TNFR2sequencesequenceHumanKCRPG142-1467P20333.3CattleKCGPG142-146280AAI05223BisonKCGPG142-146281XP_010848145MouseKCGPG144-148282AAA39752.1RatKCGPG144-148283Q80WY6 Epitopes within TNFR2 derived from the non-human mammals discussed above that may be bound by antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention are illustrated in the sequence alignment below. This sequence alignment shows partial sequences of TNFR2 derived from human, cattle, bison, mouse, and rat, as well as epitopes (highlighted in grey) equivalent to the human KCRPG motif (SEQ ID NO: 19). Alignment of partial TNFR2 sequences derived from human and select non-human mammalsHuman:1MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRL--REYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSC78(SEQ ID NO: 289)Cattle:1MAPTAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ--QEYYNQKIQMCCSKCPPGYRVQSLCNMTLDTICASC78(SEQ ID NO: 290)Bison:1MAPTAFWAALAVGLQFWAAGRAVPAQAVFTPYIPEPGSSCRQ--QEYYNHKIQMCCSKCPPGYRVQSLCNTTLDTICASC78(SEQ ID NO: 291)Mouse:1MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQIS-QEYYDRKAQMCCAKCPPGQYVKHFCNKTSDTVCADC79(SEQ ID NO: 292)Rat:1MAPAALWVALVVELQLWATGHTVPAKVVLTPYKPEPGNQCQIS-QEYYDKKAQMCCAKCPPGQYAKHFCNKTSDTVCADC79(SEQ ID NO: 293)Human: Cattle: Bison: Mouse: Rat:79 79 79 80 80157 157 157 159 159(SEQ ID NO: 294) (SEQ ID NO: 295) (SEQ ID NO: 296) (SEQ ID NO: 297) (SEQ ID NO: 298)Human:158DVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCT------STSPTRSMAPGAVHLPQPVSTRSQHTQPTP231(SEQ ID NO: 299)Cattle:158NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTASTDAVCT------SVLPTRKVARG------PATTRSQHMEPTL225(SEQ ID NO: 300)Bison:158NVICAPCGPGTFSDTTSYTDTCKPHRNCSSVAIPGTASTDAVCT------SVLPTRKVARG------PATTRSQHMEPTL225(SEQ ID NO: 301)Mouse:160NVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCAPESPTLSAIPR------TLYVSQPEPTRSQPLDQEP233(SEQ ID NO: 302)Rat:160NVICSACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDAVCASESPTPSAVPR------TIYVSQPEPTRSQPMDQEP233(SEQ ID NO: 303)Human:232EPSTAPSTSFLLPMGPSPPA----EGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADK307(SEQ ID NO: 304)Cattle:226GPSTAPSTFFLLPKVPSPPSSPVEQPNTGNISLPIELIVGVTALGLLLIVVVNCVIMTQKKKKPFCLQGDAKVPHLPANK305(SEQ ID NO: 305)Bison:226GPSTAPSTFFLLPKVPSPPSSPVEQPNAGNISLPIELIVGVTALGLLLIVVVNCVIMTQKKKKPFCLQGDAKVPHLPANK305(SEQ ID NO: 306)Mouse:234GPSQTPS---ILTSLGSTPI--IEQSTKGGISLPIGLIVGVTSLGLLMLGLVNCIILVQRKKKPSCLQRDAKVPHVPDEK308(SEQ ID NO: 307)Rat:234GPSQTPH---IPVSLGSTPI--IEPSITGGISLPIGLIVGLTTLGLLMLGLANCFILVQRKKKPSCLQRETMVPHLPDDK308(SEQ ID NO: 308)Human:308ARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVE-ASGAGEARASTGSSDSSPGGHGTQVNVTCIVN386(SEQ ID NO: 309)Cattle:306AQGAPGPEQQHLLTTAPSSSSSSLESSTSSTDKRAPTRSQLQSPGVEKASTSGEAQTGCSSSEASSGGHGTQVNVTCIVN385(SEQ ID NO: 310)Bison:306AQGAPGPEQQHLLTTAPSSSSSSLESSTSSTDKRAPTRSQLQSPGVE-ANTSGEAQTGCSSSEASSGGHGTQVNVTCIVN384(SEQ ID NO: 311)Mouse:309SQDAVGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVMAEAQGFQEARASSRISDSSHGSHGTHVNVTCIVN388(SEQ ID NO: 312)Rat:309SQDAIGLEQQHLLTTAPSSSSSSLESSASAGDRRAPPGGHPQARVTAEAQGSQEACAGSRSSDSSHGSHGTHVNVTCIVN388(SEQ ID NO: 313) The Antagonistic TNFR2 Antibody TNFRAB1 Antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention may contain the CDR-H3 sequence of TNFRAB1, also referred to herein as TNFR2 antagonist 1, which is a murine antibody that antagonizes the TNFRα-TNFR2 interaction. For instance, the CDR-H3 of TNFRAB1 and variants thereof (e.g., variants that exhibit conservative amino acid substitutions relative to this CDR-H3 sequence) can be used to make an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention, for instance, using antibody humanization methods described herein or known in the art. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may exhibit binding properties that are the same as or similar to those of TNFRAB1. These properties are as follows: In the presence of TNFR2, TNFRAB1 exhibits a high konvalue of 4.98×106M−1s−1, as well as a low koffof 2.21×10−4s−1and a KDof about 44.4 pM in complex with TNFR2. The KCRPGFGV motif (SEQ ID NO: 20), and specifically, the KCRPG sequence (SEQ ID NO: 19), has been identified as a particularly important component of the functional epitope that establishes intermolecular contacts with TNFRAB1 as determined by epitope mapping analysis (FIGS.2and3). The interaction of these residues with anti-TNFR2 antibodies of the invention selectively promotes antagonistic activity. Significantly, a TNFR2 epitope including amino acid residues 56-60 of SEQ ID NO: 7 within human TNFR2 (KCSPG, SEQ ID NO: 12) is distinctly not a part of the conformational epitope that is specifically bound by TNFRAB1 or antagonistic TNFR2 antibodies or antibody fragments of the invention, as specific binding to both of these epitopes has been shown to lead to a loss of, or significant reduction in, antagonistic activity. As such, TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) that specifically bind both of these epitopes (KCSPG (SEQ ID NO: 12) and an epitope containing at least the KCR sequence, and more specifically, the KCRPG sequence (SEQ ID NO: 19) of human TNFR2) are not considered antagonistic TNFR2 antibodies of the invention. In addition to binding an epitope contained within the sequence KCRPGFGV (SEQ ID NO: 20), TNFRAB1 also binds to a downstream epitope contained within a sequence defined by positions 161-169 of SEQ ID NO: 7 within human TNFR2 (CKPCAPGTF, SEQ ID NO: 21). TNFR2 antibodies and antibody fragments of the invention may also bind this epitope or a larger region within TNFR2 containing this epitope (e.g., a sequence that includes at least five continuous or discontinuous residues from positions 150-190 of SEQ ID NO: 7 within human TNFR2 (ARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAI, SEQ ID NO: 22). TNFRAB1 contains two heavy chains, as well as two light chains, as shown inFIGS.1A and1B. The heavy chains of TNFRAB1 contain the following amino acid sequence (CDRs are indicated in bold): (SEQ ID NO: 2)EVQLQESGGGLVKPGGSLKLSCAASGFTFSSYVMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARQRVDGYSSYWYFDVWGAGTAVTVSS The sequence of the TNFRAB1 light chain is as follows (CDRs are indicated in bold): (SEQ ID NO: 4)DIVLTQSPAIMSASPGEKVTITCSASSSVYYMYWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRRNYPYTFGGGTKLEIKRA The Antagonistic TNFR2 Antibody TNFRAB2 An antagonistic TNFR2 antibody or antibody fragment of the invention may contain, for instance, the CDR-H3 sequence of TNFRAB2, also referred to herein as TNFR2 antagonist 2, an antibody that selectively binds and inhibits TNFR2 by virtue of specifically binding various epitopes within this receptor. For instance, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention may exhibit binding properties that are the same as or similar to those of TNFRAB2. These properties are as follows: In the presence of TNFR2, TNFRAB2 exhibits a high konvalue of 3.6099×105M−1s−1, as well as a low koffof 2.24×10−4s−1and a KDof about 621 pM in complex with TNFR2. An epitope containing residues 137-144 of SEQ ID NO: 7 within human TNFR2 (CAPLRKCR, SEQ ID NO: 11) has been identified as a particularly important component of the functional epitope that establishes intermolecular contacts with TNFRAB2 as determined by epitope mapping analysis (see, e.g., Example 1 andFIGS.2B and3B). Included in the invention are TNFR2 antibodies and antibody fragments that specifically bind this epitope. In addition to binding an epitope containing residues CAPLRKCR (SEQ ID NO: 11), TNFRAB2 also binds to epitopes that include one or more residues within positions 80-86 of SEQ ID NO: 7 within human TNFR2 (DSTYTQL, SEQ ID NO: 8), positions 91-98 of SEQ ID NO: 7 within human TNFR2 (PECLSCGS, SEQ ID NO: 9), as well as positions 116-123 of SEQ ID NO: 7 within human TNFR2 (RICTCRPG, SEQ ID NO: 10). TNFR2 antibodies and antibody fragments of the invention may also bind one or more of these epitopes. Antibodies and antibody fragments of the invention can be designed and identified using the knowledge of the epitopes specifically bound by TNFRAB2. For instance, one can use any of a variety of in vitro peptide display techniques or combinatorial antibody library screens as described herein or known in the art in order to screen for antibodies capable of binding these epitopes with high affinity and selectivity. The heavy chain and light chain CDRs of TNFRAB2 are shown below: (SEQ ID NO: 257)TNFRAB2 CDR-H1:GYTFTDY(L/I)(SEQ ID NO: 258)TNFRAB2 CDR-H2:VDPEYGST(SEQ ID NO: 259)TNFRAB2 CDR-H3:ARDDGSYSPFDYWG(SEQ ID NO: 260)TNFRAB2 CDR-L1:QNINKYTNFRAB2 CDR-L2:TYS or YTS(SEQ ID NO: 261)TNFRAB2 CDR-L3:CLQYVNL(L/I)T As shown above, the CDR-H1 sequence of TNFRAB2 may contain either a leucine or isoleucine residue at the eighth position of this region. Similarly, the TNFRAB2 CDR-L2 may include a TYS or YTS tripeptide, and the TNFRAB2 CDR-L3 may contain either a leucine or isoleucine residue at the eighth position of this region. Notably, the CDR-L2 of TNFRAB2 is flanked by the N-terminal framework residues LLIR (SEQ ID NO: 262) and the C-terminal framework residues TLE. Accordingly, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments) of the invention include those that contain one or more of the above CDRs of TNFRAB2, as well as N-terminal LLIR (SEQ ID NO: 262) and C-terminal TLE residues that flank the CDR-L2 sequence of the antagonistic TNFR2 antibody or antigen-binding fragment thereof. The Antagonistic TNFR2 Antibody TNFR2A3 A representative antagonist TNFR2 antibody of the invention is TNFR2A3, a murine antibody that was discovered by immunization of a mouse with human TNFR2 and subsequent CDR mutagenesis. A precursor antibody to TNFR2A3 was identified from murine immunization experiments as a TNFR2-binding antibody that was neither antagonistic nor agonistic of TNFR2. Using recombinant gene expression techniques, TNFR2A3 was produced by replacing the CDR-H3 sequence of the precursor murine antibody with the CDR-H3 sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). Upon insertion of this CDR-H3 sequence into the precursor scaffold, the resulting TNFR2A3 antibody was capable of exhibiting an antagonistic effect on the TNFR2 target. Additionally, the TNFR2A3 antibody was found to bind epitopes within human TNFR2 that are consistent with those bound by dominant antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2. As described in Example 15, TNFR2A3 binds to two distinct epitopes within human TNFR2. The first epitope spans residues 140-150 of human TNFR2 (LRKCRPGFGVA, SEQ ID NO: 285) and contains the KCRPG motif (SEQ ID NO: 19). The second epitope is a downstream sequence that contains residues 159-171 of human TNFR2 (VVCKPCAPGTFSN, SEQ ID NO: 286). For instance, dominant antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention may exhibit binding properties that are the same as or similar to those of TNFR2A3. Importantly, the binding of TNFR2A3 to these epitopes within TNFR2 is site-specific, as the TNFR2A3 antibody does not bind a human TNFR2-derived peptide containing an unrelated, distal amino acid sequence (see, e.g., Example 15). Collectively, these findings demonstrate two significant features of the CDR-H3 sequence with respect to TNFR2 antagonism: (i) that the CDR-H3 sequence of an antagonistic TNFR2 antibody largely dictates the antigen-binding properties of the system, and (ii) that the CDR-H3 motif is a modular domain that can be substituted into anti-TNFR2 antibodies that do not exhibit antagonistic activity in order to impart such antibodies with TNFR2 dominant antagonistic features. Molecular Determinants of TNFR2 Affinity and Antagonism Notably, there are distinct sequence similarities between the CDR-H3 regions of the antagonistic TNFR2 antibodies TNFRAB1, TNFRAB2, and TNFR2A3. An analysis of the residues common to the CDR-H3 sequences of these antibodies provides insight into the molecular features of antibodies that bind TNFR2 and exhibit an antagonistic effect, such as a dominant antagonistic effect. Epitope mapping analysis has shown that both TNFRAB1, TNFRAB2, and TNFR2A3 bind epitopes within TNFR2 that contain residues 142-146 of SEQ ID NO: 7 and do not bind epitopes containing residues 56-60 of SEQ ID NO: 7. The structural similarities between corresponding CDR-H3 regions provide a basis for predicting residue substitutions that may preserve or enhance TNFR2 affinity and antagonism (e.g., dominant antagonism). Inspection of the CDR-H3 sequences of these antibodies demonstrates that several residues and physicochemical characteristics are conserved throughout this region, while other positions within this CDR can be varied significantly without loss of affinity and antagonistic function. The CDR-H3 sequences of TNFRAB1, TNFRAB2, and TNFR2A3 are shown below: (TNFRAB1 CDR-H3, SEQ ID NO: 25)QRVDGYSSYWYFDV(TNFRAB2 CDR-H3, SEQ ID NO: 259)ARDDG-S-YSPFDYWG(TNFR2A3 CDR-H3, SEQ ID NO: 284)ARDDG-S-YSPFDYFG-R-DG-S-Y--FD--- (Consensus sequence) Inspection of the CDR-H3 sequences of TNFRAB1, TNFRAB2, and TNFR2A3 reveals conserved arginine, aspartic acid, glycine, serine, tyrosine, and phenylalanine residues throughout this CDR. Notably, residues of varying steric and electrostatic properties are tolerated in the remaining positions. For instance, the first position of the CDR-H3 sequence tolerates amino acid residues of contrasting size and hydrogen bond-forming tendencies, as the first position of CDR-H3 in TNFRAB1 features a polar glutamine residue containing a carboxamide side-chain with hydrogen bond donor and acceptor moieties, while an alanine residue bearing an unfunctionalized methyl side-chain is found at the corresponding position in TNFRAB2 and TNFR2A3. Additionally, the third position in the above CDR-H3 sequences features a hydrophobic valine in TNFRAB1 and an anionic aspartic acid moiety in the corresponding position of TNFRAB2. Similarly, positions ten and eleven of the CDR-H3 of TNFRAB1 contain aromatic systems, while the corresponding residues in TNFRAB2 and TNFR2A3 contain polar and cyclic aliphatic substituents. Collectively, the shared structural features of the above CDR-H3 sequences provide insight into those residues that are important for selectively binding residues within TNFR2 that promote receptor antagonism (e.g., dominant receptor antagonism), such as residues 140-150 of human TNFR2 (LRKCRPGFGVA, SEQ ID NO: 285) and residues 159-171 of human TNFR2 (VVCKPCAPGTFSN, SEQ ID NO: 286) and demonstrate that other amino acids can be varied while retaining affinity and dominant antagonistic activity. For instance, antagonistic TNFR2 polypeptides, such a dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may contain a CDR-H3 represented by the formula JZ1JZ2Z4JZ3JZ5(J)2Z5Z2Z5or JZ1JZ2Z4Z3Z5(J)2Z5Z2Z5(J)2, wherein each J is independently a naturally occurring amino acid, each Z1is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH, each Z2is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH, each Z3is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH, each Z4is independently a glycine or alanine, and each Z5is independently a naturally occurring amino acid containing a hydrophobic side-chain. Similarly, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may contain a CDR-H3 represented by the formula JRJDGJSJY(J)2FDJ (SEQ ID NO: 278), JRJDGSY(J)2FD(J)3(SEQ ID NO: 279), QZ1VZ2Z4YZ3SZ5WYZ5Z2Z5(SEQ ID NO: 265), or AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266). For instance, the CDR-H3 may be derived from TNFRAB1 and have the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25). The CDR-H3 may be derived from TNFRAB2 and have the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259). In some embodiments, the CDR-H3 is derived from TNFR2A3 and has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). In addition to the above CDR-H3 sequences, other CDR sequences can be include in antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention. Additional CDR sequences that promote dominant TNFR2 antagonism can be determined, for instance, by alignment of the CDR sequences of TNFRAB1 and TNFRAB2. For example, the CDR-H1 sequences of TNFRAB1 and TNFRAB2 are shown below: (TNFRAB1 CDR-H1, SEQ ID NO: 23)GFTFSSY(TNFRAB2 CDR-H1, SEQ ID NO: 257)GYTFTDY(L/I)G-TF--Y- (Consensus sequence) Alignment of the sequences reveals a shared GXTFXXY motif, wherein “X” designates any amino acid. These CDR-H1 sequences feature a conserved glycine residue at the first position and conserved threonine, phenylalanine, and tyrosine residues at the third, fourth, and seventh positions, respectively. Inspection of these sequences demonstrates that the CDR-H1 region is tolerant of substitutions at the remaining positions. Side-chains of varying polarity are tolerated at the second position, for example, as both phenylalanine, containing an unsubstituted and hydrophobic phenyl moiety, and tyrosine, containing a protic hydroxyl substituent, are found in this position in the CDR-H1 region of TNFRAB1 and TNFRAB2, respectively. Additionally, while the fifth and sixth positions are occupied by polar serine residues in the CDR-H1 of TNFRAB1, these positions feature a threonine, containing an additional hydrophobic methyl substituent, and aspartic acid, which is anionic at physiological pH, in TNFRAB2. This diversity demonstrates that these positions can be substituted with amino acids of diverse electrostatic properties without loss of TNFR2 affinity and antagonism. Sequence analysis of the CDR-H2 regions of TNFRAB1 and TNFRAB2 similarly reveals a set of conserved amino acids at various positions throughout these regions: (TNFRAB1 CDR-H2, SEO ID NO: 24)SSG--GSY(TNFRAB2 CDR-H2, SEQ ID NO: 258)VDPEYGST-----GS- (Consensus sequence) Analysis of this sequence alignment demonstrates that the CDR-H2 sequences exhibit a conserved GS motif at the C-terminal end of the CDR-H2 region, with side-chains of variable molecular size, polarity, and electrostatic charge tolerated at the remaining positions. A similar analysis reveals molecular features common to the CDR-L sequences of TNFRAB1 and TNFRAB2. For instance, the CDR-L1 sequences of TNFRAB1 and TNFRAB2 are shown below: (TNFRAB1 CDR-L1, SEQ ID NO: 26)SASSSVYYMY(TNFRAB2 CDR-L1, SEQ ID NO: 260)Q-N--INK-YY (Consensus residue) Inspection of these sequences reveals that a hydroxyl-containing tyrosine residue is featured at the final position of CDR-L1, while residues of varying physicochemical properties are tolerated at the remaining positions. Similarly, analysis of the CDR-L2 regions of TNFRAB1 and TNFRAB2 reveals a conserved amino acid at the final position in both regions: (TNFRAB1 CDR-L2, SEQ ID NO: 27)STSNLASTY----S or (TNFRAB2 CDR-L2)YT----SS (Consensus residue) Analysis of the above sequence alignment demonstrates that serine residues are featured at the third position of these CDR-L2 sequences, while substitutions are widely tolerated at the remaining residues. Similarly, the CDR-L3 sequences of TNRAB1 and TNFRAB2 are as follows: (TNFRAB1 CDR-L3, SEQ ID NO: 28)Q-QRRNYPY------T(TNFRAB2 CDR-L3, SEQ ID NO: 261)CLQ---YVNL(L/I)T------Y--------T (Consensus sequence) Analysis of the CDR-L3 sequences of TNFRAB1 and TNFRAB2 reveals a preference for tyrosine and threonine residues at distinct positions within these regions, while amino acids of a wide range of physicochemical characteristics are tolerated at other positions, including residues with cationic side-chains (Arg), conformationally restricted side-chains (Pro), and side-chains of varying polarity (e.g., Gln, Asn, Leu, and Val). Collectively, the shared structural features of the above CDR-H and CDR-L sequences provide insight into those residues that are important for selectively binding one or more residues of the KCRPG epitope of TNFR2 (positions 142-146 of SEQ ID NO: 7, shown in SEQ ID NO: 19) in an anti-parallel dimer configuration and demonstrate that certain amino acids can be varied while retaining affinity and dominant antagonistic activity. Antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may therefore have heavy chain and light chain CDRs that contain the above consensus sequences. For instance, TNFR2 antagonists of the invention may have a CDR-H1 having the amino acid sequence Z4JZ3Z5(J)2Z5J; a CDR-H2 having the amino acid sequence (J)5Z4Z3J; a CDR-L1 having the amino acid sequence (J)5Z5; a CDR-L2 having the amino acid sequence (J)2Z3; and/or a CDR-L3 having the amino acid sequence (J)3Z5(J)4Z3; wherein each J is independently a naturally occurring amino acid; each Z1is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH; each Z2is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH; each Z3is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH; each Z4is independently a glycine or alanine; and each Z5is independently a naturally occurring amino acid containing a hydrophobic side-chain. In some embodiments, antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence GJTF(J)2YJ (SEQ ID NO: 277); a CDR-H2 having the amino acid sequence (J)5GSJ; a CDR-L1 having the amino acid sequence (J)5Y; a CDR-L2 having the amino acid sequence (J)2S; and/or a CDR-L3 having the amino acid sequence (J)3Y(J)4T; wherein each J is independently a naturally occurring amino acid. Antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence Z4YZ3Z5TDZ5X; a CDR-H2 having the amino acid sequence VDPEYZ4Z3T (SEQ ID NO: 264); a CDR-L1 having the amino acid sequence QNINKZ5(SEQ ID NO: 268); a CDR-L2 having the amino acid sequence TYZ3or YTZ3; and/or a CDR-L3 having the amino acid sequence CLQZ5VNLXZ3(SEQ ID NO: 271); wherein each Z1is independently an amino acid containing a cationic side-chain at physiological pH; each Z2is independently an amino acid containing an anionic side-chain at physiological pH; each Z3is independently an amino acid containing a polar, uncharged side-chain at physiological pH; each Z4is independently a glycine or alanine; each Z5is independently an amino acid containing a hydrophobic side-chain; and each X is independently leucine or isoleucine. In some embodiments, antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence GYTFTDYX (SEQ ID NO: 257), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; a CDR-H2 having the amino acid sequence VDPEYGST (SEQ ID NO: 258), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; a CDR-L1 having the amino acid sequence QNINKY (SEQ ID NO: 260), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; a CDR-L2 having the amino acid sequence TYS or YTS; and/or a CDR-L3 having the amino acid sequence CLQYVNLXT (SEQ ID NO: 261), or an amino acid sequence having up to two amino acid substitutions relative to this sequence. For example, in some embodiments, antagonistic TNFR2 polypeptides of the invention, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) may have a CDR-H1 having the amino acid sequence GYTFTDYL (SEQ ID NO: 274), or an amino acid sequence having up to two amino acid substitutions relative to this sequence; and a CDR-L3 having the amino acid sequence CLQYVNLIT (SEQ ID NO: 273), or an amino acid sequence having up to two amino acid substitutions relative to this sequence. The present invention is based in part on the discovery that this particular combination of CDR-H1 and CDR-L3 regions promote the selective killing of activated T-reg cells and potentiate augmented T effector cell proliferation. As described herein, these phenotypes are beneficial for the treatment of cancers and infectious diseases, as the ability to deplete activated T-reg cell populations in a patient suffering from such pathologies can lessen the attenuation of cytotoxic CD8+ T cells, thereby enabling effector cells to mount an immune response against cancerous and infectious cells (see, e.g., Example 17). Humanized, Primatized, and Chimeric Antibodies Antibodies of the invention include human, humanized, primatized, and chimeric antibodies that contain the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, or a CDR-H3 that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain conservative mutations relative to these CDR-H3 sequences. Antibodies of the invention also include human, humanized, primatized, and chimeric antibodies that contain the CDR-H3 of TNFRAB1, TNFAB2, or TNFR2A3, or a CDR-H3 that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain conservative mutations relative to these CDR-H3 sequences. For instance, antibodies of the invention also include human, humanized, primatized, and chimeric antibodies that contain a CDR-H3 that is identical to the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 except for conservative amino acid substitutions. In some embodiments, a humanized, primatized, or chimeric antibody may contain the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, or a CDR that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain conservative mutations relative to these CDR-H3 sequences. For example, antagonistic TNFR2 antibodies of the invention can be generated by incorporating any of the above CDR-H3 sequences into the framework regions (e.g., FW1, FW2, FW3, and FW4) of a human antibody. Exemplary framework regions that can be used for the development of a humanized anti-TNFR2 antibody containing one or more of the above CDRs include, without limitation, those described in U.S. Pat. Nos. 7,732,578, 8,093,068, and WO 2003/105782; incorporated herein by reference. One strategy that can be used to design humanized antibodies of the invention is to align the sequences of the heavy chain variable region and light chain variable region of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3, with the heavy chain variable region and light chain variable region of a consensus human antibody. Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the “VBASE” human germline sequence database; see also Kabat, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-98, 1992; and Cox et al, Eur. J. Immunol. 24:827-836, 1994; incorporated herein by reference). In this way, the variable domain framework residues and CDRs can be identified by sequence alignment (see Kabat, supra). One can substitute, for example, the CDR-H3 of the consensus human antibody with the CDR-H3 of an antagonistic TNFR2 antibody, such as a CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, in order to produce a humanized TNFR2 antagonist antibody. Exemplary variable domains of a consensus human antibody include the heavy chain variable domain: (SEQ ID NO: 32)EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS and the light chain variable domain: (SEQ ID NO: 33)DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT identified in U.S. Pat. No. 6,054,297; incorporated herein by reference (CDRs are shown in bold were determined according to the method of Chothia, et al., J. Mol. Biol, 196:901-917, 1987). These amino acid substitutions can be made, for example, by recombinant expression of polynucleotides encoding the heavy and light chains of a humanized antibody in a host cell using methods known in the art or described herein. Similarly, this strategy can also be used to produce primatized anti-TNFR2 antibodies, as one can substitute, for example, the CDR-H3 of a primate antibody consensus sequence with, for example, the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3. Consensus primate antibody sequences known in the art (see e.g., U.S. Pat. Nos. 5,658,570; 5,681,722; and 5,693,780; incorporated herein by reference). In some embodiments, it may be desirable to import particular framework residues in addition to CDR sequences from a TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3 into the heavy and/or light chain variable domains of a human antibody. For instance, U.S. Pat. No. 6,054,297 identifies several instances when it may be advantageous to retain certain framework residues from a particular antibody heavy chain or light chain variable region in the resulting humanized antibody. In some embodiments, framework residues may engage in non-covalent interactions with the antigen and thus contribute to the affinity of the antibody for the target antigen. In other cases, individual framework residues may modulate the conformation of a CDR, and thus indirectly influence the interaction of the antibody with the antigen. Alternatively, certain framework residues may form the interface between VH and VL domains, and may therefore contribute to the global antibody structure. In other cases, framework residues may constitute functional glycosylation sites (e.g., Asn-X-Ser/Thr) which may dictate antibody structure and antigen affinity upon attachment to carbohydrate moieties. In cases such as those described above, it may be beneficial to retain certain framework residues of a TNFR2 antagonist antibody (e.g., TNFRAB1, TNFRAB2, or TNFR2A3) in the antagonistic antibodies and antigen-binding fragments thereof of the invention, such as humanized antibodies, as various framework residues may promote high epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment thereof. Antibodies of the invention also include antibody fragments, Fab domains, F(ab′) molecules, F(ab′)2molecules, single-chain variable fragments (scFvs), tandem scFv fragments, diabodies, triabodies, dual variable domain immunoglobulins, multi-specific antibodies, bispecific antibodies, and heterospecific antibodies that contain the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 (e.g., QRVDGYSSYWYFDV (SEQ ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), or ARDDGSYSPFDYFG (SEQ ID NO: 284)) or a CDR-H3 that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences. Antibodies and antigen-binding fragments thereof of the invention include those that also contain CDR-H3 sequences having between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the CDR-H3 sequences of TNFRAB1, TNFRAB2, or TNFR2A3. These molecules can be expressed recombinantly, e.g., by incorporating polynucleotides encoding these proteins into expression vectors for transfection in a eukaryotic or prokaryotic cell using techniques described herein or known in the art, or synthesized chemically, e.g., by solid phase peptide synthesis methods described herein or known in the art. Polypeptides of the invention additionally include antibody-like scaffolds that contain, for example, the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, or a CDR-H3 sequence that exhibits at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the CDR-H3 sequences of TNFRAB1, TNFRAB2, or TFNR2A3. Examples of antibody-like scaffolds include proteins that contain a tenth fibronectin type III domain (10Fn3), which contains BC, DE, and FG structural loops analogous to canonical antibodies. It has been shown that the tertiary structure of the10Fn3 domain resembles that of the variable region of the IgG heavy chain, and one of skill in the art can graft, e.g., the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3, or sequences having at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences containing conserved amino acid substitutions relative to these CDR-H3 sequences onto the fibronectin scaffold by replacing residues of the BC, DE, and FG loops of10Fn3 with residues of the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3. This can be achieved by recombinant expression of a modified10Fn3 domain in a prokaryotic or eukaryotic cell (e.g., using the vectors and techniques described herein). Examples of using the10Fn3 domain as an antibody-like scaffold for the grafting of CDRs from antibodies onto the BC, DE, and FG structural loops are reported in WO 2000/034784, WO 2009/142773, WO 2012/088006, and U.S. Pat. No. 8,278,419; incorporated herein by reference. Antagonistic TNFR2 Single-Chain Polypeptides TNFR2 antagonists of the invention may be in the form of a single-chain polypeptide, such as a single-chain polypeptide that contains a CDR-H3 region described herein (e.g., the CDR-H3 region of TNFRAB1, TNFRAB2, or TNFR2A3), optionally in combination with a CDR derived from a phenotype-neutral TNFR2 antibody (i.e., an antibody that is neither antagonistic nor agonistic of TNFR2 activation). Single-chain polypeptides may be in the form of an antibody fragment, e.g., an antibody fragment described herein or known in the art, such as a scFv fragment. Single chain polypeptides may alternatively contain one or more CDRs described herein covalently bound to one another using conventional bond-forming techniques known in the art, for instance, by an amide bond, a thioether bond, a carbon-carbon bond, or by a linker, such as a peptide linker or a multi-valent electrophile (e.g., a bis(bromomethyl) arene derivative, such as a bis(bromomethyl)benzene or bis(bromomethyl)pyridine) described herein or known in the art. For instance, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 region that contains one of the above-described consensus sequences that promote selective binding to TNFR2 epitopes, such as the KCRPG motif (SEQ ID NO: 19), and induce TNFR2 antagonism. For instance, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence JZ1JZ2Z4JZ3JZ5(J)2Z5Z2Z5or JZ1JZ2Z4Z3Z5(J)2Z5Z2Z5(J)2, wherein each J is independently a naturally occurring amino acid; each Z1is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH; each Z2is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH; each Z3is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH; each Z4is independently a glycine or alanine; and each Z5is independently a naturally occurring amino acid containing a hydrophobic side-chain. In some embodiments, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence JRJDGJSJY(J)2FDJ (SEQ ID NO: 278) or JRJDGSY(J)2FD(J)3(SEQ ID NO: 279), wherein each J is independently a naturally occurring amino acid. Antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence QZ1VZ2Z4YZ3SZ5WYZ5Z2Z5(SEQ ID NO: 265) or AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266), wherein each Z1is independently an amino acid containing a cationic side-chain at physiological pH; each Z2is independently an amino acid containing an anionic side-chain at physiological pH; each Z3is independently an amino acid containing a polar, uncharged side-chain at physiological pH; each Z4is independently a glycine or alanine; each Z5is independently an amino acid containing a hydrophobic side-chain; and each X is independently leucine or isoleucine. In some embodiments, antagonistic TNFR2 single-chain polypeptides of the invention may have a CDR-H3 having the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25), ARDDGSYSPFDYWG (SEQ ID NO: 259), or an amino acid sequence having up to two amino acid substitutions relative to these sequences (e.g., one or two amino acid substitutions, such as conservative amino acid substitutions) Single-chain polypeptides can be produced by a variety of recombinant and synthetic techniques, such as by recombinant gene expression or solid-phase peptide synthesis procedures described herein or known in the art. For instance, one of skill in the art can design polynucleotides encoding, e.g., two or more CDRs operably linked to one another in frame so as to produce a continuous, single-chain peptide containing these CDRs. Optionally, the CDRs may be separated by a spacer, such as by a framework region (e.g., a framework sequence described herein or a framework region of a germline consensus sequence of a human antibody) or a flexible linker, such as a poly-glycine or glycine/serine linker described herein or known in the art. When produced by chemical synthesis methods, native chemical ligation can optionally be used as a strategy for the synthesis of long peptides (e.g., greater than 50 amino acids). Native chemical ligation protocols are known in the art and have been described, e.g., by Dawson et al. (Science, 266:776-779, 1994); incorporated herein by reference. A detailed description of techniques for the production of single-chain polypeptides, full-length antibodies, and antibody fragments is provided in the sections that follow. Nucleic Acids and Expression Systems Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention can be prepared by any of a variety of established techniques. For instance, an antagonistic TNFR2 antibody or antigen-binding fragment thereof of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. To express an antibody recombinantly, a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, optionally, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N. Y., 1989), Current Protocols in Molecular Biology (Ausubel et al., eds., Greene Publishing Associates, 1989), and in U.S. Pat. No. 4,816,397; incorporated herein by reference. Vectors for Expression of Antagonistic TNFR2 Polypeptides Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration. Examples of viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding antibody light and heavy chains or antibody fragments of the invention include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication,In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Pat. No. 5,801,030); incorporated herein by reference. Genome Editing Techniques In addition to viral vectors, a variety of additional methods have been developed for the incorporation of genes, e.g., those encoding antibody light and heavy chains, single-chain polypeptides, single-chain variable fragments (scFvs), tandem scFvs, Fab domains, F(ab′)2 domains, diabodies, and triabodies, among others, into the genomes of target cells for polypeptide expression. One such method that can be used for incorporating polynucleotides encoding anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) into prokaryotic or eukaryotic cells includes transposons. Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by excision sites at the 5′ and 3′ positions. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some embodiments, these excision sites may be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a prokaryotic or eukaryotic cell by transposase-catalyzed cleavage of similar excision sites that exist within nuclear genome of the cell. This allows the gene encoding an anti-TNFR2 antibody or fragment or domain thereof to be inserted into the cleaved nuclear DNA at the excision sites, and subsequent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the prokaryotic or eukaryotic cell genome completes the incorporation process. In some embodiments, the transposon may be a retrotransposon, such that the gene encoding the antibody is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the prokaryotic or eukaryotic cell genome. Exemplary transposon systems include the piggybac transposon (described in detail in WO 2010/085699) and the sleeping beauty transposon (described in detail in US20050112764); incorporated herein by reference. Another useful method for the integration of nucleic acid molecules encoding anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) into the genome of a prokaryotic or eukaryotic cell is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, which is a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against infection by viruses. The CRISPR/Cas system consists of palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site. In this manner, highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization. As a result, one can theoretically design a CRISPR/Cas system to cleave any target DNA molecule of interest. This technique has been exploited in order to edit eukaryotic genomes (Hwang et al., Nat. Biotech., 31:227-229, 2013) and can be used as an efficient means of site-specifically editing eukaryotic or prokaryotic genomes in order to cleave DNA prior to the incorporation of a polynucleotide encoding an anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention. The use of CRISPR/Cas to modulate gene expression has been described in U.S. Pat. No. 8,697,359, which is incorporated herein by reference. Alternative methods for site-specifically cleaving genomic DNA prior to the incorporation of a polynucleotide encoding a TNFR2 antibody or antibody fragment of the invention include the use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR/Cas system, these enzymes do not contain a guiding polynucleotide to localize to a specific target sequence. Target specificity is instead controlled by DNA binding domains within these enzymes. Zinc finger nucleases and TALENs for use in genome editing applications are described in Urnov et al. (Nat. Rev. Genet., 11:636-646, 2010); and in Joung et al., (Nat. Rev. Mol. Cell. Bio. 14:49-55, 2013); incorporated herein by reference. Additional genome editing techniques that can be used to incorporate polynucleotides encoding antibodies of the invention into the genome of a prokaryotic or eukaryotic cell include the use of ARCUS™ meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA. The use of these enzymes for the incorporation of polynucleotides encoding antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention into the genome of a prokaryotic or eukaryotic cell is particularly advantageous in view of the structure-activity relationships that have been established for such enzymes. Single-chain meganucleases can thus be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations. These single-chain nucleases have been described extensively, e.g., in U.S. Pat. Nos. 8,021,867 and 8,445,251; incorporated herein by reference. Polynucleotide Sequence Elements To express antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention, polynucleotides encoding partial or full-length light and heavy chains, e.g., polynucleotides that encode a CDR-H3 region as described herein, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Polynucleotides encoding the light chain gene and the heavy chain of a TNFR2 antibody can be inserted into separate vectors, or, optionally, both polynucleotides can be incorporated into the same expression vector using established techniques described herein or known in the art. In addition to polynucleotides encoding the heavy and light chains of an antibody (or a polynucleotide encoding a single-chain polypeptide or an antibody fragment, such as a scFv molecule), the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed or the level of expression of protein desired. For instance, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention can carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR′ host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). In order to express the light and heavy chains of a TNFR2 antibody or a TNFR2 antibody fragment, the expression vector(s) containing polynucleotides encoding the heavy and light chains can be transfected into a host cell by standard techniques. Polynucleotides Encoding Modified Antagonistic TNFR2 Polypeptides Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention may contain the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3, but feature differences in the sequence of one or more of the remaining CDRs relative to the corresponding sequence in TNFRAB1, TNFRAB2, or TNFR2A3. Similarly, polypeptides of the invention may contain the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 but may feature differences in one or more framework regions. For instance, one or more framework regions of TNFRAB1, TNFRAB2, or TNFR2A3 may be substituted with the framework region of a human antibody. Exemplary framework regions include, for example, human framework regions described in U.S. Pat. No. 7,829,086, and primate framework regions as described in EP 1945668; incorporated herein by reference. To generate nucleic acids encoding such TNFR2 antibodies, DNA fragments encoding, e.g., at least one, or both, of the light chain variable regions and the heavy chain variable regions can be produced by chemical synthesis (e.g., by solid phase polynucleotide synthesis techniques), in vitro gene amplification (e.g., by polymerase chain reaction techniques), or by replication of the polynucleotide in a host organism. For instance, nucleic acids encoding anti-TNFR2 antibodies of the invention may be obtained by amplification and modification of germline DNA or cDNA encoding light and heavy chain variable sequences so as to incorporate the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 into the framework residues of a consensus antibody. In some embodiments, a humanized antagonistic TNFR2 antibody may include the CDR-H3 of TFNRAB1, TNFRAB2, or TNFR2A3, or a variant thereof that has at least 85% sequence identity (e.g., 90%, 95%, 97%, 99%, or 100% sequence identity) to any of these CDR-H3 sequences or sequences that contain between one and three amino acid substitutions (e.g., conservative or nonconservative substitutions) relative to the CDR-H3 sequences of TNFRAB1, TNFRAB2, or TNFR2A3. This can be achieved, for example, by performing site-directed mutagenesis of germline DNA or cDNA and amplifying the resulting polynucleotides using the polymerase chain reaction (PCR) according to established procedures. Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see, e.g., the “VBASE” human germline sequence database; see also Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al., Eur. J. Immunol. 24:827-836, 1994; incorporated herein by reference). Chimeric nucleic acid constructs encoding human heavy and light chain variable regions containing the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 can be produced, e.g., using established cloning techniques known in the art. Additionally, a polynucleotide encoding a heavy chain variable region containing the CDR-H3 of TNFRAB1, TNFRAB2, or TFNR2A3 can be synthesized and used as a template for mutagenesis to generate a variant as described herein using routine mutagenesis techniques. Alternatively, a DNA fragment encoding the variant can be directly synthesized (e.g., by established solid phase nucleic acid chemical synthesis procedures). Once DNA fragments encoding VH segments containing the CDR-H3 of TNFRAB1, TNFRAB2, or TNFR2A3 are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The isolated DNA encoding the VH region of an anti-TNFR2 antibody of the invention can be converted to a full-length heavy chain gene (as well as a Fab heavy chain gene), e.g., by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant region domains (CH1, CH2, CH3, and, optionally, CH4). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, and in certain embodiments is an IgG1 constant region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 domain. Isolated DNA encoding the VL region of an anti-TNFR2 antibody can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991)) and DNA fragments encompassing these regions can be obtained, e.g., by amplification in a prokaryotic or eukaryotic cell of a polynucleotide encoding these regions, by PCR amplification, or by chemical polynucleotide synthesis. The light chain constant region can be a kappa (κ) or lambda (λ) constant region, but in certain embodiments is a kappa constant region. To create a scFv gene, the VH and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., a polynucleotide encoding a flexible, hydrophilic amino acid sequence, such as the amino acid sequence (Gly4Ser)3, such that the VHand VLsequences can be expressed as a contiguous single-chain protein, with the VLand VHregions joined by the linker (see e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990). Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to TNFR2. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention. In addition, bifunctional antibodies can be produced in which one heavy contains a CDR-H3 sequence derived from TNFRAB1, TNFRAB2, or TNFR2A3, and the other heavy chain and/or the light chains are specific for an antigen other than TNFR2. Such antibodies can be generated, e.g., by crosslinking a heavy chain containing the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3 and a light chain of an anti-TNFR2 antibody, such as an anti-TNFR2 antibody that is neither agonistic nor antagonistic, to a heavy chain and light chain of a second antibody by standard chemical crosslinking methods (e.g., by disulfide bond formation). Bifunctional antibodies can also be made by expressing a nucleic acid molecule engineered to encode a bifunctional antibody in a prokaryotic or eukaryotic cell. Dual specific antibodies, i.e., antibodies that bind TNFR2 and a different antigen using the same binding site, can be produced by mutating amino acid residues in the light chain and/or heavy chain CDRs. In some embodiments, dual specific antibodies that bind two antigens, such as TNFR2 and a second cell-surface receptor, can be produced by mutating amino acid residues in the periphery of the antigen binding site (Bostrom et al., Science 323: 1610-1614, 2009). Dual functional antibodies can be made by expressing a polynucleotide engineered to encode a dual specific antibody. Modified antagonistic TNFR2 antibodies and antibody fragments of the invention can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, 111; incorporated herein by reference). Variant antibodies can also be generated using a cell-free synthetic platform (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals); incorporated herein by reference). Host Cells for Expression of Antagonistic TNFR2 Polypeptides It is possible to express the polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) of the invention in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) is performed in eukaryotic cells, e.g., mammalian host cells, for optimal secretion of a properly folded and immunologically active antibody. Exemplary mammalian host cells for expressing the recombinant antibodies or antigen-binding fragments thereof of the invention include Chinese Hamster Ovary (CHO cells) (including DHFR CHO cells, described in Urlaub and Chasin (1980, Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, 293 cells, and SP2/0 cells. Additional cell types that may be useful for the expression of antibodies and fragments thereof include bacterial cells, such as BL-21 (DE3)E. colicells, which can be transformed with vectors containing foreign DNA according to established protocols. Additional eukaryotic cells that may be useful for expression of antibodies include yeast cells, such as auxotrophic strains ofS. cerevisiae, which can be transformed and selectively grown in incomplete media according to established procedures known in the art. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. The invention also includes methods in which the above procedure is varied according to established protocols known in the art. For example, it can be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-TNFR2 antibody of the invention in order to produce an antigen-binding fragment of the antibody. Once an anti-TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment) thereof of the invention has been produced by recombinant expression, it can be purified by any method known in the art, such as a method useful for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for TNFR2 after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the anti-TNFR2 polypeptides of the invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification or to produce therapeutic conjugates (see “Antagonistic TNFR2 polypeptide conjugates,” below). Once isolated, an anti-TNFR2 single-chain polypeptide, antibody, or antigen-binding fragments thereof can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques in Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980); incorporated herein by reference), or by gel filtration chromatography, such as on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden). Platforms for Generating and Affinity-Maturing Antagonistic Anti-TNFR2 Polypeptides Mapping Epitopes of TNFR2 that Promote Receptor Antagonism Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be produced by screening libraries of polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) for functional molecules that are capable of binding epitopes within TNFR2 that selectively promote receptor antagonism rather than receptor activation. Such epitopes can be modeled by screening antibodies or antigen-binding fragments thereof against a series of linear or cyclic peptides containing residues that correspond to a desired epitope within TNFR2. As an example, peptides containing individual fragments isolated from TNFR2 that promote receptor antagonism can be synthesized by peptide synthesis techniques described herein or known in the art. These peptides can be immobilized on a solid surface and screened for molecules that bind antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof), such as TNFRAB1, TNFRAB2, or TNFR2A3, e.g., using an ELISA-based screening platform using established procedures. Using this assay, peptides that specifically bind TNFRAB1, TNFRAB2, or TNFR2A3 with high affinity therefore contain residues within epitopes of TNFR2 that preferentially bind these antibodies. Peptides identified in this manner (e.g., peptides having the sequence of any one of SEQ ID NOs: 11, 19, 20, 34-117, 285, and 286, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids between positions 80 and 130 of SEQ ID NO: 7) can be used to screen libraries of antibodies and antigen-binding fragments thereof in order to identify anti-TNFR2 antibodies of the invention. Moreover, since these peptides act as surrogates for epitopes within TNFR2 that promote receptor antagonism, antibodies generated using this screening technique may bind the corresponding epitopes in TNFR2 and are expected to be antagonistic of receptor activity. Screening of Libraries for Antagonistic TNFR2 Polypeptides Methods for high throughput screening of polypeptide (e.g., single-chain polypeptide, antibody, or antibody fragment) libraries for molecules capable of binding epitopes within TNFR2 (e.g., peptides having the sequence of SEQ ID NO: 285 or 286) include, without limitation, display techniques including phage display, bacterial display, yeast display, mammalian display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate ligands that bind biologically relevant molecules has been reviewed, e.g., in Felici et al. (Biotechnol. Annual Rev. 1:149-183, 1995), Katz (Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997), and Hoogenboom et al. (Immunotechnology 4:1-20, 1998). Several randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind different targets, e.g., cell surface receptors or DNA (reviewed by Kay (Perspect. Drug Discovery Des. 2, 251-268, 1995), Kay et al., (Mol. Divers. 1:139-140, 1996)). Proteins and multimeric proteins have been successfully phage-displayed as functional molecules (see EP 0349578A, EP 4527839A, EP 0589877A; Chiswell and McCafferty (Trends Biotechnol. 10, 80-84 1992)). In addition, functional antibody fragments (e.g. Fab, single-chain Fv [scFv]) have been expressed (McCafferty et al. (Nature 348: 552-554, 1990), Barbas et al. (Proc. Natl. Acad Sci. USA 88:7978-7982, 1991), Clackson et al. (Nature 352:624-628, 1991)). These references are hereby incorporated by reference in their entirety. (i) Phage Display Techniques As an example, phage display techniques can be used in order to screen libraries of polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) for functional molecules capable of binding cyclic or polycyclic peptides containing epitopes within TNFR2 that promote receptor antagonism (e.g., peptides having the sequence of SEQ ID NO: 285 or 286). For instance, libraries of polynucleotides encoding single-chain antibody fragments, such as scFv fragments, that contain randomized hypervariable regions can be obtained using established procedures (e.g., solid phase polynucleotide synthesis or error-prone PCR techniques, see McCullum et al. (Meth. Mol. Biol., 634:103-109, 2010); incorporated herein by reference). These randomized polynucleotides can subsequently be incorporated into a viral genome such that the randomized antibody chains encoded by these genes are expressed on the surface of filamentous phage, e.g., by a covalent bond between the antibody chain and a coat protein (e.g., pIII coat protein on the surface of M13 phage). This provides a physical connection between the genotype and phenotype of the antibody chain. In this way, libraries of phage that display diverse antibody chains containing random mutations in hypervariable regions can be screened for the ability of the exterior antibody chains to bind TNFR2 epitopes (e.g., peptides having the sequence of SEQ ID NO: 285 or 286) that are immobilized to a surface using established procedures. For instance, such peptides can be physically bound to the surface of a microtiter plate by forming a covalent bond between the peptide and an epitope tag (e.g., biotin) and incubating the peptide in wells of a microtiter plate that have been previously coated with a complementary tag (e.g., avidin) that binds the tag attached to the peptide with high affinity. Suitable epitope tags include, without limitation, maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin. Peptides containing the epitopes presented by these molecules are capable of being immobilized on surfaces containing such complementary molecules as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. In this way, phage can be incubated with a surface containing an immobilized TNFR2-derived peptide for a time suitable to allow binding of the antibody to the constrained peptide and in the presence of an appropriate buffer system (e.g., one that contains physiological salt concentration, ionic strength, and is maintained at physiological pH by a buffering agent). The surface can then be washed (e.g., with phosphate buffer containing 0.1% Tween-20) so as to remove phage that do not present antibody chains that interact with the TNFR2-derived peptides with an affinity greater than a particular threshold value. The affinity of the polypeptides that remain after this initial panning (i.e., screening) step can be modulated by adjusting the conditions of the washing step (e.g., by including mildly acidic or basic components, or by including other TNFR2-derived peptides at a low concentration in order to compete with immobilized peptides for antigen-binding sites). In this way, the population of phage that remains bound to the surfaces of the microtiter plate following the washing step is enriched for phage that bind TNFR2-derived peptide epitopes that promote receptor antagonism. The remaining phage can then be amplified by eluting the phage from the surface containing these peptides (e.g., by altering the ambient pH, ionic strength, or temperature) so as to diminish protein-protein interaction strength. The isolated phage can then be amplified, e.g., by infecting bacterial cells, and the resulting phage can optionally be subjected to panning by additional iterations of screening so as to further enrich the population of phage for those harboring higher-affinity anti-TNFR2 polypeptides. Following these panning stages, phage that display high-affinity antibodies or antigen-binding fragments thereof can subsequently be isolated and the genomes of these phage can be sequenced in order to identify the polynucleotide and polypeptide sequences of the encoded antibodies. Phage display techniques such as this can be used to generate, e.g., antibody chains, such as scFv fragments, tandem scFv fragments, and other antigen-binding fragments of the invention that can be used as antagonists of TNFR2. Exemplary phage display protocols for the identification of antibody chains and antigen-binding fragments thereof that bind a particular antigen with high affinity are well-established and are described, e.g., in U.S. Pat. No. 7,846,892, WO 1997/002342, U.S. Pat. No. 8,846,867, and WO 2007/132917; incorporated herein by reference. Similar phage display techniques can be used to generate antibody-like scaffolds (e.g.,10Fn3 domains) of the invention that bind epitopes within TNFR2 that promote receptor antagonism (e.g., epitopes presented by peptides with the sequence of SEQ ID NO: 285 or 286). Exemplary phage display protocols for the identification of antibody-like scaffold proteins are described, e.g., in WO 2009/086116; incorporated herein by reference). (ii) Cell-Based Display Techniques Other in vitro display techniques that exploit the linkage between genotype and phenotype of a solvent-exposed polypeptide include yeast and bacterial display. Yeast display techniques are established in the art and are often advantageous in that high quantities of antibodies (often up to 30,000) can be presented on the surface of an individual yeast cell (see, e.g., Boder et al. (Nat Biotechno. 15:553, 1997); incorporated herein by reference). The larger size of yeast cells over filamentous phage enables an additional screening strategy, as one can use flow cytometry to both analyze and sort libraries of yeast. For instance, established procedures can be used to generate libraries of bacterial cells or yeast cells that express polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) containing randomized hypervariable regions (see, e.g., see U.S. Pat. No. 7,749,501 and US 2013/0085072; the teachings of each which are incorporated herein by reference). For instance, large libraries of yeast cells that express polynucleotides encoding naïve scFv fragments can be made using established procedures (de Bruin et al., Nat Biotechnol 17:397, 1999; incorporated herein by reference). Yeast cells expressing these polynucleotides can then be incubated with two different fluorescent molecules during the panning steps: one dye that binds conserved residues within the antibody and thus reflects the amount of antibody displayed, and another dye that fluoresces at a different wavelength and binds the antigen and thus indicates the amount of antigen bound. For instance, one of skill in the art can use a TNFR2-derived peptide containing the sequence of SEQ ID NO: 285 or 286 that has been conjugated to an epitope tag (e.g., biotin), optionally at the N- or C-terminus of the peptide or at a residue that is not expected to interfere with antibody-antigen binding. This enables a fluorescent dye labeled with a complementary tag (e.g., avidin) to localize to the antibody-antigen complex. This results in great flexibility and immediate feedback on the progress of a selection. In contrast to phage display, by normalizing to antibody display levels, antibodies with higher affinities, rather than greater expression levels can easily be selected. In fact, it is possible to distinguish and sort antibodies whose affinities differ by only two-fold (VanAntwerp and Wittrup (Biotechnol Prog 16:31, 2000)). (iii) Nucleotide Display Techniques Display techniques that utilize in vitro translation of randomized polynucleotide libraries also provide a powerful approach to generating anti-TNFR2 antibodies of the invention. For instance, randomized DNA libraries encoding polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) that contain mutations within designated hypervariable regions can be obtained, e.g., using established PCR-based mutagenesis techniques as described herein. The polynucleotides of these libraries may contain transcription regulating sequences, such as promoters and transcription terminating sequences, and may additionally encode sequences that increase the rate of translation of the resulting mRNA construct (e.g., IRES sequences, 5′ and 3′ UTRs, a poly-adenylation tract, etc). These polynucleotide libraries can be incubated in an appropriately buffered solution containing RNA polymerase and RNA nucleoside triphosphates (NTPs) in order to enable transcription of the DNA sequences to competent mRNA molecules, which can subsequently be translated by large and small ribosomal subunits, aminoacyl tRNA molecules, and translation initiation and elongation factors present in solution (e.g., using the PURExpress® In Vitro Protein Synthesis Kit, New England Biolabs®). Designed mRNA modifications can enable the antibody product to remain covalently bound to the mRNA template by a chemical bond to puromycin (e.g., see Keefe (Curr. Protoc. Mol. Biol., Chapter 24, Unit 24.5, 2001); incorporated herein by reference). This genotype-phenotype linkage can thus be used to select for antibodies that bind a TNFR2-derived peptide (e.g., a peptide that has the sequence SEQ ID NO: 285 or 286) by incubating mRNA:antibody fusion constructs with a peptide immobilized to a surface and panning in a fashion similar to phage display techniques (see, e.g., WO 2006/072773; incorporated herein by reference). Optionally, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be generated using a similar technique, except the antibody product may be bound non-covalently to the ribosome-mRNA complex rather than covalently via a puromycin linker. This platform, known as ribosome display, has been described, e.g., in U.S. Pat. No. 7,074,557; incorporated herein by reference. Alternatively, antibodies can be generated using cDNA display, a technique analogous to mRNA display with the exception that cDNA, rather than mRNA, is covalently bound to an antibody product via a puromycin linker. cDNA display techniques offer the advantage of being able to perform panning steps under increasingly stringent conditions, e.g., under conditions in which the salt concentration, ionic strength, pH, and/or temperature of the environment is adjusted in order to screen for antibodies with particularly high affinity for TNFR2-derived peptides. This is due to the higher natural stability of double-stranded cDNA over single-stranded mRNA. cDNA display screening techniques are described, e.g., in Ueno et al. (Methods Mol. Biol., 805:113-135, 2012); incorporated herein by reference. In addition to generating anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention, in vitro display techniques (e.g., those described herein and those known in the art) also provide methods for improving the affinity of an anti-TNFR2 polypeptide of the invention. For instance, rather than screening libraries of antibodies and fragments thereof containing completely randomized hypervariable regions, one can screen narrower libraries of antibodies and antigen-binding fragments thereof that feature targeted mutations at specific sites within hypervariable regions. This can be accomplished, e.g., by assembling libraries of polynucleotides encoding antibodies or antigen-binding fragments thereof that encode random mutations only at particular sites within hypervariable regions. These polynucleotides can then be expressed in, e.g., filamentous phage, bacterial cells, yeast cells, mammalian cells, or in vitro using, e.g., ribosome display, mRNA display, or cDNA display techniques in order to screen for antibodies or antigen-binding fragments thereof that specifically bind TNFR2 epitopes (e.g., peptides containing the sequence of SEQ ID NO: 285 or 286) with improved binding affinity. Yeast display, for instance, is well-suited for affinity maturation, and has been used previously to improve the affinity of a single-chain antibody to a KDof 48 fM (Boder et al. (Proc Natl Acad Sci USA 97:10701, 2000)). Additional in vitro techniques that can be used for the generation and affinity maturation of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention include the screening of combinatorial libraries of antibodies or antigen-binding fragments thereof for functional molecules capable of specifically binding TNFR2-derived peptides (e.g., a peptide having the amino acid sequence of SEQ ID NO: 285 or 286). Combinatorial antibody libraries can be obtained, e.g., by expression of polynucleotides encoding randomized hypervariable regions of an antibody or antigen-binding fragment thereof in a eukaryotic or prokaryotic cell. This can be achieved, e.g., using gene expression techniques described herein or known in the art. Heterogeneous mixtures of antibodies can be purified, e.g., by Protein A or Protein G selection, sizing column chromatography), centrifugation, differential solubility, and/or by any other standard technique for the purification of proteins. Libraries of combinatorial libraries thus obtained can be screened, e.g., by incubating a heterogeneous mixture of these antibodies with a peptide derived from TNFR2 that has been immobilized to a surface (e.g., a peptide having the amino acid sequence of SEQ ID NO: 285 or 286 immobilized to the surface of a solid-phase resin or a well of a microtiter plate) for a period of time sufficient to allow antibody-antigen binding. Non-binding antibodies or fragments thereof can be removed by washing the surface with an appropriate buffer (e.g., a solution buffered at physiological pH (approximately 7.4) and containing physiological salt concentrations and ionic strength, and optionally containing a detergent, such as TWEEN-20). Antibodies that remain bound can subsequently be detected, e.g., using an ELISA-based detection protocol (see, e.g., U.S. Pat. No. 4,661,445; incorporated herein by reference). Additional techniques for screening combinatorial libraries of polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) for those that specifically bind TNFR2-derived peptides (e.g., a peptide containing the amino acid sequence of SEQ ID NO: 285 or 286) include the screening of one-bead-one-compound libraries of antibody fragments. Antibody fragments can be chemically synthesized on a solid bead (e.g., using established split-and-pool solid phase peptide synthesis protocols) composed of a hydrophilic, water-swellable material such that each bead displays a single antibody fragment. Heterogeneous bead mixtures can then be incubated with a TNFR2-derived peptide that is optionally labeled with a detectable moiety (e.g., a fluorescent dye) or that is conjugated to an epitope tag (e.g., biotin, avidin, FLAG tag, HA tag) that can later be detected by treatment with a complementary tag (e.g., avidin, biotin, anti-FLAG antibody, anti-HA antibody, respectively). Beads containing antibody fragments that specifically bind a TNFR2-derived peptide (e.g., a peptide containing the amino acid sequence of SEQ ID NO: 285 or 286) can be identified by analyzing the fluorescent properties of the beads following incubation with a fluorescently-labeled antigen or complementary tag (e.g., by confocal fluorescent microscopy or by fluorescence-activated bead sorting; see, e.g., Muller et al. (J. Biol. Chem., 16500-16505, 1996); incorporated herein by reference). Beads containing antibody fragments that specifically bind TNFR2-derived peptides can thus be separated from those that do not contain high-affinity antibody fragments. The sequence of an antibody fragment that specifically binds a TNFR2-derived peptide can be determined by techniques known in the art, including, e.g., Edman degradation, tandem mass spectrometry, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), and 2D gel electrophoresis, among others (see, e.g., WO 2004/062553; incorporated herein by reference). Negative Screens of Polypeptides In addition to the above-described methods for screening for a single-chain polypeptide, antibody, or antibody fragment that specifically binds to an epitope derived from human TNFR2 that promotes receptor antagonism, one can additionally perform negative screens in order to eliminate antibodies or antibody fragments that may also bind an epitope that contains the KCSPG sequence (SEQ ID NO: 12). For instance, mixtures of antibodies or antibody fragments isolated as a result of any of the above-described screening techniques can be screened for antibodies or antibody fragments that also specifically bind to a peptide derived from human TNFR2 that contains the KCSPG motif, such as a peptide containing residues 48-67 of SEQ ID NO: 7 (QTAQMCCSKCSPGQHAKVFC, SEQ ID NO: 18). This can be accomplished using any of the above-described methods or variations thereof, e.g., such that the antibodies or antibody fragments being screened are those that were previously identified as being capable of specifically binding a peptide containing one or more residues of the KCRPG sequence (SEQ ID NO: 19)(e.g., at least the KCR sequence). Exemplary techniques useful for a negative screen include those described above or known in the art, such as phage display, yeast display, bacterial display, ribosome display, mRNA display, cDNA display, or surface-based combinatorial library screens (e.g., in an ELISA format). This screening technique represents a useful strategy for identifying an antagonistic TNFR2 antibody or antibody fragment, as antibodies or antibody fragments capable of binding TNFR2 epitopes containing the KCSPG sequence (SEQ ID NO: 12) and one or more residues of the KCRPG sequence (SEQ ID NO: 19) have been shown to lack, or to have significantly reduced, antagonistic activity. Immunization of a Non-Human Mammal Another strategy that can be used to produce antagonistic TNFR2 antibodies and antigen-binding fragments thereof of the invention includes immunizing a non-human mammal. Examples of non-human mammals that can be immunized in order to produce antagonistic TNFR2 antibodies and fragments thereof of the invention include rabbits, mice, rats, goats, guinea pigs, hamsters, horses, and sheep, as well as non-human primates. For instance, established procedures for immunizing primates are known in the art (see, e.g., WO 1986/6004782; incorporated herein by reference). Immunization represents a robust method of producing monoclonal antibodies by exploiting the antigen specificity of B lymphocytes. For example, monoclonal antibodies can be prepared by the Kohler-Millstein procedure (described, e.g., in EP 0110716; incorporated herein by reference), wherein spleen cells from a non-human animal (e.g., a primate) immunized with a peptide that presents a TNFR2-derived antigen that promotes receptor antagonism (e.g., a peptide containing the amino acid sequence of SEQ ID NO: 285 or 286). A clonally-expanded B lymphocyte produced by immunization can be isolated from the serum of the animal and subsequently fused with a myeloma cell in order to form a hybridoma. Hybridomas are particularly useful agents for antibody production, as these immortalized cells can provide a lasting supply of an antigen-specific antibody. Antibodies from such hybridomas can subsequently be isolated using techniques known in the art, e.g., by purifying the antibodies from the cell culture medium by affinity chromatography, using reagents such as Protein A or Protein G. Antagonistic TNFR2 Polypeptide Conjugates Prior to administration of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention to a mammalian subject (e.g., a human), it may be desirable to conjugate the antibody or fragment thereof to a second molecule, e.g., to modulate the activity of the antibody in vivo. Antagonistic TNFR2 antibodies and fragments thereof can be conjugated to other molecules at either the N-terminus or C-terminus of a light or heavy chain of the antibody using any one of a variety of established conjugation strategies that are well-known in the art. Examples of pairs of reactive functional groups that can be used to covalently tether an antagonistic TNFR2 antibody or fragment thereof to another molecule include, without limitation, thiol pairs, carboxylic acids and amino groups, ketones and amino groups, aldehydes and amino groups, thiols and alpha,beta-unsaturated moieties (such as maleimides or dehydroalanine), thiols and alpha-halo amides, carboxylic acids and hydrazides, aldehydes and hydrazides, and ketones and hydrazides. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can be covalently appended directly to another molecule by chemical conjugation as described. Alternatively, fusion proteins containing antagonistic TNFR2 antibodies and fragments thereof can be expressed recombinantly from a cell (e.g., a eukaryotic cell or prokaryotic cell). This can be accomplished, for example, by incorporating a polynucleotide encoding the fusion protein into the nuclear genome of a cell (e.g., using techniques described herein or known in the art). Optionally, antibodies and fragments thereof of the invention can be joined to a second molecule by forming a covalent bond between the antibody and a linker. This linker can then be subsequently conjugated to another molecule, or the linker can be conjugated to another molecule prior to ligation to the anti-TNFR2 antibody or fragment thereof. Examples of linkers that can be used for the formation of a conjugate include polypeptide linkers, such as those that contain naturally occurring or non-naturally occurring amino acids. In some embodiments, it may be desirable to include D-amino acids in the linker, as these residues are not present in naturally-occurring proteins and are thus more resistant to degradation by endogenous proteases. Fusion proteins containing polypeptide linkers can be made using chemical synthesis techniques, such as those described herein, or through recombinant expression of a polynucleotide encoding the fusion protein in a cell (e.g., a prokaryotic or eukaryotic cell). Linkers can be prepared using a variety of strategies that are well known in the art, and depending on the reactive components of the linker, can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012). Drug-Polypeptide Conjugates An antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, and antigen-binding fragment thereof) of the invention can additionally be conjugated to, admixed with, or administered separately from a therapeutic agent, such as a cytotoxic molecule. Conjugates of the invention may be applicable to the treatment or prevention of a disease associated with aberrant cell proliferation, such as a cancer described herein. Exemplary cytotoxic agents that can be conjugated to, admixed with, or administered separately from an antagonistic TNFR2 polypeptide include, without limitation, antineoplastic agents such as: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; adriamycin; aldesleukin; altretamine; ambomycin; a. metantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; camptothecin; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; combretestatin a-4; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daca (n-[2-(dimethyl-amino) ethyl] acridine-4-carboxamide); dactinomycin; daunorubicin hydrochloride; daunomycin; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; dolasatins; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; ellipticine; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; ethiodized oil i 131; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; 5-fdump; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; gold au 198; homocamptothecin; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta-i a; interferon gamma-ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peploycinsulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; rhizoxin; rhizoxin d; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium chloride sr 89; sulofenur; talisomycin; taxane; taxoid; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; thymitaq; tiazofurin; tirapazamine; tomudex; top53; topotecan hydrochloride; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine; vinblastine sulfate; vincristine; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride; 2-chlorodeoxyadenosine; 2′ deoxyformycin; 9-aminocamptothecin; raltitrexed; N-propargyl-5,8-dideazafolic acid; 2chloro-2′-arabino-fluoro-2′-deoxyadenosine; 2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R; CEP-751; linomide; sulfur mustard; nitrogen mustard (mechlor ethamine); cyclophosphamide; melphalan; chlorambucil; ifosfamide; busulfan; N-methyl-Nnitrosourea (MNU); N, N′-Bis (2-chloroethyl)-N-nitrosourea (BCNU); N-(2-chloroethyl)-N′ cyclohexyl-N-nitrosourea (CCNU); N-(2-chloroethyl)-N′-(trans-4-methylcyclohexyl-N-nitrosourea (MeCCNU); N-(2-chloroethyl)-N′-(diethyl) ethylphosphonate-N-nitrosourea (fotemustine); streptozotocin; diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C; AZQ; adozelesin; cisplatin; carboplatin; ormaplatin; oxaliplatin; C1-973; DWA 2114R; JM216; JM335; Bis (platinum); tomudex; azacitidine; cytarabine; gemcitabine; 6-mercaptopurine; 6-thioguanine; hypoxanthine; teniposide 9-amino camptothecin; topotecan; CPT-11; Doxorubicin; Daunomycin; Epirubicin; darubicin; mitoxantrone; losoxantrone; Dactinomycin (Actinomycin D); amsacrine; pyrazoloacridine; all-trans retinol; 14-hydroxy-retro-retinol; all-trans retinoic acid; N-(4-hydroxyphenyl) retinamide; 13-cis retinoic acid; 3-methyl TTNEB; 9-cis retinoic acid; fludarabine (2-F-ara-AMP); or 2-chlorodeoxyadenosine (2-Cda). Other therapeutic compounds that can be conjugated to, admixed with, or administered separately from an antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention in order to treat, prevent, or study the progression of a disease associated with aberrant cell proliferation include, but are not limited to, cytotoxic agents such as 20-pi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bleomycin A2; bleomycin B2; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives (e.g., 10-hydroxy-camptothecin); canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; 2′deoxycoformycin (DCF); deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; discodermolide; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epothilones (A, R═H; B, R=Me); epithilones; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide; etoposide 4′-phosphate (etopofos); exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; homoharringtonine (HHT); hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; ifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mithracin; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; podophyllotoxin; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single-chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. Labeled Anti-TNFR2 Polypeptides In some embodiments, antagonistic TNFR2 single-chain polypeptides, antibodies, or antigen-binding fragments thereof may be conjugated to another molecule (e.g., an epitope tag) for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is a common strategy that is employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Examples of epitope tag molecules that can be conjugated to antagonistic TNFR2 antibodies or fragments thereof for the purposes of molecular recognition include, without limitation, maltose-binding protein, glutathione-S-transferase, a poly-histidine tag, a FLAG-tag, a myc-tag, human influenza hemagglutinin (HA) tag, biotin, streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by such complementary molecules as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify an antagonistic TNFR2 antibody or fragment thereof of the invention that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc) by treating the mixture with a solid phase resin containing an complementary molecule that can selectively recognize and bind the epitope tag of the antagonistic anti-TNFR2 antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution. An antagonistic TNFR2 single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention can also be covalently appended to a fluorescent molecule, e.g., to detect the antibody or antigen-binding fragment thereof by fluorimetry and/or by direct visualization using fluorescence microscopy. Exemplary fluorescent molecules that can be conjugated to antibodies of the invention include green fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, red fluorescent protein, phycoerythrin, allophycocyanin, hoescht, 4′,6-diamidino-2-phenylindole (DAPI), propidium iodide, fluorescein, coumarin, rhodamine, tetramethylrhoadmine, and cyanine. Additional examples of fluorescent molecules suitable for conjugation to antibodies of the invention are well-known in the art and have been described in detail in, e.g., U.S. Pat. Nos. 7,417,131 and 7,413,874, each of which is incorporated by reference herein. Antagonistic TNFR2 polypeptides containing a fluorescent molecule are particularly useful for monitoring the cell-surface localization properties of antibodies and fragments thereof of the invention. For instance, one can expose cultured mammalian cells (e.g., T-reg cells) to antagonistic TNFR2 antibodies or fragments thereof of the invention that have been covalently conjugated to a fluorescent molecule and subsequently analyze these cells using conventional fluorescent microscopy techniques known in the art. Confocal fluorescent microscopy is a particularly powerful method for determining cell-surface localization of antagonistic anti-TNFR2 antibodies or fragments thereof, as individual planes of a cell can be analyzed in order to distinguish antibodies or fragments thereof that have been internalized into a cell's interior, e.g., by receptor-mediated endocytosis, from those that are bound to the external face of the cell membrane. Additionally, cells can be treated with antagonistic TNFR2 antibodies conjugated to a fluorescent molecule that emits visible light of a particular wavelength (e.g., fluorescein, which fluoresces at about 535 nm) and an additional fluorescent molecule that is known to localize to a particular site on the T-reg cell surface and that fluoresces at a different wavelength (e.g., a molecule that localizes to CD25 and that fluoresces at about 599 nm). The resulting emission patterns can be visualized by confocal fluorescence microscopy and the images from these two wavelengths can be merged in order to reveal information regarding the location of the antagonistic TNFR2 antibody or antigen-binding fragment thereof on the T-reg cell surface with respect to other receptors. Bioluminescent proteins can also be incorporated into a fusion protein for the purposes of detection and visualization of an antagonistic anti-TNFR2 polypeptide, such as a single-chain polypeptide, antibody, or fragment thereof. Bioluminescent proteins, such as Luciferase and aequorin, emit light as part of a chemical reaction with a substrate (e.g., luciferin and coelenterazine). Exemplary bioluminescent proteins suitable for use as a diagnostic sequence and methods for their use are described in, e.g., U.S. Pat. Nos. 5,292,658, 5,670,356, 6,171,809, and 7,183,092, each of which is herein incorporated by reference. Antagonistic TNFR2 antibodies or fragments thereof labeled with bioluminescent proteins are a useful tool for the detection of antibodies of the invention following an in vitro assay. For instance, the presence of an antagonistic TNFR2 antibody that has been conjugated to a bioluminescent protein can be detected among a complex mixture of additional proteins by separating the components of the mixture using gel electrophoresis methods known in the art (e.g., native gel analysis) and subsequently transferring the separated proteins to a membrane in order to perform a Western blot. Detection of the antagonistic TNFR2 antibody among the mixture of other proteins can be achieved by treating the membrane with an appropriate Luciferase substrate and subsequently visualizing the mixture of proteins on film using established protocols. The polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be conjugated to a molecule comprising a radioactive nucleus, such that an antibody or fragment thereof of the invention can be detected by analyzing the radioactive emission pattern of the nucleus. Alternatively, an antagonistic TNFR2 antibody or fragment thereof can be modified directly by incorporating a radioactive nucleus within the antibody during the preparation of the protein. Radioactive isotopes of methionine (35S), nitrogen (15N), or carbon (13C) can be incorporated into antibodies or fragments thereof of the invention by, e.g., culturing bacteria in media that has been supplemented with nutrients containing these isotopes. Optionally, tyrosine derivatives containing a radioactive halogen can be incorporated into an antagonistic TNFR2 antibody or fragment thereof by, e.g., culturing bacterial cells in media supplemented with radiolabeled tyrosine. It has been shown that tyrosine functionalized with a radioactive halogen at the C2 position of the phenol system are rapidly incorporated into elongating polypeptide chains using the endogenous translation enzymes in vivo (U.S. Pat. No. 4,925,651; incorporated herein by reference). The halogens include fluorine, chlorine, bromine, iodine, and astatine. Additionally, antagonistic TNFR2 antibodies or fragments thereof can be modified following isolation and purification from cell culture by functionalizing antibodies or fragments thereof of the invention with a radioactive isotope. The halogens represent a class of isotopes that can be readily incorporated into a purified protein by aromatic substitution at tyrosine or tryptophan, e.g., via reaction of one or more of these residues with an electrophilic halogen species. Examples of radioactive halogen isotopes include18F,75Br,77Br,122I,123I,124I,125I,129I,131I, or211At. Another alternative strategy for the incorporation of a radioactive isotope is the covalent attachment of a chelating group to the antagonistic anti-TNFR2 polypeptide, such as a single-chain polypeptide, antibody, or fragment thereof. Chelating groups can be covalently appended to an antagonistic TNFR2 antibody or fragment thereof by attachment to a reactive functional group, such as a thiol, amino group, alcohol, or carboxylic acid. The chelating groups can then be modified to contain any of a variety of metallic radioisotopes, including, without limitation, such radioactive nuclides as125I,67Ga,111In,99Tc,169Yb,186Re,123I,124I,125I,131I,99mTc,111In,64Cu,67Cu,186Re,188Re,177Lu,90Y,77As,72As, 86Y,89Zr,211At,212Bi,213Bi, or225Ac. In some embodiments, it may be desirable to covalently conjugate the polypeptides (e.g., single-chain polypeptides, antibodies, or fragments thereof) of the invention with a chelating group capable of binding a metal ion from heavy elements or rare earth ions, such as Gd3+, Fe3+, Mn3+, or Cr2+. Conjugates containing chelating groups that are coordinated to such paramagnetic metals are useful as in MRI imaging applications. Paramagnetic metals include, but are not limited to, chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), and ytterbium (III). In this way, antagonistic TNFR2 antibodies can be detected by MRI spectroscopy. For instance, one can administer antagonistic TNFR2 antibodies or fragments thereof conjugated to chelating groups bound to paramagnetic ions to a mammalian subject (e.g., a human patient) in order to monitor the distribution of the antibody following administration. This can be achieved by administration of the antibody to a patient by any of the administration routes described herein, such as intravenously, and subsequently analyzing the location of the administered antibody by recording an MRI of the patient according to established protocols. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can additionally be conjugated to other molecules for the purpose of improving the solubility and stability of the protein in aqueous solution. Examples of such molecules include PEG, PSA, bovine serum albumin (BSA), and human serum albumin (HSA), among others. For instance, one can conjugate an antagonistic TNFR2 antibody or fragment thereof to carbohydrate moieties in order to evade detection of the antibody or fragment thereof by the immune system of the patient receiving treatment. This process of hyperglycosylation reduces the immunogenicity of therapeutic proteins by sterically inhibiting the interaction of the protein with B cell receptors in circulation. Alternatively, antagonistic TNFR2 antibodies or fragments thereof can be conjugated to molecules that prevent clearance from human serum and improve the pharmacokinetic profile of antibodies of the invention. Exemplary molecules that can be conjugated to or inserted within anti-TNFR2 antibodies or fragments thereof of the invention so as to attenuate clearance and improve the pharmacokinetic profile of these antibodies and fragments include salvage receptor binding epitopes. These epitopes are found within the Fc region of an IgG immunoglobulin and have been shown to bind Fc receptors and prolong antibody half-life in human serum. The insertion of salvage receptor binding epitopes into anti-TNFR2 antibodies or fragments thereof can be achieved, e.g., as described in U.S. Pat. No. 5,739,277; incorporated herein by reference. Modified Antagonistic TFNR2 Polypeptides In addition to conjugation to other therapeutic agents and labels for identification or visualization, anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be modified so as to improve their pharmacokinetic profile, biophysical stability, or inhibitory capacity. For instance, any cysteine residue not involved in maintaining the proper conformation of the anti-TNFR2 antibody or fragment thereof may be substituted with an isosteric or isolectronic amino acid (e.g., serine) in order to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cystine bond(s) may be added to the antibody or fragment thereof to improve its stability (particularly where the antibody is an antibody fragment, such as an Fv fragment). This can be accomplished, e.g., by altering a polynucleotide encoding the antibody heavy and light chains or a polynucleotide encoding an antibody fragment so as to encode one or more additional pairs of cysteine residues that can form disulfide bonds under oxidative conditions in order to reinforce antibody tertiary structure (see, e.g., U.S. Pat. No. 7,422,899; incorporated herein by reference). Another useful modification that may be made to anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention includes altering the glycosylation profile of these antibodies and fragments thereof. This can be achieved, e.g., by substituting, inserting, or deleting amino acids in an antagonistic TNFR2 antibody so as to insert or remove a glycosylation site. Glycosylation of antibodies typically occurs in N-linked or O-linked fashion. N-linked glycosylation is a process whereby the attachment of a carbohydrate moiety to an antibody occurs at the side-chain of an asparagine residue. Consensus amino acid sequences for N-linked glycosylation include the tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X is any amino acid except proline. The insertion of either of these tripeptide sequences in a polypeptide (e.g., an anti-TNFR2 antibody) creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine are also competent substrates for glycoside formation. Addition of glycosylation sites to an anti-TNFR2 antibody can thus be accomplished by altering the amino acid sequence of the antibody (e.g., using recombinant expression techniques as described herein) such that it contains one or more of the above-described tripeptide sequences to promote N-linked glycosylation, or one or more serine or threonine residues to the sequence of the original antibody engender O-linked glycosylation (see, e.g., U.S. Pat. No. 7,422,899; incorporated herein by reference). In alternative cases, it may be desirable to modify the antibody or fragment thereof of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. For instance, cysteine residues may be introduced in the Fc region of an anti-TNFR2 antibody or fragment thereof (e.g., by recombinant expression techniques as described herein), so as to facilitate additional inter-chain disulfide bond formation in this region. The homodimeric antibody thus generated may have increased conformational constraint, which may foster improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Canc. Res., 53:2560-2565, 1993); incorporated herein by reference. Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities (see Stevenson et al. (Anti-Canc. Drug Des., 3:219-230, 1989); incorporated herein by reference). The serum half-life of anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be improved in some embodiments by incorporating one more amino acid modifications, such as by altering the CH1 or CL region of the Fab domain to introduce a salvage receptor motif, e.g., that found in the two loops of a CH2 domain of an Fc region of an IgG. Such alterations are described, for instance, in U.S. Pat. Nos. 5,869,046 and 6,121,022; incorporated herein by reference. Additional framework modifications can also be made to reduce immunogenicity of the antibody or fragment thereof or to reduce or remove T cell epitopes that reside therein, as described for instance in US2003/0153043; incorporated herein by reference. Methods of Treatment Antagonistic TNFR2 polypeptides, such a dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) that contain a CDR-H1 and CDR-H2 derived from a phenotype-neutral TNFR2 antibody and a CDR-H3 represented by the formula JZ1JZ2Z4JZ3JZ5(J)2Z5Z2Z5or JZ1JZ2Z4Z3Z5(J)2Z5Z2Z5(J)2(wherein each J is independently a naturally occurring amino acid, each Z1is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH, each Z2is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH, each Z3is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH, each Z4is independently a glycine or alanine, and each Z5is independently a naturally occurring amino acid containing a hydrophobic side-chain) can be used to treat a patient suffering from a cell proliferation disorder (such as a cancer described herein), an infectious disease (such as a viral, bacterial, fungal, or parasitic infection described herein), or another disease mediated by TNFR2 signaling. For instance, antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) that contain a CDR-H1 and CDR-H2 derived from a phenotype-neutral TNFR2 antibody and a CDR-H3 represented by the formula JRJDGJSJY(J)2FDJ (SEQ ID NO: 278), JRJDGSY(J)2FD(J)3(SEQ ID NO: 279), QZ1VZ2Z4YZ3SZ5WYZ5Z2Z5(SEQ ID NO: 265), or AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266) can be used to treat a patient suffering from a cell proliferation disorder (such as a cancer described herein), an infectious disease (such as a viral, bacterial, fungal, or parasitic infection described herein), or another disease mediated by TNFR2 signaling. In some embodiments, the CDR-H3 is be derived from TNFRAB1 and have the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25). The CDR-H3 may be derived from TNFRAB2 and have the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259). In some embodiments, the CDR-H3 is derived from TNFR2A3 and has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). Methods of Treating Cell Proliferation Disorders Antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention are useful therapeutics for the treatment of a wide array of cancers and cell proliferation disorders. Antagonistic TNFR2 polypeptides, such as dominant antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can be administered to a mammalian subject, such as a human, suffering from a cell proliferation disorder, such as cancer, e.g., to enhance the effectiveness of the adaptive immune response against the target cancer cells. In particular, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be administered to a mammalian subject, such as a human, to reduce or inhibit T-reg cell growth and activation, which allows tumor-infiltrating T-lymphocytes to localize to cells presenting tumor-associated antigens and to promote cytotoxicity. In addition, polypeptides of the invention may synergize with existing adoptive T cell therapy platforms, as one of the limitations on the effectiveness of this strategy has been the difficulty of prolonging cytotoxicity of tumor-reactive T cells following infusion into a mammalian subject (e.g., a human). Polypeptides of the invention may also promote the activity of allogeneic T-lymphocytes, which may express foreign MHC proteins and may be increasingly susceptible to inactivation by the host immune system. For example, antibodies and antigen-binding fragments thereof of the invention can mitigate the T-reg-mediated depletion of tumor-reactive T cells by suppressing the growth and proliferation of T-reg cells that typically accompanies T cell infusion. For instance, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may be capable of reducing the growth of T-reg cells by about 50% to about 200% relative to untreated cells (e.g., 50%, 75%, 100%, 125%, 150%, 175%, or 200%). The reduction in cellular growth does not require the presence of TNFα. In some embodiments, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may be capable of restricting the growth of T-reg cells in the presence of TNFα to between 90% and 150% relative to untreated cells (e.g., 90%, 100%, 110%, 120%, 130%, 140%, or 150%). Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention are also capable of restricting the proliferation of T-reg cells to less than 70% (e.g., 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%) of that of an untreated population of T-reg cells. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention are also capable of decreasing the survival of T-reg cells by about 10% (e.g., by about 20%, 30%, 40%, or 50%, or more) relative to an untreated population of T-reg cells. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be administered to a mammalian subject (e.g., a human) suffering from cancer in order to improve the condition of the patient by promoting the immune response against cancer cells and tumorogenic material. Antibodies of the invention can be administered to a subject, e.g., via any of the routes of administration described herein. Antibodies of the invention can also be formulated with excipients, biologically acceptable carriers, and may be optionally conjugated to, admixed with, or co-administered separately (e.g., sequentially) with additional therapeutic agents, such as anti-cancer agents. Cancers that can be treated by administration of antibodies or antigen-binding fragments thereof of the invention include such cancers as leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardiac cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cavity cancer, ocular cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and throat cancer. Particular cancers that can be treated by administration of antibodies or antigen-binding fragments thereof of the invention include, without limitation, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), adrenocortical carcinoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic cancer, ewing sarcoma family, osteosarcoma and malignant fibrous histiocytoma, central nervous system embryonal tumors, central nervous system germ cell tumors, craniopharyngioma, ependymoma, bronchial tumors, burkitt lymphoma, carcinoid tumor, primary lymphoma, chordoma, chronic myeloproliferative neoplasms, colon cancer, extrahepatic bile duct cancer, ductal carcinoma in situ (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, fallopian tube cancer, fibrous histiocytoma of bone, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), testicular germ cell tumor, gestational trophoblastic disease, glioma, childhood brain stem glioma, hairy cell leukemia, hepatocellular cancer, langerhans cell histiocytosis, hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, wilms tumor and other childhood kidney tumors, langerhans cell histiocytosis, small cell lung cancer, cutaneous T cell lymphoma, intraocular melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer, midline tract carcinoma, multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, myelodysplastic syndromes, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma (NHL), non-small cell lung cancer (NSCLC), epithelial ovarian cancer, germ cell ovarian cancer, low malignant potential ovarian cancer, pancreatic neuroendocrine tumors, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, kaposi sarcoma, rhabdomyosarcoma, sézary syndrome, small intestine cancer, soft tissue sarcoma, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Waldenström macroglobulinemia. For example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be administered to a patient (e.g., a mammalian patient, such as a human patient) in order to treat T cell lymphoma (e.g., Hodgkin's or cutaneous non-Hodgkin's lymphoma cells), ovarian cancer, colon cancer, multiple myeloma, or renal cell carcinoma. An anti-TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, or antigen-binding fragment thereof) of the invention can also be co-administered with a therapeutic antibody that exhibits reactivity towards a cancer cell. In this way, antagonistic TNFR2 polypeptides (e.g., antibodies or fragments thereof) of the invention may synergize not only with the adaptive immune response, e.g., by prolonging T-lymphocyte tumor reactivity, but also with other inhibitors of tumor cell growth. Examples of additional therapeutic antibodies that can be used to treat cancer and other cell proliferation disorders include those that exhibit reactivity with a tumor antigen or a cell-surface protein that is overexpressed on the surface of a cancer cell. Exemplary antibodies that can be admixed, co-administered, or sequentially administered with antagonistic TNFR2 antibodies of the invention include, without limitation, Trastuzamb (HERCEPTIN®), Bevacizumab (AVASTIN®), Cetuximab (ERBITUX®), Panitumumab (VECTIBIX®), Ipilimumab (YERVOY®), Rituximab (RITUXAN® and MABTHERA®), Alemtuzumab (CAMPATH®), Ofatumumab (ARZERRA®), Gemtuzumab ozogamicin (MYLOTARG®), Brentuximab vedotin (ADCETRIS®),90Y-Ibritumomab Tiuxetan (ZEVALIN®), and131I-Tositumomab (BEXXAR®), which are described in detail in Scott et al. (Cancer Immun., 12:14-21, 2012); incorporated herein by reference. A physician having ordinary skill in the art can readily determine an effective amount of an antagonistic TNFR2 polypeptide, such as single-chain polypeptide, antibody, or antibody fragment for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician could start prescribing doses of a polypeptide of the invention at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering an antagonistic TFNR2 polypeptide, such as a single-chain polypeptide, antibody, or antibody fragment at a high dose and subsequently administer progressively lower doses until a therapeutic effect is achieved (e.g., a reduction in the volume of one or more tumors, a decrease in the population of T-reg cells, or remission of a cell proliferation disorder). In general, a suitable daily dose of a single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention will be an amount of the compound which is the lowest dose effective to produce a therapeutic effect. An antibody or antigen-binding fragment thereof of the invention may be administered by injection, e.g., by intravenous, intramuscular, intraperitoneal, or subcutaneous injection, optionally proximal to the site of the target tissue (e.g., a tumor). A daily dose of a therapeutic composition of an antibody or antigen-binding fragment thereof of the invention may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for an antibody or fragment thereof of the invention to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents. Polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be monitored for their ability to attenuate the progression of a cell proliferation disease, such as cancer, by any of a variety of methods known in the art. For instance, a physician may monitor the response of a mammalian subject (e.g., a human) to treatment with a polypeptide, such as a single-chain polypeptide, antibody, or antibody fragment of the invention by analyzing the volume of one or more tumors in the patient. For example, polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention may be capable of reducing tumor volume by between 1% and 100% (e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%). Alternatively, a physician may monitor the responsiveness of a subject (e.g., a human) to treatment with antagonistic TNFR2 polypeptides, such as single-chain polypeptides, antibodies, or antigen-binding fragments thereof of the invention by analyzing the T-reg cell population in the lymph of a particular subject. For instance, a physician may withdraw a sample of blood from a mammalian subject (e.g., a human) and determine the quantity or density of T-reg cells (e.g., CD4+ CD25+ FOXP3+ T-reg cells or CD17+ T-reg cells) using established procedures, such as fluorescence activated cell sorting. Methods of Treating Infectious Diseases Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be used for treating infectious diseases, such as those caused by any one or more of a virus, a bacterium, a fungus, or a parasite (e.g., a eukaryotic parasite). For instance, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can be administered to a mammalian subject (e.g., a human) suffering from an infectious disease in order to treat the disease, as well as to alleviate one or more symptoms of the disease. For example, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be used for treating, or alleviating one or more symptoms of, viral infections in a mammalian subject, such as a human, that are caused by, e.g., a member of the Flaviviridae family (e.g., a member of the Flavivirus, Pestivirus, and Hepacivirus genera), which includes the hepatitis C virus, Yellow fever virus; Tick-borne viruses, such as the Gadgets Gully virus, Kadam virus, Kyasanur Forest disease virus, Langat virus, Omsk hemorrhagic fever virus, Powassan virus, Royal Farm virus, Karshi virus, tick-borne encephalitis virus, Neudoerfl virus, Sofjin virus, Louping ill virus and the Negishi virus; seabird tick-borne viruses, such as the Meaban virus, Saumarez Reef virus, and the Tyuleniy virus; mosquito-borne viruses, such as the Aroa virus, dengue virus, Kedougou virus, Cacipacore virus, Koutango virus, Japanese encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, West Nile virus, Yaounde virus, Kokobera virus, Bagaza virus, Ilheus virus, Israel turkey meningoencephalo-myelitis virus, Ntaya virus, Tembusu virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, yellow fever virus; and viruses with no known arthropod vector, such as the Entebbe bat virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis leukoencephalitis virus, Phnom Penh bat virus, Rio Bravo virus, Tamana bat virus, and the Cell fusing agent virus; a member of the Arenaviridae family, which includes the Ippy virus, Lassa virus (e.g., the Josiah, LP, or GA391 strain), lymphocytic choriomeningitis virus (LCMV), Mobala virus, Mopeia virus, Amapari virus, Flexal virus, Guanarito virus, Junin virus, Latino virus, Machupo virus, Oliveros virus, Parand virus, Pichinde virus, Pirital virus, Sabid virus, Tacaribe virus, Tamiami virus, Whitewater Arroyo virus, Chapare virus, and Lujo virus; a member of the Bunyaviridae family (e.g., a member of the Hantavirus, Nairovirus, Orthobunyavirus, and Phlebovirus genera), which includes the Hantaan virus, Sin Nombre virus, Dugbe virus, Bunyamwera virus, Rift Valley fever virus, La Crosse virus, California encephalitis virus, and Crimean-Congo hemorrhagic fever (CCHF) virus; a member of the Filoviridae family, which includes the Ebola virus (e.g., the Zaire, Sudan, Ivory Coast, Reston, and Uganda strains) and the Marburg virus (e.g., the Angola, Ci67, Musoke, Popp, Ravn and Lake Victoria strains); a member of the Togaviridae family (e.g., a member of the Alphavirus genus), which includes the Venezuelan equine encephalitis virus (VEE), Eastern equine encephalitis virus (EEE), Western equine encephalitis virus (WEE), Sindbis virus, rubella virus, Semliki Forest virus, Ross River virus, Barmah Forest virus, O‘nyong’nyong virus, and the chikungunya virus; a member of the Poxviridae family (e.g., a member of the Orthopoxvirus genus), which includes the smallpox virus, monkeypox virus, and vaccinia virus; a member of the Herpesviridae family, which includes the herpes simplex virus (HSV; types 1, 2, and 6), human herpes virus (e.g., types 7 and 8), cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster virus, and Kaposi's sarcoma associated-herpesvirus (KSHV); a member of the Orthomyxoviridae family, which includes the influenza virus (A, B, and C), such as the H5N1 avian influenza virus or H1N1 swine flu; a member of the Coronaviridae family, which includes the severe acute respiratory syndrome (SARS) virus; a member of the Rhabdoviridae family, which includes the rabies virus and vesicular stomatitis virus (VSV); a member of the Paramyxoviridae family, which includes the human respiratory syncytial virus (RSV), Newcastle disease virus, hendravirus, nipahvirus, measles virus, rinderpest virus, canine distemper virus, Sendai virus, human parainfluenza virus (e.g., 1, 2, 3, and 4), rhinovirus, and mumps virus; a member of the Picornaviridae family, which includes the poliovirus, human enterovirus (A, B, C, and D), hepatitis A virus, and the coxsackievirus; a member of the Hepadnaviridae family, which includes the hepatitis B virus; a member of the Papillamoviridae family, which includes the human papilloma virus; a member of the Parvoviridae family, which includes the adeno-associated virus; a member of the Astroviridae family, which includes the astrovirus; a member of the Polyomaviridae family, which includes the JC virus, BK virus, and SV40 virus; a member of the Calciviridae family, which includes the Norwalk virus; a member of the Reoviridae family, which includes the rotavirus; and a member of the Retroviridae family, which includes the human immunodeficiency virus (HIV; e.g., types 1 and 2), and human T-lymphotropic virus Types I and II (HTLV-1 and HTLV-2, respectively); Friend Leukemia Virus; and transmissible spongiform encephalopathy, such as chronic wasting disease. Particularly, methods of the invention include administering an antagonistic TNFR2 antibody (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7), such as a TNFR2 antibody that contains the CDR-H3 sequence or a variant thereof of TNFRAB1, TNFRAB2, or TNFR2A3) to a human in order to treat an HIV infection (such as a human suffering from AIDS). Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be used for treating, or alleviating one or more symptoms of, bacterial infections in a mammalian subject (e.g., a human). Examples of bacterial infections that may be treated by administration of an antagonistic TNFR2 polypeptide, such as a single-chain polypeptide, antibody, or antibody fragment of the invention include, without limitation, those caused by bacteria within the generaStreptococcus, Bacillus, Listeria, Corynebacterium, Nocardia, Neisseria, Actinobacter, Moraxella, Enterobacteriacece (e.g.,E. coli, such as O157:H7),Pseudomonas(such asPseudomonas aeruginosa),Escherichia, Klebsiella, Serratia, Enterobacter, Proteus, Salmonella, Shigella, Yersinia, Haemophilus, Bordetella(such asBordetella pertussis),Legionella, Pasteurella, Francisella, Brucella, Bartonella, Clostridium, Vibrio, Campylobacter, Staphylococcus, Mycobacterium(such asMycobacterium tuberculosisandMycobacterium aviumparatuberculosis, andHelicobacter(such asHelicobacter pyloriandHelicobacter hepaticus). Particularly, methods of the invention include administering an antagonistic TNFR2 polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof that contains a CDR-H3 region as described herein (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7), such as a TNFR2 antibody that contains the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3) to a human or a non-human mammal in order to treat aMycobacterium tuberculosisinfection. Particular methods of the invention include administering an antagonistic TNFR2 polypeptide (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7), such as a TNFR2 antibody that contains the CDR-H3 sequence or a variant thereof of TNFRAB1, TNFRAB2, or TNFR2A3) to bovine mammals or bison in order to treat aMycobacterium tuberculosisinfection. Additionally, methods of the invention include administering an antagonistic TNFR2 polypeptide (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7), such as a TNFR2 antibody that contains the CDR-H3 sequence or a variant thereof of TNFRAB1, TNFRAB2, or TNFR2A3) to a human or a non-human mammal in order to treat aMycobacterium aviumparatuberculosis infection. Particular methods of the invention include administering an antagonistic TNFR2 polypeptide (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7), such as a TNFR2 antibody that contains the CDR-H3 sequence or a variant thereof of TNFRAB1, TNFRAB2, or TNFR2A3) to bovine mammals or bison in order to treat aMycobacterium aviumparatuberculosis infection. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can also be administered to a mammalian subject (e.g., a human) for treating, or alleviating one or more symptoms of, parasitic infections caused by a protozoan parasite (e.g., an intestinal protozoa, a tissue protozoa, or a blood protozoa) or a helminthic parasite (e.g., a nematode, a helminth, an adenophorea, a secementea, a trematode, a fluke (blood flukes, liver flukes, intestinal flukes, and lung flukes), or a cestode). Exemplary protozoan parasites that can be treated according to the methods of the invention include, without limitation,Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Leishmania major, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Plasmodium yoelli, Trichomonas vaginalis, andHistomonas meleagridis. Exemplary helminthic parasites include richuristrichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, andDracunculus medinensis, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes,Paragonimus westermani, Taenia solium, Taenia saginata, Hymenolepis nana, andEchinococcus granulosus. Additional parasitic infections that can be treated according to the methods of the invention includeOnchocercas volvulus. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can also be administered to a mammalian subject (e.g., a human) in order to treat, or to alleviate one or more symptoms of, fungal infections. Examples of fungal infections that may be treated according to the methods of the invention include, without limitation, those caused by, e.g.,Aspergillus, Candida, Malassezia, Trichosporon, Fusarium, Acremonium, Rhizopus, Mucor, Pneumocystis, andAbsidia. Exemplary fungal infections that can be treated according to the methods of the invention also includePneumocystis carinii, Paracoccidioides brasiliensisandHistoplasma capsulatum. Pharmaceutical Compositions Pharmaceutical compositions containing an antagonistic TNFR2 polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention can be prepared using methods known in the art. Pharmaceutical compositions of the invention may contain an antagonistic TNFR2 antibody, such as a dominant antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or antigen-binding fragment thereof) that contains a CDR-H1 and CDR-H2 derived from a phenotype-neutral TNFR2 antibody and a CDR-H3 represented by the formula JZ1JZ2Z4JZ3JZ5(J)2Z5Z2Z5or JZ1JZ2Z4Z3Z5(J)2Z5Z2Z5(J)2(wherein each J is independently a naturally occurring amino acid, each Z1is independently a naturally occurring amino acid containing a cationic side-chain at physiological pH, each Z2is independently a naturally occurring amino acid containing an anionic side-chain at physiological pH, each Z3is independently a naturally occurring amino acid containing a polar, uncharged side-chain at physiological pH, each Z4is independently a glycine or alanine, and each Z5is independently a naturally occurring amino acid containing a hydrophobic side-chain). For instance, pharmaceutical compositions of the invention may contain an antagonistic TNFR2 polypeptide, such as a dominant antagonistic TNFR2 polypeptide (e.g., single-chain polypeptides, antibodies, or antigen-binding fragments thereof) that contains a CDR-H1 and CDR-H2 derived from a phenotype-neutral TNFR2 antibody and a CDR-H3 represented by the formula JRJDGJSJY(J)2FDJ (SEQ ID NO: 278), JRJDGSY(J)2FD(J)3(SEQ ID NO: 279), QZ1VZ2Z4YZ3SZ5WYZ5Z2Z5(SEQ ID NO: 265), or AZ1DZ2Z4Z3Z5SPZ5Z2Z5WG (SEQ ID NO: 266). In some embodiments, the CDR-H3 is be derived from TNFRAB1 and have the amino acid sequence QRVDGYSSYWYFDV (SEQ ID NO: 25). The CDR-H3 may be derived from TNFRAB2 and have the amino acid sequence ARDDGSYSPFDYWG (SEQ ID NO: 259). In some embodiments, the CDR-H3 is derived from TNFR2A3 and has the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284). Pharmaceutical compositions of the invention can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980); incorporated herein by reference), and in a desired form, e.g., in the form of lyophilized formulations or aqueous solutions. The compositions can also be prepared so as to contain the active agent (e.g., an antagonistic anti-TNFR2 antibody or fragment thereof) at a desired concentration. For example, a pharmaceutical composition of the invention may contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) active agent by weight (w/w). Additionally, an active agent (e.g., an antagonistic TNFR2 polypeptide of the invention, such as a dominant antagonistic TNFR2 polypeptide of the invention) that can be incorporated into a pharmaceutical formulation can itself have a desired level of purity. For example, a polypeptide, such as a single-chain polypeptide, antibody, or antigen-binding fragment thereof of the invention may be characterized by a certain degree of purity after isolating the antibody from cell culture media or after chemical synthesis, e.g., of a single-chain antibody fragment (e.g., scFv) by established solid phase peptide synthesis methods or native chemical ligation as described herein. An antagonistic TNFR2 polypeptide of the invention may be at least 10% pure prior to incorporating the antibody into a pharmaceutical composition (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure). Pharmaceutical compositions of anti-TNFR2 polypeptides of the invention can be prepared for storage as lyophilized formulations or aqueous solutions by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art, e.g., buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants, and other miscellaneous additives. See, e.g., Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980; incorporated herein by reference). Such additives must be nontoxic to the recipients at the dosages and concentrations employed. Buffering Agents Buffering agents help to maintain the pH in the range which approximates physiological conditions. They can be present at concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use with antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention include both organic and inorganic acids and salts thereof such as citrate buffers {e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers {e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers {e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers {e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer {e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers {e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers {e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Additionally, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used. Preservatives Preservatives can be added to a composition of the invention to retard microbial growth, and can be added in amounts ranging from 0.2%-1% (w/v). Suitable preservatives for use with antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides {e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonifiers sometimes known as “stabilizers” can be added to ensure isotonicity of liquid compositions of the invention and include polhydric sugar alcohols, for example trihydric or higher sugar alcohols, such as glycerin, arabitol, xylitol, sorbitol and mannitol. Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols (enumerated above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thio sulfate; low molecular weight polypeptides (e.g., peptides of 10 residues or fewer); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers can be present in the range from 0.1 to 10,000 weights per part of weight active protein. Detergents Non-ionic surfactants or detergents (also known as “wetting agents”) can be added to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stressed without causing denaturation of the protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Non-ionic surfactants can be present in a range of about 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL to about 0.2 mg/mL. Additional miscellaneous excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and cosolvents. Other Pharmaceutical Carriers Alternative pharmaceutically acceptable carriers that can be incorporated into a composition of the invention may include dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. A composition containing an antagonistic TNFR2 antibody of the invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found inRemington's Pharmaceutical Sciences(19th ed., 1995), which is incorporated herein by reference. Compositions and Methods for Combination Therapy Pharmaceutical compositions of the invention may optionally include more than one active agent. For instance, compositions of the invention may contain an antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, or antigen-binding fragment thereof) conjugated to, admixed with, or administered separately from another pharmaceutically active molecule, e.g., a cytotoxic agent, an antibiotic, or a T-lymphocyte (e.g., a gene-edited T-lymphocyte for use in CAR-T therapy). For instance, an antagonistic TNFR2 polypeptide or therapeutic conjugate thereof (e.g., a drug-antibody conjugate described herein), may be admixed with one or more additional active agents that can be used to treat cancer or another cell proliferation disorder (e.g., neoplasm). Alternatively, pharmaceutical compositions of the invention may be formulated for co-administration or sequential administration with one or more additional active agents that can be used to treat cancer or other cell proliferation disorders. Examples of additional active agents that can be used to treat cancer and other cell proliferation disorders and that can be conjugated to, admixed with, or administered separately from an antagonistic TNFR2 polypeptide of the invention include cytotoxic agents (e.g., those described herein), as well as antibodies that exhibit reactivity with a tumor antigen or a cell-surface protein that is overexpressed on the surface of a cancer cell. Exemplary antibodies that can be conjugated to, admixed with, or administered separately from antagonistic TNFR2 antibodies of the invention include, without limitation, Trastuzamb (HERCEPTIN®), Bevacizumab (AVASTIN®), Cetuximab (ERBITUX®), Panitumumab (VECTIBIX®), Ipilimumab (YERVOY®), Rituximab (RITUXAN® and MABTHERA®), Alemtuzumab (CAMPATH®), Ofatumumab (ARZERRA®), Gemtuzumab ozogamicin (MYLOTARG®), Brentuximab vedotin (ADCETRIS®),90Y-Ibritumomab Tiuxetan (ZEVALIN®), and131I-Tositumomab (BEXXAR®), which are described in detail in Scott et al. (Cancer Immun., 12:14-21, 2012); incorporated herein by reference. Additional agents that can be conjugated to, admixed with, or administered separately from antagonistic TNFR2 polypeptides of the invention include T-lymphocytes that exhibit reactivity with a specific antigen associated with a particular pathology. For instance, antagonistic TNFR2 polypeptides of the invention can be formulated for administration with a T cell that expresses a chimeric antigen receptor (CAR-T) in order to treat a cell proliferation disorder, such as a cancer described herein. Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) can synergize with CAR-T therapy by preventing T-reg cells from deactivating T-lymphocytes that have been genetically modified so as to express tumor-reactive antigen receptors. In this way, CAR-T cells can be administered to a patient prior to, concurrently with, or after administration of an antagonistic TNFR2 polypeptide in order to treat a mammalian subject (e.g., a human) suffering from a cell proliferation disorder, such as cancer. CAR-T therapy is a particularly robust platform for targeting cancer cells in view of the ability to genetically engineer T-lymphocytes to express an antigen receptor specific to a tumor-associated antigen. For instance, identification of antigens overexpressed on the surfaces of tumors and other cancer cells can inform the design and discovery of chimeric T cell receptors, which are often composed of cytoplasmic and transmembrane domains derived from a naturally-occurring T cell receptor operatively linked to an extracellular scFv fragment that specifically binds to a particular antigenic peptide. T cells can be genetically modified in order to express an antigen receptor that specifically binds to a particular tumor antigen by any of a variety of genome editing techniques described herein or known in the art. Exemplary techniques for modifying a T cell genome so as to incorporate a gene encoding a chimeric antigen receptor include the CRISPER/Cas, zinc finger nuclease, TALEN, ARCUS™ platforms described herein. Methods for the genetic engineering of CAR-T lymphocytes have been described, e.g., in WO 2014/127261, WO 2014/039523, WO 2014/099671, and WO 20120790000; the disclosures of each of which are incorporated by reference herein. CAR-T cells useful in the compositions and methods of the invention include those that have been genetically modified such that the cell does not express the endogenous T cell receptor. For instance, a CAR-T cell may be modified by genome-editing techniques, such as those described herein, so as to suppress expression of the endogenous T cell receptor in order to prevent graft-versus-host reactions in a patient receiving a CAR-T infusion. Additionally or alternatively, CAR-T cells can be genetically modified so as to reduce the expression of one or more endogenous MHC proteins. This is a particularly useful technique for the infusion of allogeneic T-lymphocytes, as recognition of foreign MHC proteins represents one mechanism that promotes allograft rejection. One of skill in the art can also modify a T-lymphocyte so as to suppress the expression of immune suppressor proteins, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). These proteins are cell surface receptors that, when activated, attenuate T cell activation. Infusion of CAR-T cells that have been genetically modified so as to diminish the expression of one or more immunosupressor proteins represents one strategy that can be used to prolong the T-lymphocyte-mediated cytotoxicity in vivo. In addition to deleting specific genes, one can also modify CAR-T cells in order to express a T cell receptor with a desired antigen specificity. For instance, one can genetically modify a T-lymphocyte in order to express a T cell receptor that specifically binds to a tumor-associated antigen in order to target infused T cells to cancer cells. An exemplary T cell receptor that may be expressed by a CAR-T cell is one that binds PD-L1, a cell surface protein that is often overexpressed on various tumor cells. As PD-L1 activates PD-1 on the surface of T-lymphocytes, targeting this tumor antigen with CAR-T therapy can synergize with antagonistic TNFR2 antibodies or antibody fragments of the invention in order to increase the duration of an immune response mediated by a T-lymphocyte in vivo. CAR-T cells can also be modified so as to express a T cell receptor that specifically binds an antigen associated with one or more infectious disease, such as an antigen derived from a viral protein, a bacterial cell, a fungus, or other parasitic organism. Other pharmaceutical compositions of the invention include those that contain an antagonistic TNFR2 antibody or antibody fragment, interferon alpha, and/or one or more antibiotics that can be administered to a patient (e.g., a human patient) suffering from an infectious disease. For instance, an antagonistic TNFR2 antibody or antibody fragment can be conjugated to, admixed with, or administered separately from an antibiotic useful for treating one or more infectious diseases, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem, meropenem, cefadroxil, cefazolin, cefazlexin, cefaclor, cefoxitin, cefprozil, cefuroxime, cefdinir, cefditoren, cefoperazone, clindamycin, lincomycin, daptomycin, erythromycin, linezolid, torezolid, amoxicillin, ampicillin, bacitracin, ciprofloxacin, doxycycline, and tetracycline, among others. Immunotherapy Agents An antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) described herein may be admixed, conjugated, administered with, or administered separately from, an immunotherapy agent, for instance, for the treatment of a cancer or infectious disease, such as a cancer or infectious disease described herein. Exemplary immunotherapy agents useful in conjunction with the compositions and methods of the invention include, without limitation, an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, an anti-TNF-α cross-linking agent, an anti-TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1 BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILRi agent, an anti-TRAILR2 agent, and an anti-TWEAKR agent, as well as, for example, agents directed toward the immunological targets described in Table 1 of Mahoney et al., Cancer Immunotherapy, 14:561-584 (2015), the disclosure of which is incorporated herein by reference in its entirety. For instance, immunological target 4-1 BB ligand may be targeted with an anti-4-1 BB ligand antibody; immunological target OX40L may be targeted with an anti-OX40L antibody; immunological target GITR may be targeted with an anti-GITR antibody; immunological target CD27 may be targeted with an anti-CD27 antibody; immunological target TL1A may be targeted with an anti-TL1A antibody; immunological target CD40L may be targeted with an anti-CD40L antibody; immunological target LIGHT may be targeted with an anti-LIGHT antibody; immunological target BTLA may be targeted with an anti-BTLA antibody; immunological target LAG3 may be targeted with an anti-LAG3 antibody; immunological target TIM3 may be targeted with an anti-TIM3 antibody; immunological target Singlecs may be targeted with an anti-Singlecs antibody; immunological target ICOS ligand may be targeted with an anti-ICOS ligand antibody; immunological target B7-H3 may be targeted with an anti-B7-H3 antibody; immunological target B7-H4 may be targeted with an anti-B7-H4 antibody; immunological target VISTA may be targeted with an anti-VISTA antibody; immunological target TMIGD2 may be targeted with an anti-TMIGD2 antibody; immunological target BTNL2 may be targeted with an anti-BTNL2 antibody; immunological target CD48 may be targeted with an anti-CD48 antibody; immunological target KIR may be targeted with an anti-KIR antibody; immunological target LIR may be targeted with an anti-LIR antibody; immunological target ILT may be targeted with an anti-ILT antibody; immunological target NKG2D may be targeted with an anti-NKG2D antibody; immunological target NKG2A may be targeted with an anti-NKG2A antibody; immunological target MICA may be targeted with an anti-MICA antibody; immunological target MICB may be targeted with an anti-MICB antibody; immunological target CD244 may be targeted with an anti-CD244 antibody; immunological target CSF1R may be targeted with an anti-CSF1R antibody; immunological target IDO may be targeted with an anti-IDO antibody; immunological target TGFβ may be targeted with an anti-TGFβ antibody; immunological target CD39 may be targeted with an anti-CD39 antibody; immunological target CD73 may be targeted with an anti-CD73 antibody; immunological target CXCR4 may be targeted with an anti-CXCR4 antibody; immunological target CXCL12 may be targeted with an anti-CXCL12 antibody; immunological target SIRPA may be targeted with an anti-SIRPA antibody; immunological target CD47 may be targeted with an anti-CD47 antibody; immunological target VEGF may be targeted with an anti-VEGF antibody; and immunological target neuropilin may be targeted with an anti-neuropilin antibody (see, e.g., Table 1 of Mahoney et al.). Additional examples of immunotherapy agents that can be used in conjunction with the compositions and methods described herein include TARGRETIN®, Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, gabapentin, antibodies to lymphoid cell surface receptors and/or lymphokines, antibodies to surface cancer proteins, and/or small molecular therapies like Vorinostat. Using the methods of the invention, an antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) described herein may be co-administered with (e.g., admixed with) or administered separately from an immunotherapy agent. For example, an antagonistic TNFR2 polypeptide of the invention (such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to a patient, such as a human patient suffering from a cancer or infectious disease, simultaneously or at different times. In some embodiments, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) is administered to the patient prior to administration of an immunotherapy agent to the patient. Alternatively, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to the patient after an immunotherapy agent. For example, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to the patient after a failed immunotherapy treatment. A physician of skill in the art can monitor the efficacy of immunotherapy treatment to determine whether the therapy has successfully ameliorated the pathology being treated (such as a cancer or infectious disease, e.g., a cancer or infectious disease described herein) using methods described herein and known in the art. For instance, a physician of skill in the art may monitor the quantity of cancer cells in a sample isolated from a patient (e.g., a blood sample or biopsy sample), such as a human patient, for instance, using flow cytometry or FACS analysis. Additionally or alternatively, a physician of skill in the art can monitor the progression of a cancerous disease in a patient, for instance, by monitoring the size of one or more tumors in the patient, for example, by CT scan, MRI, or X-ray analysis. A physician of skill in the art may monitor the progression of a cancer, such as a cancer described herein, by evaluating the quantity and/or concentration of tumor biomarkers in the patient, such as the quantity and/or concentration of cell surface-bound tumor associated antigens or secreted tumor antigens present in the blood of the patient as an indicator of tumor presence. A finding that the quantity of cancer cells, the size of a tumor, and/or the quantity or concentration of one or more tumor antigens present in the patient or in a sample isolated from the patient has not decreased, for instance, by a statistically significant amount following administration of the immunotherapy agent within a specified time period (e.g., from 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicate that the immunotherapy treatment has failed to ameliorate the cancer. Based on this indication, a physician of skill in the art may administer an antagonistic TNFR2 polypeptide of the invention, such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein. Similarly, a physician a physician of skill in the art may monitor the quantity of bacterial, fungal, or parasitic cells, or the quantity of viral particles in a sample isolated from a patient suffering from an infectious disease, such as an infectious disease described herein. Additionally or alternatively, a physician of skill in the art may monitor the progression of an infectious disease by evaluating the symptoms of a patient suffering from such a pathology. For instance, a physician may monitor the patient by determining whether the frequency and/or severity of one or more symptoms of the infectious disease have stabilized (e.g., remained the same) or decreased following treatment with an immunotherapy agent. A finding that the quantity of bacterial, fungal, or parasitic cells or viral particles in a sample isolated from the patient and/or a finding that the frequency or severity of one or more symptoms of the infectious disease have not decreased, for instance, by a statistically significant amount following administration of the immunotherapy agent within a specified time period (e.g., from 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicate that the immunotherapy treatment has failed to ameliorate the infectious disease. Based on this indication, a physician of skill in the art may administer an antagonistic TNFR2 polypeptide of the invention, such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein. Chemotherapy Agents and Radiation Therapy Additionally or alternatively, an antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) described herein may be admixed, conjugated, administered with, or administered separately from, a chemotherapy agent, for example, for the treatment of cancer, such as a cancer described herein. Exemplary chemotherapy agents useful in conjunction with the compositions and methods of the invention include, without limitation, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin), ADRIAMYCIN®, bleomycin, vinblastine, and dacarbazine (ABVD), ADRIAMYCIN®, bleomycin, vincristine sulfate, and etoposide phosphate (ABVE), ADRIAMYCIN®, bleomycin, vincristine sulfate, etoposide phosphate, prednisone, and cyclophosphamide (ABVE-PC), doxorubicin and cyclophosphamide (AC), doxorubicin, cyclophosphamide, and paclitaxel or docetaxel (AC-T), ADCETRIS® (Brentuximab Vedotin), cytarabine, daunorubicin, and etoposide (ADE), ado-trastuzumab emtansine, ADRIAMYCIN® (doxorubicin hydrochloride), afatinib dimaleate, AFINITOR® (Everolimus), AKYNZEO® (netupitant and palonosetron hydrochloride), ALDARA® (imiquimod), aldesleukin, ALECENSA® (alectinib), alectinib, alemtuzumab, ALKERAN® for Injection (Melphalan Hydrochloride), ALKERAN® tablets (melphalan), ALIMTA® (pemetrexed disodium), ALOXI® (palonosetron hydrochloride), AMBOCHLORIN® (chlorambucil), AMBOCLORIN® (Chlorambucil), aminolevulinic acid, anastrozole, aprepitant, AREDIA® (pamidronate disodium), ARIMIDEX® (anastrozole), AROMASIN® (exemestane), ARRANON® (nelarabine), arsenic trioxide, ARZERRA® (ofatumumab), asparaginaseErwinia chrysanthemi, AVASTIN® (bevacizumab), axitinib, azacitidine, BEACOPP Becenum (carmustine), BELEODAQ® (Belinostat), belinostat, bendamustine hydrochloride, bleomycin, etoposide, and cisplatin (BEP), bevacizumab, bexarotene, BEXXAR® (tositumomab and iodine131I tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, BLINCYTO® (blinatumomab), bortezomib, BOSULIF® (bosutinib), bosutinib, brentuximab vedotin, busulfan, BUSULFEX® (busulfan), cabazitaxel, cabozantinib-S-malate, CAF, CAMPATH® (alemtuzumab), CAMPTOSAR® (irinotecan hydrochloride), capecitabine, CAPOX, CARAC® (fluorouracil), carboplatin, CARBOPLATIN-TAXOL®, carfilzomib, CARMUBRIS® (carmustine), carmustine, carmustine implant, CASODEX® (bicalutamide), CEENU (lomustine), cisplatin, etoposide, and methotrexate (CEM), ceritinib, CERUBIDINE® (daunorubicin hydrochloride), CERVARIX® (recombinant HPV bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, CLAFEN® (cyclophosphamide), clofarabine, CLOFAREX® (clofarabine), CLOLAR® (Clofarabine), CMF, cobimetinib, cometriq (cabozantinib-S-malate), COPDAC, COPP, COPP-ABV, COSMEGEN® (dactinomycin), COTELLIC® (cobimetinib), crizotinib, CVP, cyclophosphamide, CYFOS® (ifosfamide), CYRAMZA® (ramucirumab), cytarabine, cytarabine liposome, CYTOSAR-U® (cytarabine), CYTOXAN® (cyclophosphamide), dabrafenib, dacarbazine, DACOGEN® (decitabine), dactinomycin, daratumumab, DARZALEX® (daratumumab), dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DEPOCYT® (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, DOXIL® (doxorubicin hydrochloride), doxorubicin hydrochloride, DOX-SL® (doxorubicin hydrochloride), DTIC-DOME® (dacarbazine), EFUDEX (fluorouracil), ELITEK® (rasburicase), ELLENCE® (epirubicin hydrochloride), elotuzumab, ELOXATIN® (oxaliplatin), eltrombopag olamine, EMEND® (aprepitant), EMPLICITI® (elotuzumab), enzalutamide, epirubicin hydrochloride, EPOCH, ERBITUX® (cetuximab), eribulin mesylate, ERIVEDGE® (vismodegib), erlotinib hydrochloride, ERWINAZE® (asparaginaseErwinia chrysanthemi), ETOPOPHOS® (etoposide phosphate), etoposide, etoposide phosphate, EVACET® (doxorubicin hydrochloride liposome), everolimus, EVISTA® (raloxifene hydrochloride), EVOMELA® (melphalan hydrochloride), exemestane, 5-FU (5-fluorouracil), FARESTON® (toremifene), FARYDAK® (panobinostat), FASLODEX® (fulvestrant), FEC, FEMARA® (letrozole), filgrastim, FLUDARA® (fludarabine phosphate), fludarabine phosphate, FLUOROPLEX® (fluorouracil), fluorouracil injection, flutamide, FOLEX® (methotrexate), FOLEX® PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, FOLOTYN® (pralatrexate), FU-LV, fulvestrant, GARDASIL® (recombinant HPV quadrivalent vaccine), GARDASIL 9® (recombinant HPV nonavalent vaccine), GAZYVA® (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, GEMZAR® (gemcitabine hydrochloride), GILOTRIF® (afatinib dimaleate), GLEEVEC® (imatinib mesylate), GLIADEL® (carmustine implant), GLIADEL® wafer (carmustine implant), glucarpidase, goserelin acetate, HALAVEN® (eribulin mesylate), HERCEPTIN® (trastuzumab), HPV bivalent vaccine, HYCAMTIN® (topotecan hydrochloride), Hyper-CVAD, IBRANCE (palbociclib), IBRITUMOMAB® tiuxetan, ibrutinib, ICE, ICLUSIG® (ponatinib hydrochloride), IDAMYCIN® (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, IFEX® (ifosfamide), ifosfamide, ifosfamidum, IL-2 (aldesleukin), imatinib mesylate, IMBRUVICA® (ibrutinib), ilmiquimod, IMLYGIC® (talimogene laherparepvec), INLYTA (axitinib), recombinant interferon alpha-2b, intron A, tositumomab, such as131I tositumomab, ipilimumab, IRESSA® (gefitinib), irinotecan hydrochloride, ISTODAX® (romidepsin), ixabepilone, ixazomib citrate, IXEMPRA® (ixabepilone), JAKAFI® (ruxolitinib phosphate), JEVTANA® (cabazitaxel), KADCYLA® (ado-trastuzumab emtansine), KEOXIFENE® (raloxifene hydrochloride), KEPIVANCE® (palifermin), KEYTRUDA® (pembrolizumab), KYPROLIS® (carfilzomib), lanreotide acetate, lapatinib ditosylate, lenalidomide, lenvatinib mesylate, LENVIMA® (lenvatinib mesylate), letrozole, leucovorin calcium, leukeran (chlorambucil), leuprolide acetate, levulan (aminolevulinic acid), LINFOLIZIN® (chlorambucil), LIPODOX® (doxorubicin hydrochloride liposome), lomustine, LONSURF® (trifluridine and tipiracil hydrochloride), LUPRON® (leuprolide acetate), LYNPARZA® (olaparib), MARQIBO® (vincristine sulfate liposome), MATULANE® (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, MEKINIST® (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, MESNEX® (mesna), METHAZOLASTONE® (temozolomide), methotrexate, methotrexate LPF, MEXATE® (methotrexate), MEXATE-AQ® (methotrexate), mitomycin C, mitoxantrone hydrochloride, MITOZYTREX® (mitomycin C), MOPP, MOZOBIL® (plerixafor), MUSTARGEN® (mechlorethamine hydrochloride), MUTAMYCIN® (mitomycin C), MYLERAN® (busulfan), MYLOSAR® (azacitidine), MYLOTARG® (gemtuzumab ozogamicin), nanoparticle paclitaxel, NAVELBINE® (vinorelbine tartrate), NECITUMUMAB, nelarabine, NEOSAR® (cyclophosphamide), netupitant and palonosetron hydrochloride, NEUPOGEN® (filgrastim), NEXAVAR® (sorafenib tosylate), NILOTINIB, NINLARO® (ixazomib citrate), nivolumab, NOLVADEX® (tamoxifen citrate), NPLATE® (romiplostim), obinutuzumab, ODOMZO® (sonidegib), OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuccinate, ONCASPAR® (pegaspargase), ondansetron hydrochloride, ONIVYDE® (irinotecan hydrochloride liposome), ONTAK® (denileukin diftitox), OPDIVO® (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, PARAPLAT® (carboplatin), PARPLATIN® (carboplatin), pazopanib hydrochloride, PCV, pegaspargase, peginterferon alpha-2b, PEG-INTRON® (peginterferon alpha-2b), pembrolizumab, pemetrexed disodium, PERJETA® (pertuzumab), pertuzumab, PLATINOL® (cisplatin), PLATINOL-AQ® (cisplatin), plerixafor, pomalidomide, POMALYST® (pomalidomide), ponatinib hydrochloride, PORTRAZZA® (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, PROLEUKIN® (aldesleukin), PROLIA® (denosumab), PROMACTA (eltrombopag olamine), PROVENGE® (sipuleucel-T), PURINETHOL® (mercaptopurine), PURIXAN® (mercaptopurine),223Ra dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HPV), recombinant interferon alpha-2b, regorafenib, R-EPOCH, REVLIMID® (lenalidomide), RHEUMATREX® (methotrexate), RITUXAN® (rituximab), rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, SCLEROSOL® intrapleural aerosol (talc), siltuximab, sipuleucel-T, somatuline depot (lanreotide acetate), sonidegib, sorafenib tosylate, SPRYCEL® (dasatinib), STANFORD V, sterile talc powder (talc), STERITALCO (talc), STIVARGA® (regorafenib), sunitinib malate, SUTENT® (sunitinib malate), SYLATRON® (peginterferon alpha-2b), SYLVANT® (siltuximab), SYNOVIR® (thalidomide), SYNRIBO® (omacetaxine mepesuccinate), thioguanine, TAC, TAFINLAR® (dabrafenib), TAGRISSO® (osimertinib), talimogene laherparepvec, tamoxifen citrate, tarabine PFS (cytarabine), TARCEVA (erlotinib hydrochloride), TARGRETIN® (bexarotene), TASIGNA® (nilotinib), TAXOL® (paclitaxel), TAXOTERE® (docetaxel), TEMODAR® (temozolomide), temsirolimus, thalidomide, THALOMID® (thalidomide), thioguanine, thiotepa, TOLAK® (topical fluorouracil), topotecan hydrochloride, toremifene, TORISEL® (temsirolimus), TOTECT® (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, TREANDA® (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, TRISENOX® (arsenic trioxide), TYKERB® (lapatinib ditosylate), UNITUXIN® (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, VARUBI® (rolapitant hydrochloride), vectibix (panitumumab), VelP, VELBAN® (vinblastine sulfate), VELCADE® (bortezomib), VELSAR (vinblastine sulfate), VEMURAFENIB, VIADUR (leuprolide acetate), VIDAZA (azacitidine), vinblastine sulfate, VINCASAR® PFS (vincristine sulfate), vincristine sulfate, vinorelbine tartrate, VIP, vismodegib, VISTOGARD® (uridine triacetate), VORAXAZE® (glucarpidase), vorinostat, VOTRIENT® (pazopanib hydrochloride), WELLCOVORIN® (leucovorin calcium), XALKORI® (crizotinib), XELODA® (capecitabine), XELIRI, XELOX, XGEVA® (denosumab), XOFIGO® (223Ra dichloride), XTANDI® (enzalutamide), YERVOY® (ipilimumab), YONDELIS® (trabectedin), ZALTRAP® (ziv-aflibercept), ZARXIO® (filgrastim), ZELBORAF® (vemurafenib), ZEVALIN® (ibritumomab tiuxetan), ZINECARD® (dexrazoxane hydrochloride), ziv-aflibercept, ZOFRAN® (ondansetron hydrochloride), ZOLADEX® (gGoserelin acetate), zoledronic acid, ZOLINZA® (vorinostat), ZOMETA® (zoledronic acid), ZYDELIG® (idelalisib), ZYKADIA® (ceritinib), and ZYTIGA (abiraterone acetate). Using the methods of the invention, an antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) described herein may be co-administered with (e.g., admixed with) or administered separately from a chemotherapy agent for the treatment of cancer. For example, an antagonistic TNFR2 polypeptide of the invention (such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to a patient, such as a human patient suffering from a cancer, simultaneously or at different times. In some embodiments, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) is administered to the patient prior to administration of a chemotherapy agent to the patient. Alternatively, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to the patient after a chemotherapy agent. For example, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to the patient after a failed chemotherapy treatment. A physician of skill in the art can monitor the efficacy of chemotherapy treatment to determine whether the therapy has successfully ameliorated the pathology being treated (such as a cancer described herein) using methods described herein and known in the art. For instance, a physician of skill in the art may monitor the quantity of cancer cells in a sample isolated from a patient (e.g., a blood sample or biopsy sample), such as a human patient, for instance, using flow cytometry or FACS analysis. Additionally or alternatively, a physician of skill in the art can monitor the progression of a cancerous disease in a patient, for instance, by monitoring the size of one or more tumors in the patient, for example, by CT scan, MRI, or X-ray analysis. A physician of skill in the art may monitor the progression of a cancer, such as a cancer described herein, by evaluating the quantity and/or concentration of tumor biomarkers in the patient, such as the quantity and/or concentration of cell surface-bound tumor associated antigens or secreted tumor antigens present in the blood of the patient as an indicator of tumor presence. A finding that the quantity of cancer cells, the size of a tumor, and/or the quantity or concentration of one or more tumor antigens present in the patient or a sample isolated from the patient has not decreased, for instance, by a statistically significant amount following administration of the chemotherapy agent within a specified time period (e.g., from 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicate that the chemotherapy treatment has failed to ameliorate the cancer. Based on this indication, a physician of skill in the art may administer an antagonistic TNFR2 polypeptide of the invention, such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein. Additionally or alternatively, an antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) described herein may be administered simultaneously with, or administered separately from, radiation therapy. For instance, a physician of skill in the art may administer radiation therapy to a patient, such as a human patient suffering from a cancer described herein, by treating the patient with external and/or internal electromagnetic radiation. The energy delivered by such radiation, which is typically in the form of X-rays, gamma rays, and similar forms of low-wavelength energy, can cause oxidative damage to the DNA of cancer cells, thereby leading to cell death, for instance, by apoptosis. External radiation therapy can be administered, for instance, using machinery such as a radiation beam to expose the patient to a controlled pulse of electromagnetic radiation. Additionally or alternatively, the patient may be administered internal radiation, for instance, by administering to the patient a therapeutic agent that contains a radioactive substituent, such as agents that contain223Ra or131I, which emit high-energy alpha and beta particles, respectively. Exemplary therapeutic agents that may be conjugated to a radiolabel include, for example, small molecule chemotherapeutics, antibodies, and antigen-binding fragments thereof, among others. For instance, an antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) of the invention may be conjugated to a radioactive substituent or a moiety that ligate such a substituent, for example, using bond-forming techniques known in the art or described herein. Such conjugates can be administered to the subject in order to deliver a therapeutic dosage of radiation therapy and a TNFR2 antagonist of the invention in a simultaneous administration (see, for example, “Antagonistic TNFR2 polypeptide conjugates,” above). In some embodiments, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) is administered to the patient after failed radiation treatment. A physician of skill in the art can monitor the efficacy of radiation treatment to determine whether the therapy has successfully ameliorated the pathology being treated (such as a cancer described herein) using, e.g., methods described herein. For instance, a physician of skill in the art may monitor the quantity of cancer cells in a sample isolated from a patient (e.g., a blood sample or biopsy sample), such as a human patient, for instance, using flow cytometry or FACS analysis. Additionally or alternatively, a physician of skill in the art can monitor the progression of a cancerous disease in a patient, for instance, by monitoring the size of one or more tumors in the patient, for example, by CT scan, MRI, or X-ray analysis. A physician of skill in the art may monitor the progression of a cancer, such as a cancer described herein, by evaluating the quantity and/or concentration of tumor biomarkers in the patient, such as the quantity and/or concentration of cell surface-bound tumor associated antigens or secreted tumor antigens present in the blood of the patient as an indicator of tumor presence. A finding that the quantity of cancer cells, the size of a tumor, and/or the quantity or concentration of one or more tumor antigens present in the patient or a sample isolated from the patient has not decreased, for instance, by a statistically significant amount following administration of the radiation therapy within a specified time period (e.g., from 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicate that the radiation treatment has failed to ameliorate the cancer. Based on this indication, a physician of skill in the art may administer an antagonistic TNFR2 polypeptide of the invention, such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein. In some embodiments, a physician of skill in the art may administer to a patient suffering from cancer a chemotherapeutic agent, radiation therapy, and a TNFR2 antagonist of the invention (such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein). The TNFR2 antagonist of the invention, chemotherapeutic agent, and radiation therapy may be administered to the patient simultaneously (for instance, in a single pharmaceutical composition or as multiple compositions administered to the patient at the same time) or at different times. In some embodiments, the TNFR2 antagonist (such as an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct of the invention) is administered to the patient first, and the chemotherapeutic agent and radiation therapy follow. Alternatively, the TNFR2 antagonist (such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct of the invention) may be administered to the patient following chemotherapy and radiation treatment. For example, the antagonistic TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein) may be administered to the patient after failed chemotherapy and/or radiation treatment. A physician of skill in the art can monitor the efficacy of chemotherapy and radiation treatment to determine whether the therapy has successfully ameliorated the pathology being treated (such as a cancer described herein) using methods described herein, such as the methods described above. For instance, a physician of skill in the art may monitor the quantity of cancer cells in a sample isolated from a patient (e.g., a blood sample or biopsy sample), such as a human patient, for instance, using flow cytometry or FACS analysis. Additionally or alternatively, a physician of skill in the art can monitor the progression of a cancerous disease in a patient, for instance, by monitoring the size of one or more tumors in the patient, for example, by CT scan, MRI, or X-ray analysis. A physician of skill in the art may monitor the progression of a cancer, such as a cancer described herein, by evaluating the quantity and/or concentration of tumor biomarkers in the patient, such as the quantity and/or concentration of cell surface-bound tumor associated antigens or secreted tumor antigens present in the blood of the patient as an indicator of tumor presence and even measure serum soluble TNFR2. On skilled in the art would expect a decrease in the number of activated T-regs, and increase in the numbers of T effectors and a decrease in the total number of cancer cells. Because of the specificity of these TNFR2 antibodies for cancer, the clinical monitoring would be expected to be most dramatic in the tumor microenvironment. A finding that the quantity of cancer cells, the size of a tumor, and/or the quantity or concentration of one or more tumor antigens present in the patient or a sample isolated from the patient has not decreased, for instance, by a statistically significant amount following administration of the chemotherapy agent and radiation within a specified time period (e.g., from 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicate that the chemotherapy and radiation treatment has failed to ameliorate the cancer. Based on this indication, a physician of skill in the art may administer an antagonistic TNFR2 polypeptide of the invention, such as a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct described herein. Blood-Brain Barrier Penetration In certain embodiments, antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compositions of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. Methods of manufacturing liposomes have been described, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, e.g., V. V. Ranade (J. Clin. Pharmacol.29:685, 1989)). Exemplary targeting moieties include, e.g., folate or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al. (Biochem. Biophys. Res. Commun. 153:1038, 1988)); antibodies (P. G. Bloeman et al. (FEBS Lett. 357:140, 1995); M. Owais et al. (Antimicrob. Agents Chemother. 39:180, 1995)); surfactant protein A receptor (Briscoe et al. (Am. J. Physiol. 1233:134, 1995)); the disclosures of each of which are incorporated herein by reference. Routes of Administration and Dosing Antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention can be administered to a mammalian subject (e.g., a human) by a variety of routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, intratumorally, parenterally, topically, intrathecally and intracerebroventricularly. The most suitable route for administration in any given case will depend on the particular polypeptide administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's excretion rate. The effective dose of an anti-TNFR2 polypeptide of the invention can range from about 0.0001 to about 100 mg/kg of body weight per single (e.g., bolus) administration, multiple administrations or continuous administration, or to achieve a serum concentration of 0.0001-5000 μg/mL serum concentration per single (e.g., bolus) administration, multiple administrations or continuous administration, or any effective range or value therein depending on the condition being treated, the route of administration and the age, weight, and condition of the subject. In certain embodiments, e.g., for the treatment of cancer, each dose can range from about 0.0001 mg to about 500 mg/kg of body weight. For instance, a pharmaceutical composition of the invention may be administered in a daily dose in the range of 0.001-100 mg/kg (body weight). The dose may be administered one or more times (e.g., 2-10 times) per day, week, month, or year to a mammalian subject (e.g., a human) in need thereof. Therapeutic compositions can be administered with medical devices known in the art. For example, in an embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well-known implants and modules useful in the invention include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medicaments through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art. Kits Containing Antagonistic Anti-TNFR2 Polypeptides This invention also includes kits that contain antagonistic anti-TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof). The kits provided herein may contain any of the antagonistic TNFR2 polypeptides described above, as well as any of the polynucleotides encoding these antibodies, vectors containing these polynucleotides, or cells engineered to express and secrete antibodies of the invention (e.g., prokaryotic or eukaryotic cells). A kit of this invention may include reagents that can be used to produce the compositions of the invention (e.g., antagonistic anti-TNFR2 polypeptides, such as single-chain polypeptides, antibodies, conjugates containing antagonistic anti-TNFR2 polypeptides, polynucleotides encoding antagonistic anti-TNFR2 polypeptides, vectors containing these polynucleotides). Optionally, kits of the invention may include reagents that can induce the expression of antagonistic TNFR2 polypeptides within cells (e.g., mammalian cells), such as doxycycline or tetracycline. In other cases, a kit of the invention may contain a compound capable of binding and detecting a fusion protein that contains an antagonistic TNFR2 antibody and an epitope tag. For instance, in such cases a kit of the invention may contain maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, biotin, or streptavidin. Kits of the invention may also include reagents that are capable of detecting an antagonistic TNFR2 polypeptide (e.g., single-chain polypeptide, antibody, or fragment thereof) directly. Examples of such reagents include secondary antibodies that selectively recognize and bind particular structural features within the Fc region of an anti-TNFR2 antibody of the invention. Kits of the invention may contain secondary antibodies that recognize the Fc region of an antagonistic TNFR2 antibody and that are conjugated to a fluorescent molecule. These antibody-fluorophore conjugates provide a tool for analyzing the localization of antagonistic anti-TNFR2 antibodies, e.g., in a particular tissue or cultured mammalian cell using established immunofluorescence techniques. In some embodiments, kits of the invention may include additional fluorescent compounds that exhibit known sub-cellular localization patterns. These reagents can be used in combination with another antibody-fluorophore conjugate, e.g., one that specifically recognizes a different receptor on the cell surface in order to analyze the localization of an anti-TNFR2 antibody relative to other cell-surface proteins. Kits of the invention may also contain a reagent that can be used for the analysis of a patient's response to treatment by administration of antagonistic TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention. For instance, kits of the invention may include an antagonistic TNFR2 antibody and one or more reagents that can be used to determine the quantity of T-reg cells in a blood sample withdrawn from a subject (e.g., a human) that is undergoing treatment with an antibody of the invention. Such a kit may contain, e.g., antibodies that selectively bind cell-surface antigens presented by T-reg cells, such as CD4 and CD25. Optionally, these antibodies may be labeled with a fluorescent dye, such as fluorescein or tetramethylrhodamine, in order to facilitate analysis of T-reg cells by fluorescence-activated cell sorting (FACS) methods known in the art. Kits of the invention may optionally contain one or more reagents that can be used to quantify tumor-reactive T-lymphocytes in order to determine the effectiveness of an antagonistic TNFR2 antibody of the invention in restoring tumor-infiltrating lymphocyte proliferation. For instance, kits of the invention may contain an antibody that selectively binds cell-surface markers on the surface of a cytotoxic T cell, such as CD8 or CD3. Optionally, these antibodies may be labeled with fluorescent molecules so as to enable quantitation by FACS analysis. A kit of the invention may also contain one or more reagents useful for determining the affinity and selectivity of an antagonistic TNFR2 polypeptide of the invention for one or more peptides derived from TNFR2 (e.g., a peptide containing the sequence of any one of SEQ ID NOs: 11, 19, 20, 34-117, 285, or 286). For instance, a kit may contain an antagonistic TNFR2 polypeptide and one or more reagents that can be used in an ELISA assay to determine the KDof an antibody of the invention for one or more peptides that present a TNFR2 epitope in a conformation similar to that of the epitope in the native protein. A kit may contain, e.g., a microtiter plate containing wells that have been previously conjugated to avidin, and may contain a library of TNFR2-derived peptides, each of which conjugated to a biotin moiety. Such a kit may optionally contain a secondary antibody that specifically binds to the Fc region of an antagonistic TNFR2 antibody of the invention, and the secondary antibody may be conjugated to an enzyme (e.g., horseradish peroxidase) that catalyzes a chemical reaction that results in the emission of luminescent light. Kits of the invention may also contain antagonistic TNFR2 polypeptides of the invention and reagents that can be conjugated to such an antibody, including those previously described (e.g., a cytotoxic agent, a fluorescent molecule, a bioluminescent molecule, a molecule containing a radioactive isotope, a molecule containing a chelating group bound to a paramagnetic ion, etc). These kits may additionally contain instructions for how the conjugation of an antagonistic TNFR2 antibody of the invention to a second molecule, such as those described above, can be achieved. A kit of the invention may also contain a vector containing a polynucleotide that encodes an antagonistic anti-TNFR2 polypeptide, such as any of the vectors described herein. Alternatively, a kit may include mammalian cells (e.g., CHO cells) that have been genetically altered to express and secrete antagonistic TNFR2 antibodies or fragments thereof from the nuclear genome of the cell. Such a kit may also contain instructions describing how expression of the antagonistic TNFR2 antibody or fragment thereof from a polynucleotide can be induced, and may additionally include reagents (such as, e.g., doxycycline or tetracycline) that can be used to promote the transcription of these polynucleotides. Such kits may be useful for the manufacture of antagonistic TNFR2 antibodies or antigen-binding fragments thereof of the invention. Other kits of the invention may include tools for engineering a prokaryotic or eukaryotic cell (e.g., a CHO cell or a BL21(DE3)E. colicell) so as to express and secrete an antagonistic TNFR2 polypeptide of the invention from the nuclear genome of the cell. For example, a kit may contain CHO cells stored in an appropriate media and optionally frozen according to methods known in the art. The kit may also provide a vector containing a polynucleotide that encodes a nuclease (e.g., such as the CRISPER/Cas, zinc finger nuclease, TALEN, ARCUS™ nucleases described herein) as well as reagents for expressing the nuclease in the cell. The kit can additionally provide tools for modifying the polynucleotide that encodes the nuclease so as to enable one to alter the DNA sequence of the nuclease in order to direct the cleavage of a specific target DNA sequence of interest. Examples of such tools include primers for the amplification and site-directed mutagenesis of the polynucleotide encoding the nuclease of interest. The kit may also include restriction enzymes that can be used to selectively excise the nuclease-encoding polynucleotide from the vector and subsequently re-introduce the modified polynucleotide back into the vector once the user has modified the gene. Such a kit may also include a DNA ligase that can be used to catalyze the formation of covalent phosphodiester linkages between the modified nuclease-encoding polynucleotide and the target vector. A kit of the invention may also provide a polynucleotide encoding an antagonistic anti-TNFR2 antibody or fragment thereof, as well as a package insert describing the methods one can use to selectively cleave a particular DNA sequence in the genome of the cell in order to incorporate the polynucleotide encoding an antagonistic TNFR2 antibody into the genome at this site. Optionally, the kit may provide a polynucleotide encoding a fusion protein that contains an antagonistic TNFR2 antibody or fragment thereof and an additional polypeptide, such as, e.g., those described herein. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods claimed herein are performed, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventor regards as her invention. Example 1. Mapping the Discrete Epitopes within TNFR2 that Interact with TNFRAB1 and TNFRAB2 Libraries of linear, cyclic, and bicyclic peptides derived from human TNFR2 were screened for distinct sequences within the protein that exhibit high affinity for TNFR2 antibody TNFRAB2. In order to screen conformational epitopes within TFNR2, peptides from distinct regions of the primary protein sequence were conjugated to one another to form chimeric peptides. These peptides contained cysteine residues at strategic positions within their primary sequences (see, e.g.,FIG.3, SEQ ID NOs: 34-117 and 134-252). This facilitated an intramolecular cross-linking strategy that was used to constrain individual peptides to a one of a wide array of three dimensional conformations. Unprotected thiols of cysteine residues were cross-linked via nucleophilic substitution reactions with divalent and trivalent electrophiles, such as 2,6-bis(bromomethyl)pyridine and 1,3,5-tris(bromomethyl)benzene, so as to form conformationally restricted cyclic and bicyclic peptides, respectively. In this way, peptides containing unique combinations of amino acids from disparate regions of the TNFR2 primary sequence were constrained so as to structurally pre-organize epitopes that may resemble those presented in the native TNFR2 tertiary structure. Libraries containing these peptides were screened by immobilizing peptides to distinct regions of a solid surface and treating the surface in turn with TNFRAB1 or TNFRAB2, secondary antibody conjugated to horseradish peroxidase (HRP), and HRP substrate (2,2′-azino-di-3-ethylbenzthiazoline sulfonate) in the presence of hydrogen peroxide. The solid surface was washed in between treatment with successive reagents so as to remove excess or non-specifically bound materials. The luminescence of each region of each surface was subsequently analyzed using a charge coupled device (CCD)—camera and an image processing system. The “Constrained Libraries of Peptides on Surfaces” (CLIPS) platform starts with the conversion of the target protein, e.g., TNFR2, into a library of up to 10,000 overlapping peptide constructs, using a combinatorial matrix design (Timmerman et al., J. Mol. Recognit., 20: 283-29, 2007). On a solid carrier, a matrix of linear peptides is synthesized, which are subsequently shaped into spatially defined CLIPS constructs. Constructs representing multiple parts of the discontinuous epitope in the correct conformation bind the antibody with high affinity, which is detected and quantified. Constructs presenting the incomplete epitope bind the antibody with lower affinity, whereas constructs not containing the epitope do not bind at all. Affinity information is used in iterative screens to define the sequence and conformation of epitopes in detail. The raw luminescence data obtained from these ELISA experiments are reported inFIGS.3A and3Bfor TNFRAB1 and TNFRAB2, respectively. These results informed the analysis of epitopes present on the surface of TNFR2 that bind antagonistic TNFR2 antibodies. Structural models of TNFR2 illustrating epitopes that bind such antibodies are shown inFIGS.4and15A-15C. Peptide Synthesis To reconstruct epitopes of the target molecule a library of peptides was synthesized. An amino functionalized polypropylene support was obtained by grafting a proprietary hydrophilic polymer formulation via reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS® liquid handling stations (Perkin Elmer). CLIPS technology allows one to structure peptides into single loops, double-loops, triple loops, sheet-like folds, helix-like folds and combinations thereof. CLIPS templates are coupled to cysteine residues. The side-chains of multiple cysteines in the peptides are coupled to one or two CLIPS templates. For example, a 0.5 mM solution of the CLIPS template (2,6-bis(bromomethyl)pyridine) is dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (1:3(v/v)). This solution is added to a surface-bound peptide array. The CLIPS template will react with side-chains of two cysteines as present in the solid-phase bound peptides of the peptide-arrays (455 wells plate with 3 μl wells). The peptide arrays are gently shaken in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrays are washed extensively with excess of H2O and sonicated in disrupt-buffer containing 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70° C. for 30 minutes, followed by sonication in H2O for another 45 minutes. Analysis of Binding Affinities of Antagonistic TNFR2 Antibodies by Surface Plasmon Resonance The affinities of antagonistic TNFR2 antibodies for recombinant human TNFR2 were measured using BIACORE™ Analysis Services (Precision Antibody). Briefly, the antibody was biotinylated at a 5:1 stoichiometric ratio using biotinyl-LC-LC-NOSE (Thermo-Fisher) in PBS. Excess biotinylation reagent was removed by centrifugation chromatography and the biotinylated antibody was captured on 3000 RU of streptavidin surface to a level of 100 RU. Theoretical maximum of signal with TNFR2 with that level of antibody capture was 26 RU and that signal was reached with a preliminary experiment using 500 nM TNFR2 in the running buffer. Analysis of the kinetics of antigen binding was performed at a flow of 60 μL/min with 2 min injections. Antibodies were injected at a concentration of 1 mg/ml to the final capture of 100 RU. The instrument used was BIACORE™ 3000 with the BioCap chip (GE Healthcare). Double reference method was used for analysis. Reference channel contained the identical level of streptavidin. The thermodynamic and kinetic parameters of the binding of antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 to TNFR2 as measured using this assay are shown inFIG.13A. ELISA Screening The binding of antibody to each of the synthesized peptides was tested in an ELISA format. Surface-immobilized peptide arrays were incubated with primary antibody solution (overnight at 4° C.). After washing, the peptide arrays were incubated with a 1/1000 dilution of an appropriate antibody peroxidase conjugate (SBA) for one hour at 25° C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 2 μl/ml of 3 percent H2O2were added. After one hour, the color development was measured. The color development was quantified with a charge coupled device (CCD)—camera and an image processing system. The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96-well plate ELISA-reader. The results are quantified and stored into the Peplab database. Occasionally a well contains an air-bubble resulting in a false-positive value, the cards are manually inspected and any values caused by an air-bubble are scored as 0. To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. These were screened with a negative control, antibody 57.9, an antibody that does not specifically bind TNFR2 (Posthumuset al. (J. Virology. 64:3304-3309, 1990)). Peptides that bound TNFRAB1 and TNFRAB2 with high affinity are highlighted inFIGS.3A and3B, respectively. These peptides therefore contain residues within TNFR2 that are structurally configured into epitopes that are preferentially bound by TNFRAB1 and TNFRAB2. Epitope Mapping ELISA was also used to determine linear epitopes present on the extracellular surface of TNFR2. Linear peptides corresponding to various regions within the TNFR2 primary sequence were purchased from GenScript (Piscataway, NJ), diluted in coating buffer and placed on Immulon 4HBX Flat Bottom Microtiter Plates (Thermo Scientific) at a concentration of 1 μg/well. Primary TNFR2 antagonistic antibodies (0.1 μg/well) were incubated with substrates. Secondary antibodies against rodent IgG were used to detect the primary antibodies. Absorbance was measured using the SPECTRAMAX® 190 Absorbance Plate Reader and analyzed with SoftMax Pro 6.3 (Molecular Devices). Results of these ELISA-based assays are shown inFIGS.13B and13C. Example 2. Antagonistic TNFR2 Antibodies Inhibit T-Reg Cell Proliferation Materials and MethodsHuman T-reg Flow™ Kit (BioLegend, Cat. No. 320401)Cocktail Anti-human CD4 PE-Cy5/CD25 PE (BioLegend, Part No. 78930)Alexa Fluor® 488 Anti-human FOXP3, Clone 259D (BioLegend, Part No. 79467)Alexa Fluor® 488 Mouse IgG1, k Isotype Ctrl (ICFC), Clone MOPC-21 (BioLegend, Part No. 79486)FOXP3 Fix/Perm Buffer (4×) (BioLegend, Cat. No. 421401)FOXP3 Perm Buffer (10×) (BioLegend, Cat. No. 421402)PE anti-human CD25, Clone: BC96 (BioLegend, Cat. No. 302606)Alexa Fluor® 488 Anti-human FOXP3, Clone 259D (BioLegend, Cat. No. 320212)PBS pH 7.4 (1×) (Gibco Cat. No. 10010-023)HBSS (1×) (Gibco Cat. No. 14175-095)FBS (heat inactivated)15 ml tubesBench top centrifuge with swing bucket rotor for 15 ml tubes (set speed 1100 rpm or 200 g) Antagonistic TNFR2 antibodies (TNFRAB1 and TNFRAB2) were tested for the ability to suppress the proliferation of T-reg cells. Cultured T-reg cells were treated with varying concentrations of the antagonistic TNFR2 antibodies in the presence and absence of stimulatory growth factors (e.g., TNFα) for set periods of time. T-reg cells were also cultured in the presence of TNFRAB1 at various concentrations ranging from 0.0008-25 μg/ml in the presence and absence of TNFα. As controls, T-reg cells were also incubated with TNFα alone at concentrations ranging from 0-40 ng/ml in order to select levels of TNFα that induce a high fractional increase in T-reg cell count. Additionally, T-reg cells were cultured in the presence of IL-2 alone and in the presence of TNFRAB2 alone. Following the incubation of T-reg cells under the conditions described above, the cell counts were determined using flow cytometry analysis. T-reg cells at a density of 0.2-1×106cells/100 μl were distributed into a 15-ml conical tube and centrifuged for 5 minutes in order to pellet the cells. The supernatant was discarded and cells were resuspended in 100 μl of wash buffer (1×HBSS containing 2% FBS). 5 μl of PE anti-human CD25 fluorophore-antibody conjugate were added to this mixture, and the cells were subsequently vortexed and incubated in the dark for 25 minutes. The cells were then washed by adding 1 ml of wash buffer and subsequently centrifuging for 5 minutes. The supernatant was then discarded and 1 ml of FoxP3 fixation/permeabilization buffer (1:4 dilution of 4×FOXP3 Fix/Perm buffer in PBS) was added to the cells. The cells were then vortexed and incubated in the dark for 20 minutes. Cells were subsequently centrifuged for 5 minutes and supernatant was discarded. Cells were then resuspended in 1 ml of fresh wash buffer, vortexed, and centrifuged for 5 minutes. Cells were subsequently resuspended in 1 ml of 1×FOXP3 Perm Buffer (1:10 dilution of 10×FOXP3 Perm Buffer in PBS), vortexed, and incubated in the dark for 15 minutes. Following incubation, cells were centrifuged for 5 minutes and supernatant was subsequently discarded. The cell pellet was then resuspended in 100 μl of 1×FOXP3 Perm Buffer. At this point, 5 μl of either Alexa Fluor® 488 anti-human FOXP3 or Alexa Fluor® 488 mouse IgG1, k isotype control were added to the cells. Cells were then vortexed and incubated in the dark for 35 minutes. Following incubation, cells were washed by adding 1 ml of fresh wash buffer to the cells, vortexing the cells and centrifuging for 5 minutes. The supernatant was then discarded and the cell pellet was resuspended in 0.2-0.5 ml of 1×HBSS free of FBS. Cell counts were then determined by flow cytometry analysis. As seen inFIG.5, incubation of antagonistic TNFR2 antibody TNFRAB1 suppressed T-reg cell proliferation in a dose dependent manner even when cells were co-incubated with 20 ng/ml TNFα. Strikingly, this concentration of TNFα was capable of inducing the largest fractional increase in T-reg cell count among the TNFα dosages analyzed in T-reg samples treated with TNFα alone. That TNFRAB1 is capable of diminishing T-reg cell proliferation even in the presence of activating levels of TNFα indicates that antagonistic TNFR2 antibodies are capable of not only competing with TNFα for receptor binding, but also exhibit the capacity to inhibit downstream signaling cascades that lead to T-reg cell growth and proliferation. For instance, when T-reg cells were incubated with TNFα alone at 20 ng/ml, the resulting T-reg population increased to 130% of untreated control cells. However, upon co-incubation of T-reg cells at 20 ng/ml with TNFRAB1, a steady dose-dependent suppression of cell proliferation is observed with concentrations ranging from 0.004-1.25 μg/ml of TNFRAB1. Example 3. Generating Antagonistic TNFR2 Antibodies by Phage Display An exemplary method for in vitro protein evolution of antagonistic TNFR2 antibodies of the invention is phage display, a technique which is well known in the art. Phage display libraries can be created by making a designed series of mutations or variations within a coding sequence for the CDRs of an antibody or the analogous regions of an antibody-like scaffold (e.g., the BC, CD, and DE loops of10Fn3 domains). The template antibody-encoding sequence into which these mutations are introduced may be, e.g., a naive human germline sequence as described herein. These mutations can be performed using standard mutagenesis techniques described herein or known in the art. Each mutant sequence thus encodes an antibody corresponding in overall structure to the template except having one or more amino acid variations in the sequence of the template. Retroviral and phage display vectors can be engineered using standard vector construction techniques as described herein or known in the art. P3 phage display vectors along with compatible protein expression vectors, as is well known in the art, can be used to generate phage display vectors for antibody diversification as described herein. The mutated DNA provides sequence diversity, and each transformant phage displays one variant of the initial template amino acid sequence encoded by the DNA, leading to a phage population (library) displaying a vast number of different but structurally related amino acid sequences. Due to the well-defined structure of antibody hypervariable regions, the amino acid variations introduced in a phage display screen are expected to alter the binding properties of the binding peptide or domain without significantly altering its structure. In a typical screen, a phage library is contacted with and allowed to bind a TNFR2-derived peptide (e.g., a peptide having the sequence SEQ ID NO: 285 or 286), or a particular subcomponent thereof. To facilitate separation of binders and non-binders, it is convenient to immobilize the target on a solid support. Phage bearing a TNFR2-binding moiety can form a complex with the target on the solid support whereas non-binding phage remain in solution and can be washed away with excess buffer. Bound phage can then liberated from the target by changing the buffer to an extreme pH (pH 2 or pH 10), changing the ionic strength of the buffer, adding denaturants, or other known means. To isolate the binding phage exhibiting the polypeptides of the present invention, a protein elution is performed. The recovered phage can then be amplified through infection of bacterial cells and the screening process can be repeated with the new pool that is now depleted in non-binding antibodies and enriched for antibodies that bind the target peptide. The recovery of even a few binding phage is sufficient to amplify the phage for a subsequent iteration of screening. After a few rounds of selection, the gene sequences encoding the antibodies or antigen-binding fragments thereof derived from selected phage clones in the binding pool are determined by conventional methods, thus revealing the peptide sequence that imparts binding affinity of the phage to the target. During the panning process, the sequence diversity of the population diminishes with each round of selection until desirable peptide-binding antibodies remain. The sequences may converge on a small number of related antibodies or antigen-binding fragments thereof, typically 10-50 out of about 109 to 1010original candidates from each library. An increase in the number of phage recovered at each round of selection is a good indication that convergence of the library has occurred in a screen. After a set of binding polypeptides is identified, the sequence information can be used to design other secondary phage libraries, biased for members having additional desired properties (See WO 2014/152660; incorporated herein by reference). Example 4. Treatment of Cancer in a Human Patient by Administration of Antagonistic Anti-TNFR2 Antibodies The antagonistic TNFR2 antibodies of the invention can be administered to a human patient in order to treat a cell proliferation disorder, such as cancer. Administration of these antibodies suppresses the growth and proliferation of T-reg cells. Antibodies of the invention can also be administered to a patient in order to suppress a T-reg-mediated immune response. For instance, a human patient suffering from cancer, e.g., a cancer described herein, can be treated by administering an antagonistic TNFR2 antibody of the invention by an appropriate route (e.g., intravenously) at a particular dosage (e.g., between 0.001 and 100 mg/kg/day) over a course of days, weeks, months, or years. If desired, the anti-TNFR2 antibody can be modified, e.g., by hyperglycosylation or by conjugation with PEG, so as to evade immune recognition and/or to improve the pharmacokinetic profile of the antibody. The progression of the cancer that is treated with an antagonistic TNFR2 antibody of the invention can be monitored by any one or more of several established methods. A physician can monitor the patient by direct observation in order to evaluate how the symptoms exhibited by the patient have changed in response to treatment. A patient may also be subjected to MRI, CT scan, or PET analysis in order to determine if a tumor has metastasized or if the size of a tumor has changed, e.g., decreased in response to treatment with an anti-TNFR2 antibody of the invention. Optionally, cells can be extracted from the patient and a quantitative biochemical analysis can be conducted in order to determine the relative cell-surface concentrations of various growth factor receptors, such as the epidermal growth factor receptor. Based on the results of these analyses, a physician may prescribe higher/lower dosages or more/less frequent dosing of the antagonistic TNFR2 antibody in subsequent rounds of treatment. Example 5. Producing an scFv TNFR2 Antagonist Antibody fragments of the invention include scFv fragments, which consist of the antibody variable regions of the light and heavy chains combined in a single peptide chain. A phenotype-neutral TNFR2 antibody can be used as a framework for the development of a scFv antibody fragment by recombinantly expressing a polynucleotide encoding the variable region of a light chain of the TNFR2 antibody operatively linked to the variable region of a heavy chain of that antibody. The polynucleotide encoding the variable region of the heavy chain may contain a CDR-H3 sequence derived, e.g., from TNFRAB1, TNFRAB2, or TNFR2A3. Recombinant polynucleotides encoding such variable regions can be produced, for example, using established mutagenesis protocols as described herein or known in the art. This polynucleotide can then be expressed in a cell (e.g., a CHO cell) and the scFv fragment can subsequently be isolated from the cell culture media. Alternatively, scFv fragments derived from a TNFR2 antagonist can be produced by chemical synthetic methods (e.g., by Fmoc-based solid-phase peptide synthesis, as described herein). One of skill in the art can chemically synthesize a peptide chain consisting of the variable region of a light chain of a phenotype-neutral TNFR2 antibody operatively linked to the variable region of a heavy chain of the antibody such that the variable region of the heavy chain contains the CDR-H3 sequence of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3. Native chemical ligation can be used as a strategy for the synthesis of long peptides (e.g., greater than 50 amino acids). Native chemical ligation protocols are known in the art and have been described, e.g., by Dawson et al. (Science, 266:776-779, 1994); incorporated herein by reference. Example 6. Producing a Humanized TNFR2 Antibody One method for producing humanized TNFR2 antibodies of the invention is to import the CDRs of a TNFR2 antibody into a human antibody consensus sequence. Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the “VBASE” human germline sequence database; Kabat et al. (Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991); Tomlinson et al. (J. Mol. Biol. 227:776-798, 1992); and Cox et al. (Eur. J. Immunol. 24:827-836, 1994); incorporated herein by reference). Using established procedures, one can identify the variable domain framework residues and CDRs of a consensus antibody sequence (e.g., by sequence alignment (see Kabat, supra)). One can substitute, e.g., the CDR-H1, CDR-H2, CDR-L1, CDR-L2, and CDR-L3 sequences of the antibody with the corresponding sequences of a phenotype-neutral anti-TNFR2 antibody, as well as substitute the CDR-H3 of the consensus human antibody with the CDR-H3 an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3 in order to produce a humanized, antagonistic TNFR2. Polynucleotides encoding the above-described CDR sequences can be produced synthetically or recombinantly, e.g., using gene editing techniques described herein or known in the art. One example of a variable domain of a consensus human antibody includes the heavy chain variable domain EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVAVISENGSDTYYADSVKGR FTISRDDSKNTLYLQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 32) and the light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 33), identified in U.S. Pat. No. 6,054,297; incorporated herein by reference (CDRs are shown in bold). In order to produce humanized, antagonistic TNFR2 antibodies, one can recombinantly express a polynucleotide encoding the above consensus sequence in which the CDR-H1, CDR-H2, CDR-L1, CDR-L2, and CDR-L3 sequences are replaced with the corresponding CDR sequences of a phenotype-neutral, TNFR2-specific antibody. The CDR-H3 sequence of the consensus human antibody can be substituted with the CDR-H3 of an antagonistic TNFR2 antibody, such as TNFRAB1, TNFRAB2, or TNFR2A3 (i.e., SEQ ID NO: 25, 259, or 284, respectively). A polynucleotide encoding the above heavy chain and light chain variable domains operatively linked to one another can be incorporated into an expression vector (e.g., an expression vector optimized for protein expression in prokaryotic or eukaryotic cells as described herein or known in the art). The single-chain antibody fragment (scFv) can thus be expressed in a host cell and subsequently purified from the host cell medium using established techniques, such as size-exclusion chromatography and/or affinity chromatography as described herein. Example 7. Treatment of HIV in a Human Patient by Administration of Antagonistic Anti-TNFR2 Antibodies The antagonistic TNFR2 antibodies of the invention can be administered to a human patient in order to treat a viral infection, such as HIV. Administration of these antibodies suppresses the growth and proliferation of T-reg cells, which enhances the immune response of a patient by allowing the growth and proliferation of cytotoxic T-lymphocytes capable of mounting an attack on infected cells. For instance, a human patient suffering from HIV can be treated by administering an antagonistic TNFR2 antibody of the invention (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7) and has a non-native constant region, such as a TNFR2 antibody that contains the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3) by an appropriate route (e.g., intravenously) at a particular dosage (e.g., between 0.001 and 100 mg/kg/day) over a course of days, weeks, months, or years. If desired, the anti-TNFR2 antibody can be modified, e.g., by hyperglycosylation or by conjugation with PEG, so as to evade immune recognition and/or to improve the pharmacokinetic profile of the antibody. The progression of HIV that is treated with an antagonistic TNFR2 antibody of the invention can be monitored by any one or more of several established methods. A physician can monitor the patient by direct observation in order to evaluate how the symptoms exhibited by the patient have changed in response to treatment. A blood sample can also be withdrawn from the patient in order to analyze the cell count of one or more white blood cells in order to determine if the quantity of infected cells has changed (e.g., decreased) in response to treatment with an anti-TNFR2 antibody of the invention. Based on the results of these analyses, a physician may prescribe higher/lower dosages or more/less frequent dosing of the antagonistic TNFR2 antibody in subsequent rounds of treatment. Example 8. Treatment ofMycobacterium tuberculosisin a Non-Human Mammal by Administration of Antagonistic Anti-TNFR2 Antibodies The antagonistic TNFR2 antibodies of the invention can be administered to a non-human mammal (e.g., a bovine mammal, pig, bison, horse, sheep, goat, cow, cat, dog, rabbit, hamster, guinea pig, or other non-human mammal) in order to treat a bacterial infection, such asMycobacterium tuberculosis. Administration of these antibodies suppresses the growth and proliferation of T-reg cells, which enhances the immune response of a patient by allowing the growth and proliferation of cytotoxic T-lymphocytes capable of mounting an attack on the pathogenic organism. For instance, a non-human mammal suffering fromMycobacterium tuberculosiscan be treated by administering an antagonistic TNFR2 antibody of the invention (e.g., a TNFR2 antibody that specifically binds an epitope containing one or more residues of the KCRPG sequence of TNFR2 (residues 142-146 of SEQ ID NO: 7) and that does not exhibit specific binding to an epitope containing the KCSPG sequence of TNFR2 (residues 56-60 of SEQ ID NO: 7) and has a non-native constant region, such as a TNFR2 antibody that contains the CDR-H3 sequence of TNFRAB1, TNFRAB2, or TNFR2A3) by an appropriate route (e.g., intravenously) at a particular dosage (e.g., between 0.001 and 100 mg/kg/day) over a course of days, weeks, months, or years. If desired, the anti-TNFR2 antibody can be modified, e.g., by hyperglycosylation or by conjugation with PEG, so as to evade immune recognition and/or to improve the pharmacokinetic profile of the antibody. The progression of theMycobacterium tuberculosisinfection that is treated with an antagonistic TNFR2 antibody of the invention can be monitored by any one or more of several established methods. A physician can monitor the patient by direct observation in order to evaluate how the symptoms exhibited by the patient have changed in response to treatment. A blood sample can also be withdrawn from the patient in order to analyze the cell count of one or more white blood cells in order to determine if the immune response has changed (e.g., increased) in response to treatment with an anti-TNFR2 antibody of the invention. Based on the results of these analyses, a physician may prescribe higher/lower dosages or more/less frequent dosing of the antagonistic TNFR2 antibody in subsequent rounds of treatment. Example 9. Antagonistic TNFR2 Antibodies Inhibit T-Reg Cell Proliferation in the Presence of IL-2 and TNF The presence of an elevated quantity of T-reg cells can hamper the ability of the immune system to combat cancer and infectious disease. TNFR2 represents an important checkpoint in T-reg proliferation and thus constitutes an ideal target for immunomodulation. To characterize the functional activity of two antagonistic TNFR2 monoclonal antibodies (mAbs), TNFRAB1 and TNFRAB2, a cell-based assay developed for homogeneous T-reg expansion was employed. It was first confirmed that normal human CD4+ cells incubated with increasing concentrations of TNFα resulted in a dose-dependent increase in T-reg cells (FIGS.6A and7A). We also confirmed that the presence of IL-2 is required for T-reg proliferation (FIG.6B). As shown inFIGS.6B-6G, relative to treatment with IL-2 alone, the effect on T-reg proliferation with TNF at 20 ng/ml is dramatic (>20% expansion) and enhanced further by co-incubation with the TNFR2 agonist (>60% expansion). Antagonistic TNFR2 antibodies elicit an opposite effect on T-reg proliferation as measured using this assay. Both TNFR2 antagonistic antibodies induced suppressive effects on T-reg proliferation that resulted in a 4-15% decrease in the percentage of remaining T-reg cells in normal blood donors. Remarkably, the TNFR2 antagonistic effect is dominant and can even overcome the presence of a generous amount of TNFα (20 mg/ml) (FIGS.6C-6G). The TNFR2 antagonistic effect on T-regs was dose dependent (FIGS.6D-6G). More remarkable was the observation the when the two TNFR2 antagonistic antibodies were studied in the presence of TNFα, both TNFR2 antagonist antibodies were able to overcome TNFα agonism in a dose dependent fashion. Both antagonistic antibodies were capable of attenuating T-reg expansion and inverting the TNFα agonistic curve (FIGS.6A and6C-6G). This assay was also performed by incubating T-reg cells with a fixed amount of TNFR2 antagonistic antibody and escalating doses of TNFα (FIGS.6D-6G). Again both TNFR2 antagonists in a dose-dependent fashion were capable of reversing TNFα agonism. It should be noted that this 48 hour T-reg expansion assay is performed with IL-2, an agonist, in the culture to prevent non-specific human CD4+ T cell death. When the data obtained from these experiments were processed to determine the relative changes in T-reg proliferation in the presence of antagonistic TNFR2 antibodies as compared to the presence of IL-2 alone, the antagonistic properties of both antibodies persisted (FIGS.7A-7E). Together these results demonstrate the functional ability of two TNFR2 antagonist antibodies to suppress T-reg proliferation in human CD4+ cells, and the effect is remarkably dominant over agonism driven by moderate to high doses of TNFα. To conduct the T-reg suppression assays described above, peripheral blood mononuclear cells (PBMCs) were used as responders. PBMCs were isolated on the day of venipuncture using a Ficoll-Plaque Plus (GE Healthcare, Piscataway, NJ) density gradient and were cryopreserved at −80′C. Cells were thawed one day prior to mixing with T-reg cells and were rested overnight in RPMI 1640 and IL-2 (10 U/ml). The next day, responder cells were stained with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). Responder cells (5×104cells) were then mixed with expanded T-reg cells at various ratios (0:1, 4:1, 2:1, 1:1), and stimulated with anti-CD3 mAb (HIT3a, BD Biosciences) and IL-2 (50 U/ml). Cells were collected after 4 days and analyzed by flow cytometry. Human Subjects Human blood samples from over 100 donors were collected according to a human studies protocol approved by the Massachusetts General Hospital Human Studies Committee (MGH-2001 P001379). All of the donors provided written informed consent. Blood was collected into BD Vacutainer EDTA tubes (BD Diagnostics) and processed within 2 hours of phlebotomy. T-reg samples isolated from ovarian cancer patients were obtained from women with newly diagnosed ovarian cancer prior to irradiation and prior to chemotherapy. These human studies were approved by the Massachusetts General Hospital Human Studies Committee (MGH-2015P002489; see Example 14 below). Blood and Cell Culture Fresh human blood was processed within 2 hours of venipuncture. Blood was washed twice with 1×HBSS (Invitrogen) plus 2% FBS (Sigma-Aldrich) and CD4+ cells were isolated using Dynabeads® CD4 Positive Isolation Kit (Invitrogen). Isolated CD4+ cells were resuspended in RPMI GlutaMAX™ (Life Technologies) plus 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Life Technologies). Cells were seeded in 96-round-bottom well plates at a concentration of 0.2-1×106cells/well, treated with either TNFR2 antagonist antibodies or TNFR2 agonists (as indicated in each example), and then incubated for up to 48 hours at 37° C. with 5% CO2−Since isolated and cultured human T cells die in the absence of IL-2 in the cell culture medium, all cell-based experiments described herein used a low level of IL-2 (100 U/ml) in the culture media to prevent IL-2 withdrawal from influencing the data. T-reg cells isolated from sterile ovarian cancer ascites were obtained by first concentrating the cells in 50-ml conical tubes and then suspending the cell pellets to obtain CD4+ cell isolations (see Example 14 below). Reagents and Flow Cytometry Recombinant human TNF was purchased from Sigma-Aldrich and recombinant human IL-2 was purchased from Life Technologies. F(ab′)2fragments of TNFRAB1 and TNFRAB2 were prepared using Pierce F(ab′)2Preparation Kit (Life Technologies). Crosslinking antibodies against rodent IgG (ab9165 and ab99670) were purchased from Abcam (Cambridge, MA). Cells were prepared for flow cytometry using Human T-reg FIow™ Kit (BioLegend) according to the manufacturer instructions. Antibodies used for flow cytometry included Alexa Fluor® 488 Anti-human FOXP3, Clone 259D for intracellular staining of FOXP3, and PE Anti-human CD25 Clone BC96 for cell surface staining of CD25 (BioLegend). Fluorescently stained cells were resuspended in 1×HBSS (Invitrogen) and analyzed using a BD FACS Calibur flow cytometer machine (Becton Dickinson). FACS data was processed using FlowJo software (Version 10.0.8). Statistical Analysis Data analysis was performed by the paired Student t test using Excel (Microsoft) or GraphPad Prism-5 software (GraphPad Software, La Jolla, CA). Significance was determined by a two-sided p-value less than 0.05. Example 10. Short Term Culture Effects on Activated T-Reg Cells by TNFR2 Antagonist Antibodies To investigate the ability of antagonistic TNFR2 antibodies to modulate TNFR2 secretion, soluble human TNFR2 was measured from cell culture supernatants using Quantikine® ELISA (R&D Systems). Briefly, supernatants were collected after 24-42 hour incubation of CD4+ cells with IL-2 (200 U/ml) alone or with TNF (20 ng/ml) or antagonistic TNFR2 antibodies (12.5 μg/ml) and either used immediately or frozen at −20° C. ELISA was performed according to the manufacturer's instructions. Absorbance was measured using the SpectraMax® 190 Absorbance Plate Reader and analyzed with SoftMax Pro 6.3 (Molecular Devices). T-reg cells exist in two states, activated (aT-reg) and resting (rT-reg); the two states can be distinguished on the basis of the expression of CD45RA. The phenotype of an aT-reg cell is CD25HiCD45RALowwhile the phenotype of a rT-reg cell is CD25MedCD45RAHiaT-reg cells are more potent suppressors of immune function and therefore ideal targets for immunotherapy To investigate the efficacy of inhibiting aT-reg cells, it was first confirmed that the total number of T-reg cells, rather than simply the proportion of T-reg cells, was reduced when CD4+ cells were treated with the TNFR2 antagonists (FIGS.8A-8H). Next, to determine which class of T-reg is inhibited by the TNFR2 antagonists, cells were stained by CD45RA. We found that while both classes of T-reg cells are inhibited, the aT-reg cells were suppressed to a greater extent than the rT-reg cells. These data suggest that the TNFR2 antagonist antibodies selectively inhibit the proliferation of aT-reg cells. High TNFR2 expression is another characteristic of suppressive T-reg cells. The level of TNFR2 expression was measured and it was found that TNFR2 antagonist treatment reduced the number of TNFR2Hi-expressing cells. In contrast, mean fluorescent intensity (MFI) of TNFR2 expression remained similar among all treatments (FIG.9A). Taken together, these data suggest that antagonistic TNFR2 antibodies, such as TNFRAB1 and TNFRAB2, are capable of selectively attenuating the proliferation of CD25Hi+, CD45RALow+T-reg cells. The immune response is often characterized by an increase in soluble TNFR2 due to high protein turnover cells The level of soluble TNFR2 was therefore measured in culture supernatant after treatment with TNFR2-modulating agents in order to determine the effect of these molecules on immune activity. It was observed that TNF and the TNFR2 agonist increased the quantity of soluble TNFR2, whereas the TNFR2 antagonist reduced the level of soluble TNFR2 (FIG.9B). Additionally, T-reg suppressor assays confirmed potent suppression of CD8+ cells by TNFR2 antagonism (FIGS.9A and9C). These data demonstrate that treatment of human CD4+ cells with a TNFR2 antagonist effectively reduces the number of activated and highly suppressive T-reg cells and had the additional benefit of inhibiting soluble decoy TNFR2 secretion. To conduct T-reg suppression assays as described above, peripheral blood mononuclear cells (PBMCs) were used as responders. PBMCs were isolated on the day of venipuncture using a Ficoll-Plaque Plus (GE Healthcare, Piscataway, NJ) density gradient and cryopreserved at −80° C. PBMCs were thawed the day before mixing with T-reg cells and were rested overnight in RPMI 1640 and IL-2 (10 U/ml). The next day, responder cells were stained with 1 μM carboxyfluorescein diacetate succinimidyl ester (CFSE). Responder cells (5×104cells) were then mixed with expanded T-reg cells at various ratios (0:1, 4:1, 2:1, 1:1), and stimulated with anti-CD3 mAb (HIT3a, BD Biosciences) and IL-2 (50 U/ml). Cells were collected after 4 days and analyzed by flow cytometry. To measure the concentration of secreted TNFR2 as described above, the QUANTIKINE® ELISA assay kit was used (R&D Systems). Briefly, supernatants were collected after 24-42 hour incubation of CD4+ cells with IL-2 (200 U/ml) alone or with TNFα (20 ng/ml) or TNFR2 antagonist antibodies (12.5 μg/ml) and either used immediately or frozen at −20° C. ELISA assays were performed according to the manufacturer's instructions. Absorbance was measured using the SPECTRAMAX® 190 Absorbance Plate Reader and analyzed with SoftMax Pro 6.3 (Molecular Devices). Example 11. TNFR2 Antagonist Activity is Independent of Non-Specific Fc Region Binding or Cross-Linking Non-specific binding by antibody Fc regions can result in arbitrary functional activity. Treatment of CD4+ cells with TNFR2 mAb F(ab′)2fragments, with or without TNF, resulted in similar dose responses in T-reg cell quantities as observed with full monoclonal antibodies (FIGS.10A-10D). This confirms that specific binding by the F(ab′)2region of the antagonistic TNFR2 antibody to TNFR2, rather than non-specific binding mediated by the Fc region, is likely responsible for the suppressive activity. Purity of F(ab′)2fragments and full antibodies were assessed by SDS-PAGE analysis (FIGS.11A-11D). Crosslinking can also result in aberrant functional activity of antibodies. To rule out the possibility that non-specific crosslinking was a cause of the observed functional activity, a dose response assay was performed in which the TNFR2 antagonist antibodies were incubated with T-reg cells with and without anti-IgG antibodies. The dose-dependent suppression of T-reg cells by TNFR2 antagonist antibodies was unaffected by the presence of anti-IgG (FIGS.10C and10D). Taken together, these data confirm that the functional activity of TNFR2 antagonist antibodies is independent of non-specific Fc region activity or crosslinking. To conduct the SDS-PAGE analysis described above, protein samples were run alongside PRECISION PLUS™ (BioRad) or PERFECT PROTEIN™ (EMD Millipore) markers on NuPAGE 4-12% Bis-Tris Gels with MOPS SDS Running Buffer (Life Technologies) at 100V for 1 hour. Gels were stained for 24 hours with SIMPLY BLUE™ Safe Stain (Invitrogen). Example 12. NFkB Activation and Gene Expression is Inhibited by Antagonistic TNFR2 Antibodies NFkB signaling is required for TNF-mediated cell response and has been proposed as a potential target for cancer therapy. Upon observing a decrease in T-reg cell proliferation following treatment with TNFR2 antagonist antibodies, it was hypothesized that the underlying signaling mechanism would involve a reduction in NFkB activation. To investigate the molecular response to TNFR2 antagonism, the expression of eight promoters of NFkB activation were measured by real-time PCR analysis. These proteins included: CHUK, NFKBIE, KNKBIA, MAP3K11, TRAF2, TRAF3, relB, cIAP2/BIRCH. The expression of TNF and lymphotoxin were additionally monitored, as well as two markers of T-reg cells, FoxP3 and CD25. In each case, treatment with TNFRAB2 resulted in down-regulation of gene expression compared to treatment with TNF (FIGS.12A and12B). Next, using phosphorylated RelA/NFkB p65 as a marker, it was demonstrated that treatment of CD4+ cells with either TNFR2 antagonist antibody reduced NFkB activation, whereas treatment with TNF increased NFkB activation (FIGS.12C-12E). This suggests the effect of the two TNFR2 antagonist antibodies on TNFR2 signal transduction is highly similar. Additionally, analysis of the kinetic parameters of binding of antagonist TNFR2 antibodies to recombinant human TNFR2 revealed that there was no significant difference in association or dissociation rates between TNFRAB1 and TNFRAB2 (FIG.13A). To conduct gene expression assays, isolated CD4+ cells were incubated for 3 hours in the presence of IL-2 (50 U/ml) and TNFα (20 ng/ml) or TNFR2 antagonist antibody (2.5 μg/ml). Cells were collected and total RNA was isolated using RNAqueous-4PCR Kit (Ambion). The total RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied-Biosystems). Real-time PCR was performed using the TaqMan® Array Human NFkB Pathway 96-well Plate with TaqMan© Gene Expression Master Mix and the ABI Prism 7000 Sequence Detection System (Applied-Biosystems). NFkB activation was measured using the cell-based Human Phospho-RelA/NFkB p65 Immunoassay (R&D Systems). Briefly, fresh CD4+ cells were cultured in 96-well flat-bottom plates (0.2×106cells/well) in the presence of IL-2 (200 U/ml) alone or in the presence of TNF or TNFR2 antagonists at the indicated concentrations for 10 minutes at 37° C. Cells were adhered to the plate by centrifugation and fixation and then stained according to the manufacturer's instructions. Fluorescence was read using the EnVision® Multilabel Plate Reader (Perkin Elmer) and normalized relative fluorescence units (RFU) were calculated. Example 13. Binding of Antagonistic TNFR2 Antibodies to TNFR2 Maps to Overlapping Regions To further investigate the specific binding properties of the TNFR2 antagonist antibodies TNFRAB1 and TNFRAB2, epitope mapping analysis was performed and epitopes within human TNFR2 that bind these antibodies were correlated with a distinct location on the receptor surface. Linear peptide mapping of the two TNFR2 antagonists revealed that while both antagonistic antibodies exhibit a high affinity for the full-length target protein, these antibodies exhibited differential affinity for linear peptides. Specifically, TNFRAB1 exhibited moderate affinity to the epitope containing amino acids 112-139 of SEQ ID NO: 7 within human TNFR2, and exhibited strong affinity to the epitope containing amino acids 128-155 of SEQ ID NO: 7. TNFRAB2 did not bind to any linear peptide region (FIG.13B). The affinity of TNFRAB1 for TNFR2 was not perturbed by the presence of TNFα, indicating that TNFα is not a competitor with this antibody for TNFR2 binding (FIG.13C). To examine the conformational epitopes that bind the two antagonist antibodies, a three-dimensional binding analysis was conducted. For TNFRAB1, the 3D data was in agreement with the linear peptide mapping analysis and indicated that the region of amino acids 142-149 of SEQ ID NO: 7 within human TNFR2 were bound by TNFRAB1 with high affinity. Additionally, the 3D analysis revealed a new binding region at amino acids 161-169 of SEQ ID NO: 7 within human TNFR2 (FIGS.13A-13C and15A-15C). For TNFRAB2, even though there was no successful linear peptide binding, four binding regions were mapped by 3D analysis (FIGS.15A-15C). Interestingly, there was partial overlap in the precise discontinuous epitopes of the two TNFR2 antagonist antibodies at amino acids 142-144 of SEQ ID NO: 7 within human TNFR2. This later data was of interest since previous attempts to raise antagonistic TNFR2 antibodies against the TNFR2 binding site of the TNFR2 trimer signaling complex and/or to the exterior region of the trimer binding site to prevent TNFα entry and prevent stabilization of the TNF trimer had been unsuccessful. The solution structure of the TNFα and TNFR2 complexes had defined a central TNFα homotrimer surrounded by three TNFR2 receptors (Mukai et al. Science Signaling 3: ra83 (2010)). This arrangement had also been observed by many previous TNF superfamily members including TNFR1 with lymphotoxin, DR5 with TRAIL ligand, and 0x40 receptor with 0x40L (Banner et al. Cell 73: 431-445 (1993); Cha et al. J. Biol. Chem. 275: 31171-31177 (2000); Compaan et al. Structure 14:1321-1330 (2006); Hymowitz et al. Mol. Cell 4:563-571 (1999); Mongkolsapaya et al. Nat. Struct. Biol. 6:1048-1053 (1999)). Previous attempts to produce antagonistic antibodies to these regions to prevent TNFα binding and TNFR2 trimers yielded recessive-antagonistic antibodies with mild TNFR2 inhibitory activity having a common trait in T-reg proliferation assays. AsFIGS.14A-14Dshow, in the 48 hour T-reg assay these recessive-antagonistic antibodies performed as antagonists in the absence of a TNFR2-stimulating agent. Indeed, both recessive-antagonist TNFR2 antibodies A and B by themselves had a demonstration of dose response T-reg antagonism with a dose response from 0-25 μg/ml (FIGS.14A and14C). Importantly, when TNFα (20 mg/ml) was added to the cultures, these TNFR2-directed recessive-antagonistic antibodies A and B competed poorly with TNFα. In each case, TNFα driven agonism was dominant over this form of antagonism, at least the partial and weak antagonism with antibodies to prevent TNFα or TNFR2 trimer formations (FIG.15C). This behavior is in contrast with the capacity of dominant antagonistic TNFR2 antibodies, such as TNFRAB1 and TNFRAB2 that bind the specific epitopes within TNFR2 identified and described herein, such as epitopes containing the KCRPG motif (SEQ ID NO: 19) of TNFR2 and other epitopes as identified in the descriptions of TNFRAB1 and TNFRAB2 provided herein. As opposed to recessive-antagonistic TNFR2 antibodies A and B, TNFR2 antagonistic antibodies TNFRAB1 and TNFRAB2 displayed the ability to attenuate TNFR2 signaling and T-reg proliferation even in the presence of a TNFR2 agonist, such as TNFα and IL-2. Importantly, the epitopes identified herein can be used to raise antagonistic TNFR2 antibodies that display an antagonistic effect that persists in the presence of TNFR2 stimulants. Since linear peptide mapping only partially answered the question of why TNFRAB1 and TNFRAB2 were superior antagonists, we next explored identifying conformational epitopes of the two TNFR2 antagonists. We conducted three-dimensional (3D) binding analysis using the Pepscan technology (Pepscan, The Netherlands). For both TNFRAB1 and TNFRAB2, the 3D data showed overlapping regions from approximately aa138-aa150. What became apparent is that these binding regions did not appear to be able to bind to the classic TNFR2 trimer with trimeric TNFα since the site would have been on the interior of the trimer (FIG.151B). Since other research groups had reported alternative forms of TNF independent forms of TNF superfamily members that were parallel or anti-parallel models, we mapped the epitopes on those sites (Naismith et al. J. Biol. Chem. 270:13303-13307 (1995); Naismith et al. Structure 4:1251-1262 (1996)). Surprisingly, only one model of TNFR2 antagonistic binding was optimal: a model in which the anti-parallel dimer (FIG.15A) contains two TNFR2 antagonistic antibody binding regions that are spaced apart for the obligatory hinged TNFR2 antagonists and would appear to stabilize this TNFα-independent complex, another functional trait of these antibodies. With further modeling, it was apparent that the anti-parallel complex, but not the parallel dimer complex, would have intracellular regions that are distant from one another and would inhibit NFkB signaling as we had observed in our functional assays. Since all members of the TNF superfamily of receptors can display these less commonly studied anti-parallel conformations, antagonistic antibodies to any receptor of the TNFR superfamily can be made by designing or screening for antibodies or antigen-binding fragments thereof that bind surface-exposed epitopes within the anti-parallel TNFRS member protein (see, e.g., Example 15, below). These antibodies or antigen-binding fragments thereof can stabilize the anti-parallel forms of these proteins and thus prevent signaling, as well as the formation of active trimeric forms of these receptors that frequently also bind soluble cognate ligands. Known members of the TNF superfamily of receptors known to exhibit anti-parallel dimer structures include: TNFR1, TNFR2, Fas, DCR3, DR3, TRAIL-R1 (DR4). TRAIL-R2 (DR5), TRAIL-R3, TRAIL-R4, DR6, EDAR, CD271, OPG, RANK, LT3R, TWEAK-R, HVEM, CD27, CD30, CD40, CD137, OX40, GITR, BCMA, TACI, BAFFR, EDAR2, TROY, and RELT, among others. Example 14. TNFR2 Antagonist Activity on Cancer Cells T-reg cells present in patients suffering from cancer are potent immune suppressors. This is particularly true for T-reg cells from tumor sites as compared to the T-reg cells of peripheral blood of the cancer patients or control subjects. To begin to understand the potency of TNFR2 antagonist antibody on ovarian cancer T-reg cells, fresh ovarian cancer T-reg cells were isolated and their proliferation characterized as described in Example 2. First with TNF and then with TNFR2 agonist, it was indeed the case that ovarian cancer residing T-reg cells expanded far greater than the T-reg cells from normal donors, a sign of hyper activation (FIGS.16A-16B and17A-17H). TNFR2 antagonistic antibodies inhibited T-reg cell proliferation in a dose-dependent fashion and with higher variability as compared to the tight dose response data obtained from analysis of proliferation of T-reg cells isolated from healthy donors. Additionally, both antagonistic antibodies more potently inhibited the proliferation of T-reg cells isolated from ovarian cancer patients as compared to healthy donors. The experiments described above demonstrate that antagonistic TNFR2 antibodies are capable of inhibiting both normal and cancer-associated T-reg cell proliferation. Such antibodies can eliminate or inactivate potent suppressive T-reg cells, both in terms of the quantity of T-reg cells in culture as well as in terms of T-reg proliferation. The traits of these antibodies include the ability to attenuate intracellular early phosphoyrlation events that precede NFkB activation and the capacity to change the phenotype of the remaining T-reg cells such that these cells expressed reduced quantities of activation markers, such as CD45RO. Additionally, antagonistic TNFR2 antibodies may diminish the secretion of soluble TNFR2, a proinflammatory protein. Remarkably even in the presence of generous amounts of agonistic TNFα or agonistic TNFR2 antibodies added to cell culture media, antagonistic TNFR2 antibodies still act as dominant antagonists. It is worthwhile contrasting antibody or ligand agonism of the TNFα receptors with antibody antagonism. The requirement for TNF and other TNF superfamily agonism with the natural ligands is well studied and requires aggregation and clustering for efficient signal transduction (Das et al. J. Mol. Endocrinol. 53:81-91 (2014); Siegel et al. J. Cell. Biol. 167:735-744 (2004); Takeda et al. Oncogene 26:3745-3757 (2007)). The most common confirmation for the tumor necrosis factor receptor superfamily is a trimeric receptor paired with trimers of the ligand (Gommerman et al. Nat. Rev. Immunol. 3:642-655 (2003); Loetscher et al. J. Biol. Chem. 266:18324-18329 (1991); Smith et al. Cell 76:959-962 (1994); Tartaglia J. Biol. Chem. 267:4304-4307 (1992); Ware Annu. Rev. Immunol. 23:787-819 (2005)). Agonist antibodies seem to follow the same principles and also often require the Fc portions of their receptors for the associated cells through antibody-dependent cell-mediated cytotoxicity (ADCC) mechanisms, something that would stabilize the extracellular lattices. This exterior receptor oligomerization and clustering observed with TNFR2 agonism permits internal oligomerization of the reciprocal TRAF lattice network for brisk NFkB signaling (Cabal-Hierro et al. Cell Signal. 26:2658-2666 (2014); Yin et al. Nat. Struct. Mol. Biol. 16:658-666 (2009); Zheng et al. Mol. Cell. 38:101-113 (2010)). The stabilization of agonist is also promoted by the cooperation of the added ligand to the receptor. This appears important for many TNF receptor superfamily members for therapeutic effectiveness (Graves et al. Cancer Cell 26:177-189 (2014)). The shared receptor binding regions of two TNFR2 antagonist antibodies studied herein leads us to a very different possible model of antibody binding to the TNFR2 receptor to cause stable and dominant antagonism. The commonly studied trimeric form of TNFR2 would not allow the newly identified antibody antagonists to bind to their amino acid sequences and explain how these antagonistic antibodies are dominant over TNFα or IL-2 (Mukai et al. Science Signal. 3:ra83 (2010)). Indeed, years of work generating TNFR2 antibodies to the binding region of TNFα in the trimer TNFR2 structure or to the exterior surface of the TNFα-binding region of the trimer did not generate potent antagonists that maintained dominant inhibition of receptor signaling in the presence of TNFR2 agonists. TNFR2 trimer-directed antibodies designed to compete with TNFα were not dominant, but exhibited neutralizing effects at times when challenged with the inflammatory environment of TNFα or IL-2. The trimeric form of TNFR2 is the most commonly studied form of the TNFα receptor, as it relates to cell growth signals involving NFkB. Previous studies have proposed that TNFR2 and similar TNFα superfamily members like the lymphotoxin receptor can also exist as parallel and anti-parallel forms. These forms lack the TNF binding site (Naismith et al. J. Biol. Chem. 270:13303-13307 (1995); Naismith et al. Structure 4:1251-1262 (1996)). The data described herein show no added benefit of adding a cross-linking reagent to either augment antagonism or to convert antagonism to agonism. It would be hard to imagine that high-affinity TNFα binding of TNFα to trimeric TNFR2 could be competitively inhibited by an antibody, even with extremely high affinity. The anti-parallel dimeric form of TNFR2 fits best with the assessable binding site for antagonistic antibodies and the functional assay but there is also another feature to support this observation—the exterior interface is considerably more extensive and thus this would also cause the intracellular tails of TNFR2 to be a further distance apart as supported by others data (Naismith et al. Structure 4:1251-1262 (1996)). With TNFR2 trimerization the intracellular signaling regions of the TNFR2 are pulled close together, thus facilitating TRAF direct or indirect recruitment of the intracellular domains of these TNF superfamily receptors (Napetschnig et al. Annu. Rev. Biophys. 42:443-468 (2013); Yin et al. Nat. Struct. Mol. Biol. 16:658-666 (2009)). This agonist signal would subsequently engage other signaling proteins to activate the inhibitor of kB (IkB) kinase (IKK) and MAP kinases ultimately activating NFkB. The fact that the post-receptor signaling pathway is blocked to varying degrees with TNFR2 antagonistic antibodies suggests the recruitment of intracellular TRAFs are inhibited by the dominant stabilization of the exterior TNFR2 structure. It has been previously purposed by others that the anti-parallel dimers of TNFR1 could be an inhibitory complex and would not be able to bind TNF (Naismith et al. Structure 4:1251-1262 (1996)). The anti-parallel model more closely substantiates how antagonistic formation of this complex would explain dominance over TNF co-culture since new activation trimers could not be formed. It is worth mentioning that as reported by others, the T-reg cells in the cancer environment are extremely potent compared to the T-reg cells of non-cancerous donors or even the T-reg cells isolated from the peripheral blood of cancerous donors (Govindaraj et al. Clin. Cancer Res. 20: 724-735 (2013)). As described herein, antagonistic TNFR2 antibodies are capable of inhibiting the proliferation of potent T-reg cells isolated from ovarian cancer patients. It has also been observed that accentuated inhibition of T-reg cells isolated from ovarian cancer patients is achieved with TNFR2 antagonist antibodies TNFRAB1 and TNFRAB2. This activity is beneficial in view of recent reports identifying the features of the TNFR2 gene sequence that imparts enhanced potency to TNFR2 signaling in cancer patients. In the setting of cutaneous T cell lymphoma, for instance, the gene for TNFR2 is a gene duplication and/or may be mutated in the intracellular region to confer constitutive agonism. This genetic pressure to constantly maintain TNFR2 growth signals is in line with exaggerated growth. The antagonist antibodies described herein bind to the restricted regions of the TNFR2 receptor that modulate T-reg inhibition and even dominant inhibition in the presence of TNFα. These antagonists are very different from the recessive antagonists also described herein that can mildly inhibit T-reg cell proliferation but, when used in an inflammatory environment of TNF and IL-2, agonism dominates (FIGS.14A-14D). Taken together, the data described above provides a new platform for the creation of additional antagonist TNFR2 antibodies beyond those described herein that would be capable of modulating T-reg cell growth in patients suffering from a cancer in which cancer cells express elevated quantities of TNFR2 (e.g., ovarian cancer) and may display antagonistic effects even in the presence of TNFR2 agonists. Remarkably, such antagonistic TNFR2 antibodies are more potent on the T-reg cells of ovarian cancer patients than on the T-reg cells of non-cancerous donors. Example 15. Development of an Antagonistic TNFR2 Antibody by CDR-H3 Sequence Substitution In order to generate anti-TNFR2 antibodies, mice were immunized with full-length human TNFR2 using established immunization procedures. Serum was subsequently isolated from the immunized mice, and twelve distinct monoclonal antibodies were purified. The affinity of these twelve antibodies for human TNFR2 was verified by ELISA (FIG.18A) using microtiter plates coated with human TNFR2. The wells of microtiter plates used in these experiments were coated with antigen at 5 μg/ml. The murine antibodies were subsequently added to each well at dilutions of 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, and 1:218799 from rows A through G of the microtiter plate. Readings derived from row H correspond to background fluorescence. Notably, a positive titer will have an optical density reading of twice the background at a 1:2700 dilution. The added antibody was detected by adding a developing antibody, such as a labeled goat anti-mouse immunoglobulin conjugated to a fluorescent agent. The microtiter plate was subsequently analyzed using a fluorescence plate reader. The ELISA screening raw data was then analyzed numerically using Excel. Any microtiter well coated with antigen that has an optical density (OD) value greater than the positive threshold is be considered a “positive hit” provided the corresponding well on the counter screen-coated plate has an OD less than the negative threshold. Based on its high affinity for TNFR2 (as evidenced, e.g., by the fluoresce reading shown in row B, column 3 inFIG.18A), murine clone 3 was selected for CDR-H3 mutagenesis. The CDR-H3 sequence of clone 3 was substituted with the amino acid sequence ARDDGSYSPFDYFG (SEQ ID NO: 284) using recombinant gene expression techniques known in the art in order to produce the antagonistic TNFR2 antibody TNFR2A3. The precursor clone 3 was not capable of antagonizing the TNFR2 target, while the recombinantly produced TNFR2A3 exhibited antagonistic characteristics. As shown in column 3 ofFIGS.18B and18C, as well as in columns two and three ofFIG.20, TNFR2A3 was capable of specifically binding two human TNFR2-derived peptides having the amino acid sequence LRKCRPGFGVA (SEQ ID NO: 285) and VVCKPCAPGTFSN (SEQ ID NO: 286), respectively. This selective binding was evidenced by a dose-dependent increase in fluorescence within increasing concentrations of TNFR2A3 in each well of the microtiter plate. TNFR2A3 binds TNFR2 in a site-specific manner, as this antibody was found to exhibit negligible binding affinity for an unrelated TNFR2-derived peptide isolated from a distal region of the human TNFR2 primary structure (see, e.g., column 3 ofFIG.19). Collectively, these findings demonstrate two important facets of the CDR-H3 sequence with respect to TNFR2 antagonism. First, the CDR-H3 sequence of an antagonistic TNFR2 antibody is the primary molecular determinant of the antigen-binding properties of the antibody. Secondly, the CDR-H3 motif is a modular domain that can be imported into an anti-TNFR2 antibody that does not exhibit antagonistic activity in order to impart such an antibody with the ability to bind regions of TNFR2 that promote receptor antagonism. Example 16. Treatment of Cancer or an Infectious Disease in a Human Patient by Administration of Antagonistic Anti-TNFR2 Antibodies in Combination with an Immunotherapy Agent The antagonistic TNFR2 antibodies, antigen-binding fragments, single-chain polypeptides, and constructs of the invention can be administered to a human patient in combination with (for instance, admixed with, co-administered with, or administered separately from) an immunotherapy agent in order to treat a cell proliferation disorder, such as cancer, or an infectious disease, such as a viral, bacterial, fungal, or parasitic infection. Administration of the antibody, antigen-binding fragment, single-chain polypeptide, or construct can suppress the growth and proliferation of T-reg cells and/or cancer cells that express TNFR2. Immunotherapy agents, such as anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, TNF-α cross-linking agents, TRAIL cross-linking agents, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agent, anti-GITR agents, anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agent, and anti-TWEAKR agents can function in tandem with antagonist TNFR2 antibodies, antigen-binding fragments thereof, single-chain polypeptides, or constructs, as immunotherapy agents are capable of downregulating the signal transduction of immune checkpoint receptors and/or ligands that would otherwise lead to tolerance toward tumor-associated antigens and downregulation of the cytotoxic T cell response. Additional examples of immunotherapy agents that may be used in conjunction with an antagonistic TNFR2 antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct include TARGRETIN®, Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, gabapentin, antibodies to lymphoid cell surface receptors and/or lymphokines, antibodies to surface cancer proteins, and/or small molecular therapies like Vorinostat. A physician of skill in the art may administer an antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct that specifically binds TNFR2 as an antagonist, for instance, as described herein, to a human patient suffering from a cancer or infectious disease in combination with an immunotherapy agent. The antibody, antigen-binding fragment thereof, single-chain polypeptide, or construct and the immunotherapy agent may be administered to the patient by an appropriate route of administration (for example, intravenously, intramuscularly, or subcutaneously, among others) at a particular dosage (for example, between 0.001 and 100 mg/kg/day, among other ranges) over a course of days, weeks, months, or years. If desired, the anti-TNFR2 antibody, antigen-binding fragment, single-chain polypeptide, or construct can be modified, for instance, by hyperglycosylation or by conjugation with PEG, so as to evade immune recognition and/or to improve the pharmacokinetic profile of the antibody, antigen-binding fragment, single-chain polypeptide, or construct. The progression of the cancer or infectious disease that is treated in this fashion can be monitored by any one or more of several established methods. A physician can monitor the patient by direct observation in order to evaluate how the symptoms exhibited by the patient have changed in response to treatment. A patient may also be subjected to MRI, CT scan, or PET analysis in order to determine if a tumor has metastasized or if the size of a tumor has changed, for example, decreased in response to treatment with an anti-TNFR2 antibody, antigen-binding fragment, single-chain polypeptide, or construct and an immunotherapy agent. Optionally, cells can be extracted from the patient and a quantitative biochemical analysis can be conducted in order to determine the relative cell-surface concentrations of various growth factor receptors, such as the epidermal growth factor receptor. Based on the results of these analyses, a physician may prescribe higher/lower dosages or more/less frequent dosing of the antagonistic TNFR2 antibody, antigen-binding fragment, single-chain polypeptide, or construct and immunotherapy agent in subsequent rounds of treatment. Example 17. Combinatorial Analysis of the Effect of CDR-H and CDR-L Regions of Antagonistic TNFR2 Polypeptides on the Killing of Activated T-Reg Cells and the Proliferation of T Effector Cells It has presently been discovered that antagonistic TNFR2 polypeptides, such as antibodies, antigen-binding fragments, single-chain polypeptides, and constructs, that contain certain combinations of CDR-H and CDR-L regions are particularly capable of promoting T-reg cell death and augmenting T effector cell (e.g., CD8+ T cell) proliferation. To test the effects of various combinations of CDR-H1 and CDR-L3 regions on the antagonistic activity of anti-TNFR2 polypeptides, a series of monoclonal antibodies was developed containing all four possible combinations of the two CDR-H1 regions: GYTFTDYL (SEQ ID NO: 274) and GYTFTDYI (SEQ ID NO: 275) and the two CDR-L3 regions: CLQYVNLLT (SEQ ID NO: 272) and CLQYVNLIT (SEQ ID NO: 273). The remaining CDR regions were the same as those described for TNFRAB2 (CDR-H2: VDPEYGST (SEQ ID NO: 258), CDR-H3: ARDDGSYSPFDYWG (SEQ ID NO: 259), CDR-L1: QNINKY (SEQ ID NO: 260), CDR-L2: TYS or YTS). The monoclonal antibodies containing each of the four possible combinations of the foregoing CDR-H1 and CDR-L3 regions were then tested for the ability to induce T-reg cell death and promote CD8+ cell proliferation using standard CD4+CD25+ and CD8+ cell counting procedures, such as those described in Example 2 above. The abilities of each monoclonal antibody to induce T-reg cell death and induce CD8+ T cell proliferation were ranked on a qualitative scale of from 1 to 5, in which 1 corresponds to measurable biological activity and 5 corresponds to robust biological activity. The results of these experiments are summarized in Table 3, below: TABLE 3Effects of CDR-H1 and CDR-L3 combinations on T-regsuppression and CD8+ T cell induction propertiesof antagonistic TNFR2 monoclonal antibodiesCombination ofCapacity to promote T-Capacity to promoteCDR-H1 and CDR-L3 Regionsreg cell deathCD8+ T cell proliferationGYTFTDYL (SEQ ID NO: 274)25andCLQYVNLLT (SEQ ID NO: 272)GYTFTDYL (SEQ ID NO: 274)55andCLQYVNLIT (SEQ ID NO: 273)GYTFTDYI (SEQ ID NO: 275)13andCLQYVNLLT (SEQ ID NO: 272)GYTFTDYI (SEQ ID NO: 275)44andCLQYVNLIT (SEQ ID NO: 273) The results reported in Table 3 demonstrate that antagonistic TNFR2 polypeptides, such as antibodies, antigen-binding fragments, single-chain polypeptides, and constructs as described herein, that contain a CDR-H1 having the amino acid sequence GYTFTDYL (SEQ ID NO: 274) and a CDR-L3 having the amino acid sequence CLQYVNLIT (SEQ ID NO: 273) are particularly effective in promoting the selective killing of activated T-reg cells and potentiating augmented T effector cell proliferation. As described herein, these phenotypes are beneficial for the treatment of cancers and infectious diseases, as the ability to deplete activated T-reg cell populations in a patient suffering from such pathologies can lessen the attenuation of cytotoxic CD8+ T cells, thereby enabling effector cells to both function and or expand to mount an immune response against cancerous and infectious cells. Example 18. Antagonistic TNFR2 Polypeptides Exhibit Effects on T-Reg Cells, T Effector Cells, and TNFR2+ Cancer Cells Antagonistic TFNR2 polypeptides, such as antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs described herein, may exert biological activities on T-reg cells and T effector cells. To investigate these effects, antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 were incubated with cultured T-reg cells at ascending concentrations of antibody, and the percentage change in the quantity of T-reg cells in culture was subsequently recorded. The results of these experiments are shown inFIGS.21A and21B, and demonstrate that antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 reduce or inhibit the proliferation of T-reg cells in culture in a dose-dependent fashion. Additionally, antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 promote the proliferation of T effector cells. To investigate this activity, antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 were incubated with cultured CD8+ T cells at ascending concentrations of antibody, and the percentage change in the quantity of CD8+ T cells in culture was subsequently recorded. The results of these experiments are shown inFIG.21C, and demonstrate that antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 increase the proliferation of T effector cells in a dose-dependent fashion. The antagonistic TNFR2 antibodies TNFRAB1 and TNFRAB2 also directly kill TNFR2-expressing cancer cells. The antagonistic TNFR2 antibody TNFRAB1, was incubated with cultured OVCAR3 cells, a line of TNFR2+ ovarian cancer cells, at ascending concentrations of antibody, and the percentage change in the quantity of CD8+ T cells in culture was subsequently recorded. The results of this experiments are shown inFIG.21D, and demonstrate that the antagonistic TNFR2 antibody TNFRAB1 suppresses the proliferation of TNFR2+ cancer cells in a dose-dependent fashion. Taken together, the data shown inFIGS.21A-21Ddemonstrate that antagonistic TNFR2 polypeptides, such as anti-TNFR2 antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs described herein, are capable of exerting therapeutic effects through several distinct mechanisms. Antagonistic TNFR2 polypeptides can suppress T-reg cell proliferation and increase the proliferation of T effector cells, which can then mount an immune response against, for example, a cancer cell or a cell of an infectious pathogen. Additionally, antagonistic TNFR2 polypeptides can directly kill cancer cells that express TNFR2. Through these mechanisms, for example, antagonistic TNFR2 polypeptides, such as those described herein, can be used to treat patients suffering from a variety of cancers and infectious diseases, such as those conditions described herein. Example 19. Antagonistic TNFR2 Polypeptides Kill T-Reg Cells and Expand T Effector Cells in the Tumor Microenvironment Antagonistic TFNR2 polypeptides, such as antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs described herein, may preferentially reduce the proliferation of T-reg cells and increase the proliferation of T effector in the tumor microenvironment of a cancer patient. To investigate this property, the antagonistic TNFR2 antibody, TNFRAB1, was incubated with either fresh human ovarian cancer ascites or a blood sample obtained from a human subject without cancer. TNFRAB1 was present in these samples in ascending doses, and the quantities of T-reg cells and T effector cells present in each sample were recorded following an incubation period. The results of these experiments are reported inFIGS.22A and22B. As shown therein, TNFRAB1 killed or reduced the proliferation of T-reg cells in the ovarian cancer ascites sample in a dose-dependent fashion. TNFRAB1 also killed or reduced proliferation of T-reg cells in the sample obtained from a human without cancer, but to a lesser extent. Conversely, TNFRAB1 increased T effector cell proliferation in the ovarian cancer ascites sample to a greater extent than in the sample obtained from a human without cancer. Collectively, these data demonstrate that antagonistic TNFR2 polypeptides, such as anti-TNFR2 antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs described herein, can preferentially deplete T-reg cells in the tumor microenvironment, such as in a patient suffering from a cancer described herein, relative to T-reg cells in an environment free of cancer cells, such as in a healthy human subject. Example 20. Antagonistic TNFR2 Polypeptides Kill T-Reg Cells, Expand T Effector Cells, and Deplete TNFR2+CD26-Cells Antagonistic TFNR2 polypeptides, such as antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs described herein, exhibit the ability to kill T-reg cells, increase T effector cell proliferation, and kill TNFR2-expressing cancer cells in the tumor microenvironment. To further investigate these activities, the antagonistic TNFR2 antibody TNFRAB1 was incubated with blood samples obtained from healthy human subjects and human patients suffering from advanced cutaneous T cell lymphoma. These patients were receiving background treatment with one or more immunotherapy agents, such as TARGRETIN®, Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, and gabapentin, among others, and were failing to respond to the immunotherapy at the time the present experiments were conducted. After an incubation period, the quantities of effector T cells, T-reg cells, and TNFR2+ CD26-cells were measured in each sample. The absence of CD26 expression by a T cell is an indicator of T cell lymphoma. Thus, effects exerted on these cells are indicative of effects on cancer cells in a T cell lymphoma patient. The results of these experiments are reported inFIGS.23A-23C. These data demonstrate that antagonistic TNFR2 polypeptides, such as TNFRAB1, can simultaneously modulate a cancer patient's T-reg, T effector, and TNFR2+ CD26-cell populations. Antagonistic TNFR2 polypeptides described herein, such as antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs, may demonstrate all three of these beneficial characteristics so as to impart a therapeutic effect on patients suffering from cancer, such as T cell lymphoma or another cancer described herein. Additionally, the results shown inFIGS.23A-23Cdemonstrate that antagonistic TNFR2 polypeptides, such as antibodies, antigen-binding fragments thereof, single-chain polypeptides, and constructs described herein, can be used in combination with immunotherapy agents for the treatment of cancer in a patient. The T cell lymphoma patients studied in the foregoing experiments were not responsive to immunotherapy alone. Upon treatment of isolated blood samples from these patients with TNFRAB1, a favorable in vitro response was observed, as the proliferation of T-reg cells and TNFR2+ CD26-cells decreased and the proliferation of T effector cells increased. These results show that antagonistic TNFR2 polypeptides can be used in combination with immunotherapy agents, such an immunotherapy agent described herein (for example, TARGRETIN®, Interferon-alpha, clobetasol, Peg Interferon (e.g., PEGASYS®), prednisone, Romidepsin, Bexarotene, methotrexate, Triamcinolone cream, anti-chemokines, Vorinostat, and gabapentin), to improve treatment efficacy in cancer patients. Moreover, the results of the above experiments demonstrate that antagonistic TNFR2 polypeptides of the invention exhibit therapeutic effects in patients having a variety of cancers. Antagonistic TNFR2 antibodies can be used to suppress ovarian cancer cell proliferation (see, e.g., Example 14, above) and T cell lymphoma cell proliferation, as presently described. Cells of both of these cancers exhibit the common property expressing TNFR2. Thus, antagonistic TNFR2 polypeptides can be used to treat a wide array of cancer types, such as those in which TNFR2 is expressed on the cancer cell surface. Other Embodiments All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims. | 339,483 |
11859003 | DETAILED DESCRIPTION A. Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, antibody engineering, immunotherapy, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art. As used herein, each of the following terms has the meaning associated with it in this section. The articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. The term “amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid but the C-terminal carboxy group, the N-terminal amino group, or side chain functional group has been chemically modified to another functional group. The term “amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid. The term “antibody” is used herein in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., a single-chain variable fragment or scFv) so long as they exhibit the desired biological activity. The term “antibody” is an art-recognized term and may refer to an antigen-binding protein (i.e, immunoglobulin) having a basic four-polypeptide chain structure consisting of two identical heavy (H) chains and two identical light (L) chains. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each heavy chain has, at the N-terminus, a variable region (abbreviated herein as VH) followed by a constant region. The heavy chain constant region is comprised of three domains, CH1, CH2and CH3. Each light chain has, at the N-terminus, a variable region (abbreviated herein as VI) followed by a constant region at its other end. The light chain constant region is comprised of one domain, CL. The VLis aligned with the VHand the CLis aligned with the first constant domain of the heavy chain (CH1). The pairing of a VHand VLtogether forms a single antigen-binding site. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called J chain, and therefore contains 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. The VHand VLregions can be further subdivided into regions of hypervariability, termed hyper-variable regions (HVR) based on the structural and sequence analysis. HVRs are interspersed with regions that are more conserved, termed framework regions (FW). For comparison, the Kabat CDR definition by Yvonne Chen, et al. (Selection and Analysis of an Optimized Anti-VEGF Antibody: Crystal Structure of an Affinity-matured Fab in Complex with Antigen, J. Mol. Biol. (1999) 293, 865-881) is listed below (see alsoFIG.1a). Each VHand VLis composed of three HVRs and four FWs, arranged from amino-terminus to carboxy-terminus in the following order: FW1, HVR1, FW2, HVR2, FW3, HVR3, FW4. Throughout the present disclosure, the three HVRs of the heavy chain are referred to as HVR_H1, HVR_H2, and HVR_H3. Similarly, the three HVRs of the light chain are referred to as HVR_L1, HVR_L2, and HVR_L3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)). The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), antibodies can be assigned to different classes or isotypes. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α (alpha), δ (delta), ε(epsilon), γ (gamma), and μ(mu), respectively. The IgG class of antibody can be further classified into four subclasses IgG1, IgG2, IgG3, and IgG4 by the gamma heavy chains, Y1-Y4, respectively. The term “antibody derivative” or “derivative” of an antibody refers to a molecule that is capable of binding to the same antigen (e.g., CD137) that the antibody binds to and comprises an amino acid sequence of the antibody linked to an additional molecular entity. The amino acid sequence of the antibody that is contained in the antibody derivative may be a full-length heavy chain, a full-length light chain, any portion or portions of a full-length heavy chain, any portion or portions of the full-length light chain of the antibody, any other fragment(s) of an antibody, or the complete antibody. The additional molecular entity may be a chemical or biological molecule. Examples of additional molecular entities include chemical groups, amino acids, peptides, proteins (such as enzymes, antibodies), and chemical compounds. The additional molecular entity may have any utility, such as for use as a detection agent, label, marker, pharmaceutical or therapeutic agent. The amino acid sequence of an antibody may be attached or linked to the additional molecular entity by chemical coupling, genetic fusion, noncovalent association, or otherwise. The term “antibody derivative” also encompasses chimeric antibodies, humanized antibodies, and molecules that are derived from modifications of the amino acid sequences of a CD137 antibody, such as conservation amino acid substitutions, additions, and insertions. The term “antigen-binding fragment” or “antigen binding portion” of an antibody refers to one or more portions of an antibody that retain the ability to bind to the antigen that the antibody bonds to (e.g., CD137). Examples of “antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CLand CH1domains; (ii) a F(ab′)2fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature341:544-546 (1989)), which consists of a VHdomain; and (vi) an isolated complementarity determining region (CDR). The term “binding molecule” encompasses (1) antibody, (2) antigen-binding fragment of an antibody, and (3) derivative of an antibody, each as defined herein. The term “binding CD137,” “binds CD137,” “binding to CD137,” or “binds to CD137” refers to the binding of a binding molecule, as defined herein, to the human CD137 in an in vitro assay, such as a Biacore assay as described in Example 4, with an affinity (KD) of 100 nM or less. The terms “CD137” and “CD137 receptor” are used interchangeably in the present application, and include the human CD137 receptor, as well as variants, isoforms, and species homologs thereof. Accordingly, a binding molecule, as defined and disclosed herein, may also bind CD137 from species other than human. In other cases, a binding molecule may be completely specific for the human CD137 and may not exhibit species or other types of cross-reactivity. The term “CD137 antibody” refers to an antibody, as defined herein, capable of binding to human CD137 receptor. The term “chimeric antibody” refers to an antibody that comprises amino acid sequences derived from different animal species, such as those having a variable region derived from a human antibody and a murine immunoglobulin constant region. The term “compete for binding” refers to the interaction of two antibodies in their binding to a binding target. A first antibody competes for binding with a second antibody if binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not, be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). The term “epitope” refers to a part of an antigen to which an antibody (or antigen-binding fragment thereof) binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope can include various numbers of amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography, 2-dimensional nuclear magnetic resonance, deuterium and hydrogen exchange in combination with mass spectrometry, or site-directed mutagenesis, or all methods used in combination with computational modeling of antigen and its complex structure with its binding antibody and its variants. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). Once a desired epitope of an antigen is determined, antibodies to that epitope can be generated, e.g., using the techniques described herein. The generation and characterization of antibodies may also elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. An approach to achieve this is to conduct cross-competition studies to find antibodies that competitively bind with one another, i.e., the antibodies compete for binding to the antigen. A high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03/48731. The term “germline” refers to the nucleotide sequences of the antibody genes and gene segments as they are passed from parents to offspring via the germ cells. The germline sequence is distinguished from the nucleotide sequences encoding antibodies in mature B cells which have been altered by recombination and hypermutation events during the course of B cell maturation. The term “glycosylation sites” refers to amino acid residues which are recognized by a eukaryotic cell as locations for the attachment of sugar residues. The amino acids where carbohydrate, such as oligosaccharide, is attached are typically asparagine (N-linkage), serine (O-linkage), and threonine (O-linkage) residues. The specific site of attachment is typically signaled by a sequence of amino acids, referred to herein as a “glycosylation site sequence”. The glycosylation site sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-, where X may be any of the conventional amino acids, other than proline. The terms “N-linked” and “O-linked” refer to the chemical group that serves as the attachment site between the sugar molecule and the amino acid residue. N-linked sugars are attached through an amino group; O-linked sugars are attached through a hydroxyl group. The term “glycan occupancy” refers to the existence of a carbohydrate moiety linked to a glycosylation site (i.e., the glycan site is occupied). Where there are at least two potential glycosylation sites on a polypeptide, either none (0-glycan site occupancy), one (1-glycan site occupancy) or both (2-glycan site occupancy) sites can be occupied by a carbohydrate moiety. The term “host cell” refers to a cellular system which can be engineered to generate proteins, protein fragments, or peptides of interest. Host cells include, without limitation, cultured cells, e.g., mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters) such as CHO, BHK, NSO, SP2/0, YB2/0; or human tissues or hybridoma cells, yeast cells, and insect cells, and cells comprised within a transgenic animal or cultured tissue. The term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell.” The term “human antibody” refers to an antibody in which the entire amino acid sequences of the light chains and heavy chains are from the human immunoglobulin genes. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Human antibodies may be prepared in a variety of ways known in the art. The term “humanized antibody” refers to a chimeric antibody that contains amino acid residues derived from human antibody sequences. A humanized antibody may contain some or all of the CDRs or HVRs from a non-human animal or synthetic antibody while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. The term “illustrative antibody” refers to any one of the antibodies described in the disclosure and designated as those listed in Tables 1a and 1b. These antibodies may be in any class (e.g., IgA, IgD, IgE, IgG, and IgM). Thus, each antibody identified above encompasses antibodies in all five classes that have the same amino acid sequences for the VLand VHregions. Further, the antibodies in the IgG class may be in any subclass (e.g., IgG1 IgG2, IgG3, and IgG4). Thus, each antibody identified above in the IgG subclass encompasses antibodies in all four subclasses that have the same amino acid sequences for the VLand VHregions. The amino acid sequences of the heavy chain constant regions of human antibodies in the five classes, as well as in the four IgG subclasses, are known in the art. The amino acid sequence of the full length heavy chain and light chain for the IgG4 subclass of each of the illustrative antibodies shown in in Table 1b is provided in the disclosure. The term “isolated antibody” or “isolated binding molecule” refers to an antibody or a binding molecule, as defined herein, that: (1) is not associated with naturally associated components that accompany it in its native state; (2) is free of other proteins from the same species; (3) is expressed by a cell from a different species; or (4) does not occur in nature. Examples of isolated antibodies include a CD137 antibody that has been affinity purified using CD137, a CD137 antibody that has been generated by hybridomas or other cell line in vitro, and a CD137 antibody derived from a transgenic animal. The term “isolated nucleic acid” refers to a nucleic acid molecule of genomic, cDNA, or synthetic origin, or a combination thereof, which is separated from other nucleic acid molecules present in the natural source of the nucleic acid. For example, with regard to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid of interest. The term “ka” refers to the association rate constant of a particular antibody-antigen interaction, whereas the term “kd” refers to the dissociation rate constant of a particular antibody-antigen interaction. The term “KD” refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. It is obtained from the ratio of kdto ka(i.e., kd/ka) and is expressed as a molar concentration (M). KDis used as a measure for the affinity of an antibody's binding to its binding partner. The smaller the KD, the more tightly bound the antibody is, or the higher the affinity between antibody and the antigen. For example, an antibody with a nanomolar (nM) dissociation constant binds more tightly to a particular antigen than an antibody with a micromolar (μM) dissociation constant. KDvalues for antibodies can be determined using methods well established in the art. One method for determining the KDof an antibody is by using surface plasmon resonance, typically using a biosensor system such as a Biacore® system. An assay procedure using the BIACORE™ system (BIAcore assay) is described in the Examples section of this disclosure. The term “mammal” refers to any animal species of the Mammalia class. Examples of mammals include: humans; laboratory animals such as rats, mice, simians and guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like. The term “prevent” or “preventing,” with reference to a certain disease condition in a mammal, refers to preventing or delaying the onset of the disease, or preventing the manifestation of clinical or subclinical symptoms thereof. As used herein, “sequence identity” between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as Bestfit, FASTA, or B LAST (see, e.g. Pearson,Methods Enzynol.183:63-98 (1990); Pearson,Methods Mol. Biol.132:185-219 (2000); Altschul et al.,J. Mol. Biol.215:403-410 (1990); Altschul et al.,Nucelic Acids Res.25:3389-3402 (1997)). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed. This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, fragments, or variants thereof disclosed herein. The term “specifically binds” or “specifically binds to,” in reference to the interaction of a binding molecule, as defined herein, (e.g., an antibody) with its binding partner (e.g., an antigen), refers to the ability of the binding molecule to discriminate between an antigen of interest from an animal species and the antigen orthologue from a different animal species under a given set of conditions. A CD137 binding molecule is said to specifically bind to human CD137 if it binds to human CD137 at an EC50 that is below 50 percent of the EC50 at which it binds CD137 of rat or mouse as determined in an in vitro assay. Binding specificity of an antibody can be determined using methods known in the art. Examples of such methods include FACS using PHA stimulated primary cells, Western blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. The term “selectively binds” or “selectively binds to,” in reference to the interaction of a binding molecule, as defined herein, (e.g., an antibody) with its binding partner (e.g., an antigen), refers to the ability of the binding molecule to discriminate between an antigen of interest from an animal species (such as human CD137) and a different antigen from the same animal species (such as human CD40) under a given set of conditions. A CD137 binding molecule is said to selectively bind to human CD137 if it binds to human CD137 at an EC50 that is below 10 percent of the EC50 at which it binds to human CD40 or human CD134 as determined in an in vitro assay. The term “treat”, “treating”, or “treatment”, with reference to a certain disease condition in a mammal, refers causing a desirable or beneficial effect in the mammal having the disease condition. The desirable or beneficial effect may include reduced frequency or severity of one or more symptoms of the disease (i.e., tumor growth and/or metastasis, or other effect mediated by the numbers and/or activity of immune cells, and the like), or arrest or inhibition of further development of the disease, condition, or disorder. In the context of treating cancer in a mammal, the desirable or beneficial effect may include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of reoccurrence of cancer, reduction of pain associated with the cancer, or improved survival of the mammal. The effect can be either subjective or objective. For example, if the mammal is human, the human may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Alternatively, the clinician may notice a decrease in tumor size or tumor burden based on physical exam, laboratory parameters, tumor markers or radiographic findings. Some laboratory signs that the clinician may observe for response to treatment include normalization of tests, such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, the clinician may observe a decrease in a detectable tumor marker. Alternatively, other tests can be used to evaluate objective improvement, such as sonograms, nuclear magnetic resonance testing and positron emissions testing. The term “vector” refers to a nucleic acid molecule capable of transporting a foreign nucleic acid molecule. The foreign nucleic acid molecule is linked to the vector nucleic acid molecule by a recombinant technique, such as ligation or recombination. This allows the foreign nucleic acid molecule to be multiplied, selected, further manipulated or expressed in a host cell or organism. A vector can be a plasmid, phage, transposon, cosmid, chromosome, virus, or virion. One type of vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Another type of vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Another specific type of vector capable of directing the expression of expressible foreign nucleic acids to which they are operatively linked is commonly referred to as “expression vectors.” Expression vectors generally have control sequences that drive expression of the expressible foreign nucleic acids. Simpler vectors, known as “transcription vectors,” are only capable of being transcribed but not translated: they can be replicated in a target cell but not expressed. The term “vector” encompasses all types of vectors regardless of their function. Vectors capable of directing the expression of expressible nucleic acids to which they are operatively linked are commonly referred to “expression vectors.” The methods and techniques of the present disclosure are generally performed according to methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Such references include, e.g., Sambrook and Russell,Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. SeeImmunology—A Synthesis(2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)). B. Binding Molecules that Bind to Human CD137 The present disclosure provides isolated binding molecules that bind to human CD137, including CD137 antibodies, antigen-binding fragments of the CD137 antibodies, and derivatives of the CD137 antibodies. In some embodiments, the binding molecules are any of the antibodies described herein, including antibodies described with reference to epitope binding and antibodies described with reference to specific amino acid sequences of HVRs, variable regions (VL, VH), and IgG (e.g., IgG4) light and heavy chains. In some embodiments, the present disclosure relates to binding molecules that bind to human CD137, and have at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, eight, or all nine) of the following functional properties: (a) bind to human CD137 with a KD of 500 nM or less; (b) have agonist activity on human CD137; (c) do not bind to human OX40, CD40, GITR and/or CD27 receptor at concentration up to 1000 nM; (d) is cross-reactive with monkey, mouse, rat, or dog CD137; (e) do not induce ADCC effects; (f) are capable of inhibiting tumor cell growth; (g) have therapeutic effect on a cancer; (h) blocks binding between CD137 and CD137L; and (i) blocks CD137 signaling stimulated by CD137L (e.g., CD137L-stimulated NF-κB-dependent transcription) in a cell that expresses CD137. In some embodiments, the antibodies disclosed herein can also block, e.g., completely block, the binding between CD137 and its ligand CD137L. Also provided herein are one or more anti-CD137 antibodies or antigen-binding fragments that cross-compete for binding to human CD137 with one or more of the antibodies or antigen-binding fragments as described herein. In some embodiments, the antibodies or the antigen-binding fragments thereof bind to one or more amino acid residues within amino acid residues 34-108 of SEQ ID NO:1. In some embodiments, the antibodies or antigen-binding fragments bind to one or more amino acid residues within amino acid residues 34-93 of SEQ ID NO:1. In some embodiments, the antibodies or antigen-binding fragments bind to one or more amino acid residues selected from the group consisting of amino acid residues 34-36, 53-55, and 92-93 of SEQ ID NO:1. In some embodiments, the antibodies or antigen-binding fragments bind to one or more of amino acid residues 34-36, one or more of amino acid residues 53-55, and one or more or amino acid residues 92-93 of SEQ ID NO:1. In some embodiments, the antibodies or antigen-binding fragments do not bind to one or more of amino acid residues selected from the group consisting of amino acid residues 109-112, 125, 126, 135-138, 150 and 151 of SEQ ID NO:1. In some embodiments, the antibodies or antigen-binding fragments do not bind to amino acid residues 109-112, 125, 126, 135-138, 150 and 151 of SEQ ID NO:1. Methods of measuring an antibody or antigen-binding fragment's ability to bind a target antigen may be carried out using any method known in the art, including for example, by surface plasmon resonance, an ELISA, isothermal titration calorimetry, a filter binding assay, an EMSA, etc. In some embodiments, the ability of the antibody or antigen-binding fragment to bind a target antigen is measured by surface plasmon resonance (See e.g., Example 1 below). In some embodiments, the antibodies or antigen-binding fragments bind to human CD137 with a KD of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 150 nM or less, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 75 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, etc.) In some embodiments, the antibodies or antigen-binding fragments bind to human CD137 with a KD of about 100 nM or less. In some embodiments, the antibodies or antigen-binding fragments bind to human CD137 with a KD of about 50 nM or less. Methods of measuring the KD of an antibody or antigen-binding fragment may be carried out using any method known in the art, including for example, by surface plasmon resonance, an ELISA, isothermal titration calorimetry, a filter binding assay, an EMSA, etc. In some embodiments, the KD is measured by surface plasmon resonance (See e.g., Example 1 below). Anti-CD137 antibodies need to be cross-linked to become agonistic. For example, cross-linking is achieved in vivo through Fcgamma receptors, while typically polyclonal anti-Fc antibodies are used in cell-based experiments in vitro. In some embodiments, the antibodies or antigen-binding fragments described herein have agonist activity on human CD137. In some embodiments, the antibodies or antigen-binding fragments induce one or more (e.g., one or more, two or more, three or more, etc.) activities of human CD137 when a cell (e.g., a human cell) expressing human CD137 is contacted by the antibody or antigen binding fragment. Various CD137 activities are known in the art and may include, without limitation, induction of NF-κB-dependent transcription, induction of T cell proliferation, prolonging T cell survival, co-stimulation of activated T cells, induction of cytokine secretion (such as IL-2), and induction of monocyte activation. In some embodiments, the one or more CD137 activities is not CD137 binding to its ligand. Methods of measuring CD137 activity (e.g., the induction of NF-κB-dependent transcription and/or T cell proliferation, etc.) are known in the art, including, for example, via the methods described in Examples 8 and 9 below. In some embodiments, the antibodies or antigen-binding fragments increase NF-κB dependent transcription in cells (e.g., human cells) expressing human CD137. In some embodiments, NF-κB dependent transcription is increased by about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 99% or more in cells (e.g., human cells) expressing CD137 contacted with the antibody or antigen-binding fragment, relative to a corresponding cell not contacted with the antibody or antigen-binding fragment (e.g., a corresponding cell not contacted with an antibody, or contacted with an isotype control antibody). In some embodiments, NF-κB dependent transcription is increased by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold or more in cells (e.g., human cells) expressing CD137 contacted with the antibody or antigen-binding fragment, relative to a corresponding cell not contacted with the antibody or antigen-binding fragment (e.g., a corresponding cell not contacted with an antibody, or contacted with an isotype control antibody). In some embodiments, the antibodies or antigen-binding fragments are cross-reactive with monkey (e.g., cynomolgus monkey), mouse, rat, and/or dog CD137. In some embodiments, the antibodies or antigen-binding fragments are cross-reactive with monkey CD137. In some embodiments, the antibodies or antigen-binding fragments are cross-reactive with mouse CD137. In some embodiments, the antibodies or antigen-binding fragments are cross-reactive with rat CD137. In some embodiments, the antibodies or antigen-binding fragments are cross-reactive with dog CD137. In some embodiments, the antibodies or antigen binding fragments are cross reactive with monkey and mouse CD137; monkey and rat CD137; monkey and dog CD137; mouse and rat CD137; mouse and dog CD137; rat and dog CD137; monkey, mouse, and rat CD137; monkey, mouse, and dog CD137; monkey, rat, and dog CD137; mouse, rat, and dog CD137; or monkey, mouse, rat, and dog CD137. In some embodiments, the antibodies or antigen-binding fragments are cross-reactive at about 100 nM (e.g., at about 1 nM, at about 10 nM, at about 25 nM, at about 50 nM, at about 75 nM, at about 100 nM). Methods of measuring antibody cross-reactivity are known in the art, including, without limitation, surface plasmon resonance, an ELISA, isothermal titration calorimetry, a filter binding assay, an EMSA, etc. In some embodiments, the cross-reactivity is measured by ELISA (See e.g., Example 2 below). In some embodiments, the antibodies do not induce ADCC effects. Methods of measuring ADCC effects (e.g., in vivo methods) are known in the art, including, without limitation, via the methods described in Example 11 below. In some embodiments, the antibodies do not ADCC effects by more than about 10% (do not induce ADCC by more than about 10%, more than about 5%, more than about 1%, more than about 0.1%, more than about 0.01%) relative to a control. In some embodiments, the antibodies or antigen-binding fragments are capable of inhibiting tumor cell growth/proliferation. In some embodiments, the tumor cell growth/proliferation is inhibited by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) when contacted with the antibodies or antigen-binding fragments relative to corresponding tumor cells not contacted with the antibodies or antigen-binding fragments. In some embodiments, the antibodies or antigen-binding fragments are capable of reducing tumor volume in a subject when the subject is administered the antibodies or antigen-binding fragments. In some embodiments, the antibodies or antigen-binding fragments are capable of reducing tumor volume in a subject by at least about 5% (e.g., at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%) relative to the initial tumor volume in the subject (e.g., prior to administration of the antibodies or antigen-binding fragments). Methods of monitoring tumor cell growth/proliferation, tumor volume, and/or tumor inhibition are known in the art, including, for example, via the methods described in Example 10 below. In some embodiments, the antibodies or antigen-binding fragments have therapeutic effect on a cancer. In some embodiments, the antibodies or antigen-binding fragments reduce one or more signs or symptoms of a cancer. In some embodiments, a subject suffering from a cancer goes into partial or complete remission when administered the antibodies or antigen-binding fragments. In another aspect, the disclosure provides isolated antibodies that compete or cross-compete for binding to human CD137 with any of the illustrative antibodies of the disclosure, such as AG10058, AG10059, and/or AG10131. In a particular embodiment, the disclosure provides isolated antibodies that compete or cross-compete for binding to the same epitope on the human CD137 with any of the illustrative antibodies of the disclosure. The ability of an antibody to compete or cross-compete for binding with another antibody can be determined using standard binding assays known in the art, such as BIAcore analysis, ELISA assays, or flow cytometry. For example, one can allow an illustrative antibody of the disclosure to bind to human CD137 under saturating conditions and then measure the ability of the test antibody to bind to the CD137. If the test antibody is able to bind to the CD137 at the same time as the illustrative antibody, then the test antibody binds to a different epitope as the illustrative antibody. However, if the test antibody is not able to bind to the CD137 at the same time, then the test antibody binds to the same epitope, an overlapping epitope, or an epitope that is in close proximity to the epitope bound by the illustrative antibody. This experiment can be performed using various methods, such as ELISA, RIA, FACS or surface plasmon resonance. In some embodiments, the antibodies or antigen-binding fragments block the binding between CD137 and its ligand (e.g., human CD137 and human CD137L). In some embodiments, the antibodies or antigen-binding fragments block the binding between CD137 and its ligand in vitro. In some embodiments, the antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC50) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for blocking binding of CD137 its ligand. In some embodiments, the antibody or antigen-binding fragment has a half maximal inhibitory concentration (IC50) of about 100 nM or less for blocking binding of CD137 its ligand. In some embodiments, the antibody or antigen-binding fragment completely blocks binding of human CD137 to its ligand when provided at a concentration of about 100 nM or greater (e.g., about 100 nM or greater, about 500 nM or greater, about 1 μM or greater, about 10 μM or greater, etc.). As used herein, the term “complete blocking” or “completely blocks” refers to the antibody or antigen-binding fragment's ability to reduce binding between a first protein and a second protein by at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, etc.). Methods of measuring the ability of an antibody or antigen-binding fragment to block binding of a first protein (e.g., a CD137) and a second protein (e.g., CD137L) are known in the art, including, without limitation, via BIAcore analysis, ELISA assays, and flow cytometry (See e.g., Example 6 below). B-1. CD137 Antibodies In some aspects, the present disclosure provides an isolated antibody that binds to human CD137 at an epitope within amino acid residues 34-108 or 34-93 of SEQ ID NO.: 1. The antibody, in some embodiments, binds human CD137 with a KDof 50 nM or less as measured by surface plasmon resonance. In certain embodiments, the antibody can be cross-reactive with at least one non-human species selected from the list consisting of cynomolgus monkey, mouse, rat and dog. In one aspect, the present disclosure provides an isolated antibody comprising a heavy chain variable region and a light chain variable region, a) wherein the heavy chain variable region comprises an HVR-H1, an HVR-H2, and an HVR-H3, wherein the HVR-H1 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (I): X1TFX2X3YX4IHWV (SEQ ID NO:2), wherein X1 is F or Y, X2 is S or T, X3 is G, N, or S, and X4 is A, G, or W; Formula (II): YSIX1SGX2X3WX4WI (SEQ ID NO:3), wherein X1 is S or T, X2 is H or Y, X3 is H or Y, and X4 is A, D, G, N, S, or T; and Formula (III): FSLSTX1GVX2VX3WI (SEQ ID NO:4), wherein X1 is G or S, X2 is A or G, and X3 is A, G, S, or T; wherein the HVR-H2 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (IV): LALIDWX1X2DKX3YSX4SLKSRL (SEQ ID NO:5), wherein X1 is A, D, or Y, X2 is D or G, X3 is R, S, or Y, and X4 is P or T; Formula (V): IGX1IYHSGX2TYYX3PSLKSRV (SEQ ID NO:6), wherein X1 is D or E, X2 is N or S, and X3 is N or S; and Formula (VI): VSX1ISGX2GX3X4TYYADSVKGRF (SEQ ID NO:7), wherein X1 is A, G, S, V, or Y, X2 is A, D, S, or Y, X3 is D, G, or S, and X4 is S or T; and wherein the HVR-H3 comprises an amino acid sequence according to Formula (VII): ARX1GX2X3X4VX5GDWFX6Y (SEQ ID NO:8), wherein X1 is E or G, X2 is E or S, X3 is D or T, X4 is A, T, or V, X5 is A, I, L, T, or V, and X6 is A, D, or G; and/or b) wherein the light chain variable region comprises an HVR-L1, an HVR-L2, and an HVR-L3, wherein the HVR-L1 comprises an amino acid sequence according to Formula (VIII): X1ASQX2X3X4X5X6X7X8 (SEQ ID NO:9), wherein X1 is Q or R, X2 is D, G, or S, X3 is I or V, X4 is G, R, S, or T, X5 is P, R, S, or T, X6 is A, D, F, S, V, or Y, X7 is L or V, and X8 is A, G, or N; wherein the HVR-L2 comprises an amino acid sequence according to Formula (IX): X1ASX2X3X4X5GX6 (SEQ ID NO:10), wherein X1 is A or D, X2 is N, S, or T, X3 is L or R, X4 is A, E, or Q, X5 is S or T, and X6 is I or V; and wherein the HVR-L3 comprises an amino acid sequence according to a formula selected from the group consisting of: Formula (X): YCQQX1YX2X3X4T (SEQ ID NO:11), wherein X1 is A, G, S, or Y, X2 is Q, S, or Y, X3 is I, L, T, or Y, and X4 is I, S, V, or W; and Formula (XI): YCX1QX2X3X4X5PX6T (SEQ ID NO:12), wherein X1 is E or Q, X2 is P, S, or Y, X3 is D, L, S, T, or Y, X4 is D, E, H, S, or T, X5 is D, L T, or W, and X6 is L, P, R, or V. In some embodiments, the antibody can comprise an HVR_H1 having the amino acid sequence selected from the group consisting of SEQ ID NO: 253-312, an HVR_H2 having the amino acid sequence selected from the group consisting of SEQ ID NO: 313-372, an HVR_H3 having the amino acid sequence selected from the group consisting of SEQ ID NO: 373-432, an HVR_L1 having the amino acid sequence selected from the group consisting of SEQ ID NO: 433-492, an HVR_L2 having the amino acid sequence selected from the group consisting of SEQ ID NO: 493-552, and/or an HVR_L3 having the amino acid sequence selected from the group consisting of SEQ ID NO: 553-612. In certain embodiments, the antibody can comprise a VL and/or VH having the amino acid sequence selected from the group consisting of SEQ ID NO:13-132, which can preferably be encoded by the DNA sequence selected from the group consisting of SEQ ID NO: 133-252, respectively. In some embodiments, the antibody can comprise an HVR_H1 having the amino acid sequence selected from the group consisting of SEQ ID NO: 709-732, an HVR_H2 having the amino acid sequence selected from the group consisting of SEQ ID NO: 733-756, an HVR_H3 having the amino acid sequence selected from the group consisting of SEQ ID NO: 757-780, an HVR_L1 having the amino acid sequence selected from the group consisting of SEQ ID NO: 781-804, an HVR_L2 having the amino acid sequence selected from the group consisting of SEQ ID NO: 805-828, and/or an HVR_L3 having the amino acid sequence selected from the group consisting of SEQ ID NO: 829-852. In certain embodiments, the antibody can comprise a light chain and/or heavy chain (e.g., those of IgG such as IgG4) having the amino acid sequences selected from the group consisting of SEQ ID NO: 613-660, which can be preferably encoded by the DNA sequence selected from the group consisting of SEQ ID NO: 661-708, respectively. In some embodiments, the HVRs are according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising an HVR-H1 comprising the sequence GFSLSTSGVGVG (SEQ ID NO:866), an HVR-H2 comprising the sequence LIDWDDDKYYSPSLKS (SEQ ID NO:867), and an HVR-H3 comprising the sequence GGSDTVLGDWFAY (SEQ ID NO:868); and/or a light chain variable (VL) domain comprising an HVR-L1 comprising the sequence RASQSVSPYLA (SEQ ID NO:869), an HVR-L2 comprising the sequence DASSLES (SEQ ID NO:870), and an HVR-L3 comprising the sequence QQGYSLWT (SEQ ID NO:871). In some embodiments, the HVRs are according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising an HVR-H1 comprising the sequence GYSITSGHYWA (SEQ ID NO:872), an HVR-H2 comprising the sequence SISGYGSTTYYADSVKG (SEQ ID NO:873), and an HVR-H3 comprising the sequence GGSDAVLGDWFAY (SEQ ID NO:874); and/or a light chain variable (VL) domain comprising an HVR-L1 comprising the sequence RASQGIGSFLA (SEQ ID NO:875), an HVR-L2 comprising the sequence DASNLET (SEQ ID NO:876), and an HVR-L3 comprising the sequence QQGYYLWT (SEQ ID NO:877). In some embodiments, the HVRs are according to Kabat. In some embodiments, the antibody comprises a heavy chain variable (VH) domain comprising an HVR-H1 comprising the sequence GFSLSTGGVGVG (SEQ ID NO:878), an HVR-H2 comprising the sequence LIDWADDKYYSPSLKS (SEQ ID NO:879), and an HVR-H3 comprising the sequence GGSDTVIGDWFAY (SEQ ID NO:880); and/or a light chain variable (VL) domain comprising an HVR-L1 comprising the sequence RASQSIGSYLA (SEQ ID NO:881), an HVR-L2 comprising the sequence DASNLET (SEQ ID NO:882), and an HVR-L3 comprising the sequence QQGYYLWT (SEQ ID NO:883). The CD137 antibodies described herein can be in any class, such as IgG, IgM, IgE, IgA, or IgD. It is preferred that the CD137 antibodies are in the IgG class, such as IgG1, IgG2, IgG3, or IgG4 subclass. A CD137 antibody can be converted from one class or subclass to another class or subclass using methods known in the art. An exemplary method for producing an antibody in a desired class or subclass comprises the steps of isolating a nucleic acid encoding a heavy chain of an CD137 antibody and a nucleic acid encoding a light chain of a CD137 antibody, isolating the sequence encoding the VHregion, ligating the VHsequence to a sequence encoding a heavy chain constant region of the desired class or subclass, expressing the light chain gene and the heavy chain construct in a cell, and collecting the CD137 antibody. Further, the antibodies provided by the present disclosure can be monoclonal or polyclonal, but preferably monoclonal. Examples of specific isolated antibodies provided by the present disclosure include those listed in Tables 1a and 1b. The nucleotide and amino acid sequences of the heavy chain variable region, full length heavy chain for the IgG2 and IgG4 subclass, light chain variable region, and full length light chain of these antibodies are also provided hereunder. Antibodies of the present disclosure can be produced by techniques known in the art, including conventional monoclonal antibody methodology e.g., the standard somatic cell hybridization technique (See e.g., Kohler and Milstein,Nature256:495 (1975), viral or oncogenic transformation of B lymphocytes, or recombinant antibody technologies as described in detail herein below. Hybridoma production is a very well-established procedure. The common animal system for preparing hybridomas is the murine system. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. One well-known method that may be used for making human CD137 antibodies provided by the present disclosure involves the use of a XenoMouse™ animal system. XenoMouse™ mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics 7:13-21 (1994) and WO2003/040170. The animal is immunized with a CD137 antigen. The CD137 antigen is isolated and/or purified CD137, preferably CD137. It may be a fragment of CD137, such as the extracellular domain of CD137, particularly a CD137 extracellular domain fragment comprising amino acid resides 34-108 or 34-93 of SEQ ID NO: 1. Immunization of animals can be carried out by any method known in the art. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane, supra, and U.S. Pat. No. 5,994,619. The CD137 antigen may be administered with an adjuvant to stimulate the immune response. Exemplary adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulating complexes). After immunization of an animal with a CD137 antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and/or splenic B cells are immortalized. Methods of immortalizing cells include, but are not limited to, transferring them with oncogenes, inflecting them with the oncogenic virus cultivating them under conditions that select for immortalized cells, subjecting them to carcinogenic or mutating compounds, fusing them with an immortalized cell, e.g., a myeloma cell, and inactivating a tumor suppressor gene. See, e.g., Harlow and Lane, supra. If fusion with myeloma cells is used, the myeloma cells preferably do not secrete immunoglobulin polypeptides (a non-secretory cell line). Immortalized cells are screened using CD137, a portion thereof, or a cell expressing CD137. CD137 antibody-producing cells, e.g., hybridomas, are selected, cloned and further screened for desirable characteristics, including robust growth, high antibody production and desirable antibody characteristics, as discussed further below. Hybridomas can be expanded in vivo in syngeneic animals, in animals that lack an immune system, e.g., nude mice, or in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those of ordinary skill in the art. Antibodies of the disclosure can also be prepared using phage display or yeast display methods. Such display methods for isolating human antibodies are established in the art, such as Achim Knappik, et al., “Fully Synthetic Human Combinatorial Antibody Libraries (HuCAL) Based on Modular Consensus Frameworks and CDRs Randomized with Trinucleotides.” J. Mol. Biol. (2000) 296, 57-86; and Michael J. Feldhaus, et al, “Flow-cytometric isolation of human antibodies from a non-immuneSaccharomyces cerevisiaesurface display library” Nat Biotechnol (2003) 21:163-170. B-2. Antigen Binding Fragments In some other aspects, the present disclosure provides antigen-binding fragments of any of the CD137 antibodies provided by the present disclosure. The antigen-binding fragment may comprise any sequences of the antibody. In some embodiments, the antigen-binding fragment comprises the amino acid sequence of: (1) a light chain of a CD137 antibody; (2) a heavy chain of a CD137 antibody; (3) a variable region from the light chain of a CD137 antibody; (4) a variable region from the heavy chain of a CD137 antibody; (5) one or more HVRs (two, three, four, five, or six HRVs) of a CD137 antibody; or (6) three HVRs from the light chain and three HVRs from the heavy chain of a CD137 antibody. In some particular embodiments, the disclosure provides an antigen-binding fragment of an antibody selected from those listed in Tables 1a and 1b. In some other particular embodiments, the antigen-binding fragments of an CD137 antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CLand CH1 domains; (ii) a F(ab′)2fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VHand CH1 domains; (iv) a Fv fragment consisting of the VLand VHdomains of a single arm of an antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VHdomain; (vi) an isolated CDR, and (vii) single chain antibody (scFv), which is a polypeptide comprising a VLregion of an antibody linked to a VHregion of an antibody. Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. In some particular embodiments, the antigen-binding fragment is a Fab fragment selected from those listed in Table 1a. B-3. Antibody Derivatives In some further aspects, the present disclosure provides derivatives of any of the CD137 antibodies provided by the present disclosure. In one aspect, the antibody derivative is derived from modifications of the amino acid sequences of an illustrative antibody (“parent antibody”) of the disclosure while conserving the overall molecular structure of the parent antibody amino acid sequence. Amino acid sequences of any regions of the parent antibody chains may be modified, such as framework regions, HVR regions, or constant regions. Types of modifications include substitutions, insertions, deletions, or combinations thereof, of one or more amino acids of the parent antibody. In some embodiments, the antibody derivative comprises a VLor VHregion that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 13-132. In some embodiments, the antibody derivative comprises an HVR_H1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 253-312. In some embodiments, the antibody derivative comprises an HVR_H2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 313-372. In some embodiments, the antibody derivative comprises an HVR_H3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 373-432. In some embodiments, the antibody derivative comprises an HVR_L1 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 433-492. In some embodiments, the antibody derivative comprises an HVR_L2 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 493-552. In some embodiments, the antibody derivative comprises an HVR_L3 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 553-612. In some particular embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions to an amino acid sequence as set forth in any of SEQ ID NO: 13-132 and 253-612. In some embodiments, the antibody derivative comprises a light chain or heavy chain that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 613-660. In some embodiments, the antibody derivative comprises an HVR_H1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 709-732. In some embodiments, the antibody derivative comprises an HVR_H2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 733-756. In some embodiments, the antibody derivative comprises an HVR_H3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 757-780. In some embodiments, the antibody derivative comprises an HVR_L1 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 781-804. In some embodiments, the antibody derivative comprises an HVR_L2 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 805-828. In some embodiments, the antibody derivative comprises an HVR_L3 amino acid sequence region that is at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence as set forth in any of SEQ ID NO: 829-852. In some particular embodiments, the derivative comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 additions and/or deletions to an amino acid sequence as set forth in any of SEQ ID NO: 613-660 and 709-852. Amino acid substitutions encompass both conservative substitutions and non-conservative substitutions. The term “conservative amino acid substitution” means a replacement of one amino acid with another amino acid where the two amino acids have similarity in certain physico-chemical properties such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, substitutions typically may be made within each of the following groups: (a) nonpolar (hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids, such as arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids, such as aspartic acid and glutamic acid. The modifications may be made in any positions of the amino acid sequences of the antibody, including the HVRs, framework regions, or constant regions. In one embodiment, the present disclosure provides an antibody derivative that contains the VHand VLHVR sequences of an illustrative antibody of this disclosure, yet contains framework sequences different from those of the illustrative antibody. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database or in the “VBase” human germline sequence database (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); Tomlinson, I. M., et al.,J. Mol. Biol.227:776-798 (1992); and Cox, J. P. L. et al.,Eur. J. Immunol.24:827-836 (1994)). Framework sequences that may be used in constructing an antibody derivative include those that are structurally similar to the framework sequences used by illustrative antibodies of the disclosure, e.g., similar to the VH3-23 framework sequences and/or the VLλ3 or λ1-13 framework sequences used by illustrative antibodies of the disclosure. For example, the HVR_H1, HVR_H2, and HVR_H3 sequences, and the HVR_L1, HVR_L2, and HVR_L3 sequences of an illustrative antibody can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence derive, or the HVR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. In a particular embodiment, the antibody derivative is a chimeric antibody which comprises an amino acid sequence of an illustrative antibody of the disclosure. In one example, one or more HVRs from one or more illustrative human antibodies are combined with HVRs from an antibody from a non-human animal, such as mouse or rat. In another example, all of the HVRs of the chimeric antibody are derived from one or more illustrative antibodies. In some particular embodiments, the chimeric antibody comprises one, two, or three HVRs from the heavy chain variable region or from the light chain variable region of an illustrative antibody. Chimeric antibodies can be generated using conventional methods known in the art. Another type of modification is to mutate amino acid residues within the HRV regions of the VHand/or VLchain. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays known in the art. Typically, conservative substitutions are introduced. The mutations may be amino acid additions and/or deletions. Moreover, typically no more than one, two, three, four or five residues within a HVR region are altered. In some embodiments, the antibody derivative comprises 1, 2, 3, or 4 amino acid substitutions in the heavy chain HVRs and/or in the light chain HVRs. In another embodiment, the amino acid substitution is to change one or more cysteines in an antibody to another residue, such as, without limitation, alanine or serine. The cysteine may be a canonical or non-canonical cysteine. In one embodiment, the antibody derivative has 1, 2, 3, or 4 conservative amino acid substitutions in the heavy chain HVR regions relative to the amino acid sequences of an illustrative antibody. Modifications may also be made to the framework residues within the VHand/or VLregions. Typically, such framework variants are made to decrease the immunogenicity of the antibody. One approach is to “back mutate” one or more framework residues to the corresponding germline sequence. An antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be “back mutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. In addition, modifications may also be made within the Fc region of an illustrative antibody, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In one example, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another case, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. Furthermore, an antibody of the disclosure may be modified to alter its potential glycosylation site or pattern in accordance with routine experimentation known in the art. In another aspect, the present disclosure provide an derivative of an CD137 antibody of the disclosure that contains at least one mutation in an variable region of a light chain or heavy chain that changes the pattern of glycosylation in the variable region. Such an antibody derivative may have an increased affinity and/or a modified specificity for binding an antigen. The mutations may add a novel glycosylation site in the V region, change the location of one or more V region glycosylation site(s), or remove a pre-existing V region glycosylation site. In one embodiment, the present disclosure provides a derivative of a CD137 antibody having a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation site in one heavy chain variable region is removed. In another embodiment, the present disclosure provides a derivative of a CD137 antibody having a potential N-linked glycosylation site at asparagine in the heavy chain variable region, wherein the potential N-linked glycosylation site in both heavy chain variable regions is removed. Method of altering the glycosylation pattern of an antibody is known in the art, such as those described in U.S. Pat. No. 6,933,368, the disclosure of which incorporated herein by reference. In another aspect, the present disclosure provides an antibody derivative that comprises a CD137 antibody, or antigen-binding fragment thereof, as described herein, linked to an additional molecular entity. Examples of additional molecular entities include pharmaceutical agents, peptides or proteins, detection agent or labels, and antibodies. In some embodiments, the antibody derivative comprises an antibody of the disclosure linked to a pharmaceutical agent. Examples of pharmaceutical agents include cytotoxic agents or other cancer therapeutic agents, and radioactive isotopes. Specific examples of cytotoxic agents include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include, but are not limited to, iodine131, indium111, yttrium90and lutetium77. Methods for linking an antibody to a pharmaceutical agent are known in the art, such as using various linker technologies. Examples of linker types include hydrazones, thioethers, esters, disulfides and peptide-containing linkers. For further discussion of linkers and methods for linking therapeutic agents to antibodies, see also Saito et al.,Adv. Drug Deliv. Rev.55:199-215 (2003); Trail, et al.,Cancer Immunol. Immunother.52:328-337 (2003); Payne,Cancer Cell3:207-212 (2003); Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan, I. and Kreitman,Curr. Opin. Investig. Drugs3:1089-1091 (2002); Senter, P. D. and Springer, C. J. (2001)Adv. Drug Deliv. Rev.53:247-264. In a particular embodiment, the antibody derivative is a CD137 antibody multimer, which is a multimeric form of a CD137 antibody, such as antibody dimers, trimers, or higher-order multimers of monomeric antibodies. Individual monomers within an antibody multimer may be identical or different. In addition, individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies may be accomplished through natural aggregation of antibodies. For example, some percentage of purified antibody preparations (e.g., purified IgG4 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art, such as through using crosslinking agents. Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate, and N-succinimidyl S-acethylthio-acetate) or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available from, for example, Pierce Chemical Company, Rockford, IL. Antibodies can also be made to multimerize through recombinant DNA techniques known in the art. In some embodiments, an antibody of the present disclosure is a multimeric antibody (e.g., a bispecific antibody). In some embodiments, an antibody of the present disclosure is an IgM antibody, e.g., comprises an IgM Fc region (e.g., a human IgM Fc region). Examples of other antibody derivatives provided by the present disclosure include single chain antibodies, diabodies, domain antibodies, nanobodies, and unibodies. A “single-chain antibody” (scFv) consists of a single polypeptide chain comprising a VLdomain linked to a VHdomain wherein VLdomain and VHdomain are paired to form a monovalent molecule. Single chain antibody can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). A “diabody” consists of two chains, each chain comprising a heavy chain variable region connected to a light chain variable region on the same polypeptide chain connected by a short peptide linker, wherein the two regions on the same chain do not pair with each other but with complementary domains on the other chain to form a bispecific molecule. Methods of preparing diabodies are known in the art (See, e.g., Holliger P. et al., (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448, and Poljak R. J. et al., (1994) Structure 2:1121-1123). Domain antibodies (dAbs) are small functional binding units of antibodies, corresponding to the variable regions of either the heavy or light chains of antibodies. Domain antibodies are well expressed in bacterial, yeast, and mammalian cell systems. Further details of domain antibodies and methods of production thereof are known in the art (see, for example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European Patents 0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609). Nanobodies are derived from the heavy chains of an antibody. A nanobody typically comprises a single variable domain and two constant domains (CH2 and CH3) and retains antigen-binding capacity of the original antibody. Nanobodies can be prepared by methods known in the art (See e.g., U.S. Pat. Nos. 6,765,087, 6,838,254, WO 06/079372). Unibodies consist of one light chain and one heavy chain of an IgG4 antibody. Unibodies may be made by the removal of the hinge region of IgG4 antibodies. Further details of unibodies and methods of preparing them may be found in WO2007/059782. C. Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of Producing CD137 Antibodies Another aspect of the disclosure provides an isolated nucleic acid molecule that comprises a nucleotide sequence encoding an amino acid sequence of a binding molecule provided by the present disclosure. The amino acid sequence encoded by the nucleotide sequence may be any portion of an antibody, such as a HVR, a sequence comprising one, two, or three HVRs, a variable region of a heavy chain, variable region of a light chain, or may be a full-length heavy chain or full length light chain. A nucleic acid of the disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. Typically, the nucleic acid is a cDNA molecule. In some embodiments, the disclosure provides an isolated nucleic acid molecule that comprises or consists of a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (1) amino acid sequence of a HVR_H3 or HVR_L3 of an illustrative antibody; (2) a variable region of a heavy chain or variable region of a light chain of an illustrative antibody; or (3) a full length heavy chain or full length light chain of an illustrative antibody. In other embodiments, the nucleic acid molecule comprises or consists of a nucleotide sequence that encodes an amino acid sequence as set forth in any one of SEQ ID NO: 13-132, 253-612, 613-660 and 709-852. In still other embodiments, the nucleic acid molecule comprises or consists of nucleotide sequence selected from the group consisting of SEQ ID NO: 133-252 and 661-708. Nucleic acids of the disclosure can be obtained using any suitable molecular biology techniques. For antibodies expressed by hybridomas, cDNAs encoding the light and heavy chains of the antibody made by the hybridoma can be obtained by PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (e.g., using phage display techniques), the nucleic acid encoding the antibody can be recovered from the library. The isolated DNA encoding the VHregion can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgG4 or IgG2 constant region without ADCC effect. The IgG4 constant region sequence can be any of the various alleles or allotypes known to occur among different individuals. These allotypes represent naturally occurring amino acid substitution in the IgG4 constant regions. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region. The isolated DNA encoding the VLregion can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. To create a scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VHand VLsequences can be expressed as a contiguous single-chain protein, with the VLand VHregions joined by the flexible linker (see e.g., Bird et al.,Science242:423-426 (1988); Huston et al.,Proc. Natl. Acad. Sci. USA85:5879-5883 (1988); and McCafferty et al.,Nature348:552-554 (1990)). The present disclosure further provides a vector that comprises a nucleic acid molecule provided by the present disclosure. The nucleic acid molecule may encode a portion of a light chain or heavy chain (such as a CDR or a HVR), a full-length light or heavy chain, polypeptide that comprises a portion or full-length of a heavy or light chain, or an amino acid sequence of an antibody derivative or antigen-binding fragment. In some embodiments, the vector is an expression vector useful for the expression of a binding molecule, such as an antibody or an antigen binding fragment thereof. In some embodiments, provided herein are vectors, wherein a first vector comprises a polynucleotide sequence encoding a heavy chain variable region as described herein, and a second vector comprises a polynucleotide sequence encoding a light chain variable region as described herein. In some embodiments, a single vector comprises polynucleotides encoding a heavy chain variable region as described herein and a light chain variable region as described herein. To express a binding molecule of the disclosure, DNAs encoding partial or full-length light and heavy chains are inserted into expression vectors such that the DNA molecules are operatively linked to transcriptional and translational control sequences. In this context, the term “operatively linked” means that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the DNA molecule. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by any suitable methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or homologous recombination-based DNA ligation). The light and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype and subclass by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype and subclass such that the VHsegment is operatively linked to the CHsegment(s) within the vector and the VLsegment is operatively linked to the CLsegment within the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein). In addition to the antibody chain genes, the expression vectors of the disclosure typically carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or β-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SR promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472). In addition to the antibody chain genes and regulatory sequences, the expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by any suitable techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the antibodies of the disclosure in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and typically mammalian host cells, is most typical. The present disclosure further provides a host cell containing a nucleic acid molecule provided by the present disclosure. The host cell can be virtually any cell for which expression vectors are available. It may be, for example, a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant nucleic acid construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, electroporation or phage infection. Suitable prokaryotic hosts for transformation includeE. coli, Bacillus subtilis, Salmonella typhimuriumand various species within the generaPseudomonas, Streptomyces, andStaphylococcus. Mammalian host cells for expressing a binding molecule of the disclosure include, for example, Chinese Hamster Ovary (CHO) cells (including dhfr-CHO cells, described in Urlaub and Chasin,Proc. Natl. Acad. Sci. USA77:4216-4220 (1980), used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp,J. Mol. Biol.159:601-621 (1982), NS0 myeloma cells, COS cells and Sp2 cells. In particular, for use with NS0 myeloma or CHO cells, another expression system is the GS (glutamine synthetase) gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using any suitable protein purification methods. D. Compositions In other aspects, the present disclosure provides a composition containing a binding molecule provided by the disclosure. In one aspect, the composition is a pharmaceutical composition comprising a binding molecule and a pharmaceutically acceptable carrier. The compositions can be prepared by conventional methods known in the art. In some embodiments, present disclosure provides a composition comprising an antibody, or an antigen-binding fragment thereof, provided by the present disclosure and a pharmaceutically acceptable carrier, wherein said antibody comprises a variable domain comprising the HVR amino acid sequence disclosed herein, and wherein said composition comprises not more than about 11%, 10%, 8%, 5%, 3%, or 2% of said antibody, or antigen-binding portion, that is glycosylated at the asparagine of said amino acid sequence compared with the total amount of antibody, or antigen-binding portion thereof, present in said composition. In another embodiment, the composition comprises at least about 2% of said antibody, or antigen-binding portion, that is glycosylated at the asparagine of said amino acid sequence compared with the total amount of antibody, or antigen-binding portion thereof, present in said composition. The term “pharmaceutically acceptable carrier” refers to any inactive substance that is suitable for use in a formulation for the delivery of a binding molecule. A carrier may be an antiadherent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial, or antifungal agent), sweetener, absorption delaying agent, wetting agent, emulsifying agent, buffer, and the like. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) dextrose, vegetable oils (such as olive oil), saline, buffer, buffered saline, and isotonic agents such as sugars, polyalcohols, sorbitol, and sodium chloride. The compositions may be in any suitable forms, such as liquid, semi-solid, and solid dosage forms. Examples of liquid dosage forms include solution (e.g., injectable and infusible solutions), microemulsion, liposome, dispersion, or suspension. Examples of solid dosage forms include tablet, pill, capsule, microcapsule, and powder. A particular form of the composition suitable for delivering a binding molecule is a sterile liquid, such as a solution, suspension, or dispersion, for injection or infusion. Sterile solutions can be prepared by incorporating the antibody in the required amount in an appropriate carrier, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and other carriers. In the case of sterile powders for the preparation of sterile liquid, methods of preparation include vacuum drying and freeze-drying (lyophilization) to yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The various dosage forms of the compositions can be prepared by conventional techniques known in the art. The relative amount of a binding molecule included in the composition will vary depending upon a number of factors, such as the specific binding molecule and carriers used, dosage form, and desired release and pharmacodynamic characteristics. The amount of a binding molecule in a single dosage form will generally be that amount which produces a therapeutic effect, but may also be a lesser amount. Generally, this amount will range from about 0.01 percent to about 99 percent, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent relative to the total weight of the dosage form. In addition to the binding molecule, one or more additional therapeutic agents may be included in the composition. Examples of additional therapeutic agents are described herein below. The suitable amount of the additional therapeutic agent to be included in the composition can be readily selected by a person skilled in the art, and will vary depending on a number of factors, such as the particular agent and carriers used, dosage form, and desired release and pharmacodynamic characteristics. The amount of the additional therapeutic agent included in a single dosage form will generally be that amount of the agent which produces a therapeutic effect, but may be a lesser amount as well. E. Use of the Binding Molecules and Pharmaceutical Compositions Binding molecules and pharmaceutical compositions provided by the present disclosure are useful for therapeutic, diagnostic, or other purposes, such as modulating an immune response, treating cancer, enhancing efficacy of other cancer therapy, enhancing vaccine efficacy, or treating autoimmune diseases. Thus, in other aspects, the present disclosure provides methods of using the binding molecules or pharmaceutical compositions. In one aspect, the present disclosure provides a method of treating a disorder in a mammal, which comprises administering to the mammal in need of treatment a therapeutically effective amount of a binding molecule provided by the disclosure. The binding molecule may be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In some embodiments, the mammal is a human. In some embodiments, the disorder is a cancer. A variety of cancers where CD137 is implicated, whether malignant or benign and whether primary or secondary, may be treated or prevented with a method provided by the disclosure. Examples of such cancers include lung cancers such as bronchogenic carcinoma (e.g., squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (noncancerous), and sarcoma (cancerous); heart cancer such as myxoma, fibromas, and rhabdomyomas; bone cancers such as osteochondromas, condromas, chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcomas, malignant fibrous histiocytomas, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; brain cancer such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, astrocytomas, oligodendrogliomas, medulloblastomas, chordoma, Schwannomas, ependymomas, meningiomas, pituitary adenoma, pinealoma, osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germinomas, teratomas, dermoid cysts, and angiomas; cancers in digestive system such as leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, stomach adenocarcinomas, intestinal lipomas, intestinal neurofibromas, intestinal fibromas, polyps in large intestine, and colorectal cancers; liver cancers such as hepatocellular adenomas, hemangioma, hepatocellular carcinoma, fibrolamellar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as kidney adenocarcinoma, renal cell carcinoma, hypernephroma, and transitional cell carcinoma of the renal pelvis; bladder cancers; hematological cancers such as acute lymphocytic (lymphoblastic) leukemia, acute myeloid (myelocytic, myelogenous, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myelocytic (myeloid, myelogenous, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocythemia, and chronic myelocytic leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancers; eye-related cancers such as retinoblastoma and intraoccular melanocarcinoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancers (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial carcinoma), cervical cancer (cervical carcinoma), cancer of the ovaries (ovarian carcinoma), vulvar carcinoma, vaginal carcinoma, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary cancer); pheochromocytomas (adrenal gland); noncancerous growths of the parathyroid glands; pancreatic cancers; and hematological cancers such as leukemias, myelomas, non-Hodgkin's lymphomas, and Hodgkin's lymphomas. In some other embodiments, the disorder is an autoimmune disease. Examples of autoimmune diseases that may be treated with the binding molecules include autoimmune encephalomyelitis, lupus erythematosus, and rheumatoid arthritis. The binding molecule may also be used to treat inflammation (such as allergic asthma) and chronic graft-versus-host disease, In another aspect, the present disclosure provides a method of enhancing an immune response in a mammal, which comprises administering to the mammal a therapeutically effective amount of a binding molecule provided by the disclosure. In some embodiments, the binding molecule is a CD137 antibody or antigen-binding fragment thereof and the mammal is a human. In a further embodiment, the binding molecule is CD137 agonist antibody or an antigen-binding fragment thereof. The term “enhancing immune response” or its grammatical variations, means stimulating, evoking, increasing, improving, or augmenting any response of a mammal's immune system. The immune response may be a cellular response (i.e. cell-mediated, such as cytotoxic T lymphocyte mediated) or a humoral response (i.e. antibody mediated response), and may be a primary or secondary immune response. Examples of enhancement of immune response include increased CD4+ helper T cell activity and generation of cytolytic T cells. The enhancement of immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including, but not limited to, cytotoxic T lymphocyte assays, release of cytokines (for example IL-2 production), regression of tumors, survival of tumor bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, methods of the disclosure enhance the immune response by a mammal when compared to the immune response by an untreated mammal or a mammal not treated using the claimed methods. In one embodiment, the binding molecule is used to enhance the immune response of a human to a microbial pathogen (such as a virus). In another embodiment, the binding molecule is used to enhance the immune response of a human to a vaccine. The binding molecule may be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In one embodiment, the method enhances a cellular immune response, particularly a cytotoxic T cell response. In another embodiment, the cellular immune response is a T helper cell response. In still another embodiment, the immune response is a cytokine production, particularly IL-2 production. The binding molecule may be used to enhance the immune response of a human to a microbial pathogen (such as a virus) or to a vaccine. The binding molecule may be a CD137 agonist or antagonist. In some embodiments, the binding molecule is a CD137 agonist. In practicing the therapeutic methods, the binding molecules may be administered alone as monotherapy, or administered in combination with one or more additional therapeutic agents or therapies. Thus, in another aspect, the present disclosure provides a combination therapy, which comprises a binding molecule in combination with one or more additional therapies or therapeutic agents for separate, sequential or simultaneous administration. The term “additional therapy” refers to a therapy which does not employ a binding molecule provided by the disclosure as a therapeutic agent. The term “additional therapeutic agent” refers to any therapeutic agent other than a binding molecule provided by the disclosure. In one particular aspect, the present disclosure provides a combination therapy for treating cancer in a mammal, which comprises administering to the mammal a therapeutically effective amount of a binding molecule provided by the disclosure in combination with one or more additional therapeutic agents. In a further embodiment, the mammal is a human. A wide variety of cancer therapeutic agents may be used in combination with a binding molecule provided by the present disclosure. One of ordinary skill in the art will recognize the presence and development of other cancer therapies which can be used in combination with the methods and binding molecules of the present disclosure, and will not be restricted to those forms of therapy set forth herein. Examples of categories of additional therapeutic agents that may be used in the combination therapy for treating cancer include (1) chemotherapeutic agents, (2) immunotherapeutic agents, and (3) hormone therapeutic agents. The term “chemotherapeutic agent” refers to a chemical or biological substance that can cause death of cancer cells, or interfere with growth, division, repair, and/or function of cancer cells. Examples of chemotherapeutic agents include those that are disclosed in WO 2006/129163, and US 20060153808, the disclosures of which are incorporated herein by reference. Examples of particular chemotherapeutic agents include: (1) alkylating agents, such as chlorambucil (LEUKERAN), mcyclophosphamide (CYTOXAN), ifosfamide (IFEX), mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX), streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine (CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or plant vinca alkaloids, including cytotoxic antibiotics, such as doxorubicin (ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin (CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN PFS, ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE). dactinomycin (actinomycin D, COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin (BLENOXANE), vinorelbine tartrate (NAVELBINE)), vinblastine (VELBAN), vincristine (ONCOVIN), and vindesine (ELDISINE); (3) antimetabolites, such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine (FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate (FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate (NEUTREXIN), and pemetrexed (ALIMTA); (4) Pyrimidine antagonists, such as 5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed (TOMUDEX), tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) taxanes, such as docetaxel (TAXOTERE), paclitaxel (TAXOL); (6) platinum drugs, such as cisplatin (PLATINOL) and carboplatin (PARAPLATIN), and oxaliplatin (ELOXATIN); (7) topoisomerase inhibitors, such as irinotecan (CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID, TOPOSAR), and teniposide (VUMON); (8) epipodophyllotoxins (podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID, TOPOSAR); (9) folic acid derivatives, such as leucovorin (WELLCOVORIN); (10) nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11) inhibitors of receptor tyrosine kinase, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), insulin receptor, insulin-like growth factor receptor (IGFR), hepatocyte growth factor receptor (HGFR), and platelet-derived growth factor receptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA), bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib, lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib, rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab (ERBITUX), bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1 (ONCOLYM), antibodies to insulin-like growth factor-1 receptor (IGF-1R) that are disclosed in WO2002/053596); (12) angiogenesis inhibitors, such as bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416, thalidomide, and matrix metalloproteinase inhibitors (such as batimastat and marimastat), and those that are disclosed in WO2002055106; and (13) proteasome inhibitors, such as bortezomib (VELCADE). The term “immunotherapeutic agents” refers to a chemical or biological substance that can enhance an immune response of a mammal. Examples of immunotherapeutic agents include:bacillusCalmette-Guerin (BCG); cytokines such as interferons; vaccines such as MyVax personalized immunotherapy, Onyvax-P, Oncophage, GRNVAC1, Favld, Provenge, GVAX, Lovaxin C, BiovaxID, GMXX, and NeuVax; and antibodies such as alemtuzumab (CAMPATH), bevacizumab (AVASTIN), cetuximab (ERBITUX), gemtuzunab ozogamicin (MYLOTARG), ibritumomab tiuxetan (ZEVALIN), panitumumab (VECTIBIX), rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), tositumomab (BEXXAR), ipilimumab (YERVOY) tremelimumab, CAT-3888, agonist antibodies to OX40 receptor (such as those disclosed in WO2009/079335), agonist antibodies to CD40 receptor (such as those disclosed in WO2003/040170, and TLR-9 agonists (such as those disclosed in WO2003/015711, WO2004/016805, and WO2009/022215). The term “hormone therapeutic agent” refers to a chemical or biological substance that inhibits or eliminates the production of a hormone, or inhibits or counteracts the effect of a hormone on the growth and/or survival of cancerous cells. Examples of such agents suitable for the methods herein include those that are disclosed in US20070117809. Examples of particular hormone therapeutic agents include tamoxifen (NOLVADEX), toremifene (Fareston), fulvestrant (FASLODEX), anastrozole (ARIMIDEX), exemestane (AROMASIN), letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX), and leuprolide (LUPRON). The binding molecules of this disclosure may also be used in combination with non-drug hormone therapies such as (1) surgical methods that remove all or part of the organs or glands which participate in the production of the hormone, such as the ovaries, the testicles, the adrenal gland, and the pituitary gland, and (2) radiation treatment, in which the organs or glands of the patient are subjected to radiation in an amount sufficient to inhibit or eliminate the production of the targeted hormone. The combination therapy for treating cancer also encompasses the combination of a binding molecule with surgery to remove a tumor. The binding molecule may be administered to the mammal before, during, or after the surgery. The combination therapy for treating cancer also encompasses combination of a binding molecule with radiation therapy, such as ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) and particle beam radiation therapy (e.g., high linear energy radiation). The source of radiation can be external or internal to the mammal. The binding molecule may be administered to the mammal before, during, or after the radiation therapy. The binding molecules and compositions provided by the present disclosure can be administered via any suitable enteral route or parenteral route of administration. The term “enteral route” of administration refers to the administration via any part of the gastrointestinal tract. Examples of enteral routes include oral, mucosal, buccal, and rectal route, or intragastric route. “Parenteral route” of administration refers to a route of administration other than enteral route. Examples of parenteral routes of administration include intravenous, intramuscular, intradermal, intraperitoneal, intratumor, intravesical, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, transtracheal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal, subcutaneous, or topical administration. The antibodies and compositions of the disclosure can be administered using any suitable method, such as by oral ingestion, nasogastric tube, gastrostomy tube, injection, infusion, implantable infusion pump, and osmotic pump. The suitable route and method of administration may vary depending on a number of factors such as the specific antibody being used, the rate of absorption desired, specific formulation or dosage form used, type or severity of the disorder being treated, the specific site of action, and conditions of the patient, and can be readily selected by a person skilled in the art The term “therapeutically effective amount” of a binding molecule refers to an amount that is effective for an intended therapeutic purpose. For example, in the context of enhancing an immune response, a “therapeutically effective amount” is any amount that is effective in stimulating, evoking, increasing, improving, or augmenting any response of a mammal's immune system. In the context of treating a disease, a “therapeutically effective amount” is any amount that is sufficient to cause any desirable or beneficial effect in the mammal being treated. Specifically, in the treatment of cancer, examples of desirable or beneficial effects include inhibition of further growth or spread of cancer cells, death of cancer cells, inhibition of reoccurrence of cancer, reduction of pain associated with the cancer, or improved survival of the mammal. The therapeutically effective amount of a CD137 antibody usually ranges from about 0.001 to about 500 mg/kg, and more usually about 0.01 to about 100 mg/kg, of the body weight of the mammal. For example, the amount can be about 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg, or 100 mg/kg of body weight of the mammal. In some embodiments, the therapeutically effective amount of a CD137 antibody is in the range of about 0.01-30 mg/kg of body weight of the mammal. In some other embodiments, the therapeutically effective amount of a CD137 antibody is in the range of about 0.05-15 mg/kg of body weight of the mammal. The precise dosage level to be administered can be readily determined by a person skilled in the art and will depend on a number of factors, such as the type, and severity of the disorder to be treated, the particular binding molecule employed, the route of administration, the time of administration, the duration of the treatment, the particular additional therapy employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A binding molecule or composition is usually administered on multiple occasions. Intervals between single doses can be, for example, weekly, monthly, every three months or yearly. An exemplary treatment regimen entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months or once every three to six months. Typical dosage regimens for a CD137 antibody include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kg body weight every three weeks. The present disclosure will be more fully understood by reference to the following examples. The examples should not, however, be construed as limiting the scope of the present disclosure. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. The contents of all figures and all references, patents and published patent applications cited throughout this disclosure are expressly incorporated herein by reference in their entirety. EXAMPLES Example 1 Generation of Primary Fabs that Specifically Binds to Human CD137 Proprietary phagemid libraries (See PCT International Application titled “Dynamic Human Antibody Light Chain Libraries” filed concurrently herewith under Attorney Docket No. 69540-2000140, incorporated herein by reference in its entirety; See also PCT International Application titled “Dynamic Human Heavy Chain Antibody Libraries” filed concurrently herewith under Attorney Docket No. 69540-2000240, incorporated herein by reference in its entirety) were employed to pan against human CD137 antigens. A total of three or four rounds of panning were conducted. After the final round of panning, single-colony supernatant ELISA was performed to identify the primary hits that specifically recognize human CD137. The primary hits were defined as those whose ELISA signals were at least twice that of background. They were sequenced, the unique clones were expressed and purified for affinity measurement by ForteBio and Biacore. The list was refined to 124 in Fab with both ELISA positive hits and unique sequences. Following the criteria of KDresponse signal R>0.1, R2>0.9 and affinity KD<100 nM, the list was further refined to 60 hits (Table 1a). 24 of them were then converted into IgG (Table 1b) for detailed biophysical and functional characterization. The Fabs corresponding to the unique hits were expressed inE. coliand purified. Their affinities against human CD137 were measured by ForteBio Octet RED96 Systems. Briefly, the AHC sensors (Anti-Human gG Fe Capture Dip and Read Biosensors) were used to capture CD137-hisFc fusion protein (Sino Biological #Cat 10041-H03H), and dipped into wells containing purified Fabs that were diluted to 5-10 μg/ml with kinetic buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, pH 7.4). The acquired ForteBio data were processed with Data Acquisition software 7.1, and kinetic data were fitted to a 1:1 Langmuir binding model. The affinity and kinetic parameters (with background subtracted) are listed in Table 1a. The affinity of their corresponding IgGs to human CD137 was measured by Biacore and shown in Table 1b. TABLE 1aAffinity of selected Fabs against human CD137 andcorresponding amino acid sequences (in SEQ ID NO.)SEQ ID NO.Hit(upper row VH;IDKD (nM)kon(1/Ms)koff(1/s)lower row VL)37601.26E−083.52E+054.44E−03131440726.95E−091.54E+051.07E−03151640741.95E−084.80E+049.37E−04171840767.44E−096.38E+044.75E−04192040793.15E−086.93E+042.19E−03212241341.30E−085.93E+047.69E−0423244137<1.0E−128.56E+04<1.0E−07252641391.65E−094.96E+048.17E−0527284140<1.0E−122.57E+04<1.0E−07293042179.67E−085.64E+055.45E−02313252991.37E−085.55E+057.60E−03333453001.53E−085.96E+059.10E−03353653021.21E−093.54E+054.26E−04373853035.12E−099.95E+055.09E−03394053105.72E−098.13E+054.65E−03414253148.39E−092.10E+051.77E−03434453161.14E−081406000.001605454653181.90E−081.41E+052.69E−03474853231.04E−087.82E+058.12E−03495053412.93E−086.42E+041.88E−03515253423.89E−081.57E+056.12E−03535453461.61E−086.05E+059.77E−03555653481.02E−081.31E+061.33E−02575853496.20E−091.62E+051.01E−03596053517.29E−094.66E+053.40E−03616253531.61E−083.70E+055.97E−03636453597.10E−104.64E+053.30E−04656653602.41E−081.20E+052.89E−03676853639.87E−098.37E+048.26E−04697053652.56E−097.01E+051.79E−03717253671.49E−084.07E+056.08E−03737453701.91E−095.24E+051.00E−03757653713.97E−091.21E+064.79E−03777854043.30E−093.95E+051.30E−03798054071.76E−092.48E+054.37E−04818254082.36E−083.18E+057.50E−03838454091.70E−082.51E+054.27E−03858654139.93E−105.55E+055.51E−04878854174.04E−085.72E+042.31E−03899070771.88E−084.98E+059.34E−03919270782.52E−083.45E+058.70E−03939470792.99E−081.00E+053.00E−03959670802.44E−083.06E+057.46E−03979870814.31E−082.87E+051.23E−029910070876.96E−081.23E+058.55E−0310110270884.36E−082.55E+051.11E−0210310470905.55E−083.12E+051.73E−0210510670924.57E−084.31E+051.97E−0210710870972.43E−085.42E+051.32E−0210911071003.50E−084.62E+051.62E−0211111271053.33E−083.30E+051.10E−0211311471093.20E−081.73E+055.55E−0311511671203.45E−082.64E+059.11E−0311711871283.97E−083.09E+051.23E−0211912071313.04E−082.66E+058.10E−0312112271334.03E−081.01E+054.05E−0312312471353.17E−081.02E+053.22E−0312512671593.79E−081.06E+054.03E−0312712871631.26E−082.99E+053.78E−0312913071661.24E−083.45E+054.29E−03131132 The corresponding DNA sequences encoding the amino acid sequences of SEQ ID NO: 13-132 can be found in SEQ ID NO: 133-252, respectively. The HVR_H1 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 253-312, respectively. The HVR_H2 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 313-372, respectively. The HVR_H3 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 373-432, respectively. The HVR_L1 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 433-492, respectively. The HVR_L2 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 493-552, respectively. The HVR_L3 amino acid sequences for all Fab hits shown in Table 1a can be found in SEQ ID NO: 553-612, respectively (See also, Table 1c). TABLE 1bAffinity of Fabs and the corresponding IgGs against human CD137IgG SEQ ID NO.(upper row heavyFabIgGchain; lower rowHits IDKD (M)ka(1/Ms)kd(1/s)IgG IDKD (M)Ka(1/Ms)kd (1/s)light chain)40727.0E−091.5E+051.1E−03AG100541.3E−081.4E+051.9E−0361361453035.1E−091.0E+065.1E−03AG100577.9E−097.7E+056.1E−0361561653105.7E−098.1E+054.7E−03AG100585.9E−094.1E+052.4E−0361761853517.3E−094.7E+053.4E−03AG100593.8E−081.6E+056.3E−0361962053597.1E−104.6E+053.3E−04AG100601.1E−092.2E+052.5E−0462162253701.9E−095.2E+051.0E−03AG100613.6E−092.2E+057.8E−0462362454043.3E−094.0E+051.3E−03AG100625.9E−091.6E+059.4E−0462562654139.9E−105.6E+055.5E−04AG100639.9E−103.9E+053.9E−0462762840742.0E−084.8E+049.4E−04AG100791.4E−091.9E+052.7E−0462963042179.7E−085.6E+055.5E−02AG100801.0E−081.2E+061.2E−0263163252991.4E−085.6E+057.6E−03AG100816.9E−092.4E+051.7E−0363363453001.5E−086.0E+059.1E−03AG100821.3E−085.6E+057.2E−0363563653231.0E−087.8E+058.1E−03AG100831.2E−085.7E+056.9E−0363763853602.4E−081.2E+052.9E−03AG100844.3E−086.6E+042.8E−0363964053671.5E−084.1E+056.1E−03AG100855.4E−081.5E+057.9E−0364164254091.7E−082.5E+054.3E−03AG100864.6E−081.0E+054.5E−0364364453021.2E−093.5E+054.3E−04AG101246.0E−095.0E+053.0E−0364564653148.4E−092.1E+051.8E−03AG101251.5E−081.1E+051.7E−0364764853161.1E−081.4E+051.6E−03AG101261.4E−085.4E+097.3E+0164965053181.9E−081.4E+052.7E−03AG101279.6E−093.0E+052.9E−0365165253423.9E−081.6E+056.1E−03AG101283.0E−091.2E+053.7E−0465365453531.6E−083.7E+056.0E−03AG101291.9E−083.1E+056.0E−0365565653652.6E−097.0E+051.8E−03AG101313.7E−095.1E+051.9E−0365765854082.4E−083.2E+057.5E−03AG101326.9E−082.0E+051.4E−02659660 The corresponding DNA sequences encoding the amino acid sequences of SEQ ID NO: 613-660 can be found in SEQ ID NO: 661-708, respectively. The HVR_H1 amino acid sequences for all IgG sequences shown in Table 1b can be found in SEQ ID NO: 709-732, respectively. The HVR_H2 amino acid sequences for all IgG sequences shown in Table 1b can be found in SEQ ID NO: 733-756, respectively. The HVR_H3 amino acid sequences for all IgG sequences shown in Table 1b can be found in SEQ ID NO: 757-780, respectively. The HVR_L1 amino acid sequences for all IgG sequences shown in Table 1b can be found in SEQ ID NO: 781-804, respectively. The HVR_L2 amino acid sequences for all IgG sequences shown in Table 1b can be found in SEQ ID NO: 805-828, respectively. The HVR_L3 amino acid sequences for all IgG sequences shown in Table 1b can be found in SEQ ID NO: 829-852, respectively. TABLE 1cCDR sequences of FabsHitHVR-H1HVR-H2HVR-H3HVR-L1HVR-L2HVR-L31IDVH/VLSEQ ID NO.SEQ ID NO.SEQ ID NO.SEQ ID NO.SEQ ID NO.SEQ ID NO.3760VH1/VL12533133734334935534072VH2/VL22543143744344945547074VH3/VL32553153754354955554076VH4/VL42563163764364965564079VH5/VL52573173774374975574134VH6/VL62583183784384985584137VH7/VL72593193794394995594139VH8/VL82603203804405005604140VH9/VL92613213814415015614217VH10/VL102623223824425025625299VH11/VL112633233834435035635300VH12/VL122643243844445045645302VH13/VL132653253854455055655303VH14/VL142663263864465065665310VH15/VL152673273874475075675314VH16/VL162683283884485085685316VH17/VL172693293894495095695318VH18/VL182703303904505105705323VH19/VL192713313914515115715341VH20/VL202723323924525125725342VH21/VL212733333934535135735346VH22/VL222743343944545145745348VH23/VL232753353954555155755349VH24/VL242763363964565165765351VH25/VL252773373974575175775353VH26/VL262783383984585185785359VH27/VL272793393994595195795360VH28/VL282803404004605205805363VH29/VL292813414014615215815365VH30/VL302823424024625225825367VH31/VL312833434034635235835370VH32/VL322843444044645245845371VH33/VL332853454054655255855404VH34/VL342863464064665265865407VH35/VL352873474074675275875408VH36/VL362883484084685285885409VH37/VL372893494094695295895413VH38/VL382903504104705305905417VH39/VL392913514114715315917077VH40/VL402923524124725325927078VH41/VL412933534134735335937079VH42/VL422943544144745345947080VH43/VL432953554154755355957081VH44/VL442963564164765365967087VH45/VL452973574174775375977088VH46/VL462983584184785385987090VH47/VL472993594194795395997092VH48/VL483003604204805406007097VH49/VL493013614214815416017100VH50/VL503023624224825426027105VH51/VL513033634234835436037109VH52/VL523043644244845446047120VH53/VL533053654254855456057128VH54/VL543063664264865466067131VH55/VL553073674274875476077133VH56/VL563083684284885486087135VH57/VL573093694294895496097159VH58/VL583103704304905506107163VH59/VL593113714314915516117166VH60/VL60312372432492552612 Example 2 Selection of Fab Hits that are Cross-Reactive with Mouse CD137 The species cross-reactivity of Fab hits was determined using ELISA. Briefly, 200 μL 5 μg/mL anti-human IgG (Fab specific) (Sigma #I5260) was coated on Maxisorp microplate (Thermo Scientific 446469) at 4° C. overnight. After blocking, 100 μL Fab 5310 (5 μg/mL), 5351 (2.8 μg/mL) and 5365 (5 μg/mL) were added and incubated for 1 hr. After washing for three times, serial dilutions of human or mouse CD137 antigens fused with human FC fragments were added and incubated for 1 hr. After washing, HRP labelled goat anti-human FC were diluted 1:2000 with PBS, and added to each well for 1 hr incubation. Plates were washed three times and incubated with TMB substrate for 20 min at room temperature. Absorbance at 450 nm was measured after the reaction was stopped. The result is presented inFIG.1b, lower panel showing that Fab 5310 and 5365 bind to both human and mouse CD137, whereas Fab 5351 binds to human CD137, but not to mouse CD137. Example 3 IgG Conversion and Expression: AG10058, AG10059 and AG10131 The heavy chains and light chains of the Fabs 5310, 5351, and 5365 were cloned into the mammalian expression vector pCDNA3.3 (Thermo Fisher Scientific) separately in IgG4 isotype with S241P mutation. The heavy and light chains of two reference antibodies were also cloned into pCDNA3.3 in IgG4 and IgG2 isotype respectively. The heavy chain variable region used in reference antibody AC1097 comprised the sequence EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSS (SEQ ID NO: 862), and the light chain variable region in reference antibody AC1097 comprised the sequence SYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGS NSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVL (SEQ ID NO: 863). The heavy chain variable region used in reference antibody AC1121 comprised the sequence QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQSPEKGLEWIGEINHGGYVTYNPS LESRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDYGPGNYDWYFDLWGRGTLVTVSS (SEQ ID NO: 864), and the light chain variable region in reference antibody AC1121 comprised the sequence EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGS GSGTDFTLTISSLEPEDFAVYYCQQRSNWPPALTFGGGTKVEIK (SEQ ID NO: 865). IgGs used in herein are shown in Table 2. TABLE 2List of IgGsIgGFabIsotypeDescriptionAC1097Reference 1IgG2Reference AbAC1121Reference 2IgG4Reference Ab(S241P)AG100585310IgG4Adagene mAb(S241P)AG100595351IgG4Adagene mAb(S241P)AG101315365IgG4Adagene mAb(S241P)AG10154IgG4Isotype control(S241P) Pairs of plasmids were transiently transfected into HEK293F cells following manufacturer's instructions. The supernatants were harvested, cleared by centrifugation and filtration, and IgGs were purified with standard protein A affinity chromatography (MabSelect SuRe, GE Healthcare). The proteins were eluted and neutralized, and buffer exchanged into PB buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). Protein concentrations were determined by UV-spectrophotometry, and IgG purity was analyzed under denaturing, reducing and non-reducing conditions by SDS-PAGE or SEC-HPLC. Example 4 Binding Affinity to Human, Monkey and Mouse CD137 The binding affinity of IgGs to human, monkey and mouse CD137 were measured by BIAcore, ELISA and flow cytometry. The results were summarized in Table 3. TABLE 3Binding affinity of antibodies to human, monkey and mouse CD137BiacoreELISAHEK293F Cell surfaceKD (nM)HumanCynoMouseHumanCynoMouseHumanCynoMouseAG101313.712.564.50.20.323.91.31.249.4AG100585.99.315.20.20.30.31.8210.1AG1005924.223.1NC0.80.4NC52.6NCAC109720.937.6NC0.20.4NC1.92.9NCAC11219.6NCNC0.2NC3.3NCNCNC: not cross-reactive 4a. Measurement of Binding Affinity and Kinetics by SPR Binding affinity and kinetics of antibodies against human, monkey and mouse CD137 protein were examined by surface plasmon resonance (SPR) analysis using a Biacore™ T200 instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines. Anti-Human IgG (Fc) antibody from Human Antibody Capture Kit (GE BR-1008-39) was immobilized on CM5 chips by coupling of its amine groups onto carboxylated surfaces of sensor chips according to the instructions of Amine Coupling kit (GE Biacore #BR-1000-50). The immobilized Anti-Human IgG (Fc) antibody was used to capture AG10058, AG10059, AG10131, AC1121 and AC1097. Finally, six concentrations (3.13, 6.25, 12.5, 25, 50, 100) (nM) (diluted in running buffer) of human CD137-His6 (Sino Biological #10041-H08H) were injected at a flow rate of 30 I/min for 300 seconds, and the dissociation time was 300 seconds. The running buffer used was 1×HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, pH 7.4 at 25° C.). Corresponding controls were conducted in each case using a blank flow cell with no protein immobilized for “background” subtraction. The association and dissociation curves were fitted to a 1:1 Langmuir binding model using Biacore T200 Evaluation Software (Biacore AB, Uppsala, Sweden) according to the manufacturer's guidelines. As shown in Table 3, all antibodies bind to human CD137. AG10058 and AG10059 show higher affinity than both reference antibodies. Except AC1121 reference mAb, all antibodies bind to monkey CD137. Only AG10058 and AG10131 bind to mouse and rat CD137. AG10058 has higher affinity (15.2 nM) than AG10131 (64.5 nM). 4b. Measurement of Binding Affinity to Soluble CD137 Using ELISA Assay A serial dilution of human, monkey or mouse CD137 fused with human FC fragment were prepared and used to coat the ELISA plate at 37° C. for 1 hr. After blocking, 100 μL IgGs (5 μg/mL) were added and incubated at 37° C. for 1 hr. Plates were washed for three times and then incubated with HRP-conjugated protein L (1:2000 dilution) at 37° C. for 1 hr. Plates were washed again for three times and incubated with TMB substrate for 20 min at room temperature. Absorbance at 450 nm was measured after the reaction was stopped. The data was analyzed by Graphpad Prism 6 with nonlinear fitting. As shown inFIG.2, all antibodies bind to human CD137 (FC fusion protein) with similar sub nM affinity. Except AC1121 reference mAb, all antibodies bind to monkey CD137 with similar sub nM affinity. Consistent with results from Biacore, only AG10058 and AG10131 bind to mouse CD137. AG10058 has higher affinity (0.3 nM) than AG10131 (23.9 nM). 4c. Measurement of Binding Affinity to CD137 Overexpressed on Cell Surface by Flow Cytometry The affinity of antibodies were also assessed against human, monkey and mouse CD137 that are transiently expressed on the surface of HEK293F cells. Briefly, HEK293F cells were transfected with a plasmid expressing full-length human, monkey or mouse CD137 from a bicistronic IRES vector, EGFP was used to identify the transfected cells. After 48 hrs, the transfected cells were harvested and then washed once with cold FACS buffer (PBS supplemented with 1% BSA). Cells were then incubated with various IgGs (each at 100 nM) for 1 hr on ice, washed twice with pre-chilled FACS buffer, and incubated with Alexa Fluor® 647 conjugated mouse anti-human FC antibodies for 30 min on ice. The cells were washed once prior to analysis by flow cytometry (Beckman® CytoFlex). As shown inFIG.3a, all antibodies bind to human CD137 expressed on cell surface with low nM affinity. AG10058, AG10059 and AG10131 are slightly better than both reference antibodies. Except AC1121 reference mAb, all antibodies bind to monkey CD137 with low nM affinity, AG10058, AG10059 and AG10131 are slightly better than AC1097 reference antibody. Consistent with results from Biacore and ELISA, only AG10058 and AG10131 bind to mouse and rat CD137. AG10058 has higher affinity than AG10131 against mouse CD137. Additionally, AG10058 and AG10131 (each at 100 nM) also bind to rat and canine CD137 overexpressed on HEK293F cell surface (FIG.3b). 4d. Binding of IgGs to Activated Human, Monkey, Mouse and Rat T Cells. The species-cross reactivity of the exemplary antibodies was further confirmed using PMA and Ionomycin stimulated PBMC or T cells of human, monkey, mouse and rat. Human and cynomolgus monkey PBMC were isolated by Ficoll-density gradient centrifugation. Briefly, fresh whole blood from healthy donors or cynomolgus monkeys were diluted with equal volume of PBS and carefully loaded on to the top of Histopaque 1077 (14 ml in 50 ml centrifuge tube). Centrifuge at 1,200×g for 30 minutes at room temperature with brake off. After centrifugation, carefully aspirate the upper layer with a pipette to within 0.5 cm of the opaque interface containing mononuclear cells. Discard upper layer. Carefully transfer the opaque interface (about 3-5 ml) with a pipette into a clean 50 ml conical centrifuge tube. Wash the cells with 20 ml of PBS, collect cells by centrifuge at 400×g for 5 minutes, and resuspend cells into 20 ml of PBS. Count the cells with hemocytometer, and collect cells again by centrifuge at 400×g for 5 minutes. Mouse or rat splenocytes were isolated by passing spleens through a 45 μm cell strainer attached to a 50-mL conical tube to get single cell suspension, and wash cells through the strainer with PBS. Centrifuge at 1600 rpm for 5 min and discard the supernatant. Resuspend cell pellet in 2 ml red blood cell lysing solution for 2 min. Add in excess of 10-fold volume PBS and collect cells at 1600 rpm centrifugation for 5 min. Discard supernatant and resuspend splenocytes in RPMI1640/10% FBS. Pan-T cells were enriched from PBMC (human/monkey), or splenocytes (mouse/rat) by negative selection with magnetic beads in commercial kits (Stemcell Technologies) specific for human, monkey, mouse and rat, respectively. Activation of human/monkey PBMC, or mouse/rat splenocytes was performed by incubating cells with 50 ng/ml PMA+1 μM Ionomycin at 37° C., 5% CO2 for overnight. Activated cells (˜2×105cells/tube) were washed in pre-chilled staining buffer (PBS supplemented with 2% FBS) and incubated with 100 nM tested antibodies for 1 hr on ice. Cells were then washed twice using 1 mL staining buffer and resuspended in 100 μL staining buffer containing Alexa Fluor® 647 conjugated mouse anti-human FC antibody and the species-specific T cell marker antibodies. The T cell marker antibodies were used as follows: CD3, CD4 or CD8. The cells were washed twice with staining buffer after incubation in dark for 30 minutes. Finally, cells were resuspended in 300 μL staining buffer and analyzed by Beckman CytoFlex. The data analysis was performed using Flowjo 10 software. As shown inFIG.4a, all tested antibodies bind to both activated human and monkey T cells, but not to the naïve human T cells. The binding ability of AG10131 to activated mouse and rat T cells were further evaluated (FIG.4b). AG10131 binds to both activated mouse and rat T cells. In summary, AG10058 and AG10131 antibodies show higher affinity to human and monkey CD137. They exhibit broad species-cross reactivity, including human, cynomolgus monkey, mouse, rat and dog for AG10131, but human, cynomolgus monkey, mouse, and dog for AG10058, allowing quick assessment of in vivo efficacy in mouse syngeneic models. Example 5 Binding Selectivity of Antibodies for CD137 The selectivity of antibodies for CD137 was evaluated using flow cytometry analysis of their binding ability to members of TNFR superfamily. The TNFRSF receptors including CD137, OX40, CD40, GITR and CD27 were transiently overexpressed on the surface of HEK293F cells. Transfected cells were washed in pre-chilled staining buffer (PBS supplemented with 2% FBS), then incubated with 100 nM test antibodies for 1 hr on ice. Cells were washed twice with staining buffer, and Alexa Fluor® 647 conjugated mouse anti-human FC antibodies were added and incubated for 30 min on ice. Samples were washed once with staining buffer prior to analysis by flow cytometry. As shown inFIG.5, AG10058, AG10059 and AG10131 bind specifically to CD137, not to any other tested family members or parent cells transfected with empty vectors. Example 6 Ligand Competition Using ELISA and Flow Cytometry Antibodies were tested for their ability to block the binding of CD137 to its cognate ligand CD137L by both ELISA and flow cytometry assay. As shown inFIGS.6aand6b, all tested antibodies block the binding of CD137 and CD137L. 6a. Ligand Competition Binding by ELISA Recombinant human CD137 (fused with human Fc and His tag) was diluted to 1 μg/mL in PBS and coated on Maxisorp plate at 4° C. overnight. Plates were blocked with PBS supplemented with 3% non-fat milk at 37° C. for 1 hr. After washing, a total volume of 100 uL mixture of 50 uL biotinylated CD137L (4 μg/mL) and various concentrations of test antibodies (eight 1:2 serial dilutions ranging from 500 μg/mL to 2 μg/mL) were added to each well and incubated at 37° C. for 1 hr. Plates were washed three times and 100 μL HRP conjugated neutravidin (1:1000) were added to each well and incubated at 37° C. for 1 hr. Plates were washed as previously described and 50 μL TMB substrate solution was added and incubated at room temperature for 20 minutes before the reaction was stopped by 50 μL H2SO4. As shown inFIG.6a, all test antibodies AG10058, AG10059 and AG10131 block the binding of CD137 to CD137L. AG10131 shows the strongest or complete blocking ability, at about uM range, followed by AG10058 for significant blocking at >uM; and AG10059 for effective blocking at uM range. These data suggest that, under the conditions tested and with the reagents used, the broad species cross-reactive antibodies AG10131 and AG10058 are highly effective inhibitors of the interaction between CD137 and its ligand CD137L, whereas AG10059 only shows moderately effective blocking of the interaction between CD137 and its ligand CD137L. It should be noted that the reference antibody AC1097, which cross-reacts with both human and monkey CD137, while AC1121, which only reacts with human CD137, shows almost no blocking at all. 6b. Ligand Competition Binding by Flow Cytometry The plasmid encoding full-length human CD137 was transiently expressed in HEK293F cells. Cells were washed with staining buffer (PBS supplemented with 1% BSA) and resuspended in staining buffer containing 100 nM test antibodies. After incubation on ice for 30 min, 33 nM biotinylated CD137L were added to each well and incubated for another 1 hr on ice. Cells were washed with staining buffer twice, and 50 μL staining buffer containing Alexa fluor 647 conjugated streptavidin were added and incubated on ice for 30 min. Cells were washed once and analyzed by CytoFlex flow cytometry. As shown inFIG.6b, all three tested antibodies can block binding between CD137 and CD137L in a concentration dependent manner. AG10131 shows the strongest blocking capability, followed by AG10058 with significant blocking; and AG10059 with less effective blocking. These data suggest that the broad species cross-reactive antibodies AG10131 and AG10058 are highly effective in blocking the interaction between CD137 and its ligand CD137L, while AG10059 shows partial blocking of the interaction between CD137 and its ligand CD137L. In contrast, the AC1097 reference antibody, which cross-reacts with both human and monkey CD137, shows only partial blocking, while the AC1121 reference antibody, which only reacts with human CD137, shows no blocking. Example 7 Epitope Mapping To determine the binding regions of the tested antibodies at amino acid residue level, a series of mutations (Table 5) were made at the extracellular domain of human CD137. These CD137 mutation plasmids were used to transfect HEK293F cells. The binding of antibodies to the human CD137 mutants were assessed by flow cytometry analysis as previously described in Example 5 and shown inFIG.7A. The results are summarized in Table 5, together with the cross-reactivity of these antibodies with human, monkey, mouse, and rat CD137 in interesting differentiation, indicating the fine epitopes from hits derived from Adagene libraries. AG10131 binds to all 4 species, whereas AG10058 binds all 3 CD137 but not rat CD137. AG10058, AG10059 and AG10131 lost the binding ability to GFT34AAA, FSS53AAA, and FH92AA mutations, indicating that their binding epitopes are within these regions, e.g., amino acid residues 34-93 or 34-108 of SEQ ID NO.: 1 (See also,FIG.7B). AG10058 and AG10131 may bind the same or highly similar epitope, while AG10059 may bind different epitopes from AG10058 and AG10131. The mutant constructs were meant to differentiate the epitopes by AG10058, AG10059 and AG10131 from the reference antibodies by AC1121 and AC1097. It is clear that all three antibodies AG10058, AG10059 and AG10131 target very different epitopes from AC1121 and AC1097. AG10058, AG10059 and AG10131 differ from AC1121 in regions defined by mutants Hu_FH92AA and Hu_FSS53AAA and possibly Hu_GTF34AAA, whereas AG10058, AG10059 and AG10131 differ from AC1097 in regions defined by most of the mutants used, except for Hu_FH92AA and their species cross-reactivity with Monkey but different in other species cross-reactivity such as mouse, rat and dog CD137. In some embodiments, AG10058, AG10059 and AG10131 or other antibodies disclosed herein do not bind to an epitope located within amino acid residues 115-156 of SEQ ID NO.: 1. Also shown inFIG.7Aand Table 5 is that binding of the human CD137 ligand to the wild-type vs. mutant human CD137 matches well with binding pattern of the tested antibodies, consisting with the observation that these antibodies block CD137 ligand binding to its receptor. TABLE 5Epitope MappingMutationsAG10058AG10059AG10131AC1121AC1097HuCD137LHu_WT++++++Cyno_WT+++−+Mouse_WT+−+−−Rat_WT−−+−−Hu_GTF34AAA−−−−/++−Hu_FSS53AAA−−−++−Hu_FH92AA−−−+−−Hu_GQ109AA++++−+Hu_EL111AA++++−+Hu_F125A++++−+Hu_FN125AA++++−+Hu_PW135AA++++−+Hu_TN137AA++++−+Hu_GT150AA++++−+ Example 8 Agonist Activity of Antibodies in NFκB Luciferase Reporter Assay Agonist activity of antibodies was evaluated using NFκB reporter assay. 293T cells were transfected with plasmid expressing human, monkey or mouse CD137 along with NFκB luciferase reporter plasmids. After 4 h, 50 μL cells were plated into each assay well of a 96-well plate at density of 0.4×106/mL. A total volume of 50 μL antibody mixture containing test antibodies and 3:1 ratio of cross linking antibody (Fab′ goat anti-human IgG FC) was added and incubated for 18 h. After medium is removed, 50 μL Passive Lysis Buffer (Promega E1980) were added and incubated at 37° C. for 30 min. 20 μL lysate were transferred to a white plate and the luciferase substrates were added. The luminescence signal of firefly and Renina was measured and their ratio was used for data analysis by GraphPad Prism 6.0 software. As shown inFIG.8, compare to isotype control antibody, all test antibodies activate NFκB reporter gene expression when human and monkey CD137 is expressed. When mouse CD137 is expressed, AG10058 and AG10131, but not AG10059, activate NFκB reporter gene expression. This is consistent with the prior observation that AG10058 and AG10131 bind to mouse CD137 whereas AG10059 does not. Example 9 Agonist Activity of Antibodies in T Cell Activation Assay Agonist activity of antibodies was further confirmed in the T cell activation assay. 96-well cell culture plates were coated with 50 μL of the anti-CD3 antibody (2 μg/ml) alone or along with 50 μL of the test antibodies (60 μg/mL, 20 μg/mL, 6 μg/mL, 2 μg/mL, and 0 μg/mL) in 1×PBS at 4° C. overnight. CD8+ T cells were isolated using protocols according to the manufacture's instruction. Cells were prepared at density of 1×107cells/mL in RPMI1640 media supplemented with 10% FBS. 200 μL cells were plated to each assay well and incubated for 4 days in a 37° C., 5% CO2 incubator. Cells were checked daily under microscope for proliferation. After 96 hr incubation, 100 μL of supernatant were transferred to a new 96-well plate for IFN-γ detection. T cell proliferation was assayed using Cell Titer Glow kit (Promega). As shown inFIG.9, compared to isotype control antibody, all tested antibodies induced both CD8+ T cell proliferation and IFN-γ secretion in a dose-dependent manner. Example 10 Anti-Tumor Activity in Mouse Syngeneic Models The species cross-reactivity with mouse CD137 allows quick in vivo functional assessment. AG10058 and AG10131 have been tested in multiple mouse syngeneic models. BALB/c mice (n=8 per group) were transplanted subcutaneously with 2×106H22 liver cancer cells (Xiao et. al, Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo. Clin Cancer Res. 2007; 13(6):1823-30), 5×105CT26 colon cancer cells, or 5×105EMT6 breast cancer cells. When tumors were established (>50 mm3), treatment began with isotype control antibody, AG10058, or AG10131 by intraperitoneal injection, twice a week for up to 3 weeks. Tumor growth was monitored twice a week and reported as the mean tumor volume±s.e.m. over time. As shown inFIGS.10-13, compared to the isotype control antibody, both AG10058 and AG10131 exhibited potent in vivo anti-tumor activity in these different syngeneic mouse tumor models. 10a. CD137 Agonist Antibodies Exhibit Anti-Tumor Efficacy in H22 Mouse Liver Cancer Model First, AG10058 or AG10131 was administrated twice a week for 3 weeks at dosage of 50 mg/kg. Both molecules showed almost 100% TGI (tumor growth inhibition) (FIG.10, panel a). Immunohistochemistry staining of CD4 and CD8 markers showed that AG10131 significantly increased the infiltration of both CD4+ and CD8+ T cells in H22 tumor (Xiao et. al, Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo. Clin Cancer Res. 2007; 13(6):1823-30) microenvironment (FIG.10, panel b). Further dose titrations down to 3 mg/kg still showed ˜100% TGI, suggesting both molecules have potent anti-tumor activity (FIG.10, panels c and d). Further dose titration of AG10131 down to 1 and 0.1 mg/kg showed greater than 50% TGI at 0.1 mg/kg and 1 mg/kg (FIG.10, panel e). 10b. CD137 Agonist Antibodies Exhibit Anti-Tumor Efficacy in CT26 Mouse Colon Cancer Model As shown inFIG.10, both AG10058 and AG10131 showed almost 100% TGI (tumor growth inhibition) at dose of 50 mg/kg (FIG.11, panel a) in CT26 mouse colon cancer model (Martinez-Forero et. al, T cell costimulation with anti-CD137 monoclonal antibodies is mediated by K63-polyubiquitin-dependent signals from endosomes. J Immunol. 2013; 190(12):6694-706). Further dose titration of AG10131 (FIG.11, panel b) showed almost 100% TGI at doses of 5 mg/kg and 1 mg/kg. At 0.1 mg/kg dosage, approximately 40% TGI was achieved, indicating a dose-dependent anti-tumor activity. 10c. EMT6 Breast Cancer Model Anti-tumor activity is further evaluated in EMT6 mouse breast cancer syngeneic model (Shi and Siemann, Augmented antitumor effects of radiation therapy by 4-1BB antibody (BMS-469492) treatment. Anticancer Res. 2006; 26:3445-53) (FIG.12). Both AG10058 and AG10131 exhibited almost ˜100% tumor growth inhibition. 10d. Mice with Complete Response to CD137 Agonist Antibody Treatment Maintain Tumor Free after Re-Challenge with New Tumor Cells After treatment with AG10058 or AG10131 for 3 weeks in the CT26 tumor model, the mice with complete tumor regression were maintained without treatment for over an additional month. Mice that maintained complete response were then re-challenged on Day 62 subcutaneously with 5×105CT26 tumor cells in the opposite flank, and monitored for tumor growth. Re-challenge control group was set up at the same time with naïve mice inoculated with the same number of CT26 tumor cells. As shown inFIGS.13, treatment with AG10131 (at 1 and 5 mg/kg, seeFIG.13, top and bottom panel, respectively) exhibited potent antitumor activity in CT26 tumor model, 5/8 in AG10131 (1 mg/kg group), 6/8 in AG10131 (5 mg/kg group) showing complete response over 60 days before re-challenged with CT26 tumor cells again. Furthermore, these mice remained tumor free after re-challenge with the same tumor cells, suggesting that specific anti-tumor memory was developed in these mice. To prove this hypothesis, splenocytes were collected from these tumor-rejecting re-challenged mice and control mice and co-cultivated with the mitomycin C-arrested CT26 tumor cells in vitro for 7 days to amplify the tumor-specific memory T cells. These splenocytes were then recovered and mixed with fluorescence labeled live CT26 tumor cells at different E/T ratio for 4 h and tumor cell killing was detected by the live/dead staining and FACS analysis. As shown inFIG.14, significantly increased tumor cell killing was observed with splenocytes from tumor-rejecting re-challenged mice with prior treatment of both AG10058 and AG10131. Example 11 AG10131-IgG4 does not Induce ADCC Effect Human CD8+ T cells were isolated from peripheral blood from a healthy donor with the EasySep human CD8+ T cell enrichment kit (StemCell Technologies), and then stimulated with PMA (50 ng/ml)+Ionomycin (1 uM) for 18 hours in vitro. These activated CD8+ T cells were then labeled with Calcein-AM and served as the target cells. NK cells from different healthy donors were isolated with the human NK isolation kit (StemCell Technologies), and served as effector cells. For the antibody-dependent cytotoxicity (ADCC) assay, effector (NK) and target (activated CD8+T) cells were mixed at 5:1 ratio in a 96-well plate in the absence and presence of serially diluted antibodies for 4 hours under culture condition. Supernatant from each well was then collected, and fluorescence signal was detected by plate-reader SpectraMax i3x (Ex 488 nm, Em 520 nm). An isotype hIgG4 mAb was used as a negative control, whereas the humanized OKT3 (an anti-CD3 hIgG1 from Novoprotein) was used as a positive control. The % lysis was then calculated using the following formula: % Lysis=[(Experimental Release)−Ave (Target+NK)]/[Ave (Target Max)−Ave (Target only)]×100% (FIG.15). Example 12 Developability Profile of Antibodies For developability assessment, purified AG10058, AG10059, AG10131 and AC1097 were exchanged into PB buffer (20 mM PB, 150 mM NaCl, pH 7.0). All experiments, including filtration, concentration, accelerated stress tests, were performed in PB buffer. For all the SEC-HPLC analyses, the TSKgel columns (Tosoh Bioscience G3000SW×1) were used. 12a. Solubility All three antibodies can be concentrated to higher than 100 mg/ml in PB buffer without obvious precipitation (Table 6). Antibodies then were adjusted to 20 mg/ml in PB buffer. Samples (10 g each) were then assayed through SEC-HPLC for detection of high molecular weight (HMW) aggregate. As shown in the chromatograms (FIG.16), no increase of HMW aggregate was observed at high concentration (20 mg/ml) for all test antibodies. TABLE 6Solubility of antibodiesSampleConcentration/(mg/mL)Aggregation (HMW %)AG100581081.0AG100591341.4AG101311102.0 12b. Antibody Stability Under Accelerated Stress Conditions Antibody stabilities were also examined under accelerated stress conditions, result is summarized in Table 7. All antibodies remain stable after six cycles of freezing (−80° C.) and thawing (Room temperature) (FIG.17). After seven days at 50° C., there was little change of HMW aggregate or LMW fragments (FIG.17). In longer-term time course experiments (40° C. for up to 28 days), all antibodies remain stable, and there were no significant increase of HMW aggregate or LMW fragments (FIG.17). TABLE 7Changes of HMW under accelerated conditionsAG10058AG10059AG10131Freeze-thaw 6X4.6%1.2%0.4%50° C. 7 d0.7%1.2%0%40° C. 28 d0.9%0%0% Furthermore, thermostability as measured by differential scanning calorimetry (DSC) shows that both AG10131 and AG10058 are stable up to at least about 59° C. The transition midpoint, Tm (the characteristic temperature at which the unfolding transition for almost all protein domains occur) is shown inFIG.18and Table 8 below. TABLE 8Thermostability by DSCTm onset (° C.)Tm1 (° C.)Tm2 (° C.)AG1005861.567.376.9AG1013159.367.681.5 In addition, the highest achievable concentration of AG10131 and AG10058 after centrifugation was over 180 mg/mL and over 220 mg/mL, respectively. Example 13 Safety Profile in Relevant Species: Mouse and Cynomolgus Monkey 13a. Repeated Dosing Toxicity Studies of AG10131 in Normal C57BL/6 Mice. Repeated dosing toxicity of AG10131 was conducted in normal C57BL/6 mice. Vehicle, AG10131 (100 mg/kg) was administered i.p. (10 mL/kg) on Day1, Day4, Day8, and Day11. Five female mice (7-8 weeks old) were included in each group. Mice were monitored daily for abnormal behaviors and symptoms, and measured daily for food intake and body weight. On day 14, animals were euthanized for post-mortem examination and other analysis. Blood was collected from each animal, with 2 blood samples per group used for hematology (RBC, platelet, WBC, WBC differential) and the other 3 blood samples in the group for blood biochemistry (AL, AST, ALB, GLB, A/G, TBIL, ALP, GGT, and LDH) analysis. The following organs from each mouse were collected and preserved in FFPE: Heart, lung, thymus, liver, spleen, and kidneys. FFPE blocks for liver tissues were prepared, sectioned and H&E stained for histopathology analysis. During the in-life period of the whole study, there was no abnormal behavior observed or un-scheduled animal death. Compared to the vehicle treatment, AG10131 did not affect the food intake and body weight. Post-mortem examination also did not show any obvious lesions in mice of the treatment groups with both AG10131. Hematology analysis did not show any significant changes, so as to the blood biochemistry parameters tested in mice treated with AG10131 (FIG.19). No obvious abnormalities were found in the histopathology sections of the liver from all these mice (FIG.20). Overall, AG10131 was well tolerated in this study and no significant toxicity was observed in mice. 13b. Repeated Dosing Studies of AG10131 in Cynomolgus Monkeys Repeated dosing study of AG10131 was conducted in normal cynomolgus monkeys. Human IgG4 Isotype control (10 mg/kg), AG10131 (0.5 and 10 mg/kg) was administered i.v. (1 mL/kg) on Day0, Day7, Day14, and Day22. One male and one female cynomolgus monkeys (3-5 years old) were included in each group. Animals were monitored daily for abnormal behaviors and clinical signs, and measured daily for food intake. Body weight was measured on predose Day (−15), Day (−5), and postdose Day6, Day13, Day18 and Day26. Hematology and blood chemistry parameters were measured on predose Day (−12), Day (−5), and postdose Day7, Day14, Day19, and Day27 (10 mg/kg group only), urinalysis was conducted on predose Day (−12), Day (−5), and postdose Day6, Day13, Day18. Animals in the 10 mg/kg groups were euthanized for post-mortem examination and other analysis on Day27. Major organs were dissected and weighed. FFPE liver tissue blocks were prepared, sectioned and H&E stained for histopathology analysis. During the in-life period of the whole study, there was no abnormal behavior observed or un-scheduled animal death in all groups. Compared to the vehicle treatment, AG10131 treatment at 10 mg/kg did not affect the food intake and body weight. No clinical signs were noted including injection site reactions as well. Post-mortem examinations did not show any obvious lesions and weight abnormalities in all organs examined in cynomolgus monkeys treated with AG10131 at 10 mg/kg. Hematology, blood chemistry and urine parameters are also within normal ranges in all treatment groups (FIG.21). Histopathology analysis of the liver did not show any obvious abnormalities including lymphocyte infiltration after repeated dosing of AG10131 at 10 mg/kg (FIG.22). Overall, AG10131 was well tolerated at up to 10 mg/kg weekly doses in cynomolgus monkeys and no obvious toxicity was detected. Example 14 Pharmacokinetics of AG10131 in Cynomolgus Monkey 14a. Pharmacokinetics of AG10131 in Cynomolgus Monkey A pharmacokinetics study of AG10131 was conducted in naive cynomolgus monkeys. Three dose levels of AG10131 (10 mg/kg, 30 mg/kg and 100 mg/kg) were intravenous bolus administrated to three groups of monkeys. Each group contains 3 males and 3 females. Serum samples were collected pre-dose, 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, 336, 408, 504, 672 and 840 hours post dosing. Serum concentrations of AG10131 were determined by ELISA. AG10131 was rapidly cleared at day 14 (336 hrs) in 12 out of 16 animals, i.e., all animals from low and medium dose groups and 2 of 6 animals from high dose group. On day 21, 2 more animals from high dose group showed a rapid clearance. Serum concentrations in these 14 animals are low or below the limit of the quantification. This is consistent with the observation of anti-drug antibody generation in these animals. Data from the two animals with potentially unaffected pharmacokinetics from the high dose group were fitted to predict the pharmacokinetics parameters (FIG.23). The half-life of AG10131 ranges from 7.3 to 8.8 days. 14b. Pharmacokinetics of AG10131 in Rat A pharmacokinetics study of AG10131 was conducted in naïve SD rats. Three dose levels of AG10131 (10 mg/kg, 30 mg/kg and 100 mg/kg) were intravenous bolus administrated to three groups of animals. Each group contains 15 males and 15 females. Serum samples were collected from 3 animals each time point: pre-dose, 0.083, 0.25, 0.5, 1, 2, 6, 12, 24, 36, 48, 72, 96, 120, 144, 168, 240, 336, 408, 504, 672 and 840 hours post dosing. Serum concentrations of AG10131 were determined by ELISA and the data were analyzed by Phoenix Professional V6.3. Results: The PK parameters from low, mid and high doses are similar (FIG.24). The clearance rate of AG10131 is about 0.004 ml/kg/min. The half-life of AG10131 ranges from 11.5 to 14.6 days. 14c. Pharmacokinetics of AG10131 in Mouse A pharmacokinetics study of AG10131 was conducted in BALB/c mice at about 8-week age. 3 female BALB/c mice per dosing group were intravenously injected the test antibodies including AG10131 at 1 mg/kg through the tail vein. Blood samples (˜100 ul per sample) were collected at 1 h, 8, 48, 168, and 336 hours post dosing. Blank control blood was collected from 3 naive female mice without antibody administration. Serum concentrations of each test antibody including AG10131 were determined by ELISA, in which the anti-human IgG (Fc specific) antibody was used for capture and the HRP-labeled anti-human IgG (Fab specific) antibody for detection. All test antibodies including the isotype control (AG10154), two benchmark antibodies (AC1020 and AC1021), and three Adagene antibodies (AG10131, AG10058, and AG10059) exhibit comparable pharmacokinetics in mice (FIG.25). Example 15 Further Epitope Mapping To determine the binding epitope of the antibodies shown herein by Adagene and other reference antibodies, we have taken a systematic approach to dissecting the epitopes by three levels of resolution: domain, motif, and residues. The extra cellular domain of CD137 containing 4 CRD motifs and CD137 from 4 different species such as human, monkey, mouse, and rat CD137 were used (Table 9). A series of human CD137 CRD motif (cysteine rich domain) and their constructs containing one, two, and three units of human CRD motifs (Table 9) were displayed. A low copy number, CEN/ARS-based vector was used to express the human CD137 CRDs under the control of the inducible GAL1-10 promoter in the yeastS. cerevisiae(Boder and Wittrup (1997) Nat Biotechnol 15(6):553-7). The binding of antibodies to the human CD137 CRDs were assessed by flow cytometry analysis and other technology as previously described in Example 5 and shown inFIG.27. The results are summarized in Table 9, these antibodies only bind selectively to CD137 target, but none of the non-CD137 targets listed in the table; however, their species-specific cross-reactivity by these antibodies with CD137 from human, monkey, mouse, and rat species are striking, highlighting the fine epitope coverage by diverse hits screened from Adagene Dynamic Precision Libraries. For example, AG10131 binds to CD137 from human, monkey, mouse, rat, and dog (not shown); AG10058 binds human, monkey, mouse CD137, but not rat CD137; whereas AG10059 binds both human and monkey CD137. In contrast, the reference antibody AC1121 from transgenic mouse only binds to human CD137; whereas another reference antibody by morphosys phage library binds to both human and monkey CD137. For comparison, it should be noted that the human ligand CD137L only interacts with human CD137 receptor, not mouse CD137, and mouse CD137L only interact with mouse CD137 not human CD137 (see table 9). TABLE 9HumanMouseAG10058AG10059AG10131AC1121AC1097CD137LCD137LHuman WT++++++−Mouse_WT+−+−−−+Cyno_WT+++−+NARat_WT−−+−−NA To further dissect the binding motifs of these antibodies to human CD137 target as summarized in Table 9, distinguished binding sites by Adagene, other reference antibodies, in comparison with CD137 ligand binding with the dissected CD137 CRD motifs and their combination are well separated and noted: AG10058, AG10059 and AG10131 antibodies and human CD137 ligand does not bind to the single CRD or two CRD units (CRD2-CRD3) of human CD137. AG10058, AG10059, and AG10131 antibodies, similar to the human CD137 ligand, can bind to three CRD units (CRD1-CRD2-CRD3) of human CD137. Although the reference antibody AC1121 can also bind to three CRD units (CRD1-CRD2-CRD3) of human CD137, however, it is the specific two CRD units (CRD1-CRD2) of human CD137. In comparison, the three CRD units (CRD1-CRD2-CRD3) of human CD137 are required for binding by AG10058, AG10059, AG10131 antibodies, and the human CD137 ligand. Reference antibody AC1097 can bind two CRD units (CRD3-CRD4), including three CRD units (CRD2-CRD3-CRD4) of human CD137. These indicate Adagene AG10058, AG10059, AG10131 antibodies bind the epitopes covered by the three CRD units (CRD1-CRD2-CRD3) of human CD137, similar to human CD137 ligand, but they are very different from the reference antibody AC1121 that binds to two CRD units (CRD1-CRD2) of human CD137 and reference antibody AC1097 which binds to two CRD units (CRD3-CRD4) of human CD137. In conclusion, the binding epitope of CD137 by Adagene's antibodies is different from the epitope by the two reference antibodies (AC1121 with CRD1-CRD2; and AC1097 with CRD3-CRD4) as shown by the distinction in terms of the specific CRDs used and their number of CRD units required (see Table 9B) by highly similar, if not overlapping, to the CD137 epitope by human CD137L ligand; the epitope between CD137-CD137L is confirmed by the recently reported crystal structure complex, as is shown inFIG.26(Gilbreth, R. N., Oganesyan, V. Y., Amdouni, H., Novarra, S., Grinberg, L., Barnes, A., Baca, M. (2018) J. Biol. Chem. 293: 9880-9891). TABLE 9BHumanMouseAG10058AG10059AG10131AC1121AC1097CD137LCD137LHu_CRD1−−−−−−−Hu_CRD2−−−−−−−Hu_CRD3−−−−−−−Hu_CRD4−−−−−−−Hu_CRD1-CRD2−−−+−−−Hu_CRD2-CRD3−−−−−−−Hu_CRD1-CRD2-CRD3++++−+−Hu_CRD3-CRD4−−−−+−−Hu_CRD2-CRD3-CRD4−−−−+−− To determine the binding epitope of Adagene's and reference antibodies at amino acid residue level, a series of mutations (Table 5) were made at the extracellular domain of human CD137. These CD137 mutation plasmids were used to transfect HEK293F cells. The binding of antibodies to the human CD137 mutants were assessed by flow cytometry analysis as previously described in Example 5 and shown inFIG.7A. The results are summarized in Table 5, together with the cross-reactivity of these antibodies with human, monkey, mouse, and rat CD137 in interesting differentiation, indicating the fine epitopes from hits derived from Adagene libraries. AG10131 binds to human, monkey, mouse, and rat CD137, whereas AG10058 binds human, monkey and mouse CD137, but not rat CD137. The binding epitope of CD137 by AG10058, AG10059 and AG10131 was mapped onto CRD1-CRD2-CRD3 units of CD137, they lost their binding ability to GFT34AAA, FSS53AAA, and FH92AA mutations, indicating that their binding epitopes are within these regions, e.g., amino acid residues 34-93 or 34-108 of SEQ ID NO.: 1 (See also,FIG.7B). AG10058 and AG10131 may bind the same or highly similar epitope, while AG10059 may bind different epitopes from AG10058 and AG10131. Single mutants such as T35A, F36A, F53A, R66A, F72A, N83A, and F92A show that the loss of binding by Adagene antibodies AG10058, AG10059 and AG10131 with human CD137, together with the binding by CD137L except for R66A which still maintains the binding of CD137 by its ligand. Single mutants, P32A and P49A, however, that the binding between CDL137L and CD137 is lost but its impact on the interaction between antibody and CD137 are varied. F125A shows AC1097 does not bind to CD137 anymore, but with no effects on the binding by other antibodies including human CD137L. In conclusion, the overall binding pattern by mutants across CD137 does show a clear message that Adagene antibodies and their reference antibodies are distinguished in terms of their binding sites. The mutant constructs were meant to differentiate the epitopes by AG10058, AG10059 and AG10131 from the reference antibodies by AC1121 and AC1097. Three antibodies AG10058, AG10059 and AG10131 target very different epitopes from AC1121 and AC1097. AG10058, AG10059 and AG10131 differ from AC1121 in regions defined by mutants Hu_FH92AA and Hu_FSS53AAA and possibly Hu_GTF34AAA, whereas AG10058, AG10059 and AG10131 differ from AC1097 in regions defined by most of the mutants used, except for Hu_FH92AA and their species cross-reactivity with Monkey but different in other species cross-reactivity such as mouse, rat and dog CD137. In some embodiments, AG10058, AG10059 and AG10131 or other antibodies disclosed herein do not bind to an epitope located within amino acid residues 115-156 of SEQ ID NO.: 1. Also shown inFIG.7Aand Table 5 is that binding of the human CD137 ligand to the wild-type vs. mutant human CD137 matches well with binding pattern of the tested antibodies, consisting with the observation that these antibodies block CD137 ligand binding to its receptor. HumanMouseAG10058AG10059AG10131AC1121AC1097CD137LCD137LHuman WT++++++−Mouse_WT+−+−−−+Cyno_WT+++−+NARat_WT−−+−−NAHu_N30A++++++−Hu_P32A+++−+−−Hu_GTF34AAA−−−−/++−−Hu_T35A−−−−+−−Hu_F36A−−−−+−−Hu_P49A+−+++−−Hu_P50A++++++−Hu_F53A−−−−+−−Hu_FSS53AAA−−−++−−Hu_Q59A++++++−Hu_I64A++++++−Hu_R66A−−−+++−Hu_F72A−−−++−−Hu_N83A−−−++−−Hu_F92A−−−++−−Hu_L95A++++++−Hu_FH92AA−−−+−−−Hu_GQ109AA++++−+−Hu_EL111AA++++−+−Hu_F125A++++−+−Hu_FN125AA++++−+−Hu_PW135AA++++−+−Hu_TN137AA++++−+−Hu_GT150AA++++−+−Hu_CRD1−−−−−−−Hu_CRD2−−−−−−−Hu_CRD3−−−−−−−Hu_CRD4−−−−−−−Hu_CRD1-CRD2−−−+−−−Hu_CRD2-CRD3−−−−−−−Hu_CRD1-CRD2-CRD3++++−+−Hu_CRD3-CRD4−−−−+−−Hu_CRD2-CRD3-CRD4−−−−+−− Example 16 Native CD137L Signaling is Blocked by AG10131 An in vitro binding assay by ELISA demonstrated that AG10131 can block recombinant CD137 and its ligand interaction. To further functionally validate this ligand-blocking activity of AG10131, a cellular NFκB luciferase reporter assay was conducted. Briefly, 293T cells stably expressing an NFκB luciferase reporter were transfected with a DNA construct expressing human CD137, and the cells were co-cultivated with the human B-cell lymphoma cells Daudi or Raji at different ratios. The cell mixture was incubated with serial dilutions of isotype control or ligand-blocking anti-CD137 antibodies overnight, and luciferase activity was measured using the Promega luciferase assay kit according to manufacturer's instructions. Relative luciferase units (RLUs) were calculated vs. the levels of luciferase expressed in 293T cells in the absence of antibody treatment. As shown inFIG.28, both Daudi (top row) and Raji (bottom row) cells expressed functional CD137 ligand to activate the NFκB luciferase reporter in 293T cells. Compared to the isotype control antibody (left column), addition of AG10131 to the co-culture system (right column) significantly inhibited the NFκB signaling stimulated by both cell types, suggesting that AG10131 antibody can functionally block the CD137 signaling stimulated from CD137 ligand expressed on both Daudi and Raji B lymphoma cells. Example 17 Anti-CD137 Antibody Crosslinking in an NFκB Reporter Assay Using the functional cellular NFκB reporter assay, three anti-CD137 antibodies (AG10131, AC1121 and AC1097) were tested. As shown inFIG.29, when crosslinked, all three anti-CD137 antibodies were capable of stimulating human CD137 receptor signaling in a dose-dependent manner at comparable levels. The EC50s of the antibody induced NFκB signaling activation response were at similar range for all three anti-CD137 antibodies. However, AC1121 displayed a unique property that is different from AG10131 and AC1097. AC1121 was able to activate human CD137 receptor signaling significantly in the absence of crosslinking, whereas AG10131 and AC1097 were unable to do so. The EC50 of AC1121 with or without crosslinking in the stimulation of the CD137 receptor signaling was found to be at similar levels. Example 18 AG10131 does not Induce CDC The CDC activity of AG10131 was determined by the direct binding of AG10131 with the purified C1q component of the human complement with ELISA. As shown inFIG.30, AG10131 and its human IgG4 isotype control antibody lack the ability to bind to human complement C1q component in the concentration range tested, whereas a human IgG1 isotype control antibody is able to bind to C1q. This result suggests that AG10131 is also unlikely to induce complement dependent cytotoxicity, consistent with its IgG4 isotype framework. Example 19 Anti-CD137 Antibody AG10131 Enhances Tumor-Infiltrating T-Lymphocytes The in vivo anti-tumor efficacy studies in the syngeneic mouse H22 liver cancer, EMT6 breast cancer, and CT26 colon cancer models shown in Example 10 demonstrated that AG10131 treatment strongly inhibits tumor growth. AG10131 is an agonistic antibody that activates T cells, and thus AG10131 treatment is expected to stimulate tumor infiltrating T cells into the tumor micro-environment, thereby mediating an anti-tumor effect in vivo. To evaluate the effect of AG10131 treatment on the tumor infiltrating lymphocytes, tumors from the in vivo anti-tumor efficacy study of AG10131 in mouse H22, EMT6, and CT26 cancer models were collected at the end of studies. FIG.31shows representative IHC staining images of mouse CD4+ and CD8+ T cells in H22 tumors (top left), EMT6 tumors (top right), and CT26 tumors (bottom center). As shown inFIG.32, few T lymphocytes (either CD4+ or CD8+ T cells) were present in the vehicle control treated H22, EMT6, and CT26 tumors, whereas AG10131 treatment significantly stimulated the infiltration of both CD4+ and CD8+ T cells (indicated by black arrows inFIG.31) into the tumors. These data are consistent with the function of AG10131 as an immune agonist by stimulating T cell proliferation, activation, and infiltration into the tumor micro-environment to mediate an antitumor effect. Example 20 Enhanced Anti-Tumor Efficacy by Combining Anti-CD137 Antibody AG10131 and Anti-PD1 Antibody in the CT26 Colon Cancer Model The effect of combining the anti-CD137 antibody AG10131 with an anti-PD1 antibody was next tested in the CT26 colon cancer model. Each female BALB/c mouse was inoculated subcutaneously at the right lower flank region with CT26 tumor cells (3×105) for tumor development. When the mean tumor volume reached 98 mm3, 10 mice were assigned to each experimental group. These groups received either vehicle (PBS), AG10131 at 5 or 10 mg/kg, anti-PD-1 at 10 mg/kg, or a combination of 5 or 10 mg/kg of AG10131 and 10 mg/kg of anti-PD-1 mAb by i.p. injection twice weekly for 3 weeks. Tumor volumes were measured and each mouse was euthanized when its tumor reached the endpoint volume of 2000 mm3, or on the final day (Day 42), whichever came first. As shown inFIG.33, both AG10131 (5 mg/kg or 20 mg/kg) and anti-PD1 (10 mg/kg) delayed tumor progression, though AG10131 delayed tumor progression by a few more days, and in rare cases, resulted in tumor shrinkage. However, nearly all mice treated with either AG10131 or anti-PD1 eventually died of tumor progression. Importantly, when AG10131 (5 mg/kg or 20 mg/kg) was administered in combination with anti-PD1 (10 mg/kg), most of the mice were essentially cured of tumor, with only 2 (out of 10) or 1 (out of 10) escaped tumor suppression in the combinations of AG10131 (5 mg/kg) with anti-PD1 (10 mg/kg) or AG10131 (20 mg/kg) with anti-PD1 (10 mg/kg) respectively. These results demonstrated the strong synergistic effect of AG10131 and anti-PD1, suggesting that the combination of AG10131 with anti-PD1 could be effective in anti-PD1 resistance tumors. | 167,742 |
11859004 | CERTAIN DEFINITIONS In the description that follows, a number of terms used in recombinant DNA and immunology are extensively utilized. In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided. About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value. Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. Affinity: As is known in the art, “affinity” is a measure of the tightness with a particular ligand binds to its partner. Affinities can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, binding partner concentration may be fixed to be in excess of ligand concentration so as to mimic physiological conditions. Alternatively or additionally, in some embodiments, binding partner concentration and/or ligand concentration may be varied. In some such embodiments, affinity may be compared to a reference under comparable conditions (e.g., concentrations). Agonist: Those skilled in the art will appreciate that the term “agonist” may be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with an increased level or activity of another agent (i.e., the agonized agent). In general, an agonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant activating activity. In some embodiments, an agonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an agonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered). Animal: as used herein refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, of either sex and at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically engineered animal, and/or a clone. Antagonist: Those skilled in the art will appreciate that the term “antagonist”, as used herein, may be used to refer to an agent condition, or event whose presence, level, degree, type, or form correlates with decreased level or activity of another agent (i.e., the inhibited agent, or target). In general, an antagonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity. In some embodiments, an antagonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an antagonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered). Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)—an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3 (located at the base of the Y's stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains—an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab′ fragments, F(ab′)2 fragments, Fd′ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); humabodies, VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.] Antibody fragment: As used herein, an “antibody fragment” refers to a portion of an antibody or antibody agent as described herein, and typically refers to a portion that includes an antigen-binding portion or variable region thereof. An antibody fragment may be produced by any means. For example, in some embodiments, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody or antibody agent. Alternatively, in some embodiments, an antibody fragment may be recombinantly produced (i.e., by expression of an engineered nucleic acid sequence. In some embodiments, an antibody fragment may be wholly or partially synthetically produced. In some embodiments, an antibody fragment (particularly an antigen-binding antibody fragment) may have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids or more, in some embodiments at least about 200 amino acids. Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts—including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). Cancer: The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma”, are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like. CDR: as used herein, refers to a complementarity determining region within an antibody variable region. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. A “set of CDRs” or “CDR set” refers to a group of three or six CDRs that occur in either a single variable region capable of binding the antigen or the CDRs of cognate heavy and light chain variable regions capable of binding the antigen. Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.); those skilled in the art appreciate the differences between and among these systems and are capable of understanding CDR boundaries to the extent required to understand and to practice the claimed invention. Chemotherapeutic Agent: The term “chemotherapeutic agent”, has used herein has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example specifically including agents utilized and/or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g. microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and/or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or analogs thereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine. Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more therapeutic regimens may be administered simultaneously. In some embodiments, the two or more therapeutic regimens may be administered sequentially (e.g., a first regimen administered prior to administration of any doses of a second regimen). In some embodiments, the two or more therapeutic regimens are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more therapeutic agents or modalities to a subject receiving the other agent(s) or modality. Corresponding to: As used herein, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure. Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered” when the polypeptide sequence manipulated by the hand of man. For example, in some embodiments of the present invention, an engineered polypeptide comprises a sequence that includes one or more amino acid mutations, deletions and/or insertions that have been introduced by the hand of man into a reference polypeptide sequence. Comparably, a cell or organism is considered to be “engineered” if it has been manipulated so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). As is common practice and is understood by those in the art, derivatives and/or progeny of an engineered polypeptide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity. Epitope: as used herein, includes any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Ex vivo: as used herein refers to biologic events that occur outside of the context of a multicellular organism. For example, in the context of cell-based systems, the term may be used to refer to events that occur among a population of cells (e.g., cell proliferation, cytokine secretion, etc.) in an artificial environment. Framework or framework region: as used herein, refers to the sequences of a variable region minus the CDRs. Because a CDR sequence can be determined by different systems, likewise a framework sequence is subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FRs within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub-regions, FR1, for example, represents the first framework region closest to the amino terminal end of the variable region and 5′ with respect to CDR1, and FRs represents two or more of the sub-regions constituting a framework region. Humanized: as is known in the art, the term “humanized” is commonly used to refer to antibodies (or antibody components) whose amino acid sequence includes VH and VL region sequences from a reference antibody raised in a non-human species (e.g., a mouse), but also includes modifications in those sequences relative to the reference antibody intended to render them more “human-like”, i.e., more similar to human germline variable sequences. In some embodiments, a “humanized” antibody (or antibody component) is one that immunospecifically binds to an antigen of interest and that has a framework (FR) region having substantially the amino acid sequence as that of a human antibody, and a complementary determining region (CDR) having substantially the amino acid sequence as that of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor immunoglobulin) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody also may include a CH1, hinge, CH2, CH3, and, optionally, a CH4 region of a heavy chain constant region. In vitro: The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism. In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). Isolated: as used herein, refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients. To give but one example, in some embodiments, a biological polymer such as a polypeptide or polynucleotide that occurs in nature is considered to be “isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a polypeptide that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an “isolated” polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an “isolated” polypeptide to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced. KD: as used herein, refers to the dissociation constant of a binding agent (e.g., an antibody or binding component thereof) from a complex with its partner (e.g., the epitope to which the antibody or binding component thereof binds). Operably linked: as used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element “operably linked” to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, “operably linked” control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest. Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof. Prevent or prevention: as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset and/or severity of one or more characteristics or symptoms of the disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. Recombinant: as used herein, is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc). Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. A binding agent that interacts with one particular target when other potential targets are present is said to “bind specifically” to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations. Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered. Therapeutic agent: As used herein, the phrase “therapeutic agent” in general refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans. Therapeutically Effective Amount: As used herein, the term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, stabilizes one or more characteristics of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. For example, in some embodiments, term “therapeutically effective amount”, refers to an amount which, when administered to an individual in need thereof in the context of inventive therapy, will block, stabilize, attenuate, or reverse a cancer-supportive process occurring in said individual, or will enhance or increase a cancer-suppressive process in said individual. In the context of cancer treatment, a “therapeutically effective amount” is an amount which, when administered to an individual diagnosed with a cancer, will prevent, stabilize, inhibit, or reduce the further development of cancer in the individual. A particularly preferred “therapeutically effective amount” of a composition described herein reverses (in a therapeutic treatment) the development of a malignancy such as a pancreatic carcinoma or helps achieve or prolong remission of a malignancy. A therapeutically effective amount administered to an individual to treat a cancer in that individual may be the same or different from a therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the therapeutic methods described herein are not to be interpreted as, restricted to, or otherwise limited to a “cure” for cancer; rather the methods of treatment are directed to the use of the described compositions to “treat” a cancer, i.e., to effect a desirable or beneficial change in the health of an individual who has cancer. Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, a stabilization of patient condition, a decrease in tumor size (tumor regression), an improvement in vital functions (e.g., improved function of cancerous tissues or organs), a decrease or inhibition of further metastasis, a decrease in opportunistic infections, an increased survivability, a decrease in pain, improved motor function, improved cognitive function, improved feeling of energy (vitality, decreased malaise), improved feeling of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as the result of treatments described herein) may also be assessed by taking samples of cancer cells from the site of a tumor such as a pancreatic adenocarcinoma (e.g., over the course of treatment) and testing the cancer cells for the level of metabolic and signaling markers to monitor the status of the cancer cells to verify at the molecular level the regression of the cancer cells to a less malignant phenotype. For example, tumor regression induced by employing the methods of this invention would be indicated by finding a decrease in any of the pro-angiogenic markers discussed above, an increase in anti-angiogenic markers described herein, the normalization (i.e., alteration toward a state found in normal individuals not suffering from cancer) of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer. Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen. Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence. In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. Vector: as used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS The present disclosure relates, inter alia, to 4-1BB, which is an inducible co-stimulatory molecule, and therapeutic antibodies that bind thereto that have been engineered to have improved characteristics over a reference anti-4-1BB antibody. For example, engineered antibodies provided herein have been modified to enhance antigen affinity relative to that of a reference agonist antibody that specifically recognizes an epitope within the extracellular domain of human 4-1BB (Korean Patent No. 10-0500286, Accession No: KCTC 0952BP). Specifically, as described herein, the inventors engineered a reference humanized anti-human 4-1BB antibody, 94G1 (U.S. Pat. No. 7,932,045). As described examples herein, the light chain and heavy chain CDR sequences of a reference antibody 94G1, were separately engineered to improve the affinity of each chain. Moreover, as described herein, exemplary engineered anti-4-1BB antibodies can effectively induce proliferation of activated T cells. Notably, exemplary engineered anti-4-1BB antibodies are capable of inducing surprisingly improved activity of CD8+T cells due to the stimulation caused by the 4-1BB humanized antibody binding to a 4-1BB molecule and inhibiting activation-induced cell death (AICD). Thus the present disclosure provides engineered anti-human 4-1BB antibodies with improved properties over a reference antibody, and moreover demonstrate that these antibodies have surprisingly beneficial activity in vitro and in vivo. 4-1BB 4-1BB (also referred to as CD137, TNFRSF9, etc) is a receptor belonging to the tumor necrosis factor receptor (TNFR) superfamily. 4-1BB is a co-stimulatory molecule generally expressed in activated T lymphocytes and involved in immunity and autoimmune diseases (Kwon et al.PNAS84:2896, 1987; Kwon et al.PNAS(1989) 86:1963; Son et al.Journal of Immunological Methods(2004) 286(1-2):187-201, each of which is herein incorporated by reference in its entirety). Human 4-1BB is a 255 amino acid protein (Accession No. NM 001561; NP 001552). The complete human 4-1BB amino acid sequence is provided in SEQ ID NO: 44. 4-1BB is expressed on the cell surface in monomer (30 kDa) and dimer (55 kDa) forms and likely trimerizes with 4-1BB ligand to signal. Current understanding of 4-1BB suggests that it is constitutively expressed on a number of cells, albeit at low levels, including Foxp3+Tregs and dendritic cells (DC). (See, Vinay and Kwon (2014)BMB Rep.47(3): 122-129, which is incorporated by reference herein.) Activation with a number of agonists, such as cytokines (e.g., IL-2, IL-4), polyclonal activators (e.g., Con A and PHA), cell surface molecules (e.g., anti-CD3 and anti-CD28) and promoters of Ca2+induction and PKC activity (e.g., ionomycin and photbol myristate acetate) further enhance expression of 4-1BB. Id. Numerous studies of murine and human T cells indicate that 4-1BB promotes enhanced cellular proliferation, survival, and cytokine production (Croft, 2009, Nat. Rev. Immunol.9:271-285). Studies have indicated that some 4-1BB agonist monoclonal antibodies can increase costimulatory molecule expression and markedly enhance cytolytic T lymphocyte responses, resulting in anti-tumor efficacy in various models. 4-1BB agonist monoclonal antibodies have demonstrated efficacy in prophylactic and therapeutic settings. Further, 4-1BB monotherapy and combination therapy tumor models have established durable anti-tumor protective T cell memory responses (Lynch (2008)Immunol. Rev.22: 277-286). 4-1BB agonists also have been shown to inhibit autoimmune reactions in a variety of art-recognized autoimmunity models (Vinay (2006)J. Mol. Med.84:726-736). This dual activity of 4-1BB offers the potential to provide anti-tumor activity while dampening autoimmune side effects that can be associated with immunotherapy approaches. 4-1BB Antibodies and Fragments Thereof The present disclosure provides, at least in part, engineered anti-human 4-1BB antibodies and fragments thereof that exhibit markedly, and unexpectedly, superior characteristics in vitro and/or in vivo. For example, certain provided antibodies have increased affinity relative to a reference humanized anti-human 4-1BB antibody. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 heavy chain CDR sequences that are or include a sequence of SEQ ID NOs: 5 to 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes one or more of: a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes each of: a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes 1, 2, or 3 light chain CDR sequences that are or include a sequence of SEQ ID NOs: 1-4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes one or more of: a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes each of: a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR1 that is or includes a sequence of SEQ ID NO: 5, a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 and a heavy chain CDR3 that is or includes a sequence of SEQ ID NO: 7 or 8 and/or a light chain variable domain that includes a light chain CDR1 that is or includes a sequence of SEQ ID NO: 1, a light chain CDR2 that is or includes a sequence of SEQ ID NO: 2 and a light chain CDR3 that is or includes a sequence of SEQ ID NO: 4. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 where the 5thamino acid, asparagine (N), was substituted with glutamine (Q), glutamic acid (E) or serine (S). In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain CDR2 that is or includes a sequence of SEQ ID NO: 6 where the 5thamino acid, asparagine (N), was substituted with valine (V), glycine (G), or proline (P). In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that includes a light chain CDR3 that is or includes a sequence of SEQ ID NO: 3 or 4 where the 6thamino acid position of LCDR3 is mutated. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework 1 (FR1) region comprising a sequence of SEQ ID NO: 16 or 17. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework 3 (FR3) region comprising a sequence of any one of SEQ ID NOs: 18-20. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that includes a heavy chain framework 1 (FR1) region comprising a sequence of SEQ ID NO: 16 or 17 and a heavy chain framework 3 (FR3) region comprising a sequence of any one of SEQ ID NOs: 18-20. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID NOs: 11-14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a light chain variable domain that has or includes a sequence of SEQ ID NO: 9 or 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence of SEQ ID NO: 9 or 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a light chain variable domain that is or includes a sequence of SEQ ID NO: 9 or 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes substantial homology to an antibody or antibody fragment that includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14 and a light chain variable domain that is or includes a sequence of SEQ ID NO: 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID NOs: 11-14 and a light chain variable domain that is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to a sequence of SEQ ID NO: 10. In some embodiments, an anti-4-1BB antibody or antigen-binding antibody fragment includes a heavy chain variable domain that is or includes a sequence selected from SEQ ID NOs: 11-14 and a light chain variable domain that is or includes a sequence of SEQ ID NO: 10. Amino acid sequences of anti-human 4-1BB antibody or antigen-binding fragment binds of the present disclosure may be substituted through conservative substitution. The term “conservative substitution” used herein refers to modification of a polypeptide in which one or more amino acids are substituted with an amino acid having a similar biochemical property so as not to cause the loss of a biological or biochemical function of the corresponding polypeptide. The term “conservative sequence variant” or “conservative amino acid substitution” used herein is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Amino acid residues having a similar side chain are defined in the art. Those residues encompass amino acids with a basic side chain (e.g., lysine, arginine, and histidine), amino acids with an acidic side chain (e.g., aspartic acid and glutamate), amino acids with a non-charged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids with a non-polar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids with a beta-branched side chain (e.g., threonine, valine, and isoleucine) and amino acids with an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is expected that the antibody of the present invention can have conservative amino acid substitution, and still ensure an activity. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure may include a constant region selected from an IgG1 constant domain, an IgG2 constant domain, an IgG1/IgG2 hybrid constant domain, a human IgG4 constant domain, an IgA constant domain, an IgE constant domain, an IgM constant domain, and an IgD constant domain. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is or includes an IgA, IgD, IgE, IgM, IgG, or variants thereof. In some embodiments, an anti-human 4-1BB antibody of the present disclosure includes a variant Fc-region that has an amino acid mutations and/or substitutions at one or more positions of 234, 235, 236, 237, 238, 239, 253, 254, 265, 266, 267, 268, 269, 270, 288, 297, 298, 299, 307, 311, 322, 327, 328, 329, 330, 331, 332, 434 and 435. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is human IgG1 isotype. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure includes a variant IgG1. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide that has amino acid mutation at one or more positions of 233, 234, 235, 236, 265, 297, 329, 331 and 322. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide containing one or more mutations in L234, L235, D270, N297, E318, K320, K322, P331 and P329. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide containing two, three, four, or more mutations in L234, L235, D270, N297, E318, K320, K322, P331 and P329. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes an IgG1 polypeptide with mutations in L234A and L235A. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes a light chain constant region. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment includes a kappa (κ) and/or lambda (λ) light chain and/or a variant thereof. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is a monoclonal antibody. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, a disulfide-bonded Fv fragment, a scFv fragment, a single domain antibody, humabody, nanobody, and/or a diabody. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is a monovalent antibody. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is a multivalent antibody. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is a multi-specific antibody (e.g., a bispecific antibody). In some embodiments, the present disclosure encompasses methods of modifying the carbohydrate content of an antibody of the disclosure by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and encompassed within the disclosure, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S. Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No. 2003/0115614; U.S. Pat. Nos. 6,218,149; 6,472,511; all of which are incorporated herein by reference in their entirety. In other embodiments, the present disclosure encompasses methods of modifying the carbohydrate content of an antibody of the present disclosure by deleting one or more endogenous carbohydrate moieties of the antibody. In a specific embodiment, the present disclosure encompasses deleting the glycosylation site of the Fc region of an antibody, by modifying position 297 from asparagine to alanine. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises a N297A mutation in the CH2 domain. In some embodiments, the N297A mutation results in aglycosylation, which reduces FcR or Clq binding. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising a N297A mutation and a K322A mutation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising a N297A mutation and a D265A mutation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises a heavy chain comprising an Fc region comprising a N297A mutation, a D265A mutation, and a K322A mutation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises an Fc region with a L234A mutation and/or a L235A mutation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises an Fc region with one or more mutations selected from L234A, L235A, N297A, D265A, and K322A. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises Fc region with two or more mutations selected from L234A, L235A, N297A, D265A, and K322A. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment comprises Fc region with three, four, or five mutations selected from L234A, L235A, N297A, D265A, and K322A. Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to one skilled in the art, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example DI N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed. Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et al, 1999, Nat. Biotechnol17:176-180; Davies et al., 20017Biotechnol Bioeng74:288-294; Shields et al, 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton, N.J.); GLYCOMAB™glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, e.g., WO 00061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB,336: 1239-49 each of which is incorporated herein by reference in its entirety. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is as an agonist for human 4-1BB. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-1 BB molecule. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure specifically binds to a human 4-1 BB molecule. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment binds to a sequence that is or includes that of SEQ ID NO: 15. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment binds to an epitope of 4-1BB extracellular domain that is or includes a sequence of SEQ ID NO: 15. In some embodiments, binding of an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure with human 4-1BB extracellular domain is abrogated by one or more mutations of SEQ ID NO: 44 selected from N30, D38, N39, R41, A56, G57, R60 or T61. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-1 BB molecule with a binding affinity (KD) of 1×10−7to 1×10−12M. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-1 BB molecule with a binding affinity (KD) of 1×10−8to 1×10−12M. Binding affinity (KD) may be measured, for example, by surface plasmon resonance, for example, using a BIACORE system. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-1BB molecule or a fragment thereof at a binding affinity (KD) of less than 1.0×10−8M. In some embodiments, an anti-humanized 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-1BB molecule or a fragment thereof at a binding affinity (KD) of less than 1.0×10−9M. In some embodiments, an anti-humanized 4-1BB antibody or antigen-binding fragment of the present disclosure binds to a human 4-1BB molecule or a fragment thereof at a binding affinity (KD) of less than 1.0×10−10M. In some embodiments, an anti-4-1BB antibody or antigen-binding fragment of the present disclosure fails to bind or weakly binds a non-primate 4-1BB polypeptide (e.g., a canine, mouse and rat 4-1BB polypeptide). In some embodiments, an anti-4-1BB antibody or antigen-binding fragment of the present disclosure binds efficiently to human or monkey 4-1BB. This binding affinity suggests that the structure and/or sequence of epitope for a primate 4-1BB antibody may be quite different from canine, mouse and rat. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is an agonistic antibody. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure mediates T cell activation. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure binds CD8+and/or CD4+T cells expressing human 4-1BB. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure does not have or has low ADCC activity. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure does not have or has low CDC activity. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure does not have or has low ADCC activity and CDC activity. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure has an ADCC cell killing activity of less than about less than about 20%, less than about 10%, less than about 8%, or less than about 5%. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure has an ADCC cell killing activity of less than about 10%. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure has a CDC cell killing activity of less than about 30%, less than about 20%, less than about 10%, less than about 8%, or less than about 5%. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure has a CDC cell killing activity of less than about 20%. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is characterized by low toxicity (e.g., a low degree of post administration cell death). In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is characterized by low hepatoxicity. In some embodiments, a subject that has been administered an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure at a therapeutic dose has levels of one or more of ALT, AST and total bilirubin in a normal range. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is characterized by an ability to treat patients for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, can contribute to the therapeutic results achieved. “Low immunogenicity” is defined herein as raising significant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (Elliott et al.,Lancet344:1125-1127 (1994), entirely incorporated herein by reference). Nucleic Acids The disclosure provides polynucleotides comprising a nucleotide sequence encoding anti-human 4-1BB antibodies of the present disclosure and fragments thereof. Anti-human 4-1BB antibodies and fragments thereof as described herein may be produced from nucleic acid molecules using molecular biological methods known to the art. Nucleic acids of the present disclosure include, for example, DNA and/or RNA. In some embodiments, nucleic acid constructs include regions that encode an anti-human 4-1BB antibody or fragment thereof (e.g., 94K, 94 KV, 94KVT, EU101). In some embodiments, such antibodies or fragments thereof will include VHand/or VLregions. An anti-human 4-1BB antibody or fragment thereof may be identified and/or selected for a desired binding and/or functional properties, and variable regions of said antibody isolated, amplified, cloned and/or sequenced. Modifications may be made to the VHand VLnucleotide sequences, including additions of nucleotide sequences encoding amino acids and/or carrying restriction sites, and/or substitutions of nucleotide sequences encoding amino acids. In some embodiments, a nucleic acid sequence may or may not include an intron sequence. Where appropriate, nucleic acid sequences that encode anti-human 4-1BB antibodies and fragments thereof (e.g., 94K, 94 KV, 94KVT, EU101) may be modified to include codons that are optimized for expression in a particular cell type or organism (e.g., see U.S. Pat. Nos. 5,670,356 and 5,874,304). Codon optimized sequences are synthetic sequences, and preferably encode the identical polypeptide (or a biologically active fragment of a full length polypeptide which has substantially the same activity as the full length polypeptide) encoded by the non-codon optimized parent polynucleotide. In some embodiments, the coding region of the genetic material encoding antibody components, in whole or in part, may include an altered sequence to optimize codon usage for a particular cell type (e.g., a eukaryotic or prokaryotic cell). For example, a coding sequence for a humanized heavy (or light) chain variable region as described herein may be optimized for expression in a bacterial cells. Alternatively, the coding sequence may be optimized for expression in a mammalian cell (e.g., a CHO cell). Such a sequence may be described as a codon-optimized sequence. Nucleic acid constructs of the present disclosure may be inserted into an expression vector or viral vector by methods known to the art, and nucleic acid molecules may be operably linked to an expression control sequence. A vector comprising any of the above-described nucleic acid molecules, or fragments thereof, is further provided by the present disclosure. Any of the above nucleic acid molecules, or fragments thereof, can be cloned into any suitable vector and can be used to transform or transfect any suitable host. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, “Recombinant DNA Part D,”Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)). In some embodiments, conventionally used techniques, such as, fore example, electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, lipofection, etc. may be used to introduce a foreign nucleic acid (DNA or RNA) into a prokaryotic or eukaryotic host cell. Desirably, a vector may include regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA. In some embodiments, a vector comprises regulatory sequences that are specific to the genus of the host. Preferably, a vector comprises regulatory sequences that are specific to the species of the host. In addition to the replication system and the inserted nucleic acid, a nucleic acid construct can include one or more marker genes, which allow for selection of transformed or transfected hosts. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable vectors include those designed for propagation and expansion or for expression or both. For example, a cloning vector is selected from the group consisting of the pUC series, the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Examples of plant expression vectors include pBI110, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM and pMAMneo (Clontech). The TOPO cloning system (Invitrogen, Carlsbad, Calif.) also can be used in accordance with the manufacturer's recommendations. An expression vector can comprise a native or nonnative promoter operably linked to an isolated or purified nucleic acid molecule as described above. Selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the skill in the art. Similarly, combining of a nucleic acid molecule, or fragment thereof, as described above with a promoter is also within the skill in the art. Suitable viral vectors include, for example, retroviral vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors, and lentiviral vectors, such as Herpes simplex (HSV)-based vectors. These viral vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al.,Molecular Cloning, a Laboratory Manual,2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates andJohn Wiley & Sons, New York, N.Y. (1994). A retroviral vector is derived from a retrovirus. Retrovirus is an RNA virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into the genome of its host cell and is replicated along with host cell DNA, thereby constantly producing viral RNA and any nucleic acid sequence incorporated into the retroviral genome. As such, long-term expression of a therapeutic factor(s) is achievable when using retrovirus. Retroviruses contemplated for use in gene therapy are relatively non-pathogenic, although pathogenic retroviruses exist. When employing pathogenic retroviruses, e.g., human immunodeficiency virus (HIV) or human T-cell lymphotrophic viruses (HTLV), care must be taken in altering the viral genome to eliminate toxicity to the host. A retroviral vector additionally can be manipulated to render the virus replication-deficient. As such, retroviral vectors are considered particularly useful for stable gene transfer in vivo. Lentiviral vectors, such as HIV-based vectors, are exemplary of retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non-dividing cells and, therefore, can be of use in treating persistent forms of disease. Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra). In some embodiments, nucleic acids and vectors of the present disclosure may be isolated and/or purified. The present disclosure also provides a composition comprising an above-described isolated or purified nucleic acid molecule, optionally in the form of a vector. Isolated nucleic acids and vectors may be prepared using standard techniques known in the art including, for example, alkali/SDS treatment, CsCl binding, column chromatography, agarose gel electrophoresis and other techniques well known in the art. The composition can comprise other components as described further herein. In some embodiments, nucleic acid molecules are inserted into a vector that is able to express an anti-human 4-1BB antibody or fragment thereof when introduced into an appropriate host cell. Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Exemplary host cells include prokaryotes (e.g.,E. coli) and eukaryotes (e.g., a COS or a CHO cell). Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells (e.g., DG44 cells). In some embodiments, a mammalian host cell suitable for the expression of the antibody may be a Chinese Hamster Ovary (CHO) cell (for example, including DHFR-CHO cells used along with a DHFR-selectable marker), an NSO myeloma cell, a COS cell or an SP2 cell. Any method(s) known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding an anti-human 4-1BB antibody or fragment thereof of the present disclosure under control of transcriptional/translational control signals. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (See, e.g., Ausubel, supra; or Sambrook, supra). Production of Antibodies Antibodies and antigen-binding fragments of the present invention may be prepared and/or purified by any technique known in the art, which allows for the subsequent formation of a stable antibody or antibody fragment. A nucleic acid encoding an anti-human 4-1BB antibody and/or antigen-binding fragment of the present disclosure may be easily isolated and sequenced by conventional procedures. For example, an oligonucleotide primer designed to specifically amplify corresponding heavy chain and light chain-coding regions from a hybridoma or phage template DNA may be used. Isolated nucleic acids may be inserted into an expression vector, and then desired monoclonal antibodies may be produced from a suitable host cell (that is, transformant) transformed by introducing the expression vector to the host cell. In some embodiments, a method for preparing anti-human 4-1BB antibody and/or antigen-binding fragment of the present disclosure may include amplifying an expression vector including a nucleic acid encoding the antibody, but is not limited thereto. In some embodiments, a host cell is eukaryotic host cell, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, antibodies and antibody fragments of the present disclosure can be glycosylated or can be non-glycosylated. In some embodiments, a recombinant expression vector encoding an anti-human 4-1BB antibody and/or antigen-binding fragment of the present disclosure is introduced into a mammalian host cell and an antibody may be prepared by culturing the host cell for a sufficient time to express the antibody. In some embodiments, a mammalian host cell is cultured for a sufficient time to secrete an antibody or antibody fragment of the present disclosure in a culture medium. In some embodiments, an expressed antibody of the present disclosure may be uniformly purified after being isolated from the host cell. Isolation and/or purification of an antibody of the present disclosure may be performed by a conventional method for isolating and purifying a protein. For example, not wishing to be bound by theory, an anti-human 4-1BB antibody and/or antigen-binding fragment of the present disclosure can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, protein A purification, protein G purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan,Current Protocols in Immunology, orCurrent Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, each entirely incorporated herein by reference. In some embodiments, an antibody of the present disclosure may be isolated and/or purified by additionally combining filtration, superfiltration, salting out, dialysis, etc. Purified anti-human 4-1BB antibodies and/or antigen-binding fragments of the present disclosure can be characterized by, for example, ELISA, ELISPOT, flow cytometry, immunocytology, BIACORE™ analysis, SAPIDYNE KINEXA™ kinetic exclusion assay, SDS-PAGE and Western blot, or by HPLC analysis as well as by a number of other functional assays disclosed herein. Therapeutic Applications The present disclosure encompasses a recognition that engineered anti-human 4-1BB antibodies and antigen-binding fragments may be useful for diagnosis, prevention, and/or treatment of certain diseases such as, for example, cancer. Any of the anti-4-1BB antibodies or antigen-binding fragments provided herein may be used in therapeutic methods. For example, an anti-4-1BB antibody or antigen-binding fragment of the present disclosure can be used as immunotherapeutic agents, for example in the treatment of a malignant disease (e.g., cancer). The present disclosure provides methods for treating and/or preventing a malignant disease, said methods including administering an anti-4-1BB antibody or antigen-binding fragment of the present disclosure to a subject. Methods for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, include, but are not limited to, cancer and/or and the treatment of inflammatory diseases. Cancer treatments in the context of the present disclosure may be mediated through increasing cytotoxic T cells and anti-cancer cytokines. Generally, antigen-specific cell-mediated immunity is caused by cytotoxic T cells, and includes two signaling events: a first signaling event is induced when a T cell recognizes an antigen from an antigen-presenting cell via a receptor, and a second signaling is induced by co-stimulatory molecules. Due to the first and second stimuli, T cell activity and related factors are increased, thereby forming T cells specifically functioning in cancer treatment, and the formed T cells are increased in cytotoxicity, cell division, cell viability and anti-cancer cytokine secretion due to stimulation with the co-stimulatory molecules. Specifically, it has been demonstrated that stimulation by 4-1BB can enhance the activity of CD8+T cells, increase secretion of anti-cancer cytokines such as interferon gamma (IFNγ), increase expression of anti-apoptotic molecules such as Bcl-2, BclXL and Bfl-1, and/or inhibits activation-induced cell death (AICD). In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure can enhance or increase one or more of CD8+T cell activity, secretion of anti-cancer cytokines such as interferon gamma (IFNγ), expression of anti-apoptotic molecules such as Bcl-2, BclXL and Bfl-1, and inhibition of activation-induced cell death (AICD). In some embodiments, therapeutic treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure can reduce and/or inhibit growth of cancer cells. In some embodiments, the present disclosure provides a method for delaying or inhibiting tumor growth, comprising regulation of cytokine secretion in vivo or in vitro by administering an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure. In some embodiments, the present disclosure provides a method for reducing tumor burden, comprising regulation of cytokine secretion in vivo or in vitro by administering an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure. In some embodiments, the present disclosure provides a method for treating cancer or tumor by monitoring to a biological subject of cancer or tumor to be treated, comprising: (i) administrating an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure to a subject, (ii) separating then isolating a biological sample from the subject, (iii) measuring a secretion amount of INFγ or TGFβ from the sample and estimating a proportion ratio and (iv) determining a therapeutically effective amount of the antibody or antigen-binding fragment thereof by comparing the control samples which are administrated or not administrated with the anti-human 4-1BB antibody or antigen-binding fragment thereof. In some embodiments, the present disclosure provides a method of treating a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or delivers an anti-4-1BB antibody or antigen-binding fragment of the present disclosure and/or a nucleic acid the same. In some embodiments, a subject has or is at risk for developing cancer. In some embodiments, the present disclosure provides a method for preventing or treating cancer or tumor of a patient, which includes administering a therapeutically effective amount of the humanized 4-1BB antibody or the antigen-binding fragment thereof to a patient with cancer or tumor. In some embodiments, the present disclosure provides a method of inducing an immune response in a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or delivers an anti-4-1BB antibody or antigen-binding fragment of the present disclosure and/or a nucleic acid the same. In some embodiments, a subject has or is at risk for developing cancer. In some embodiments, the present disclosure provides a method of enhancing an immune response or increasing the activity of an immune cell in a subject in need thereof, the method comprising a step of administering to the subject a composition that comprises or delivers an anti-4-1BB antibody or antigen-binding fragment of the present disclosure and/or a nucleic acid the same. In some embodiments, a subject has or is at risk for developing cancer. Cancers suitable for treatment with method of the present disclosure can include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, a cancer for treatment with an anti-4-1BB antibody or antigen-binding fragment of the present disclosure may include, but is not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphomas), blastoma, sarcoma and leukemia. In some embodiments, cancer may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer. A composition including an anti-4-1BB antibody or antigen-binding fragment of the present disclosure may be administered at a pharmaceutically effective amount to treat cancer cells or metastasis thereof, or inhibit the growth of cancer. For use in therapeutic methods, an anti-4-1BB antibody or antigen-binding fragment of the present disclosure would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the age of the patient, the weight of the patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The present disclosure provides high affinity anti-human 4-1BB antibodies that may have superior properties relative to a reference antibody. The present disclosure encompasses a recognition that these antibodies may have improved ability to induce T cell activation and/or secretion of cytokines such as IFNγ. Accordingly, the present disclosure encompasses a recognition that an anti-human 4-1BB antibody or antigen binding fragment of the present disclosure may be administered a dose lower than reference antibody. In some embodiments composition that includes an anti-4-1BB antibody or antigen-binding fragment of the present disclosure may be administered to a patient as a bolus or by continuous injection when needed. In some embodiments, bolus administration is of an anti-4-1BB Fab of the present disclosure and may be administered at a dose of 0.0025 to 100 mg/kg, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg, or 0.10 to 0.50 mg/kg. In the case of the continuous injection, the antibody of the present invention presented as a Fab fragment may be administered at a dose of 0.001 to 100 mg/kg/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min or 0.10 to 0.50 mg/kg/min for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours. In some embodiment, an antibody of the present disclosure is a full-length antibody (having a complete constant domain). In some embodiments, a full-length antibody is administered at a dose of approximately 0.01 to 10 mg/kg, 1 to 8 mg/kg, or 2 to 6 mg/kg. In some embodiments, a full-length antibody is administered by injection for 30 to 35 minutes. Administration frequency may vary depending on the severity of a condition. For example, the frequency may be once every 2 to 7 days, once a week, or once every 1, 2, 3 or 4 weeks. In some embodiments, a composition may be administered to a patient by subcutaneous injection. Specifically, the antibody may be administered to a patient at a dose of 0.1 to 100 mg by subcutaneous injection once every 2 to 7 days, every week, once every two weeks, or every month. Combination Therapies The present disclosure provides therapeutic methods that include administration of an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure in combination with one or more other therapies. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered in combination with one or more therapies that have been approved for treatment of cancer. For example, combination treatment of with an anti-4-1BB antibody and a conventional chemotherapeutic, cisplatin, has been shown to have synergistic activity in tumor killing and prevention of organ-specific toxicity. (Kim et al.,Cancer Research(2008) 68(18):7264-9) In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered in combination with a second therapy selected from an immune checkpoint inhibitor, Interleukin 12 (IL-12), Granulocyte-macrophage colony-stimulating factor (GM-CSF), an anti-CD4 agent, and a chemotherapeutic agent, such that the subject receives treatment with both. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition comprising a chemotherapeutic agent, such that the subject receives treatment with both. Therapeutic methods of the present disclosure may include administration of any chemotherapeutic agent known in the art. In some embodiments, chemotherapeutic agent is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising fluorouracil. In some embodiments, fluorouracil is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising doxorubicin. In some embodiments, doxorubicin is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising irinotecan. In some embodiments, irinotecan is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising paclitaxel. In some embodiments, paclitaxel is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising cisplatin. In some embodiments, cisplatin is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment is administered to a subject that been administered or will be administered a composition comprising cyclophosphamide. In some embodiments, cyclophosphamide is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition comprising GM-CSF, such that the subject receives treatment with both. In some embodiments, GM-CSF is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition comprising IL-12, such that the subject receives treatment with both. In some embodiments, IL-12 is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition comprising an anti-CD4 agent, such that the subject receives treatment with both. In some embodiments, an anti-CD4 agent is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. In some embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to a subject that been administered or will be administered a composition comprising a checkpoint inhibitor (e.g., an immune checkpoint inhibitor), such that the subject receives treatment with both. In some embodiments, an immune checkpoint inhibitor is administered to a subject that been administered or will be administered a composition comprising an anti-human 4-1BB antibody or antigen-binding fragment. A checkpoint inhibitor used in combination with an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure can be, for example, any immune checkpoint inhibitor. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3, TIGIT and VISTA. An immune checkpoint inhibitor may refer to any compound that inhibits the function of an immune inhibitory checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, an immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. A number of immune checkpoint inhibitors are known and in analogy of these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. Immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molecules and small molecules. In some embodiments, an immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, a checkpoint inhibitor is an antibody that targets CTLA-4, such as, for example, ipilimumab. In some embodiments, a checkpoint inhibitor targets CD366, which is a transmembrane protein also known as T cell immunoglobulin and mucin domain containing protein-3 (TIM-3). In some embodiments, an immune checkpoint inhibitor is an agent that inhibits PD-1 signaling. PD-1 (i.e. programmed cell death protein-1), is a protein that is distributed on the surface of an immune cell such as a T or B cell and is also known as CD279. In a human, PD-1 is expressed by a PDCD1 gene located at the 2p37.3 position on chromosome 2. PD-1 is known to bind two ligands, PD-L1 and PD-L2. In some embodiments, an anti-PD-1 agent is administered to patient who is receiving, has received or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure. In some certain embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to patient who is receiving, has received or will receive treatment with an anti-PD-1 agent. In some embodiments, an anti-PD-L1 agent is administered to patient who is receiving, has received or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure. In some certain embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to patient who is receiving, has received or will receive treatment with an anti-PD-L1 agent. In some embodiments, agents that inhibit PD-L1 include, for example, AMP-244, MEDI-4736, MPDL328 OA, MIH1. In some embodiments, an anti-PD-1 agent is an agent that inhibits PD-1. In some embodiments, an anti-PD-1 agent is an agent that inhibits PD-L1 and/or PD-L2. In some embodiments, an antibody agent that inhibits PD-1 signaling is a monoclonal antibody or a fragment thereof. In some embodiments, an antibody agent that inhibits PD-1 signaling is an anti-PD-1 antibody or fragment thereof. In some embodiments, an anti-PD-1 antibody is administered to patient who is receiving, has received or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure. In some certain embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to patient who is receiving, has received or will receive treatment with an anti-PD-1 antibody. Anti-PD-1 antibodies include, for example, nivolumab, pembrolizumab, atezolizumab, durvalumab, and avelumab. Pembrolizumab (Keytruda, Merck) is an antibody therapeutic that inhibits PD-1 activity. As described in the Examples of the present application, administration of an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure in combination with an anti-PD-1 antibody may enhance efficacy relative to either treatment alone, and further may also reduce conventionally known side effects. In some certain embodiments, pembrolizumab is administered to patient who is receiving, has received or will receive treatment with an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure. In some certain embodiments, an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is administered to patient who is receiving, has received or will receive treatment with pembrolizumab. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD-1 agent) is administered to a patient in an amount of from about 0.01 mg/kg to about 100 mg/kg. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD-1 agent) is administered to a patient in an amount within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. In some embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD-1 agent) may be administered to a patient in an amount of from about 1 mg/kg to about 20 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 4 mg/kg, from about 3 mg/kg to about 5 mg/kg, or from about 3 mg/kg to about 4 mg/kg. In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD-1 agent) may be administered to a patient in an amount of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, or about 5 mg/kg. In some embodiments, treatment with a combination of an immune checkpoint inhibitor and an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure may enhance proliferation, migration, persistence and/or cytoxic activity of CD8+T cells in a subject. Cell-Based Applications Yet another object of the present invention is to provide a method for proliferating activated T cells ex vivo by administering the 4-1BB humanized antibody or antigen-binding fragment thereof. In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes contacting a population of T cells with an anti-4-1BB antibody or antigen-binding fragment of the present disclosure, thereby increasing proliferation of activated T cells. In some embodiments, a method for proliferating activated T cells ex vivo includes administering an anti-4-1BB antibody or antigen-binding fragment of the present disclosure. In some embodiments, activated T cells are proliferated and/or isolated from a sample of peripheral blood mononuclear cells (PBMC). PBMCs can be obtained/isolated using methods known in the art. In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes administration of an anti-CD3 monoclonal antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, a method for ex vivo proliferation and/or isolation of activated T cells includes administration of IL-2 and/or IL-15 to the culture medium (e.g., at concentration that is at least about 10 units/ml). In some embodiments, a method for isolating antigen-specific activated T cells includes (a) culturing peripheral blood mononuclear cells (PBMC) in a medium together with a peptide of an epitope of interest and IL-2; (b) inducing 4-1BB expression in the cultured cells by adding the peptide of the epitope of interest; (c) contacting the cultured cells with a surface coated with an anti-4-1BB antibody or antigen-binding fragment, wherein cultured cells expressing 4-1BB adhere to the coated surface; and (d) removing unattached cells, thereby isolating antigen-specific activated T cells. In some embodiments, the activated T cells are CD8+T cells. In some embodiments, lymphocytes (e.g., T cells) are cultured at a temperature of at least about 25° C., preferably at least about 30° C., more preferably about 37° C. The present disclosure encompasses the recognition that activated T cells (e.g., CD8+T cells), generated by the methods described herein may be therapeutically useful (e.g., for the treatment of cancer). Cell-Based Therapies The present disclosure provides methods to selectively isolate and mass culture CD8+T cells which recognize an autologous cancer antigen (self-tumor antigen), for example, an autologous cancer antigen that overexpressed in cancer cells while present in a low ratio in normal cells. The present disclosure that cells (e.g., CD8+T) isolated by these methods may be useful for the treatment of cancer. In some embodiments, a method for treating and/or preventing cancer in a subject in need thereof includes administering to the subject a therapeutically effective amount of activated T cells produced by an ex vivo method such as those described herein. Upon appropriate reactivation, tumor antigen specific T cells can recognize and eliminate autologous tumor cells. For example, tumor antigen specific T cells can be generated ex vivo using methods as described herein. Upon adoptive transfer, specifically reactivated T cells from cancer patients can efficiently reject autologous human tumors in vivo. The present disclosure provides methods for preventing and/or treating cancer and/or tumor of a patient, which include administering a therapeutically effective amount of activated T cells prepared ex vivo by administering an anti-4-1BB antibody or antigen-binding fragment of the present disclosure. In some embodiments, T cells for using in a therapeutic method are allogenic (from the same species but different donor) as the recipient subject. In some embodiments, T cells for using in a therapeutic method are autologous (the donor and the recipient are the same). In some embodiments, T cells for using in a therapeutic method are syngeneic (the donor and the recipients are different but are identical twins). In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a “pharmaceutically acceptable” carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amount of cells in the composition is at least 108, typically greater than 108, at least 109cells, and generally more than 1010. The number of cells will depend upon the ultimate use for which the composition is intended as will the type of cells included therein. For example, if cells that are specific for a particular antigen are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less. In some embodiments, cells for administration are in a volume of less than 500 ml, less than 250 ml, or 100 ml or less. In some embodiments, a density of the desired cells is typically greater than 106cells/ml and generally is greater than 107cells/ml, generally 108cells/ml or greater. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 108cells, 109cells, 1010cells. 1011cells, or 1012cells. Compositions Provided herein are compositions comprising antibodies and antigen binding fragments that specifically bind to an epitope of a human 4-1BB polypeptide. Compositions of the present disclosure (e.g., compositions that deliver an anti-human 4-1BB antibody or antibody fragment) may include any suitable and effective amount of a composition for use in delivering a provided anti-human 4-1BB antibody or antibody fragment to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. Also provided herein are compositions that include activated cell populations (e.g., activated T cell population) that have been generated via a method of the present disclosure (e.g., a method that includes a step contacting a cell with an anti-human 4-1BB antibody or antibody fragment). Compositions of the present disclosure include pharmaceutical compositions that include an anti-human 4-1BB antibody or antigen-binding fragment disclosed herein and/or a cell population obtained by a method disclosed herein. In some embodiments, a pharmaceutical composition can include a buffer, a diluent, an excipient, or any combination thereof. In some embodiments, a composition, if desired, can also contain one or more additional therapeutically active substances. In some embodiments, an anti-4-1BB antibody, antigen-binding fragment and/or cell population of the present disclosure are suitable for administration to a mammal (e.g., a human). Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. In some embodiments, compositions are formulated for parenteral administration. For example, a pharmaceutical composition provided herein may be provided in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, a pharmaceutical compositions is provided in a liquid dosage form that is suitable for injection. In some embodiments, a pharmaceutical composition is provided as powders (e.g., lyophilized and/or sterilized), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, buffer, salt solution, etc.) prior to injection. In some embodiments, a pharmaceutical composition is diluted and/or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, a powder should be mixed gently with the aqueous diluent (e.g., not shaken). In some embodiments, an anti-4-1BB antibody, antigen-binding fragment, and/or cell population of the present disclosure is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. A vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). In some embodiments, a formulation is sterilized by known or suitable techniques. Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a diluent or another excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. In some embodiments, a pharmaceutical composition including an anti-4-1BB antibody, antigen-binding fragment, and/or cell population of the present disclosure can be included in a container for storage or administration, for example, an vial, a syringe (e.g., an IV syringe), or a bag (e.g., an IV bag). A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The examples below describe, in part, dosing of an exemplary anti-human 4-1BB antibody to a rodent. Standard methods are known in the art of how to scale dosing in animal systems. See, for example,J Basic Clin Pharm. March 2016-May 2016; 7(2): 27-31, which is incorporated herein by reference in its entirety. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. In some embodiments, a composition comprises or delivers an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure at a dose of 0.01 mg/kg to 100 mg/kg. In some embodiments, a composition comprises or delivers an anti-human 4-1BB antibody or antigen-binding fragment at a dose in an amount within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. In some embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or 100 mg/kg. A pharmaceutical composition may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia. Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator. In some embodiments, a provided pharmaceutical composition comprises one or more pharmaceutically acceptable excipients (e.g., preservative, inert diluent, dispersing agent, surface active agent and/or emulsifier, buffering agent, etc.). In some embodiments, a pharmaceutical composition comprises one or more preservatives. In some embodiments, pharmaceutical compositions comprise no preservative. In some embodiments, a composition including an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure is stably formulated. In some embodiments, a stable formulation of an anti-human 4-1BB antibody or antigen-binding fragment of the present disclosure may comprise a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use. Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like. In some embodiments, a pharmaceutical composition is provided in a form that can be refrigerated and/or frozen. In some embodiments, a pharmaceutical composition is provided in a form that cannot be refrigerated and/or frozen. In some embodiments, reconstituted solutions and/or liquid dosage forms may be stored for a certain period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, a month, two months, or longer). In some embodiments, storage of antibody compositions for longer than the specified time results in antibody degradation. Liquid dosage forms and/or reconstituted solutions may comprise particulate matter and/or discoloration prior to administration. In some embodiments, a solution should not be used if discolored or cloudy and/or if particulate matter remains after filtration. General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005. Kits The present disclosure further provides a pharmaceutical pack or kit comprising one or more containers filled with at least one anti-human 4-1BB antibody or antibody fragment as described herein. Kits may be used in any applicable method, including, for example, therapeutic methods, diagnostic methods, cell proliferation and/or isolation methods, etc. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human administration, (b) directions for use, or both. In some embodiments, a kit may include one or more reagents for detection (e.g, detection of an anti-human 4-1BB antibody or antibody fragment). In some embodiments, a kit may include an anti-human 4-1BB antibody or antibody fragment in a detectable form (e.g., covalently associated with detectable moiety or entity). In some embodiments, an anti-human 4-1BB antibody or antibody fragment as provided herein may be included in a kit used for treatment of subjects. In some embodiments, an anti-human 4-1BB antibody or antibody fragment as provided herein may be included in a kit used for proliferation and/or isolation of T cells (e.g., CD8+T cells). The contents of all cited references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments. However, the following examples are merely provided to illustrate the present invention, but the scope of the present invention is not limited to the following examples. EXAMPLES The present disclosure provides, at least in part, humanized anti-human 4-1BB antibodies and fragments thereof with improved properties that contain one or more structural features that are not found in a reference humanized anti human 4-1BB antibody, 94G1. 94G1 was generated by humanization of the murine anti human 4-1BB antibody BBK-4 antibody. Antigen-recognizing sites (CDR regions) were determined using CDR loop assignment (IMGT: Lefranc, 1997) and a 3-D model (Swiss-Pdb Viewer (www.expasy.org)). A phage display library was prepared with diversity in a total of 10 sites including 4 sites on the amino acid sequence of the light chain and 6 sites of the heavy chain was constructed. After panning, approximately 14 humanized antibody clones out of 1,000 clones were selected (for a total of six humanized scFvs), and among the selected clones, 94G1, was obtained (Son et al.J. Immunol. Methods(2004) 286: 187-201). These humanized antibodies, including 94G1, had affinities for human 4-1BB antigen that were less than 1/10ththat of BBK-4, but were active in vitro. The present disclosure encompassed the recognition that structural variants of 94G1, may have improved properties. Generation and characterization of variant humanized anti-human 4-1BB antibodies and fragments thereof is described in further detail in the following examples. Example 1—Preparation of Humanized Anti-Human 4-1BB Antibodies This example describes the production of an exemplary anti-human 4-1BB antibodies with improved affinity over a reference 94G1 antibody. 94G1 was generated by humanizing a murine anti human 4-1BB antibody (BBK 4) as described in Son et al.J. Immunol. Methods(2004) 286: 187-201, which is herein incorporated by reference in its entirety. Also used herein is a H4-1BB antigen (Accession No: KCTC 0952BP) that is specifically isolated from activated T cells (e.g., activated T cell line), and has not been identified from unstimulated T cells. For example, a H4-1BB antigen can be isolated from T cells that have been matured by phorbol myristate acetate (PMA), ionomycin, Concanavalin A, or anti CD3i, This H4-1BB antigen has a size of 1.4 kb, and 60% homology with mouse 4-1BB (Garni-Wagner et al.,Cellular Immunology(1996) 169: 91-98, which is herein incorporated by reference in its entirety). In this example, 94G1 was divided into a light chain and a heavy chain vectors, each of optimized to generate improved humanized antibodies. The present disclosure encompasses a recognition that a suitable method for generating improved humanized anti-human 4-1BB antibodies or fragments thereof is through single, stepwise amino acid substitutions and/or combinations thereof. The present disclosure provides various structural variants of humanized anti-human 4-1BB antibodies and fragments thereof with one or more structural features (e.g., amino acid substitutions) that are not found in a 94G1 antibody. The present disclosure further encompasses a recognition that structural features can be combined for stepwise improvements in one or more antibody properties (e.g., increased antigen affinity). First, a humanized anti-human 4-1BB antibody with increased affinity relative to 94G1 reference antibody was obtained by changing a CDR region of a light chain, rather than a heavy chain. This light chain structural variant was fixed, and combined with humanized anti-human 4-1BB antibody heavy chains structural variants with, e.g., mutations in the CDR region of 94G1. Further structural features were integrated to generate humanized anti-human 4-1BB antibodies with high affinity and/or other improved characteristics. 1.1 Construction of Vectors Vectors with a 94G1 light chain and 94G1 heavy chain, respectively, were constructed by changing pComb3H-HA to be expressed in a Fab type to improve a heavy chain and a light chain of a humanized antibody inE. coli(J. Immunol Methods(2008) 329(1-2):176-83; Virology(2004) 318: 598). Specifically, a 94G1 light chain was inserted into a vector designed by changing an AP2 tag (SEQ ID NO: 42—NANNPDWDFNP) with a flag tag (SEQ ID NO: 43—DYKDDDDK), the flag tag is designed to be located downstream thereof, and has a human heavy chain sequence (Accession No. AB019438) obtained from known data of the NCBI GenBank was placed as a constant domain in a heavy chain position. In addition, after a 94G1 light chain sequence was cloned into the vector, it was transferred toE. coli(e.g., TG1) (F′ [traD36 proAB+lacIqlacZΔM15]supE thi−1 Δ(lac−proAB) Δ(mcrB−hsdSM)5, (rK−mK−) by transformation, followed by selection of a transformed vector called pCOM-Fab-94G1-L, which was used as a backbone to induce affinity maturation of the light chain (Table 1). The above-described method was similarly carried out for the 94G1 heavy chain, and a selected vector was called pCOM-Fab-94G1-H. An improved light chain, 94/w, was designed as the light chain of pCOM-Fab-94G1, which served as backbone for production of heavy chain variants with improved affinity. TABLE 194G1 and 94/w LCDR amino acid sequencesSEQ ID NO.Amino acid sequenceCDRSEQ ID NO: 1QTISDYLCDR 1SEQ ID NO: 2YASLCDR 2SEQ ID NO: 3QDGHSFPPTLCDR 3SEQ ID NO: 4QDGHSWPPTLCDR 3.6variant 94/w 1.2 Affinity Maturation of Humanized Anti-Human 4-1BB Antibody Light Chain Described herein is the development a humanized anti-human 4-1BB antibody with a light chain variant that has improved binding affinity. An antibody with a high affinity was obtained by changing LCDR3 (SEQ ID NO: 3) of a 94G1 light chain in the context of the pCOM-Fab-94G1-L vector described above as follows. Various DNA sequences encoding a light chain were amplified by PCR using primers [using NNS (N: A, T, C, G; S: C, G)] designed to insert 19 different amino acids into each amino acid position of the 9 amino acids SEQ ID NO: 3, constituting the LCDR3 part of the 94G1 light chain. Amplified products were ligated to a light chain position of the vector and then transformed intoE. coliTG1. All clones with light chain structure variants of LCDR3 were substituted in different forms and collected to prepare nine position mixes. To assess whether each amino acid position was substituted with a different amino acid, two clones changed in respective positions were randomly chosen and analyzed by sequencing using an ABI-3730xl sequencer, which showed that the amino acid residues at respective positions were substituted at various positions. To see whether 94G1 Fab variants with mutations at different LCDR3 positions had increased antibody affinity, each position mix was expressed by adding IPTG (to a final concentration of 1 mM) toE. coliTG1, and then Fab antibody present in a supernatant was subjected to ELISA. Specifically, each position mix was cultured with shaking in a 2YT medium in a 37° C. incubator until the culture had an absorbance at 600 nm of 0.8 or more, then overnight cultured at 30° C. with IPTG (e.g., at a final concentration of 1 mM). ELISA was performed the following day on a supernatant obtained by centrifugation at 12,000 rpm for 10 minutes at 4° C. Binding affinities were determined for the various 94G1 LCDR3 variant Fabs by dividing the binding activities of each clones with respect to 4-1BB Fab by the expression levels for the respective mutant clone. A 94G1 LCDR3 variant with a mutation position 6 of LCDR3 (LCDR3.6) showed the highest binding affinity. Subsequently, to determine how various mutations of 94G1 at the LCDR3.6 position impacted antibody affinity, 25 monoclonal antibodies were isolated from pCOM-Fab94G1-LCDR3.6 position mix and expressed by adding IPTG (e.g., at a final concentration of 1 mM) toE. coli(e.g., TG1), cultured, and ELISA was performed on a Fab antibody present in a supernatant. Binding affinities were determined for the various 94G1 LCDR3.6 clones by dividing the 4-1BB Fab binding activities of each clones by the expression levels for each. A 94G1 LCDR3.6 variant with phenylalanine at the LCDR3.6 position substituted with tryptophan exhibited the highest binding affinity. A Fab antibody prepared by substituting the constant heavy chain of pCOM-Fab94G1-L with the heavy chain of the backbone 94G1 on the improved 94G1 light chain was called 94/w. Thus, a 94/w variant includes an improved 94G1 light chain in which the 6thamino acid of LCDR3 is substituted with tryptophan (W) (QDGHSWPPT—SEQ ID NO: 4) and a 94G1 heavy chain. IPTG-induced expression inE. coliand ELISA of a 94/w Fab was used to determine binding affinity as described above. Using this method, it was determined that a 94/w Fab antibody has a binding activity 3.5 times higher than that of 94G1 (Fab antibody) (data not shown). 1.3 Affinity Maturation of Humanized Anti-Human 4-1BB Antibody Heavy Chain CDRs Described herein is the development humanized anti-human 4-1BB antibodies with heavy chain structural variants that have improved binding affinity. To achieve further improved anti-human 4-1BB antibodies, a 94/w light chain as described above was used and the 94G1 heavy chain was affinity matured. Provided in Table 2 below are the HCDR amino acid sequences for a reference 94G1 antibody heavy chain. TABLE 294G1 and 94K HCDR amino acid sequencesSEQ ID NO.Amino acid sequenceCDRSEQ ID NO: 5GYTFSSYWHCDR 1SEQ ID NO: 6INPGNGHTHCDR 2SEQ ID NO: 7ARSFTTARAFAYHCDR 3SEQ ID NO: 8ARSFKTARAFAYHCDR 3.5variant 94K Improvement of a heavy chain using 94/w as a starting sequence was performed by similar methods as described for the 94G1 light chain above. Particularly, to improve a 94G1 heavy chain, amino acid residues were substituted with various amino acids at respective amino acid positions of HDR2 and/or HCDR3. In the case of the third CDR of the heavy chain (HCDR3, SEQ ID NO: 7), clones were produced with random substitution amino acid residues of 94/w HCDR3 by different amino acids were collected to prepare 12 position mixes. A mutant clone that increases the length of HCDR3 was also prepared. When the 5thamino acid residue of HCDR3 was substituted with a different amino acid, an affinity increase was observed. Subsequently, to determine how various mutations at the HCDR3.5 position impacted affinity of the 94/w antibody, 19 monoclonal antibodies were isolated from a position mix in which the HCDR3.5 position of the 94/w antibody was randomly substituted. HCDR3.5 variant Fabs were expressed inE. coliby adding IPTG (e.g., to a concentration of 1 mM) and ELISA was performed using a Fab antibody present in a supernatant. Sequencing identified that when threonine was substituted with lysine at HCDR3.5 (5thposition) position (SEQ ID NO: 8—ARSFKTARAFAY), the highest affinity was shown, and the resulting product was called 94K/w. In the case of the second CDR of the heavy chain (HCDR2), a position mix was prepared by random substitution of each of 9 amino acids of a 94G1 HCDR2 (SEQ ID NO: 6) for ELISA. The ELISA results showed that when amino acid residues at 2nd, 5thand 6thpositions were changed, the affinity increased. From each of the 94/w HCDR2.2, HCDR2.5 and HCDR2.6 position mixes, 22, 19, and 36 monoclonal antibodies were isolated, respectively, and the binding activity of each clone with respect to 4-1BB was analyzed depending on an Fab expression level. In the case of HCDR2.5, an ELISA value was relatively higher than those when asparagine was substituted with valine (V), glycine (G), or proline (P). In addition, according to sequencing data for antibody heavy chains, there was a risk of deamination at the 5thamino acid, asparagine (N), of HCDR2 (SEQ ID NO: 6), and variant HCDR2 sequences were also prepared with substitutions at this residue with each of glutamine (Q), glutamic acid (E), and serine (S). DNAs of 94G1 structural variants with mutations in HCDR3 and/or HDR2 of the heavy chain prepared as described above, were amplified by PCR using a three-base sequence NNS, ligated to the heavy chain position of a vector having a constant domain of the 94/w light chain, and then transformed intoE. coliTG1 by the method used in improvement of the light chain as described above. 1.4 Optimization of Humanized Anti-Human 4-1BB Antibody Heavy Chain Framework Regions Heavy chain variants were also produced with optimized framework sequences. For example, heavy chain framework 1 (FR1) regions were produced where the heavy chain FR1 (SEQ ID NO: 16) was modified so that the 5thamino acid, glutamine (Q), was substituted with valine (V). Exemplary FR1 regions are provided in Table 3 below. TABLE 394G1 heavy chain FR1 and variations thereofSEQ ID NO.Amino acid sequenceSEQ ID NO: 16QVQLQQSGAEVKKPGASV94G1 FR1KLSCKASSEQ ID NO: 17QVQLVQSGAEVKKPGASVFR1 Gln 5 ValKLSCKAS Also, framework 3 (FR3) regions were produced where the heavy chain FR3 (SEQ ID NO: 18) was modified as such: the 10thamino acid, alanine (A), and/or the 33rdamino acid, serine (S), which were murine sequences, were substituted with valine (V) and threonine (T), respectively. Exemplary FR3 regions are provided in Table 4 below. TABLE 494G1 heavy chain FR3 and variations thereofSEQ ID NO.Amino acid sequenceSEQ ID NO: 18NYNEKFKSRATMTRDTSTST94G1 FR3AYMELSSLRSEDSAVYYCSEQ ID NO: 19NYNEKFKSRVTMTRDTSTSTFR3 Ala 10 ValAYMELSSLRSEDSAVYYCSEQ ID NO: 20NYNEKFKSRVTMTRDTSTSTFR3 Ala 10 Val;AYMELSSLRSEDTAVYYCFR3 Ser 33 Thr 1.5 Preparation of Humanized Anti-Human 4-1BB Variable Regions and Full-Length Antibodies Anti-human 4-1BB antibody variable regions were produced that include various combinations of the above described heavy chain and light chain CDRs and framework regions. For example, a Fab-type 94KVT/w antibody was produced with the 5thamino acid, threonine, at CDR3 of a heavy chain was substituted with lysine (K), and the 10thamino acid of heavy chain FR3, alanine, and the 33rdamino acid of heavy chain FR3, serine, were substituted with valine (V) and threonine (T), respectively to produce heavy chain and light chain variable region sequence that are or include SEQ ID NO: 30 and SEQ ID NO: 34, respectively. In addition, 94KVT heavy chain variants were produced where the 5thamino acid of HCDR2 (SEQ ID NO: 6), asparagine (N), was substituted with glutamine (Q), glutamic acid (E) or serine (S). Exemplary heavy chain and light chain variable domain sequences are provided in Table 5 below (CDR sequences underlined). TABLE 5Exemplary humanized anti-human 4-1BB antibody variable domainsAntibodyLight chain variable domainHeavy chain variable domain94G1DIVMTQSPAFLSVTPGEKVTITQVQLQQSGAEVKKPGASVKLSCRASQTISDYLHWYQQKPDQAPCKASGYTFSSYWMHWVRQAPGKLLIKYASQSISGIPSRFSGSGQGLEWIGEINPGNGHTNYNEKSGTDFTFTISSLEAEDAATYYCFKSRATMTRDTSTSTAYMELSQDGHSFPPTFGQGTKLEIKSLRSEDSAVYYCARSFTTARA(SEQ ID NO: 9)FAYWGQGTLVTVSS(SEQ ID NO: 11)94wDIVMTQSPAFLSVTPGEKVTITQVQLQQSGAEVKKPGASVKLSCRASQTISDYLHWYQQKPDQAPCKASGYTFSSYWMHWVRQAPGKLLIKYASQSISGIPSRFSGSGQGLEWIGEINPGNGHTNYNEKSGTDFTFTISSLEAEDAATYYCFKSRATMTRDTSTSTAYMELSQDGHSWPPTFGQGTKLEIKSLRSEDSAVYYCARSFTTARA(SEQ ID NO: 10)FAYWGQGTLVTVSS(SEQ ID NO: 11)94K/wDIVMTQSPAFLSVTPGEKVTITQVQLQQSGAEVKKPGASVKLSCRASQTISDYLHWYQQKPDQAPCKASGYTFSSYWMHWVRQAPGKLLIKYASQSISGIPSRFSGSGQGLEWIGEINPGNGHTNYNEKSGTDFTFTISSLEAEDAATYYCFKSRATMTRDTSTSTAYMELSQDGHSWPPTFGQGTKLEIKSLRSEDSAVYYCARSFKTARA(SEQ ID NO: 10)FAYWGQGTLVTVSS(SEQ ID NO: 12)94KV/wDIVMTQSPAFLSVTPGEKVTITQVQLVQSGAEVKKPGASVKLSCRASQTISDYLHWYQQKPDQAPCKASGYTFSSYWMHWVRQAPGKLLIKYASQSISGIPSRFSGSGQGLEWIGEINPGNGHTNYNEKSGTDFTFTISSLEAEDAATYYCFKSRVTMTRDTSTSTAYMELSQDGHSWPPTFGQGTKLEIKSLRSEDSAVYYCARSFKTARA(SEQ ID NO: 10)FAYWGQGTLVTVSS(SEQ ID NO: 13)94KVT/w,DIVMTQSPAFLSVTPGEKVTITQVQLVQSGAEVKKPGASVKLSEU101CRASQTISDYLHWYQQKPDQAPCKASGYTFSSYWMHWVRQAPGKLLIKYASQSISGIPSRFSGSGQGLEWIGEINPGNGHTNYNEKSGTDFTFTISSLEAEDAATYYCFKSRVTMTRDTSTSTAYMELSQDGHSWPPTFGQGTKLEIKSLRSEDTAVYYCARSFKTARA(SEQ ID NO: 10)FAYWGQGTLVTVSS(SEQ ID NO: 14) For conversion to a full-length anti-human 4-1BB antibodies (whole Ig type), an Fc domain was connected to the respective Fab. For example, a 94K/w Fab composed of a heavy chain in which threonine is substituted with lysine at HCDR3.5 and a light chain with 94/w variant in which the 6thamino acid of LCDR3 is substituted with tryptophan (W), and respective regions extended from CH2 and CH3 domains and a sequence of human IgG1 were amplified by PCR to overlap and subjected to splice PCR to produce full IgG DNA, and then the resulting DNA was cloned in a mammalian expression vector. Full length antibodies for other humanized anti-human 4-1BB antibodies described herein were produced in a similar manner. Exemplary immunoglobulin constant region sequences are provided in Table 6 below. TABLE 6Exemplary immunoglobulin constant domainsSEQ ID NO.Amino acid sequenceDescriptionSEQ ID NO:RTVAAPSVFIFPPSDEQLKSGTASVk constant21VCLLNNFYPREAKVQWKVDNALQSGdomainNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO:ASTKGPSVFPLAPSSKSTSGGTAALIgG122GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO:ASTKGPSVFPLAPSSKSTSGGTAALIgG123GCLVKDYFPEPVTVSWNSGALTSGVvariantHTFPAVLQSSGLYSLSSVVTVPSSS(L234;LGTQTYICNVNHKPSNTKVDKKVEPL235;KSCDKTHTCPPCPAPEAAGGPSVFLK322)FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK As used herein a full length 94KVT/w antibody includes an IgG1 sequence, such as that of SEQ ID NO: 22. Additionally, a full length antibody, referred to herein as EU101, was produced that includes 94KVT/w variable domains describe above (SEQ ID NOs: 10 and 14, for light chain and heavy chain variable domains, respectively), with a variant IgG1 constant domain that includes 3 mutations: L234, L235, and K322 (SEQ ID NO: 23). Thus, example provides a number of exemplary humanized anti-human 4-1BB antibodies and antibody fragments that have been engineered to potentially enhance antigen binding affinity. These exemplary antibodies and fragments are characterized in the following examples. Example 2—Characterization of Binding of Humanized Anti-Human 4-1BB Antibodies 2.1 Determining Binding Epitope of Anti-Human 4-1BB Antibodies The present disclosure encompasses a recognition that humanized anti-human 4-1BB antibodies provided herein may be useful for 4-1BB co-stimulation. Therapeutic applications of antibodies of the present disclosure may include promoting anti-cancer immunity and/or anti-viral immunity. However, for clinical applications, it is important to identify which part of human 4-1BB is recognized by and/or reacts with an anti-humanized 4-1BB antibody (i.e., a binding epitope). 4-1BB antibodies that recognize different epitopes of 4-1BB molecule have identified, and these antibodies can have been shown to have different clinical effects. (See, e.g., Kwon et al. Eur. J. Immunogenetics (2002) 29: 449-452, herein incorporated by reference in its entirety). Epitope mapping encompasses methods for identifying a molecular determinant of antibody-antigen recognition. This example describes epitope mapping of an exemplary anti-human 4-1BB antibody as engineered in Example 1 above. Specifically, this example assesses the binding epitope of a humanized anti-human 4-1BB antibody with 94KVT/w variable domains, EU101. A human 4-1BB antigen for investigating an epitope of the humanized 4-1BB antibody is derived from a cDNA library manufactured from human peripheral blood lymphocytes that was generated by at least some of the inventors of the present application (See, e.g., Kwon et al. Cellular Immunology (1996) 169: 91-98; Immunol. Lett. (1995) 45: 67-73; and Korean Patent No. 10-0500286, each of which is incorporated herein by reference). cDNA encoding an extracellular domain (ECD) of the obtained human homologue of 4-1BB cDNA (hereinafter, referred to as H4-1BB) was selected, fused with GST, and then inserted into a vector (pGEX-6T) to express. A cell line producing a GST-4-1BB fusion polypeptide as used herein, was deposited as part of the disclosure for Korean Patent No. 10-0500286, Accession No: KCTC 0952BP. A full length sequence of human 4-1BB is provided as SEQ ID NO: 44, below. The extracellular domain of human 4-1BB corresponds to amino acids 1 to 167 of the full length H4-1BB sequence. Full length human 4-1BB sequenceSEQ ID NO: 44MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL To determine an epitope of 4-1BB recognized by humanized anti-human 4-1BB antibodies of the present disclosure, constructs were generated with fragments of a 4-1BB extracellular domain of various sizes (e.g., R1, R2, R3), fused to GST, and replicated. A schematic of GST-4-1BB polypeptides as used in the present example is provided inFIG.1A. and exemplary primer sequences used herein for generating different 4-1BB extracellular domain constructs are provided in Table 7 below. Individual recombinant GST-H-4-1BB constructs were cultured with 1 mM IPTG and produced inE. coliBL21DX5α cells, and the fusion polypeptides were purified using a glutathione-agarose column. TABLE 7Exemplary primers used to generate human4-1BB extracellular domain fragmentsuseful for epitope mappingForwardReverseR15'-5'-GGATCCACAAGATCATTGCATTGAGCTCGAGCCTGGTCCTGAAG-3' (SEQ ID NO. 24)AACA-3' (SEQ ID NO. 25)R25'-5'-CGCGTGGATCCAAGGAGTGTTTGAGCTCGAGACGTTTCTGATCTCCTCCA-3'GTTA-3' (SEQ ID NO. 27)(SEQ ID NO. 26)R35'-5'-CGCGTGGATCCGGCATCTGTTTGAGCTCGAGGATCTGCGGAGACGACCCT-3'GTGT-3' (SEQ ID NO. 29)(SEQ ID NO. 28)R1.15'-5'-GGATCCACAAGATCATTGCACTCGAGGCATATGTCACAGG-3' (SEQ ID NO. 30)GT-3' (SEQ ID NO. 31)R1.25'-5'-GGATCCACAAGATCATTGCACTCGAGGCTGGAGAAACTG-3' (SEQ ID NO. 32)AT-3' (SEQ ID NO. 33)R1.35'-5'-GGATCCTGCCCAGCTGGTATTGAGCTCGAGCCTGGTCCTGAAC-3' (SEQ ID NO. 34)AACA-3' (SEQ ID NO. 35)R1.45'-5'-GGATCCAGGAATCAGATTTGTTGAGCTCGAGCCTGGTCCTGAAC-3' (SEQ ID NO. 36)AACA-3' (SEQ ID NO. 37)R1.55'-5'-GGATCCACAAGATCATTGCACTCGAGGCAAATCTGATTCG-3' (SEQ ID NO. 38)CT-3' (SEQ ID NO. 39)R1.65'-5'.GGATCCACAAGATCATTGCACTCGAGTGGAGGACAGGGAG-3' (SEQ ID NO. 40)CT-3' (SEQ ID NO. 41) Purified protein samples were obtained from transformed bacterial cells by a lysis buffer (e.g., 10 mM Tris-HCl-pH 7.4, 50 mM NaCl, 5 mM EDTA, 30 mM NaF, 0.1 mM Na3VO4, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM PMSF, and protease inhibitor mixture). Approximately 20 μg of each fusion polypeptide sample was diluted in a 4×SDS sample buffer, subjected to electrophoresis on SDS-PAGE gels, and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). On the cellulose membranes, anti-human 4-1BB mAb was reacted with anti-mouse IgG horseradish peroxide (HRP). Binding antibodies were recognized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Little Chalfont, UK). As described above and shown inFIG.1B, when each of three non-overlapping H4-1BB ECD fragment-GST fusion polypeptides, R1, R2, and R3, were treated with GST-binding, respectively. It was determined that an exemplary humanized anti-4-1BB antibody encompassed by the present disclosure (EU101) binds to an N-terminal fragment construct (R1) fusion construct of approximately 32 kDa (amino acids 1 to 55 of 4-1BB) by western blotting. Moreover, this binding was specific, as no binding was observed with either of the R2 or R3 fusion constructs. SeeFIG.1B. Furthermore, to determine the minimal binding site of the humanized anti-4-1BB antibody, an R1 extracellular domain fragment was further divided into 6 smaller fragments: R1.1 (amino acids 1 to 45 of 4-1BB), R1.2 (amino acids 1 to 35 of 4-1BB), R1.3 (amino acids 11 to 55 of 4-1BB), R1.4 (amino acids 21 to 55 of 4-1BB), R1.5 (amino acids 1 to 25 of 4-1BB), and R1.6 (amino acids 1 to 30 of 4-1BB) polypeptide fragments, as depicted inFIG.1A, and fused to GST (Glutathione S-Transferase, 27 kDa). Exemplary primer pairs used for the generation of these constructs are provided in Table 7 above. Fusion polypeptide constructs were produced inE. coliBL21 cells with IPTG induction (e.g., 1 mM IPTG) and bacterial whole cell extract was resolved by 12% SDS-PAGE. As shown inFIG.2A, SDS-PAGE confirmed that individual 4-1BB fusion polypeptides are well expressed. SDS-PAGE was transferred to a nitrocellulose membrane and immunoblotting was performed using an exemplary anti-human 4-1BB antibody, EU101. As shown inFIG.2B, it was confirmed that a sequence of amino acids 10 to 30 of the extracellular domain of H4-1BB is significant for binding an exemplary humanized anti-4-1BB antibody. This analysis indicates that an exemplary anti-human 4-1BB antibody of the present disclosure (EU101) binds to an epitope of human 4-1BB whose sequence is or includes CPAGTFCDNNRNQICSPCPP (SEQ ID NO: 15). It was also confirmed that a sequence including amino acids 35 to 50 of the 4-1BB extracellular domain is not significant for binding an exemplary humanized antibody described herein (FIG.2B). 2.2 Assessing Binding Affinity of Exemplary Humanized Anti-Human 4-1BB Antibodies to 4-1BB Antigen Binding Ability of Exemplary Anti-Human 4-1BB Antibodies To examine the binding ability of exemplary humanized anti-human 4-1BB antibodies described in Example 1 to a human 4-1BB antigen (H4-1BB), ELISA was performed.E. coli-expressed recombinant human 4-1BB was used for antigen. A murine BBK-4 antibody, a reference 94G1 humanized antibody, and exemplary engineered antibodies 94K, 94 KV, 94KVT and EU101 as described in Example 1 were each treated on 96 well plates coated with histidine-tagged 4-1BB extracellular domain recombinant protein (H4-1BB). Exemplary ELISA affinity analysis employed a total volume of 100 μl at a concentration of 1.0 μg/ml, and the reaction was allowed to proceed at room temperature for 1 hour. Horseradish peroxidase (HRP)-labeled anti-human IgG and anti-mIgG-HRP, as appropriate, recognizing an antibody was treated thereto, and allow to react at room temperature for 40 minutes. After washing, treatment with an ABTS solution (Sigma-Aldrich), which is a substrate for a coloring reaction, and the reaction to allow to proceed at room temperature for 30 minutes, and an absorbance at 450 nm in the coloring reaction was detected using an ELISA reader to analyze a binding activity of the antibodies. Results are shown inFIG.3. As shown inFIG.3, as antibody concentration increases, binding between each antibody and 4-1BB antigen (H4-1BB) is improved. This data confirms that antibodies encompassed by the present disclosure specifically bind to 4-1BB. Binding of Exemplary Anti-Human 4-1BB Antibodies to Cell-Expressed Antigen The ability of exemplary humanized anti-human 4-1BB antibodies to bind a human 4-1BB antigen (H4-1BB) in a cellular context was assessed. Jurkat 8-1 cells were genetically engineered for overexpressing 4-1BB. Exemplary engineered antibodies 94K, 94 KV, 94KVT and EU101 as described in Example 1, along with that of a murine BBK-4 antibody, and a reference 94G1 humanized antibody were each assessed for binding to Jurkat 8-1 cells using an anti-mIgG-HRP or anti-hIgG-HRP secondary antibody, as appropriate, and analyzed by FACS. As shown inFIG.4, each of the antibodies were able to effectively bind 4-1BB expressed by Jurkat 8-1 cells and the affinity of 94KVT and EU101 was higher than BBK-4 and 94G1. In Vitro Binding Affinity of Exemplary Anti-Human 4-1BB Antibodies to Antigen In vitro binding affinity of exemplary engineered antibody EU101 as described in Example 1, along with that of a reference 94G1 humanized antibody were each determined by Biacore analysis. Anti-human IgG was immobilized on a CMS chip, and coupled to the Fab antibodies prepared above by flowing over the chip, and ultimately reacted with a human 4-1BB antigen (H4-1BB) to measure the binding activity between the antibody and the antigen (Biacore3000, sensor chip CMS). Affinity measurement results are shown inFIG.5. Ka (1/Ms) and Kd (1/s) values represent how fast an antibody associates with and dissociates from an antigen, respectively. A dissociation constant (KD) is obtain by dividing Kd by Ka (Kd/Ka=KD). As a dissociation constant decreases, it can be interpreted that dissociation occurs at a lower concentration and that affinity is increasing. As shown inFIG.5, the exemplary engineered anti-human 4-1BB antibody had improved binding affinity relative to a reference 94G1. Exemplary Anti-Human 4-1BB Antibodies Recognize 4-1BB Expressed by Activated CD8+T Cells CD8+T cells were isolated from human PBMCs and activated with 1 μg/ml anti-CD3 antibody for 2 day. The ability of exemplary humanized anti-human 4-1BB antibodies (94K, 94 KV, 94KVT and EU101) described in Example 1 to detect a 4-1BB on the surface of activated CD8+T cells was assessed relative to an exemplary commercially available anti-4-1BB antibody (4-1BB-PE). Also shown is detection with a BBK-4 a murine anti-human 4-1BB antibody and a 94G1 reference humanized antibody. Treatment with 4-1BB antibodies was at a concentration of 25 ng/ml. Exemplary antibodies were detected with an anti-mIgG-Dylight488 or anti-hIgG-Dylight488 as appropriate, and analyzed by FACS. Results are shown inFIG.6. While a reference 94G1 antibody detected 4-1BB on 17.93% of CD8+T cells, each of a 94KVT and EU101 antibody showed robust detect of 25.3% and 28.33%, respectively. Demonstrating that exemplary antibodies 94KVT and EU101both had improved binding properties over BBK-4 and 94G1. Thus, humanized variant antibodies of the present disclosure have superior binding to activated T cells in vitro. Example 3—Analysis of In Vitro Efficacy of Humanized Anti-Human 4-1BB Antibodies Anti-4-1BB antibodies have previously been demonstrated to provide signal stimulation to a co-stimulation molecule expressed in activated CD8+T cells, 4-1BB, to activate the CD8+T cells, induce proliferation and increase TH1-type cytokine expression. In this example, activity of exemplary humanized anti-human 4-1BB antibodies described in Example 1 to induce proliferation of CD8+T cells and TH1-type cytokine expression was examined. 3.1 Exemplary Anti-Human 4-1BB Antibodies Induce Cell Proliferation of CD8+T Cells To assess proliferation of CD8+T cells, cells were stained with WST-1 (water-soluble tetrazolium salt) is a cell proliferation reagent. WST-1-labeled CD8+T cells were prepared and activated with 0.5 μg/ml of anti-CD3 antibody. The activated CD8+T cells were treated with 1.0 μg/ml of iso-type control antibody, murine BBK-4 antibody, reference 94G1 antibody, and exemplary humanized anti-human 4-1BB antibodies (94K, 94 KV, 94KVT and EU101) described in Example 1. Cells were analyzed using a MACS system and results are shown inFIG.7. Referring toFIG.7, it was confirmed that exemplary humanized anti-human 4-1BB antibodies of the present disclosure induce cell proliferation of CD8+T cells. Moreover, a degree of CD8+T cell activation increases in an order of 94G1<94K/94 KV<94KVT/EU101. 3.2 Exemplary Anti-Human 4-1BB Antibodies Stimulate Cytokine Secretion IFN-γ is a representative cytokine primarily secreted from a T lymphocyte or a natural killer cell (NK cell) and exhibiting proliferation and anti-viral activities. In addition, IFN-γ is a major activator for a macrophage, and particularly, a major cytokine distinguishing TH1 cells from other types of cells. IFN-γ secretion plays a major role in the activation of cytotoxic T cells, phagocytes and B cells. Consequently, efficiency of an anticancer agent can be evaluated with an increased amount of TH1 inducing IFN-γ. For this reason, measurement of secretion of IFN-γ by specific stimulation may be an optimal standard which can be used as a quantitative criterion for a functional change of T cells. CD8+T cells were isolated from human PBMCs and treated with 0.5 μg/ml of an anti-CD3 mAb antibody and then treated with either no antibody, or with 1.0 μg/ml of an anti-4-1BB antibody: BBK-4, 94G1, 94K, 94 KV, 94KVT and EU101. IFNγ secretion was evaluated on days 1, 3, and 5. Results are shown inFIG.8. As can be seen inFIG.8, IFNγ secretion increased in all anti-4-1BB antibody treated samples, and this increase correlated with duration of antibody treatment. Treatment with 94KVT and EU101 antibody reached a secretion level that was 13-fold higher than the control group as day 5. Accordingly, exemplary humanized antibodies 94KVT and EU101 can both induce IFNγ secretion more efficiently than 94G1 reference antibody. 3.3 Increase in IFN-γ Level According to Treatment of Activated CD4+T Cells or CD8+T Cells with an Exemplary Anti-Human 4-1BB Antibody Blood was collected from three healthy donors, PBMCs obtained there from were isolated by Ficoll-plaque gradient centrifugation, and active T cells present in the PBMCs were rested in a RPMI-1640+2% FBS medium for 24 hours. The rested PBMCs were treated with an iron beads-attached anti-CD4 antibody or anti-CD8 antibody, and CD4+cells or CD8+cells were isolated using an MACS magnetic separator. The isolated CD4+T cells or CD8+T cells were treated with a T cell activator, anti-CD3, to induce 4-1BB expression, and treated with EU101 at different concentrations (0.5, 1.0, 2.5, and 5.0 μg/ml) for 3 days. After 3 days, a culture medium excluding the cells was obtained, and fluorescence of human IFN-γ in the culture medium was assessed by ELISA (ebioscience), and the result was compared with the standard curve provided in an IFN-γ ELISA kit (FIG.9). As shown inFIG.9, expression levels of IFN-γ in the CD4+T cells and CD8+T cells dose-dependently increased. Particularly, when 5.0 μg/ml of EU101 was treated, compared with a 278% increase in the CD4+T cells, the expression level of IFN-γ increased 612% in the CD8+T cells. According to the T-cell specific expression pattern of IFN-γ involved in the conversion of the T cells into TH1, an exemplary anti-human 4-1BB antibody of the present disclosure, EU101, has sufficient in vitro activity to suggest it may be effective for prevention and/or treatment of cancer. 3.4 Measurement of ADCC and CDC Activities of an Exemplary Anti-Human 4-1BB Antibody An immune system recognizes and attacks virus-infected cells or cancer cells, and antibodies may be used to induce cytotoxicity mediated apoptosis. For such an immune system, two types of mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) may be used. In both cases, apoptosis may be mediated by an antibody binding to a target on a cell surface. That is, when an antibody has an ADCC activity, a cell recognized by the antibody results in apoptosis mediated by a natural killer (NK) cell, and when an antibody has a CDC activity, killing is mediated by a complement protein. Therefore, in the case of the development of an antagonistic antibody therapeutic, a degree of killing cells recognized by an antibody can be identified through analyses of the ADCC and CDC activities. However, a target for the humanized 4-1BB antibody disclosed in the present disclosure is T cells, not cancer cells. That is, in consideration of a mechanism for inducing activation of T cells by binding a 4-1BB antibody as an agonistic antibody, an antibody that does not have the ADCC and CDC activities may be preferably for therapeutic uses. In the present disclosure, for an ADCC assay, human PBMCs were isolated by Ficoll centrifugation using the same density difference. The PBMCs were incubated into RPMI (Thermo Fisher Scientific) and 10% FBS with IL-2 (100 U/ml) for overnight cultured. Target cells (4-1BB expressing cell lines) were harvested, resuspended in a culture medium at 1 ml, and labeled with 5 uM CFSE at 37° C. for 5 min. Effector/Target cells of the present disclosure were washed in a ratio of 10:1, counted and then dispensed. For analysis, an antibody of the present disclosure was prepared for a final concentration of 10 nM (1.5 μg/ml), and cultured on a plate at 37° C. for 4 hours. 5 μl of 7-AAD was added to each well and transferred to a FACS tube, and then the sample was analyzed by FACS manufactured by BDFACScan. Frequencies of non-viable target cells (CFSE+7-AAD+) viable target cells (CFSE+7-AAD−) were measured. ADCC was assessed with a frequency of viable cells of the total cells (FIG.10A). A complement-dependent cytotoxicity (CDC) assay was conducted similarly to the ADCC assay described above using FACS as a read-out value, with the above Target cells incubated with anti-4-1BB antibodies at ice for 30 min and then added the human supplemented serum at a final concentration of 20% at 37° C. for 30 minutes. Afterward, resulting samples were each transferred to a FACS tube, and assessed by FACS manufactured by BDFACScan (FIG.10B). The results inFIG.10AandFIG.10Bconfirm that an exemplary humanized 4-1BB antibody, EU101, has almost no ADCC and CDC effects. Therefore, it can be said that an exemplary EU101 antibody of the present disclosure has beneficial ADCC and CDC properties for an agonist antibody, and is a good candidate for anti-cancer treatment in vivo. Example 4—Confirmation of In Vivo Efficiency of an Exemplary Humanized Anti-Human 4-1BB Antibody The anti-human 4-1BB antibody, EU101, of the present disclosure showed a dose-dependent effect in an in vitro example, and showed a considerably superior effect to a conventional antibody. This example is to check if the anti-human 4-1BB antibody, EU101, is able to be used alone or in combination with a different composition to diagnose, prevent or treat cancer or tumor in vivo, and to effectively inhibit the growth of tumor. 4.1 NOD-Scid IL2RgammanullMouse Engraftment of Human Peripheral Blood Mononuclear Cells and Anti-Tumor Activity of Anti-Human 4-1BB Antibody Peripheral venous blood collected from a HLA-A24-type healthy donor was treated with heparin, and subjected to concentration-gradient centrifugation on Ficoll-paque (GE Healthcare, Piscataway, NJ) to harvest PBMCs. The PBMCs were washed with an RPMI-1640 medium, and 3×106of the cells were intraperitoneally injected into immunodeficient mice, that is, NSG mice (NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ; NOD-scid IL2rγnull, Jackson Laboratory). Analysis of humanized mice was performed by flow cytometry to check whether human T cells were present in the mouse blood collected by mouse orbital blood collection after 5 weeks of the engraftment of human PBMCs. 7-week-old NSG mice (Jackson Laboratory, Barharbor, ME) were raised under a specific pathogen-free (SPF) environment. Flow cytometry was performed to check ratios of CD4 and CD8 after the cells were stained with human blood cell markers such as an APC-cy7 fluorescence-labeled CD45 antibody and a FITC fluorescence-labeled CD4 antibody, and a BV510 fluorescence-labeled CD8 antibody. After orbital blood collection from each mouse, human T cells from mouse blood samples were observed to check if a human immune system is engrafted into the mouse. Human tumor cells were prepared in an HLA type humanized mouse model and 1×107cells were subcutaneously injected into the back of each mouse. When a tumor size reached 100 to 200 mm3, a preparation of exemplary anti-human 4-1BB antibody (EU101) was intravenously administered at 1.0 mg, 5.0 mg or 10.0 mg per 1 kg of body weight once every 5 days total 3 times. As a control, human IgG was used. Tumor volume (mm3) of each mouse was measured in every 3 days (FIG.11). Results shownFIG.11confirm that tumor size in mice treated with an exemplary anti-human 4-1BB antibody (EU101) was reduced relative to mice treated with human IgG, and moreover that this reduction was proportional to antibody concentration. Particularly, tumor regression in a 5 mg/kg antibody-administered group occurred rapidly. Within a week after administration at a 5 mg/kg dose, tumor size settled in a humanized mouse and tumor growth was eradicated. Therefore, an exemplary antibody EU101 of the present disclosure shows an anticancer effect in vivo. Consequently, the above results show that an exemplary anti-human 4-1BB antibody (EU101) that specifically recognizes an epitope (SEQ ID NO: 15) of H4-1BB, but due to improved characteristics of this exemplary antibody, such as, for example, improved affinity, this antibody shows superior effects in an in vivo mouse model. Thus, the example suggests that an antibody encompassed by the present disclosure can be used as an anticancer agent at a lower dosage than reference antibody. 4.2 Effects of Inhibiting Tumor Growth with an Exemplary Anti-Human 4-1BB Antibody and an Anti-PD-1 Agent Comparison of Effects Caused by Individual Treatment of an Exemplary Anti-Human 4-1BB Antibody (EU101) and an Exemplary Anti-PD-1 Agent after Tumor Injection to Humanized Mice Humanized mice were prepared by the same method described in Example 4.1 above. To perform an experiment confirming an increase in anti-cancer effect according to doses of an exemplary anti-human 4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda)(purchased from MSD, GER), 1×107cells of a HLA-A-type matched human colorectal adenocarcinoma cell line, HT29, were subcutaneously injected into the previously-prepared humanized mice. When the volume of the injected tumor reached 100 to 150 mm3, the mice were divided into a total 5 groups of three mice, and to compare the effect of EU101 on tumor inhibition, each group of mice were treated with each of three administration conditions (Control: IgG, Treated group 1: 5 mg/kg, and Treated group 2: 10 mg/kg) at 5 day intervals 5-3 times, and for anti-PD-1, the same procedures were carried out (FIG.12). As a result of the experiment, in both cases of EU101 and keytruda (anti-PD1), tumor volumes were dose-dependently reduced. However, inFIG.12, 5 mg/kg of EU101 did not have an influence on the tumor growth, but according to the treatment with 5 mg/kg and 10 mg/kg of EU101, an anti-tumor activity was dose-dependently exhibited. In addition, it was confirmed that EU101 exhibited higher efficiency at a lower dose than keytruda (anti-PD-1), and the tumor growth was completely blocked particularly by the treatment with 5 mg/kg of EU101. Treatment of Humanized Mice with Combination of EU101 and an Anti-PD-1 Agent after Tumor Injection Since co-inhibitory receptors (PD-1 and CTLA-4) signals and a co-stimulation (CD137) T cell signal are differentiated for the same purpose of inhibiting tumor growth, stimulation of the two receptors can expect a synergyic effect (Chen et al.,Cancer Immunol. Res. (2015) 3:149-160; Bartkowiak et al.,Front. Oncol. (2015) 5: 117, both of which are incorporated by reference herein). In addition, PD1 immunotherapy showed a possibility of an anticancer treatment effect for some of cancer patient populations, but the administration of a low dose in combination therapy with a different anticancer agent may still be required in more extensive patient population. To investigate the anti-tumor effect caused by a combination therapy of an exemplary anti-human 4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda), tumor-bearing humanized mice were treated with the combination therapy of EU101 and Keytruda. Preparation of humanized mice was performed by the same method as described in Example 4.1 Eye bleeding was performed to identify humanized mice. Among the humanized mice, HT29, colon carcinoma were subcutaneously injected into HLA-A24 mice maintaining a normal condition at 1×107cells/mice. When a tumor size was 300 to 450 mm3, an experiment was performed as follows. As known from this example, although tumor growth was not delayed with individual injection at the minimum concentration or less (EU101: 2.5 mg/kg, Keytruda (manufactured from MSD, GER): 2.5 mg/kg), tumor was greatly regressed with combination therapy of EU101 and Keytruda. This is the result showing that exemplary anti-human 4-1BB antibodies provided herein (e.g., EU101) are good candidates for combination therapy with different anticancer agents, including in combination with one or more immune checkpoint inhibitors (FIG.13). Analyses of T Cell Infiltrating Lymphocytes (TILs) in Normal Tissue and Human Colorectal Adenocarcinoma Tissue after Individual and Combination Treatment of an Exemplary Anti-Human 4-1BB Antibody and an Exemplary Anti-PD-1 Agent After individual administration of an exemplary anti-human 4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda) (purchased from MSD, GER) and combination administration of EU101 and Keytruda to HT29-implanted humanized mice, on the day when the effect analysis is terminated, all groups were dissected to separate tumor and blood. After the separated tumor was treated with collagenase IV at 37° C. for 30 minutes, cells in the tumor tissue were dissociated by a mechanical method and then washed with 1×PBS. PBMCs were separated from the separated blood by Ficoll gradient centrifugation, and separated tumor cells and PBMCs were subjected to the following experiment. Red blood cells (RBCs) were removed from washed cells using RBC lysis buffer and then washed with 1×PBS. Tangled cell debris was removed from the washed cells using a 40-μm nylon cell strainer to create a single cell state, and the single cells were washed with 1×PBS, followed by counting T cells separated from each group using a cell counter. The separate T cells were stained with human blood cell markers such as a CD45 antibody (fluorescent APC-cy7 labeled), a fluorescent FITC-labeled human CD4 antibody and a fluorescent BV510-labeled human CD8 antibody, and then subjected to FACS assay. The FACS assay was carried out based on a ratio (%) of CD4 and CD8 cell groups, which were gated from the CD45 group (FIG.14A). Particularly, to identify a Treg group among the separated T cells, the surfaces of cells were stained with human blood cell markers such as a CD45 antibody (fluorescent APC-cy7 labeled), a human fluorescent FITC-labeled CD4 antibody and a human fluorescent PE-cy5-labeled CD25 antibody, and intracellular and intranuclear staining with a cell transcription factor Foxp3 (human fluorescent APC-labeled Foxp3 antibody) were performed using a Foxp3/Transcription Factor Staining Buffer Set kit (ebioscience). In the FACS assay, a CD45 group was separated to gating R1, a CD4+CD25highgroup was separated to gating R2, and a ratio (%) of a Foxp3highgroup was measured in the R1 and R2 groups. To identify IFN-γ+CD8+T cells in the separated cells, the cell surfaces were stained with the blood cell markers such as the fluorescent APC-cy7-labeled human CD45 antibody and the fluorescent BV510-labeled human CD8 antibody, fixed with 2% PFA, and reacted with a 0.5% saponin solution and a fluorescent PE-cy7-labeled human IFN-γ antibody. Afterward, cytokine IFN-γ+cells in the CD8 T cell group were measured by FACS assay. The cells were identified in a ratio by the same method as described above and a proportional ratio of the CD8+IFN-γ+ratio and the Treg ratio was calculated, shown inFIG.14B. According to the result of this embodiment, other than the individual administration, the combination administration of EU101 and Keytruda greatly increased infiltration of the combination of tumor tissue and a T lymphocyte. Further more specific results of the combination treatment are as follows. When the combination treatment was performed on PBMCs in the healthy humanized mouse as a control, the number of lymphocytes increased approximately 3 times, and the infiltrated lymphocytes per 1 g of tumor increased 76 times in tumor tissue. This means that most of tumor-specific lymphocytes were activated and recruited to tumor tissue to kill target cells. Particularly, when PBMCs in the combination therapy group were measured, as shown inFIG.14A, the CD4+T cells do not highly increased, but cytotoxic CD8+T cells were increased approximately 5 times. Moreover, the combination therapy group showed a 100-fold increase in CD8+Tcell count per 1 g of tumor tissue. In addition, as a result, a ratio of CD8+T cells secreting IFN-γ and regulatory T cells was also greatly increased (FIG.14B). That is, it can be said that the combination treatment of EU101 and anti-PD-1 agent gives a sharp increase of effector T cells and thus tumor inhibition is effectively performed. Analyses of IFN-γ in Serum or Tumor Fluid Obtained from Human Colorectal Adenocarcinoma Tissue after Individual and Combination Treatment with an Exemplary Anti-Human 4-1BB Antibody (EU101) and an Exemplary Anti-PD-1 Agent (Keytruda) After individual administration and combination administration of an exemplary anti-human 4-1BB antibody (EU101) and an exemplary anti-PD-1 agent (Keytruda) to HT29-implanted humanized mice. On the day when effect analyses were terminated, all groups were dissected to separate tumor and blood. In tumor dissection to separate a tumor fluid present in the separated tumor, 300 μl of 1×PBS was injected into the upper portion of a tumor membrane using a 1cc-syringe, and a flowing solution is taken from the lower portion of the tumor membrane using an insulin syringe. In addition, in dissociation of the tumor tissue, the taken solution was added to dissociate the tumor tissue, and then stored. In addition, as a serum, the serum stored when PBMCs were separated from blood by Ficoll gradient centrifugation. The stored serum and a tumor fluid were dissolved and filtered using a 0.22 μm filter unit (manufacturer: corning). 10 μl of serum was used for each group, and 100 μl of the tumor fluid was used to measure human IFN-γ and human TGF-β using a human IFN-γ ELISA Ready-SET-Go kit (eBioscience) and a Human TGF beta 1 ELISA Ready-SET-Go kit (eBioscience). Results were analyzed by comparing the standard curve provided in each ELISA kit. As a result, compared to the individual administration of EU101 and Keytruda, in the combination administration, the concentration of interferon in serum of the tumor group was the highest. Since a EU101 mechanism can be explained with a correlation between IFN-γ and an anti-tumor effect, expression levels of IFN-γ and TGF-β in serum of a healthy donor and serum of a tumor group, to which the combination therapy had been applied, were evaluated. According to the material of the example on the serum of the healthy donor, in the combination therapy group shown inFIG.15A, IFN-γ was increased approximately 16 times, but a cytokine secreted from Treg cells, TGF-β, was decreased approximately 65%. In addition, inFIG.15B, the IFN-γ concentration caused by the combination administration in the tumor fluid was considerably higher (approximately 213-fold) than that in the control. As a result of the examples, due to EU101, particularly, compared to the control group, the combination group showed sharp increases in IFN-γ secretion. Therefore, it can be confirmed that the anti-cancer effect caused by an improved anti-humanized 4-1BB antibody of the present disclosure gives effective tumor-infiltration of effector T cells directly related to apoptosis of cancer cells, and a considerably specific effect in the tumor tissue, compared to the non-treated group. In other words, in the present disclosure, it was confirmed that EU101, as an anti-cancer agent, has the optimal conditions for apoptosis of cancer cells. Conventionally, in cancer patients, anti-cancer cytokine and anti-cancer cellular immunity were considerably reduced, but it can be expected in the present disclosure that EU101 induces the increases in anti-cancer cytokine and anti-cancer cellular immunity, resulting in a considerable therapeutic effect. Thus, an exemplary anti-human 4-1BB antibody EU101 exhibits an anti-tumor effect mediated by the high expression of IFN-γ, and such an effect is dose-dependently exhibited, as such, an IFN-γ concentration in a serum of a cancer patient can be used as a biomarker to diagnose and estimate tumor. Therefore, according to effective treatment of cancer or tumor through the combination treatment of EU101 and anti-PD-1 and prognosis through the measurement of an IFN-γ concentration, it is expected to perform more effective treatment with respect to each patient. Example 5—Separation and Massive Proliferation of 4-1BB+CD8+T Cells Ex Vivo Using an Exemplary Humanized Anti-Human 4-1BB Antibody The inventors used 4-1BB expression in antigen-specifically activated CD8+T cells in isolation and purification of 4-1BB+CD8+T cells specific to various antigens using an anti-4-1BB antibody (Korean Patent No. 10-1503341). A subsequent experiment was performed to examine if the EU101 antibody developed herein is also used for isolation and mass-proliferation of antigen-specific CD8+T cells. Construction of PBMCs from peripheral blood of a cancer patient was performed as described in Example 4.1. However, in this example, cancer antigen-specific undifferentiated T cells may be obtained by the method described in Korean Patent Application No. 10-2016-0165224, filed by the inventors. In this example, for effective separation of 4-1BB+CD8+T cells and mass-production of the 4-1BB+CD8+T cells with high purity, a panning method using an anti-human 4-1BB antibody (EU101) was used. 10 μg/ml of the anti-human 4-1BB antibody (EU101) antibody diluted in PBS was added to a 10 ml flask, and then stored at 4° C. for 20 to 24 hours. After storage, a supernatant containing the antibody was removed, and without washing, a solution of BSA dissolved at 2.5% in PBS was added to cell pellets in the 10 ml flask and then stored at 4° C. for 20 to 24 hours. Afterward, the BSA solution was removed, and each flask was washed twice with 15 ml of PBS. The previously-prepared cells were suspended in an X-VIVO 10 medium, added to a EU101 antibody-coated flask, and then incubated at 37° C. in a CO2incubator for 1 hour. After incubation, a supernatant was removed, and cell pellets were washed twice with 10 ml of RPMI1640 medium to remove non-specifically binding cells. 1% of self serum and a 1000 IU/ml IL-2-containing X-VIVO 10 medium were added to the flask, followed by culturing for 14 days. In the example, some cells were harvested and then stained to measure the purity and phenotypes of the isolated cells. As shown inFIGS.16A and16B, it was confirmed that, before panning with the 94 kvt antibody, a ratio of antigen-specific 4-1BB+CD8+T cells increased 43.2% (CD8+T cell ratio: 58.6%), and after panning with the EU101 antibody, a ratio of antigen-specific pCMV+CD8+T cells increased 60.0% (CD8+T cell ratio: 79.3%). This means that the antigen-specific 4-1BB+CD8+T cells can be isolated with high purity using a EU101. Antigen-specific 4-1BB+CD8+T cells isolated as described above may be easily mass-produced by the method described in Korean Patent Application No. 10-2016-0165224 filed by the inventors. From the above description, it will be understood by those of ordinary skill in the art that the present invention can be realized in different specific forms without changing the technical idea or essential characteristics of the present invention. However, there is no intention to limit the present invention to the specific exemplary embodiments, and it should be understood that all modifications or modified forms deduced from the meaning and range of the following claims and equivalents thereof are included in the scope of the present disclosure, rather than the detailed description. Anti-human 4-1BB antibodies encompassed by the present disclosure demonstrated a number of beneficial properties, such as, for example, superior affinity to a reference antibody, and/or can be used alone or in combination with another anticancer agent to diagnose, prevent or treat cancer or tumor, or used to inhibit the growth of cancer. Above, the present invention has been described with reference to examples, but it can be understood by those of ordinary skill in the art that the present invention may be changed and modified in various forms without departing from the spirit and scope of the present invention, which is described in the accompanying claims. EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims. | 181,466 |
11859005 | DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the analogs, pharmaceutical compositions and methods, the preferred methods and materials are described herein. Moreover, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.” Definitions As used herein, “about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence similarity, time frame, temperature, volume, etc. Such a value or range can be within an order of magnitude typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art. As used herein, and in reference to one or more of receptors, “activity,” “activate,” “activating” and the like means a capacity of a compound, such as a fusion herein, to bind to and induce a response at the receptor(s), as measured using assays known in the art, such as the in vitro assays described below. As used herein, “amino acid” means a molecule that, from a chemical standpoint, is characterized by a presence of one or more amine groups and one or more carboxylic acid groups and may contain other functional groups. As is known in the art, there is a set of twenty amino acids that are designated as standard amino acids and that can be used as building blocks for most of the peptides/proteins produced by any living being. The amino acid sequences in the disclosure contain the standard single letter or three letter codes for the twenty naturally occurring amino acids. As used herein, “analog” means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and that elicits at least one in vivo or in vitro effect elicited by a native agonist for that receptor. The term “antibody,” as used herein, refers to an immunoglobulin molecule that binds an antigen. Embodiments of an antibody include a monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, or conjugated antibody. The antibodies can be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). An exemplary antibody is an immunoglobulin G (IgG) type antibody comprised of four polypeptide chains: two heavy chains (HC) and two light chains (LC) that are cross-linked via inter-chain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 or more amino acids primarily responsible for antigen recognition. The carboxyl-terminal portion of each of the four polypeptide chains contains a constant region primarily responsible for effector function. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The IgG isotype may be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). The VH and VL regions can be further subdivided into regions of hyper-variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). The CDRs are exposed on the surface of the protein and are important regions of the antibody for antigen binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as “HCDR1, HCDR2, and HCDR3” and the three CDRs of the light chain are referred to as “LCDR1, LCDR2 and LCDR3”. The CDRs contain most of the residues that form specific interactions with the antigen. Assignment of amino acid residues to the CDRs may be done according to the well-known schemes, including those described in Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable regions of immunoglobulins”, Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available on the internet; see Lefranc et al., Nucleic Acids Res. 1999; 27:209-212). The North CDR definitions are used for the anti-human GITR antibodies described herein. The term “antigen-binding fragment” refers to a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen. Examples of antigen binding fragments include, but are not limited to, Fab or Fab′. A “Fab” fragment consists of an entire antibody light chain comprising the light chain variable region (VL) and the light chain constant region (CL), along with the heavy chain variable region (VH) and the heavy chain first constant domain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. A Fab′ fragment differs from the Fab fragment by having a few additional residues at the carboxyl terminus of the CH1 domain including one or more residues from the antibody hinge region. A Fab or Fab′ described herein can be a human Fab or Fab′ or a chimeric Fab or Fab′ that comprises a human CL and CH1. The terms “bind” and “binds” as used herein are intended to mean, unless indicated otherwise, the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, which results in proximity of the two proteins or molecules as determined by common methods known in the art. As used herein, “biotherapeutic” and the like means an amino acid- or nucleic acid-based compounds such as antibodies, coagulation factors, clotting factors, cytokines, enzymes, growth factors, hormones, and fragments thereof, having at least one therapeutic activity/applicability, as well as therapeutic DNA and/or RNA molecules. As used herein, “conservative substitution” means a variant of a reference peptide or polypeptide that is identical to the reference molecule, except for having one or more conservative amino acid substitutions in its amino acid sequence. In general, a conservatively modified variant includes an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference amino acid sequence. More specifically, a conservative substitution refers to substitution of an amino acid with an amino acid having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) and having minimal impact on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitutions of functionally similar amino acids are well known in the art and thus need not be exhaustively described herein. As used herein, “effective amount” means an amount or dose of one or more of the compounds herein, or a pharmaceutically acceptable salt thereof that, upon single or multiple dose administration to an individual in need thereof, provides a desired effect in such an individual under diagnosis or treatment (i.e., may produce a clinically measurable difference in a condition of the individual, for example, reduction in one or more clinical disease activity measures such as American College of Rheumatology (ACR) 20, ACR50, ACR70; Disease Activity Score (DAS); Psoriasis Area and Severity Index (PASI) 50, PASI75, PASI90, PASI100; Systemic Lupus erythematosus disease activity index (SLEDAI); Mayo Score Disease activity index (DAI); Crohn's disease activity index (CDAI); Geboes score (GS); Robarts Histopathology index (RHI); Atopic dermatitis Severity Index (ADSI); and EULAR Sjogren's syndrome disease activity index (ESSDAI)). An effective amount can be readily determined by one of skill in the art by using known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for an individual, a number of factors are considered, including, but not limited to, the species of mammal, its size, age and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances. As used herein, “extended duration of action” means that binding affinity and activity for a fusion including at least one compound herein and a biotherapeutic herein continues for a period of time greater than a native biotherapeutic, allowing for dosing at least as infrequently as once daily or even thrice-weekly, twice-weekly or once-weekly. The time action profile may be measured using known pharmacokinetic test methods such as those utilized in the Examples below. The term “Fc region” as used herein refers to a region of an antibody, which comprises the CH2 and CH3 domains of the antibody heavy chain. Optionally, the Fc region may include a portion of the hinge region or the entire hinge region of the antibody heavy chain. As used herein, “glucocorticoid-induced TNFR-related protein” or “GITR”, also known as tumor necrosis factor receptor superfamily member 18 (TNFRSF18), means a GITR protein obtained or derived from any species, such as a mammalian species, especially a human. GITR includes both native GITR (i.e., full-length) and variations thereof (i.e., additions, deletions, insertions and/or substitutions of native GITR). One sequence for human GITR full-length (but without the signal peptide) is set forth in SEQ ID NO:20 (see also, UniProt/SwissProt Database Accession No. Q9Y5U5). One sequence for human GITR ECD (but without the signal peptide) is set forth in SEQ ID NO:21. As used herein, “half-life” or “t½” means the time it takes for one-half of a quantity of a compound, such as a fusion protein described herein, to be removed from a fluid or other physiological space such as serum or plasma of an individual by biological processes. Alternatively, t½ also can mean a time it takes for a quantity of such a fusion protein to lose one-half of its pharmacological, physiological or radiological activity. As used herein, “half-maximal effective concentration” or “EC50” means a concentration of compound that results in 50% activation/stimulation of an assay endpoint, such as a dose-response curve. As used herein, “in combination with” means administering at least one of the fusion proteins herein either simultaneously, sequentially or in a single combined formulation with one or more additional therapeutic agents. As used herein, “individual in need thereof” means a mammal, such as a human, with a condition, disease, disorder or symptom requiring treatment or therapy, including for example, those listed herein. In particular, the preferred individual to be treated is a human. As used herein, “long-acting” means that binding affinity and activity of a composition herein continues for a period of time greater than native peptide or protein, allowing for dosing at least as infrequently as once daily or even thrice-weekly, twice-weekly, once-weekly or monthly. The time action profile of the compounds herein may be measured using known pharmacokinetic test methods such as those described in the Examples below. The terms “nucleic acid” or “polynucleotide”, as used interchangeably herein, refer to polymers of nucleotides, including single-stranded and/or double-stranded nucleotide-containing molecules, such as DNA, cDNA and RNA molecules, incorporating native, modified, and/or analogs of, nucleotides. Polynucleotides of the present disclosure may also include substrates incorporated therein, for example, by DNA or RNA polymerase or a synthetic reaction. As used herein, “non-standard amino acid” means an amino acid that may occur naturally in cells but does not participate in peptide synthesis. Non-standard amino acids can be constituents of a peptide and oftentimes are generated by modification of standard amino acids in the peptide (i.e., via post-translational modification). Non-standard amino acids can include D-amino acids, which have an opposite absolute chirality of the standard amino acids above. As used herein, “oligomer” means a molecule having a few similar or identical repeating units that could be derived from copies of a smaller molecule, its monomer. These monomers can be joined by bonds that are either strong or weak, covalent or non-covalent (e.g., intramolecular). As used herein, “patient,” “subject” and “individual,” are used interchangeably herein, and mean a mammal, especially a human. In certain instances, the individual is further characterized with a condition, disease, disorder or symptom that would benefit from administering a compound or composition herein. As used herein, “pharmaceutically acceptable buffer” means any of the standard pharmaceutical buffers known to one of skill in the art. As used herein, “sequence similarity” means a quantitative property of two or more nucleic acid sequences or amino acid sequences of biological compounds such as, for example, a correspondence over an entire length or a comparison window of the two or more sequences. Sequence similarity can be measured by (1) percent identity or (2) percent similarity. Percent identity measures a percentage of residues identical between two biological compounds divided by the length of the shortest sequence, whereas percent similarity measures identities and, in addition, includes sequence gaps and residue similarity in the evaluation. Methods of and algorithms for determining sequence similarity are well known in the art and thus need not be exhaustively described herein. A specified percentage of identical nucleotide or amino acid positions is at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. As used herein, “treating” or “to treat” means managing and caring for an individual having a condition, disease, disorder or symptom for which administration of a compound herein is indicated for the purpose of attenuating, restraining, reversing, slowing or stopping progression or severity of the condition, disease, disorder or symptom. Treating includes administering a compound herein or composition containing a compound herein to the individual to prevent the onset of symptoms or complications, alleviating the symptoms or complications, or eliminating the condition, disease, disorder or symptom. Treating includes administering a compound herein or composition containing a compound herein to the individual to result in such as, for example, decreased (or prevented) autoimmunity and/or reduced (or prevented) allergic disease, asthma, atopic dermatitis, joint inflammation, arthritis, rheumatoid arthritis, or other inflammatory disorders such as, IBD, CD and/or UC. The individual to be treated is a mammal, especially a human. The term “therapeutically effective amount,” as used herein, refers to an amount of a protein or nucleic acid or vector or composition that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In a non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of a protein or nucleic acid or vector or composition that, when administered to a subject, is effective to at least partially alleviate, inhibit, prevent and/or ameliorate a condition, or a disorder or a disease. As used herein, “variable heavy homodimer,” “VHH” or “VHH moiety” means a form of single-domain antibody, especially an antibody fragment of a single, monomeric variable region of a heavy chain only antibody (HcAb), which has a very small size of about 15 kDa. It has been found herein that engineered/modified VHH-based compounds can be used as a pharmacokinetic enhancer to extend the duration of action of and/or to improve the t½ of biotherapeutics. The VHH-based compounds bind serum albumin; however, the VHH-based compounds can be used to bind IgG (including Fc domain), neonatal Fc receptor (FcRn) or other long-lasting serum proteins. The VHH-based compound therefore can be used to improve the t½ of a compound such as a peptide or protein or even other molecules such as, for example, small molecules. Certain abbreviations are defined as follows: “ACR” refers to urine albumin/urine creatinine ratio; “AUC” refers to area under the curve; “AUC0-inf” refers to area under the curve from time 0 hours to infinity; “cAMP” refers to cyclic adenosine monophosphate; “C0” refers to estimated plasma concentration at time zero; “CL” refers to clearance following IV administration; “CL/F” refers to apparent clearance following SQ administration; “Cmax” refers to maximum observed plasma concentration “CMV” refers to cytomegalovirus; “DNA” refers to deoxyribonucleic acid; “ECD” refers to extracellular domain; “EDC” refers to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; “ETA” refers to ethanolamine; “GS” refers to glutamine synthetase; “HC” refers to heavy chain; “HIC” refers to hydrophobic interaction chromatography; “hr” refers to hour or hours; “IV” refers to intravenous; “kDa” refers to kilodaltons; “LC” refers to light chain; “LC-MS” refers to liquid chromatography-mass spectrometry; “min” refers to minute or minutes; “MS” refers to mass spectrometry; “MSX” refers to methionine sulfoximine; “NHS” refers to N-hydroxysuccinimide; “OtBu” refers to O-tert-butyl; “PEI” refers to polyethylenimine; “RP-HPLC” refers to reversed-phase high performance liquid chromatography; “sec” refers to second or seconds; “NaOAc” refers to sodium acetate; “rcf” means relative centrifugal force; “RT” means room temperature; “RU” means resonance units; “SQ” refers to subcutaneous; “SEC” refers to size-exclusion chromatography; “SEM” refers to standard error of the mean; “SPR” means surface plasmon resonance; “t½” refers to half-life; “TFA” refers to trifluoroacetic acid; “Tmax” refers to time of maximum observed concentration; and “Trt” refers to trityl. Anti-Human GITR Antigen Binding Fragment Compounds The present disclosure provides an anti-human GITR antigen binding fragment compound comprising: 1) a heavy chain variable region (HCVR) comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 1, an HCDR2 having the amino acid sequence of SEQ ID NO: 2 or 7, an HCDR3 having the amino acid sequence of SEQ ID NO: 3; and 2) a light chain variable region (LCVR) comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 4, an LCDR2 having the amino acid sequence of SEQ ID NO: 5, and an LCDR3 having the amino acid sequence of SEQ ID NO: 6. In some instances, the anti-human GITR antigen binding fragment compound comprises a Fab comprising a human kappa CL and a human IgG1 CH1. Compounds Having VHH-Based Half-Life Extenders Briefly, certain compounds herein are of the formula (from N-terminus to C-terminus): X-L-M, wherein M is a compound acting as a t½-extending moiety, L (if present) is a linker, and X is an antibody Fab fragment that binds human GITR. In some instances, L can have an amino acid sequence comprising (GGGGQ)n, (GGGQ)n, (GGGGS)n, (PGPQ)n, (PGPA)n, GGGG(AP)nGGGG, (GGE)n, (GGGGE)n, (GGK)n, (GGGGK)n, GGGG(EP)nGGGG, GGGG(KP)nGGGG, (PGPE)nor (PGPK)n, where n can be from 1 to 15, especially from about 5 to about 10. In some instances, M is an albumin-binding VHH, and L is a peptide comprising an amino acid sequence of (GGGGQ)nwhere n can be from 1 to 15, especially from about 4 to about 8. In other instances, M comprises an amino acid sequence as shown in SEQ ID NO: 13, and L can have an amino acid sequence selected from SEQ ID NOS:11 or 12. In still other instances, L can have one or more additions, deletions, insertions or substitutions such that L has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOS:11 or 12. In still other instances, L can be a polymer such as a polyethylene glycol (PEG), especially (PEG)n, where n can be from 1 to 20. In some instances, the compounds disclosed herein are of the formula (from N-terminus to C-terminus): X-L-M, wherein M comprises an amino acid sequence as shown in SEQ ID NO:13 or having an amino acid sequence having at least about 90% to about 99% sequence similarity thereto, where L (if present) is a linker, and wherein X is a Fab that binds human GITR. In some instances, L can have an amino acid sequence comprising (GGGGQ)nwhere n can be from 1 to 15, especially from about 4 to about 8. In other instances, L can have an amino acid sequence selected from SEQ ID NOS:11 or 12. In still other instances, L can have one or more additions, deletions, insertions or substitutions such that L has an amino acid sequence having at least about 90% to about 99% sequence similarity to any one of SEQ ID NOS:11 or 12. In still other instances, L can be a polymer such as a polyethylene glycol (PEG), especially (PEG)n, where n can be from 1 to 20. Preferably, the compound is a human GITR antagonist. Pharmaceutical Compositions and Kits In some instances, the anti-human GITR antigen binding fragment compounds disclosed herein such as the anti-human GITR Fabs, the anti-human GITR Fab fusions or conjugates to albumin-binding VHHs thereof (such as Antibody I and Antibody II disclosed herein) can be formulated as pharmaceutical compositions, which can be administered by parenteral routes (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal). Such pharmaceutical compositions and techniques for preparing the same are well known in the art. See, e.g., Remington, “The Science and Practice of Pharmacy” (D. B. Troy ed., 21stEd., Lippincott, Williams & Wilkins, 2006). In particular instances, the compositions are administered SQ or IV. Alternatively, however, the compositions can be formulated in forms for other pharmaceutically acceptable routes such as, for example, tablets or other solids for oral administration; time release capsules, and any other form currently used, including creams, lotions, inhalants and the like. As noted above, and to improve their in vivo compatibility and effectiveness, the VHH-based fusions or VHH-based conjugates herein may be reacted with any number of inorganic and organic acids/bases to form pharmaceutically acceptable acid/base addition salts. Pharmaceutically acceptable salts and common techniques for preparing them are well known in the art (See, e.g., Stahl et al., “Handbook of Pharmaceutical Salts: Properties, Selection and Use” (2ndRevised Ed. Wiley-VCH, 2011)). Pharmaceutically acceptable salts for use herein include sodium, trifluoroacetate, hydrochloride and acetate salts. The compounds herein may be administered by a physician or self-administered using an injection. It is understood the gauge size and amount of injection volume can be readily determined by one of skill in the art. However, the amount of injection volume can be ≤about 2 mL or even ≤about 1 mL, and the needle gauge can be ≥about 27 G or even ≥about 29 G. The disclosure also provides and therefore encompasses novel intermediates and methods useful for synthesizing the compounds herein, or a pharmaceutically acceptable salt thereof. The intermediates and compounds can be prepared by a variety of techniques that are well known in the art. For example, a method using recombinant synthesis is illustrated in the Examples below. The specific steps for each of the techniques described may be combined in different ways to prepare the compounds. The reagents and starting materials are readily available to one of skill in the art. The compounds herein are generally effective over a wide dosage range. Exemplary doses of the compounds or of pharmaceutical compositions including the same can be milligram (mg) or microgram (μg) amounts per kilogram (kg) of an individual. In this manner, a daily dose can be from about 1 μg to about 1000 mg. Here, the effective amount of the compound in a pharmaceutical composition can be a dose of about 2.5 mg to about 1000 mg. One of skill in the art, however, understands that in some instances the effective amount (i.e., dose/dosage) may be below the lower limit of the aforesaid range and be more than adequate, while in other cases the effective amount may be a larger dose and may be employed with acceptable side effects. In addition to a compound herein, the pharmaceutical composition also can include at least one additional therapeutic agent such as, for example, a therapeutic agent typically used as the standard of care in of a particular condition, disease and disorder (e.g., a cardiovascular, neurological, immunological, metabolic, oncological, psychological, pulmonological and/or renal condition, disease or disorder). In this manner, a pharmaceutical composition can include an effective amount of one or more compounds herein, a pharmaceutically acceptable carrier and optionally at least one additional therapeutic agent. Alternatively, the compounds herein can be provided as part of a kit. In some instances, the kit includes a device for administering at least one compound (and optionally at least one additional therapeutic agent) to an individual. In certain instances, the kit includes a syringe and needle for administering the at least one compound (and optionally at least one additional therapeutic agent). In particular instances, the compound (and optionally at least one additional therapeutic agent) is pre-formulated in aqueous solution within the syringe. Methods of Making and Using VHH-Based Compounds Acting as Half-Life Extenders or Fusions and Conjugates Thereof The compounds herein can be made via any number of standard recombinant DNA methods or standard chemical peptide synthesis methods known in the art. With regard to recombinant DNA methods, one can use standard recombinant techniques to construct a polynucleotide having a nucleic acid sequence that encodes an amino acid sequence for a compound (i.e., fusion peptide or fusion protein or fusion conjugate), incorporate that polynucleotide into recombinant expression vectors, and introduce the vectors into host cells, such as bacteria, yeast and mammalian cells, to produce the compound. (See, e.g., Green & Sambrook, “Molecular Cloning: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, 4thed. 2012)). With regard to recombinant DNA methods, the compounds herein can be prepared by producing a protein or precursor protein molecule using recombinant DNA techniques. DNA, including cDNA and synthetic DNA, may be double-stranded or single-stranded, and the coding sequences therein encoding a compound herein may vary as a result of the redundancy or degeneracy of the genetic code. Briefly, the DNA sequences encoding the compounds herein are introduced into a host cell to produce the compound or precursor thereof. The host cells can be bacterial cells such as K12 or B strains ofEscherichia coli, fungal cells such as yeast cells, or mammalian cells such as Chinese hamster ovary (CHO) cells. An appropriate host cell is transiently or stably transfected or transformed with an expression system, such as expression vectors, for producing a compound herein or a precursor thereof. Expression vectors typically are replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers such as, for example, tetracycline, neomycin, G418 and dihydrofolate reductase, to permit selection of those cells transformed with the desired DNA sequences. The specific biosynthetic or synthetic steps for each of the steps described herein may be used, not used or combined in different ways to prepare the compounds herein. With regard to chemical peptide synthesis methods, one can use standard manual or automated solid-phase synthesis procedures. For example, automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial sources. Solid-phase synthesizers can be used according to the manufacturer's instructions for blocking interfering groups, protecting amino acids during reaction, coupling, deprotecting and capping of unreacted amino acids. The methods can include the steps described herein, and these maybe be, but not necessarily, carried out in the sequence as described. Other sequences, however, also are conceivable. Moreover, individual or multiple steps may be carried out either in parallel and/or overlapping in time and/or individually or in multiply repeated steps. Furthermore, the methods may include additional, unspecified steps. Such methods therefore can include selecting an individual having, for example, a autoimmune condition, disease or disorder, or who is predisposed to the same. The methods also can include administering to the individual an effective amount of at least one compound herein, which may be in the form of a pharmaceutical composition as also described herein. In some instances, the compound/pharmaceutical composition can include an additional therapeutic agent. The concentration/dose/dosage of the compound and optional additional therapeutic agent are discussed elsewhere herein. With regard to a route of administration, the compound or pharmaceutical composition including the same can be administered in accord with known methods such as, for example, orally; by injection (i.e., intra-arterially, intravenously, intraperitoneally, intracerebrally, intracerebroventricularly, intramuscularly, intraocularly, intraportally or intralesionally); by sustained release systems, or by implantation devices. In certain instances, the compound or pharmaceutical composition including the same can be administered SQ by bolus injection or continuously. With regard to a dosing frequency, the compound or pharmaceutical composition including the same can be administered daily, every other day, three times a week, two times a week, one time a week (i.e., weekly), biweekly (i.e., every other week), or monthly. In certain instances, the compound or pharmaceutical composition including the same is administered SQ every other day, SQ three times a week, SQ two times a week, SQ one time a week, SQ every other week or SQ monthly. In particular instances, the compound or pharmaceutical composition including the same is administered SQ one time a week (QW). Alternatively, and if administered IV, the compound or pharmaceutical composition including the same is administered IV every other day, IV three times a week, IV two times a week, IV one time a week, IV every other week or IV monthly. In particular instances, the compound or pharmaceutical composition including the same is administered IV one time a week (IW). With regard to those instances in which the compound or pharmaceutical composition including the same is administered in combination with an effective amount of at least one additional therapeutic agent, the additional therapeutic agent can be administered simultaneously, separately or sequentially with the compound or pharmaceutical composition including the same. Moreover, the additional therapeutic agent can be administered with a frequency same as the compound or pharmaceutical composition including the same (i.e., every other day, twice a week, or even weekly). Alternatively, the additional therapeutic agent can be administered with a frequency distinct from the compound or pharmaceutical composition including the same. In other instances, the additional therapeutic agent can be administered SQ. In other instances, the additional therapeutic agent can be administered IV. In still other instances, the additional therapeutic agent can be administered orally. It is further contemplated that the methods may be combined with additional therapeutic agents other than those discussed above. EXAMPLES The following non-limiting examples are offered for purposes of illustration, not limitation. Polypeptide Expression Example 1: Recombinant Expression of Antibody I Antibody I is an anti-human GITR Fab—albumin-binding VHH fusion having a heavy chain amino acid sequence of: (SEQ ID NO: 14)QVQLVESGGGVVQPGRSLRLSCAASGYTFSSYVMHWVRQAPGKGLEWVAVTSYDGTHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWAPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP; and a light chain amino acid sequence of: (SEQ ID NO: 16)DIQMTQSPSSLSASVGDRVTITCRASQDISNSLAWYQQKPGKAPKRLIYAAIFSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCYQYYNYPSAFGQGTKLEIKRTVAAPSVFIFPPSDFQLKSGTASVVCLLNNFYPREAKVQWIKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. The antibody with the heavy chain of SEQ ID NO:14 and the light chain of SEQ ID NO:16 is generated in a mammalian cell expression system using CHOK1 cell derivatives (Lonza Biologics Inc.). The cDNA sequences encoding SEQ ID NO:14 and SEQ ID NO:16 are sub-cloned into GS-containing expression plasmid backbones (pEE12.4-based plasmids; Lonza Biologics Inc.). The cDNA sequence is fused in frame with the coding sequence of a signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO:22), to enhance secretion of the antibody into the tissue culture medium. The expression is driven by the viral CMV promoter. For generating the antibody via transient transfection, CHOK1 cells are transfected with an equal stoichiometric ratio of the recombinant expression plasmids using a PEI-based method. Briefly, the appropriate volume of CHOK1 suspension cells at a density of 4×106cells/mL is transferred in shake flasks, and both PEI and recombinant plasmid DNA are added to the cells. Cells are incubated in a suspension culture at 32° C. for 6 days. At the end of the incubation period, cells are removed by low-speed centrifugation and the antibody is purified from the conditioned medium. Alternatively, and for generating the antibody via stable transfections, CHOK1 cells are stably transfected using electroporation and the appropriate amount of recombinant expression plasmid, and the transfected cells are maintained in suspension culture at an adequate cell density. Selection of the transfected cells is accomplished by growth in 25 μM MSX-containing serum-free medium and incubated at about 35° C.-37° C. and about 5%-7% CO2. Subsequently, cells are removed by low-speed centrifugation and the antibody is purified from the conditioned medium. The antibody is secreted into the media from the CHO cells, which is purified by Protein A affinity chromatography followed by cation exchange chromatography. Specifically, the antibody from harvested media is captured onto MabSelect PrismA Protein A resin (Cytiva). The resin then is briefly washed with a wash buffer, such as a phosphate-buffered saline (PBS; pH 7.4) or a buffer containing Tris, to remove non-specifically bound material. The protein is eluted from the resin with a low pH solution, such as 10 mM citric acid pH 3. Fractions containing the antibody are pooled and may be held at a low pH to inactivate potential viruses. The pH may be neutralized by adding a base such as 0.1 M Tris pH 8.0. The antibody may be further purified by ion exchange chromatography using resins such as POROS 50 HS (ThermoFisher). The antibody may be eluted from the column using a 0 to 1M NaCl gradient in 20 mM sodium acetate, pH 5.0 over 20 column volumes. Purified antibody may be buffer exchanged into phosphate buffered saline or, alternatively, passed through a viral retention filter such as Planova 20N (Asahi Kasei Medical) followed by concentration/diafiltration into phosphate buffered saline using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore). The antibody therefore is prepared in this manner or in a similar manner that would be readily determined by one of skill in the art. TABLE 1SEQ ID NOs for Antibody IAntibody ISEQ ID NO:Amino Acid Sequence for:HCDR11HCDR22HCDR33LCDR14LCDR25LCDR36HCVR8LCVR10Linker11Albumin-binding VHH13HC anti-GITR Fab-VHH Fusion14LC16DNA Sequence for:HC anti-GITR Fab-VHH Fusion17LC19 Example 2: Recombinant Expression of Antibody II Antibody II is an anti-human GITR Fab—albumin-binding VHH fusion having a heavy chain amino acid sequence of: (SEQ ID NO: 15)QVQLVESGGGVVQPGRSLRLSCAASGYTFSSYVMHWVRQAPGKGLEWVAVTSYDGTHELYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWAPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAATGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYIQMNSLRPEDTAVYNCAARPGRPLTSKVADLYPYWGQGTLVTVSSPP and a light chain amino acid sequence of: (SEQ ID NO: 16)DIQMTQSPSSLSASVGDRVTITCRASQDISNSLAWYQQKPGKAPKRLIYAAFSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCYQYYNYPSAFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. The antibody with the heavy chain of SEQ ID NO:15 and the light chain of SEQ ID NO:16 is generated essentially as described for Example 1 except that cDNA sequences encoding SEQ ID NOs:15 and 16 are used in the expression plasmids. TABLE 2SEQ ID NOs for Antibody IIAntibody IISEQ ID NO:Amino Acid Sequence for:HCDR11HCDR27HCDR33LCDR14LCDR25LCDR36HCVR9LCVR10Linker11Albumin-binding VHH13HC anti-GITR Fab-VHH Fusion15LC16DNA Sequence for:HC anti-GITR Fab-VHH Fusion18LC19 Example 3: Antibody Binding to Albumin Orthologs by SPR In vitro binding of the antibodies (i.e., anti-human GITR Fab—albumin-binding VHH fusions) to human, cynomolgus monkey, mouse, rat, pig, dog, cow and rabbit serum albumin is determined by SPR at 25° C. In particular, the affinity of Antibodies I and II to serum albumin of these species is summarized below in Tables 3 and 4, respectively. Binding of Antibody I and Antibody II to various serum albumins is carried out on Biacore 8K instrument. The immobilization of the serum albumin to a Series S Sensor Chip CM5 (Cytiva 29149603) surface is performed according to the manufacturer's instructions (Amine Coupling Kit BR-1000-50). Briefly, carboxyl groups on the sensor chip surfaces (flow cell 1 and 2) are activated by injecting 70 μL of a mixture containing 75 mg/ml 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and 11.5 mg/ml N-Hydroxysuccinimide (NHS) at 10 μl/min. Human, cynomolgus monkey, rat, mouse, dog, pig, cow, and rabbit serum albumin are diluted in 10 mM sodium acetate pH 4.0 (BR-1003-49) at 0.8, 0.8, 0.8, 2.5, 0.8, 1, 1, and 1.5 μg/ml then injected over the activated chip surfaces (flow cell 2, channel 1 to 8) at 10 μl/min for 100 seconds. Human serum albumin was obtained from Sigma Aldrich (St. Louis, MO) (Cat. No. A8763). Cynomolgus monkey serum albumin was obtained from Athens R&T (Athens, GA) (Cat. No. 16-16-011202-CM). Rat serum albumin was obtained from Sigma Aldrich (St. Louis, MO) (Cat. No. A4538). Mouse serum albumin was obtained from Sigma Aldrich (St. Louis, MO) (Cat. No. A3139). Dog serum albumin was obtained from Molecular Innovations (Novi, MI) (Cat. No. DSA-1213 NC0739153). Pig serum albumin was obtained from Sigma Aldrich (St. Louis, MO) (Cat. No. A4414). Cow serum albumin was obtained from Sigma Aldrich (St. Louis, MO) (Cat. No. A7030). Rabbit serum albumin was obtained from Fitzgerald Industries International (Acton, MA) (Cat. No. 30R-3303). The serum albumins are covalently immobilized through free amines onto a carboxymethyl dextran-coated sensor chip CM5 at surface densities of 29-52 resonance units (RU) for human, cynomolgus monkey, rat, mouse, dog, pig, and cow as well as 118 resonance units (RU) for rabbit serum albumin. Excess reactive groups on the surfaces (flow cell 1 and 2) are deactivated by injecting 70 μl of 1 M Ethanolamine hydrochloride-NaOH pH 8.5. The antibodies of Examples 1 and 2 (i.e., Antibody I and II, respectively) are diluted in HBS-EP+buffer (10 mM HEPES pH 7.6, 150 mM NaCl, 3 mM EDTA, 0.05% Polysorbate 20) at concentrations of 1000, 333.3, 111.1, 37.04, 12.35, 4.12, 1.37, 0.457, 0.152, 0.051 and 0.017 nM. 180 μl of sample are individually injected sequentially across the immobilized serum albumins on the chip's surface and dissociated for 600 sec at 60 μl/min flow rate at 25° C. The surface is regenerated by injecting 10 mM glycine-HCl pH 1.5 (BR-1003-54) at 60 μl/min for 100 sec. The resulting sensorgrams are analyzed using Biacore 8K Insight Evaluation Software (version 3.0.11.15423) 1:1 binding kinetics model fitting to calculate the binding kinetic parameters: association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD). KDis determined as 0.25, 4.4, 53, 44, 100, 20, and 460 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding, respectively, with Antibody I (Table 3). KD is determined as 0.4, 5.9, 42, 51, 99, 20 and 430 nM for human, cynomolgus monkey, mouse, rat, pig, dog and cow serum albumin binding, respectively, with Antibody II (Table 4). TABLE 3Binding Kinetics of Antibody I to Human, Cynomolgus Monkey,Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C.Binding to ImmobilizedSerum Albumins (SA)ka (1/Ms)kd (1/s)KD(M)Human SA2.6E+056.5E−052.5E−10Cyno SA1.9E+058.2E−044.4E−09Mouse SA1.8E+059.4E−035.3E−08Rat SA1.6E+057.0E−034.4E−08Pig SA1.2E+051.3E−021.0E−07Dog SA1.7E+053.5E−032.0E−08Cow SA2.0E+059.1E−024.6E−07Rabbit SANo binding TABLE 4Binding Kinetics of Antibody II to Human, Cynomolgus Monkey,Mouse, Rat, Pig, Dog, Cow and Rabbit Serum Albumin at 25° C.Binding to ImmobilizedSerum Albumins (SA)ka (1/Ms)kd (1/s)KD(M)Human SA1.9E+057.8E−054.0E−10Cyno SA1.5E+058.7E−045.9E−09Mouse SA2.3E+059.6E−034.2E−08Rat SA1.4E+057.3E−035.1E−08Pig SA1.2E+051.2E−029.9E−08Dog SA1.7E+053.4E−032.0E−08Cow SA2.0E+058.7E−024.3E−07Rabbit SANo binding Example 4: Antibody Binding to GITR Expressing Cells Jurkat human GITR/NFkB-Luc2 cells (CS184004) were purchased from Promega and maintained in RPMI1640 media+10% FBS (HyClone SH30070.3)+400 μg/mL hygromycin B+600 μg/mL G418. The NFkB reporter pNiFty2-Luc (InvivoGen) is introduced into Jurkat cells (ATCC) by electroporation and transfectants are selected with 400 μg/mL zeocin for 2 weeks. The resulting Jurkat NFkBluc cell line is transduced with cynomolgus monkey GITR lentivirus and selected with 0.5 μg/mL puromycin for 1 week. Jurkat cyno GITR/NFkBluc cells are maintained in RPMI1640 media+10% FBS (HyClone SH30070.3)+400 μg/mL zeocin+0.5 μg/mL puromycin. Jurkat human GITR/NFkB-Luc2 and Jurkat cynomolgus monkey GITR/NFkBluc cells are resuspended in FACS buffer (PBS+1% BSA+0.01% sodium azide) at 1 million cells/mL and 5 μl Fc block/mL (BioLegend) is added and incubated at 4° C. for 20 min. Cells are washed with FACS buffer and resuspended at 1e6 cells/mL in FACS buffer and added to a polypropylene 96-well plate at 100 μl/well for 100,000 cells/well. 100 μl/well of 2X antibody dose titrations diluted in FACS buffer are added to cells and incubated 45 minutes at 4° C. Cells are washed twice with 200 μl/well FACS buffer and resuspended in 12.5 μg/mL anti-human Ig light chain kappa/PE secondary antibody (Invitrogen #PA1-74408) in FACS buffer and incubated for 45 minutes at 4° C. Cells are washed twice with 200 μl/well FACS buffer and resuspended in 200 μl FACS buffer. Samples are read on a Millipore EasyCyte Cytometer and median fluorescence intensity values are calculated using GuavaSoft 3.3 InCyte software. EC50values are calculated for each of 3 experiment replicates and the EC50Geometric Mean +/−Error (Delta Method) is calculated from these 3 results. The cell binding results shown in Table 5 indicate that both Antibody I and Antibody II bind potently to human GITR and cynomolgus monkey GITR (“cyno GITR”). On the other hand, an adalimumab Fab-albVHH used as a negative control for this study demonstrated negligible binding to human GITR and cynomolgus monkey GITR (data not shown). TABLE 5In Vitro Potency of Antibodies Binding to Jurkat GITR cell lines.HumanCynoGITRGITRcell bindingErrorcell bindingErrorCom-EC50nM(deltaEC50nM(deltapoundGeoMeanmethod)NGeoMeanmethod)NAntibody I6.800.3033.060.043Antibody II3.250.1232.340.063 Example 5: Antibody In Vitro GITRL Antagonism at Human and Cyno GITR Jurkat human GITR/NFkB-Luc2 or Jurkat cyno GITR/NFkBluc cells are starved overnight in assay media (RPMI1640+1% FBS) at 37° C., 5% CO2and resuspended the next day in assay media (RPMI1640+1% FBS) at 2e6 cells/mL. 25 μL/well of cell suspension is added to white opaque 96-well plates (Corning Costar) for 5e5 cells/well. 50 μL/well of 2X antibody dose titrations diluted in assay media are added, immediately followed by addition of 25 μL/well of 4X (12 nM) human GITRL or cyno GITRL, respectively, diluted in assay media. Plates are incubated at 37° C., 5% CO2for 6 hours and then placed at room temperature for 15 minutes. 100 μL/well of BioGlo luciferase reagent (Promega) is added per well and incubated with shaking for 5 minutes at room temperature. Luminescence is measured with a BioTek SynergyNeo2 plate reader with Gen5 software. IC50values are calculated for GITRL inhibition by GITR antagonist antibody treatments from each of 3 experiment replicates and the IC50Geometric Mean with Error (delta method) is calculated from these 3 results. TABLE 6In Vitro Potency of GITR Antagonist Antibodies for Inhibition ofGITRL Stimulation of Jurkat GITR/NFkB luciferase Cells.HumanCynoGITRErrorGITRErrorIC50nM(deltaIC50nM(deltaCompoundGeoMeanmethod)NGeoMeanmethod)NAntibody I4.100.9832.270.373Antibody II1.680.1831.260.093 The luciferase reporter functional bioassay results in Table 6 demonstrate that both Antibody I and Antibody II potently inhibit human GITRL stimulation of human GITR and cyno GITRL stimulation of cyno GITR. Example 6: Antibody In Vitro Agonism at Human and Cyno GITR Jurkat human GITR/NFkB-Luc2 or Jurkat cyno GITR/NFkBluc cells are starved overnight in assay media (RPMI1640+1% FBS) at 37° C., 5% CO2and resuspended the next day in assay media (RPMI1640+1% FBS) at 2e6 cells/mL. 50 μL/well of cell suspension is added to white opaque 96-well plates (Corning Costar) for 5e5 cells/well. 50 μL/well of 2X antibody dose titrations diluted in assay media are added and plates are incubated at 37° C., 5% CO2for 6 hours and then placed at room temperature for 15 minutes. 100 μL/well of BioGlo luciferase reagent (Promega) is added per well and incubated with shaking for 5 minutes at room temperature. Luminescence is measured with a BioTek SynergyNeo2 plate reader with Gen5 software. EC50values are calculated for GITR antibody treatments from each of 2 or 3 experiment replicates and the EC50Geometric Error (delta method) is calculated from these results. TABLE 7In Vitro GITR Antibody Agonism of Jurkat GITR/NFkB luciferase Cells.Human GITRCyno GITREC50nMIC50nMCompoundGeoMeanNGeoMeanNAntibody Ino activity3no activity3Antibody IIno activity3no activity3Antibody III8.5020.522 The luciferase reporter functional bioassay results in Table 7 demonstrate that treatment of Jurkat human GITR/NFkB luciferase or Jurkat cyno GITR/NFkB luciferase cells with Antibody I or Antibody II in the absence of GITRL did not result in agonist stimulation. In contrast, treatment with a bivalent IgG variant of Antibody I (i.e., Antibody III) did induce GITR pathway activation. Example 7: Antibody In Vitro Antagonism of GITRL Co-Stimulation of T cell Proliferation One aspect of GITRL biology is its ability to co-stimulate T cells, resulting in increased proliferation. In this example, it is demonstrated that Antibody I and Antibody II treatment potently inhibits plate-bound GITRL co-stimulation of human T cell proliferation. Human CD3+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated at 1.5e6 cells/mL with plate-bound 0.2 μg/mL anti-CD3 antibody for 4 days to increase GITR expression on T cells. T cells were then rested in culture medium for 2 days to prepare cells for restimulation. Activated and rested T cells at 1.5e6 cells/mL were then co-stimulated with plate-bound 2 μg/mL anti-CD3 antibody and 1 nM human GITRL for 5 days in the presence of Antibody I or Antibody II dose titrations. T cell proliferation was measured by H3-thymidine uptake during the last 18 hours of incubation. Each antibody was tested with triplicate dose titration conditions and tested on four different donors over three separate experiments. TABLE 8In Vitro Potency of GITR Antagonist Antibodies for Inhibition ofGITRL co-stimulation of T cell proliferation.Human GITRCompoundIC50nM GeoMeanError (delta method)NAntibody I4.761.424Antibody II1.840.524 The GITRL co-stimulation assay results shown in Table 8 demonstrate that Antibody I and Antibody II potently inhibit GITRL co-stimulation of T cell proliferation. In contrast, an albVHH antibody fragment used as a negative control demonstrated negligible ability to inhibit GITRL co-stimulation of T cell proliferation (data not shown). Example 8: Antibody Restoration of In Vitro Regulatory T Cell Suppression of Effector T Cell Proliferation in the Presence of GITRL One aspect of GITRL biology is its ability to inhibit regulatory T cell suppression of effector T cell proliferation through binding and activation of GITR. In this example, we demonstrate that Antibody I and Antibody II treatment potently restores the suppressive activity of regulatory T cells in the presence of plate-bound GITRL. Human T cells and CD4+CD127lowCD25+ regulatory T cells were isolated from peripheral blood mononuclear cells (PBMCs) and were labeled with CellTrace CFSE or CellTrace Violet (Invitrogen), respectively. T cells and CD4+CD127lowCD25+ regulatory T cells were combined at a 2:1 ratio in the presence of Antibody I or Antibody II dose titrations and stimulated with 2 μg/mL anti-CD28 and plate-bound 1 μg/mL anti-CD3 and 2 nM GITRL. After incubation for four days, proliferation of CD4+ T cells was evaluated by flow cytometry tracking CFSE labeling, excluding CellTrace Violet labeled CD4+CD127lowCD25+ regulatory T cells. The assay was performed with four individual donors on four separate occasions. 10,000 events were acquired per treatment (single biological replicate per antibody concentration tested) and used to calculate the percent proliferation of T effector cells. TABLE 9In Vitro Potency of GITR Antagonist Antibodies for Restoration ofRegulatory T cell Suppression of Effector T cell Proliferationin the presence of GITRLHuman GITRCompoundIC50nM MeanError (delta method)NAntibody I4.690.204Antibody II2.720.834 The assay results shown in Table 9 demonstrate that Antibody I and Antibody II potently restore regulatory T cell suppression of effector T cell proliferation in the presence of GITRL. Example 9: Antibody Pharmacokinetics in Mice Male C57BL/6 mice are administered a single IV or SQ dose of 10 mg/kg of Antibody I or Antibody II in PBS (pH 7.4) at a volume of 0.1 mL/animal. For pharmacokinetic characterization, blood is collected from 3 animals/group/timepoint at 1, 6, 24, 48, 72, 96, 120, 168, 240, and 336 hours post IV dose or at 3, 6, 12, 24, 48, 96, 120, 168, 240, and 336 hours post SQ dose and processed to plasma. Plasma concentrations of Antibodies I and II are determined by a plate-based GITR antigen capture enzyme-linked immunosorbent assay (ELISA) method. A recombinant human GITR Fc chimera (rh GITR/TNFRSF18 Fc) is coated on the ELISA plate as a capture reagent at 1 mg/mL. After incubation with plasma standards, control and samples, a goat anti-human Ig Fab horseradish peroxidase is used to detect plate-bound Antibody I or Antibody II. Pharmacokinetic parameters are calculated using non-compartmental analysis (NCA) of mean concentrations determined at each time point (N=1 to 3 animals/group/time point). NCA is performed using Watson Bioanalytical LIMS. As shown in Table 10, Antibodies I and II demonstrate an extended pharmacokinetic profile in C57BL/6 mice. TABLE 10Composite Plasma Pharmacokinetic Parametersfor Antibodies I and II Following aSingle 10 mg/kg IV or SQ Dose to Male C57BL/5 Mice.C0CmaxAUC0-infCL orAnti-(μg/(μg/Tmax(hr*μg/CL/Ft1/2bodyRoutemL)mL)(hr)mL)(mL/hr/kg)(hr)% FIIV86.8NANA56301.7850.6NAISQNA42.41232703.0640.758.1IIIV124NANA54301.8429.3NAIISQNA69.21235002.8634.664.5 Example 10: Antibody Pharmacokinetics in Rats Male Sprague Dawley rats are administered a single IV or SQ dose of 10 mg/kg Antibody I or Antibody II in PBS (pH 7.4) at volumes of 2.5 mL/kg and 1 mL/kg, respectively. For pharmacokinetic characterization, blood is collected from 3 animals/group/timepoint at 1, 6, 24, 48, 72, 96, 120, 168, 240, 336, 432 and 504 hours post IV dose or at 3, 6, 12, 24, 48, 96, 120, 168, 240, 336, 432 and 504 hours post SQ dose and processed to plasma. Plasma concentrations of Antibody Antibodies I and II are determined by a plate-based GITR antigen capture enzyme-linked immunosorbent assay (ELISA) method. A recombinant human GITR Fc chimera (rh GITR/TNFRSF18 Fc) was coated on the ELISA plate as a capture reagent at 1 μg/mL. After incubation with plasma standards, control and samples, a goat anti-human Ig Fab horseradish peroxidase (diluted 1:10,000) was used to detect plate-bound Antibody I or Antibody II. Pharmacokinetic parameters are calculated using NCA for each animal (N=2-3) and parameters are summarized by the mean and standard deviation (SD), where appropriate. Pharmacokinetic data were available for N=2 rats dosed subcutaneously with Antibody II and therefore mean parameters are reported. NCA and summary statistic calculations are performed using Watson Bioanalytical LIMS. As shown in Table 11 and Table 12, Antibody Antibodies I and II has demonstrate an extended pharmacokinetic profile in Sprague Dawley rats. TABLE 11Plasma Pharmacokinetic Parameters for Antibody I Following aSingle 10 mg/kg IV (N = 3) or 10 mg/kg SQ (N = 3)Dose to Male Sprague Dawley Rats.C0CmaxAUC0-infCL orAnti-(μg/(μg/Tmax(hr*μg/CL/Ft1/2bodyRoutemL)mL)(hr)mL)(mL/hr/kg)(hr)% FIIV289NANA146000.6954.0NA(24.3)(1710)(0.08)(1.36)ISQNA48.84866301.5155.445.4(6.87)(0)(267)(0.06)(1.99)NOTE:Parameters are reported as mean (SD). TABLE 12Plasma Pharmacokinetic Parameters for Antibody II Following aSingle 10 mg/kg IV (N = 3) or 10 mg/kg SQ (N = 2)Dose to Male Sprague Dawley Rats.C0CmaxAUC0-infCL orAnti-(μg/(μg/Tmax(hr*μg/CL/Ft1/2bodyRoutemL)mL)(hr)mL)(mL/hr/kg)(hr)% FIIIV188NANA109000.9253.0NA(19.4)(473)(0.04)(6.90)IISQNA24.24835402.8655.632.5NOTE:Parameters are reported as mean (SQ route) or mean (SD) (IV route). Example 11: Antibody Pharmacokinetics in Cynomolgus Monkey Cynomolgus monkeys are administered a single 0.1, 1, or 10 mg/kg IV dose or 10 mg/kg SQ dose of Antibody I or Antibody II in PBS (pH 7.2) at volumes of 1 mL/kg (IV) or 0.2 mL/kg (SC). For pharmacokinetic characterization, blood is collected from 3 animals/group/timepoint at 0.5, 3, 6, 24, 48, 72, 96, 168, 240, 336, 432, 504 and 672 hours post dose and processed to plasma. Plasma concentrations of Antibodies I and II are determined by plate-based GITR antigen capture enzyme-linked immunosorbent assay (ELISA) methods. A recombinant human GITR Fc chimera (rh GITR/TNFRSF18 Fc) is coated on the ELISA plate as a capture reagent at 1 μg/mL. After incubation with plasma standards, control and samples, detections antibodies are used to determine the stability of the antibodies. In one method, a goat anti-human Ig Fab horseradish peroxidase is used to detect the Fab-portion of plate-bound Antibody I or Antibody II. In the second method, a Biotin-SP AffiniPure Goat Anti-Alpaca IgG, VHH domain is added as a secondary antibody to detect metabolically stable antibody with intact linker of plate-bound Antibody I or Antibody II. Following this incubation, a peroxidase-conjugated streptavidin is added as a reagent to detect plate-bound Antibody I or Antibody II. Pharmacokinetic parameters are calculated using NCA for each animal and parameters are summarized by the mean and standard deviation (SD), where appropriate. NCA and summary statistic calculations are performed using Watson Bioanalytical LIMS. As shown in Table 13 and Table 14, Antibodies I and II demonstrate an extended pharmacokinetic profile in cynomolgus monkeys. When comparing concentrations of Antibodies I or II measured with both ELISA methods, similar exposure and pharmacokinetics was observed, indicating metabolic stability of these antibodies in vivo following IV or SQ administration. TABLE 13Plasma Pharmacokinetic Parameters for Antibody I Following a Single0.1 (N = 1), 1 (N = 2), or 10 mg/kg IV (N = 2) or10 mg/kg SQ (N = 2) Dose to Cyno Monkeys.CL orCL/FDoseC0CmaxAUC0-inf(mL/ELISA(mg/(μg/(μg/Tmax(hr*μg/hr/t1/2%MethodRoutekg)mL)mL)(hr)mL)kg)(hr)Fanti-FabIV0.12.25NANA2500.4078.8NAdetec-IV142.2NANA60900.17236NAtionIV10386NANA471000.21247NASC10NA10672339000.3025472anti-IV0.12.93NANA2860.3596.2NAVHHIV135.1NANA48700.21180NAdetec-IV10282NANA415000.24186NAtionSQ10NA11748343000.3020983NOTE:Parameters are reported as mean. TABLE 14Plasma Pharmacokinetic Parameters for Antibody II Following a Single 0.1(N = 2), 1 (N = 2), or 10 mg/kg IV (N = 2) or 10 mg/kg SQ (N = 3) Dose to Cynomolgus Monkeys.CL orDoseC0CL/FELISA(mg/(μg/CmaxTmaxAUC0-inf(mL/hr/t1/2MethodRoutekg)mL)(μg/mL)(hr)(hr*μg/mL)kg)(hr)% Fanti-FabIV0.12.47NANA1890.5482.9NAdetectionIV125.9NANA32800.32194NAIV10263NANA465000.22291NASC10NA95.9112356000.2822076.6(12.7)(111)(1760)(0.01)(64.3)anti-IV0.12.28NANA1900.5377.4NAVHHIV127.6NANA36200.29224NAdetectionIV10349NANA381000.26218NASQ10NA99.964343000.3019490.0(16.8)(14)(5460)(0.04)(23.9)NOTE:Parameters are reported as mean (IV route) or mean (SD) (SQ route). Listing of SequencesSEQ ID NO: 1 HCDR1AASGYTFSSYVMHSEQ ID NO: 2 HCDR2.1VTSYDGTHEYSEQ ID NO: 3 HCDR3ARENNWAPDYSEQ ID NO: 4 LCDR1RASQDISNSLASEQ ID NO: 5 LCDR2YAAFSLQSSEQ ID NO: 6 LCDR3YQYYNYPSASEQ ID NO: 7 HCDR2.2VTSYDGTHELSEQ ID NO: 8 HC Variable Region (VH1)QVQLVESGGGVVQPGRSLRLSCAASGYTFSSYVMHWVRQAPGKGLEWVAVTSYDGTHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWAPDYWGQGTLVTVSSSEQ ID NO: 9 HC Variable Region (VH2)QVQLVESGGGVVQPGRSLRLSCAASGYTFSSYVMHWVRQAPGKGLEWVAVTSYDGTHELYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWAPDYWGQGTLVTVSSSEQ ID NO: 10 LC Variable Region (VL)DIQMTQSPSSLSASVGDRVTITCRASQDISNSLAWYQQKPGKAPKRLIYAAFSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCYQYYNYPSAFGQGTKLEIKSEQ ID NO: 11 Linker 1DKTHTGGGGQGGGGQGGGGQGGGGQGGGGQSEQ ID NO: 12 Linker 2DKTGGGGQGGGGQGGGGQGGGGQGGGGQSEQ ID NO: 13 Albumin-binding VHHEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPPSEQ ID NO: 14 HC anti-Fab - albumin-binding VHHFusion 1QVQLVESGGGVVQPGRSLRLSCAASGYTFSSYVMHWVRQAPGKGLEWVAVTSYDGTHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWAPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPPSEQ ID NO: 15 HC anti-GITR Fab - albumin-bindingVHH Fusion 2QVQLVESGGGVVQPGRSLRLSCAASGYTFSSYVMHWVRQAPGKGLEWVAVTSYDGTHELYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENNWAPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPPSEQ ID NO: 16 LCDIQMTQSPSSLSASVGDRVTITCRASQDISNSLAWYQQKPGKAPKRLIYAAFSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCYQYYNYPSAFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 17 DNA Encoding HC anti-GITR Fab-albumin-binding VHH Fusion 1CAAGTGCAGCTCGTGGAGTCGGGTGGCGGAGTGGTGCAGCCCGGAAGGTCCTTGCGGCTCTCCTGTGCCGCTTCCGGCTACACCTTCTCGAGCTACGTGATGCACTGGGTCAGACAGGCACCGGGAAAGGGTCTGGAATGGGTGGCCGTGACTTCCTACGACGGCACCCACGAGTATTACGCCGACTCAGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCAAAGAACACCCTGTATCTGCAAATGAACTCACTGCGGGCCGAGGACACTGCCGTGTACTACTGCGCGCGCGAAAACAACTGGGCCCCTGACTACTGGGGACAGGGGACTCTGGTCACTGTGTCGTCCGCCTCGACCAAGGGACCCTCCGTGTTTCCGCTGGCGCCAAGCAGCAAGAGCACCTCGGGGGGAACTGCAGCCTTGGGGTGCCTCGTGAAGGATTACTTCCCCGAACCAGTGACCGTGTCCTGGAACTCTGGGGCCCTCACCAGTGGAGTGCACACTTTCCCTGCGGTGCTGCAGTCCTCCGGACTGTACAGCCTGTCCAGCGTGGTCACGGTGCCCAGCTCCTCACTGGGCACCCAGACCTACATTTGCAACGTGAACCATAAGCCGTCCAATACCAAAGTCGATAAGAAAGTCGAGCCGAAGTCCTGCGACAAGACACACACCGGTGGAGGAGGCCAGGGTGGAGGTGGACAAGGCGGCGGAGGTCAAGGCGGAGGAGGACAGGGTGGC GGAGGACAGGAAGTGCAGCTGCTGGAGTCCGGGGGCGGACTGGTGCAGCCTGGCGGATCATTGCGGCTGTCGTGCGCGGCCTCCGGACGCTACATCGACGAGACAGCAGTGGCCTGGTTCAGACAGGCTCCCGGAAAGGGAAGAGAGTTCGTGGCCGGAATTGGCGGGGGAGTCGACATTACCTACTACGCCGATTCCGTGAAGGGTCGCTTTACCATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCGCTGAGGCCGGAAGATACCGCGGTGTATTACTGTGCCGCCCGCCCGGGACGCCCGCTGATCACGTCCAAAGTCGCCGACCTGTACCCGTACTGGGGACAGGGTACCCTCGTGACCGTGTCCAGCCCTCCCSEQ ID NO: 18 DNA Encoding HC GITR- and albumin-binding VHH Fusion 2CAGGTGCAGCTCGTGGAGTCGGGTGGCGGAGTGGTGCAGCCCGGAAGGTCCTTGCGGCTCTCCTGTGCCGCTTCCGGCTACACCTTCTCGAGCTACGTGATGCACTGGGTCAGACAGGCACCAGGAAAGGGTCTGGAATGGGTGGCCGTGACCTCCTACGACGGCACCCACGAGCTGTACGCCGACTCAGTGAAGGGCCGCTTCACTATCTCCCGGGACAACTCAAAGAACACCCTGTATCTGCAAATGAACTCACTGCGGGCCGAGGACACTGCCGTGTACTACTGCGCGCGCGAAAATAACTGGGCCCCTGACTACTGGGGACAGGGGACTCTGGTCACTGTGTCGTCCGCCTCGACCAAGGGACCCTCCGTGTTTCCGCTGGCGCCAAGCAGCAAGAGCACCTCGGGGGGAACTGCAGCCTTGGGGTGCCTCGTGAAGGATTACTTCCCCGAACCAGTGACCGTGTCCTGGAACTCTGGGGCCCTCACCAGTGGAGTGCACACTTTCCCTGCGGTGCTGCAGTCCTCCGGACTGTACAGCCTGTCCAGCGTGGTCACGGTGCCCAGCTCCTCACTGGGCACCCAGACCTACATTTGCAACGTGAACCATAAGCCGTCCAATACCAAAGTCGATAAGAAAGTCGAGCCGAAGTCCTGCGACAAGACACACACCGGTGGAGGAGGCCAGGGTGGAGGTGGACAAGGCGGCGGAGGTCAAGGCGGAGGAGGACAGGGTGGCGGAGGACAGGAAGTGCAGCTGCTGGAGTCCGGGGGCGGACTGGTGCAGCCTGGCGGATCATTGCGGCTGTCGTGCGCGGCCTCCGGACGCTACATCGACGAGACAGCAGTGGCCTGGTTCAGACAGGCTCCCGGAAAGGGAAGAGAGTTCGTGGCCGGAATTGGCGGGGGAGTCGACATTACCTACTACGCCGATTCCGTGAAGGGTCGCTTTACCATCTCCCGGGACAATTCGAAGAACACCCTGTACCTCCAAATGAACTCGCTGAGGCCGGAAGATACCGCGGTGTATTACTGTGCCGCCCGCCCGGGACGCCCGCTGATCACGTCCAAAGTCGCCGACCTGTACCCGTACTGGGGACAGGGTACCCTCGTGACCGTGTCCAGCCCTCCCSEQ ID NO: 19 DNA Encoding LCGATATCCAGATGACCCAGTCCCCGAGCTCGCTGTCCGCTTCCGTGGGAGACAGAGTGACGATCACTTGTCGGGCCAGCCAAGACATTAGCAACTCCCTGGCCTGGTACCAGCAGAAGCCCGGCAAAGCACCCAAGAGGTTGATCTACGCGGCCTTTTCACTGCAATCCGGAGTGCCGAGCCGGTTCTCCGGATCCGGTTCAGGGACCGAGTTCACCTTGACCATTAGCAGCCTGCAGCCCGAAGATTTCGCCACTTACTACTGCTACCAGTATTACAATTACCCATCGGCGTTCGGCCAAGGCACCAAGCTCGAGATCAAGCGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCSEQ ID NO: 20 Human GITR Full-length (w/o signalpeptide)QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWVSEQ ID NO: 21 Human GITR ECD (w/o signal peptide)QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAESEQ ID NO: 22: Signal Peptide amino acid sequenceMETDTLLLWVLLLWVPGSTG | 64,895 |
11859006 | DETAILED DESCRIPTION OF THE INVENTION The present invention will be described in more detail. 1. Definition As used herein, the term “gene” includes not only DNA but mRNA, cDNA, and cRNA. As used herein, the term “polynucleotide” is used with the same meaning as a nucleic acid and also includes, for example, DNA, RNA, probes, oligonucleotides, and primers. As used herein, the “polypeptide” and the “protein” are used interchangeably with each other. As used herein, the “RNA fraction” refers to a fraction containing RNA. As used herein, the “cell” also includes cells within animal individuals and cultured cells. As used herein, “ALK2” is used with the same meaning as ALK2 protein and includes wild-type ALK2 and mutants thereof (also referred to as “mutant”). As used herein, the “antigen-binding fragment of an (the) antibody”, also called “functional fragment of an (the) antibody”, means a partial fragment of the antibody having an activity binding to the antigen and includes, for example, F(ab′)2, diabodies, linear antibodies, single-chain Fvs, and multispecific antibodies formed from antibody fragments. However, the antigen-binding fragment is not limited to these molecules as long as the antigen-binding fragment has an ability to bind to ALK2 or a property of binding to the ALK2) and has an ability to cross-link ALK2 or a property of cross-linking ALK2), as in the anti-ALK2 antibody. Preferably, the antigen-binding fragment of the antibody further has an ability to inhibit BMP signal transduction (or a property of inhibiting BMP signal transduction), as in the anti-ALK2 antibody. Such an antigen-binding fragment includes not only a fragment obtained by treating a full-length molecule of the antibody protein with an appropriate enzyme but a protein produced in appropriate host cells using a genetically engineered antibody gene. As used herein, the “epitope”, also called “antigenic determinant”, generally refers to an antibody-binding antigenic site consisting of at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, or at least 10 amino acids, of an antigen. As used herein, the “epitope” means a partial peptide or a partial conformation of ALK2 to which a particular anti-ALK2 antibody binds. The epitope as a partial peptide of ALK2 may be determined by a method well known to those skilled in the art such as immunoassay and may be determined, for example, by the following method in which various partial structures of ALK2 are prepared. For the preparation of the partial structures, an oligopeptide synthesis technique known in the art may be used. For example, a series of polypeptide fragments having an appropriate length are prepared in order from the C or N terminus of ALK2 using gene recombination techniques well known to those skilled in the art. Then, the reactivity of the antibody with the polypeptide fragments is studied to roughly determine recognition sites. Then, shorter peptides are synthesized, and the reactivity of the antibody with these peptides may be studied to determine the epitope. Alternatively, the epitope as a partial conformation of ALK2 to which a particular ALK2 antibody binds may be determined by identifying amino acid residues of ALK2 adjacent to the antibody by X-ray crystal structure analysis. If a second anti-ALK2 antibody binds to a partial peptide or a partial conformation that is bound by a first anti-ALK2 antibody, then the first antibody and the second antibody may be determined to share an epitope. In addition, even if a specific sequence or structure of an epitope is not determined, the first antibody and the second antibody may be determined to share the epitope by confirming that the second anti-ALK2 antibody (cross-)competes with the first anti-ALK2 antibody for binding to ALK2 i.e., that the second antibody interferes with binding of the first antibody to ALK2). Furthermore, when the first antibody and the second antibody bind to a common epitope and the first antibody has an activity such as inhibitory activity against ALK2-mediated BMP signal transduction, the second antibody can also be expected to have similar activity. The heavy and light chains of an antibody molecule are known to each have three complementarity determining regions (CDRs). The complementarity determining regions, also called hypervariable domains, are located in the variable regions of the antibody heavy and light chains. These sites have a particularly highly variable primary structure and are separated into three places on the respective primary structures of heavy and light chain polypeptide chains. As used herein, the complementarity determining regions of an antibody are referred to as CDRH1, CDRH2, and CDRH3 from the amino terminus of the heavy chain amino acid sequence for the complementarity determining regions of the heavy chain and as CDRL1, CDRL2, and CDRL3 from the amino terminus of the light chain amino acid sequence for the complementarity determining regions of the light chain. These sites are proximal to each other on the conformation and determine specificity for the antigen to be bound. In the present invention, the term “hybridizing under stringent conditions” means hybridization under conditions involving hybridization at approximately 50 to 70° C. (e.g., 68° C.) in a commercially available hybridization solution ExpressHyb Hybridization Solution (manufactured by Clontech Laboratories, Inc.), or hybridization at approximately 50 to 70° C. (e.g., 68° C.) in the presence of approximately 0.7 to 1.0 M NaCl using a DNA-immobilized filter, followed by washing at approximately 50 to 70° C. (e.g., 68° C.) using an SSC solution having an approximately 0.1 to 2× concentration (SSC having a 1× concentration consists of 150 mM NaCl and 15 mM sodium citrate; if necessary, the solution may contain approximately 0.1 to 0.5% SDS) which permits identification, or hybridization under conditions equivalent thereto. As used herein, the term “several” in the phrase “one or several” refers to 2 to 10. The term “several” is preferably 10 or less, more preferably 5 or 6 or less, far more preferably 2 or 3. In the present invention, the “cross-linking ability” or the “ability to cross-link” refers to the ability of one antibody or an antigen-binding fragment to bind to the respective extracellular regions in two molecules of the ALK2 protein, thereby cross-linking these molecules. Typically, ALK2 forms a complex in the presence of a BMP ligand to activate downstream SMAD1/5/8. The anti-ALK2 antibody induces the cross-link between two molecules of ALK2, probably leading to complex-like formation even in the absence of the ligand. The present inventors have now found that an anti-ALK2 antibody that binds to ALK2 inhibits the BMP signal transduction when the amino acid residue at position 330 in a mutant of human ALK2 protein is proline and, in some cases, when a mutant of human ALK2 protein has no G328V mutation, but that the anti-ALK2 antibody promotes (or activates) the BMP signal transduction when the proline at position 330 is a different amino acid residue such as serine, aspartic acid, glutamic acid, or alanine. On the basis of this finding, when a patient has proline at position 330 in a mutant of human ALK2 protein, and in some cases, has no G328V mutation in the mutant of human ALK2 protein, the patient identified so may be effectively treated with the anti-ALK2 antibody. As used herein, the promotion of BMP signal transduction refers to activating the downstream intracellular signaling pathway via the ALK2 receptor molecule. In the present invention, the “patient” is not only a human affected by (or suffered from) a disease, but may also be a human suspected of being affected by a disease. The “biological sample” as used herein is not particularly limited as long as the presence or absence of a mutation in ALK2 is detectable in a biological sample. The biological sample is, for example, a blood sample or a tumor sample. The biological sample may be protein extracts or nucleic acid extracts (e.g., mRNA extracts, and a cDNA preparation and a cRNA preparation prepared from the mRNA extracts) obtained from these samples. 2. ALK2 The ALK2 gene is a causative gene for FOP encoding a receptor of BMP that induces ectopic bone formation in soft tissues including skeletal muscle tissues. Mutant ALK2 having amino acid substitutions has been found in familial and sporadic FOP cases. For example, L196P (i.e., the mutation that substitutes leucine at position 196 by proline), delP197_F198insL (also referred to as “PF197-8L”) (i.e. the mutation that deletes proline at position 197 and phenylalanine at position 198 and, instead, inserts leucine between them), R2021 i.e., the mutation that substitutes arginine at position 202 by isoleucine), R206H (i.e., the mutation that substitutes arginine at position 206 by histidine), Q207E (i.e., the mutation that substitutes glutamine at position 207 by glutamic acid), R258S (i.e., the mutation that substitutes arginine at position 258 by serine), R258G (i.e., the mutation that substitutes arginine at position 258 by glycine), G325A (i.e., the mutation that substitutes glycine at position 325 by alanine), G328E (i.e., the mutation that substitutes glycine at position 328 by glutamic acid), G328R (i.e., the mutation that substitutes glycine at position 328 by arginine), G328W (i.e., the mutation that substitutes glycine at position 328 by tryptophan), G356D (i.e., the mutation that substitutes glycine at position 356 by aspartic acid), and R375P (i.e., the mutation that substitutes arginine at position 375 by proline) in the amino acid sequence of SEQ ID NO: 1 are known as active mutations in human ALK2. Mutant ALK2 having an amino acid substitution(s) has also been found in DIPG cases. R206H, R258G, G328E, G328V (which is the mutation that substitutes glycine at position 328 by valine), G328W, G356D, and the like in the amino acid sequence of SEQ ID NO: 1 are known as active mutations in human ALK2. ALK2 used herein may be obtained by in vitro synthesis or by production from host cells through gene manipulation. Specifically, ALK2 cDNA is inserted into a vector that permits its expression. Then, the ALK2 protein may be obtained by synthesis in solutions containing enzymes, substrates, and energy substances necessary for transcription and translation, or by expression in other prokaryotic or eukaryotic host cells transformed with the vector. ALK2 used herein is from a mammal including human or mouse. For example, the amino acid and nucleotide sequences of human ALK2 are available with reference to GenBank Accession No. NM_001105. Herein, similarly the amino acid sequence is disclosed as SEQ ID NO: 1, and the nucleotide sequence is disclosed as SEQ ID NO: 2. The amino acid and nucleotide sequences of mouse ALK2 are available with reference to GenBank Accession No. NP_001103674. Herein, similarly the amino acid sequence is disclosed as SEQ ID NO: 3, and the nucleotide sequence is disclosed as SEQ ID NO: 4. Furthermore, the amino acid sequences of monkey, rat and dog ALK2s are available with reference to GenBank Accession Nos. NM-001260761 (SEQ ID NO: 40), NP_077812 (SEQ ID NO: 42), and XM_549615.5 (SEQ ID NO: 41), respectively. ALK2 is also called ACVR1 Activin A type I receptor 1) or ACTR1 Activin receptor type 1), and all of these terms represent the same molecules. The ALK2 cDNA may be obtained by a so-called PCR method which involves carrying out polymerase chain reaction (hereinafter, referred to as “PCR”) (Saiki, R. K., et al., Science, (1988) 239, 487-49), for example, using a cDNA library expressing the ALK2 cDNA as a template and primers specifically amplifying the ALK2 cDNA. 3. Detection of Mutation in ALK2 Herein, the term “detecting a mutation” means detecting a mutation on genomic DNA as a rule. Alternatively, when the mutation on the genomic DNA is reflected in change of a base(s) in a transcribed product or in change of an amino acid(s) in a translated product, this term also means including detecting this change in the transcribed product or the translated product (i.e., indirect detection). In a preferred embodiment, the method of the present invention is a method of directly determining a nucleotide sequence of an ALK2 gene region of a patient, thereby detecting a mutation. As used herein, the “ALK2 gene region” means a certain region on genomic DNA containing the ALK2 gene. The region also contains the expression control regions (e.g., a promoter region and an enhancer region) of the ALK2 gene, a 3′-terminal untranslated region of the ALK2 gene, and the like. A mutation in these regions may influence, for example, the transcription activity of the ALK2 gene. In this method, first, a DNA sample is prepared from a biological sample derived from a patient. Examples of the DNA sample include genomic DNA samples, and cDNA samples prepared from RNA by reverse transcription. A method for extracting genomic DNA or RNA from the biological sample is not particularly limited, and approaches known in the art may be appropriately selected for use in the extraction. Examples of the method for extracting genomic DNA include a SDS phenol method (i.e., a method which involves: denaturing proteins in tissues preserved in a urea-containing solution or in ethanol, using a proteolytic enzyme (proteinase K), a surfactant (SDS), and phenol; and extracting DNA by precipitation from the tissues using ethanol), and DNA extraction methods using Clean Columns® (manufactured by NextTec Biotechnolgie GmbH), AquaPure®) (manufactured by Bio-Rad Laboratories, Inc.), ZR Plant/Seed DNA Kit (manufactured by Zymo Research Corp.), Aqua Genomic Solution® (manufactured by MoBiTec GmbH), prepGEM® (manufactured by ZyGEM NZ Ltd.) or BuccalQuick® (manufactured by TrimGen Corp.). Examples of the method for extracting RNA include extraction methods using phenol and a chaotropic salt (more specifically, extraction methods using a commercially available kit such as TRIzol (manufactured by Invitrogen Corp.) or ISOGEN (manufactured by Wako Pure Chemical Industries, Ltd.)), and methods using other commercially available kits (RNAPrep Total RNA Extraction Kit (manufactured by Beckman Coulter, Inc.), RNeasy Mini (manufactured by Qiagen N.V.), RNA Extraction Kit (manufactured by Pharmacia Biotech Inc.), etc.). Examples of reverse transcriptase for use in the preparation of cDNA from the extracted RNA include, but are not particularly limited to, reverse transcriptase derived from retrovirus such as RAV (Rous associated virus) or AMV (avian myeloblastosis virus), and reverse transcriptase derived from mouse retrovirus such as MMLV (Moloney murine leukemia virus). In this aspect, DNA containing a mutation site in the ALK2 gene region is subsequently isolated, and the nucleotide sequence of the isolated DNA is determined. The isolation of the DNA may be performed by, for example, PCR using a pair of oligonucleotide primers designed so as to flank on the both sides of the mutation in the ALK2 gene region, and using the genomic DNA or the RNA as a template. The determination of the nucleotide sequence of the isolated DNA may be performed by, for example, a method known to those skilled in the art, such as Maxam-Gilbert method or Sanger method, or a method using a next-generation sequencer. The determined nucleotide sequence of the DNA or the cDNA may be compared with a control (e.g., a nucleotide sequence of the corresponding DNA or cDNA derived from biological samples of healthy people), thereby determining the presence or absence of the mutation in the ALK2 gene region of the patient. The method for detecting a mutation in the ALK2 gene region may be performed by various methods capable of detecting a mutation, in addition to the method of directly determining the nucleotide sequence of DNA or cDNA. The detection of a mutation according to the present invention may also be performed by, for example, the following method. Specifically, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, a reporter fluorescent dye- and quencher fluorescent dye-labeled oligonucleotide probe having a nucleotide sequence complementary to a nucleotide sequence containing the mutation in the ALK2 gene region is prepared. Then, the oligonucleotide probe is hybridized to the DNA sample under stringent conditions. The nucleotide sequence containing the mutation in the ALK2 gene region is further amplified using the DNA sample hybridized with the oligonucleotide probe as a template. Then, fluorescence (signals) emitted by the reporter fluorescent dye through the decomposition of the oligonucleotide probe associated with the amplification is detected. Subsequently, the detected fluorescence is compared with a control. Examples of such a method include double die probe method and TaqMan® probe method. In an alternative method, a DNA or cDNA sample is prepared from a biological sample. Subsequently, the nucleotide sequence containing the mutation in the ALK2 gene region is amplified using the DNA sample as a template in a reaction system containing an intercalator that emits fluorescence upon insertion between two strands of DNA. Then, the temperature of the reaction system is changed, and variation in the intensity of the fluorescence emitted by the intercalator is detected. The detected variation in the intensity of the fluorescence caused by the change in the temperature is compared with a control. Examples of such a method include HRM (high resolution melting) method. In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Subsequently, DNA containing a mutation site in the ALK2 gene region is amplified. The amplified DNA is further cleaved with restriction enzymes, and the cleaved DNA fragments are separated according to their sizes. Then, the detected sizes of the DNA fragments are compared with a control. Examples of such a method include a method using restriction fragment length polymorphism (RFLP) and PCR-RFLP. In a further alternative method, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, DNA containing a mutation site in the ALK2 gene region is amplified. The amplified DNA is further dissociated into single-stranded DNAs, which are then separated on a non-denaturing gel. Subsequently, the mobility of the separated single-stranded DNAs on the gel is compared with a control. Examples of such a method include PCR-SSCP (single-strand conformation polymorphism). In a further alternative method, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, DNA containing a mutation site in the ALK2 gene region is amplified. Then, the amplified DNA is separated on a gel in which the concentration of a DNA denaturant is gradually elevated. Subsequently, the mobility of the separated DNA on the gel is compared with a control. Examples of such a method include denaturant gradient gel electrophoresis (DGGE). A further alternative method is a method using DNA containing a mutation site in the ALK2 gene region prepared from the biological sample, and a substrate with immobilized oligonucleotide probes hybridizing to the DNA under stringent conditions. Examples of such a method include a DNA array method. In a further alternative method, a DNA or cDNA sample is first prepared from the biological sample. Also, an “oligonucleotide primer having a nucleotide sequence complementary to a 3′-side nucleotide downstream by one nucleotide from the base at the mutation site in the ALK2 gene region and to a 3′-side nucleotide sequence downstream of the 3′-side nucleotide” is prepared. Subsequently, ddNTP primer extension reaction is performed using the DNA as a template and the primer. Subsequently, the primer extension reaction product is applied to a mass spectrometer to conduct mass spectrometry. Subsequently, the genotype is determined from the mass spectrometry results. The determined genotype is then compared with a control. Examples of such a method include MALDI-TOF/MS. In a further alternative method, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, an oligonucleotide probe consisting of 5′—(a nucleotide sequence complementary to the nucleotide at the mutation site in the ALK2 gene region and to a 5′-side nucleotide sequence upstream of the nucleotide)—(a nucleotide sequence that does not hybridize to 3′-side nucleotide downstream by one nucleotide from the mutation site in the ALK2 gene region and to a 3′-side nucleotide sequence downstream of the 3′-side nucleotide)—3′ (i.e., flap) is prepared. Also, an “oligonucleotide probe having a nucleotide sequence complementary to the nucleotide at the mutation site in the ALK2 gene region, and to a 3′-side nucleotide sequence downstream of the nucleotide” is prepared. Subsequently, the prepared DNA is hybridized to the two types of oligonucleotide probes, and the hybridized DNA is cleaved with a single-stranded DNA-cleaving enzyme to release the flap. Examples of the single-stranded DNA-cleaving enzyme include, but are not particularly limited to, cleavase. In this method, a fluorescent reporter- and fluorescent quencher-labeled oligonucleotide probe having a sequence complementary to the flap is then hybridized to the flap. Subsequently, the intensity of the generated fluorescence is measured. Subsequently, the measured intensity of the fluorescence is compared with a control. Examples of such a method include the Invader method. In a further alternative method, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, DNA containing a mutation site in the ALK2 gene region is amplified. Then, the amplified DNA is dissociated into single strands, and only one of the single strands of the dissociated DNA is separated. Extension reaction is then performed one by one from a nucleotide in the vicinity of the nucleotide at the mutation site in the ALK2 gene region. Pyrophosphoric acid generated during this reaction is enzymatically allowed to develop light. The intensity of the light is measured. The measured intensity of the fluorescence is compared with a control. Examples of such a method include the Pyrosequencing method. In a further alternative method, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, DNA containing a mutation site in the ALK2 gene region is amplified. Then, an “oligonucleotide primer having a nucleotide sequence complementary to a 3′-side nucleotide downstream by one nucleotide from the nucleotide at the mutation site in the ALK2 gene region and to a 3′-side nucleotide sequence downstream of the 3′-side nucleotide” is prepared. Subsequently, single-base extension reaction is performed using the amplified DNA as a template and the prepared primer in the presence of fluorescently labeled nucleotides. Then, the degree of polarization of fluorescence is measured. The measured degree of polarization of fluorescence is compared with a control. Examples of such a method include the AcycloPrime method. In a further alternative method, a DNA or cDNA sample is first prepared from a biological sample. Subsequently, DNA containing a mutation site in the ALK2 gene region is amplified. Then, an “oligonucleotide primer having a nucleotide sequence complementary to a 3′-side nucleotide downstream by one nucleotide from the nucleotide at the mutation site in the ALK2 gene region and to a 3′-side nucleotide sequence downstream of the 3′-side nucleotide” is prepared. Subsequently, single-nucleotide extension reaction is performed using the amplified DNA as a template and the prepared primer in the presence of fluorescently labeled nucleotides. Subsequently, the nucleotide species used in the single-nucleotide extension reaction are determined. Then, the determined nucleotide species are compared with a control. Examples of such a method include the SNuPE method. The sample prepared from the above-mentioned biological sample may be a protein. In such a case, a method using a molecule (e.g., an antibody) specifically binding to a site having a change of amino acid caused by the mutation may be used for detecting the mutation. 4. Detection of Ectopic Ossification and/or Brain Tumor Ectopic ossification and/or brain tumor is induced by ALK2-mediated BMP signal transduction. The “ectopic ossification” means bone formation at a site where the bone is originally absent. Examples of the “ectopic ossification” may include fibrodysplasia ossificans progressiva (FOP) and progressive osseous heteroplasia (POH), though the ectopic ossification is not limited thereto as long as the ectopic ossification is induced by BMP signal transduction mediated by ALK2 having an active mutation. The “brain tumor” means a tumor that develops in a tissue in the skull. Examples of the “brain tumor” may include diffuse intrinsic pontine glioma (DIPG), brain stem glioma, glioblastoma, glioblastoma multiforme (GBM), non-glioblastoma brain tumor, meningioma, central nervous system lymphoma, glioma, astroglioma, anaplastic astrocytoma, oligodendroglioma, oligoastrocytoma, medulloblastoma, and ependymoma, though the brain tumor is not limited thereto as long as the brain tumor is induced by BMP signal transduction mediated by ALK2 having an active mutation. ALK2 is a transmembrane serine/threonine kinase receptor binding to BMP. ALK2 binds to BMP at the N-terminal extracellular region and activates a downstream intracellular signaling pathway through intracellular serine/threonine kinase. Bone morphogenetic protein (BMP) is a multifunctional growth factor belonging to the transforming growth factor 13 TGF-(3) superfamily, and approximately 20 BMP family members have been identified. BMP has been confirmed to induce ectopic bone formation in soft tissues including skeletal muscle tissues and is therefore considered to participate in diseases promoting abnormal bone formation. BMP-2 and BMP-4 are considered to have higher affinity for ALK3 than that for ALK2. Since ALK3 is expressed ubiquitously as compared with ALK2, BMP-2 or BMP-4 seems to be often used in general in experiments of inducing ectopic ossification at various sites. On the other hand, BMP-7 has relatively high affinity for ALK2. BMP-9 is generally considered to have high affinity for ALK1 and has also been found to have relatively high affinity for ALK2. In FOP, ectopic ossification occurs via ALK2. Therefore, the presence or absence of therapeutic and/or prophylactic effects on FOP may probably be confirmed by testing efficacy for ectopic osteoinduction caused by the activation of ALK2-mediated signals by BMP-7 and BMP-9. The culture of myoblasts (C2C12 cells) in the presence of BMP suppresses their differentiation into mature muscle cells through an intracellular signal transduction mechanism specific for BMP and instead induces the differentiation into osteoblasts. Thus, ALK2-mediated BMP signal transduction may be analyzed with models of induction of differentiation of C2C12 cells into osteoblasts by BMP. 5. Production of Anti-ALK2 Antibody The antibody used in the present invention against ALK2 may be obtained according to a method known in the art (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497; and Kennet, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, N.Y. (1980)). Specifically, the monoclonal antibody may be obtained by fusing antibody-producing cells that produce the antibody against ALK2 with myeloma cells to establish hybridomas. The obtained antibody may be tested for its binding activity and cross-linking ability to ALK2 to select an antibody applicable to human diseases. Herein, positions of amino acids assigned to CDR/FR characteristic of an antibody are laid out according to the KABAT numbering (KABAT et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)). The antibody used in the present invention includes monoclonal antibodies against ALK2 described above as well as, for example, polyclonal antibodies similarly having therapeutic and/or prophylactic effects, recombinant antibodies artificially engineered for the purpose of, for example, reducing heterogeneous antigenicity against humans, for example, chimeric antibodies, humanized antibodies, human antibodies, and the like. These antibodies may be produced by use of known methods. Examples of the chimeric antibody may include chimeric antibodies comprising variable regions and constant regions (Fc) of antibodies derived from different species, for example, the variable regions of a mouse- or rat-derived antibody joined to human-derived constant regions (see Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984)). Examples of the humanized antibody may include an antibody comprising CDRs alone integrated into a human-derived antibody (see Nature (1986) 321, p. 522-525), and an antibody comprising the CDR sequences as well as amino acid residues of a portion of frameworks grafted into a human antibody by a CDR grafting method (International Publication No. WO 90/07861). Examples of the anti-ALK2 antibody that may be used in the present invention may include, but are not limited to, the following anti-ALK2 antibodies a comprising heavy chain variable region sequence and a light chain variable region sequence. An anti-ALK2 antibody in which a heavy chain sequence of the anti-ALK2 antibody or the antigen-binding fragment thereof comprises a variable region having CDRH1, CDRH2, and CDRH3, wherein the CDRH1, the CDRH2, and the CDRH3 consist of the amino acid sequences ofSEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively;SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively;SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; orSEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively, and a light chain sequence thereof comprises a variable region having CDRL1, CDRL2, and CDRL3, wherein the CDRL1, the CDRL2, and the CDRL3 consist of the amino acid sequences ofSEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively;SEQ ID NO: 8, SEQ ID NO: 17, and SEQ ID NO: 10, respectively;SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively;SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; orSEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively, and an antibody that competes, for binding to the ALK2, with the anti-ALK2 antibody, and has a property of cross-linking the ALK2 and a property of inhibiting BMP signal transduction. Alternatively, an anti-ALK2 antibody in which the heavy chain variable region sequence of the anti-ALK2 antibody or the antigen-binding fragment thereof is:a1) an amino acid sequence consisting of amino acid residues from position 20 to position 142 of the amino acid sequence of SEQ ID NO: 31;a2) an amino acid sequence consisting of amino acid residues from position 20 to position 142 of the amino acid sequence of SEQ ID NO: 33;a3) an amino acid sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 34;a4) an amino acid sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 36;a5) an amino acid sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 38;a6) an amino acid sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 39;a7) an amino acid sequence having at least 95% identity to any one amino acid sequence selected from the amino acid sequences a1) to a6);a8) an amino acid sequence having at least 99% identity to any one amino acid sequence selected from the amino acid sequences a1) to a6); ora9) an amino acid sequence comprising a substitution(s), a deletion(s), or an addition(s) of one or several amino acid residues in any one amino acid sequence selected from the amino acid sequences a1) to a6), and the light chain variable region sequence isb1) an amino acid sequence consisting of amino acid residues from position 21 to position 133 of the amino acid sequence of SEQ ID NO: 32;b2) an amino acid sequence consisting of amino acid residues from position 21 to position 129 of the amino acid sequence of SEQ ID NO: 35;b3) an amino acid sequence consisting of amino acid residues from position 21 to position 129 of the amino acid sequence of SEQ ID NO: 37;b4) an amino acid sequence having at least 95% identity to any one amino acid sequence selected from the amino acid sequences b1) to b3);b5) an amino acid sequence having at least 99% identity to any one amino acid sequence selected from the amino acid sequences b1) to b3); orb6) an amino acid sequence comprising a substitution(s), a deletion(s), or an addition(s) of one or several amino acid residues in any one amino acid sequence selected from the amino acid sequences b1) to b3), and an antibody that competes, for binding to the ALK2, with the anti-ALK2 antibody, and has a property of cross-linking the ALK2 and a property of inhibiting BMP signal transduction. Further specifically, examples of the anti-ALK2 antibody that may be used in the present invention may include anti-ALK2 antibodies disclosed in WO 2016/121908 by the present inventors. Examples of the rat anti-ALK2 antibody may include A2-11E, A2-15A, A2-25C, and A2-27D described in Example 1 of WO 2016/121908. Examples of the human chimeric anti-ALK2 antibody may include cA2-15A and cA2-27D described in Example 5 of WO 2016/121908. The humanized antibody derived from the A2-15A antibody is included in the antibody used in the present invention as long as the humanized antibody contains all of the 6 CDR sequences of A2-15A and has binding activity and cross-linking ability to ALK2. The heavy chain variable region of the A2-15A antibody comprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 5 (GFTFSHYYMA), CDRH2 consisting of the amino acid sequence of SEQ ID NO: 6 (SITNSGGSINYRDSVKG), and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 7 (EGGENYGGYPPFAY). The light chain variable region of the A2-15A antibody comprises CDRL1 consisting of the amino acid sequence of SEQ ID NO: 8 (RANQGVSLSRYNLMH), CDRL2 consisting of the amino acid sequence of SEQ ID NO: 9 (RSSNLAS), and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 10 (QQSRESPFT). Further, an antibody that competes, for binding to the ALK2, with the A2-15A antibody, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included in the present invention. The humanized antibody derived from the A2-27D antibody is included in the antibody used in the present invention as long as the humanized antibody contains all of the 6 CDR sequences of A2-27D and has binding activity and cross-linking ability to ALK2. The heavy chain variable region of the A2-27D antibody comprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 11 (GSTFSNYGMK), CDRH2 consisting of the amino acid sequence of SEQ ID NO: 12 (SISRSSTYIYYADTVKG), and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 13 (AISTPFYWYFDF). The light chain variable region of the A2-27D antibody comprises CDRL1 consisting of the amino acid sequence of SEQ ID NO: 14 (LASSSVSYMT), CDRL2 consisting of the amino acid sequence of SEQ ID NO: 15 (GTSNLAS), and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 16 (LHLTSYPPYT). Further, an antibody that competes, for binding to the ALK2, with the A2-27D antibody, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included in the present invention. The humanized antibody derived from the A2-11E antibody is included in the antibody used in the present invention as long as the humanized antibody contains all of the 6 CDR sequences of A2-11E and has binding activity and cross-linking ability to ALK2. The heavy chain variable region of the A2-11E antibody comprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 18 (GFTFSNYYMY), CDRH2 consisting of the amino acid sequence of SEQ ID NO: 19 (SINTDGGSTYYPDSVKG), and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 20 (STPNIPLAY). The light chain variable region of the A2-11E antibody comprises CDRL1 consisting of the amino acid sequence of SEQ ID NO: 21 (KASQNIYKYLN), CDRL2 consisting of the amino acid sequence of SEQ ID NO: 22 (YSNSLQT), and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 23 (FQYSSGPT). Further, an antibody that competes, for binding to the ALK2, with the A2-11E antibody, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included therein. The humanized antibody derived from the A2-25C antibody is included in the antibody used in the present invention as long as the humanized antibody contains all of the 6 CDR sequences of A2-25C and has binding activity and cross-linking ability to ALK2. The heavy chain variable region of the A2-25C antibody comprises CDRH1 consisting of the amino acid sequence of SEQ ID NO: 24 (GFTFSYYAMS), CDRH2 consisting of the amino acid sequence of SEQ ID NO: 25 (SISRGGDNTYYRDTVKG), and CDRH3 consisting of the amino acid sequence of SEQ ID NO: 26 (LNYNNYFDY). The light chain variable region of the A2-25C antibody comprises CDRL1 consisting of the amino acid sequence of SEQ ID NO: 27 (QASQDIGNWLS), CDRL2 consisting of the amino acid sequence of SEQ ID NO: 28 (GATSLAD), and CDRL3 consisting of the amino acid sequence of SEQ ID NO: 29 (LQAYSAPFT). Further, an antibody that competes, for binding to the ALK2, with the A2-25C antibody, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included in the present invention. A CDR-modified humanized antibody prepared by substitution of 1 to 3 amino acid residues in each CDR by other amino acid residues is also included in the antibody used in the present invention as long as the humanized antibody has binding activity and cross-linking ability to ALK2. Examples of the amino acid substitution in CDRL2 may include the substitution of one amino acid of CDRL2 in the amino acid sequence of SEQ ID NO: 30 (humanized hA2-15A-L4). CDRL2 consisting of the amino acid sequence of SEQ ID NO: 17 (RSSNLAQ) is preferred. Actual examples of the humanized antibody derived from the A2-15A antibody may include: an antibody consisting of a heavy chain comprising a heavy chain variable region sequence consisting of amino acid residues from position 20 to position 142 of the amino acid sequence of SEQ ID NO: 31 (humanized hA2-15A-H4) and a light chain comprising a light chain variable region sequence consisting of amino acid residues from position 21 to position 133 of the amino acid sequence of SEQ ID NO: 32 (humanized hA2-15A-L6), and an antibody consisting of a heavy chain comprising a heavy chain variable region sequence consisting of amino acid residues from position 20 to position 142 of the amino acid sequence of SEQ ID NO: 33 (humanized hA2-15A-H4 IgG2) and a light chain comprising a light chain variable region sequence consisting of amino acid residues from position 21 to position 133 of the amino acid sequence of SEQ ID NO: 32, and an antibody that competes, for binding to the ALK2, with any of the A2-15A antibodies, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included in the present invention. Preferred examples of the combination may include: an antibody consisting of a heavy chain comprising an amino acid sequence consisting of amino acid residues from position 20 to 472 of the amino acid sequence of SEQ ID NO: 31 and a light chain comprising an amino acid sequence consisting of amino acid residues from position 21 to position 238 of the amino acid sequence of SEQ ID NO: 32, and an antibody consisting of a heavy chain comprising an amino acid sequence consisting of amino acid residues from position 20 to position 468 of the amino acid sequence of SEQ ID NO: 33 and a light chain comprising an amino acid sequence consisting of amino acid residues from position 21 to position 238 of the amino acid sequence of SEQ ID NO: 32, and an antibody that competes, for binding to the ALK2, with any of the antibodies, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included therein. Actual examples of the humanized antibody derived from the A2-27D antibody may include: an antibody consisting of a heavy chain comprising a heavy chain variable region sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 34 (humanized hA2-27D-H2) and a light chain comprising a light chain variable region sequence consisting of amino acid residues from position 21 to position 129 of the amino acid sequence of SEQ ID NO: 35 (humanized hA2-27D-L2); an antibody consisting of a heavy chain comprising a heavy chain variable region sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 36 (humanized hA2-27D-H3) and a light chain comprising a light chain variable region sequence consisting of amino acid residues from position 21 to position 129 of the amino acid sequence of SEQ ID NO: 37 (humanized hA2-27D-L4); an antibody consisting of a heavy chain comprising a heavy chain variable region sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 38 (humanized hA2-27D-H2-LALA) and a light chain comprising a light chain variable region sequence consisting of amino acid residues from position 21 to position 129 of the amino acid sequence of SEQ ID NO: 35; and an antibody consisting of a heavy chain comprising a heavy chain variable region sequence consisting of amino acid residues from position 20 to position 140 of the amino acid sequence of SEQ ID NO: 39 (humanized hA2-27D-H3-LALA) and a light chain comprising a light chain variable region sequence consisting of amino acid residues from position 21 to position 129 of the amino acid sequence of SEQ ID NO: 37; and an antibody that competes, for binding to the ALK2, with any of the antibodies, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included in the present invention. Preferred examples of the combination may include: an antibody consisting of a heavy chain comprising an amino acid sequence consisting of amino acid residues from position 20 to position 470 of the amino acid sequence of SEQ ID NO: 34 and a light chain comprising an amino acid sequence consisting of amino acid residues from position 21 to position 234 of the amino acid sequence of SEQ ID NO: 35; an antibody consisting of a heavy chain comprising an amino acid sequence consisting of amino acid residues from position 20 to position 470 of the amino acid sequence of SEQ ID NO: 36 and a light chain comprising an amino acid sequence consisting of amino acid residues from position 21 to position 234 of the amino acid sequence of SEQ ID NO: 37; an antibody consisting of a heavy chain comprising an amino acid sequence consisting of amino acid residues from position 20 to position 470 of the amino acid sequence of SEQ ID NO: 38 and a light chain comprising an amino acid sequence consisting of amino acid residues from position 21 to position 234 of the amino acid sequence of SEQ ID NO: 35; and an antibody consisting of a heavy chain comprising an amino acid sequence consisting of amino acid residues from position 20 to position 470 of the amino acid sequence of SEQ ID NO: 39 and a light chain comprising an amino acid sequence consisting of amino acid residues from position 21 to position 234 of the amino acid sequence of SEQ ID NO: 37;and an antibody that competes, for binding to the ALK2, with any of the antibodies, and has a property of cross-linking the ALK2 and a property of inhibiting the BMP signal transduction is also included in the present invention. Further examples of the antibody used in the present invention may include a human antibody. The anti-ALK2 human antibody means a human antibody produced from only human chromosome-derived antibody gene sequences. The anti-ALK2 human antibody may be obtained by a method using human antibody-producing mice carrying human chromosome fragments that comprise human antibody heavy and light chain genes (see e.g., Tomizuka, K. et al., Nature Genetics (1997), 16, p. 133-143; Kuroiwa, Y. et al., Nuc. Acids Res. (1998), 26, p. 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects vol. 10, p. 69-73 Kitagawa, Y., Matsuda, T. and lijima, S. eds.), Kluwer Academic Publishers, 1999; and Tomizuka, K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, p. 722-727). Specifically, such a human antibody-producing mouse may be created as a recombinant animal in which the endogenous immunoglobulin heavy and light chain gene loci have been disrupted and instead human immunoglobulin heavy and light chain gene loci are integrated via a vector, for example, a human artificial chromosome (HAC) vector or a mouse artificial chromosome (MAC) vector, by preparing a knockout animal or a transgenic animal or by crossing these animals. Alternatively, eukaryotic cells may be transformed with cDNAs encoding the heavy and light chains, respectively, of such a human antibody, preferably with vectors comprising the cDNAs, by gene recombination techniques. The transformed cells producing a recombinant human monoclonal antibody may be cultured to obtain this antibody from the culture supernatant. In this context, for example, eukaryotic cells, preferably mammalian cells such as CHO cells, lymphocytes, or myeloma cells, may be used as hosts. Also, a method for obtaining a phage display-derived human antibody selected from a human antibody library (see e.g., Wormstone, I. M. et al., Investigative Ophthalmology & Visual Science (2002), 43 (7), p. 2301-2308; Carmen, S. et al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; and Siriwardena, D. et al., Ophthalmology (2002), 109 (3), p. 427-431) is known. For example, a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105-1116) may be used, which involves allowing the variable regions of a human antibody to be expressed as single-chain Fv (scFv) on phage surface and selecting a phage binding to the antigen. The phage selected on the basis of its ability to bind to the antigen may be subjected to gene analysis to determine DNA sequences encoding the variable regions of the human antibody binding to the antigen. If the DNA sequence of scFv binding to the antigen is determined, an expression vector having this sequence may be prepared and transferred to appropriate hosts, followed by expression to obtain the human antibody (WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388, Annu. Rev. Immunol (1994), 12, p. 433-455; and Nature Biotechnology (2005), 23 (9), p. 1105-1116). Antibodies binding to the same epitope as that for an anti-ALK2 antibody disclosed in WO 2016/121908 are also included in the anti-ALK2 antibody that may be used in the present invention. Examples thereof include antibodies binding to the same epitope as that for the A2-11E antibody, the A2-15A antibody, the A2-25C antibody, and/or the A2-27D antibody. When an antibody binds to or recognizes a partial conformation of an antigen, the epitope for this antibody may be determined by identifying amino acid residues on the antigen adjacent to the antibody by use of X-ray structure analysis. For example, the antibody or a fragment thereof and the antigen or a fragment thereof may be bound to each other, crystallized, and structurally analyzed to identify amino acid residues on the antigen having an interaction distance between the amino acid residue and the antibody. The interaction distance is 8 angstroms or smaller, preferably 6 angstroms or smaller, more preferably 4 angstroms or smaller. One or more amino acid residues having such an interaction distance with the antibody may constitute an epitope (or an antigenic determinant) for the antibody. When the number of such amino acid residues is two or more, these amino acids may not be adjacent to each other on the primary sequence. Examples of the antibody or an antigen-binding fragment thereof binding to the epitope of the ALK2 protein are as described below. The anti-ALK2 antibody or the antigen-binding fragment thereof may specifically bind to a polypeptide consisting of amino acid residues from position 21 to position 123 in the amino acid sequence (SEQ ID NO: 1) of human ALK2. The A2-27D antibody recognizes a partial conformation on human ALK2. In the amino acid sequence (SEQ ID NO: 1) of human ALK2, the amino acid residues having an interaction distance with the A2-27D antibody, i.e., the epitope, is constituted by each of the residues of glutamic acid (Glu) at position 38, glycine (Gly) at position 39, isoleucine (Ile) at position 59, asparagine (Asn) at position 60, aspartic acid (Asp) at position 61, glycine (Gly) at position 62, phenylalanine (Phe) at position 63, histidine (His) at position 64, valine (Val) at position 65, tyrosine (Tyr) at position 66, asparagine (Asn) at position 102, threonine (Thr) at position 104, glutamine (Gln) at position 106, and leucine (Leu) at position 107. The antibody, an antigen-binding fragment thereof, or a modified form of the antibody or the fragment which binds to this epitope or has an interaction distance between the antibody or the fragment and each of the amino acid residues are also encompassed in the antibody used in the present invention. The A2-25C antibody recognizes a partial conformation on human ALK2. In the amino acid sequence (SEQ ID NO: 1) of human ALK2, the amino acid residues having an interaction distance with the A2-25C antibody, i.e., the epitope, is constituted by each of the residues of glutamic acid (Glu) at position 38, glycine (Gly) at position 39, leucine (Leu) at position 40, isoleucine (Ile) at position 59, asparagine (Asn) at position 60, aspartic acid (Asp) at position 61, glycine (Gly) at position 62, phenylalanine (Phe) at position 63, histidine (His) at position 64, valine (Val) at position 65, tyrosine (Tyr) at position 66, and threonine (Thr) at position 104. The antibody, an antigen-binding fragment thereof, or a modified form of the antibody or the fragment which binds to this epitope or has an interaction distance with these amino acid residues are also encompassed in the antibody used in the present invention. Alternatively, the anti-ALK2 antibody or the antigen-binding fragment thereof may be an antibody or an antigen-binding fragment thereof that competes, for binding to ALK2, with the anti-ALK2 antibody or the antigen-binding fragment thereof described above (e.g., the A2-27D antibody and the A2-25C antibody). The antibody described above may be evaluated for its binding activity to the antigen by, for example, a method described in Example 2, 6, 9, or 10 of WO 2016/121908 to select suitable antibodies. The dissociation constant (KD) of the antibody is, for example, 1×10−6to 1×10−12M or less, but is not limited to this range as long as the therapeutic or prophylactic effects of interest are obtained. The dissociation constant of the antibody for the antigen (ALK2) may be measured using Biacore T200 GE Healthcare Bio-Sciences Corp.) based on surface plasmon resonance (SPR) as detection principles. For example, the antibody set to an appropriate concentration is reacted as an analyte with the antigen immobilized as a ligand on a solid phase. The association and dissociation between the antibody and the antigen may be measured to obtain an association rate constant ka1, a dissociation rate constant kd1, and a dissociation constant (KD; KD=kd1/ka1). The evaluation of binding activity to ALK2 is not limited to use of Biacore T200 and may be conducted using, for example, an instrument based on surface plasmon resonance (SPR) as detection principles, KinExA (Sapidyne Instruments Inc.) based on kinetic exclusion assay as detection principles, BLItz system (Pall Corp.) based on bio-layer interferometry as detection principles, or ELISA (enzyme-linked immunosorbent assay). The antibody described above may be evaluated for its cross-linking ability to the antigen by, for example, a method described in Example 4 mentioned later to select suitable antibodies. Specifically, a fusion body of ALK2 and LgBiT or SmBiT is expressed in in vitro cells using NanoLuc® Binary Technology: NanoBiT® (Promega Corp.), and the interaction of the ALK2 protein with the antibody may be detected from luminescence brought about by structural complementarity of LgBiT and SmBiT. One example of another indicator for comparing the properties of antibodies may include the stability of the antibodies. Differential scanning calorimetry (DSC) is a method that may rapidly and accurately measure a transition midpoint (Tm), which serves as a good indicator for the relative structural stability of proteins. Tm values may be measured using DSC and compared to determine difference in thermal stability. The preservation stability of an antibody is known to correlate with the thermal stability of the antibody to some extent (Lori Burton, et al., Pharmaceutical Development and Technology (2007) 12, p. 265-273). A suitable antibody may be selected using its thermal stability as an indicator. Examples of other indicators for selecting the antibody may include high yields in appropriate host cells and low aggregation in an aqueous solution. For example, an antibody having the highest yield does not always exhibit the highest thermal stability. Therefore, it is necessary to select an antibody most suitable for administration to humans by comprehensive judgment based on the indicators mentioned above. A method for obtaining a single-chain immunoglobulin by linking the full-length sequences of antibody heavy and light chains via an appropriate linker is also known (Lee, H-S, et al., Molecular Immunology (1999) 36, p. 61-71; and Shirrmann, T. et al., mAbs (2010), 2, (1) p. 1-4). Such single-chain immunoglobulins may be dimerized to retain a structure and activity similar to those of antibodies which are originally tetramers. Alternatively, the antibody used in the present invention may be an antibody that has a single heavy chain variable region and lacks a light chain sequence. Such an antibody, which is called a single-domain antibody (sdAb), a nanobody, or an antibody of Camelidae family (heavy chain antibody), has actually been observed in camels or llamas and reported to have an ability to bind to an antigen (Muyldemans S. et al., Protein Eng. (1994) 7 (9), 1129-35; and Hamers-Casterman C. et al., Nature (1993) 363 (6428) 446-8). These antibodies may also be interpreted as an antigen-binding fragment of the antibody according to the present invention. The antibody-dependent cellular cytotoxic activity of the antibody used in the present invention may be enhanced by controlling the modification of the sugar chain bound with the antibody. For example, methods described in WO 99/54342, WO 2000/61739, and WO 2002/31140 are known as such a technique of controlling the sugar chain modification of the antibody, though this technique is not limited thereto. In the case of preparing an antibody by isolating the antibody genes and then transferring the genes to an appropriate host, the appropriate host may be used in combination with an expression vector. Specific examples of the antibody genes may include a gene (or a polynucleotide) encoding a heavy chain sequence and a gene (or a polynucleotide) encoding a light chain sequence of the antibody as described in WO 2016/121908, and a combination of these genes (or polynucleotides). For the transformation of host cells, a heavy chain sequence gene (or polynucleotide) and a light chain sequence gene (or polynucleotide) may be inserted in a same expression vector or may be inserted in distinct expression vectors. When eukaryotic cells are used as hosts, animal cells, plant cells, or eukaryotic microorganisms may be used. Examples of the animal cells may include mammalian cells, for example, simian COS cells (Gluzman, Y., Cell (1981) 23, p. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), and dihydrofolate reductase-deficient cell lines (Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. U.S.A. (1980) 77, p. 4126-4220) of Chinese hamster ovary cells (CHO cells, ATCC CCL-61). In the case of using prokaryotic cells, examples thereof may includeE. coliandBacillus subtilis. The antibody gene of interest is transferred to these cells by transformation, and the transformed cells are cultured in vitro to obtain antibodies. Such culture methods may differ in yield depending on the sequences of the antibodies. An antibody that is easy to produce as a drug may be selected using its yield as an indicator from among antibodies having equivalent binding activity. The isotype of the antibody used in the present invention may be any isotype having an ability to cross-link ALK2. Examples thereof may include, but are not limited to, IgGs (IgG1, IgG2, IgG3, and IgG4), IgM, IgAs (IgA1 and IgA2), IgD, and IgE. Preferred examples of the isotypes may include IgG and IgM, more preferably IgG1, IgG2, and IgG4. When IgG1 is used as an isotype of the antibody used in the present invention, the effector functions may be controlled by substituting a part of amino acid residues in constant regions (see WO 88/07089, WO 94/28027, and WO9 4/29351). Examples of such variants of IgG1 include IgG1 LALA (IgG1-L234A, L235A) and IgG1 LAGA (IgG1-L235A, G237A). IgG1 LALA is preferred. When IgG4 is used as an isotype of the antibody used in the present invention, splitting unique to IgG4 can be suppressed to extend the half-life by substituting a part of amino acid residues in constant regions (see Molecular Immunology, 30, 1 105-108 (1993)). An example of such mutant of IgG4 includes IgG4 pro (IgG4-S241P). The antibody used in the present invention may be an antigen-binding fragment of the antibody having antigen-binding sites, or a modified form of the antibody. The fragment of the antibody may be obtained by treating the antibody with a proteolytic enzyme such as papain or pepsin or by expressing a genetically engineered antibody gene in appropriate cultured cells. Among such antibody fragments, a fragment that maintains the whole or a portion of the functions possessed by the full-length molecule of the antibody can be referred to as an antigen-binding fragment of the antibody. Examples of the functions of the antibody may generally include an antigen binding activity, an activity of inhibiting the activity of the antigen, an activity of enhancing the activity of the antigen, an antibody-dependent cellular cytotoxic activity, a complement-dependent cytotoxic activity, and a complement-dependent cellular cytotoxic activity. The function possessed by the antigen-binding fragment of the antibody according to the present invention is an activity to bind ALK2 and an ability to cross-link ALK2. The binding activity to ALK2 is antibody's or antigen-binding fragment's property of (preferably, specifically) binding to the ALK2 molecule, and is preferably an activity of inhibiting the activity of ALK2, more preferably an activity of inhibiting ALK2-mediated BMP signal transduction, most preferably an activity of suppressing, mitigating or causing the regression of ectopic ossification and/or brain tumor. Examples of the fragment of the antibody may include F(ab′)2and the like. The antibody used in the present invention may have enhanced affinity for an antigen by multimerization. A single antibody may be multimerized, or a plurality of antibodies recognizing a plurality of epitopes, respectively, of the same antigen may be multimerized. Examples of a method for multimerizing these antibodies may include the binding of two scFvs to an IgG CH3 domain, the binding to streptavidin, and the introduction of a helix-turn-helix motif. The antibody used in the present invention may be a polyclonal antibody which is a mixture of plural types of anti-ALK2 antibodies, whose amino acid sequences are different from one another. An example of the polyclonal antibody may include a mixture of plural types of antibodies that are different in CDRs. An antibody obtained by culturing a mixture of cells that produce different antibodies, followed by purification from the cultures, may be used as such a polyclonal antibody (see WO 2004/061104). The antibody used in the present invention may be an antibody having 80% to 99% identity when compared with the heavy and/or light chains of the antibody. In this context, the term “identity” has general definition used in the art. The % identity refers to the percentage of the number of identical amino acids relative to the total number of amino acids (including gaps) when two amino acid sequences are aligned so as to give the largest consistency of amino acids. Antibodies that have an ability to bind to the antigen, an inhibitory effect on BMP signal transduction, and cross-linking ability at analogous levels to the antibodies described above may be selected by combining sequences that exhibit high identity to the amino acid sequences of the heavy and light chains. Such identity is generally 80% or 85% or higher identity, preferably 90% or higher, 91% or higher, 92% or higher, 93% or higher or 94% or higher identity, more preferably 95% or higher, 96% or higher, 97% or higher or 98% or higher identity, most preferably 99% or higher identity. Alternatively, antibodies that have various effects equivalent to the antibodies described above may be selected by combining amino acid sequences that comprise a substitution(s), a deletion(s), and/or an addition(s) of one or several amino acid residues in the amino acid sequences of the heavy and/or light chains. The number of amino acid residues to be substituted, deleted, and/or added is generally 10 or less amino acid residues, preferably 5 or 6 or less amino acid residues, more preferably two or three or less amino acid residues, most preferably one amino acid residue. The heavy chain of an antibody produced by cultured mammalian cells is known to lack a carboxyl-terminal lysine residue (Journal of Chromatography A, 705: 129-134 (1995)). Also, the heavy chain of such an antibody is known to lack two carboxyl-terminal amino acid residues (glycine and lysine) and instead have an amidated proline residue at the carboxy terminus (Analytical Biochemistry, 360: 75-83 (2007)). An N-terminal glutamine or glutamic acid residue in the heavy or light chain of an antibody is known to be modified by pyroglutamylation during preparation of the antibody, and the antibody used in the present invention may have such a modification (WO 2013/147153). Such deletion in the heavy chain sequence or modification in the heavy or light chain sequence does not influence the ability of the antibody to bind to the antigen and its effector functions (complement activation, antibody-dependent cytotoxic effects, etc.). Thus, the antibody used in the present invention also encompasses an antibody that has received the deletion or the modification. Examples thereof may include a deletion variant derived from a heavy chain by the deletion of one or two amino acids at its carboxyl terminus, an amidated form of the deletion variant (e.g., a heavy chain having an amidated proline residue at the carboxyl-terminal site), and an antibody having a pyroglutamylated N-terminal amino acid residue in a heavy or light chain thereof. However, the deletion variant at the carboxyl terminus of the antibody heavy chain used in the present invention is not limited to the types described above as long as the deletion variant maintains the ability to bind to the antigen and the effector functions. Two heavy chains constituting the antibody used in the present invention may be heavy chains of any one type selected from the group consisting of the full-length heavy chain and the deletion variants described above, or may be a combination of heavy chains of any two types selected therefrom. The quantitative ratio of each deletion variant may be influenced by the type of cultured mammalian cells producing the antibody according to the present invention, and culture conditions. Examples of such a case may include the deletion of one carboxyl-terminal amino acid residue each in both the two heavy chains as main components of the antibody. The identity between two types of amino acid sequences may be determined using the default parameters of Blast algorithm version 2.2.2 Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402). The Blast algorithm is also available by access to www.ncbi.nlm.nih.gov/blast on the Internet. Two types of percentage values, Identity (or Identities) and Positivity (or Positivities), are calculated according to the Blast algorithm. The former is a value that indicates identical amino acid residues between two types of amino acid sequences that the identity should be determined. The latter is a numerical value determined by also taking into consideration similar amino acid residues in terms of their chemical structures. Herein, the value of identity is defined as the value of “Identity” when amino acid residues are identical between the amino acid sequences. An antibody conjugated with any of various molecules such as polyethylene glycol (PEG) may also be used as a modified form of the antibody. The antibody used in the present invention may further be any of conjugates formed by these antibodies with other drugs (immunoconjugates). Examples of such an antibody may include the antibody conjugated with a radioactive material or a compound having a pharmacological effect (Nature Biotechnology (2005) 23, p. 1137-1146). The obtained antibodies may be purified until becoming homogeneous. Protein separation and purification methods conventionally used may be used for the separation and purification of the antibodies. The antibodies may be separated and purified by appropriately selected or combined approaches, for example, column chromatography, filtration through a filter, ultrafiltration, salting-out, dialysis, preparative polyacrylamide gel electrophoresis, and/or isoelectric focusing (Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R. Marshak et al. eds., Cold Spring Harbor Laboratory Press (1996); and Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory (1988)), though the separation and purification method is not limited thereto. Examples of the chromatography may include affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse-phase chromatography, and adsorption chromatography. These chromatography approaches may be carried out using liquid chromatography such as HPLC or FPLC. Examples of the column for use in the affinity chromatography may include protein A columns and protein G columns. Examples of the protein A columns may include Hyper D, POROS, and Sepharose F. F. (GE Healthcare Bio-Sciences Corp.). Also, the antibody may be purified by exploiting its binding activity to the antigen using an antigen-immobilized carrier. The KDvalue that indicates the binding affinity of the anti-ALK2 antibody according to the present invention for ALK2 is preferably 10−6M or less, for example, 10−7M or less, 10−8M or less, 10−9M or less, 10−10M or less, 10−11M or less, or 10−12M or less. 6. Method for Treating Ectopic Ossification and/or Brain Tumor and Pharmaceutical Composition for Use in the Method The present invention provides a method for treating and/or preventing a disease caused by an active mutation in ALK2, comprising using a biological sample from a patient, detecting the presence or absence of the active mutation in ALK2 in the biological sample, and administering an anti-ALK2 antibody to a patient having the active mutation in ALK2 and having no mutation of an amino acid residue at position 330 proline residue in the human ALK2 sequence). Examples of the disease caused by an active mutation in ALK2 may include fibrodysplasia ossificans progressiva (FOP), progressive osseous heteroplasia (POH), traumatic ectopic ossification, ectopic ossification after implant arthroplasty, diffuse intrinsic pontine glioma (DIPG), spondyloarthritis (SpA), ankylosing spondylitis (AS), anemia, and thinning hair. The disease is preferably fibrodysplasia ossificans progressiva (FOP), progressive osseous heteroplasia (POH), traumatic ectopic ossification, or ectopic ossification after implant arthroplasty, more preferably fibrodysplasia ossificans progressiva (FOP), though the disease is not limited thereto as long as the disease is caused by an active mutation in ALK2. In FOP patients, finger or toe fusion or deformity, cervical fusion or deformity, or the like is also found, and hearing loss is also manifested. These conditions are also included in the disease caused by an active mutation in ALK2. The present invention also provides a pharmaceutical composition for use in a method for treating and/or preventing a patient having ectopic ossification, wherein the patient has an active mutation in ALK2 protein which is responsible for ectopic ossification; an amino acid residue at position 330 of the ALK2 is proline; and an active ingredient of the composition is an anti-ALK2 antibody or an antigen-binding fragment thereof comprising a property of binding to the ALK2, a property of cross-linking the ALK2, and a property of inhibiting BMP signal transduction. In an embodiment, the method comprises the steps of: (a) detecting the presence or absence of an active mutation in ALK2 in patients; (b) selecting a patient having the active mutation in ALK2; c) confirming that the patient has no mutation of an amino acid residue at position 330 of ALK2; and (d) administering the anti-ALK2 antibody or the antigen-binding fragment thereof to the selected patient. In another embodiment, the step (c) further comprises the step of confirming that the ALK2 of the patient has no G328V mutation. In a further alternative embodiment, the selection of the patient to which the anti-ALK2 antibody or the antigen-binding fragment thereof is to be administered comprises the steps of: (a) detecting the presence or absence of an active mutation in ALK2 in ectopic ossification patients; (b) selecting a patient having the active mutation in ALK2; and (c) excluding a patient having a mutation of an amino acid residue at position 330 of ALK2. In another embodiment, the step (c) further comprises the step of excluding a patient having G328V mutation in ALK2. Examples of the “ectopic ossification” according to the present invention may include fibrodysplasia ossificans progressiva (FOP). Fibrodysplasia ossificans progressiva (FOP) is preferred. Active mutations in ALK2 have been confirmed in all FOP patients, and 10 or more types of mutations have been reported so far. All of these mutations have been found to be amino acid mutations (missense mutations) present in the intracellular region of the ALK2 protein and do not cause any change in the amino acid sequence of the extracellular region. Thus, use of the anti-ALK2 antibody binding to the extracellular region of ALK2 produces therapeutic and/or prophylactic effects on FOP, irrespective of the types of mutations. The treatment of FOP means cure of FOP symptoms, amelioration of the symptoms, mitigation of the symptoms, or suppression of progression of the symptoms. The prevention of FOP means circumvention or suppression of onset of flare-up or ectopic ossification. Alternatively, the present invention provides a method for treating and/or preventing brain tumor, comprising using a biological sample derived from a patient, detecting the presence or absence of an active mutation in ALK2 in the biological sample, and administering an anti-ALK2 antibody to a patient having the active mutation other than G328V mutation in ALK2. The present invention further provides a pharmaceutical composition for use in a method for treating and/or preventing a patient having brain tumor, wherein the patient has an active mutation in ALK2 protein which is responsible for brain tumor; and an active ingredient of the composition is an anti-ALK2 antibody or an antigen-binding fragment thereof comprising a property of binding to the ALK2, a property of cross-linking the ALK2, and a property of inhibiting BMP signal transduction. Examples of the “brain tumor” according to the present invention may include diffuse intrinsic pontine glioma (DIPG), brain stem glioma, glioblastoma, glioblastoma multiforme (GBM), non-glioblastoma brain tumor, meningioma, central nervous system lymphoma, glioma, astroglioma, anaplastic astrocytoma, oligodendroglioma, oligoastrocytoma, medulloblastoma, and ependymoma. Diffuse intrinsic pontine glioma (DIPG) is preferred. Active mutations in ALK2 have also been confirmed in DIPG patients. R206H, R258G, G328E, G328V, G328W, and G356D mutants are known as mutants of human ALK2. These mutations, except for the G328V mutation, are also common in FOP patients. The anti-ALK2 antibody exhibits ALK2 inhibitory activity except that the G328V mutation is present. Therefore, the anti-ALK2 antibody used in the present invention has therapeutic and/or prophylactic effects on DIPG in a patient having an active mutation other than G328V mutation in ALK2. The inhibition of the biological activity of ALK2 (BMP signal inhibitory activity) with the anti-ALK2 antibody may be confirmed in vitro, for example, by luciferase assay using reporter plasmids having an insert of a BMP-responsive sequence, SMAD1/5/8 phosphorylation, expression analysis of BMP target genes, or measurement of alkaline phosphatase activity in mouse myoblasts C2C12 induced to differentiate into osteoblasts by stimulation with a BMP ligand. The therapeutic or prophylactic effects of the anti-ALK2 antibody on ectopic ossification may be confirmed in vivo using laboratory animals, for example, by subcutaneously or intravenously administering the anti-ALK2 antibody to ectopic ossification-induced models with BMP ligand-containing pellets transplanted to mouse muscle, or FOP mouse models harboring mutated ALK2, and analyzing ectopic bone formation. Alternatively, the therapeutic or prophylactic effects on brain tumor may be confirmed, for example, by subcutaneously or intravenously administering the anti-ALK2 antibody to models prepared by the administration of patient-derived tumor cells to the brain or under the skin of immunodeficient mice, and analyzing tumor growth or the number of days of survival of the mice. In the method of the present invention, the patient to be treated or prevented is a patient having an active mutation in ALK2, the patient having no mutation of an amino acid residue at position 330 of ALK2 the patient having proline at position 330) or the patient having no G328V mutation (the patient having no substitution of an amino acid residue at position 328 by valine), preferably a patient having no mutation of an amino acid residue at position 330 of ALK2 and having an active mutation other than G328V mutation in ALK2. Examples of the active mutation in ALK2 include L196P, delP197_F198insL (also referred to as “PF-197-8L”), R2021, R206H, Q207E, R258S, R258G, G325A, G328E, G328R, G328W, G356D, and R375P, though the mutation is not limited thereto as long as the mutation activates ALK2. The anti-ALK2 antibody used in the present invention may be administered alone or in combination with at least one additional therapeutic drug for ectopic ossification in the treatment or prevention of ectopic ossification, and can be administered alone or in combination with at least one additional therapeutic drug for brain tumor, radiotherapy, immunotherapy or chemotherapy, etc. in the treatment or prevention of brain tumor. Examples of the additional therapeutic drug for ectopic ossification that may be administered in combination with the anti-ALK2 antibody may include, but are not limited to, anti-inflammatory drugs, steroids, bisphosphonates, muscle relaxants, and retinoic acid receptor (RAR) γ agonists. Examples of the anti-inflammatory drug may include aspirin, diclofenac, indomethacin, ibuprofen, ketoprofen, naproxen, piroxicam, rofecoxib, celecoxib, azathioprine, penicillamine, methotrexate, sulfasalazine, leflunomide, infliximab, and etanercept. Indomethacin, ibuprofen, piroxicam, or celecoxib is preferred. Examples of the steroid may include prednisolone, beclomethasone, betamethasone, fluticasone, dexamethasone, and hydrocortisone. Prednisolone is preferred. Examples of the bisphosphonate may include alendronate, cimadronate, clodronate, etidronate, ibandronate, incadronate, minodronate, neridronate, olpadronate, pamidronate, piridronate, risedronate, tiludronate, and zoledronate. Pamidronate or zoledronate is preferred. Examples of the muscle relaxant may include cyclobenzaprine, metaxalone, and baclofen. Baclofen is preferred. Examples of the retinoic acid receptor γ agonist may include palovarotene. Examples of the additional therapeutic drug for brain tumor that may be administered in combination with the anti-ALK2 antibody may include temozolomide, bevacizumab, carmustine, lomustine, procarbazine hydrochloride, and vincristine. Depending on the condition of ectopic ossification or brain tumor or the intended degree of treatment and/or prevention, two or three or more additional therapeutic drugs may be administered, and these additional therapeutic drugs may be included in the same preparation and thereby administered at the same time. The additional therapeutic drug and the anti-ALK2 antibody may also be included in the same preparation and thereby administered at the same time. Also, the anti-ALK2 antibody and the additional therapeutic drug may be included in distinct preparations and administered at the same time. Alternatively, the additional agent and the anti-ALK2 antibody may be separately administered one after another. Specifically, a therapeutic drug comprising the anti-ALK2 antibody or the antigen-binding fragment thereof as an active ingredient may be administered after administration of the additional therapeutic drug, or the additional therapeutic drug may be administered after administration of the therapeutic drug containing the anti-ALK2 antibody or the antigen-binding fragment thereof as an active ingredient. For administration in gene therapy, a gene for a protein serving as a therapeutic drug for ectopic ossification or brain tumor and the gene for the anti-ALK2 antibody may be inserted at a site downstream of distinct promoter regions or the same promoter region and may be introduced into distinct vectors or the same vector. The anti-ALK2 antibody or the fragment thereof may be conjugated with a therapeutic drug for ectopic ossification or brain tumor to produce a targeted drug conjugate described in M.C. Garnet “Targeted drug conjugates: principles and progress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216. For this purpose, an antibody molecule as well as any antibody fragment is applicable unless their ability to bind to ALK 2 ALK2-recognizing properties) and ability to cross-link ALK2 are completely deleted. Examples of the antibody fragment may include fragments such as F(ab′)2. The conjugation manner of the anti-ALK2 antibody or the fragment of the antibody with the therapeutic drug for FOP may take various forms described in, for example, M.C. Garnet “Targeted drug conjugates: principles and progress”, Advanced Drug Delivery Reviews, (2001) 53, 171-216, G. T. Hermanson “Bioconjugate Techniques” Academic Press, California (1996), Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, 55-123. Specific examples thereof may include a manner in which the anti-ALK2 antibody is chemically conjugated with the therapeutic drug for ectopic ossification or brain tumor either directly or via a spacer such as an oligopeptide, and a manner in which the anti-ALK2 antibody is conjugated with the therapeutic drug for ectopic ossification or brain tumor via an appropriate drug carrier. Examples of the drug carrier may include drug delivery systems (e.g., X. Yu et al., J Nanomater. 2016; 2016:doi:10.1155/2016/1087250; and J. Wang et al., Drug Delivery, 25: 1, 1319-1327, DOI:10.1080/10717544.2018.1477857) such as liposomes, nanoparticles, nanomicelles, and water-soluble polymers. Examples of such a manner via the drug carrier may more specifically include a manner in which the therapeutic drug for ectopic ossification or brain tumor is encapsulated in a liposome and the liposome is conjugated with the antibody, and a manner in which the therapeutic drug for ectopic ossification or brain tumor is chemically conjugated with a water-soluble polymer (compound having a molecular weight on the order of 1000 to 100,000) either directly or via a spacer such as an oligopeptide and the water-soluble polymer is conjugated with the antibody. The conjugation of the antibody (or the fragment) with the therapeutic drug for ectopic ossification or brain tumor or the drug carrier (e.g., a liposome or a water-soluble polymer) may be carried out by a method well known to those skilled in the art, such as a method described in G. T. Hermanson “Bioconjugate Techniques” Academic Press, California (1996), and Putnam and J. Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, 55-123. The encapsulation of the therapeutic drug for ectopic ossification or brain tumor in the liposome may be carried out by a method well known to those skilled in the art, such as a method described in, for example, D. D. Lasic “Liposomes: From Physics to Applications”, Elsevier Science Publishers B. V., Amsterdam (1993). The conjugation of the therapeutic drug for ectopic ossification or brain tumor with the water-soluble polymer may be carried out by a method well known to those skilled in the art, such as a method described in D. Putnam and J Kopecek “Polymer Conjugates with Anticancer Activity” Advances in Polymer Science (1995) 122, 55-123. The conjugate of the antibody (or the fragment) with the protein as a therapeutic drug for ectopic ossification or brain tumor (e.g., an antibody or a fragment thereof) may be prepared by any of the methods described above or a genetic engineering method well known to those skilled in the art. For the administration of the human type anti-ALK2 antibody to a patient, the dose of the anti-ALK2 antibody used in the present invention is, for example, approximately 0.1 to 100 mg/kg body weight, which may be administered once or twice or more per 1 to 180 days. However, the dose and the number of doses should generally be determined in consideration of the sex, body weight, and age of a patient, symptoms, severity, adverse reactions, etc., and therefore, are not limited to the dose or usage described above. Non-limiting examples of formulations comprising the anti-ALK2 antibody used in the present invention may include injections including intravenous drips, suppositories, transnasal formulations, sublingual formulations, and transdermal absorption formulations. The administration route is an oral administration route or a parenteral administration route. Non-limiting examples of the parenteral administration route include intravenous, intraarterial, intramuscular, intrarectal, transmucosal, intradermal, intraperitoneal, and intraventricular routes. 7. Determination of Eligibility of Patient for Treatment and/or Prevention In the present invention, the following methods may be carried out in order to effectively treat and/or prevent a patient having a mutation in ALK2 protein (e.g., an active mutation in ALK2) by the administration of the anti-ALK2 antibody or the pharmaceutical composition comprising the antibody. A first method is a method for predicting a risk of developing an adverse reaction ascribable to the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof, comprising the steps of:(a) detecting the presence or absence of an active mutation in ALK2 and a mutation of an amino acid residue at position 330 of ALK2 of a patient; and(b) determining that when the patient has the active mutation in ALK2 and has no mutation of an amino acid residue at position 330 of ALK2, the patient has a low risk of developing an adverse reaction ascribable to the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof. A second method is a method for predicting responsiveness to treatment and/or prevention by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof, comprising the steps of:(a) detecting the presence or absence of an active mutation in ALK2 and a mutation of an amino acid residue at position 330 of ALK2 of a patient; and(b) determining that when the patient has the active mutation in ALK2 and has no mutation of an amino acid residue at position 330 of ALK2, the patient has responsiveness to treatment and/or prevention by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof. A third method is a method for selecting a patient to be treated and/or prevented by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof, comprising the steps of:(a) detecting the presence or absence of an active mutation in ALK2 and a mutation of an amino acid residue at position 330 of ALK2 of a patient; and(b) selecting the patient as a patient to be treated and/or prevented by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof when the patient has the active mutation in ALK2 and having no mutation of an amino acid residue at position 330 of ALK2. A fourth method is a method for treating and/or preventing a disease by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof, comprising the steps of:(a) detecting the presence or absence of an active mutation in ALK2 and a mutation of an amino acid residue at position 330 of ALK2 of a patient; and(b) administering to the patient the anti-ALK2 antibody or the antigen-binding fragment thereof when the patient has the active mutation in ALK2 and has no mutation of an amino acid residue at position 330 of ALK2. The fourth method may further comprise performing any of the steps (b) of the first to third methods, i.e., (Step (b) of the First Method) the step of determining that when a patient has the active mutation in ALK2 and has no mutation of an amino acid residue at position 330 of ALK2, the patient has a low risk of developing an adverse reaction ascribable to the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof, (Step (b) of the Second Method) the step of determining that when a patient has the active mutation in ALK2 and has no mutation of an amino acid residue at position 330 of ALK2, the patient has responsiveness to treatment and/or prevention by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof, and (Step (b) of the Third Method) the step of selecting the patient as a patient to be treated and/or prevented by the administration of an anti-ALK2 antibody or an antigen-binding fragment thereof when the patient has the active mutation in ALK2 and has no mutation of an amino acid residue at position 330 of ALK2. Through such further step, whether a patient is eligible for treatment and/or prevention by the administration of the anti-ALK2 antibody or the antigen-binding fragment thereof, whether a patient has an adverse reaction, or the like is determined, and, as a result, the anti-ALK2 antibody or the antigen-binding fragment thereof can then be administered to a patient confirmed to be eligible, thereby to elicit therapeutic effects in the patient, thus a so-called personalized medicine can be performed for the patient. As used herein, the term “determination” includes decision, evaluation, or assistance for determination. In the first to fourth methods, the administration of the anti-ALK2 antibody or the antigen-binding fragment thereof is preferably the administration of a pharmaceutical composition described in the section 6. In the first to fourth methods, the step (b) may further comprise a step of confirming that the active mutation in ALK2 is not G328V mutation. In the first to fourth methods, the active mutation in ALK2 is preferably at least one selected from L196P, delP197_F198insL, R2021, R206H, Q207E, R258S, R258G, G325A, G328E, G328R, G328W, G356D, and R375P, or at least one selected from R206H, R258G, G328E, G328W, and G356D. Moreover, in the first to fourth methods, the above-mentioned patient is a subject having an unidentified disease, or a subject suspected of having a disease caused by an active mutation in ALK2. The disease to be treated is, for example, a disease caused by an active mutation in ALK2, preferably ectopic ossification or brain tumor, more preferably ectopic ossification. Specific examples of these diseases are described in the section 6. The disease is further preferably fibrodysplasia ossificans progressiva (FOP) or diffuse intrinsic pontine glioma (DIPG), still further preferably fibrodysplasia ossificans progressiva (FOP), although the disease is not intended to be limited thereto. EXAMPLES The present invention will be specifically described hereinafter with reference to Examples; however, the invention is not limited thereto. In the following Examples, unless otherwise specified, any procedures concerning genetic manipulation were performed in accordance with methods described in “Molecular Cloning” (Sambrook, J., Fritsch, E. F., and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989), or where commercially available reagents or kits were used, they were used in accordance with the manuals for such commercial products. Example 1 Evaluation of BMP signal transduction-activating effect of Anti-ALK2 antibody (27D-H2L2_LALA) by luciferase reporter assay The anti-ALK2 antibody (27D-H2L2_LALA) used in the experiment was prepared by the method described in Example 12 of WO 2016/121908. The BMP intracellular signal transduction-activating effect mediated by the anti-ALK2 antibody prepared was analyzed using a BMP-specific luciferase reporter. HEK293A cells were seeded into a 96-well white plate for luciferase assay (manufactured by Corning, Inc.) at 1×104cells/well, and cultured overnight in 10% FBS-containing DMEM medium under the conditions of 5% CO2at 37° C. On the next day, each of human or mouse wild-type ALK2-expressing or R206H mutant-expressing plasmids was introduced together with pGL4.26/Id1WT4F-luc (Genes Cells, 7, 949 (2002)), into the cells using Lipofectamine 2000 manufactured by Invitrogen Corp.). After 3 hours, the medium was exchanged with fresh OPTI-MEM I (manufactured by Life Technologies Corp.). Then, the serially diluted antibody was added, and the cells were further cultured overnight. On the next day, the luciferase activity was measured using the plate reader SpectraMaxM4 manufactured by Molecular Devices, LLC) and using One-Glo Luciferase Assay System (manufactured by Promega Corp.). The results are shown inFIG.1. 27D-H2L2_LALA was confirmed to elevate BMP-specific luciferase activity in a concentration-dependent manner only in HEK293 cells that express the R206H mutant of mouse ALK2 lower panel ofFIG.1A). On the other hand, this antibody was not confirmed to elevate BMP reporter activity in cells that express the R206H mutant of human ALK2 lower panel ofFIG.1B) or human or mouse wild-type ALK2 upper panels ofFIGS.1A and1B). Example 2 Preparation of Fab (27D-H2L2_FAB) and F(ab′)2(27D-H2L2_F(ab)2) of anti-ALK2 antibody (27D-H2L2_LALA) 2)-1 Preparation of Fab from 27D-H2L2_LALA 27D-H2L2_LALA was restrictively cleaved with Papain from Papaya latex (Sigma-Aldrich Co. LLC), to remove Fc fragments and the like using HiLoad 26/600 Superdex 200 pg (GE Healthcare Japan Corp.). Then, unreacted 27D-H2L2_LALA was separated using HiTrap Mab Select SuRe, 1 mL (GE Healthcare Japan Corp.) to collect Fab. 2)-2 Preparation of F(ab′)2from 27D-H2L2_LALA 27D-H2L2_LALA was restrictively cleaved with Endoproteinase Glu-C (Sigma-Aldrich Co. LLC), and unreacted 27D-H2L2_LALA was separated using HiTrap Mab Select SuRe, 10 mL (GE Healthcare Japan Corp.). Then, F(ab′)2was collected using Bio-Scale CHT Type I, 5 mL (Bio-Rad Laboratories, Inc.). Example 3 Evaluation of BMP signal transduction-activating effects of Fab (27D-H2L2_Fab) and F(ab′)2(27D-H2L2_F(ab)2) of anti-ALK2 antibody by luciferase reporter assay The BMP intracellular signal transduction-activating effects mediated by 27D-H2L2_Fab and 27D-H2L2_F(ab)2prepared in Example 2 were analyzed using a BMP-specific luciferase reporter. The comparative control used was the full-length anti-ALK2 antibody 27D-H2L2_LALA. The luciferase reporter assay was conducted by the same way as in Example 1. The results are shown inFIG.2. 27D-H2L2_F(ab)2was confirmed to elevate BMP-specific luciferase activity in a concentration-dependent manner only in HEK293 cells that express the R206H mutant of mouse ALK2, as with 27D-H2L2_LALA. On the other hand, 27D-H2L2_Fab was not confirmed to elevate BMP reporter activity under any of the conditions. The results are shown inFIG.2. 27D-H2L2_F(ab)2was confirmed to elevate BMP-specific luciferase activity in a concentration-dependent manner only in HEK293 cells that express the R206H mutant of mouse ALK2, as in 27D-H2L2_LALA. On the other hand, 27D-H2L2_Fab was not confirmed to elevate BMP reporter activity under any of the conditions. Example 4 Evaluating in vitro activity of cross-linking ALK2 molecules by anti-ALK2 antibody NanoBiT assay (manufactured by Promega Corp.) was conducted in order to verify the possibility that the effect of activating the BMP-specific luciferase reporter by 27D-H2L2_LALA and 27D-H2L2_F(ab)2, confirmed in Examples 1 and 3, was mediated by the cross-link between two ALK2 molecules. A nucleotide sequence encoding the full-length human ALK2 was inserted into pBit1.1-C [TK/LgBiT] and pBit2.1-C [TK/SmBiT] Vectors (manufactured by Promega Corp.) to construct expression vectors. C2C12 cells were seeded into a 96-well white plate for luciferase assay (manufactured by Greiner Group AG) at 5×103cells/well, and cultured overnight in 15% FBS-containing DMEM medium under the conditions of 5% CO2at 37° C. On the next day, two types of ALK2 expression plasmids were introduced into the cells using Lipofectamine 2000 manufactured by Invitrogen Corp.). After 2.5 hours, the medium was replaced with fresh OPTI-MEM I (manufactured by Life Technologies Corp.), and the cells were further cultured overnight. On the next day, the serially diluted antibody was added together with a substrate of Nano-Glo Live Cell Assay System (manufactured by Promega Corp.), and the cells were cultured for 15 minutes. Then, the luciferase activity was measured using a plate reader GENios (manufactured by Tecan Trading AG). The results are shown inFIG.3. It was confirmed that A2-27D, 27D-H2L2_LALA and 27D-H2L2_F(ab)2promoted the cross-link formation of ALK2 or the formation of ALK2 complex) in an antibody concentration-dependent manner, whereas 27D-H2L2_Fab did not induce the cross-link formation of ALK2 or the complex formation of ALK2). Example 5 Verifying influence of amino acid substitutions at positions 182 and 330 on the effect of activating the BMP-specific luciferase reporter by anti-ALK2 antibody 5)-1 Alignment of amino acid sequences of full-length ALK2 among human, cynomolgus monkey, dog, rat, and mouse Results of the sequence alignment are shown inFIG.4. When the amino acids of the human, cynomolgus monkey, dog, rat and mouse ALK2 intracellular regions were compared with one another, they were different in amino acid residues at positions 182 and 330. 5)-2 Verifying influence of amino acid substitutions at positions 182 and 330 on the effect of activating the BMP-specific luciferase reporter by anti-ALK2 antibody In order to analyze the roles of D182E and P330S differing between the human and mouse ALK2 intracellular regions, expression vectors were constructed using pcDEF3 such that D182E or P330S mutation was introduced into each of wild-type human ALK2 and R206H mutants of human ALK2. HEK293A cells were seeded into a 96-well white plate for luciferase reporter assay (manufactured by Greiner Group AG) at 1×104cells/well, and cultured overnight in 10% FBS-containing DMEM medium under the conditions of 5% CO2at 37° C. On the next day, each of ALK2 expression vector, pGL4.26/Id1WT4F-luc (Genes Cells, 7, 949 (2002)), and phRL SV40 manufactured by Promega Corp.) was introduced into the cells using Lipofectamine 2000 manufactured by Invitrogen Corp.). After 2.5 hours, the medium was exchanged with fresh OPTI-MEM I (manufactured by Life Technologies Corp.) containing the serially diluted antibody A2-27D, and the cells were further cultured overnight. On the next day, the firefly andRenillaluciferase activities were measured using a plate reader GENios (manufactured by Tecan Trading AG), and using Dual-Glo Luciferase Assay System (manufactured by Promega Corp.). The results are shown inFIG.5. A2-27D was confirmed to elevate activity in a concentration-dependent manner only for the R206H mutants of human ALK2 harboring P330S mutation, as in the R206H mutant of mouse ALK2. Example 6 Verifying influence of amino acid substitutions at position 330 on the effect of activating the BMP-specific luciferase reporter by anti-ALK2 antibody In order to analyze the role of P330 of human ALK2, expression vectors were constructed using pcDEF3 such that P330D, P330E, P330A, or P330V mutation was introduced into each of wild-type human ALK2 and R206H mutants of human ALK2. In order to analyze the role of 5330 of mouse ALK2, expression vectors were constructed such that S330P mutation was introduced into each of wild-type mouse ALK2 and R206H mutants of mouse ALK2. HEK293A cells were transfected with these expression vectors by the same way as in Example 5 and cultured overnight in a medium containing A2-27D, followed by luciferase activity measurement. The results are shown inFIG.6. A2-27D inhibited the activity for the mouse R206H mutant harboring the introduced S330P mutation (i.e., antagonistic activity), whereas A2-27D promoted the activity when the amino acid at this position was 5330 where the amino acid residue at position 330 is serine.) (i.e., agonistic activity). On the other hand, it was revealed that A2-27D promoted the activity for the human R206H mutants harboring the introduced P33 0 S, P330D, P330E, or P330A mutation (i.e., agonistic activity), whereas the antibody inhibited the activity when the mutation was P330V. Example 7 Evaluation of BMP signal transduction-activating effects of four types of anti-ALK2 antibodies (27D-H2L2_LALA, 15A-H4L6_IgG2, A2-11E, and A2-25C) by luciferase reporter assay The anti-ALK2 antibodies (27D-H2L2_LALA, 15A-H4L6_IgG2, A2-11E, and A2-25C) used in the experiment were prepared by the methods described in Examples 12, 11 and 1 of WO 2016/121908. The BMP intracellular signal transduction-activating effects mediated by the anti-ALK2 antibodies prepared were analyzed by the same way as in Example 1 using a BMP-specific luciferase reporter. The results are shown inFIG.7. 15A-H4L6_IgG2, A2-11E, and A2-25C were confirmed to elevate BMP-specific luciferase activity in a concentration-dependent manner only in HEK293 cells that express the R206H mutant of mouse ALK2, as in 27D-H2L2_LALA. On the other hand, none of these antibodies were confirmed to elevate BMP reporter activity in cells expressing the R206H mutant of human ALK2. Example 8 Verifying effect of activating the BMP-specific luciferase reporter by anti-ALK2 antibody on various ALK2 mutants other than R206H mutant Expression vectors were constructed using pcDEF3 such that each of fourteen types of human ALK2 mutants (L196P, P197F198del_insL (also referred to as PF197-8L), R2021, R206H, Q207E, R258G, R258S, G325A, G328E, G328R, G328V, G328W, G356D, and R375P mutants) found in FOP and DIPG, and a constitutively active Q207D mutant, were introduced into each vector. HEK293 cells were caused to overexpress these mutants by the same way as in Examples 5 and 6, and cultured overnight in a medium containing serially diluted A2-27D, followed by luciferase activity measurement. In this experiment, the G328V mutant and the Q207D mutant were used in the assay such that their amounts were 1/3 of the amount of the other mutants (e.g., 12.5 ng/well relative to 37.5 ng each of the other mutants/well) and 1/20 e.g., 1.875 ng/well relative to 37.5 ng each of the other mutants/well). The results are shown inFIG.8. It was confirmed that A2-27D promoted the activity in a concentration-dependent manner for the G328V mutant found only in DIPG and the constitutively active Q207D mutant among the human ALK2 mutants, but that A2-27D inhibited the activity in a concentration-dependent manner for the other human ALK2 mutants. INDUSTRIAL APPLICABILITY The present invention has revealed that ectopic ossification and/or brain tumor may be effectively treated and/or prevented by administering an anti-ALK2 antibody having an ability to bind to ALK2 and an ability to cross-link ALK2 to a patient having an active mutation in ALK2 and having no mutation of an amino acid residue at position 330 of ALK2, preferably the patient having no G328V mutation. The present invention has also revealed: that a risk of developing an adverse reaction ascribable to the administration of an anti-ALK2 antibody may be predicted; that responsiveness to treatment and/or prevention by the administration of an anti-ALK2 antibody may be predicted; and that a subject to be treated and/or prevented by the administration of an anti-ALK2 antibody may be selected. Free Text of Sequence Listing SEQ ID NO: 17: Gln is a substituted amino acid residue.SEQ ID NO: 30: Amino acid sequence of humanized hA2-15A-L4SEQ ID NO: 31: Amino acid sequence of humanized hA2-15A-H4SEQ ID NO: 32: Amino acid sequence of humanized hA2-15A-L6SEQ ID NO: 33: Amino acid sequence of humanized hA2-15A-H4 IgG2 typeSEQ ID NO: 34: Amino acid sequence of humanized hA2-27D-H2SEQ ID NO: 35: Amino acid sequence of humanized hA2-27D-L2SEQ ID NO: 36: Amino acid sequence of humanized hA2-27D-H3SEQ ID NO: 37: Amino acid sequence of humanized hA2-27D-L4SEQ ID NO: 38: Amino acid sequence of humanized hA2-27D-H2_LALASEQ ID NO: 39: Amino acid sequence of humanized hA2-27D-H3 LALA All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. | 103,974 |
11859007 | DETAILED DESCRIPTION OF THE INVENTION Definitions While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose. Antibodies are large, complex molecules (molecular weight of −150,000 or about 1320 amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The light and heavy chain variable regions come together in 3-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions (“CDRs”). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3-dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework (“FR”), which forms the environment for the CDRs. The terms “CDR L1”, “CDR L2” and “CDR L3” as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable light (L) chain of an antibody. Likewise, the terms “CDR H1”, “CDR H2” and “CDR H3” as provided herein refer to the complementarity determining regions (CDR) 1, 2, and 3 of the variable heavy (H) chain of an antibody. The term “antibody” is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1by a disulfide bond. The F(ab)′2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al.,Nature348:552-554 (1990)). For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein,Nature256:495-497 (1975); Kozbor et al.,Immunology Today4:72 (1983); Cole et al., pp. 77-96 inMonoclonal Antibodies and Cancer Therapy(1985)). “Monoclonal” antibodies (mAb) refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al.,Nature348:552-554 (1990); Marks et al.,Biotechnology10:779-783 (1992)). The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1×, 5×, 10×, 20× or 100× excess of one antibody inhibits binding of the other by at least 30% but preferably 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al.,Cancer Res.50:1495, 1990). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. A “ligand” refers to an agent, e.g., a polypeptide or other molecule, capable of binding to a receptor. A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. Any appropriate method known in the art for conjugating an antibody to the label may be employed, e.g., using methods described in Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., San Diego. “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents which can be produced in the reaction mixture. The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be, for example, a biotin domain as described herein and a biotin-binding domain. In embodiments contacting includes, for example, allowing a humanized antibody as described herein to interact with CD73 antigen. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may In embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety. The term “peptidyl” and “peptidyl moiety” means a monovalent peptide. The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M)(see, e.g., Creighton,Proteins(1984)). “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity over a specified region, e.g., of the entire polypeptide sequences of the invention or individual domains of the polypeptides of the invention), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length. The present invention includes polypeptides that are substantially identical to any of SEQ ID NOs:30-51. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970)Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970)J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Nat'l. Acad. Sci. USA85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al.,Current Protocols in Molecular Biology(1995 supplement)). An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nuc. Acids Res.25:3389-3402, and Altschul et al. (1990)J Mol. Biol.215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001. An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence. An amino acid residue in an antibody “corresponds” to a given residue when it occupies the same essential structural position within the antibody as the given residue. For example, a selected residue in a comparison antibody corresponds to position 48 (according to the Kabat numbering system as described herein) in an antibody provided herein when the selected residue occupies the same essential spatial or structural relationship to Kabat position 48 as assessed using applicable methods in the art. For example, a comparison antibody may be aligned for maximum sequence homology with the antibody provided herein and the position in the aligned comparison antibody that aligns with Kabat position 48 may be determined to correspond to it. Alternatively, instead of (or in addition to) a primary sequence alignment as described above, a three dimensional structural alignment can also be used, e.g., where the structure of the comparison antibody is aligned for maximum correspondence with an antibody provided herein and the overall structures compared. In this case, an amino acid that occupies the same essential position as Kabat position 48 in the structural model may be said to correspond. The term “isolated,” when applied to a protein, denotes that the protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. The term “purified” denotes that a protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. The phrase “specifically (or selectively) binds” to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background. A “cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor (e.g., humanized 1E9 antibody-CD73) interaction means negatively affecting (e.g., decreasing) the activity or function of the protein (e.g., decreasing the catalytic activity of CD73) relative to the activity or function of the protein in the absence of the inhibitor (e.g., humanized 1E9 antibody). In some embodiments inhibition refers to reduction of a disease or symptoms of disease. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. Similarly an “inhibitor” is a compound or protein that inhibits CD73 activity, e.g., by binding, partially or totally blocking, decreasing, preventing, delaying, inactivating, desensitizing, or down-regulating enzymatic activity (e.g., CD73 catalytic activity). Agents of the invention are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. SeeRemington's Pharmaceutical Science(15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. The compositions can be administered for therapeutic or prophylactic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease (e.g., cancer) in a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. A “patient” or “subject” for the purposes of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is human. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art. Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants). The compositions provided herein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons. Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above. The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents. The combined administrations contemplates co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Effective doses of the compositions provided herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. However, a person of ordinary skill in the art would immediately recognize appropriate and/or equivalent doses looking at dosages of approved compositions for treating and preventing cancer for guidance. The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with a compound, pharmaceutical composition, or method provided herein. In embodiments, the disease is cancer (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma). The disease may be an autoimmune, inflammatory, cancer, infectious, metabolic, developmental, cardiovascular, liver, intestinal, endocrine, neurological, or other disease. As used herein, the term “cancer” refers to all types of cancer, neoplasm or malignant tumors found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound, pharmaceutical composition, or method provided herein include lymphoma, sarcoma, bladder cancer, bone cancer, brain tumor, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer (e.g. triple negative, ER positive, ER negative, chemotherapy resistant, herceptin resistant, HER2 positive, doxorubicin resistant, tamoxifen resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), ovarian cancer, pancreatic cancer, liver cancer (e.g. hepatocellular carcinoma), lung cancer (e.g. non-small cell lung carcinoma, squamous cell lung carcinoma, adenocarcinoma, large cell lung carcinoma, small cell lung carcinoma, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma. Additional examples include, cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, esophagus, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus or Medulloblastoma, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's Disease of the Nipple, Phyllodes Tumors, Lobular Carcinoma, Ductal Carcinoma, cancer of the pancreatic stellate cells, cancer of the hepatic stellate cells, or prostate cancer. The term “leukemia” refers broadly to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood-leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia. The term “sarcoma” generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound, pharmaceutical composition, or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma. The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma. The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound, pharmaceutical composition, or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, ductal carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lobular carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tubular carcinoma, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum. As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non-metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast. The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g., diabetes, cancer (e.g. prostate cancer, renal cancer, metastatic cancer, melanoma, castration-resistant prostate cancer, breast cancer, triple negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., head, neck, or esophagus), colorectal cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, or multiple myeloma)) means that the disease (e.g. lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. Humanized Antibodies A humanized antibody is a genetically engineered antibody in which at least one CDR (or functional fragment thereof) from a mouse antibody (“donor antibody”, which can also be rat, hamster or other non-human species) are grafted onto a human antibody (“acceptor antibody”). The human antibody is a non-natural (e.g. not naturally occurring or not naturally produced by a human) antibody that does not elicit an immune response in a human, does not elicit a significant immune response in a human, or elicits an immune response that is less than the immune response elicited in a mouse. In embodiments, more than one mouse CDR is grafted (e.g. all six mouse CDRs are grafted). The sequence of the acceptor antibody can be, for example, a mature human antibody sequence (or fragment thereof), a consensus sequence of a human antibody sequence (or fragment thereof), or a germline region sequence (or fragment thereof). Thus, a humanized antibody may be an antibody having one or more CDRs from a donor antibody and a variable region framework (FR). The FR may form part of a constant region and/or a variable region within a human antibody. In addition, in order to retain high binding affinity, amino acids in the human acceptor sequence may be replaced by the corresponding amino acids from the donor sequence, for example where: (1) the amino acid is in a CDR; (2) the amino acid is in the human framework region (e.g. the amino acid is immediately adjacent to one of the CDR's). See, U.S. Pat. Nos. 5,530,101 and 5,585,089, incorporated herein by reference, which provide detailed instructions for construction of humanized antibodies. Although humanized antibodies often incorporate all six CDRs (e.g. as defined by Kabat, but often also including hypervariable loop H1 as defined by Chothia) from a mouse antibody, they can also be made with fewer mouse CDRs and/or less than the complete mouse CDR sequence (e.g. a functional fragment of a CDR) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology, 164:1432-1441, 2000). Typically a humanized antibody as provided herein may include (i) a light chain comprising at least one CDR (often three CDRs) from a mouse antibody (also referred to herein as a mouse CDR) and a human variable region framework; and (ii) a heavy chain comprising at least one CDR (often three CDRs) from the mouse antibody and a human variable region framework (FR). The light and heavy chain variable region frameworks (FRs) may each be a mature human antibody variable region framework sequence (or fragment thereof), a germline variable region framework sequence (combined with a J region sequence) (or fragment thereof), or a consensus sequence of a human antibody variable region framework sequence (or fragment thereof). In embodiments, the humanized antibody includes a light chain as described in (i), a heavy chain as described in (ii) together with a light chain human constant region and a heavy chain constant region. A chimeric antibody is an antibody in which the variable region of a mouse (or other rodent) antibody is combined with the constant region of a human antibody; their construction by means of genetic engineering is well-known. Such antibodies retain the binding specificity of the mouse antibody, while being about two-thirds human. The proportion of nonhuman sequence present in mouse, chimeric and humanized antibodies suggests that the immunogenicity of chimeric antibodies is intermediate between mouse and humanized antibodies. Other types of genetically engineered antibodies that may have reduced immunogenicity relative to mouse antibodies include human antibodies made using phage display methods (Dower et al., WO91/17271; McCafferty et al., WO92/001047; Winter, WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of which is incorporated herein by reference) or using transgenic animals (Lonberg et al., WO93/12227; Kucherlapati WO91/10741, each of which is incorporated herein by reference). Other approaches to design humanized antibodies may also be used to achieve the same result as the methods in U.S. Pat. Nos. 5,530,101 and 5,585,089 described above, for example, “superhumanization” (see Tan et al. J. Immunol. 169: 1119, 2002, and U.S. Pat. No. 6,881,557) or the method of Studnicak et al., Protein Eng. 7:805, 1994. Moreover, other approaches to produce genetically engineered, reduced-immunogenicity mAbs include “reshaping”, “hyperchimerization” and veneering/resurfacing, as described, e.g., in Vaswami et al., Annals of Allergy, Asthma and Immunology 81:105, 1998; Roguska et al. Protein Eng. 9:895, 1996; and U.S. Pat. Nos. 6,072,035 and 5,639,641. A “CD73 protein” or “CD73 antigen” as referred to herein includes any of the recombinant or naturally-occurring forms of the Cluster of Differentiation 73 (CD73) also known as 5′-nucleotidase (5′-NT) or ecto-5′-nucleotidase or variants or homologs thereof that maintain CD73 nucleotidase activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD73). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD73 protein. In embodiments, the CD73 protein is substantially identical to the protein identified by the UniProt reference number 21589 or a variant or homolog having substantial identity thereto. In embodiments, the CD73 protein is substantially identical to the protein identified by the UniProt reference number Q61503 or a variant or homolog having substantial identity thereto. Humanized 1E9 Antibodies Provided herein are, inter alia, humanized 1E9 antibodies including a humanized light chain variable region and a humanized heavy chain variable region. The humanized 1E9 antibodies as provided herein are capable of binding a CD73 protein and include at least one CDR or a functional fragment thereof of the mouse monoclonal antibody 1E9 (Thomson L F et al. Tissue Antigens 2008, Volume 35, Issue 1: Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5′-nucleotidase (CD73)). A functional fragment of a CDR is a portion of a complete CDR amino acid sequence that is capable of binding to an antigen (e.g., CD73). Thus, a functional fragment of a CDR typically includes the amino acid residues required for CDR binding to the antigen (e.g., CD73). A “mouse CDR” is a complete CDR amino acid sequence or a functional fragment thereof derived from a mouse antibody that is capable of binding CD73. Thus, a functional fragment of a mouse CDR typically includes the amino acid residues required for CDR binding to CD73. Where a humanized 1E9 antibody includes at least one mouse CDR, the at least one mouse CDR or a functional fragment thereof is derived from a donor antibody. In embodiments, the donor antibody is a mouse 1E9 antibody. A person of skill in the art will immediately recognize that a humanized 1E9 antibody including at least one mouse CDR is a humanized antibody with at least one mouse CDR derived from a donor 1E9 antibody and the additional CDRs are derived from the acceptor antibody (e.g. where the light chain includes a total of three CDRs and the heavy chain includes a total of three CDRs). In embodiments, the humanized light chain variable region and the humanized heavy chain variable region include combined one mouse CDR or functional fragment of a mouse CDR. Thus, in some embodiments, the humanized light chain variable region and the humanized heavy chain variable region include combined six CDRs wherein at least one of the six CDRs is a mouse CDR. Where the humanized light chain variable region and the humanized heavy chain variable region include combined one mouse CDR, the humanized light chain variable region or the humanized heavy chain variable region include one mouse CDR. For example, a humanized antibody may include CDR L3 derived from the donor antibody (e.g. mouse, also referred to herein as a mouse CDR L3) and CDR L1, CDR L2, CDR H1, CDR H2, and CDR H3 derived from the acceptor antibody (i.e. human). In embodiments, the humanized light chain variable region and the humanized heavy chain variable region include combined two mouse CDRs. Where the humanized light chain variable region and the humanized heavy chain variable region include combined two mouse CDRs, the humanized light chain variable region and the humanized heavy chain variable region each include one mouse CDR (i), the humanized light chain variable region includes two mouse CDRs (ii), or the humanized heavy chain variable region includes two mouse CDRs (iii). For example, a humanized antibody may include CDR L3 and CDR H3 derived from the donor antibody (also referred to herein as a mouse CDR L3 and a mouse CDR H3, respectively), and CDR L1, CDR L2, CDR H1, and CDR H2 derived from the acceptor antibody (i.e. human). In embodiments, the humanized light chain variable region and the humanized heavy chain variable region include combined three mouse CDRs. Where the humanized light chain variable region and the humanized heavy chain variable region include combined three mouse CDRs, the humanized light chain variable region may include one mouse CDR and the humanized heavy chain variable region may include two mouse CDRs (i), the humanized light chain variable region includes two mouse CDRs and the humanized heavy chain variable region includes one mouse CDR (ii), the humanized light chain variable region includes three mouse CDRs (iii), or the humanized heavy chain variable region includes three mouse CDRs (iv). For example, a humanized antibody may include CDR L3, CDR H3 and CDR L2 derived from the donor antibody (e.g. mouse, also referred to herein as a CDR L3, mouse CDR H3, and mouse CDR L2 respectively) and CDR L1, CDR H1, and CDR H2 derived from the acceptor antibody (i.e. human). In embodiments, the humanized light chain variable region and the humanized heavy chain variable region include combined four mouse CDRs. Where the humanized light chain variable region and the humanized heavy chain variable region include combined four mouse CDRs, the humanized light chain variable region includes one mouse CDR and the humanized heavy chain variable region includes three mouse CDRs (i), the humanized light chain variable region includes three mouse CDRs and the humanized heavy chain variable region includes one mouse CDR (ii), or the humanized light chain variable region includes two mouse CDRs and the humanized heavy chain variable region includes two mouse CDRs (iii). For example, a humanized antibody may include CDR L3, CDR H3, CDR L2 and CDR L1 derived from the donor antibody (e.g. mouse, also referred to herein as a mouse CDR L3, mouse CDR H3, mouse CDR L2 and mouse CDR L1 respectively) and CDR H1 and CDR H2 derived from the acceptor antibody (i.e. human). In embodiments, the humanized light chain variable region and the humanized heavy chain variable region each include at least one mouse CDR. Where the humanized light chain variable region and the humanized heavy chain variable region each include at least one mouse CDR, the humanized light chain variable region includes at least one mouse CDR and the humanized heavy chain variable region includes at least one mouse CDR. Thus, in some embodiments, the humanized light chain variable region includes mouse CDR L1 and the humanized heavy chain includes mouse CDR H1. In embodiments, mouse CDR L1 includes the amino acid sequence of SEQ ID NO:1 and mouse CDR H1 includes the amino acid sequence of SEQ ID NO:4. In embodiments, mouse CDR L1 is the amino acid sequence of SEQ ID NO:1 and mouse CDR H1 is the amino acid sequence of SEQ ID NO:4. In embodiments, the humanized light chain variable region includes mouse CDR L2 and the humanized heavy chain variable region includes mouse CDR H2. In embodiments, mouse CDR L2 includes the amino acid sequence of SEQ ID NO:2 and mouse CDR H2 includes the amino acid sequence of SEQ ID NO:5. In embodiments, mouse CDR L2 is the amino acid sequence of SEQ ID NO:2 and mouse CDR H2 is the amino acid sequence of SEQ ID NO:5. In embodiments, the humanized light chain variable region includes mouse CDR L3 and the humanized heavy chain variable region includes mouse CDR H3. In embodiments, mouse CDR L3 includes the amino acid sequence of SEQ ID NO:3 and mouse CDR H3 includes the amino acid sequence of SEQ ID NO:6. In embodiments, CDR L3 is the amino acid sequence of SEQ ID NO:3 and mouse CDR H3 is the amino acid sequence of SEQ ID NO:6. In embodiments, the presence of mouse CDR L3 and mouse CDR H3 may be sufficient for binding of a humanized antibody to CD73. Thus, in embodiments, the humanized antibody does not include mouse CDR L1, mouse CDR L2, CDR H1 or mouse CDR H2. Where the humanized antibody does not include mouse CDR L1, mouse CDR L2, mouse CDR H1 or mouse CDR H2, the humanized antibody includes CDR L1, CDR L2, CDR H1 or CDR H2 derived from the acceptor antibody (i.e. human). Thus, a humanized antibody that does not include mouse CDR L1, mouse CDR L2, mouse CDR H1 or mouse CDR H2, does not include CDR L1, CDR L2, CDR H1 or CDR H2 from a donor antibody (e.g. mouse, rat, rabbit), but includes CDR L1, CDR L2, CDR H1 or CDR H2 from the acceptor antibody (i.e. human). Thus, in embodiments the humanized light chain variable region does not include mouse CDR L1 or mouse CDR L2 and the humanized heavy chain variable region does not include mouse CDR H1 or mouse CDR H2. In embodiments, the humanized light chain variable region does not include mouse CDR L1 and mouse CDR L2 and the humanized heavy chain variable region does not include mouse CDR H1 and mouse CDR H2. In embodiments, the humanized light chain variable region includes mouse CDR L2 and mouse CDR L3 and the humanized heavy chain variable region includes mouse CDR H2 and mouse CDR H3. In embodiments, the humanized light chain variable region includes mouse CDR L1, mouse CDR L2 and mouse CDR L3 and the humanized heavy chain variable region includes mouse CDR H1, mouse CDR H2 and mouse CDR H3. In embodiments, the humanized light chain variable region includes mouse CDR L1 as set forth in SEQ ID NO:1, mouse CDR L2 as set forth in SEQ ID NO:2 and mouse CDR L3 as set forth in SEQ ID NO:3, and the humanized heavy chain variable region includes mouse CDR H1 as set forth in SEQ ID NO:4, mouse CDR H2 as set forth in SEQ ID NO:5, and mouse CDR H3 as set forth in SEQ ID NO:6. The position of CDRs and FRs may be defined by the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)). Likewise, the positions occupied by individual residues within the light or the heavy chain of an antibody may be defined by the Kabat numbering system. Therefore, the location of residues required for binding within a humanized light chain and a humanized heavy chain of a humanized antibody may be defined by the position of the residue according to the Kabat numbering system as is well known in the art. As described above, a humanized antibody may be an antibody having CDRs from a donor antibody (e.g. mouse) and variable region framework (FR) from a human antibody. The framework regions (FRs) are said to hold the CDRs in place in a humanized antibody. Proceeding from the amino-terminus, these regions are designated FR L1, FR L2, FR L3, and FR L4 for the light chain and FR H1, FR H2, FR H3, and FR H4, for the heavy chain, respectively. Surprisingly, the present invention provides for humanized antibodies that include one or more residues within the framework regions that are important for epitope binding of the humanized antibody. A framework region residue involved in (or important for) epitope binding (e.g. CD73 binding) is referred to herein as a binding framework region residue. The binding framework region residues may reside in the framework region of a humanized light chain variable region (i.e. FR L1, FR L2, FR L3, FR L4) or they may reside in the framework of a humanized heavy chain variable region (i.e. FR H1, FR H2, FR H3, FR H4). A binding framework residue residing in the FR L3 region of a humanized light chain is referred to herein as a FR L3 binding framework region residue. Thus, a binding framework region residue residing in the FR H3 region of a humanized heavy chain is referred to herein as a FR H3 binding framework region residue. In embodiments, the humanized antibody includes at least one binding framework region residue. In embodiments, the humanized light chain variable region includes at least one binding framework region residue. In embodiments, the humanized light chain variable region includes one or more FR L1, FR L2, FR L3 or FR L4 binding framework region residues. In embodiments, the humanized light chain variable region includes one or more FR L1 binding framework region residues. In embodiments, the humanized light chain variable region includes one or more FR L2 binding framework region residues. In embodiments, the humanized light chain variable region includes one or more FR L3 binding framework region residues. In embodiments, the humanized light chain variable region includes one or more FR L4 binding framework region residues. In embodiments, the humanized heavy chain variable region includes one or more FR H1, FR H2, FR H3 or FR H4 binding framework region residues. In embodiments, the humanized heavy chain variable region includes one or more FR H1 binding framework region residues. In embodiments, the humanized heavy chain variable region includes one or more FR H2 binding framework region residues. In embodiments, the humanized heavy chain variable region includes one or more FR H3 binding framework region residues. In embodiments, the humanized heavy chain variable region includes one or more FR H4 binding framework region residues. In embodiments, the humanized light chain variable region includes at least one binding framework region residue (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42 43, 44, 45, 46, 47, 48, 49, 50 or more residues) and the humanized heavy chain variable region includes at least one binding framework region residue (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 37, 38, 39, 40, 41, 42 43, 44, 45, 46, 47, 48, 49, 50 or more residues). The position of a binding framework region residue within a humanized antibody may be defined by the Kabat numbering system similar to the positions CDR residues. In one aspect is provided a humanized 1E9 antibody including a humanized light chain variable region including a mouse CDR L1, mouse CDR L2, or mouse CDR L3 and a humanized heavy chain variable region including a mouse CDR H1, mouse CDR H2, or mouse CDR H3. The humanized light chain variable region may include a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, or a mouse CDR L3 as set forth in SEQ ID NO:3. The humanized light chain variable region may include a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, and a mouse CDR L3 as set forth in SEQ ID NO:3. The humanized heavy chain variable region may include a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, or a mouse CDR H3 as set forth in SEQ ID NO:6. The humanized heavy chain variable region may include a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments, the humanized light chain variable region includes a mouse CDR L1 as set forth in SEQ ID NO:1. In embodiments, the humanized light chain variable region includes a mouse CDR L2 as set forth in SEQ ID NO:2. In embodiments, the humanized light chain variable region includes a mouse CDR L3 as set forth in SEQ ID NO:3. In embodiments, the humanized heavy chain variable region includes a mouse CDR H1 as set forth in SEQ ID NO:4. In embodiments, the humanized heavy chain variable region includes a mouse CDR H2 as set forth in SEQ ID NO:5. In embodiments, the humanized light chain variable region includes a mouse CDR H3 as set forth in SEQ ID NO:6. In further embodiments, the humanized light chain variable region includes at least one binding framework region residue. In other further embodiments, the humanized heavy chain variable region includes at least one binding framework region residue. In one aspect, a humanized 1E9 antibody is provided. The 1E9 antibody includes a humanized light chain variable region and a humanized heavy chain variable region. The humanized light chain variable region includes:(i) a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3 and(ii) a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a proline or a serine at a position corresponding to Kabat position 12, a lysine or a proline at a position corresponding to Kabat position 18, a alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100, a valine at a position corresponding to Kabat position 104, a glutamic acid or an alanine at a position corresponding to Kabat position 1, a glutamine at a position corresponding to Kabat position 3, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, a tyrosine at a position corresponding to Kabat position 85, or a phenylalanine at a position corresponding to Kabat position 87. The humanized heavy chain variable region includes:(i) a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6 and(ii) an isoleucine at a position corresponding to Kabat position 37, an alanine or a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, an isoleucine or a threonine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, an arginine or a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, a valine or a methionine at a position corresponding to Kabat position 89, a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 87, a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a leucine at a position corresponding to Kabat position 80, or a glutamic acid at a position corresponding to Kabat position 81. In embodiments, the humanized light chain variable region provided herein includes a binding framework region residue that is a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a proline or a serine at a position corresponding to Kabat position 12, a lysine or a proline at a position corresponding to Kabat position 18, a alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100, a valine at a position corresponding to Kabat position 104, a glutamic acid or an alanine at a position corresponding to Kabat position 1, a glutamine at a position corresponding to Kabat position 3, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, a tyrosine at a position corresponding to Kabat position 85, or a phenylalanine at a position corresponding to Kabat position 87. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a valine at a position corresponding to Kabat position 2. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a methionine at a position corresponding to Kabat position 4. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an aspartic acid or a leucine at a position corresponding to Kabat position 9. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a proline or a serine at a position corresponding to Kabat position 12. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a lysine or a proline at a position corresponding to Kabat position 18. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a alanine at a position corresponding to Kabat position 43. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a proline or a serine at a position corresponding to Kabat position 60. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a threonine at a position corresponding to Kabat position 74. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an asparagine or a serine at a position corresponding to Kabat position 76. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an asparagine or a serine at a position corresponding to Kabat position 77. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an isoleucine or a leucine at a position corresponding to Kabat position 78. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a serine or an alanine at a position corresponding to Kabat position 80. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a glutamine at a position corresponding to Kabat position 100. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a valine at a position corresponding to Kabat position 104. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a glutamic acid or an alanine at a position corresponding to Kabat position 1. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a glutamine at a position corresponding to Kabat position 3. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a phenylalanine or a threonine at a position corresponding to Kabat position 10. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a glutamine at a position corresponding to Kabat position 11. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an alanine or a leucine at a position corresponding to Kabat position 13. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a threonine at a position corresponding to Kabat position 14. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a valine or a proline at a position corresponding to Kabat position 15. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a lysine at a position corresponding to Kabat position 16. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a threonine at a position corresponding to Kabat position 22. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a lysine at a position corresponding to Kabat position 42. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an arginine at a position corresponding to Kabat position 45. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an isoleucine at a position corresponding to Kabat position 58. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a tyrosine at a position corresponding to Kabat position 67. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a phenylalanine at a position corresponding to Kabat position 73. In embodiments, the humanized light chain variable region includes a binding framework region residue that is an isoleucine at a position corresponding to Kabat position 78. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a tyrosine at a position corresponding to Kabat position 85. In embodiments, the humanized light chain variable region includes a binding framework region residue that is a phenylalanine at a position corresponding to Kabat position 87. The humanized heavy chain variable region provided herein may include a binding framework region residue that is an isoleucine at a position corresponding to Kabat position 37, an alanine or a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, an isoleucine or a threonine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, an arginine or a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, a valine or a methionine at a position corresponding to Kabat position 89, a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 87, a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a leucine at a position corresponding to Kabat position 80, or a glutamic acid at a position corresponding to Kabat position 81. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an isoleucine at a position corresponding to Kabat position 37. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an alanine or a proline at a position corresponding to Kabat position 40. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a lysine at a position corresponding to Kabat position 43. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a serine at a position corresponding to Kabat position 70. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an isoleucine or a threonine at a position corresponding to Kabat position 75. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a tryptophan at a position corresponding to Kabat position 82. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an arginine or a lysine at a position corresponding to Kabat position 83. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a alanine at a position corresponding to Kabat position 84. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a serine at a position corresponding to Kabat position 85. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a valine or a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a valine at a position corresponding to Kabat position 5. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a serine at a position corresponding to Kabat position 7. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a valine at a position corresponding to Kabat position 11. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a glutamic acid or a lysine at a position corresponding to Kabat position 12. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an isoleucine or a valine at a position corresponding to Kabat position 20. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an arginine at a position corresponding to Kabat position 38. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an arginine at a position corresponding to Kabat position 66. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an valine at a position corresponding to Kabat position 67. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an isoleucine at a position corresponding to Kabat position 69. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is an alanine at a position corresponding to Kabat position 71. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a lysine at a position corresponding to Kabat position 73. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a threonine at a position corresponding to Kabat position 87. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a glutamic acid at a position corresponding to Kabat position 1. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a valine at a position corresponding to Kabat position 24. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a arginine at a position corresponding to Kabat position 44. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a methionine at a position corresponding to Kabat position 48. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a leucine at a position corresponding to Kabat position 80. In embodiments, the humanized heavy chain variable region includes a binding framework region residue that is a glutamic acid at a position corresponding to Kabat position 81. In embodiments, the humanized light chain variable region includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, a leucine at a position corresponding to Kabat position 9, a proline at a position corresponding to Kabat position 12, or a proline at a position corresponding to Kabat position 18; and the humanized heavy chain variable region includes an isoleucine at a position corresponding to Kabat position 37, a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, a isoleucine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, or a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, a leucine at a position corresponding to Kabat position 9, a proline at a position corresponding to Kabat position 12, and a proline at a position corresponding to Kabat position 18; and the humanized heavy chain variable region includes an isoleucine at a position corresponding to Kabat position 37, a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, a isoleucine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, or a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, a leucine at a position corresponding to Kabat position 9, a proline at a position corresponding to Kabat position 12, or a proline at a position corresponding to Kabat position 18; and the humanized heavy chain variable region includes an isoleucine at a position corresponding to Kabat position 37, a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, a isoleucine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, and a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, a leucine at a position corresponding to Kabat position 9, a proline at a position corresponding to Kabat position 12, and a proline at a position corresponding to Kabat position 18; and the humanized heavy chain variable region includes an isoleucine at a position corresponding to Kabat position 37, a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, a isoleucine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, and a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes a proline or a serine at a position corresponding to Kabat position 12, an alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100 or a valine at a position corresponding to Kabat position 104; and the humanized heavy chain variable region includes a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine or a proline at a position corresponding to Kabat position 40, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, an isoleucine or a threonine at a position corresponding to Kabat position 75, an arginine or a lysine at a position corresponding to Kabat position 83 or a threonine at a position corresponding to Kabat position 87. In embodiments, the humanized light chain variable region includes a proline or a serine at a position corresponding to Kabat position 12, an alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100 and a valine at a position corresponding to Kabat position 104; and the humanized heavy chain variable region includes a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine or a proline at a position corresponding to Kabat position 40, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, an isoleucine or a threonine at a position corresponding to Kabat position 75, an arginine or a lysine at a position corresponding to Kabat position 83 or a threonine at a position corresponding to Kabat position 87. In embodiments, the humanized light chain variable region includes a proline or a serine at a position corresponding to Kabat position 12, an alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100 or a valine at a position corresponding to Kabat position 104; and the humanized heavy chain variable region includes a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine or a proline at a position corresponding to Kabat position 40, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, an isoleucine or a threonine at a position corresponding to Kabat position 75, an arginine or a lysine at a position corresponding to Kabat position 83 and a threonine at a position corresponding to Kabat position 87. In embodiments, the humanized light chain variable region includes a proline or a serine at a position corresponding to Kabat position 12, an alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100 and a valine at a position corresponding to Kabat position 104; and the humanized heavy chain variable region includes a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine or a proline at a position corresponding to Kabat position 40, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, an isoleucine or a threonine at a position corresponding to Kabat position 75, an arginine or a lysine at a position corresponding to Kabat position 83 and a threonine at a position corresponding to Kabat position 87. In embodiments, humanized light chain variable region includes a glutamic acid or an alanine at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 2, a glutamine at a position corresponding to Kabat position 3, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, a serine or a proline at a position corresponding to Kabat position 12, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a lysine or a proline at a position corresponding to Kabat position 18, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a proline or a serine at a position corresponding to Kabat position 60, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a tyrosine at a position corresponding to Kabat position 85 or a phenylalanine at a position corresponding to Kabat position 87; and the humanized heavy chain variable region includes a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, an isoleucine at a position corresponding to Kabat position 37, a lysine at a position corresponding to Kabat position 43, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a serine at a position corresponding to Kabat position 70, a leucine at a position corresponding to Kabat position 80, a glutamic acid at a position corresponding to Kabat position 81, a tryptophan at a position corresponding to Kabat position 82, an alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85 or a valine or a methionine at a position corresponding to Kabat position 89. In embodiments, humanized light chain variable region includes a glutamic acid or an alanine at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 2, a glutamine at a position corresponding to Kabat position 3, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, a serine or a proline at a position corresponding to Kabat position 12, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a lysine or a proline at a position corresponding to Kabat position 18, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a proline or a serine at a position corresponding to Kabat position 60, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a tyrosine at a position corresponding to Kabat position 85 and a phenylalanine at a position corresponding to Kabat position 87; and the humanized heavy chain variable region includes a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, an isoleucine at a position corresponding to Kabat position 37, a lysine at a position corresponding to Kabat position 43, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a serine at a position corresponding to Kabat position 70, a leucine at a position corresponding to Kabat position 80, a glutamic acid at a position corresponding to Kabat position 81, a tryptophan at a position corresponding to Kabat position 82, an alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85 or a valine or a methionine at a position corresponding to Kabat position 89. In embodiments, humanized light chain variable region includes a glutamic acid or an alanine at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 2, a glutamine at a position corresponding to Kabat position 3, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, a serine or a proline at a position corresponding to Kabat position 12, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a lysine or a proline at a position corresponding to Kabat position 18, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a proline or a serine at a position corresponding to Kabat position 60, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a tyrosine at a position corresponding to Kabat position 85 or a phenylalanine at a position corresponding to Kabat position 87; and the humanized heavy chain variable region includes a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, an isoleucine at a position corresponding to Kabat position 37, a lysine at a position corresponding to Kabat position 43, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a serine at a position corresponding to Kabat position 70, a leucine at a position corresponding to Kabat position 80, a glutamic acid at a position corresponding to Kabat position 81, a tryptophan at a position corresponding to Kabat position 82, an alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85 and a valine or a methionine at a position corresponding to Kabat position 89. In embodiments, humanized light chain variable region includes a glutamic acid or an alanine at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 2, a glutamine at a position corresponding to Kabat position 3, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, a serine or a proline at a position corresponding to Kabat position 12, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a lysine or a proline at a position corresponding to Kabat position 18, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a proline or a serine at a position corresponding to Kabat position 60, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a tyrosine at a position corresponding to Kabat position 85 and a phenylalanine at a position corresponding to Kabat position 87; and the humanized heavy chain variable region includes a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, an isoleucine at a position corresponding to Kabat position 37, a lysine at a position corresponding to Kabat position 43, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a serine at a position corresponding to Kabat position 70, a leucine at a position corresponding to Kabat position 80, a glutamic acid at a position corresponding to Kabat position 81, a tryptophan at a position corresponding to Kabat position 82, an alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85 and a valine or a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized heavy chain variable region includes a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid at a position corresponding to Kabat position 12, a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine at a position corresponding to Kabat position 40, a methionine at a position corresponding to Kabat position 48, an arginine at a position corresponding to Kabat position 66, a valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, a lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 75, a glutamic acid at a position corresponding to Kabat position 81, an arginine at a position corresponding to Kabat position 83, a threonine at a position corresponding to Kabat position 87, or a valine at a position corresponding to Kabat position 89. In embodiments, the humanized heavy chain variable region includes a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid at a position corresponding to Kabat position 12, a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine at a position corresponding to Kabat position 40, a methionine at a position corresponding to Kabat position 48, an arginine at a position corresponding to Kabat position 66, a valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, a lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 75, a glutamic acid at a position corresponding to Kabat position 81, an arginine at a position corresponding to Kabat position 83, a threonine at a position corresponding to Kabat position 87, and a valine at a position corresponding to Kabat position 89. In embodiments, the humanized heavy chain variable region includes the sequence of SEQ ID NO:7. In embodiments, the humanized heavy chain variable region is SEQ ID NO:7. In embodiments, the humanized heavy chain variable region includes the sequence of SEQ ID NO:53. In embodiments, the humanized heavy chain variable region is SEQ ID NO:53. In embodiments, the humanized light chain variable region includes the sequence of SEQ ID NO:55. In embodiments, the humanized light chain variable region is SEQ ID NO:55. Thus, in another aspect, provided is a humanized 1E9 antibody including a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized heavy chain variable region includes the sequence of SEQ ID NO:53 and the humanized light chain variable region includes the sequence of SEQ ID NO:55. Further provided herein are humanized 1E9 antibodies capable of binding CD73 and including a humanized light chain variable region and a humanized heavy chain variable region including the sequence of SEQ ID NO:7. Thus, in another aspect, provided is a humanized 1E9 antibody including a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized heavy chain variable region includes the sequence of SEQ ID NO:7. The humanized 1E9 antibodies as provided herein may be Fab′ fragments. Where the humanized 1E9 antibodies are Fab′ fragments, the humanized 1E9 antibodies include a humanized heavy chain (e.g. including a constant and a variable region) and a humanized light chain (e.g. including a constant and a variable region). In embodiments, the humanized 1E9 antibody is a Fab′ fragment. In embodiments, the humanized 1E9 antibody includes a human constant region. In embodiments, the humanized 1E9 antibody is an IgG. In embodiments, the humanized 1E9 antibody is an IgG1. In embodiments, the humanized 1E9 antibody is an IgG4. In embodiments, the humanized 1E9 antibody is an IgA. In other embodiments, the humanized antibody is an IgM. In embodiments, the humanized 1E9 antibody is a single chain antibody. A single chain antibody includes a variable light chain and a variable heavy chain. A person of skill in the art will immediately recognize that a single chain antibody includes a single light chain and a single heavy chain, in contrast to an immunoglobulin antibody, which includes two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable (“V”) region (i.e. variable light chain and variable heavy chain) involved in binding the target antigen, and a constant (“C”) region that interacts with other components of the immune system. The variable light chain and the variable heavy chain in a single chain antibody may be linked through a linker peptide. Examples for linker peptides of single chain antibodies are described in Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S. M., Lee, T., Pope, S. H., Riordan, G. S. and Whitlow, M. (1988). Methods of making scFv antibodies have been described. See, Huse et al.,Science246:1275-1281 (1989); Ward et al.,Nature341:544-546 (1989); and Vaughan et al.,Nature Biotech.14:309-314 (1996). Briefly, mRNA from B-cells from an immunized animal is isolated and cDNA is prepared. The cDNA is amplified using primers specific for the variable regions of heavy and light chains of immunoglobulins. The PCR products are purified and the nucleic acid sequences are joined. If a linker peptide is desired, nucleic acid sequences that encode the peptide are inserted between the heavy and light chain nucleic acid sequences. The nucleic acid which encodes the scFv is inserted into a vector and expressed in the appropriate host cell. The ability of an antibody to bind a specific epitope (e.g., CD73) can be described by the equilibrium dissociation constant (KD). The equilibrium dissociation constant (KD) as defined herein is the ratio of the dissociation rate (K-off) and the association rate (K-on) of a humanized 1E9 antibody to a CD73 protein. It is described by the following formula: KD=K-off/K-on. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 0.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 1 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 1.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 2 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 2.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 3 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 3.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 4 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH below 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 4.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH from about 6.0 to about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.1. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.2. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.3. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.4. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.6. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.7. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.8. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.9. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 4.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 5.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 6 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 6.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 7 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 7.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 8 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH below 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 4.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH from about 6.0 to about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.1. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.2. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.3. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.4. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.6. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.7. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.8. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.9. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 8.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 9 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 9.5 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 10 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 11 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 12 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 13 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 14 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 15 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 16 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH below 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 4.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH from about 6.0 to about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.1. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.2. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.3. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.4. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.6. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.7. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.8. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.9. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 17 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 18 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 19 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 20 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 21 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 22 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 23 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) from about 24 to about 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 0.5, 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH below 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 4.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH from about 6.0 to about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.1. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.2. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.3. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.4. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.6. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.7. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.8. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.9. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 7.1 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 6.9 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 9.4 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 19.5 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 17.8 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) of about 15.9 nM. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH below 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of less than about 4.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH from about 6.0 to about 7.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.0. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.1. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.2. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.3. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.4. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.5. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.6. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.7. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.8. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 6.9. In embodiments, the humanized antibody is capable of binding a CD73 antigen with an equilibrium dissociation constant (KD) in this paragraph at a pH of about 7.0. In one aspect, an antibody capable of binding CD73 at a pH of less than about 7.5 is provided. In embodiments, the antibody, is capable of binding a CD73 antigen at a pH of less than about 7.0. In embodiments, the antibody, is capable of binding a CD73 antigen at a pH of less than about 6.5. In embodiments, the antibody, is capable of binding a CD73 antigen at a pH of less than about 6.0. In embodiments, the antibody, is capable of binding a CD73 antigen at a pH of less than about 5.5. In embodiments, the antibody, is capable of binding a CD73 antigen at a pH of less than about 5. In embodiments, the antibody, is capable of binding a CD73 antigen at a pH of less than about 4.5. In embodiments, the antibody is capable of binding a CD73 antigen at a pH from about 6.0 to about 7.0. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.0. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.1. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.2. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.3. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.4. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.5. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.6. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.7. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.8. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.9. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 7.0. In embodiments, the antibody as set forth in this paragraph is a humanized antibody. In embodiments, the antibody includes a light chain (e.g. humanized light chain) variable region and a heavy chain (e.g. humanized heavy chain) variable region. The light chain variable region includes:(i) a CDR L1 (e.g. mouse CDR L1) as set forth in SEQ ID NO:1, a CDR L2 (e.g. a mouse CDR L2) as set forth in SEQ ID NO:2, a CDR L3 (e.g. a mouse CDR L3) as set forth in SEQ ID NO:3 and(ii) a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a proline or a serine at a position corresponding to Kabat position 12, a lysine or a proline at a position corresponding to Kabat position 18, a alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100, a valine at a position corresponding to Kabat position 104, a glutamic acid or an alanine at a position corresponding to Kabat position 1, a glutamine at a position corresponding to Kabat position 3, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a tyrosine at a position corresponding to Kabat position 85, or a phenylalanine at a position corresponding to Kabat position 87. The heavy chain variable region includes:(i) a CDR H1 (e.g. a mouse CDR H1) as set forth in SEQ ID NO:4, a CDRH2 (e.g. a mouse CDR H2) as set forth in SEQ ID NO:5, a CDR H3 (e.g. a mouse CDR H3) as set forth in SEQ ID NO:6 and(ii) an isoleucine at a position corresponding to Kabat position 37, an alanine or a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, an isoleucine or a threonine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, an arginine or a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, a valine or a methionine at a position corresponding to Kabat position 89, a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 87, a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a leucine at a position corresponding to Kabat position 80, or a glutamic acid at a position corresponding to Kabat position 81. The humanized 1E9 antibodies provided herein are capable of binding CD73 at a pH below 7.5. Thus, in embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 7.5. In embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 7.0. In embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 6.5. In embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 6.0. In embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 5.5. In embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 5. In embodiments, the humanized antibody, is capable of binding a CD73 antigen at a pH of less than about 4.5. In embodiments, the antibody is capable of binding a CD73 antigen at a pH from about 6.0 to about 7.0. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.0. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.1. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.2. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.3. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.4. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.5. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.6. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.7. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.8. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 6.9. In embodiments, the antibody is capable of binding a CD73 antigen at a pH of about 7.0. The humanized 1E9 antibody provided herein including embodiments thereof may include a glutamine at a position corresponding to Kabat position 297. In embodiments, the humanized 1E9 antibody is bound to a CD73 antigen. In embodiments, the CD73 antigen forms part of a cell. In embodiments, the cell is a lymphoid cell. In embodiments, the cell is a T cell. In embodiments, the cell is a cancer cell. In one aspect, a humanized 1E9 antibody bound to a CD73 antigen at a pH of less than about 7.5 is provided. In embodiments, the humanized 1E9 antibody includes a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region includes an isoleucine at a position corresponding to Kabat position 2, a leucine at a position corresponding to Kabat position 4, a serine or alanine at a position corresponding to Kabat position 9, a serine or a threonine at a position corresponding to Kabat position 10, a leucine at a position corresponding to Kabat position 11, a serine at a position corresponding to Kabat position 14, a glycine at a position corresponding to Kabat position 16, an arginine at a position corresponding to Kabat position 18, a threonine at a position corresponding to Kabat position 20 or a glutamine at a position corresponding to Kabat position 42; and wherein the humanized heavy chain variable region includes a glutamine at a position corresponding to Kabat position 1, a valine or glutamic acid at a position corresponding to Kabat position 12, a serine at a position corresponding to Kabat position 17, a methionine or valine at a position corresponding to Kabat position 20, a alanine at a position corresponding to Kabat position 24, a valine at a position corresponding to Kabat position 37, an arginine or alanine at a position corresponding to Kabat position 40, a proline at a position corresponding to Kabat position 41, a glutamine at a position corresponding to Kabat position 43, a glycine at a position corresponding to Kabat position 44, a threonine at a position corresponding to Kabat position 70, a threonine at a position corresponding to Kabat position 75, a methionine at a position corresponding to Kabat position 80, a threonine or arginine at a position corresponding to Kabat position 83, a serine at a position corresponding to Kabat position 84, a glutamic acid at a position corresponding to Kabat position 85, or a valine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes an isoleucine at a position corresponding to Kabat position 2, a leucine at a position corresponding to Kabat position 4, a serine or threonine at a position corresponding to Kabat position 10, a leucine at a position corresponding to Kabat position 11, a threonine at a position corresponding to Kabat position 20 and a glutamine at a position corresponding to Kabat position 42; and the humanized heavy chain variable region includes a glutamine at a position corresponding to Kabat position 1, a serine at a position corresponding to Kabat position 17, a methionine or valine at a position corresponding to Kabat position 20, a alanine at a position corresponding to Kabat position 24, a valine at a position corresponding to Kabat position 37, an arginine or alanine at a position corresponding to Kabat position 40, a proline at a position corresponding to Kabat position 41, a glutamine at a position corresponding to Kabat position 43, a glycine at a position corresponding to Kabat position 44, a threonine at a position corresponding to Kabat position 70, a threonine at a position corresponding to Kabat position 75, a methionine at a position corresponding to Kabat position 80, a threonine or arginine at a position corresponding to Kabat position 83, a serine at a position corresponding to Kabat position 84, a glutamic acid at a position corresponding to Kabat position 85, and a valine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes an isoleucine at a position corresponding to Kabat position 2, a leucine at a position corresponding to Kabat position 4, a serine or threonine at a position corresponding to Kabat position 10, a leucine at a position corresponding to Kabat position 11, a threonine at a position corresponding to Kabat position 20 or a glutamine at a position corresponding to Kabat position 42; and the humanized heavy chain variable region includes a glutamine at a position corresponding to Kabat position 1, a serine at a position corresponding to Kabat position 17, a methionine or valine at a position corresponding to Kabat position 20, a alanine at a position corresponding to Kabat position 24, a valine at a position corresponding to Kabat position 37, an arginine or alanine at a position corresponding to Kabat position 40, a proline at a position corresponding to Kabat position 41, a glutamine at a position corresponding to Kabat position 43, a glycine at a position corresponding to Kabat position 44, a threonine at a position corresponding to Kabat position 70, a threonine at a position corresponding to Kabat position 75, a methionine at a position corresponding to Kabat position 80, a threonine or arginine at a position corresponding to Kabat position 83, a serine at a position corresponding to Kabat position 84, a glutamic acid at a position corresponding to Kabat position 85 or a valine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes an isoleucine at a position corresponding to Kabat position 2, a leucine at a position corresponding to Kabat position 4, a serine or threonine at a position corresponding to Kabat position 10, a leucine at a position corresponding to Kabat position 11, a threonine at a position corresponding to Kabat position 20 and a glutamine at a position corresponding to Kabat position 42; and the humanized heavy chain variable region includes a glutamine at a position corresponding to Kabat position 1, a serine at a position corresponding to Kabat position 17, a methionine or valine at a position corresponding to Kabat position 20, a alanine at a position corresponding to Kabat position 24, a valine at a position corresponding to Kabat position 37, an arginine or alanine at a position corresponding to Kabat position 40, a proline at a position corresponding to Kabat position 41, a glutamine at a position corresponding to Kabat position 43, a glycine at a position corresponding to Kabat position 44, a threonine at a position corresponding to Kabat position 70, a threonine at a position corresponding to Kabat position 75, a methionine at a position corresponding to Kabat position 80, a threonine or arginine at a position corresponding to Kabat position 83, a serine at a position corresponding to Kabat position 84, a glutamic acid at a position corresponding to Kabat position 85 or a valine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes an isoleucine at a position corresponding to Kabat position 2, a leucine at a position corresponding to Kabat position 4, a serine or threonine at a position corresponding to Kabat position 10, a leucine at a position corresponding to Kabat position 11, a threonine at a position corresponding to Kabat position 20 or a glutamine at a position corresponding to Kabat position 42; and the humanized heavy chain variable region includes a glutamine at a position corresponding to Kabat position 1, a serine at a position corresponding to Kabat position 17, a methionine or valine at a position corresponding to Kabat position 20, a alanine at a position corresponding to Kabat position 24, a valine at a position corresponding to Kabat position 37, an arginine or alanine at a position corresponding to Kabat position 40, a proline at a position corresponding to Kabat position 41, a glutamine at a position corresponding to Kabat position 43, a glycine at a position corresponding to Kabat position 44, a threonine at a position corresponding to Kabat position 70, a threonine at a position corresponding to Kabat position 75, a methionine at a position corresponding to Kabat position 80, a threonine or arginine at a position corresponding to Kabat position 83, a serine at a position corresponding to Kabat position 84, a glutamic acid at a position corresponding to Kabat position 85 and a valine at a position corresponding to Kabat position 89. In embodiments, the humanized light chain variable region includes a serine or alanine at a position corresponding to Kabat position 9, a serine at a position corresponding to Kabat position 14, a glycine at a position corresponding to Kabat position 16 and an arginine at a position corresponding to Kabat position 18; and the humanized heavy chain variable region includes a valine or glutamic acid at a position corresponding to Kabat position 12. In embodiments, the humanized light chain variable region includes a serine or alanine at a position corresponding to Kabat position 9, a serine at a position corresponding to Kabat position 14, a glycine at a position corresponding to Kabat position 16 or an arginine at a position corresponding to Kabat position 18; and the humanized heavy chain variable region includes a valine or glutamic acid at a position corresponding to Kabat position 12. In embodiments, the pH is from about 6.0 to about 7.0. In embodiments, the pH is about 6.7. In embodiments, the pH is about 6.3. In embodiments, the antibody inhibits catalytic activity of said CD73 antigen. In embodiments, the antibody includes a humanized light chain variable region including the sequence of SEQ ID NO:36 or SEQ ID NO:37. In embodiments, the antibody includes a humanized heavy chain variable region including the sequence of SEQ ID NO:7. In embodiments, the CD73 antigen forms part of a cell. In embodiments, the CD73 antigen is bound to a solid support. In another aspect an anti-CD73 antibody is provided. The anti-CD73 binds the same epitope as a 1E9 antibody, wherein the 1E9 antibody includes a humanized light chain variable region including a mouse CDR L1, mouse CDR L2, or mouse CDR L3 and a humanized heavy chain variable region including a mouse CDR H1, mouse CDR H2, or mouse CDR H3. In another aspect an anti-CD73 antibody is provided. The anti-CD73 binds the same epitope as a 1E9 antibody, wherein the 1E9 antibody includes a humanized light chain variable region and a humanized heavy chain variable region. The humanized light chain variable region includes:(i) a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3 and(ii) a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a proline or a serine at a position corresponding to Kabat position 12, a lysine or a proline at a position corresponding to Kabat position 18, a alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100, a valine at a position corresponding to Kabat position 104, a glutamic acid or an alanine at a position corresponding to Kabat position 1, a glutamine at a position corresponding to Kabat position 3, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a tyrosine at a position corresponding to Kabat position 85, or a phenylalanine at a position corresponding to Kabat position 87. The humanized heavy chain variable region includes:(i) a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6 and(ii) an isoleucine at a position corresponding to Kabat position 37, an alanine or a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, an isoleucine or a threonine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, an arginine or a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, a valine or a methionine at a position corresponding to Kabat position 89, a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 87, a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a leucine at a position corresponding to Kabat position 80, or a glutamic acid at a position corresponding to Kabat position 81. Humanized IgG1 Antibodies In one aspect, provided herein is a humanized IgG1 antibody including a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region includes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3 and wherein the humanized heavy chain variable region includes a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. In one aspect, provided herein is a humanized IgG4 antibody including a humanized light chain variable region and a humanized heavy chain variable region, wherein the humanized light chain variable region includes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3 and wherein the humanized heavy chain variable region includes a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. The humanized light chain variable region may include a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, or a mouse CDR L3 as set forth in SEQ ID NO:3. The humanized light chain variable region may include a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, and a mouse CDR L3 as set forth in SEQ ID NO:3. The humanized heavy chain variable region may include a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, or a mouse CDR H3 as set forth in SEQ ID NO:6. The humanized heavy chain variable region may include a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments, the humanized light chain variable region includes a mouse CDR L1 as set forth in SEQ ID NO:1. In embodiments, the humanized light chain variable region includes a mouse CDR L2 as set forth in SEQ ID NO:2. In embodiments, the humanized light chain variable region includes a mouse CDR L3 as set forth in SEQ ID NO:3. In embodiments, the humanized heavy chain variable region includes a mouse CDR H1 as set forth in SEQ ID NO:4. In embodiments, the humanized heavy chain variable region includes a mouse CDR H2 as set forth in SEQ ID NO:5. In embodiments, the humanized light chain variable region includes a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments, the humanized light chain variable region further includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a proline or a serine at a position corresponding to Kabat position 12, a lysine or a proline at a position corresponding to Kabat position 18, a alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100, a valine at a position corresponding to Kabat position 104, a glutamic acid or an alanine at a position corresponding to Kabat position 1, a glutamine at a position corresponding to Kabat position 3, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a tyrosine at a position corresponding to Kabat position 85 or a phenylalanine at a position corresponding to Kabat position 87. In embodiments, the humanized light chain variable region further includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, an aspartic acid or a leucine at a position corresponding to Kabat position 9, a proline or a serine at a position corresponding to Kabat position 12, a lysine or a proline at a position corresponding to Kabat position 18, a alanine at a position corresponding to Kabat position 43, a proline or a serine at a position corresponding to Kabat position 60, a threonine at a position corresponding to Kabat position 74, an asparagine or a serine at a position corresponding to Kabat position 76, an asparagine or a serine at a position corresponding to Kabat position 77, an isoleucine or a leucine at a position corresponding to Kabat position 78, a serine or an alanine at a position corresponding to Kabat position 80, a glutamine at a position corresponding to Kabat position 100, a valine at a position corresponding to Kabat position 104, a glutamic acid or an alanine at a position corresponding to Kabat position 1, a glutamine at a position corresponding to Kabat position 3, a phenylalanine or a threonine at a position corresponding to Kabat position 10, a glutamine at a position corresponding to Kabat position 11, an alanine or a leucine at a position corresponding to Kabat position 13, a threonine at a position corresponding to Kabat position 14, a valine or a proline at a position corresponding to Kabat position 15, a lysine at a position corresponding to Kabat position 16, a glutamic acid or an aspartic acid at a position corresponding to Kabat position 17, a threonine at a position corresponding to Kabat position 22, a lysine at a position corresponding to Kabat position 42, an arginine at a position corresponding to Kabat position 45, an isoleucine at a position corresponding to Kabat position 58, a tyrosine at a position corresponding to Kabat position 67, a phenylalanine at a position corresponding to Kabat position 73, an isoleucine at a position corresponding to Kabat position 78, a tyrosine at a position corresponding to Kabat position 85 and a phenylalanine at a position corresponding to Kabat position 87. In embodiments, the humanized heavy chain variable region further includes an isoleucine at a position corresponding to Kabat position 37, an alanine or a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, an isoleucine or a threonine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, an arginine or a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, a valine or a methionine at a position corresponding to Kabat position 89, a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 87, a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a leucine at a position corresponding to Kabat position 80 or a glutamic acid at a position corresponding to Kabat position 81. In embodiments, the humanized heavy chain variable region further includes an isoleucine at a position corresponding to Kabat position 37, an alanine or a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, an isoleucine or a threonine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, an arginine or a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, a valine or a methionine at a position corresponding to Kabat position 89, a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid or a lysine at a position corresponding to Kabat position 12, an isoleucine or a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an arginine at a position corresponding to Kabat position 66, an valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, an lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 87, a glutamic acid at a position corresponding to Kabat position 1, a valine at a position corresponding to Kabat position 24, a arginine at a position corresponding to Kabat position 44, a methionine at a position corresponding to Kabat position 48, a leucine at a position corresponding to Kabat position 80 and a glutamic acid at a position corresponding to Kabat position 81. In embodiments, the humanized light chain variable region further includes a valine at a position corresponding to Kabat position 2, a methionine at a position corresponding to Kabat position 4, a leucine at a position corresponding to Kabat position 9, a proline at a position corresponding to Kabat position 12, and a proline at a position corresponding to Kabat position 18. In embodiments, the humanized heavy chain variable region further comprises an isoleucine at a position corresponding to Kabat position 37, a proline at a position corresponding to Kabat position 40, a lysine at a position corresponding to Kabat position 43, a serine at a position corresponding to Kabat position 70, a isoleucine at a position corresponding to Kabat position 75, a tryptophan at a position corresponding to Kabat position 82, a lysine at a position corresponding to Kabat position 83, a alanine at a position corresponding to Kabat position 84, a serine at a position corresponding to Kabat position 85, and a methionine at a position corresponding to Kabat position 89. In embodiments, the humanized heavy chain variable region comprises a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid at a position corresponding to Kabat position 12, a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine at a position corresponding to Kabat position 40, a methionine at a position corresponding to Kabat position 48, an arginine at a position corresponding to Kabat position 66, a valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, a lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 75, a glutamic acid at a position corresponding to Kabat position 81, an arginine at a position corresponding to Kabat position 83, a threonine at a position corresponding to Kabat position 87, or a valine at a position corresponding to Kabat position 89. In embodiments, the humanized heavy chain variable region comprises a valine at a position corresponding to Kabat position 5, a serine at a position corresponding to Kabat position 7, a valine at a position corresponding to Kabat position 11, a glutamic acid at a position corresponding to Kabat position 12, a valine at a position corresponding to Kabat position 20, an arginine at a position corresponding to Kabat position 38, an alanine at a position corresponding to Kabat position 40, a methionine at a position corresponding to Kabat position 48, an arginine at a position corresponding to Kabat position 66, a valine at a position corresponding to Kabat position 67, an isoleucine at a position corresponding to Kabat position 69, an alanine at a position corresponding to Kabat position 71, a lysine at a position corresponding to Kabat position 73, a threonine at a position corresponding to Kabat position 75, a glutamic acid at a position corresponding to Kabat position 81, an arginine at a position corresponding to Kabat position 83, a threonine at a position corresponding to Kabat position 87, and a valine at a position corresponding to Kabat position 89. In embodiments, the humanized IgG1 antibody further includes a glutamine at a position corresponding to Kabat position 297. In embodiments, the humanized IgG1 antibody provided herein including embodiments thereof is bound to a CD73 antigen. In embodiments, the CD73 antigen forms part of a cell. In embodiments, the cell is a T cell. In embodiments, the cell is a cancer cell. Nucleic Acid Compositions In one aspect, an isolated nucleic acid encoding a humanized 1E9 antibody provided herein including embodiments thereof is provided. The humanized 1E9 antibody encoded by the isolated nucleic acid is described in detail throughout this application (including the description above and in the examples section). Thus, the humanized antibody encoded by the isolated nucleic acid includes all of the embodiments described herein. For example, the nucleic acid may encode at least one CDR, specific residues involved in binding the epitope, or binding framework residues. For instance, the nucleic acid may encode a humanized light chain including a valine at a position corresponding to Kabat position 2. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 or SEQ ID NO:17. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:8 and the sequence of SEQ ID NO:18. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:9 and the sequence of SEQ ID NO:19. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:10 and the sequence of SEQ ID NO:20. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:11 and the sequence of SEQ ID NO:21. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:12 and the sequence of SEQ ID NO:22. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:13 and the sequence of SEQ ID NO:23. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:14 and the sequence of SEQ ID NO:24. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:15 and the sequence of SEQ ID NO:25. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:16 and the sequence of SEQ ID NO:26. In embodiments, the isolated nucleic acid includes the sequence of SEQ ID NO:17 and the sequence of SEQ ID NO:27. In embodiments, the isolated nucleic acid includes a codon-optimized sequence. In embodiments, the isolated nucleic acid includes SEQ ID NO:52 or SEQ ID NO:54. In embodiments, the isolated nucleic acid includes SEQ ID NO:52. In embodiments, the isolated nucleic acid includes SEQ ID NO:54. In embodiments, the isolated nucleic acid is SEQ ID NO:52 or SEQ ID NO:54. In embodiments, the isolated nucleic acid is SEQ ID NO:52. In embodiments, the isolated nucleic acid is SEQ ID NO:54. In another aspect, an isolated nucleic acid encoding a humanized IgG1 antibody provided herein including embodiments is provided. The humanized IgG1 antibody encoded by the isolated nucleic acid is described in detail throughout this application (including the description above and in the examples section). Thus, the humanized antibody encoded by the isolated nucleic acid includes all of the embodiments described herein. For example, the nucleic acid may encode at least one CDR, specific residues involved in binding the epitope, or binding framework residues. Thus, in embodiments the nucleic acid encodes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3. In embodiments, the nucleic acid encodes a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments the nucleic acid encodes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3, a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, or a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments the nucleic acid encodes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3, a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. In another aspect, an isolated nucleic acid encoding a humanized IgG4 antibody provided herein including embodiments is provided. The humanized IgG4 antibody encoded by the isolated nucleic acid is described in detail throughout this application (including the description above and in the examples section). Thus, the humanized antibody encoded by the isolated nucleic acid includes all of the embodiments described herein. For example, the nucleic acid may encode at least one CDR, specific residues involved in binding the epitope, or binding framework residues. Thus, in embodiments the nucleic acid encodes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3. In embodiments, the nucleic acid encodes a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments the nucleic acid encodes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3, a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, or a mouse CDR H3 as set forth in SEQ ID NO:6. In embodiments the nucleic acid encodes a mouse CDR L1 as set forth in SEQ ID NO:1, a mouse CDR L2 as set forth in SEQ ID NO:2, a mouse CDR L3 as set forth in SEQ ID NO:3, a mouse CDR H1 as set forth in SEQ ID NO:4, a mouse CDR H2 as set forth in SEQ ID NO:5, and a mouse CDR H3 as set forth in SEQ ID NO:6. Pharmaceutical Compositions In another aspect, a pharmaceutical composition including a therapeutically effective amount of a humanized 1E9 antibody provided herein including embodiments thereof and a pharmaceutically acceptable excipient is provided. In another aspect, a pharmaceutical composition including a therapeutically effective amount of a humanized IgG1 antibody provided herein including embodiments thereof and a pharmaceutically acceptable excipient is provided. In another aspect, a pharmaceutical composition including a therapeutically effective amount of a humanized IgG4 antibody provided herein including embodiments thereof and a pharmaceutically acceptable excipient is provided. A therapeutically effective amount as provided herein refers to an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods to treat a disease, the pharmaceutical compositions described herein will contain an amount of active humanized antibody effective to achieve the desired result, e.g., modulating the activity of a target molecule (e.g., CD73), and/or reducing, eliminating, or slowing the progression of disease symptoms (e.g., cancer). Determination of a therapeutically effective amount of a humanized antibody provided herein is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure herein. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or acetate at a pH typically of 5.0 to 8.0, most often 6.0 to 7.0; salts such as sodium chloride, potassium chloride, etc. to make isotonic; antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers such as polysorbate 80, amino acids such as glycine, carbohydrates, chelating agents, sugars, and other standard ingredients known to those skilled in the art (Remington's Pharmaceutical Science 16thedition, Osol, A. Ed. 1980). The mAb is typically present at a concentration of 0.1-100 mg/ml, e.g., 1-10 mg/ml or 10-50 mg/ml, for example 5, 10, 20, 30, 40, 50 or 60 mg/ml. A pharmaceutical composition including a humanized antibody as described herein can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. In embodiments, administration is intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. Pharmaceutically acceptable excipients can be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Pharmaceutical compositions of the humanized antibody can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington:The Science and Practice of Pharmacy, Mack Publishing Co., 20thed., 2000; andSustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the humanized antibody is employed in the pharmaceutical compositions of the invention. The humanized antibodies provided can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate the humanized antibodies in combination with other therapies or agents. It can be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of humanized antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical excipient. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular antibody being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors. A physician or veterinarian can start doses of the humanized antibodies of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the present invention vary depending upon many different factors, including the specific disease or condition to be treated, means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. For administration with an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. The humanized antibody provided herein can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the humanized antibody in the patient. In some methods, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, humanized antibodies show longer half-life than that of chimeric antibodies and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime. Methods In one aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a humanized 1E9 antibody provided herein including embodiments thereof, thereby treating cancer in the subject. In embodiments, the cancer is a lymphoid cancer. In one aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a humanized IgG1 antibody provided herein including embodiments thereof, thereby treating cancer in the subject. In embodiments, the cancer is a lymphoid cancer. Methods of Inhibition In one aspect, a method of inhibiting proliferation of a cell is provided. The method includes (i) contacting a cell with a humanized IgG1 antibody as provided herein including embodiments thereof, thereby forming a contacted cell. (ii) The humanized IgG1 antibody is allowed to bind a CD73 antigen on the contacted cell, thereby inhibiting proliferation of the cell. In embodiments, the cell is a lymphoid cell. In embodiments, the lymphoid cell is a T cell. In one aspect, a method of inhibiting proliferation of a cell is provided. The method includes (i) contacting a cell with a humanized IgG4 antibody as provided herein including embodiments thereof, thereby forming a contacted cell. (ii) The humanized IgG4 antibody is allowed to bind a CD73 antigen on the contacted cell, thereby inhibiting proliferation of the cell. In embodiments, the cell is a lymphoid cell. In embodiments, the lymphoid cell is a T cell. Methods of Detecting In one aspect, a method of detecting a humanized 1E9 antibody bound to a CD73 antigen is provided. The method includes, (i) contacting a humanized 1E9 antibody with a CD73 antigen at a pH of less than about 7.5 and (ii) detecting binding of the humanized 1E9 antibody to the CD73 antigen. In embodiments, the pH is from about 6.0 to about 7.0. In embodiments, the pH is about 6.7. in embodiments, the pH is about 6.3. In embodiments, the detecting binding of step (ii) includes detecting inhibition of CD73 catalytic activity. In embodiments, the CD73 antigen forms part of a cell. In embodiments, the CD73 antigen is bound to a solid support. In embodiments, the humanized 1E9 antibody includes a detectable moiety. Methods of T-Cell Activation Provided herein are methods of activating an immunosuppressed (non-activated, non-proliferating) T cell in a cancer environment. Thus, in one aspect a method of activating an immunosuppressed T cell is provided. The method includes, (i) contacting a T cell with a humanized 1E9 antibody as provided herein including embodiments thereof, thereby forming a contacted T cell. (ii) The humanized 1E9 antibody is allowed to bind a CD73 antigen on the contacted T cell, thereby activating the immunosuppressed T cell. In embodiments, the T cell is in a cancer environment. In embodiments, the IFN-gamma secretion of the contacted T cell is increased relative to the absence of the antibody. In embodiments, the proliferation of the contacted T cell is increased relative to the absence of the antibody. An “immunosuppressed T cell” as provided herein is a T cell residing in a cancer environment (in the close vicinity to and/or in physiological contact with a cancer cell or solid tumor), which does not proliferate or secrete detectable amounts of cytokines or express cell surface markers characteristic of activated T cells (e.g., IFN-gamma, CD25, CD38). Combination Treatment Methods The methods of treating provided herein including embodiments thereof, may include administration of a second therapeutic agent. Therefore, the methods of treatment as provided herein include administering a humanized 1E9 antibody as provided herein or a humanized IgG1 or IgG4 antibody as provided herein in combination with a second therapeutic agent. The second therapeutic agent may be any composition useful in treating or preventing cancer. In one aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a humanized 1E9 antibody provided herein including embodiments thereof and an effective amount of a second therapeutic agent, thereby treating cancer in the subject. In another aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a humanized IgG1 antibody provided herein including embodiments thereof and an effective amount of a second therapeutic agent, thereby treating cancer in the subject. In another aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a humanized IgG4 antibody provided herein including embodiments thereof and an effective amount of a second therapeutic agent, thereby treating cancer in the subject. The second therapeutic agent useful for the methods provided herein may be a compound, drug, antagonist, inhibitor, or modulator, having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In embodiments, the second therapeutic agent is a chemotherapeutic. “Chemotherapeutic” or “chemotherapeutic agent” is used in accordance with its plain ordinary meaning and refers to a chemical composition or compound having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In embodiments, the second therapeutic agent is radiation therapy. In embodiments, the second therapeutic agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. In embodiments, the second therapeutic agent is a compound. In embodiments, the compound is a purine receptor antagonist. In embodiments, the compound is an A2Aadenosine receptor antagonist or A2Badenosine receptor antagonist. In embodiments, the compound is an A2Aadenosine receptor antagonist. In embodiments, the compound is an A2Badenosine receptor antagonist. In embodiments, the compound is any one of the compounds disclosed in U.S. Pat. Nos. 9,120,807, 8,450,328 or 8,354,415, which are hereby incorporated by reference and for all purposes. In embodiments, the compound is a thienopyrimidine compound. In embodiments, the compound as the structure of formula: The term “A2Aadenosine receptor” as provided herein includes any of the recombinant or naturally-occurring forms of the A2Aadenosine receptor (ADORA2A) or variants or homologs thereof that maintain ADORA2A protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ADORA2A). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ADORA2A polypeptide. In embodiments, ADORA2A is the protein as identified by the NCBI sequence reference GI:5921992, homolog or functional fragment thereof. The term “A2Badenosine receptor” as provided herein includes any of the recombinant or naturally-occurring forms of the A2Badenosine receptor (ADORA2B) or variants or homologs thereof that maintain ADORA2B protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to ADORA2B). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring ADORA2B polypeptide. In embodiments, ADORA2B is the protein as identified by the NCBI sequence reference GI:4501951, homolog or functional fragment thereof. In embodiments, the therapeutic agent is a second humanized antibody. In embodiments, the second humanized antibody is an antibody capable of binding protein programmed cell death ligand 1 (PD-L1). In embodiments, the second humanized antibody is atezolizumab. In embodiments, the second humanized antibody is an antibody capable of binding protein programmed cell death protein 1 (PD-1). In embodiments, the second humanized antibody is an antibody capable of binding CTLA-4. The term “atezolizumab” or “MPDL3280A” refers a fully humanized, engineered monoclonal antibody of IgG1 isotype against the protein programmed cell death ligand 1 (PD-L1). In the customary sense, atezolizumab refers to CAS Registry number 1380723-44-3. The term “PDL-1” as provided herein includes any of the recombinant or naturally-occurring forms of the protein programmed cell death ligand 1 (PD-L1) or variants or homologs thereof that maintain PDL-1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PDL-1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PDL-1 polypeptide. In embodiments, PDL-1 is the protein as identified by the NCBI sequence reference GI:390979639, homolog or functional fragment thereof. The term “PD-1” as provided herein includes any of the recombinant or naturally-occurring forms of the protein programmed cell death protein 1 (PD-1) or variants or homologs thereof that maintain PD-1 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to PD-1). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring PD-1 polypeptide. In embodiments, PD-1 is the protein as identified by the NCBI sequence reference GI:167857792, homolog or functional fragment thereof. The term “CTLA-4” or “CTLA-4 protein” as provided herein includes any of the recombinant or naturally-occurring forms of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or variants or homologs thereof that maintain CTLA-4 protein activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CTLA-4). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CTLA-4 polypeptide. In embodiments, CTLA-4 is the protein as identified by the NCBI sequence reference GI:83700231, homolog or functional fragment thereof. In the provided methods of treatment, additional therapeutic agents can be used that are suitable to the disease (e.g., cancer) being treated. Thus, in some embodiments, the provided methods of treatment further include administering a second therapeutic agent to the subject. Suitable additional therapeutic agents include, but are not limited to analgesics, anesthetics, analeptics, corticosteroids, anticholinergic agents, anticholinesterases, anticonvulsants, antineoplastic agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, antihelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antiviral agents, dopaminergics, hematological agents, immunological agents, muscarinics, protease inhibitors, vitamins, growth factors, and hormones. The choice of agent and dosage can be determined readily by one of skill in the art based on the given disease being treated. Combinations of agents or compositions can be administered either concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., one agent is administered first followed by administration of the second agent). Thus, the term combination is used to refer to concomitant, simultaneous or sequential administration of two or more agents or compositions. The course of treatment is best determined on an individual basis depending on the particular characteristics of the subject and the type of treatment selected. The treatment, such as those disclosed herein, can be administered to the subject on a daily, twice daily, bi-weekly, monthly or any applicable basis that is therapeutically effective. The treatment can be administered alone or in combination with any other treatment disclosed herein or known in the art. The additional treatment can be administered simultaneously with the first treatment, at a different time, or on an entirely different therapeutic schedule (e.g., the first treatment can be daily, while the additional treatment is weekly). According to the methods provided herein, the subject is administered an effective amount of one or more of the therapeutic agents provided herein (i.e. a humanized 1E9 antibody or a humanized IgG1 or IgG4 antibody in combination with, for example, a compound or a second humanized antibody). The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response (e.g., reduction of inflammation). Effective amounts and schedules for administering the agent may be determined empirically by one skilled in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, for the given parameter, an effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. The exact dose and formulation will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar, Dosage Calculations (1999)). EXAMPLES Example 1 Switching Anti-CD73 (1E9) to Human IgG1 Isotype Eliminates Direct Effects on T Cell Activation Seen with Mouse IgG3 Isotype. The antibody prepared from the 1E9 hybridoma cell line is a mIgG3 isotype. This antibody has been shown to synergize with PMA to activate T cell proliferation, IL-2 secretion, and upregulation of IL-2 receptor expression (PMID: 2550543). This effect is thought to be through a direct signaling mechanism, as 1E9 mediates these effects on cells that express catalytically inactive CD73 or CD73 that is attached to the cell membrane through a transmembrane region as opposed to the GPI-anchoring mechanism that is used endogenously (PMID: 9113412, 7697732, 8027539). Applicants swapped the variable regions from 1E9 onto a human IgG1 framework, creating a chimeric 1E9 antibody. Chimeric 1E9 does not mediate activation of T cell proliferation or IL-2 receptor (CD25 expression). Thus, Applicants have discovered that switching of the isotype can alter antibody-mediated effects on CD73 signaling. A Cellular Assay to Evaluate Catalytic Activity of CD73 CD73 is expressed on the cell surface and is an ectonucleotidase that hydrolyzes AMP to adenosine and phosphate. In order to assess the catalytic activity of CD73 and the ability of Applicants' anti-CD73 antibodies to inhibit this activity, Applicants used a cellular assay. Cells endogenously expressing CD73 were incubated with anti-CD73 antibodies or an isotype control over a range of concentrations at 37° C. prior to addition of AMP. Cells were incubated with AMP at 37° C. for 20 minutes. Phosphate levels in the media were measured with a commercially available reagent (Sensolyte MG phosphate assay kit, AnaSpec) and are directly proportional to CD73 activity. This assay was adapted from the literature and has been previously described for use in measuring CD73 activity (PMID: 21506751). To Applicants' knowledge, this assay is always performed at a physiological pH of approximately 7.2. Surprisingly, Applicants' were able to show that if the assay was performed at a lower pH to screen for anti-CD73 activities, the ability to block CD73 activity at lower pH was retained. Robust activity at slightly acidic pH would be a desired property of a therapeutic antibody to be used for solid tumor indications as the solid tumor microenvironment is known to be slightly acidic. Applicants found that some antibodies (CPX-002, CPX-005, CPX-006) retain potency in blocking of CD73 activity at lower pH (6.3 or 6.7), while other antibodies (CPX-003, CPX-004) lose potency at lower pH. Affinity Measurements Applicants obtained affinity measurements for 5 humanized anti-CD73 candidates (CPX-003, CPX-004, CPX-005, CPX-006, CPX-007) as well as a chimeric antibody (CPX-002) to measure binding of these antibodies to CD73. The results of these affinity measurements are summarized in Table 1. TABLE 1Affinity measurements of humanizedand chimeric anti-CD73 antibodies.AntibodykDCPX-0029.4 nMCPX-00319.5 nMCPX-00417.8 nMCPX-0056.9 nMCPX-0067.1 nMCPX-00715.9 nM Specific Chain Associated with Highest CD73 Affinity and Best Potency at Low pH. Two humanized antibodies (CPX-005, CPX-006) have higher affinity for CD73 and improved potency for inhibition of CD73 activity compared to other candidates. These two antibodies use the same heavy chain and differ only in the light chain. Thus, this particular heavy chain may be important for achieving high affinity and potent inhibition of CD73 activity. In embodiments, the humanized heavy chain variable region includes the sequence of SEQ ID NO:7. In embodiments, the humanized heavy chain variable region is the sequence of SEQ ID NO:7. Example 2 Humanization of Clone BAP094-01 Applicants have completed the construction of the humanization library of clone BAP094-01 (chimeric 1E9). Double stranded DNA fragments coding for the light chain and heavy chain CDR sequences of BAP094-01 (SEQ ID NO:28 and SEQ ID NO:29, respectively) were combined with pools of human frameworks. Full length variable domains were then cloned into mammalian expression vector. Light chain variable domains were cloned in frame with a secretion signal and a human kappa constant domain. Heavy chain variable domains were cloned in frame with a leader sequence and a human IgG1 constant domain. The quality of the library (diversity of the synthesized variable domains) was confirmed by sequencing (data not shown). Screening of Humanized Variants The humanized clones were arrayed into 96 well plates. Each plate also contains two wells of positive control (BAP094-01, i.e., chimeric 1E9) and negative control (vector only). Plasmid DNA was prepared for each plate and transfected into CHO-S cells in 96 well format. Supernatant was collected at 48 hours post transfection. IgG concentration was determined using ELISA protocol for quantitation of human IgGs. Binding of the humanized clones to CD73 expressed on the surface of MDA-MB-231 cells was determined using a cell based ELISA. Top hits from the primary screening were rearrayed, re-transfected and screened again by cell-based ELISA (FIG.5). Sequence Analysis of Top Humanized Variant Hits The light chain and heavy chain variable domains of the selected top humanized variant clones were sequenced and aligned with the parental murine sequences of clone BAP094-01 (1E9). Sequence analysis shows that there are four different heavy chains and 5 different light chains within the top 10 clones (FIG.4AandFIG.4B). Each top hit has a unique combination of humanized light and heavy chain. FORMAL SEQUENCE LISTINGSEQ ID NO: 1:RASKNVSTSGYSYMHSEQ ID NO: 2:LASNLESSEQ ID NO: 3:QHSRELPFTSEQ ID NO: 4:GYTFTSYWITSEQ ID NO: 5:PGSGNTNYNEKFKTSEQ ID NO: 6:EGGLTTEDYALDYSEQ ID NO: 7:QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVBAP094-hum01-LC SEQ ID NO: 8:GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGGTTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum02-LC SEQ ID NO: 9:GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCACCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum03-LC, CPX-003 SEQ ID NO: 10:GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCACCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum04-LC SEQ ID NO: 11:GAAATTGTGCTGACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTCACCATCACCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum05-LC, CPX-004 SEQ ID NO: 12:GATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum06-LC, CPX-005 SEQ ID NO: 13:GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACCTTTACCATCAGTAGCCTGGAAGCTGAAGATGCTGCAACATATTACTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum07-LC, CPX-006, CPX-007 SEQ ID NO: 14:GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum08-LC SEQ ID NO: 15:GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum09-LC SEQ ID NO: 16:GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum10-LC SEQ ID NO: 17:GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCCAAGGGACCAAGGTGGAAATCAAABAP094-hum01-HC SEQ ID NO: 18:CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum02-HC SEQ ID NO: 19:CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum03-HC, CPX-003, CPX-007 SEQ ID NO: 20:CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum04-HC SEQ ID NO: 21:GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum05-HC, CPX-004 SEQ ID NO: 22:GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum06-HC, CPX-005, CPX-006 SEQ ID NO: 23:CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum07-HC SEQ ID NO: 24:CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGGAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum08-HC SEQ ID NO: 25:GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum09-HC SEQ ID NO: 26:CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGCGACAGGCTCGTGGACAACGCCTTGAGTGGATAGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-hum10-HC SEQ ID NO: 27:GAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCTACAGTGAAAATCTCCTGCAAGGTTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGTGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAGAGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCA[0253]BAP094-01-LC CPX-002 (chimeric) SEQ ID NO: 28:GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAATGTCAGTACATCTGGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTACCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAGTAGGGAGCTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA[0254]BAP094-01-HC CPX-002 (chimeric) SEQ ID NO: 29:CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATAACCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGATATTTATCCTGGTAGTGGTAATACTAACTACAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAAAGAGGGAGGTCTTACTACGGAGGATTATGCTTTGGACTACTGGGGCCAGGGAACGCTGGTCACCGTCAGCTCABAP094-01-LC SEQ ID NO: 30:DIVLTQSPASLAVSLGQRATISCRASKNVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPTRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGSGTKLEIKBAP094-hum01-LC SEQ ID NO: 31:AIQLTQSPSSLSASVGDRVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum02-LC SEQ ID NO: 32:EIVLTQSPDFQSVTPKEKVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum03-LC SEQ ID NO: 33:EIVLTQSPDFQSVTPKEKVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIBAP094-hum04-LC SEQ ID NO: 34:EIVLTQSPDFQSVTPKEKVTITCRASKNVSTSGYSYMHWYQQKPGKAPKLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum05-LC SEQ ID NO: 35:DVVMTQSPLSLPVTLGQPASISCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum06-LC SEQ ID NO: 36:AIQLTQSPSSLSASVGDRVTITCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQHSRELPFTFGQGTKVEIKBAP094-hum07-LC SEQ ID NO: 37:EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum08-LC SEQ ID NO: 38:EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum09-LC SEQ ID NO: 39:EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-hum10-LC SEQ ID NO: 40:EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKBAP094-01-HC SEQ ID NO: 41:QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWITWVKQRPGQGLEWIGDIYPGSGNTNYNEKFKTKATLTVDTSSSTAYMQLSSLTSEDSAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum01-HC SEQ ID NO: 42:QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum02-HC SEQ ID NO: 43:QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum03-HC SEQ ID NO: 44:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITWVRQARGQRLEWIGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum04-HC SEQ ID NO: 45:EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWIRQPPGKGLEWIGDIYPGSGNTNYNEKFKTRVTISADKSISTAYLQWSSLKASDTAMYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum05-HC SEQ ID NO: 46:EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWIRQPPGKGLEWIGDIYPGSGNTNYNEKFKTRVTISADKSISTAYLQWSSLKASDTAMYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum06-HC SEQ ID NO: 47:QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum07-HC SEQ ID NO: 48:QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum08-HC SEQ ID NO: 49:EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum09-HC SEQ ID NO: 50:QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWITWVRQARGQRLEWIGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSBAP094-hum10-HC SEQ ID NO: 51:EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWITWIRQPPGKGLEWIGDIYPGSGNTNYNEKFKTRVTISADKSISTAYLQWSSLKASDTAMYYCAKEGGLTTEDYALDYWGQGTLVTVSSCPX-006_HC (codon-optimized) SEQ ID NO: 52:AAGCTTGCCGCCACCATGGAATGGTCCTGGGTGTTCCTGTTCTTCCTGTCCGTGACCACCGGCGTGCACTCCCAGGTGCAGCTGGTGCAGTCTGGCGCCGAGGTGGAAAAGCCTGGCGCCTCTGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTTACCAGCTACTGGATCACCTGGGTGCGACAGGCTCCTGGACAGGGCCTGGAATGGATGGGCGACATCTACCCTGGCTCCGGCAACACCAACTACAACGAGAAGTTCAAGACCCGCGTGACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGAACTGTCCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCTAAAGAGGGCGGCCTGACCACCGAGGACTACGCCCTGGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTTCTACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACTGCTGGGCGGACCCTCTGTGTTTCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACCAGTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCTCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTTCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGATGAATTCCPX-006_HC (codon-optimized) SEQ ID NO: 53:MEWSWVFLFFLSVTTGVHSQVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQGLEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKEGGLTTEDYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCPX-006_LC (codon-optimized) SEQ ID NO: 54:AAGCTTGCCGCCACCATGTCCGTGCCTACCCAGGTGCTGGGACTGCTGCTGCTGTGGCTGACCGATGCCAGATGCGAGATCGTGCTGACCCAGTCCCCTGCCACCCTGTCACTGTCTCCAGGCGAGAGAGCCACCCTGAGCTGCCGGGCCTCCAAGAACGTGTCCACCTCCGGCTACTCCTACATGCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACCTGGCCTCCAACCTGGAATCCGGCATCCCCCCTAGATTCTCCGGCTCTGGCTACGGCACCGACTTCACCCTGACCATCAACAACATCGAGTCCGAGGACGCCGCCTACTACTTCTGCCAGCACTCCAGAGAGCTGCCCTTCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGCTGATGAATTCCPX-006_LC (codon-optimized) SEQ ID NO: 55:MSVPTQVLGLLLLWLTDARCEIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQKPGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRELPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 222,026 |
11859008 | In contrast to normal tissues, strong and homogenous staining was observed on tissue sections from ovarian and testis cancers. A very strong membraneous staining of the malignant epithelial cell populations was detected, whereas adjacent stromal and non-malignant epithelial cells were not stained. These results clearly show that our CLDN6-specific antibodies bind specifically to malignant cells derived from tumor patients. (Explanation: number of tissues that were stained by antibody/number of analysed tissues.) Definition of Terms In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. Claudins are a family of proteins that are the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between cells of an epithelium. Claudins are transmembrane proteins spanning the membrane 4 times with the N-terminal and the C-terminal end both located in the cytoplasm. The first extracellular loop consists on average of 53 amino acids and the second one of around 24 amino acids. CLDN6 and CLDN9 are the most similar members of the CLDN family. The term “CLDN” as used herein means claudin and includes CLDN6, CLDN9, CLDN4 and CLDN3. Preferably, a CLDN is a human CLDN. The term “CLDN6” preferably relates to human CLDN6, and, in particular, to (i) a nucleic acid comprising a nucleic acid sequence encoding the amino sequence of SEQ ID NO: 2 or encoding the amino sequence of SEQ ID NO: 8 such as a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1 or (ii) a protein comprising the amino acid sequence of SEQ ID NO: 2 or comprising the amino acid sequence of SEQ ID NO: 8. The first extracellular loop of CLDN6 preferably comprises amino acids 28 to 80, more preferably amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 8, such as the amino acid sequence shown in SEQ ID NO: 7. The second extracellular loop of CLDN6 preferably comprises amino acids 138 to 160, preferably amino acids 141 to 159, more preferably amino acids 145 to 157 of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 8, such as the amino acid sequence shown in SEQ ID NO: 6. Said first and second extracellular loops preferably form the extracellular portion of CLDN6. The term “CLDN9” preferably relates to human CLDN9, and, in particular, to (i) a nucleic acid comprising a nucleic acid sequence encoding the amino sequence of SEQ ID NO: 9 or (ii) a protein comprising the amino acid sequence of SEQ ID NO: 9. The first extracellular loop of CLDN9 preferably comprises amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 9. The second extracellular loop of CLDN9 preferably comprises amino acids 141 to 159 of the amino acid sequence shown in SEQ ID NO: 9. Said first and second extracellular loops preferably form the extracellular portion of CLDN9. The term “CLDN4” preferably relates to human CLDN4, and, in particular, to (i) a nucleic acid comprising a nucleic acid sequence encoding the amino sequence of SEQ ID NO: 10 or (ii) a protein comprising the amino acid sequence of SEQ ID NO: 10. The first extracellular loop of CLDN4 preferably comprises amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 10. The second extracellular loop of CLDN4 preferably comprises amino acids 141 to 159 of the amino acid sequence shown in SEQ ID NO: 10. Said first and second extracellular loops preferably form the extracellular portion of CLDN4. The term “CLDN3” preferably relates to human CLDN3, and, in particular, to (i) a nucleic acid comprising a nucleic acid sequence encoding the amino sequence of SEQ ID NO: 11 or (ii) a protein comprising the amino acid sequence of SEQ ID NO: 11. The first extracellular loop of CLDN3 preferably comprises amino acids 27 to 75 of the amino acid sequence shown in SEQ ID NO: 11. The second extracellular loop of CLDN3 preferably comprises amino acids 140 to 158 of the amino acid sequence shown in SEQ ID NO: 11. Said first and second extracellular loops preferably form the extracellular portion of CLDN3. The above described CLDN sequences include any variants of said sequences, in particular mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those which are naturally present. An allelic variant relates to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence. The term “CLDN” shall encompass (i) CLDN splice variants, (ii) CLDN-posttranslationally modified variants, particularly including variants with different glycosylation such as N-glycosylation status, (iii) CLDN conformation variants, (iv) CLDN cancer related and CLDN non-cancer related variants. Preferably, a CLDN is present in its native conformation. CLDN6 has been found to be expressed, for example, in ovarian cancer, lung cancer, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, melanomas, head neck cancer, sarcomas, bile duct cancer, renal cell cancer, and urinary bladder cancer. CLDN6 is a particularly preferred target for the prevention and/or treatment of ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, cancer of the urinary bladder, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small bowel cancer, including cancer of the ileum, in particular small bowel adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonic testicular cancer, uterine cancer, a germ cell tumor such as a teratocarcinoma or an embryonal carcinoma, in particular a germ cell tumor of the testis, and the metastatic forms thereof. In one embodiment, the cancer disease associated with CLDN6 expression is selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer and metastatic lung cancer. Preferably, the ovarian cancer is a carcinoma or an adenocarcinoma. Preferably, the lung cancer is a carcinoma or an adenocarcinoma, and preferably is bronchiolar cancer such as a bronchiolar carcinoma or bronchiolar adenocarcinoma. In one embodiment, the tumor cell associated with CLDN6 expression is a cell of such a cancer. The term “portion” refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term “portion” thereof may designate a continuous or a discontinuous fraction of said structure. Preferably, a portion of an amino acid sequence comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, preferably at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the amino acids of said amino acid sequence. Preferably, if the portion is a discontinuous fraction said discontinuous fraction is composed of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure, each part being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8, or more, preferably not more than 4 parts of said amino acid sequence, wherein each part preferably comprises at least 5 continuous amino acids, at least 10 continuous amino acids, preferably at least 20 continuous amino acids, preferably at least 30 continuous amino acids of the amino acid sequence. The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. A portion, a part or a fragment of a structure preferably comprises one or more functional properties of said structure. For example, a portion, a part or a fragment of an epitope or peptide is preferably immunologically equivalent to the epitope or peptide it is derived from. The term “an extracellular portion of a CLDN” in the context of the present invention refers to a part of a CLDN facing the extracellular space of a cell and preferably being accessible from the outside of said cell, e.g., by antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or a part thereof or any other extracellular part of a CLDN which is preferably specific for said CLDN. Preferably, said part comprises at least 5, at least 8, at least 10, at least 15, at least 20, at least 30, or at least 50 amino acids or more. The term “CLDN associated with the surface of a cell” is to be understood to relate to native CLDN, i.e. CLDN in its non-denatured, preferably naturally folded state. Preferably, the term “CLDN associated with the surface of a cell” means that the CLDN is associated with and located at the plasma membrane of said cell, wherein at least a part of the CLDN, preferably the extracellular portion, faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, and/or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a CLDN associated with the surface of a cell may be a transmembrane protein, i.e. an integral membrane protein, having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein. CLDN6 is associated with the surface of a cell if it is located at the surface of said cell and is accessible to binding by CLDN6-specific antibodies added to the cell. In preferred embodiments, a cell being characterized by association of CLDN6 with its cell surface is a cell expressing CLDN6. It is to be understood that in the case where CLDN6 is expressed by cells, the CLDN6 associated with the surface of said cells may only be a portion of the expressed CLDN6. The term “a cell carrying a CLDN” preferably means that said cell carries a CLDN on its surface, i.e., that the CLDN is associated with the surface of said cell. “Cell surface” or “surface of a cell” is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. The expression “CLDN expressed on the surface of a cell” means that the CLDN expressed by a cell is found in association with the surface of said cell. According to the invention CLDN6 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression and association is lower compared to expression and association in placenta cells or placenta tissue. Preferably, the level of expression and association is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of the expression and association in placenta cells or placenta tissue or even lower. Preferably, CLDN6 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression and association exceeds the level of expression and association in non-tumorigenic, non-cancerous tissue other than placenta tissue by no more than 2-fold, preferably 1.5-fold, and preferably does not exceed the level of expression and association in said non-tumorigenic, non-cancerous tissue. Preferably, CLDN6 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression or association is below the detection limit and/or if the level of expression or association is too low to allow binding by CLDN6-specific antibodies added to the cells. According to the invention CLDN6 is expressed in a cell and is associated with a cell surface if the level of expression and association exceeds the level of expression and association in non-tumorigenic, non-cancerous tissue other than placenta tissue, preferably by more than 2-fold, preferably 10-fold, 100-fold, 1000-fold, or 10000-fold. Preferably, CLDN6 is expressed in a cell and is associated with a cell surface if the level of expression and association is above the detection limit and/or if the level of expression and association is high enough to allow binding by CLDN6-specific antibodies added to the cells. Preferably, CLDN6 expressed in a cell is expressed or exposed on the surface of said cell. The term “raft” refers to the sphingolipid- and cholesterol-rich membrane microdomains located in the outer leaflet area of the plasma membrane of a cell. The ability of certain proteins to associate within such domains and their ability of forming “aggregates” or “focal aggregates” can effect the protein's function. For example, the translocation of CLDN6 molecules into such structures, after being bound by antibodies of the present invention, creates a high density of CLDN6 antigen-antibody complexes in the plasma membranes. Such a high density of CLDN6 antigen-antibody complexes can enable efficient activation of the complement system during CDC. According to the invention, the term “disease” refers to any pathological state, including cancer, in particular those forms of cancer described herein. “Diseases involving cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface” means according to the invention that expression and association in cells of a diseased tissue or organ is preferably increased compared to the state in a healthy tissue or organ. An increase refers to an increase by at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more. In one embodiment, expression and association with the cell surface is only found in a diseased tissue, while expression in a healthy tissue is repressed. According to the invention, diseases associated with cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface include tumor diseases such as cancer diseases. Furthermore, according to the invention, tumor diseases such as cancer diseases preferably are those wherein the tumor cells or cancer cells express CLDN6 and are characterized by association of CLDN6 with their cell surface. As used herein, a “tumor disease”, “tumor-related disease” or “tumorigenic disease” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and/or migration, which may result in the production of or tendency to produce tumors and/or tumor metastasis. By “tumor cell” is meant an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. By “tumor” is meant an abnormal group of cells or a tissue growing by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be either benign, pre-malignant or malignant. Preferably, a “tumor disease”, “tumor-related disease” or “tumorigenic disease” according to the invention is a cancer disease, i.e. a malignant disease and a tumor cell is a cancer cell. Preferably, a “tumor disease”, “tumor-related disease” or “tumorigenic disease” is characterized by cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface and a tumor cell expresses CLDN6 and is characterized by association of CLDN6 with its cell surface. A cell expressing CLDN6 and being characterized by association of CLDN6 with its cell surface preferably is a tumor cell or cancer cell, preferably of the tumors and cancers described herein. Preferably, such cell is a cell other than a placental cell. Preferred cancer diseases or cancers according to the invention are selected from the group consisting of ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma, lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung carcinoma and adenocarcinoma, gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer, in particular basal cell carcinoma and squamous cell carcinoma, malignant melanoma, head and neck cancer, in particular malignant pleomorphic adenoma, sarcoma, in particular synovial sarcoma and carcinosarcoma, bile duct cancer, cancer of the urinary bladder, in particular transitional cell carcinoma and papillary carcinoma, kidney cancer, in particular renal cell carcinoma including clear cell renal cell carcinoma and papillary renal cell carcinoma, colon cancer, small bowel cancer, including cancer of the ileum, in particular small bowel adenocarcinoma and adenocarcinoma of the ileum, testicular embryonal carcinoma, placental choriocarcinoma, cervical cancer, testicular cancer, in particular testicular seminoma, testicular teratoma and embryonic testicular cancer, uterine cancer, a germ cell tumor such as a teratocarcinoma or an embryonal carcinoma, in particular a germ cell tumor of the testis, and the metastatic forms thereof. The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). There are three main sub-types of the non-small cell lung carcinomas: squamous cell lung carcinoma, adenocarcinoma, and large cell lung carcinoma. Adenocarcinomas account for approximately 10% of lung cancers. This cancer usually is seen peripherally in the lungs, as opposed to small cell lung cancer and squamous cell lung cancer, which both tend to be more centrally located. Skin cancer is a malignant growth on the skin. The most common skin cancers are basal cell cancer, squamous cell cancer, and melanoma. Malignant melanoma is a serious type of skin cancer. It is due to uncontrolled growth of pigment cells, called melanocytes. According to the invention, a “carcinoma” is a cancer that begins in the lining layer (epithelial cells) of organs. “Bronchiolar carcinoma” is a carcinoma of the lung, thought to be derived from epithelium of terminal bronchioles, in which the neoplastic tissue extends along the alveolar walls and grows in small masses within the alveoli. Mucin may be demonstrated in some of the cells and in the material in the alveoli, which also includes denuded cells. “Adenocarcinoma” is a cancer that originates in glandular tissue. This tissue is also part of a larger tissue category known as epithelial tissue. Epithelial tissue includes skin, glands and a variety of other tissue that lines the cavities and organs of the body. Epithelium is derived embryologically from ectoderm, endoderm and mesoderm. To be classified as adenocarcinoma, the cells do not necessarily need to be part of a gland, as long as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well differentiated adenocarcinomas tend to resemble the glandular tissue that they are derived from, while poorly differentiated may not. By staining the cells from a biopsy, a pathologist will determine whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinomas can arise in many tissues of the body due to the ubiquitous nature of glands within the body. While each gland may not be secreting the same substance, as long as there is an exocrine function to the cell, it is considered glandular and its malignant form is therefore named adenocarcinoma. Malignant adenocarcinomas invade other tissues and often metastasize given enough time to do so. Ovarian adenocarcinoma is the most common type of ovarian carcinoma. It includes the serous and mucinous adenocarcinomas, the clear cell adenocarcinoma and the endometrioid adenocarcinoma. “Cystadenocarcinoma” is a malignant form of a surface epithelial-stromal tumor, a type of ovarian cancer. Surface epithelial-stromal tumors are a class of ovarian neoplasms that are thought to be derived from the ovarian surface epithelium (modified peritoneum) or from ectopic endometrial or Fallopian tube (tubal) tissue. This group of tumors accounts for the majority of all ovarian tumors. Teratocarcinoma refers to a germ cell tumor that is a mixture of teratoma with embryonal carcinoma, or with choriocarcinoma, or with both. Choriocarcinoma is a malignant, trophoblastic and aggressive cancer, usually of the placenta. It is characterized by early hematogenous spread to the lungs. A sarcoma is a cancer of the connective tissue (bone, cartilage, fat) resulting in mesoderm proliferation. This is in contrast to carcinomas, which are of epithelial origin. A synovial sarcoma is a rare form of cancer which usually occurs near to the joints of the arm or leg. It is one of the soft tissue sarcomas. Renal cell carcinoma also known as renal cell cancer or renal cell adenocarcinoma is a kidney cancer that originates in the lining of the proximal convoluted tubule, the very small tubes in the kidney that filter the blood and remove waste products. Renal cell carcinoma is by far the most common type of kidney cancer in adults and the most lethal of all the genitorurinary tumors. Distinct subtypes of renal cell carcinoma are clear cell renal cell carcinoma and papillary renal cell carcinoma. Clear cell renal cell carcinoma is the most common form of renal cell carcinoma. When seen under a microscope, the cells that make up clear cell renal cell carcinoma appear very pale or clear. Papillary renal cell carcinoma is the second most common subtype. These cancers form little finger-like projections (called papillae) in some, if not most, of the tumors. A germ cell tumor is a neoplasm derived from germ cells. Germ cell tumors can be cancerous or non-cancerous tumors. Germ cells normally occur inside the gonads (ovary and testis). Germ cell tumors that originate outside the gonads (e.g. in head, inside the mouth, neck, pelvis; in fetuses, babies, and young children most often found on the body midline, particularly at the tip of the tailbone) may be birth defects resulting from errors during development of the embryo. The two major classes of germ cell tumors are the seminomas and non-seminomas, wherein non-seminomas include: teratocarcinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma and differentiated teratoma. Most cell lines from non-seminomas are equivalent to embryonal carcinomas, that is, they are composed almost entirely of stem cells which do not differentiate under basal conditions, though some may respond to inducers of differentiation such as retinoic acid. By “metastasis” is meant the spread of cancer cells from its original site to another part of the body. The formation of metastasis is a very complex process and depends on detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood, infiltration of target organs. Finally, the growth of a new tumor at the target site depends on angiogenesis. Tumor metastasis often occurs even after the removal of the primary tumor because tumor cells or components may remain and develop metastatic potential. In one embodiment, the term “metastasis” according to the invention relates to “distant metastasis” which relates to a metastasis which is remote from the primary tumor and the regional lymph node system. The cells of a secondary or metastatic tumor are like those in the original tumor. This means, for example, that, if ovarian cancer metastasizes to the liver, the secondary tumor is made up of abnormal ovarian cells, not of abnormal liver cells. The tumor in the liver is then called metastatic ovarian cancer, not liver cancer. By “treat” is meant to administer a compound or composition as described herein to a subject in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; arrest or slow a disease in a subject; inhibit or slow the development of a new disease in a subject; decrease the frequency or severity of symptoms and/or recurrences in a subject who currently has or who previously has had a disease; and/or prolong, i.e. increase the lifespan of the subject. The term “treatment of a disease” includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof. By “being at risk” is meant a subject, i.e. a patient, that is identified as having a higher than normal chance of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had, or who currently has, a disease, in particular cancer is a subject who has an increased risk for developing a disease, as such a subject may continue to develop a disease. Subjects who currently have, or who have had, a cancer also have an increased risk for cancer metastases. The term “immunotherapy” relates to a treatment involving a specific immune reaction. In the context of the present invention, terms such as “protect”, “prevent”, “prophylactic”, “preventive”, or “protective” relate to the prevention or treatment or both of the occurrence and/or the propagation of a tumor in an individual. The term “immunotherapy” in the context of the present invention preferably refers to active tumor immunization or tumor vaccination. A prophylactic administration of an immunotherapy, for example, a prophylactic administration of the composition of the invention, preferably protects the recipient from the development of tumor growth. A therapeutic administration of an immunotherapy, for example, a therapeutic administration of the composition of the invention, may lead to the inhibition of the progress/growth of the tumor. This comprises the deceleration of the progress/growth of the tumor, in particular a disruption of the progression of the tumor, which preferably leads to elimination of the tumor. A therapeutic administration of an immunotherapy may protect the individual, for example, from the dissemination or metastasis of existing tumors. The term “immunization” or “vaccination” describes the process of administering antigen to a subject with the purpose of inducing an immune response for therapeutic or prophylactic reasons. The terms “subject”, “individual”, “organism” or “patient” are used interchangeably and relate to vertebrates, preferably mammals. For example, mammals in the context of the present invention are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc. as well as animals in captivity such as animals of zoos. The term “animal” as used herein also includes humans. The term “subject” may also include a patient, i.e., an animal, preferably a human having a disease, preferably a disease associated with expression of CLDN6, preferably a tumorigenic disease such as a cancer. The term “adjuvant” relates to compounds which prolongs or enhances or accelerates an immune response. The composition of the present invention preferably exerts its effect without addition of adjuvants. Still, the composition of the present application may contain any known adjuvant. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such asBordetella pertussistoxin), liposomes, and immune-stimulating complexes. Examples for adjuvants are monophosphoryl-lipid-A (MPL SmithKline Beecham). Saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18, and QS-L1 (So et al., 1997, Mol. Cells 7: 178-186), incomplete Freund's adjuvants, complete Freund's adjuvants, vitamin E, montanid, alum, CpG oligonucleotides (Krieg et al., 1995, Nature 374: 546-549), and various water-in-oil emulsions which are prepared from biologically degradable oils such as squalene and/or tocopherol. According to the invention, a sample may be any sample useful according to the present invention, in particular a biological sample such a tissue sample, including bodily fluids, and/or a cellular sample and may be obtained in the conventional manner such as by tissue biopsy, including punch biopsy, and by taking blood, bronchial aspirate, sputum, urine, feces or other body fluids. According to the invention, the term “biological sample” also includes fractions of biological samples. The term “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof. The term “antibody” includes monoclonal antibodies and fragments or derivatives thereof, including, without limitation, human monoclonal antibodies, humanized monoclonal antibodies, chimeric monoclonal antibodies, single chain antibodies, e.g., scFv's and antigen-binding antibody fragments such as Fab and Fab′ fragments and also includes all recombinant forms of antibodies, e.g., antibodies expressed in prokaryotes, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. According to the invention, the term “at least one of the CDR sequences” preferably means at least the CDR3 sequence. The term “CDR sequences of an antibody chain” preferably relates to CDR1, CDR2 and CDR3 of the heavy chain or light chain of an antibody. According to the invention, a reference to an antibody chain comprising a particular CDR sequence such as a particular CDR3 sequence means that said particular CDR sequence either forms the CDR region such as the CDR3 region of said antibody chain, i.e. the CDR region consists of said particular CDR sequence, or forms a part of the CDR region such as the CDR3 region of said antibody chain, i.e. the CDR region comprises said particular CDR sequence. If according to the invention reference is made to an antibody comprising a particular antibody heavy chain and/or a particular antibody light chain, such as a chain comprising particular CDR sequences, it is preferred that both heavy chains and/or both light chains of the antibody are each composed of the particular antibody heavy chain and/or the particular antibody light chain. The term “humanized antibody” refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin from a non-human species, wherein the remaining immunoglobulin structure of the molecule is based upon the structure and/or sequence of a human immunoglobulin. The antigen binding site may either comprise complete variable domains fused onto constant domains or only the complementarity determining regions (CDR) grafted onto appropriate framework regions in the variable domains. Antigen binding sites may be wild-type or modified by one or more amino acid substitutions, e.g. modified to resemble human immunoglobulins more closely. Some forms of humanized antibodies preserve all CDR sequences (for example a humanized mouse antibody which contains all six CDRs from the mouse antibody). Other forms have one or more CDRs which are altered with respect to the original antibody. The term “chimeric antibody” refers to those antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chain is homologous to corresponding sequences in another. Typically the variable region of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to sequences of antibodies derived from another. One clear advantage to such chimeric forms is that the variable region can conveniently be derived from presently known sources using readily available B-cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and the specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response from a human subject when the antibodies are injected than would the constant region from a non human source. However the definition is not limited to this particular example. The term “antigen-binding portion” of an antibody (or simply “binding portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH domains; (ii) F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546), which consist of a VH domain; (vi) isolated complementarity determining regions (CDR), and (vii) combinations of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. The term “epitope” refers to an antigenic determinant in a molecule, i.e., to the part in a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are the discrete, three-dimensional sites on an antigen, which are recognized by the immune system. In the context of the present invention, the epitope is preferably derived from a CLDN protein. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope of a protein such as a CLDN preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, for example, the epitope may be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. The term “discontinuous epitope” as used herein, means a conformational epitope on a protein antigen which is formed from at least two separate regions in the primary sequence of the protein. The term “bispecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities. For example, the molecule may bind to, or interact with (a) a cell surface antigen, and (b) an Fc receptor on the surface of an effector cell. The term “multispecific molecule” or “heterospecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule may bind to, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to CLDN6, and to other targets, such as Fc receptors on effector cells. The term “bispecific antibodies” also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). As used herein, the term “heteroantibodies” refers to two or more antibodies, derivatives thereof, or antigen binding regions linked together, at least two of which have different specificities. These different specificities include a binding specificity for an Fc receptor on an effector cell, and a binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell. The antibodies described herein may be human antibodies. The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The term “monoclonal antibody” as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. In one embodiment, the monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a non-human animal, e.g., mouse, fused to an immortalized cell. The term “recombinant antibody”, as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal with respect to the immunoglobulin genes or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences. The term “transfectoma”, as used herein, includes recombinant eukaryotic host cells expressing an antibody, such as CHO cells, NS/0 cells, HEK293 cells, HEK293T cells, plant cells, or fungi, including yeast cells. As used herein, a “heterologous antibody” is defined in relation to a transgenic organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic organism, and being generally derived from a species other than the transgenic organism. As used herein, a “heterohybrid antibody” refers to an antibody having light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. The invention includes all antibodies and derivatives of antibodies as described herein which for the purposes of the invention are encompassed by the term “antibody”. The term “antibody derivatives” refers to any modified form of an antibody, e.g., a conjugate of the antibody and another agent or antibody, or an antibody fragment. The antibodies described herein are preferably isolated. An “isolated antibody” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to CLDN6 is substantially free of antibodies that specifically bind antigens other than CLDN6). An isolated antibody that specifically binds to an epitope, isoform or variant of human CLDN6 may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CLDN6 species homologs). Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the invention, a combination of “isolated” monoclonal antibodies relates to antibodies having different specificities and being combined in a well defined composition. According to the present invention, an antibody is capable of binding to a predetermined target if it has a significant affinity for said predetermined target and binds to said predetermined target in standard assays such as the assays described herein. Preferably, an antibody is capable of binding to a target if it detectably binds to said target in a flow cytometry analysis (FACS analysis) wherein binding of said antibody to said target expressed on the surface of intact cells is determined. Preferably, the antibody detectably binds to said target if present in a concentration of 10 μg/ml or lower, 5 μg/ml or lower or 2 μg/ml or lower. Preferably, the antibody detectably binds to said target if present in a concentration of 50 nM or lower, 30 nM or lower or 15 nM or lower. “Affinity” or “binding affinity” is often measured by equilibrium dissociation constant (KD). Preferably, the term “significant affinity” refers to the binding to a predetermined target with a dissociation constant (KD) of 10−5M or lower, 10−6M or lower, 10−7M or lower, 10−8M or lower, 10−9M or lower, 10−10M or lower, 10−11M or lower, or 10−12M or lower. Antibodies of the present invention preferably have EC50 values for binding to CLDN6 of 6500 ng/ml or lower, 3000 ng/ml or lower, 2500 ng/ml or lower, 2000 ng/ml or lower, 1500 ng/ml or lower, 1000 ng/ml or lower, 500 ng/ml or lower, 400 ng/ml or lower, 300 ng/ml or lower, 200 ng/ml or lower, or 100 ng/ml or lower. An antibody is not (substantially) capable of binding to a target if it has no significant affinity for said target and does not bind significantly to said target in standard assays. Preferably, an antibody is not (substantially) capable of binding to a target if it does not detectably bind to said target in a flow cytometry analysis (FACS analysis) wherein binding of said antibody to said target expressed on the surface of intact cells is determined. Preferably, the antibody does not detectably bind to said target if present in a concentration of up to 2 μg/ml, preferably up to 5 μg/ml, preferably up to 10 μg/ml, preferably up to 20 μg/ml, more preferably up to 50 μg/ml, in particular up to 100 μg/ml, or up to 150 μg/ml, up to 200 μg/ml or higher. Preferably, the antibody does not detectably bind to said target if present in a concentration of up to 15 nM, preferably up to 30 nM, preferably up to 50 nM, preferably up to 100 nM, preferably up to 150 nM, or up to 170 nM, up to 300 mM, up to 600 nM, up to 1000 nM, up to 1300 nM or higher. Preferably, the antibody does not detectably bind to said target if present in a concentration that saturates binding to the target to which the antibody binds, i.e. CLDN6. Preferably, an antibody has no significant affinity for a target if it binds to said target with a KD that is at least 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, or 106-fold higher than the KD for binding to the predetermined target to which the antibody is capable of binding. For example, if the KD for binding of an antibody to the target to which the antibody is capable of binding is 10−7M, the KD for binding to a target for which the antibody has no significant affinity would be is at least 10−6M, 10−5M, 10−4M, 10−3M, 10−2M, or 10−1M. An antibody is specific for a predetermined target if it is capable of binding to said predetermined target while it is not capable of binding to other targets, i.e. has no significant affinity for other targets and does not significantly bind to other targets in standard assays. According to the invention, an antibody is specific for CLDN6 if it is capable of binding to CLDN6 but is not capable of binding to other targets, in particular claudin proteins other than CLDN6 such as CLDN9, CLDN4, CLDN3 and CLDN1. Preferably, an antibody is specific for CLDN6 if the affinity for and the binding to a claudin protein other than CLDN6 such as CLDN9, CLDN4, CLDN3 and CLDN1 does not significantly exceed the affinity for or binding to claudin-unrelated proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA) or non-claudin transmembrane proteins such as MHC molecules or transferrin receptor or any other specified polypeptide. Preferably, an antibody is specific for a predetermined target if it binds to said target with a KD that is at least 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, or 106-fold lower than the KD for binding to a target for which it is not specific. For example, if the KD for binding of an antibody to the target for which it is specific is 10−7M, the KD for binding to a target for which it is not specific would be at least 10−6M, 10−5M, 10−4M, 10−3M, 10−2M, or 10−1M. Binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., “Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein. Affinities may be readily determined using conventional techniques, such as by equilibrium dialysis; by using the BIAcore 2000 instrument, using general procedures outlined by the manufacturer; by radioimmunoassay using radiolabeled target antigen; or by another method known to the skilled artisan. The affinity data may be analyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, e.g., KD, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer. A unique feature of the antibody of the present invention is the ability to bind cell surface claudin 6. This is demonstrated by flow cytometry analysis of cells expressing claudin 6. To test the binding of monoclonal antibodies to live cells expressing claudins, flow cytometry can be used. Briefly, cell lines expressing membrane-associated claudins (grown under standard growth conditions) are mixed with various concentrations of antibodies in PBS containing 2% heat inactivated FCS and 0.1% NaN3at 4° C. for 30 min. After washing, the cells are reacted with a fluorescently labeled secondary antibody under the same conditions as the primary antibody staining. The samples can be analyzed by FACS using light and side scatter properties to gate on single cells and binding of the labeled antibodies is determined. The term “binding” according to the invention preferably relates to a specific binding as defined herein. As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) that is encoded by heavy chain constant region genes. As used herein, “isotype switching” refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes. The term “naturally occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term “rearranged” as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element. The term “unrearranged” or “germline configuration” as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment. The term “nucleic acid molecule”, as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid molecule can be employed for introduction into, i.e. transfection of, cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation. The nucleic acids described according to the invention have preferably been isolated. The term “isolated nucleic acid” means according to the invention that the nucleic acid was (i) amplified in vitro, for example by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example by cleavage and gel-electrophoretic fractionation, or (iv) synthesized, for example by chemical synthesis. An isolated nucleic acid is a nucleic acid which is available for manipulation by recombinant DNA techniques. Nucleic acids may, according to the invention, be present alone or in combination with other nucleic acids, which may be homologous or heterologous. In preferred embodiments, a nucleic acid is functionally linked to expression control sequences which may be homologous or heterologous with respect to said nucleic acid wherein the term “homologous” means that the nucleic acid is also functionally linked to the expression control sequence naturally and the term “heterologous” means that the nucleic acid is not functionally linked to the expression control sequence naturally. A nucleic acid, such as a nucleic acid expressing RNA and/or protein or peptide, and an expression control sequence are “functionally” linked to one another, if they are covalently linked to one another in such a way that expression or transcription of said nucleic acid is under the control or under the influence of said expression control sequence. If the nucleic acid is to be translated into a functional protein, then, with an expression control sequence functionally linked to a coding sequence, induction of said expression control sequence results in transcription of said nucleic acid, without causing a frame shift in the coding sequence or said coding sequence not being capable of being translated into the desired protein or peptide. The term “expression control sequence” comprises according to the invention promoters, ribosome binding sites, enhancers and other control elements which regulate transcription of a gene or translation of a mRNA. In particular embodiments of the invention, the expression control sequences can be regulated. The exact structure of expression control sequences may vary as a function of the species or cell type, but generally comprises 5′-untranscribed and 5′- and 3′-untranslated sequences which are involved in initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, and the like. More specifically, 5′-untranscribed expression control sequences comprise a promoter region which includes a promoter sequence for transcriptional control of the functionally linked nucleic acid. Expression control sequences may also comprise enhancer sequences or upstream activator sequences. According to the invention the term “promoter” or “promoter region” relates to a nucleic acid sequence which is located upstream (5′) to the nucleic acid sequence being expressed and controls expression of the sequence by providing a recognition and binding site for RNA-polymerase. The “promoter region” may include further recognition and binding sites for further factors which are involved in the regulation of transcription of a gene. A promoter may control the transcription of a prokaryotic or eukaryotic gene. Furthermore, a promoter may be “inducible” and may initiate transcription in response to an inducing agent or may be “constitutive” if transcription is not controlled by an inducing agent. A gene which is under the control of an inducible promoter is not expressed or only expressed to a small extent if an inducing agent is absent. In the presence of the inducing agent the gene is switched on or the level of transcription is increased. This is mediated, in general, by binding of a specific transcription factor. Promoters which are preferred according to the invention include promoters for SP6, T3 and T7 polymerase, human U6 RNA promoter, CMV promoter, and artificial hybrid promoters thereof (e.g. CMV) where a part or parts are fused to a part or parts of promoters of genes of other cellular proteins such as e.g. human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and including or not including (an) additional intron(s). According to the invention, the term “expression” is used in its most general meaning and comprises the production of RNA or of RNA and protein/peptide. It also comprises partial expression of nucleic acids. Furthermore, expression may be carried out transiently or stably. According to the invention, the term expression also includes an “aberrant expression” or “abnormal expression”. “Aberrant expression” or “abnormal expression” means according to the invention that expression is altered, preferably increased, compared to a reference, preferably compared to the state in a non-tumorigenic normal cell or a healthy individual. An increase in expression refers to an increase by at least 10%, in particular at least 20%, at least 50% or at least 100%. In one embodiment, expression is only found in a diseased tissue, while expression in a healthy tissue is repressed. In a preferred embodiment, a nucleic acid molecule is according to the invention present in a vector, where appropriate with a promoter, which controls expression of the nucleic acid. The term “vector” is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and/or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and/or expressed in the cells. Vectors comprise plasmids, phagemids, bacteriophages or viral genomes. The term “plasmid” as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA. As the vector for expression of an antibody, either of a vector type in which the antibody heavy chain and light chain are present in different vectors or a vector type in which the heavy chain and light chain are present in the same vector can be used. The teaching given herein with respect to specific nucleic acid and amino acid sequences, e.g. those shown in the sequence listing, is to be construed so as to also relate to modifications, i.e. variants, of said specific sequences resulting in sequences which are functionally equivalent to said specific sequences, e.g. amino acid sequences exhibiting properties identical or similar to those of the specific amino acid sequences and nucleic acid sequences encoding amino acid sequences exhibiting properties identical or similar to those of the amino acid sequences encoded by the specific nucleic acid sequences. One important property is to retain binding of an antibody to its target or to sustain effector functions of an antibody such as CDC and/or ADCC. Preferably, a sequence modified with respect to a specific sequence, when it replaces the specific sequence in an antibody retains binding of said antibody to the target and preferably functions of said antibody as described herein. Similarly, the teaching given herein with respect to specific antibodies or hybridomas producing specific antibodies is to be construed so as to also relate to antibodies characterized by an amino acid sequence and/or nucleic acid sequence which is modified compared to the amino acid sequence and/or nucleic acid sequence of the specific antibodies but being functionally equivalent. One important property is to retain binding of an antibody to its target or to sustain effector functions of an antibody. Preferably, a sequence modified with respect to a specific sequence, when it replaces the specific sequence in an antibody retains binding of said antibody to the target and preferably functions of said antibody as described herein, e.g. CDC mediated lysis or ADCC mediated lysis. It will be appreciated by those skilled in the art that in particular the sequences of the CDR, hypervariable and variable regions can be modified without losing the ability to bind to a target. For example, CDR regions will be either identical or highly homologous to the regions of antibodies specified herein. By “highly homologous” it is contemplated that from 1 to 5, preferably from 1 to 4, such as 1 to 3 or 1 or 2 substitutions may be made in the CDRs. In addition, the hypervariable and variable regions may be modified so that they show substantial homology with the regions of antibodies specifically disclosed herein. It is to be understood that the specific nucleic acids described herein also include nucleic acids modified for the sake of optimizing the codon usage in a particular host cell or organism. Differences in codon usage among organisms can lead to a variety of problems concerning heterologous gene expression. Codon optimization by changing one or more nucleotides of the original sequence can result in an optimization of the expression of a nucleic acid, in particular in optimization of translation efficacy, in a homologous or heterologous host in which said nucleic acid is to be expressed. According to the invention, a variant, derivative, modified form or fragment of a nucleic acid sequence, amino acid sequence, or peptide preferably has a functional property of the nucleic acid sequence, amino acid sequence, or peptide, respectively, from which it has been derived. Such functional properties comprise the interaction with or binding to other molecules. In one embodiment, a variant, derivative, modified form or fragment of a nucleic acid sequence, amino acid sequence, or peptide is immunologically equivalent to the nucleic acid sequence, amino acid sequence, or peptide, respectively, from which it has been derived. Preferably the degree of identity between a specific nucleic acid sequence and a nucleic acid sequence which is modified with respect to or which is a variant of said specific nucleic acid sequence will be at least 70%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. Regarding CLDN6 nucleic acid variants, the degree of identity is preferably given for a region of at least about 300, at least about 400, at least about 450, at least about 500, at least about 550, at least about 600 or at least about 630 nucleotides. In preferred embodiments, the degree of identity is given for the entire length of the reference nucleic acid sequence, such as the nucleic acid sequences given in the sequence listing. Preferably, the two sequences are capable of hybridizing and forming a stable duplex with one another, with hybridization preferably being carried out under conditions which allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 or Current Protocols in Molecular Biology, F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York and refer, for example, to hybridization at 65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH2PO4(pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH 7. After hybridization, the membrane to which the DNA has been transferred is washed, for example, in 2×SSC at room temperature and then in 0.1-0.5×SSC/0.1×SDS at temperatures of up to 68° C. The term “variant” according to the invention also includes mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those which are naturally present. An allelic variant relates to an alteration in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies numerous allelic variants for a given gene. A species homolog is a nucleic acid or amino acid sequence with a different species of origin from that of a given nucleic acid or amino acid sequence. For the purposes of the present invention, “variants” of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C-terminal truncation variants. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. With respect to the amino acid sequence according to SEQ ID NO: 37 the term variant relates in particular to a sequence wherein the cysteine at position 46 is replaced by another amino acid other than cysteine such as an amino acid as mentioned above, preferably glycine, alanine, serine, threonine, valine, or leucine. Preferably the degree of similarity, preferably identity between a specific amino acid sequence and an amino acid sequence which is modified with respect to or which is a variant of said specific amino acid sequence such as between amino acid sequences showing substantial homology will be at least 70%, preferably at least 80%, even more preferably at least 90% or most preferably at least 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably continuous amino acids. Regarding CLDN6 polypeptide variants, the degree of similarity or identity is given preferably for a region of at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, or at least about 210 amino acids. In preferred embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence such as the amino acid sequences given in the sequence listing. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS: needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. “Sequence similarity” indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. “Sequence identity” between two polypeptide or nucleic acid sequences indicates the percentage of amino acids or nucleotides that are identical between the sequences. The “percentage identity” is obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleotide or amino acid sequences are conventionally carried out by comparing these sequences after having aligned them optimally, said comparison being carried out by segment or by “window of comparison” in order to identify and compare local regions of sequence similarity. The optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 so as to obtain the percentage identity between these two sequences. “Conservative substitutions,” may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example: (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d). In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants. The present invention comprises antibodies in which alterations have been made in the Fc region in order to change the functional or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of C1q binding and CDC or of FcγR binding and ADCC. Substitutions can, for example, be made in one or more of the amino acid residues of the heavy chain constant region, thereby causing an alteration in an effector function while retaining the ability to bind to the antigen as compared with the modified antibody, cf. U.S. Pat. Nos. 5,624,821 and 5,648,260. The in vivo half-life of antibodies can be improved by modifying the salvage receptor epitope of the Ig constant domain or an Ig-like constant domain such that the molecule does not comprise an intact CH2 domain or an intact Ig Fc region, cf. U.S. Pat. Nos. 6,121,022 and 6,194,551. The in vivo half-life can furthermore be increased by making mutations in the Fc region, e.g., by substituting threonine for leucine at position 252, by substituting threonine for serine at position 254, or by substituting threonine for phenylalanine at position 256, cf. U.S. Pat. No. 6,277,375. Furthermore, the glycosylation pattern of antibodies can be modified in order to change the effector function of the antibodies. For example, the antibodies can be expressed in a transfectoma which does not add the fucose unit normally attached to Asn at position 297 of the Fc region in order to enhance the affinity of the Fc region for Fc-Receptors which, in turn, will result in an increased ADCC of the antibodies in the presence of NK cells, cf. Shield et al. (2002) JBC, 277: 26733. Furthermore, modification of galactosylation can be made in order to modify CDC. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a anti-CLDN6 antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti-CLDN6 antibodies can be screened for binding activity. According to the invention the term “cell” or “host cell” preferably relates to an intact cell, i.e. a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles, or genetic material. An intact cell preferably is a viable cell, i.e. a living cell capable of carrying out its normal metabolic functions. Preferably said term relates according to the invention to any cell which can be transformed or transfected with an exogenous nucleic acid. The term “cell” includes according to the invention prokaryotic cells (e.g.,E. coli) or eukaryotic cells (e.g., dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, yeast cells, and insect cells). The exogenous nucleic acid may be found inside the cell (i) freely dispersed as such, (ii) incorporated in a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, and primates. The cells may be derived from a large number of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In further embodiments, the cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte, or macrophage. The term “host cell”, as used herein, preferably is intended to refer to a cell into which a recombinant expression vector has been introduced. A cell which comprises a nucleic acid molecule preferably express the peptide or protein encoded by the nucleic acid. The terms “transgenic animal” refers to an animal having a genome comprising one or more transgenes, preferably heavy and/or light chain transgenes, or transchromosomes (either integrated or non-integrated into the animal's natural genomic DNA) and which is preferably capable of expressing the transgenes. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces human anti-CLDN6 antibodies when immunized with CLDN6 antigen and/or cells expressing CLDN6. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic mice, e.g., HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice as described in WO 02/43478. Such transgenic and transchromosomal mice may be capable of producing multiple isotypes of human monoclonal antibodies to CLDN6 (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching. “Reduce” or “inhibit” as used herein means the ability to cause an overall decrease, preferably of 5% or greater, 10% or greater, 20% or greater, more preferably of 50% or greater, and most preferably of 75% or greater, in the level, e.g. in the level of proliferation of cells. The term “inhibit” or similar phrases includes a complete or essentially complete inhibition, i.e. a reduction to zero or essentially to zero. Terms such as “increasing” or “enhancing” preferably relate to an increase or enhancement by about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%. These terms may also relate to circumstances, wherein at time zero there is no detectable signal for a certain compound or condition and at a particular time point later than time zero there is a detectable signal for a certain compound or condition. The term “immunologically equivalent” means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect such as induction of a humoral and/or cellular immune response, the strength and/or duration of the induced immune reaction, or the specificity of the induced immune reaction. In the context of the present invention, the term “immunologically equivalent” is preferably used with respect to the immunological effects or properties of a peptide or peptide variant used for immunization. A particular immunological property is the ability to bind to antibodies and, where appropriate, generate an immune response, preferably by stimulating the generation of antibodies. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction, preferably antibodies, having a specificity of reacting with the reference amino acid sequence, such as the reference amino acid sequence forming part of CLDN6. The term “immune effector functions” in the context of the present invention includes any functions mediated by components of the immune system that result in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis. Preferably, immune effector functions result in killing of tumor cells. Preferably, the immune effector functions in the context of the present invention are antibody-mediated effector functions. Such functions comprise complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), induction of apoptosis in the cells carrying the tumor-associated antigen, for example, by binding of the antibody to a surface antigen, and/or inhibition of proliferation of the cells carrying the tumor-associated antigen, preferably ADCC and/or CDC. Thus, antibodies that are capable of mediating one or more immune effector functions are preferably able to mediate killing of cells by inducing CDC-mediated lysis, ADCC-mediated lysis, apoptosis, homotypic adhesion, and/or phagocytosis, preferably by inducing CDC-mediated lysis and/or ADCC-mediated lysis. Antibodies may also exert an effect simply by binding to tumor-associated antigens on the surface of a tumor cell. For example, antibodies may block the function of the tumor-associated antigen or induce apoptosis just by binding to the tumor-associated antigen on the surface of a tumor cell. DETAILED DESCRIPTION OF THE INVENTION Mechanisms of mAb Action Although the following provides considerations regarding the mechanism underlying the therapeutic efficacy of antibodies of the invention it is not to be considered as limiting to the invention in any way. The antibodies described herein may interact with components of the immune system, preferably through ADCC or CDC. Antibodies of the invention can also be used to target payloads (e.g., radioisotopes, drugs or toxins) to directly kill tumor cells or can be used synergistically with traditional chemotherapeutic agents, attacking tumors through complementary mechanisms of action that may include anti-tumor immune responses that may have been compromised owing to a chemotherapeutic's cytotoxic side effects on T lymphocytes. However, antibodies of the invention may also exert an effect simply by binding to CLDN6 on the cell surface, thus, e.g. blocking proliferation of the cells. Antibody-Dependent Cell-Mediated Cytotoxicity ADCC describes the cell-killing ability of effector cells as described herein, in particular lymphocytes, which preferably requires the target cell being marked by an antibody. ADCC preferably occurs when antibodies bind to antigens on tumor cells and the antibody Fc domains engage Fc receptors (FcR) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be viewed as a mechanism to directly induce a variable degree of immediate tumor destruction that leads to antigen presentation and the induction of tumor-directed T-cell responses. Preferably, in vivo induction of ADCC will lead to tumor-directed T-cell responses and host-derived antibody responses. Complement-Dependent Cytotoxicity CDC is another cell-killing method that can be directed by antibodies. IgM is the most effective isotype for complement activation. IgG1 and IgG3 are also both very effective at directing CDC via the classical complement-activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the uncloaking of multiple C1q binding sites in close proximity on the CH2 domains of participating antibody molecules such as IgG molecules (C1q is one of three subcomponents of complement C1). Preferably these uncloaked C1q binding sites convert the previously low-affinity C1q-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of the effector-cell chemotactic/activating agents C3a and C5a. Preferably, the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate free passage of water and solutes into and out of the cell. Production of Antibodies Antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of antibody genes. The preferred animal system for preparing hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Other preferred animal systems for preparing hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (e.g. described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al., Am. J. Clin. Pathol. 124: 295 (2005)). In yet another preferred embodiment, human monoclonal antibodies directed against CLDN6 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.” The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO2004 035607 Yet another strategy for generating monoclonal antibodies is to directly isolate genes encoding antibodies from lymphocytes producing antibodies of defined strategy e.g. see Babcock et al., 1996; A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined strategy. For details of recombinant antibody engineering see also Welschof and Kraus, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny K. C. Lo Antibody Engineering ISBN 1-58829-092-1. Immunizations To generate antibodies to CLDN6, mice can be immunized with carrier-conjugated peptides derived from the CLDN6 sequence, an enriched preparation of recombinantly expressed CLDN6 antigen or fragments thereof and/or cells expressing CLDN6 or fragments thereof, as described. Alternatively, mice can be immunized with DNA encoding full length human CLDN6 or fragments thereof. In the event that immunizations using a purified or enriched preparation of the CLDN6 antigen do not result in antibodies, mice can also be immunized with cells expressing CLDN6, e.g., a cell line, to promote immune responses. The immune response can be monitored over the course of the immunization protocol with plasma and serum samples being obtained by tail vein or retroorbital bleeds. Mice with sufficient titers of anti-CLDN6 immunoglobulin can be used for fusions. Mice can be boosted intraperitonealy or intravenously with CLDN6 expressing cells 3-5 days before sacrifice and removal of the spleen to increase the rate of specific antibody secreting hybridomas. Generation of Hybridomas Producing Monoclonal Antibodies To generate hybridomas producing monoclonal antibodies to CLDN6, cells from lymph nodes or spleens obtained from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can then be screened for the production of antigen-specific antibodies. Individual wells can then be screened by ELISA for antibody secreting hybridomas. By Immunofluorescence and FACS analysis using CLDN6 expressing cells, antibodies with specificity for CLDN6 can be identified. The antibody secreting hybridomas can be replated, screened again, and if still positive for anti-CLDN6 monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization. Generation of Transfectomas Producing Monoclonal Antibodies Antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as are well known in the art (Morrison, S. (1985) Science 229: 1202). For example, in one embodiment, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO cells, NS/0 cells, HEK293T cells or HEK293 cells or alternatively other eukaryotic cells like plant derived cells, fungal or yeast cells. The method used to introduce these genes can be methods described in the art such as electroporation, lipofectine, lipofectamine or others. After introduction of these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells. Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, e.g.E. coli. Furthermore, the antibodies can be produced in transgenic non-human animals, such as in milk from sheep and rabbits or in eggs from hens, or in transgenic plants; see e.g. Verma, R., et al. (1998) J. Immunol. Meth. 216: 165-181; Pollock, et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839. Use of Partial Antibody Sequences to Express Intact Antibodies (i.e. Humanization and Chimerisation). a) Chimerization Murine monoclonal antibodies can be used as therapeutic antibodies in humans when labeled with toxins or radioactive isotopes. Nonlabeled murine antibodies are highly immunogenic in man when repetitively applied leading to reduction of the therapeutic effect. The main immunogenicity is mediated by the heavy chain constant regions. The immunogenicity of murine antibodies in man can be reduced or completely avoided if respective antibodies are chimerized or humanized. Chimeric antibodies are antibodies, the different portions of which are derived from different animal species, such as those having a variable region derived from a murine antibody and a human immunoglobulin constant region. Chimerisation of antibodies is achieved by joining of the variable regions of the murine antibody heavy and light chain with the constant region of human heavy and light chain (e.g. as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918-8). In a preferred embodiment chimeric antibodies are generated by joining human kappa-light chain constant region to murine light chain variable region. In an also preferred embodiment chimeric antibodies can be generated by joining human lambda-light chain constant region to murine light chain variable region. The preferred heavy chain constant regions for generation of chimeric antibodies are IgG1, IgG3 and IgG4. Other preferred heavy chain constant regions for generation of chimeric antibodies are IgG2, IgA, IgD and IgM. b) Humanization Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V (D) J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequences spanning the CDR regions are typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons. The nucleotide sequences of heavy and light chain transcripts from hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266: 19867-19870); and HindIII sites are engineered upstream of the translation initiation sites. For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding, strand sequences are broken down into 30-50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150-400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs. The reconstructed chimerized or humanized heavy and light chain variable regions are then combined with cloned promoter, leader, translation initiation, constant region, 3′ untranslated, polyadenylation, and transcription termination sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains. Plasmids for use in construction of expression vectors for human IgGκ are described. The plasmids can be constructed so that PCR amplified V heavy and V kappa light chain cDNA sequences can be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human, or chimeric IgG1, Kappa or IgG4, Kappa antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains. Thus, in another aspect of the invention, the structural features of the anti-CLDN6 antibodies of the invention, are used to create structurally related humanized anti-CLDN6 antibodies that retain at least one functional property of the antibodies of the invention, such as binding to CLDN6. More specifically, one or more CDR regions of mouse monoclonal antibodies can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, humanized anti-CLDN6 antibodies of the invention. Binding to Antigen Expressing Cells The ability of the antibody to bind CLDN6 can be determined using standard binding assays, such as those set forth in the examples (e.g., ELISA, Western Blot, Immunofluorescence and flow cytometric analysis) Isolation and Characterization of Antibodies To purify anti-CLDN6 antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Alternatively, anti-CLDN6 antibodies can be produced in dialysis based bioreactors. Supernatants can be filtered and, if necessary, concentrated before affinity chromatography with protein G-sepharose or protein A-sepharose. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be aliquoted and stored at −80° C. To determine if the selected anti-CLDN6 monoclonal antibodies bind to unique epitopes, site-directed or multi-site directed mutagenesis can be used. Isotype Determination To determine the isotype of purified antibodies, isotype ELISAs with various commercial kits (e.g. Zymed, Roche Diagnostics) can be performed. Wells of microtiter plates can be coated with anti-mouse Ig. After blocking, the plates are reacted with monoclonal antibodies or purified isotype controls, at ambient temperature for two hours. The wells can then be reacted with either mouse IgG1, IgG2a, IgG2b or IgG3, IgA or mouse IgM-specific peroxidase-conjugated probes. After washing, the plates can be developed with ABTS substrate (1 mg/ml) and analyzed at OD of 405-650. Alternatively, the IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche, Cat. No. 1493027) may be used as described by the manufacturer. Flow Cytometric Analysis In order to demonstrate presence of anti-CLDN6 antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing CLDN6, flow cytometry can be used. Cell lines expressing naturally or after transfection CLDN6 and negative controls lacking CLDN6 expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS, and can be incubated at 4° C. for 30 min. After washing, the APC- or Alexa647-labeled anti IgG antibody can bind to CLDN6-bound monoclonal antibody under the same conditions as the primary antibody staining. The samples can be analyzed by flow cytometry with a FACS instrument using light and side scatter properties to gate on single, living cells. In order to distinguish CLDN6-specific monoclonal antibodies from non-specific binders in a single measurement, the method of co-transfection can be employed. Cells transiently transfected with plasmids encoding CLDN6 and a fluorescent marker can be stained as described above. Transfected cells can be detected in a different fluorescence channel than antibody-stained cells. As the majority of transfected cells express both transgenes, CLDN6-specific monoclonal antibodies bind preferentially to fluorescence marker expressing cells, whereas non-specific antibodies bind in a comparable ratio to non-transfected cells. An alternative assay using fluorescence microscopy may be used in addition to or instead of the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. Immunofluorescence Microscopy In order to demonstrate presence of anti-CLDN6 antibodies in sera of immunized mice or binding of monoclonal antibodies to living cells expressing CLDN6, immunofluorescence microscopy analysis can be used. For example, cell lines expressing either spontaneously or after transfection CLDN6 and negative controls lacking CLDN6 expression are grown in chamber slides under standard growth conditions in DMEM/F12 medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells can then be fixed with methanol or paraformaldehyde or left untreated. Cells can then be reacted with monoclonal antibodies against CLDN6 for 30 min. at 25° C. After washing, cells can be reacted with an Alexa555-labeled anti-mouse IgG secondary antibody (Molecular Probes) under the same conditions. Cells can then be examined by fluorescence microscopy. Total CLDN6 levels in cells can be observed when cells are methanol fixed or paraformaldehyde fixed and permeabilized with Triton X-100. In living cells and non-permeabilized, paraformaldehyde fixed cells surface localization of CLDN6 can be examined. Additionally targeting of CLDN6 to tight junctions can be analyzed by co-staining with tight junction markers such as ZO-1. Furthermore, effects of antibody binding and CLDN6 localization within the cell membrane can be examined. Western Blot Anti-CLDN6 IgG can be further tested for reactivity with CLDN6 antigen by Western Blotting. Briefly, cell extracts from cells expressing CLDN6 and appropriate negative controls can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. IgG binding can be detected using anti-mouse IgG peroxidase and developed with ECL substrate. Immunohistochemistry Anti-CLDN6 mouse IgGs can be further tested for reactivity with CLDN6 antigen by Immunohistochemistry in a manner well known to the skilled person, e.g. using paraformaldehyde or acetone fixed cryosections or paraffin embedded tissue sections fixed with paraformaldehyde from non-cancer tissue or cancer tissue samples obtained from patients during routine surgical procedures or from mice carrying xenografted tumors inoculated with cell lines expressing spontaneously or after transfection CLDN6. For immunostaining, antibodies reactive to CLDN6 can be incubated followed by horseradish-peroxidase conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO) according to the vendors instructions. Phagocytic and Cell Killing Activities of Antibodies In Vitro In addition to binding specifically to CLDN6, anti-CLDN6 antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface. The testing of monoclonal antibody activity in vitro will provide an initial screening prior to testing in vivo models. Antibody Dependent Cell-Mediated Cytotoxicity (ADCC): Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes, mononuclear cells or other effector cells, from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed effector cells can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum or, alternatively with 5% heat-inactivated human serum and mixed with51Cr labeled target cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface, at various ratios of effector cells to target cells. Alternatively, the target cells may be labeled with a fluorescence enhancing ligand (BATDA). A highly fluorescent chelate of Europium with the enhancing ligand which is released from dead cells can be measured by a fluorometer. Another alternative technique may utilize the transfection of target cells with luciferase. Added lucifer yellow may then be oxidated by viable cells only. Purified anti-CLDN6 IgGs can then be added at various concentrations. Irrelevant human IgG can be used as negative control. Assays can be carried out for 4 to 20 hours at 37° C. depending on the effector cell type used. Samples can be assayed for cytolysis by measuring51Cr release or the presence of the EuTDA chelate in the culture supernatant. Alternatively, luminescence resulting from the oxidation of lucifer yellow can be a measure of viable cells. Anti-CLDN6 monoclonal antibodies can also be tested in various combinations to determine whether cytolysis is enhanced with multiple monoclonal antibodies. Complement Dependent Cytotoxicity (CDC): Monoclonal anti-CLDN6 antibodies can be tested for their ability to mediate CDC using a variety of known techniques. For example, serum for complement can be obtained from blood in a manner known to the skilled person. To determine the CDC activity of mAbs, different methods can be used.51Cr release can for example be measured or elevated membrane permeability can be assessed using a propidium iodide (PI) exclusion assay. Briefly, target cells can be washed and 5×105/ml can be incubated with various concentrations of mAb for 10-30 min. at room temperature or at 37° C. Serum or plasma can then be added to a final concentration of 20% (v/v) and the cells incubated at 37° C. for 20-30 min. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be analyzed immediately by flow cytometry analysis using FACSArray. In an alternative assay, induction of CDC can be determined on adherent cells. In one embodiment of this assay, cells are seeded 24 h before the assay with a density of 3×104/well in tissue-culture flat-bottom microtiter plates. The next day growth medium is removed and the cells are incubated in triplicates with antibodies. Control cells are incubated with growth medium or growth medium containing 0.2% saponin for the determination of background lysis and maximal lysis, respectively. After incubation for 20 min. at room temperature supernatant is removed and 20% (v/v) human plasma or serum in DMEM (prewarmed to 37° C.) is added to the cells and incubated for another 20 min. at 37° C. All cells from each sample are added to propidium iodide solution (10 μg/ml). Then, supernatants are replaced by PBS containing 2.5 μg/ml ethidium bromide and fluorescence emission upon excitation at 520 nm is measured at 600 nm using a Tecan Safire. The percentage specific lysis is calculated as follows: % specific lysis=(fluorescence sample-fluorescence background)/(fluorescence maximal lysis-fluorescence background)×100. Inhibition of Cell Proliferation by Monoclonal Antibodies: To test for the ability to initiate apoptosis, monoclonal anti-CLDN6 antibodies can, for example, be incubated with CLDN6 positive tumor cells or CLDN6 transfected tumor cells at 37° C. for about 20 hours. The cells can be harvested, washed in Annexin-V binding buffer (BD biosciences), and incubated with Annexin V conjugated with FITC or APC (BD biosciences) for 15 min. in the dark. All cells from each sample can be added to PI solution (10 μg/ml in PBS) in a FACS tube and assessed immediately by flow cytometry (as above). Alternatively, a general inhibition of cell-proliferation by monoclonal antibodies can be detected with commercially available kits. The DELFIA Cell Proliferation Kit (Perkin-Elmer, Cat. No. AD0200) is a non-isotopic immunoassay based on the measurement of 5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis of proliferating cells in microplates. Incorporated BrdU is detected using europium labeled monoclonal antibody. To allow antibody detection, cells are fixed and DNA denatured using Fix solution. Unbound antibody is washed away and DELFIA inducer is added to dissociate europium ions from the labeled antibody into solution, where they form highly fluorescent chelates with components of the DELFIA Inducer. The fluorescence measured—utilizing time-resolved fluorometry in the detection—is proportional to the DNA synthesis in the cell of each well. Preclinical Studies Monoclonal antibodies which bind to CLDN6 also can be tested in an in vivo model (e.g. in immune deficient mice carrying xenografted tumors inoculated with cell lines expressing CLDN6, possibly after transfection) to determine their efficacy in controlling growth of CLDN6-expressing tumor cells. In vivo studies after xenografting CLDN6 expressing tumor cells into immunocompromised mice or other animals can be performed using antibodies of the invention. Antibodies can be administered to tumor free mice followed by injection of tumor cells to measure the effects of the antibodies to prevent formation of tumors or tumor-related symptoms. Antibodies can be administered to tumor-bearing mice to determine the therapeutic efficacy of respective antibodies to reduce tumor growth, metastasis or tumor related symptoms. Antibody application can be combined with application of other substances as cystostatic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or other antibodies to determine synergistic efficacy and potential toxicity of combinations. To analyze toxic side effects mediated by antibodies of the invention animals can be inoculated with antibodies or control reagents and thoroughly investigated for symptoms possibly related to CLDN6-antibody therapy. Possible side effects of in vivo application of CLDN6 antibodies particularly include toxicity at CLDN6 expressing tissues including placenta. Antibodies recognizing CLDN6 in human and in other species, e.g. mice, are particularly useful to predict potential side effects mediated by application of monoclonal CLDN6 antibodies in humans. Epitope Mapping Mapping of epitopes recognized by antibodies of invention can be performed as described in detail in “Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in “Epitope Mapping: A Practical Approach” Practical Approach Series, 248 by Olwyn M. R. Westwood, Frank C. Hay. I. Bispecific/Multispecific Molecules which Bind to CLDN6 In yet another embodiment of the invention, antibodies to CLDN6 can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., an Fab′ fragment) to generate a bispecific or multispecific molecule which binds to multiple binding sites or target epitopes. For example, an antibody of the invention can be functionally linked (e.g. by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, peptide or binding mimetic. Accordingly, the present invention includes bispecific and multispecific molecules comprising at least one first binding specificity for CLDN6 and a second binding specificity for a second target epitope. In a particular embodiment of the invention, the second target epitope is an Fc receptor, e.g. human Fc-gammaRI (CD64) or a human Fc-alpha receptor (CD89), or a T cell receptor, e.g. CD3. Therefore, the invention includes bispecific and multispecific molecules capable of binding both to Fc-gammaR, Fc-alphaR or Fc-epsilonR expressing effector cells (e.g. monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface. These bispecific and multispecific molecules may target cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface to effector cells and may trigger Fc receptor-mediated effector cell activities, such as phagocytosis of cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface, antibody dependent cellular cytotoxicity (ADCC), cytokine release, or generation of superoxide anion. Bispecific and multispecific molecules of the invention can further include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-CLDN6 binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g. a molecule which binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. The “anti-enhancement factor portion” can be an antibody, functional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The “anti-enhancement factor portion” can bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor portion can bind a cytotoxic T cell (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell). In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al., U.S. Pat. No. 4,946,778. The antibody may also be a binding-domain immunoglobulin fusion protein as disclosed in US2003/0118592 and US 2003/0133939. In one embodiment bispecific and multispecific molecules of the invention comprise a binding specificity for an Fc-gammaR or an Fc-alphaR present on the surface of an effector cell, and a second binding specificity for a target cell antigen, e.g., CLDN6. In one embodiment, the binding specificity for an Fc receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term “IgG receptor” refers to any of the eight gamma-chain genes located on chromosome 1. These genes encode a total of twelve transmembrane or soluble receptor isoforms which are grouped into three Fc-gamma receptor classes: Fc-gammaRI (CD64), Fc-gammaRII (CD32), and Fc-gammaRIII (CD16). In one preferred embodiment, the Fc-gamma receptor is a human high affinity Fc-gammaRI. In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (Fc-alphaRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term “IgA receptor” is intended to include the gene product of one alpha-gene (Fc-alphaRI) located on chromosome 19. This gene is known to encode several alternatively spliced transmembrane isoforms of 55 to 110 kDa. Fc-alphaRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. Fc-alphaRI has medium affinity for both IgA1 and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology 16: 423-440). Four Fc-alphaRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind Fc-alphaRI outside the IgA ligand binding domain, have been described (Monteiro, R. C. et al. (1992) J. Immunol. 148: 1764). In another embodiment the bispecific molecule is comprised of two monoclonal antibodies according to the invention which have complementary functional activities, such as one antibody predominately working by inducing CDC and the other antibody predominately working by inducing apoptosis. An “effector cell specific antibody” as used herein refers to an antibody or functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immunoglobulin. As used herein, the term “effector cell” refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, e.g, lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTLs), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody-dependent cellular cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of Fc-gammaRI has been found to be up-regulated by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of Fc-gammaRI-bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell. “Target cell” shall mean any undesirable cell in a subject (e.g., a human or animal) that can be targeted by an antibody of the invention. In preferred embodiments, the target cell is a cell expressing or overexpressing CLDN6 and being characterized by association of CLDN6 with its cell surface. Cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface typically include tumor cells. II. Immunoconjugates In another aspect, the present invention features an anti-CLDN6 antibody conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates which include one or more cytotoxins are referred to as “immunotoxins”. A cytotoxin or cytotoxic agent includes any agent that is detrimental to and, in particular, kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Suitable therapeutic agents for forming immunoconjugates of the invention include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cyclophosphamide or ricin A. Antibodies of the present invention also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-90 or indium-111, to generate cytotoxic radiopharmaceuticals for treating a CLDN6-related disorder, such as a cancer. The antibody conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A,Pseudomonasexotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors. Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et. al.. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62: 119-58 (1982). In a further embodiment, the antibodies according to the invention are attached to a linker-chelator, e.g., tiuxetan, which allows for the antibody to be conjugated to a radioisotope. III. Pharmaceutical Compositions In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing one or a combination of antibodies of the present invention. The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. In one embodiment, the compositions include a combination of multiple (e.g., two or more) isolated antibodies of the invention which act by different mechanisms, e.g., one antibody which predominately acts by inducing CDC in combination with another antibody which predominately acts by inducing apoptosis. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one anti-inflammatory agent or at least one immunosuppressive agent. In one embodiment such therapeutic agents include one or more anti-inflammatory agents, such as a steroidal drug or a NSAID (nonsteroidal anti-inflammatory drug). Preferred agents include, for example, aspirin and other salicylates, Cox-2 inhibitors, such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such as ibuprofen (Motrin, Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen), etodolac (Lodine), oxaprozin (Daypro), and indomethacin (Indocin). In another embodiment, such therapeutic agents include agents leading to the depletion or functional inactivation of regulatory T cells like low dose cyclophosphamid, anti-CTLA4 antibodies, anti-IL2 or anti-IL2-receptor antibodies. In yet another embodiment, such therapeutic agents include one or more chemotherapeutics, such as Taxol derivatives, taxotere, gemcitabin, 5-Fluoruracil, doxorubicin (Adriamycin), cisplatin (Platinol), cyclophosphamide (Cytoxan, Procytox, Neosar). In another embodiment, antibodies of the present invention may be administered in combination with chemotherapeutic agents, which preferably show therapeutic efficacy in patients suffering from cancer, e.g. cancer types as described herein. In yet another embodiment, the antibodies of the invention may be administered in conjunction with radiotherapy and/or autologous peripheral stem cell or bone marrow transplantation. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, e.g., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7: 27). Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). In one embodiment, the antibodies of the invention may be administered by infusion, preferably slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects. The administration may also be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. The dosage can be determined or adjusted by measuring the amount of circulating monoclonal anti-CLDN6 antibodies upon administration in a biological sample by using anti-idiotypic antibodies which target the anti-CLDN6 antibodies. In yet another embodiment, the antibodies are administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more. In still another embodiment, the antibodies according to the invention may be administered by a regimen including one infusion of an antibody against CLDN6 followed by an infusion of an antibody against CLDN6 conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9 days later. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes. In a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area, e.g., the site of a tumor. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. In a further embodiment, antibodies of the invention can be formulated to prevent or reduce their transport across the placenta. This can be done by methods known in the art, e.g., by PEGylation of the antibodies or by use of F(ab)2′ fragments. Further references can be made to “Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation. J. Immunol. Methods, 152: 177-190; and to “Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann. Allergy Asthma Immunol. 74: 279-283. A “therapeutically effective dosage” for tumor therapy can be measured by objective tumor responses which can either be complete or partial. A complete response (CR) is defined as no clinical, radiological or other evidence of disease. A partial response (PR) results from a reduction in aggregate tumor size of greater than 50%. Median time to progression is a measure that characterizes the durability of the objective tumor response. A “therapeutically effective dosage” for tumor therapy can also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth or apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier. IV. Uses and Methods of the Invention The antibodies (including immunoconjugates, bispecifics/multispecifics, compositions and other derivatives described herein) of the present invention have numerous therapeutic utilities involving the treatment of disorders involving cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface. For example, the antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat or prevent a variety of disorders such as those described herein. Preferred subjects include human patients having disorders that can be corrected or ameliorated by killing diseased cells, in particular cells characterized by an altered expression pattern of CLDN6 and/or an altered pattern of association of CLDN6 with their cell surface compared to normal cells. For example, in one embodiment, antibodies of the present invention can be used to treat a subject with a tumorigenic disorder, e.g., a disorder characterized by the presence of tumor cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface. Examples of tumorigenic diseases which can be treated and/or prevented encompass all CLDN6 expressing cancers and tumor entities including those described herein. The pharmaceutical compositions and methods of treatment described according to the invention may also be used for immunization or vaccination to prevent a disease described herein. In another embodiment, antibodies of the invention can be used to detect levels of CLDN6 or particular forms of CLDN6, or levels of cells which contain CLDN6 on their membrane surface, which levels can then be linked to certain diseases or disease symptoms such as described above. Alternatively, the antibodies can be used to deplete or interact with the function of cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface, thereby implicating these cells as important mediators of the disease. This can be achieved by contacting a sample and a control sample with the anti-CLDN6 antibody under conditions that allow for the formation of a complex between the antibody and CLDN6. Any complexes formed between the antibody and CLDN6 are detected and compared in the sample and a control sample, i.e. a reference sample. Antibodies of the invention can be initially tested for their binding activity associated with therapeutic or diagnostic uses in vitro. For example, the antibodies can be tested using flow cytometric assays as described herein. The antibodies of the invention can be used to elicit in vivo or in vitro one or more of the following biological activities: to inhibit the growth of and/or differentiation of a cell expressing CLDN6 and being characterized by association of CLDN6 with its cell surface; to kill a cell expressing CLDN6 and being characterized by association of CLDN6 with its cell surface; to mediate phagocytosis or ADCC of a cell expressing CLDN6 and being characterized by association of CLDN6 with its cell surface in the presence of effector cells; to mediate CDC of a cell expressing CLDN6 and being characterized by association of CLDN6 with its cell surface in the presence of complement; to mediate apoptosis of a cell expressing CLDN6 and being characterized by association of CLDN6 with its cell surface; to induce homotypic adhesion; and/or to induce translocation into lipid rafts upon binding CLDN6. In a particular embodiment, the antibodies are used in vivo or in vitro to treat, prevent or diagnose a variety of CLDN6-related diseases. Examples of CLDN6-related diseases include, among others, cancers such as those described herein. As described above, anti-CLDN6 antibodies of the invention can be co-administered with one or other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxic agent, antiangiogeneic agent or and immunosuppressive agent to reduce the induction of immune responses against the antibodies of invention. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as listed above. Co-administration of the anti-CLDN6 antibodies of the present invention with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms yielding a cytotoxic effect to tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody. The compositions (e.g., antibodies, multispecific and bispecific molecules and immunoconjugates) of the invention which have complement binding sites, such as portions from IgG1, -2, or -3 or IgM which bind complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of the invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions of the invention can also be lysed by complement. In yet another embodiment, the compositions of the invention do not activate complement. The compositions of the invention can also be administered together with complement. Accordingly, within the scope of the invention are compositions comprising antibodies, multispecific or bispecific molecules and serum or complement. These compositions are advantageous in that the complement is located in close proximity to the antibodies, multispecific or bispecific molecules. Alternatively, the antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately. Binding of the compositions of the present invention to target cells may cause translocation of the CLDN6 antigen-antibody complex into lipid rafts of the cell membrane. Such translocation creates a high density of antigen-antibody complexes which may efficiently activate and/or enhance CDC. Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., antibodies and immunoconjugates) and instructions for use. The kit can further contain one or more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent, or one or more additional antibodies of the invention (e.g., an antibody having a complementary activity). Accordingly, patients treated with antibody compositions of the invention can be additionally administered (prior to, simultaneously with, or following administration of a antibody of the invention) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the antibodies of the invention. In other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fc-gamma or Fc-alpha receptors by, for example, treating the subject with a cytokine. Preferred cytokines include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Other important agents for increasing the therapeutic efficacy of the antibodies and pharmaceutical compositions described herein are β-glucans which are homopolysaccharides of branched glucose residues and are produced by a variety of plants and microorganisms, for example, bacteria, algae, fungi, yeast and grains. Fragments of β-glucans produced by organisms may be also be used. Preferably, the β-glucan is a polymer of β(1,3) glucose wherein at least some of the backbone glucose units, e.g. 3-6% of the backbone glucose units, possess branches such as β(1,6) branches. In a particular embodiment, the invention provides methods for detecting the presence of CLDN6 antigen in a sample, or measuring the amount of CLDN6 antigen, comprising contacting the sample, and a control sample, with an antibody which specifically binds to CLDN6, under conditions that allow for formation of a complex between the antibody or portion thereof and CLDN6. The formation of a complex is then detected, wherein a difference complex formation between the sample compared to the control sample is indicative for the presence of CLDN6 antigen in the sample. In still another embodiment, the invention provides a method for detecting the presence or quantifying the amount of cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface in vivo or in vitro. The method comprises (i) administering to a subject a composition of the invention conjugated to a detectable marker; and (ii) exposing the subject to a means for detecting said detectable marker to identify areas containing cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface. Methods as described above are useful, in particular, for diagnosing CLDN6-related diseases and/or the localization of CLDN6-related diseases such as cancer diseases. Preferably an amount of CLDN6 in a sample which is higher than the amount of CLDN6 in a control sample is indicative for the presence of a CLDN6-related disease in a subject, in particular a human, from which the sample is derived. When used in methods as described above, an antibody described herein may be provided with a label that functions to: (i) provide a detectable signal; (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET (Fluorescence Resonance Energy Transfer); (iii) affect mobility, e.g. electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provide a capture moiety, e.g., affinity, antibody/antigen, or ionic complexation. Suitable as label are structures, such as fluorescent labels, luminescent labels, chromophore labels, radioisotopic labels, isotopic labels, preferably stable isotopic labels, isobaric labels, enzyme labels, particle labels, in particular metal particle labels, magnetic particle labels, polymer particle labels, small organic molecules such as biotin, ligands of receptors or binding molecules such as cell adhesion proteins or lectins, label-sequences comprising nucleic acids and/or amino acid residues which can be detected by use of binding agents, etc. Labels comprise, in a nonlimiting manner, barium sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate and radio diagnostic, including positron emitters such as fluorine-18 and carbon-11, gamma emitters such as iodine-123, technetium-99m, iodine-131 and indium-111, nuclides for nuclear magnetic resonance, such as fluorine and gadolinium. In yet another embodiment immunoconjugates of the invention can be used to target compounds (e.g., therapeutic agents, labels, cytotoxins, radiotoxins immunosuppressants, etc.) to cells which have CLDN6 associated with their surface by linking such compounds to the antibody. Thus, the invention also provides methods for localizing ex vivo or in vitro cells expressing CLDN6 and being characterized by association of CLDN6 with their cell surface, such as circulating tumor cells. The present invention is further illustrated by the following examples which are not be construed as limiting the scope of the invention. EXAMPLES The techniques and methods used herein are described herein or carried out in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All methods including the use of kits and reagents are carried out according to the manufacturer's information unless specifically indicated. Example 1: Quantification of CLDN6 Expression in Normal Tissues, Cancerous Tissues and Cell Lines Using Real-Time RT-PCR Total cellular RNA was extracted from frozen tissue specimens and cancer cell lines using RNeasy Mini Kit (Qiagen), primed with a dT18oligonucleotide and reverse-transcribed with Superscript II (GIBCO/Lifetech) according to the manufacturer's instructions. Integrity of the obtained cDNA was tested by amplification of p53 transcripts in a 30 cycle PCR. After normalization to HPRT expression of CLDN6 was quantified using ΔΔCT calculation. Tissues from three individuals were tested for each normal tissue type. Only trace amounts of CLDN6 transcripts could be detected in normal tissues after 40 cycles of RT-PCR. The only normal tissue slightly exceeding the expression cutoff was placenta. In contrast to normal tissues, we found high expression of CLDN6 in samples from ovarian cancer (adenocarcinomas), lung cancer (NSCLC, with highest frequency and expression levels in adenocarcinomas), gastric cancer, breast cancer, hepatic cancer, pancreatic cancer, skin cancer (basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer (malignant pleomorphic adenoma), sarcoma (synovial sarcoma and carcinosarcoma), bile duct cancer, renal cell cancer (clear cell carcinoma and papillary carcinoma), uterine cancer and cancer cell lines A2780 (ovarian cancer), NIH-OVCAR3 (ovarian cancer), HCT-116 (colon cancer), EFO-27 (ovarian cancer), CPC-N (SCLC), NCI-H552 (NSCLC), SNU-1 (gastric cancer), KATOIII (gastric cancer), YAPC (pancreatic cancer), AGS (gastric cancer), FU97 (gastric cancer), MKN7 (gastric cancer). Example 2: Quantification of CLDN6 Expression in Normal Tissues, Cancerous Tissues and Cell Lines Using Western Blot Analysis For Western blot analysis 20 μg of total protein extracted from cells lyzed with Laemmli-lysis buffer was used. Extracts were diluted in reducing sample buffer (Roth), subjected to SDS-PAGE and subsequently electrotransferred onto PVDF membrane (Pall). Immunostaining was performed with polyclonal antibodies reactive to CLDN6 (ARP) and beta-Actin (Abcam) followed by detection of primary antibodies with horseradish-peroxidase conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Dako). Tissue lysates from up to five individuals were tested for each normal tissue type. No CLDN6 protein expression was detected in any of the normal tissues analyzed. In contrast to normal tissues, high expression of CLDN6 protein was detected in samples from ovarian cancer and lung cancer. CLDN6 expression was detected in NIH-OVCAR3 (ovarian cancer), MKN7 (gastric cancer), AGS (gastric cancer), CPC-N (SCLC), HCT-116 (colon cancer), FU97 (gastric cancer), NEC8 (testicular embryonal carcinoma), JAR (placental choriocarcinoma), JEG3 (placental choriocarcinoma), BEWO (placental choriocarcinoma), and PA-1 (ovarian teratocarcinoma). Example 3: Immunohistochemical (IHC) Analysis of CLDN6 Expression in Normal Tissues and Cancerous Tissues Paraffin-embedded tissue sections (4 μm) were incubated for 1 hour at 58° C. on a heating plate (HI 1220, Leica). Paraffin was removed from the sections by incubating the slides in Roticlear (Roth) for 2×10 min at RT. Afterwards the sections were rehydrated in graded alcohol (99%, 2×96%, 80% and 70%, 5 min each). Antigen retrieval was performed by boiling slides at 120° C. (15 psi) for 15 min in 10 mM citrate buffer (pH 6.0)+0.05% Tween-20. Directly after boiling slides were incubated in PBS for 5 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in MeOH for 15 min at RT. To avoid non-specific binding the slides were blocked with 10% goat serum in PBS for 30 min at RT. Thereafter, the slides were incubated with CLDN6-specific polyclonal antibody (1 μg/ml) (ARP) overnight at 4° C. On the next day the slides were washed with PBS at RT (3×5 min) and incubated with 100 μl of the secondary antibodies (PowerVision poly HRP-Anti-Rabbit IgG ready-to-use (ImmunoLogic)) for one hour at RT. Afterwards, slides were washed with PBS at RT (3×5 min). Final staining was performed by using the VECTOR NovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame). Sections were counterstained with haematoxylin for 90 sec at RT. After dehydration with graded alcohol (70%, 80%, 2×96% and 99%, 5 min each) and 10 min incubation in xylol slides were mounted with X-tra Kit (Medite Histotechnic). No CLDN6 protein expression was detectable in normal tissues from lung, ovary, stomach, colon, pancreas, liver, duodenum or kidney. In contrast to normal tissues, strong or at least significant staining was observed on tissue sections from ovarian cancer, lung cancer, skin cancer, pancreatic cancer, gastric cancer, breast cancer, urinary bladder cancer (transitional cell carcinoma), cervical cancer, testicular cancer (seminoma) and uterine cancer. Staining was clearly accentuated at the plasma membrane of the malignant epithelial cell populations, whereas adjacent stromal and non-malignant epithelial cells were negative. These results indicate that CLDN6 protein is localized at the plasma membrane of malignant cells. Example 4: Generation of Murine Antibodies Against CLDN6 a. Generation of Expression Vectors Encoding Full Length CLDN6 and CLDN6 Fragments A non-natural, codon-optimized DNA sequence (SEQ ID NO: 3) encoding full length CLDN6 (NCBI accession number NP 067018.2, SEQ ID NO: 2) was prepared by chemical synthesis (GENEART AG, Germany) and cloned into the pcDNA3.1/myc-His vector (Invitrogen, USA) yielding the vector p3953. Insertion of a stop codon allowed the expression of CLDN6 protein without being fused to the vector encoded myc-His tag. Expression of CLDN6 was tested by Western blot, flow cytometry and immunofluorescence analyzes using commercially available anti-CLDN6 antibodies (ARP, 01-8865; R&D Systems, MAB3656). In addition, a codon-optimized DNA sequence (SEQ ID NO: 4) coding for the putative extracellular domain 2 (EC2) fragment of CLDN6 (SEQ ID NO: 6) as a fusion with an N-terminal Ig kappa leader derived signal peptide followed by 4 additional amino acids to ensure a correct signal peptidase cleavage site (SEQ ID NO: 5) was prepared and cloned into the pcDNA3.1/myc-His vector yielding the vector p3974. Prior to immunization, expression of the EC2 fragment was confirmed by immunofluorescence microscopy on transiently transfected and paraformaldehyde (PFA)-fixed CHO-K1 cells using a commercially available anti-myc antibody (Cell Signaling, MAB 2276). b. Generation of Cell Lines Stably Expressing CLDN6 HEK293 and P3X63Ag8U.1 cell lines stably expressing CLDN6 were generated by standard techniques using the vector p3953. c. Immunizations Balb/c mice were immunized with 25 μg of p3974 plasmid DNA together with 4 μl PEI-mannose (PEI-Man; in vivo-jetPEI™-Man from PolyPlus Transfection) (150 mM PEI-Man in H2O with 5% glucose) by intraperitoneal injection on days 0, 16 and 36. On days 48 and 62 mice were immunized by intraperitoneal injection with P3X63Ag8U.1 myeloma cells transfected with p3953 vector to stably express CLDN6. The cells administered on day 62 had been irradiated with 3000 rad prior to injection. The presence of antibodies directed against CLDN6 in sera of mice was monitored by immunofluorescence microscopy between days 20 and 70 using CHO-K1 cells co-transfected with nucleic acids encoding CLDN6 and GFP. To this end, 24 h following transfection, PFA-fixed or non-fixed cells were incubated with a 1:100 dilution of sera from immunized mice for 45 min at room temperature (RT). Cells were washed, incubated with an Alexa555-labeled anti-mouse Ig antibody (Molecular Probes) and subjected to fluorescence microscopy. Anti-CLDN6 specific antibodies were detected in serum samples obtained from a mouse on the basis of which the hybridoma F3-6C3-H8 was produced; seeFIG.2. For generation of monoclonal antibodies, mice with detectable anti-CLDN6 immune responses were boosted four days prior to splenectomy by intraperitonal injection of 2×107HEK293 cells stably transfected with p3953 vector. d. Generation of Hybridomas Producing Murine Monoclonal Antibodies Against CLDN6 6×107splenocytes isolated from an immunized mouse were fused with 3×107cells of the mouse myeloma cell line P3X63Ag8.653 (ATCC, CRL 1580) using PEG 1500 (Roche, CRL 10783641001). Cells were seeded at approximately 5×104cells per well in flat bottom microtiter plates and cultivated for about two weeks in RPMI selective medium containing 10% heat inactivated fetal bovine serum, 1% hybridoma fusion and cloning supplement (HFCS, Roche, CRL 11363735), 10 mM HEPES, 1 mM sodium pyruvate, 4.5% glucose, 0.1 mM 2-mercaptoethanol, 1× penicillin/streptomycin and 1×HAT supplement (Invitrogen, CRL 21060). After 10 to 14 days, individual wells were screened by flow cytometry for anti-CLDN6 monoclonal antibodies. Antibody secreting hybridomas were subcloned by limiting dilution and again tested for anti-CLDN6 monoclonal antibodies. The stable subclones were cultured to generate small amounts of antibody in tissue culture medium for characterization. At least one clone from each hybridoma which retained the reactivity of the parent cells (tested by flow cytometry) was selected. Nine-vial-cell banks were generated for each clone and stored in liquid nitrogen. Example 5: Binding Characteristics of Hybridoma Supernatants and Monoclonal Antibodies a. Quality Control of Transiently Transfected HEK293T Cells by (i) Western Blot and (ii) Flow Cytometry Analyzes (i) HEK293T cells were transfected with nucleic acids encoding CLDN3, CLDN4, CLDN6, and CLDN9, respectively, or mock-transfected. Expression of CLDN3, CLDN4, CLDN6 or CLDN9 in HEK293T cells was determined by Western blotting. To this end, cells were harvested 24 hours post transfection and subjected to lysis. The lysate was subjected to SDS-PAGE, blotted onto nitrocellulose membrane and stained with anti-CLDN3(A) (Invitrogen, 34-1700), anti-CLDN4(A) (Zymed, 32-9400), anti-CDLN6(A) (ARP, 01-8865) or anti-CLDN9(A) (Santa Cruz, sc-17672) antibodies which specifically bind to the C-terminus of the corresponding claudin under denaturing conditions. Following incubation with a peroxidase-labeled secondary antibody and developing with ECL reagent, a LAS-3000 imager (Fuji) was used for visualization. Bands of the expected molecular weights of CLDN3, CLDN4, CLDN6 and CLDN9, respectively, were observed only in the transfected cells but not in the control cells (FIG.3) demonstrating that HEK293T cells do not endogenously express any of the claudins investigated and thus, are a suitable tool for determining the cross reactivity of CLDN6 antibodies. (ii) The HEK293T cells of (i) were further analyzed by flow cytometry using anti-CLDN antibodies recognizing native epitopes (mouse anti-CLDN3 IgG2a (R&D, MAB4620), mouse anti-CLDN4 IgG2a (R&D, MAB4219), mouse anti-CLDN6 IgG2b (R&D, MAB3656)). The antibodies obtainable from Sigma under the product numbers M9144 and M8894 served as isotype controls. Specificity of these anti-CLDN antibodies was analyzed using HEK293T cells transiently transfected with nucleic acids encoding CLDN3, CLDN4, CLDN6, and CLDN9, respectively. The anti-CLDN6 antibody shows cross-reactivity with CLDN3, CLDN4 and CLDN9. The anti-CLDN4 antibody shows cross-reactivity with CLDN3, CLDN6 and CLDN9. The anti-CLDN3 antibody binds specifically to CLDN3 (FIG.4). b. Determination of the Specificity of Monoclonal Antibodies Produced According to the Invention Using Flow Cytometry HEK293T cells were co-transfected with a vector encoding different CLDN proteins and a vector encoding a fluorescence marker. 24 h post transfection cells were harvested using 0.05% trypsin/EDTA solution and washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide). Cells were transferred into U-bottom microtiter plates at 2×105cells per well and incubated for 60 min at 4° C. with hybridoma supernatants. Following washing three times with FACS buffer, cells were incubated with an allophycocyanin (APC)-conjugated anti-mouse IgG 1+2a+2b+3 specific secondary antibody (Dianova, 115-135-164). Thereafter, cells were washed twice and binding was assessed by flow cytometry using a BD FACSArray (FIG.5). The expression of the fluorescence marker is plotted on the horizontal axis against the antibody binding on the vertical axis. A commercially available mouse anti-CLDN6 IgG2b antibody (R&D, MAB3656) served as a positive control and the antibody obtainable from Sigma under the product number M8894 served as an isotype control. Antibodies in the supernatants from the monoclonal hybridoma subclones F3-6C3-H2, F3-6C3-H8, F3-6C3-H9, F3-6C3-D8 and F3-6C3-G4, all derived from hybridoma F3-6C3, were specific for CLDN6 and did not bind to CLDN9, CLDN3 and CLDN4.FIG.5Aexemplarily shows the results for the monoclonal hybridoma subclone F3-6C3-H8. Antibodies in the supernatant from the monoclonal hybridoma subclone F3-6C3-H8 also bind to cells transfected with the (I143V)-SNP variant of CLDN6. Antibodies in the supernatant from the monoclonal hybridoma subclone F4-4F7-F2 bind to both CLDN6 and CLDN9 (FIG.5A). Antibodies in the supernatant from the monoclonal hybridoma subclone F3-7B3-B4 bind to CLDN6, CLDN3 and CLDN9 (FIG.5B). Antibodies in the supernatant from the monoclonal hybridoma subclone F3-3F7-A5 bind to CLDN6, CLDN4 and CLDN9 (FIG.5B). Example 6: Generation and Testing of Monoclonal Antibodies Against CLDN6 a. Generation of Expression Vectors Encoding the Extracellular Domain 1 of CLDN6 A codon-optimized DNA sequence (SEQ ID NO: 12) coding for the putative extracellular domain 1 (EC1) fragment of CLDN6 (SEQ ID NO: 7) as a fusion with an N-terminal Ig kappa leader derived signal peptide followed by 4 additional amino acids to ensure a correct signal peptidase cleavage site (SEQ ID NO: 13) was prepared and cloned into the pcDNA3.1/myc-His vector yielding the vector p3973. Prior to immunization, expression of the EC1 fragment was confirmed by immunofluorescence microscopy on transiently transfected and paraformaldehyde (PFA)-fixed CHO-K1 cells using a commercially available anti-myc antibody (Cell Signaling, MAB 2276). b. Immunization Balb/c mice were immunized with 25 μg of p3973 plasmid DNA together with 4 μl PEI-mannose (PEI-Man; in vivo-jetPEI™-Man from PolyPlus Transfection) (150 mM PEI-Man in H2O with 5% glucose) by intraperitoneal injection on days 0 and 14. On days 28 and 44 mice were immunized subcutaneously with KLH-conjugated peptides SEQ ID NO: 14 and SEQ ID NO: 15 (100 μg each in PBS, JPT Peptide Technologies GmbH, Germany) together with HPLC-purified PTO-CpG-ODN (25 μg in PBS; 5′-TCCATGACGTTCCTGACGTT; Eurofins MWG Operon, Germany). On days 64, 77 and 97 mice were immunized by intraperitoneal injection with 2×107P3X63Ag8U.1 myeloma cells transfected with p3953 vector to stably express CLDN6. Prior to administration, cells were treated with mitomycin-C (2.5 μg/ml, Sigma-Aldrich, M4287). On days 64 and 97 cells were administered together with HPLC-purified PTO-CpG-ODN (50 μg in PBS), on day 77 together with incomplete Freund's adjuvant. For generation of monoclonal antibodies, mice with detectable anti-CLDN6 immune responses were boosted four days prior to splenectomy by intraperitonal injection of 2×107HEK293 cells stably transfected with p3953 vector. c. Testing of Monoclonal Antibodies Against CLDN6 Flow Cytometry To test the binding of monoclonal antibodies to CLDN6 and its homologous HEK293T cells were transiently transfected with the corresponding claudin-coding plasmid and the expression was analyzed by flow cytometry. In order to differentiate between transfected and non-transfected cells, HEK293T cells were co-transfected with a fluorescence marker as a reporter. 24 h post transfection cells were harvested with 0.05% trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2×106cells/ml. 100 μl of the cell suspension were incubated with the appropriate antibody at indicated concentrations for 30 min at 4° C. A cross-reactive antibody was used to detect CLDN6 and CLDN9 expression. The commercially available mouse anti-claudin antibodies anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219) served as positive controls, whereas mouse IgG2a (Sigma, M9144) and IgG2b (Sigma, M8894), respectively, served as isotype control. The cells were washed three times with FACS buffer and incubated with an APC-conjugated anti-mouse IgG 1+2a+2b+3a specific secondary antibody (Dianova, 115-135-164) for 30 min at 4° C. The cells were washed twice and resuspended in FACS buffer. The binding was analyzed by flow cytometry using a BD FACSArray. The expression of the fluorescence marker was plotted on the horizontal axis against the antibody binding on the vertical axis. CDC The complement dependent cytotoxicity (CDC) was determined by measuring the content of intracellular ATP in non-lysed cells after the addition of human complement to the target cells incubated with anti-CLDN6 antibodies. As a very sensitive analytical method the luminescent reaction of luciferase was used for measuring ATP. CHO-K1 cells stably transfected with CLDN6 (CHO-K1-CLDN6) were harvested with 0.05% trypsin/EDTA, washed twice with X-Vivo 15 medium (Lonza, BE04-418Q) and suspended at a concentration of 1×107cells/ml in X-Vivo 15 medium. 250 μl of the cell suspension were transferred into a 0.4 cm electroporation cuvette and mixed with 7 μg of in vitro transcribed RNA encoding for luciferase (luciferase IVT RNA). The cells were electroporated at 200 V and 300 μF using a Gene Pulser Xcell (Bio Rad). After electroporation, the cells were suspended in 2.4 ml pre-warmed D-MEM/F12 (1:1) with GlutaMax-I medium (Invitrogen, 31331-093) containing 10% (v/v) FCS, 1% (v/v) penicillin/streptomycin and 1.5 mg/ml G418. 50 μl of the cell suspension per well were seeded into a white 96-well PP-plate and incubated at 37° C. and 7.5% CO2. 24 h post electroporation 50 μl monoclonal murine anti-CLDN6 antibodies in 60% RPMI (containing 20 mM HEPES) and 40% human serum (serum pool obtained from six healthy donors) were added to the cells at indicated concentrations. 10 μl 8% (v/v) Triton X-100 in PBS per well were added to total lysis controls, whereas 10 μl PBS per well were added to max viable cells controls and to the actual samples. After an incubation of 80 min at 37° C. and 7.5% CO250 μl luciferin mix (3.84 mg/ml D-luciferin, 0.64 U/ml ATPase and 160 mM HEPES in ddH2O) were added per well. The plate was incubated in the dark for 45 min at RT. The luminescence was measured using a luminometer (Infinite M200, TECAN). Results are given as integrated digital relative light units (RLU). NEC8 cells were electroporated at 200 V and 400 μF and cultivated in RPMI 1640 with GlutaMAX-I medium (Invitrogen, 61870) containing 10% (v/v) FCS. The specific lysis is calculated as: specificlysis[%]=100-[(sample-totallysis)(maxviablecells-totallysis)×100]max viable cells: 10 μl PBS, without antibodytotal lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without antibody Early Treatment For early antibody treatments 2×107NEC8 cells in 200 μl PBS were subcutaneously inoculated into the flank of athymic Nude-Foxn1numice. Each experimental group consisted of ten 6-8 week-old female mice. Three days after inoculation 200 μg of purified murine monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A were applied for 46 days by alternating intravenous and intraperitoneal injections twice a week. Experimental groups treated with PBS served as a negative controls. The tumor volume (TV=(length×width2)/2) was monitored bi-weekly. TV is expressed in mm3, allowing construction of tumor growth curves over time. When the tumor reached a volume greater than 1500 mm3mice were killed. d. Results Murine monoclonal antibodies muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A showed strong binding to human CLDN6 and the CLDN6 SNP (single nucleotide polymorphism) variant I143V while no binding to CLDN3, 4, and 9 was observed (FIG.6). MuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A exhibited very low EC50 values (EC50 200-500 ng/ml) and saturation of binding was achieved at low concentrations (FIG.7). MuMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A exhibited dose-dependent CDC activity and induced CDC at low concentrations (FIG.8). The anti-CLDN6 antibodies muMAB 65A and 66B induced CDC on NEC8 cells in a dose dependent manner (FIG.9). Target specificity of muMAB 65A and 66B was proved by using NEC8 LVTS2 54 cells (CLDN6 knock-down). Furthermore, muMAB 59A, 60A, 61D, 64A, 65A, 66B and 67A showed tumor growth inhibition in mice engrafted with NEC8 cells (FIG.10). Example 7: Generation and Testing of Chimeric Monoclonal Antibodies Against CLDN6 a. Generation of Mouse/Human Chimeric Monoclonal Antibodies For chimerization, the murine heavy chain and light chain variable region including leader sequences were amplified by PCR using primers listed in the table below. The murine heavy chains were fused by an ApaI restriction site (5′-GGGCCC-3′) to the N-terminal part of the human Fcgamma1 chain, which was encoded by the expression vector. Variable domains of the murine kappa chain including leader sequences were cloned in front of the constant region using a BsiWI restriction site. The correct orientation of the constant region in the vector, i.e. suitable for the preceding promoter of the vector, was verified by sequencing. Due to the position of the ApaI restriction site, any amplification of a variable region including leader sequence for this purpose has to include the first 11 nucleotides of the sequence of the human gamma-1 constant region in addition to the sequence of the ApaI site. The nucleotide sequence of human gamma-1 heavy chain constant region is listed as SEQ ID NO: 24, the amino acid sequence of the thus expressed human gamma-1 constant region is listed as SEQ ID NO: 25. The nucleotide sequence encoding the constant part of the kappa light chain is listed as SEQ ID NO: 26, the respective amino acid sequence is listed as SEQ ID NO: 27. TABLE 1Mouse hybridoma cell lines used for antibody cloningPrimermuMABIsotypeSEQ ID NOs:heavy chain64AIgG2a17, 1889AIgG2a17, 1961DIgG2a17, 2067AIgG2a17, 20light chain64AIgK21, 2289AIgK21, 2361DIgK21, 2267AIgK21, 22 Corresponding to their murine counterparts the chimeric monoclonal antibodies were named adding the prefix “chim”, e.g. chimAB 64A. Amplification of the murine variable regions of light and heavy chains including leader sequences was carried out according to the “step-out PCR” method described in Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6). For this, total RNA was prepared from monoclonal hybridoma cell lines (see Tab. 1) by standard methods known to those skilled in the art, for example with the use of RNeasy Mini Kit (Qiagen). Single stranded cDNA was prepared according to the “template-switch” method also described in Matz et al. (Nucleic Acids Research, 1999, Vol. 27, No. 6, 1558). In addition to a (dT)30 oligomer (SEQ ID NO: 28), it included a DNA/RNA hybrid oligomer (SEQ ID NO: 29) serving as an 5′ adaptor for template switching during polymerization of the cDNA strand. In this adaptor oligomer the last three nucleotides were ribo-instead of deoxyribonucleotides. The subsequent “step-out PCR” used an antisense oligomer targeted to the constant region of the mouse kappa chain or to the constant region of the subclass 2a of the gamma chain (SEQ ID NO: 30 and 31, respectively). The IgG subclass of the murine monoclonal antibody produced by the hybridoma cell lines was afore immunologically analyzed with IsoStrip (Roche), and the appropriate antisense oligomer was chosen accordingly (see Tab. 1). A primer mix served as the sense oligomer in the “step-out PCR”, comprising the two oligomers listed in SEQ ID NO: 32 and 33. The identified murine variable regions including leader sequences were then amplified by PCR omitting the 5′ UTR and the 3′ mouse constant region, adding restriction sites to the ends which allowed subcloning into the prepared expression vectors carrying the human constant regions. In addition, the sense oligomers provided a consensus Kozak sequence (5′-GCCGCCACC-3′) and the antisense oligomers for heavy chain variable regions included the first 11 nucleotides of the human gamma-1 constant region in addition to the ApaI restriction site (see Tab. 1, SEQ ID NOs: 17 to 23). Kappa light chain variable regions including leader sequences were cloned using HindIII and BsiWI restriction enzymes, gamma heavy chain variable regions demanded HindIII and ApaI restriction enzymes. Further murine variable regions of light and heavy chains including leader sequences were amplified and further chimeric monoclonal antibodies against CLDN6 generated in accordance to the protocol disclosed above. b. Production of Chimeric Monoclonal Anti-CLDN6 Antibodies Chimeric monoclonal antibodies were transiently expressed in HEK293T cells (ATCC CRL-11268) transfected with plasmid DNA coding for the light and heavy chains of the corresponding antibody. 24 h before transfection 8×107cells were seeded on 145 mm cell culture plates and cultivated in 25 ml HEK293T-medium (DMEM/F12+GlutaMAX-I, 10% FCS, 1% penicillin/streptomycin). 20 μg plasmid DNA were dissolved in 5 ml HEK293T-medium without supplements per cell culture plate. After adding 75 μl linear polyethylenimine (PEI) (1 mg/ml) (Polyscience, 23966) the (DNA:PEI)-mixture was incubated 15 min at RT. Thereafter, the transfection-mix was added dropwise to the cells. 24 h post transfection the HEK293T-medium was replaced with Pro293a-medium (Lonza, BE12-764Q) containing 1% penicillin/streptomycin. For optimal expression, the transfected cells were cultivated at 37° C. and 7.5% CO2for additional 96 to 120 h. The supernatant was harvested and the chimeric antibody was purified by FPLC using protein-A columns. The concentration of the antibody was determined and quality was tested by SDS-PAGE. c. Testing of Chimeric Monoclonal Antibodies Against CLDN6 Flow Cytometry To test the specificities and affinities of CLDN6-specific chimeric monoclonal antibodies binding to HEK293 cells stably transfected with CLDN3, 4, 6 or 9, respectively, and tumor cell lines that endogenously express CLDN6 was analyzed by flow cytometry. Therefore, cells were harvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2×106cells/ml. 100 μl of the cell suspension were incubated with the appropriate antibody at indicated concentrations for 60 min at 4° C. A chimeric cross-reactive antibody (chimAB 5F2D2) was used to detect CLDN6 and CLDN9 expression. The commercially available mouse anti-claudin antibodies anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219) served as positive controls, whereas human IgG1-kappa (Sigma, I5154) served as a negative control. The cells were washed three times with FACS buffer and incubated for 30 min at 4° C. with an APC-conjugated goat anti-human IgG Fc-gamma (Dianova, 109-136-170) or an APC-conjugated anti-mouse IgG 1+2a+2b+3a (Dianova, 115-135-164) specific secondary antibody, respectively. The cells were washed twice and resuspended in FACS buffer. The binding was analyzed by flow cytometry using a BD FACSArray. CDC The complement dependent cytotoxicity (CDC) was determined by measuring the content of intracellular ATP in non-lysed cells after the addition of human complement to the target cells incubated with anti-CLDN6 antibodies. As a very sensitive analytical method the bioluminescent reaction of luciferase is used for measuring ATP. In this assay, NEC8 wildtype cells (CLDN6 positive) and NEC8 CLDN6 knock-down cells (CLDN6 negative) were used which both were stably transduced with luciferase expression construct. The cells were harvested with 0.05% Trypsin/EDTA and adjusted to a concentration of 2×105cells/ml in RPMI with GlutaMax-I medium (Invitrogen, 61870-010) containing 10% (v/v) FCS. 1×104cells were seeded into a white 96-well PP-plate and incubated for 24 h at 37° C. and 5% CO2. After incubation, 50 μl monoclonal chimeric anti-CLDN6 antibodies in 60% RPMI (containing 20 mM HEPES) and 40% human serum (serum pool obtained from six healthy donors) were added to the cells at indicated concentrations. 10 μl 8% (v/v) Triton X-100 in PBS per well were added to total lysis controls, whereas 10 μl PBS per well were added to max viable cells controls and to the actual samples. After a further incubation of 80 min at 37° C. and 5% CO250 μl luciferin mix (3.84 mg/ml D-luciferin, 0.64 U/ml ATPase and 160 mM HEPES in ddH2O) was added per well. The plate was incubated in the dark at RT for 45 min. The bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are given as integrated digital relative light units (RLU). The specific lysis is calculated as: specificlysis[%]=100-⌊(sample-totallysis)(maxviablecells-totallysis)×100⌋max viable cells: 10 μl PBS, without antibodytotal lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without antibody ADCC The antibody dependent cellular cytotoxicity (ADCC) was determined by measuring the content of intracellular ATP in non-lysed cells after the addition of human PBMC to the target cells incubated with anti-CLDN6 antibodies. As a very sensitive analytical method the bioluminescent reaction of luciferase is used for measuring ATP. In this assay, NEC-8 wildtype cells (CLDN6 positive) and NEC-8 CLDN6 knock-down cells (CLDN6 negative) were used which both were stably transduced with luciferase expression construct. The cells were harvested with 0.05% Trypsin/EDTA and adjusted to a concentration of 2×105cells/ml in RPMI with GlutaMax-I medium (Invitrogen, 61870-010) containing 10% (v/v) FCS and 20 mM Hepes. 1×104cells were seeded into a white 96-well PP-plate and incubated 4 h at 37° C. and 5% CO2. PBMC were isolated from human donor blood samples by density gradient centrifugation using Ficoll Hypaque (GE Healthcare, 17144003). The PMBC containing interphase was isolated and cells were washed twice with PBS/EDTA (2 mM). 1×108PBMC were seeded in 50 ml X-Vivo 15 medium (Lonza, BE04-418Q) containing 5% heat-inactivated human serum (Lonza, US14-402E) and incubated for 2 h at 37° C. and 5% CO2. 4 h post seeding of the target cells (NEC-8) 25 μl monoclonal chimeric anti-CLDN6 antibodies in PBS were added to the cells at indicated concentrations. Nonadherent PBMC, which separated within the 2 h incubation from adherent monocytes, were harvested and adjusted to 8×106cells/ml in X-vivo 15 medium. 25 μl of this cell suspension was added to the target cells and the monoclonal chimeric anti-CLDN6 antibodies. The plates were incubated for 24h at 37° C. and 5% CO2. After the 24 h incubation 10 μl 8% (v/v) Triton X-100 in PBS per well were added to total lysis controls, whereas 10 μl PBS per well were added to max viable cells controls and to the actual samples. 50 μl luciferin mix (3.84 mg/ml D-luciferin, 0.64 U/ml ATPase and 160 mM HEPES in ddH2O) was added per well. The plate was incubated in the dark at RT for 30 min. The bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are given as integrated digital relative light units (RLU). The specific lysis is calculated as: specificlysis[%]=100-[(sample-totallysis)(maxviablecells-totallysis)×100]max viable cells: 10 μl PBS, without antibodytotal lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without antibody d. Results Anti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89A showed strong binding to human CLDN6 while no binding to CLDN3, 4, and 9 was observed (FIG.11). Regarding binding to human CLDN6 stably expressed on the surface of HEK293 cells, anti-CLDN6 chimeric monoclonal antibodies chimAB 64A and 89A exhibit very low EC50 values (EC50 450-600 ng/ml) and saturation of binding was achieved at low concentrations. ChimAB 67A and 61D showed low (EC50 1000 ng/ml) and medium (EC50 2300 ng/ml) EC50 values, respectively (FIG.12). Regarding binding to CLDN6 endogenously expressed in NEC8 cells, anti-CLDN6 chimeric monoclonal antibodies chimAB 64A and 89A exhibited very low EC50 values (EC50 600-650 ng/ml) and saturation of binding was achieved at low concentrations, whereas chimAB 61D and 67A showed medium (EC50 1700 ng/ml) and high (EC50 6100 ng/ml) EC50 values, respectively (FIG.13). Regarding binding to CLDN6 endogenously expressed in OV90 cells, anti-CLDN6 chimeric monoclonal antibodies chimAB 64A and 89A exhibited very low EC50 values (EC50 550-600 ng/ml) and saturation of binding was achieved at low concentrations. ChimAB 61D and 67A showed medium EC50 values (EC50 1500 ng/ml and EC50 2300 ng/ml, respectively) (FIG.14). Anti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89A exhibited CDC activity in a dose-dependent manner on NEC-8 cells (FIG.15). Anti-CLDN6 chimeric monoclonal antibodies chimAB 61D, 64A, 67A and 89A exhibited dose-dependent ADCC activity on NEC-8 cells and induced ADCC even at low antibody concentrations (FIG.16). These results clearly show the specificity of these chimeric monoclonal antibodies for CLDN6. Example 8: Treatment Using Monoclonal Antibodies Against CLDN6 Early Treatment For early antibody treatments 2×107NEC8 cells in 200 μl RPMI medium (Gibco) were subcutaneously inoculated into the flank of athymic Nude-Foxn1numice. Each experimental group consisted of ten 6-8 week-old female mice. Three days after tumor cell inoculation 200 μg of purified murine monoclonal antibody muMAB 89A was applied for seven weeks by alternating intravenous and intraperitoneal injections twice a week. Experimental group treated with PBS served as negative control. The tumor volume (TV=(length×width2)/2) was monitored bi-weekly. TV is expressed in mm3, allowing construction of tumor growth curves over time. When the tumors reached a volume greater than 1500 mm3mice were sacrificed. Advanced Treatments For antibody treatments of advanced xenograft tumors 2×107NEC8 cells in 200 μl RPMI medium (Gibco) were subcutaneously inoculated into the flank of 6-8 week-old female athymic Nude-Foxn1numice. The tumor volume (TV=(length×width2)/2) was monitored bi-weekly. TV is expressed in mm3, allowing construction of tumor growth curves over time. 15 to 17 days after tumor cell inoculation mice were divided into treatment groups of eight animals per cohorte with homogenous tumor sizes of above 80 mm3. 200 μg of purified murine monoclonal antibodies muMAB 61D, 64A, 67A and 89A were applied for five weeks by alternating intravenous and intraperitoneal injections twice a week. Experimental groups treated with PBS and an unspecific antibody served as negative controls. When the tumors reached a volume bigger than 1500 mm3mice were sacrificed. In an early treatment xenograft model using mice engrafted with the tumor cell line NEC8, mice treated with murine monoclonal antibodies muMAB 61D, 64A and 67A did not show any tumor growth even after stopping the immunotherapy (FIG.17). In an early treatment xenograft model using mice engrafted with the tumor cell line NEC8, muMAB 89A showed tumor growth inhibition and no tumors were detectable in mice treated with muMAB89A at the end of the study (FIG.18). In an advanced treatment xenograft model using mice engrafted with the tumor cell line NEC8, muMAB 64A showed an inhibition of tumor growth (FIG.19). In an advanced treatment xenograft model using mice engrafted with the tumor cell line NEC8, mice treated with muMAB 64A showed prolonged survival (FIG.20). In an advanced treatment xenograft model using mice engrafted with the tumor cell line NEC8, inhibition of tumor growth was achieved with the murine monoclonal anti-CLDN6 antibodies muMAB 61D, 67A and 89A (FIG.21). In an advanced treatment xenograft model using mice engrafted with the tumor cell line NEC8, mice treated with the CLDN6 specific antibody muMAB 61D or 67A showed prolonged survival (FIG.22). In an advanced treatment xenograft model using mice engrafted with NEC8 wildtype and NEC8 cells with a stable CLDN6 knock-down, muMAB 64A and 89A only show a therapeutic effect in mice engrafted with NEC8 wildtype but not in mice engrafted with NEC8 CLDN6 knock-down cells demonstrating CLDN6-specificity of the antibody in vivo (FIG.23). Example 9: High-Resolution Epitope Mapping of Monoclonal Antibodies Against CLDN6 The CLDN6 specific monoclonal antibodies only show very weak (if any) binding to linear peptides in ELISA epitope-mapping studies, implying that their epitopes are conformational. To analyze the interaction between antibodies described herein and CLDN6 in its native conformation site-directed mutagenesis in mammalian cell culture was used as an epitope-mapping technique. Alanine scanning mutagenesis of amino acids 27-81 and 137-161 within the first and second extracellular domain, respectively, was performed. Following transient expression in HEK293T cells, CLDN6 mutants were assessed for their ability to be bound by specific monoclonal antibodies. Impaired binding of a specific monoclonal antibody to a CLDN6 mutant suggest that the mutated amino acid is an important contact and/or conformational residue. The binding was analyzed by flow cytometry. To discriminate between transfected and non-transfected cell populations, cells were co-transfected with a fluorescence marker. The amino acid residues of CLDN6 that are important for the interaction with CLDN6 specific chimeric antibodies have been systematically identified by alanine-scanning. Alanine and glycine mutations were generated by site-directed mutagenesis (GENEART AG, Germany). To test the binding of monoclonal chimeric antibodies to wild-type CLDN6 and its mutants HEK293T cells were transiently transfected with the corresponding claudin-coding plasmid and the expression was analyzed by flow cytometry. In order to differentiate between transfected and non-transfected cells, HEK293T cells were co-transfected with a fluorescence marker as a reporter. 24 h post transfection cells were harvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2×106cells/ml. 100 μl of the cell suspension were incubated with 10 μg/ml antibody for 45 min at 4° C. The commercially available mouse anti-CLDN6 (R&D, MAB3656) was used as a control to detect cell-surface expression of CLDN6 mutants. The cells were washed three times with FACS buffer and incubated with an APC-conjugated goat anti-human IgG Fc-gamma (Dianova, 109-136-170) or an APC-conjugated anti-mouse IgG 1+2a+2b+3a specific secondary antibody (Dianova, 115-135-164) for 30 min at 4° C. The cells were washed twice and resuspended in FACS buffer. The binding within the transfected cell population was analyzed by flow cytometry using a BD FACSArray. Therefore, the expression of the fluorescence marker was plotted on the horizontal axis against the antibody binding on the vertical axis. The average signal intensity of a monoclonal chimeric CLDN6 specific antibody bound to mutant CLDN6 was expressed as the percentage of wild-type binding. Amino acids that are essential for binding showed no binding after being mutated whereas amino acids that support binding only showed reduced binding compared to wild-type. High resolution epitope-mapping demonstrated that the amino acids F35, G37, S39 and possibly T33 of the first extracellular domain of CLDN6 are important for the interaction with the CLDN6 specific chimeric antibodies chimAB 61D, 64A, 67A and 89A. Residue 140 is essential for the binding of chimAB 89A and it contributes to the binding of chimAB 61D and 67A. In addition, L151 of the second extracellular domain of CLDN6 is important for the interaction with chimAB 67A (FIG.24). Example 10: Generation and Testing of Improved Monoclonal Antibodies Against CLDN6 It turned out that antibody 64A although being excellent in its properties regarding binding to CLDN6 and efficacy regarding tumor treatment had a free cysteine residue at position 46 of the light chain which was found to potentially compromise its properties regarding solubility, stability and aggregate formation. Such free cysteine may also be undesired for other reasons such as regulatory provisions. We thus tried to create antibodies on the basis of 64A maintaining its desired properties while avoiding a free cysteine residue at position 46. The chimeric monoclonal antibody mAb206-LCC was generated by combining the heavy chain of chimAB 64A with the light chain of chimAB 61D. MAb206-SUBG and mAb206-SUBS were constructed by amino acid substitution. To this end, the cysteine at position 46 within the light chain of chimAB 64A was substituted to glycine and serine, respectively. Production of Chimeric Monoclonal Anti-CLDN6 Antibodies in HEK293T Chimeric monoclonal antibodies were transiently expressed in HEK293T cells (ATCC CRL-11268) transfected with plasmid DNA coding for the light and heavy chains of the corresponding antibody as disclosed in section b of Example 7. Production of Chimeric Monoclonal Anti-CLDN6 Antibodies in Suspension Adapted CHO-Cells Suspension adapted CHO-cells were sub-cultivated in serum-free media in a humidified CO2shaker. One day prior transfection, cells were seeded in serum-free media in shaker flasks. On the day of transfection cells were centrifuged (5 min at 200×g) and resuspended in fresh DMEM-Medium (Invitrogen, 41965-039) in shaker flasks. DNA and transfection reagent were added to the cells and gently mixed by shaking. After static incubation in a CO2-incubator, the cells were diluted with serum free growth media and further cultivated for expression in an incubation shaker. Cells were fed at day 0, 2, 4 and 6 with CHO CD EfficientFeed™ A and B, respectively (Invitrogen, A1023401 and A1024001). Chimeric antibodies were harvested after the viability of the cells decreased below 90%. The antibodies were purified by FPLC using protein-A columns. The antibody concentration was determined by absorbance at 280 nm and the purity was analysed by SDS-PAGE. Flow Cytometry The binding of CLDN6-specific chimeric monoclonal antibodies to cells expressing the target was analyzed by flow cytometry. Therefore, cells were harvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2×106cells/ml. 100 μl of the cell suspension were incubated with the appropriate antibody at indicated concentrations for 30 min at 4° C. ChimAB 5F2D2 was used to detect CLDN6 and CLDN9 expression. The commercially available mouse anti-claudin antibodies anti-CLDN3 (R&D, MAB4620) and anti-CLDN4 (R&D, MAB4219) served as positive controls, whereas human IgG1-kappa (Sigma, I5154) served as a negative control. The cells were washed three times with FACS buffer and incubated for 30 min at 4° C. with an APC-conjugated goat anti-human IgG Fc-gamma (Dianova, 109-136-170) or an APC-conjugated anti-mouse IgG 1+2a+2b+3a (Dianova, 115-135-164) specific secondary antibody, respectively. The cells were washed twice and resuspended in FACS buffer. The binding was analyzed by flow cytometry using a BD FACSArray. CDC The complement dependent cytotoxicity (CDC) was determined by measuring the content of intracellular ATP in non-lysed cells after the addition of human complement to the target cells incubated with anti-CLDN6 antibodies as disclosed in section c of Example 7 ADCC The antibody dependent cellular cytotoxicity (ADCC) was determined by measuring the content of intracellular ATP in non-lysed cells after the addition of human PBMC to the target cells incubated with anti-CLDN6 antibodies. As a very sensitive analytical method the bioluminescent reaction of luciferase is used for measuring ATP. In this assay, NEC-8 wildtype cells (CLDN6 positive) and NEC-8 CLDN6 knock-down cells (CLDN6 negative) were used which both were stably transduced with luciferase RNA whereas OV90 cells were transiently transfected with IVT-RNA coding for luciferase. The cells were harvested with 0.05% Trypsin/EDTA and adjusted to a concentration of 2×105cells/ml (NEC-8 wildtype and CLDN6 knock-down) or 1×106cells/ml (OV90) in the respective growth medium containing additionally 20 mM Hepes. 1×104or 5×104cells, respectively, were seeded into a white 96-well PP-plate and incubated at 37° C. and 5% CO2. PBMC were isolated from human donor blood samples by density gradient centrifugation using Ficoll Hypaque (GE Healthcare, 17144003). The PMBC containing interphase was isolated and cells were washed twice with PBS/EDTA (2 mM). 1×108PBMC were seeded in 50 ml X-Vivo 15 medium (Lonza, BE04-418Q) containing 5% human serum (serum pool obtained from six healthy donors) and incubated for 2 h at 37° C. and 5% CO2. 4 h post seeding of the target cells 25 μl monoclonal chimeric anti-CLDN6 antibodies in PBS were added to the cells at indicated concentrations. Nonadherent PBMC, which separated within the 2 h incubation from adherent monocytes, were harvested and adjusted to 1.6×107cells/ml (for NEC-8 wildtype or CLDN6 knock-down experiments) or 4×107cells/ml (for OV90 experiment) in X-vivo 15 medium. 25 μl of this cell suspension were added to the target cells and the monoclonal chimeric anti-CLDN6 antibodies. The plates were incubated for 24 h at 37° C. and 5% CO2. After the 24 h incubation 10 μl 8% (v/v) Triton X-100 in PBS per well were added to total lysis controls, whereas 10 μl PBS per well were added to max viable cells controls and to the actual samples. 50 μl luciferin mix (3.84 mg/ml D-luciferin, 0.64 U/ml ATPase and 160 mM HEPES in ddH2O) were added per well. The plate was incubated in the dark at RT for 30 min. The bioluminescence was measured using a luminometer (Infinite M200, TECAN). Results are given as integrated digital relative light units (RLU). The specific lysis is calculated as: specificlysis[%]=100-[(sample-totallysis)(max[viablecells-totallysis)]×100]max viable cells: 10 μl PBS, without antibodytotal lysis: 10 μl 8% (v/v) Triton X-100 in PBS, without antibody Advanced Treatments For antibody treatments of advanced xenograft tumors 2×107NEC8 cells in 200 μl RPMI medium (Gibco) were subcutaneously inoculated into the flank of 6-8 week-old female athymic Nude-Foxn1numice. The tumor volume (TV=(length×width2)/2) was monitored bi-weekly. TV is expressed in mm3, allowing construction of tumor growth curves over time. 17 days after tumor cell inoculation mice were divided into treatment groups of eight animals per cohorte with homogenous tumor sizes of above 80 mm3. 200 μg of purified chimeric monoclonal antibodies were applied for five weeks by alternating intravenous and intraperitoneal injections twice a week. The experimental group treated with an unspecific antibody served as a negative control. Mice were sacrificed when the tumors reached a volume bigger than 1500 mm3. Metastasis Assay For metastasis assay NEC8 cells were harvested and filtered through a 70 μm cell strainer to exclude large cell aggregates. 4×106NEC8 cells were injected into the tail vein of 6 week old female athymic Nude-Foxn1numice. 3 days after cell injection 200 μg of purified murine monoclonal antibody muMAB 64A in PBS or PBS without antibody were applied twice a week by alternating intravenous and intraperitoneal injections. After 8 weeks mice were sacrificed, lungs were prepared and snap-frozen in liquid nitrogen. Genomic DNA was isolated from frozen tissue following the instructions of the “Blood & Cell Culture DNA Midi Kit” (Qiagen, 13343). Lung tissues were homogenised using the Ultra Torax T8.10 (IKA-Werke). In order to avoid contamination with human genomic DNA the quantitative PCR (qPCR) of the genomic lung DNA was prepared under sterile conditions. To evaluate the tumor load of human NEC8 cells in murine lung tissue samples a standard curve was generated using a defined amount of human NEC8 genomic DNA and murine lung genomic DNA. Therefore, the following dilution series was used (NEC8 DNA/mouse lung DNA): 200/0, 40/160, 8/192, 1,6/198,4, 0,32/199,68, 0,064/199,94, 0,013/200 and 0/200 ng. For qPCR 200 ng genomic DNA, 2×SYBR Green (Qiagen, 204145), 16 nmol sense primer (GGGATAATTTCAGCTGACTAAACAG, Eurofins) and 16 nmol antisense primer (TTCCGTTTAGTTAGGTGCAGTTATC, Eurofins) in a total volume of 50 μl were analysed using the 7300 Real Time PCR-System from Applied Biosystems. As a negative control water was used instead of DNA. QPCR was performed under following conditions: 15 min at 95° C. (1 reps), 30 sec at 95° C./30 sec at 62° C./30 sec at 72° C. (40 reps), 15 sec at 95° C./30 sec at 60° C./15 sec at 95° C. (1 reps). All qPCR reactions were carried out in triplicate. The tumor load of each lung tissue sample was quantified in correlation to the standard curve using the 7300 System software (Applied Biosystems). High-Resolution Epitope Mapping To analyse the interaction between our antibodies and CLDN6 in its native conformation we used site-directed mutagenesis in mammalian cell culture as an epitope-mapping technique. Therefore, we performed alanine scanning mutagenesis of amino acid 27-81 and 137-161 within the first and second extracellular domain of CLDN6, respectively. Alanine mutations were generated by site-directed mutagenesis (GENEART AG, Germany). Following transient expression in HEK293T cells, CLDN6 mutants were assessed for their ability to be bound by specific monoclonal antibodies. Impaired binding of a specific monoclonal antibody to a CLDN6 mutant suggest that the mutated amino acid is an important contact and/or conformational residue. The binding was analysed by flow cytometry. To discriminate between transfected and non-transfected cell populations, cells were co-transfected with a fluorescence marker as a reporter. 24 h post transfection cells were harvested with 0.05% Trypsin/EDTA, washed with FACS buffer (PBS containing 2% FCS and 0.1% sodium azide) and resuspended in FACS buffer at a concentration of 2×106cells/ml. 100 μl of the cell suspension were incubated with 10 μg/ml antibody for 30 min at 4° C. The commercially available mouse anti-Cldn6 (R&D, MAB3656) was used as a control to detect cell-surface expression of CLDN6 mutants. The cells were washed three times with FACS buffer and incubated with an APC-conjugated goat anti-human IgG Fc-gamma (Dianova, 109-136-170) or an APC-conjugated anti-mouse IgG 1+2a+2b+3a specific secondary antibody (Dianova, 115-135-164) for 30 min at 4° C. The cells were washed twice and resuspended in FACS buffer. The binding within the transfected cell population was analysed by flow cytometry using a BD FACSArray. Therefore, the expression of the fluorescence marker was plotted on the horizontal axis against the antibody binding on the vertical axis. The average signal intensity of a monoclonal chimeric CLDN6 specific antibody bound to mutant CLDN6 was expressed as the percentage of wild-type binding. Amino acids that are essential for binding showed no binding after being mutated whereas amino acids that support binding showed definite reduced binding compared to wild-type. Immunohistochemistry Cryo tissue sections (4 μm) were fixed directly after sectioning with acetone for 10 min at −20° C. Afterwards the sections were dried for 10 min at RT and stored at −80° C. Before usage the sections were thawn (10 min at RT) and rehydrated in PBS for 5 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in PBS for 15 min at RT. To avoid non-specific binding the slides were blocked with 10% goat serum in PBS for 30 min at RT. Thereafter, the slides were incubated with the CLDN6-specific murine monoclonal antibody muMAB 64A (0.2 μg/ml diluted in 10% goat serum/PBS) overnight at 4° C. On the next day, the slides were washed with PBS at RT (3×5 min) and incubated with 100 μl of the secondary antibodies (PowerVision poly HRP-Anti-mouse IgG ready-to-use (ImmunoLogic)) for one hour at RT. Afterwards, slides were washed with PBS at RT (3×5 min). Final staining was performed for 1:30 min by using the VECTOR NovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame). Sections were counterstained with haematoxylin for 90 sec at RT. After dehydration with graded alcohol (70%, 80%, 2×96% and 99%, 5 min each) and 10 min incubation in Xylol slides were mounted with X-tra Kit (Medite Histotechnic). In order to use the chimeric monoclonal antibodies mAb206-LCC and mAb206-SUBG on human tissues they were labeled with FITC (Squarix GmbH, Germany). Cryo sections were prepared and treated as described above regarding fixation, blocking of endogenous peroxidases and blocking of non-specific binding sites. Thereafter, slides were incubated with the antibodies mAb206-LCC-FITC and mAb206-SUBG-FITC (1 μg/ml in 10% goat serum/PBS) for 1 h at RT in a dark chamber. Afterwards the slides were washed with PBS at RT (3×5 min) and incubated with 200 μl of the rabbit anti-FITC-HRP antibody (AbD Serotec, diluted 1:300 in 10% goat serum/PBS) for 30 min at RT. Following washing with PBS at RT (3×5 min), the substrate reaction was performed for 2:30 min by using the VECTOR NovaRED Substrate Kit SK-4800 from Vector Laboratories (Burlingame). Counterstaining, dehydration and mounting were performed as described above. Analysis of the binding specificity of anti-CLDN6 chimeric monoclonal antibodies by flow cytometry using HEK293T cells transiently transfected with human CLDN6, 3, 4 and 9, respectively revealed that the chimeric monoclonal antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS showed binding to CLDN6 without interacting with CLDN3, 4 and 9, respectively (FIG.27). Regarding the binding to human CLDN6 stably expressed on the surface of HEK293 cells the anti-CLDN6 chimeric monoclonal antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS exhibited similar low EC50 values and saturation of binding was achieved at low concentrations (FIG.28). Binding affinities of anti-CLDN6 chimeric monoclonal antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to tumor cells that endogenously express human CLDN6 was assessed by analyzing the binding to the testicular cancer cell line NEC8 by flow cytometry. Compared to the CLDN6-specific antibodies chimAB 64A, mAb206-SUBG and mAb206-SUBS the light-chain combination variant mAb206-LCC showed a threefold stronger binding affinity to NEC8 cells. In all cases the saturation of binding was achieved at low concentrations (FIG.29). An analysis of the binding affinities of anti-CLDN6 chimeric monoclonal antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS to the human ovarian cancer cell line OV90 by flow cytometry revealed that the CLDN6-specific antibodies exhibited similar low EC50 values. The light-chain combination variant mAb206-LCC showed the strongest binding to OV90 cells (FIG.30). On NEC-8 cells anti-CLDN6 chimeric monoclonal antibodies chimAB 64A, mAb206-LCC and mAb206-SUBG exhibited CDC activity in a dose-dependent manner, whereas on NEC-8 CLDN6 knock-down cells none of these antibodies induced unspecific cell lysis. This result demonstrated target specific effector functions of chimAB 64A, mAb206-LCC and mAb206-SUBG (FIG.31a). Antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS exhibited CDC activity on NEC8 cells in a dose-dependent manner. Compared to chimAB 64A the amino acid substitution variants mAb206-SUBG and mAb206-SUBS showed similar CDC activities on NEC8 cells (FIG.31b). Anti-CLDN6 chimeric monoclonal antibodies chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS showed dose-dependent ADCC activity and induced ADCC even at low antibody concentrations in NEC8 and OV90 tumor cell lines (FIG.32a). FIG.32bshows the antibody-dependent cellular cytotoxicity (ADCC) activity of anti-CLDN6 chimeric monoclonal antibodies chimAB 64A, mAb206-LCC and mAb206-SUBG on NEC8 wildtype and NEC8 knock-down cells. To demonstrate target specificity NEC8 cells with a stable CLDN6 knock-down were used. An advanced treatment xenograft model using mice engrafted with the tumor cell line NEC8 showed that compared to the antibody control group the CLDN6 specific antibodies mAb206-LCC, mAb206-SUBG and mAb206-SUBS inhibit tumor growth (FIG.33). The growth curves inFIG.34ademonstrate that the anti-CLDN6 chimeric monoclonal antibodies mAb206-LCC, mAb206-SUBG and mAb206-SUBS are able to inhibit tumor growth. The survival plot inFIG.34bshows prolonged survival of mice treated with CLDN6 specific antibodies. High resolution epitope-mapping of chimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS demonstrated that the amino acids F35, G37 and S39 and potentially T33 of the first extracellular domain of CLDN6 are important for the interaction with the CLDN6 specific chimeric antibodies. ChimAB 64A, mAb206-LCC, mAb206-SUBG and mAb206-SUBS showed identical binding patterns (FIG.35). To test the therapeutic effect of the anti-CLDN6 murine monoclonal antibody muMAB 64A in a metastasis xenograft model, NEC8 cells were injected into the tail vain of athymic Nude-Foxn1numice. 3 days after engraftment mice were treated with the CLDN6 specific antibody muMAB 64A. After 8 weeks lungs were prepared and the tumor load was analysed by PCR. Compared to the PBS control group the murine monoclonal anti-CLDN6 antibody muMAB 64A clearly showed inhibition of tumor growth (FIG.36). Immunohistochemical staining of human cancer and normal tissues using monoclonal antibodies muMAB 64A, mAb206-LCC and mAb206-SUBG revealed that in contrast to normal tissues, strong and homogenous staining was observed on tissue sections from ovarian and testis cancers. A very strong membraneous staining of the malignant epithelial cell populations was detected, whereas adjacent stromal and non-malignant epithelial cells were not stained (FIG.37). These results clearly show that our CLDN6-specific antibodies bind specifically to malignant cells derived from tumor patients. (Explanation: number of tissues that were stained by antibody/number of analysed tissues.) Additional Sheet for Biological Material Identification of Further Deposits: 1) The Name and Address of depository institution for the deposits are:DSMZ-Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbHInhoffenstr. 7 B38124 BraunschweigDE The indications madebelow relate to thedeposited microorganism inthe description on theDate of despositsAccession Numbersfollowing page(s)Jun. 21, 2010DSM ACC3067page 14, line 1Jun. 21, 2010DSM ACC3068page 14, line 2Jun. 21, 2010DSM ACC3069page 14, line 3Jun. 21, 2010DSM ACC3070page 14, line 4Jun. 21, 2010DSM ACC3071page 14, line 5Jun. 21, 2010DSM ACC3072page 14, line 6Jun. 21, 2010DSM ACC3073page 14, line 7Aug. 31, 2010DSM ACC3089page 14, line 8Aug. 31, 2010DSM ACC3090page 14, line 9 Additional Indications for All Above Mentioned Deposits:Mouse (Mus musculus) myeloma P3X63Ag8.653 fused with mouse (Mus musculus) splenocytesHybridoma secreting antibody against human CLDN6 2) Depositor:All above mentioned depositions were made by:Ganymed Pharmaceuticals AGFreiligrathstraße 1255131 MainzDE | 216,049 |
11859009 | EXAMPLES Example 1: Single Chain TCR (scTCR Format) Example 1.1: Generation of Stable scTCR For the present invention, the TCR R11P3D3(SEQ ID NOs: 1 and 2, full length) was converted into a single chain TCR construct (scTCR R11P3D3, SEQ ID NO: 5) using the variable alpha (SEQ ID NO: 3) and beta (SEQ ID NO: 4) domains and an appropriate glycine-serine linker sequence (SEQ ID NO: 61). For TCR maturation via yeast surface display, the DNA of the corresponding sequence was synthesized and transformed intoSaccharomyces cerevisiaeEBY100 (MATa AGA1::GAL1¬AGA1::URA3 ura3¬52 trp1 leu2¬delta200 his3¬delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC® MYA¬4941 ™) together with a yeast display vector based on pCT302 (Boder and Wittrup, Methods Enzymol. 2000; 328:430-44). The resulting fusion protein after homologous recombination in the yeast (SEQ ID NO: 325) contains a leader peptide at the N-terminus of the Aga2p protein (SEQ ID NO: 88) (Boder and Wittrup, Nat Biotechnol. 1997 June; 15(6):553-7), the protein of interest, namely the scTCR R11P3D3(SEQ ID NO: 5) or its variants and additional peptide tags (FLAG and Myc (SEQ ID NOs 99 and 288)) to determine the expression level of the fusion protein. Libraries of scTCR variants were generated via PCR using degenerate primers and the transformation of yeast cells was performed as described in WO 2018/091396 and resulted in up to 109yeast clones per library. The selection process for the yeast clones bearing mutant scTCR variants with improved binding to PRAME-004 in the context of HLA-A*02 was essentially performed as described in Smith et al. (Methods Mol Biol. 2015; 1319:95-141). Expression as determined by Myc tag-FITC staining and in particular functional binding via HLA-A*02/PRAME-004 tetramer staining was applied to select for most promising candidates (FIG.1). The scTCR conversion by yeast surface display revealed nine framework mutations in combination with three single point CDR mutations, resulting in the stabilized scTCR R11P3D3SD (SEQ ID NO: 6) showing improved expression as well as HLA-A*02/PRAME-004 tetramer binding. Example 1.2: Affinity Maturation of Stabilized scTCR, Binding Motif and Specificity Assessment To generate scTCR molecules with higher binding affinity towards HLA-A*02/PRAME-004, all CDRs were maturated individually, using the previously identified stabilized scTCR R11P3D3SD (SEQ ID NO: 6). The CDR residues were randomized by using degenerate DNA oligo primers essentially as described previously (Smith et al., Methods Mol Biol. 2015; 1319:95-141). The resulting DNA libraries were transformed as described in example 1. For the selection of affinity enhanced and specific R11P3D3SD scTCR variants, decreasing concentrations of HLA-A*02/PRAME-004 tetramer or monomer were used for each selection round. After four selection rounds, single scTCR clones were isolated and sequenced, resulting in a multitude of affinity maturated CDR sequences. As exemplarily shown for scTCR with maturated CDRa1 sequences (SEQ ID NOs: 16 to 28,FIG.2), a strong improvement in binding of HLA-A*02/PRAME-004 monomers could also be demonstrated for scTCR with maturated CDRa2 and CDRb2 (SEQ ID NOs 29 to 32 and 35 to 45, Table 3). The selectivity of HLA-A*02/PRAME-004 binding was retained during maturation as confirmed by the low binding of the scTCR to a mix of HLA-A*02 tetramers containing peptides (similar peptides or SimPeps) with high degree of sequence similarity to PRAME-004 peptide (SEQ ID NO: 50). All selected scTCR maturation variants showed substantial staining with HLA-A*02/PRAME-004 monomers at a concentration of 10 nM, while the non-maturated stabilized scTCR R11P3D3SD as reference did not show staining (FIG.2and Table 3). Furthermore, binding of maturated scTCR to a mix of similar peptides, applied in a high avidity format of HLA-A*02 tetramers at a concentration of 10 nM, could not be detected or showed only low signals in comparison to HLA-A*02/PRAME-004 monomer binding, which confirms the capability of the scTCR maturation variants to bind the PRAME-004 target peptide in a highly specific manner. TABLE 3Binding data of yeast-bearing scTCRs with mutant CDR2s. Stabilized scTCR comprising non-modifiedand maturated CDR2 alpha and CDR2 beta were stained with 10 nM HLA-A*02/PRAME-004 monomer andcounterstained with a mix of HLA-A*02 tetramers, each applied at a concentration of 10 nM,containing peptides (similar peptides or SimPeps, SEQ ID NO: 51 to 59) with high sequence similarityto PRAME-004 (SEQ ID NO: 50)Yeast cells stained positive withYeast cells stained positive withCDRa2HLA-A*02/HLA-CDRb2HLA-A*02/HLA-SEQPRAME-004,A*02/SimPep,SEQPRAME-A*02/SimPep,SequenceID NOmonomertetramer mixSequenceID NO004, monomertetramer mix,FGPYGKE3261.0%8.1%YQNTAV3766.9%3.8%FGPYGRE3059.0%6.6%YQNTAL3851.6%3.3%FGPYGTE3164.5%10.9%FQNTAT3957.4%3.8%FGPYGVE2954.5%5.7%MQNSAV4069.2%4.2%MTSNGDE*143.6%3.3%FQNTAL4162.0%5.5%MQNTAI4260.7%4.6%LQNTAV4360.5%3.3%MQNTAV4458.0%4.4%YQNTAI3551.7%2.9%FQNTAV3666.9%3.3%FNNNEP*151.9%2.5%*: corresponding CDR from R11P3D3SD_scTCR (SEQ ID NO: 6) To further increase the affinity of scTCR clones, maturated CDRs identified in above-described CDR libraries were systematically combined in one DNA library and transformed intoSaccharomyces cerevisiaeEBY100 as described in example 1.1. This library was selected using HLA-A*02/PRAME-004 monomer and scTCR from single yeast clones were sequenced and analyzed regarding their binding towards HLA-A*02 monomers containing either the PRAME-004 target peptide or one peptide derived from the group of 26 peptides (similar peptides) sharing sequence similarities with PRAME-004 (SEQ ID NOs: 51-60, 62 to 69 and 71 to 78). All the selected high affinity scTCR variants (SEQ ID NOs 79 to 87 and 89 to 92) bound strongly to HLA-A*02/PRAME-004 monomer with binding EC50values in the low nanomolar or sub-nanomolar range (Table 4), as calculated by non-linear 4-point curve fitting. With the exception of SMARCD1-001 (SEQ ID NO: 76) that provoked a binding signal slightly above background (FIG.4), none of the scTCR variants (SEQ ID NOs 79 to 87 and 89 to 92) exhibited binding above background levels to any of the similar peptides (SEQ ID NOs: 51-60, 62 to 69 and 71 to 78) in the context of HLA-A*02 monomers applied at a concentration of 100 nM (FIG.3,FIG.4, Table 4). The presented data confirm the high binding specificity of the scTCR variants with combined CDR mutations whose binding properties were superior to the reference scTCR (R16P1C10_CDR6_scTCR, SEQ ID NO 357) that showed strong binding to IFT17-003 (SEQ ID NO 60) at a level indistinguishable from PRAME-004 binding (FIG.4). A set of selected high affinity scTCRs from yeast surface display was further examined regarding their functional epitope on the target peptide in context of the HLA-A*02 presentation, called binding motif. This was addressed by single alanine substitutions of positions 1, 3, 4, 5, 6, 7 and 8 of the PRAME-004 target peptide (SEQ ID NOs 318 to 324) and assessment of binding of scTCR-bearing yeast cells to the respective PRAME-004 peptide variants in context of HLA-A*02. Four concentrations (10 nM, 3 nM, 1 nM, 0.3 nM) of HLA-A*02 monomers with PRAME-004 or the respective alanine-substituted peptides were used to stain the high affinity scTCR-bearing yeast cells and revealed a broad binding motif for all scTCR variants with strong recognition of positions 3, 5 and 7 as confirmed by the lack of staining signals at all tested monomer concentrations. For positions 6 and 8 of the PRAME-004 peptide, a contribution to the binding motif can be assumed since alanine replacements at these positions significantly reduced the staining signals, even if this was observed with lower stringency than for the positions 3, 5 and 7. For positions 1 and 4 of the PRAME-004 peptide, only a marginal or no contribution to the binding motif could be determined since alanine substitutions resulted in staining intensities nearly comparable to those observed with the PRAME-004 target peptide (FIG.5and Table 4). For further analysis, the five scTCR clones R11P3D3SDA7_A02_scTCR (SEQ ID NO: 79), R11P3D3SDA7_A09_scTCR (SEQ ID NO: 82), R11P3D3SDA7_A10_scTCR (SEQ ID NO: 83), R11P3D3SDA7_B03_scTCR (SEQ ID NO: 85) and R11P3D3SDA7_B06_scTCR (SEQ ID NO: 87) were subject to conversion into scTCR-Fab bispecific format in order to determine further protein features (see following example). TABLE 4Binding data of yeast-bearing scTCR and soluble scTCR-Fab molecules and respective variable chainsequences. For scTCR-bearing yeast cells, binding towards HLA-A*02/PRAME-004 monomers is presentedas EC50values and binding towards 26 similar peptides (SEQ ID NOs 51 to 60, 62-69 and 71 to 79) incontext of 100 nM HLA-A*02 monomer is presented as number of peptides showing no binding. Bindingmotif positions constituting the functional epitope of PRAME-004 were determined by alanine scanningand positions with strong and medium (positions in brackets) impact on scTCR binding are indicated.Five soluble scTCR-Fab molecules (TPP-70 to TPP-74) were assessed for binding affinity (KD) towardsHLA-A*02/PRAME-004 monomer and for binding towards a set of 14 similar peptides (see example 2).FRa mutationsFRb mutationsMoleculeSequenceparental TCR)parental TCR)nameID NOs(compared toCDRa1CDRa2CDRa3(compared toCDRb1CDRb2CDRb3R11 P3D3SD_6W44K, A52F, V55Y,SSNFYNMTSNGDEALYNNNDMRL11E, Q44E,SGHNSFNNNEPASSPGSTDTQYstablizedK92T, G93DM46P, R48QscTCRR11P3D3SDA779,W44K, A52F, V55Y,TREFQDFGPYGVEALYNNNDMRL11E, Q44E,SGHNSFQNTAVASSPGSTDTQY_A02_scTCR93 andK92T, G93DM46P, R48Qand TPP-7094R11P3D3SDA780W44K, A52F, V55Y,TKEFQDFGPYGVEALYNNNDMRL11E, Q44E,SGHNSFQNTAVASSPGATDTQY_A05_scTCRK92T, G93DM46P, R48QR11P3D3SDA781W44K, A52F, V55Y,TREFQDFGPYGKEALYNNNDMRL11E, R22H,SGHNSFQNTAVASSPGSTDTQY_A06_scTCRK92T, G93DQ44E, M46P,R48QR11P3D3SDA782,W44K, A52F, V55Y,TKEFQDFGPYGREALYNNNDMRL11E, Q44E,SGHNSFQNTAVASSPGATDTQY_A09_scTCR93 andK92T, G93DM46P, R48Qand TPP-7195R11P3D3SDA783,W44K, A52F, V55Y,SSNFYNFGPYGVEALYNNNDMRL11E, Q44E,SGHNSFNSETVASSPGATDTQY_A10_scTCR93 andK92T, G93DM46P, R48Qand TPP-7296R11P3D3SDA784W44K, A52F, V55Y,NKEFQFGPYGVEALYNNNDMRL11E, Q44E,SGHNSYQNTAVASSPGATDTQY_B01_scTCRK92T, G93DDM46P, R48QR11P3D3SDA785,W44K, A52F, V55Y,NKEFQFGPYGTEALYNNNDMRL11E, Q44E,SGHNSFQNTAVASSPGSTDTQY_B03_scTCR93 andK92T, G93DDM46P, R48Qand TPP-7397R11P3D3SDA786W44K, A52F, V55Y,SSNFYNFGPYGKEALYNNNDMRL11E, R22H,SGHNSYQNTAIASSPGSTDTQY_B04_scTCRK92T, G93DQ44E, M46P,R48QR11P3D3SDA787,W44K, A52F, V55Y,VKEFQDFGPYGKEALYNNNDMRL11E, Q44E,SGHNSFQNTAVASSPGATDTQY_B06_scTCR93 andK92T, G93DM46P, R48Qand TPP-7498R11P3D3SDA789W44K, A52F, V55Y,VKEFQDFGPYGKEALYNNNDMRH10N, R22H,SGHNSFNSETVASSPGSTDTQY_F11_scTCRK92T, G93DL11E, Q44E,M46P, R48QR11P3D3SDA790W44K, A47D, A52F,NKEFQFGPYGREALYNNNDMRL11E, R43K,SGHNSYQNTAVASSPGATDTQY_G11_scTCRV55Y, K92T, G93DDQ44E, M46P,R48QR11P3D3SDA791W44K, A52F, V55Y,TREFQDFGPYGTEALYNNNDMRL11E, Q44E,SGHNSYQNTAVASSSGATDTQY_H08_scTCRK92T, G93DM46P, R48QR11P3D3SDA792L39M, W44K, A52F,TKEFQDFGPYGVEALYNNNDMRL11E, Q44E,SGHNSFQNTAVASSPGSTDTQY_H09_scTCRV55Y, K92T, G93DM46P, R48QscTCR on yeast cellssoluble scTCR-FabEC50for HLA-KDforA*02HLA-A*02/PRAME-SimilarBinding/PRAME-Similar004peptidesmotif004peptidesMoleculebindingwithoutposi-bindingwithoutname[nM]bindingtionsnM]bindingR11 P3D3SD_n.d.n.d.n.d.n.d.n.d.stablizedscTCRR11P3D3SDA70.5325/263, 5, 711.714/14_A02_scTCR(6, 8)and TPP-70R11P3D3SDA70.2825/263, 5, 7n.d.n.d._A05_scTCR(6, 8)R11P3D3SDA70.3325/26n.d.n.d.n.d._A06_scTCRR11P3D3SDA70.2925/263, 5, 711.114/14_A09_scTCR(6, 8)and TPP-71R11P3D3SDA70.425/263, 5, 74.3814/14_A10_scTCR(6, 8)and TPP-72R11P3D3SDA70.2425/26n.d.n.d.n.d._B01_scTCRR11P3D3SDA70.3125/263, 5, 712.514/14_B03_scTCR(6, 8)and TPP-73R11P3D3SDA72.2625/263, 5, 7n.d.n.d._B04_scTCR(6, 8)R11P3D3SDA70.8125/263, 5, 76.4114/14_B06_scTCR(6, 8)and TPP-74R11P3D3SDA71.4225/26n.d.n.d.n.d._F11_scTCRR11P3D3SDA70.6525/26n.d.n.d.n.d._G11_scTCRR11P3D3SDA70.6725/26n.d.n.d.n.d._H08_scTCRR11P3D3SDA70.9125/26n.d.n.d.n.d._H09_scTCRn.d.: not determined Example 2: Production and Characterization of Soluble scTCR-Fab Molecules TCRs consisting of Valpha and Vbeta domains were designed, produced and tested in a single-chain (scTCR) format coupled to a Fab-fragment of a humanized UCHT1-antibody (Table 5 and Table 18). Vectors for the expression of recombinant proteins were designed as mono-cistronic, controlled by HCMV-derived promoter elements, pUC19-derivatives. Plasmid DNA was amplified inE. coliaccording to standard culture methods and subsequently purified using commercial-available kits (Macherey & Nagel). Purified plasmid DNA was used for transient transfection of CHO cells. Transfected CHO-cells were cultured for 10-11 days at 32° C. to 37° C. Conditioned cell supernatant was cleared by filtration (0.22 μm) utilizing Sartoclear Dynamics® Lab Filter Aid (Sartorius). Bispecific molecules were purified using an Äkta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size-exclusion chromatography in line. Affinity chromatography was performed on protein L columns (GE Lifesciences) following standard affinity chromatographic protocols. Size exclusion chromatography was performed directly after elution (pH 2.8) from the affinity column to obtain highly pure monomeric protein using Superdex 200 pg 16/600 columns (GE Lifesciences) following standard protocols. Protein concentrations were determined on a NanoDrop system (Thermo Scientific) using calculated extinction coefficients according to predicted protein sequences. Concentration was adjusted, if needed, by using Vivaspin devices (Sartorius). Finally, purified molecules were stored in phosphate-buffered saline at concentrations of about 1 mg/mL at temperatures of 2-8° C. Final product yield was calculated after completed purification and formulation. Quality of purified bispecific molecules was determined by HPLC-SEC on MabPac SEC-1 columns (5 μm, 4×300 mm) running in 50 mM sodium-phosphate pH 6.8 containing 300 mM NaCl within a Vanquish uHPLC-System. Stress stability testing was performed by incubation of the molecules formulated in PBS for up to two weeks at 40° C. Integrity, aggregate-content as well as monomer-recovery was analyzed by HPLC-SEC analyses as described above. TABLE 5Summary of productivity and stress stabilitydata obtained for scTCR-Fab molecules.FinalMonomerMonomerscTCR-productcontent aftercontent afterFabyieldproduction14 days at 40° C.variant(mg/L)(%)(%)TPP-7014.397.1287.82TPP-7110.085.8764.15TPP-7251.498.2148.41TPP-7359.498.3392.76TPP-7478.098.6995.62 scTCR-Fab molecules TPP-70-TPP-74 were analyzed for their binding affinity to HLA-A*02 monomers containing the PRAME-004 target peptide via bio-layer interferometry. Measurements were performed on an Octet RED384 system using settings recommended by the manufacturer. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz. Binding kinetics were measured at 30° C. and 1000 rpm shake speed using PBS, 0.05% Tween-20, 0.1% BSA as buffer. His-tagged HLA-A*02/PRAME-004 monomers were loaded onto HIS1K biosensors prior to analyzing serial dilutions of the scTCR-Fab molecules. Data evaluation was done using Octet Data Analysis HT Software. Strong binding affinities were determined for the scTCR-Fab molecules with KDvalues ranging from 4 nM to 12 nM (Table 4). Furthermore, the scTCR-Fab variants were screened for binding to 14 similar peptides (SEQ ID NOs: 187, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210 and 212). Screening with similar peptides was performed by bio-layer interferometry essentially as described above analyzing a high concentration of scTCR-Fab molecules of 1 μM to allow detection of weak binding signals. None of the maturated scTCR variants showed binding to any of the tested similar peptides (FIG.6). The scTCR from TPP-74 was used for generation of bispecific molecules with alternative formats, such as the TCER® format. Example 3: T Cell Engaging Receptor (TCER®) Format Example 3.1: Production and Characterization of Soluble scTCR in Bispecific TCER® Format For construction of TCER® molecules, DNA-sequences coding for VH and VL, derived from either hUCHT1(Var17), a newly humanized version of the anti-CD3 antibody UCHT1, BMA031(V36), a humanized antibody binding to TCR/CD3 complex, or the anti-CD3 antibody ID4 as well as sequences coding for and Valpha and Vbeta and respective linkers were obtained by gene synthesis. Resulting DNA-sequences were cloned in frame into expression vectors coding for hinge region, CH2 and CH3 domain derived from human IgG1 [Accession #: P01857]. The CH2 and CH3 domains were engineered to contain different mutations (including N297Q mutation) to ablate binding to Fc gamma receptors and complement and to incorporate a knob-into-hole structure into CH3 domains with an additional interchain disulfide bond stabilization. Production, purification and characterization of TCER® molecules (Table 6, Table 18) was performed as outlined in example 2. TABLE 6Summary of productivity and stress stability dataobtained for TCER ® molecules.Mono-Finalmer (%)Va, VbproductMono-after 14TCER ®(SEQyieldmerdays atvariantID NO)Recruiter(mg/L)(%)40° C.TPP-93129, 130UCHT1-V1718.894.49n/aTPP-79129, 130BMA031(V36)66.299.47n/aTPP-105129, 130ID454.298.5097.91 Functionality of TCER® molecules, with respect to killing of an HLA-A*02-positive tumor cell line presenting PRAME-004 target peptide on their cell surface (e.g. Hs695T), was assessed in LDH-release assays. In addition, an HLA-A*02-positive but PRAME-004-negative tumor cell line (e.g. T98G) was assessed to characterize unspecific or off-target activity of the TCER® variants. Tumor cell lines were co-incubated with PBMC from a healthy HLA-A*02-positive donor at a ratio of 1:10 in the presence of increasing TCER® concentrations. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measurement of released LDH. EC50values of dose-response curves were calculated utilizing non-linear 4-point curve fitting. Results representative for 3 TCER® molecules (Table 6, Table 18) are shown inFIG.7andFIG.8. The results demonstrate that all 3 TCER® molecules utilizing different recruiting antibody domains are functional and induce T cell-mediated cytotoxicity in a strictly PRAME-004 dependent manner. Example 3.2: Slot I TCER® molecules were constructed utilizing VHand VLdomains derived from hUCHT1(Var17) or BMA031(V36) as well as Valpha and Vbeta as described above (example 3.1). Production, purification and characterization of TCER® molecules (Table 7, Table 18) was performed as outlined in example 2. TABLE 7Summary of productivity and stress stability data obtainedfor TCER ® molecules of slot I.Final productMonomer (%)TCER ®yieldMonomerafter 14 daysvariantRecruiter(mg/L)(%)at 40° C.TPP-106UCHT1-V172.9296.9794.11TPP-108UCHT1-V174.3095.4494.10TPP-109BMA031(V36)34.0097.893.82TPP-110BMA031(V36)50.0097.1292.70TPP-111BMA031(V36)61.3098.0494.46TPP-112UCHT1-V172.4796.7592.71TPP-113UCHT1-V172.2497.7995.95TPP-114UCHT1-V172.6497.6895.37TPP-115UCHT1-V171.8097.8494.15TPP-116UCHT1-V173.2697.5494.13TPP-117UCHT1-V173.0297.2994.33TPP-118UGHT1-V172.1398.0995.11TPP-119UCHT1-V173.0497.5695.18TPP-120UCHT1-V172.5897.5794.52TPP-121UCHT1-V172.7497.9292.80TPP-122UCHT1-V173.2296.992.77TPP-123UCHT1-V172.4897.1692.62TPP-124UCHT1-V172.6896.3890.73TPP-125UCHT1-V172.4896.5692.33TPP-126UCHT1-V171.7696.7190.62TPP-127UCHT1-V172.6896.3790.95TPP-128UCHT1-V171.8197.2590.44TPP-129UCHT1-V171.4796.9489.55 TCER® Slot I variants TPP-106, TPP-108-TPP-129 were analyzed for their binding affinity to the target peptide-HLA complex (HLA-A*02/PRAME-004) via bio-layer interferometry. Measurements were performed on an Octet RED384 system as described above. Strong binding affinities were determined with KDvalues ranging from 3 nM to 10 nM (Table 8). These data show the additional affinity-improving effects of TCR mutations bA84D and aN114Y, while mutations bT115L/K, bL11E, bP46M, bQ48R, aN20K do not seem to affect binding affinity. Furthermore, binding affinities were determined for three selected similar peptides serving as potential off-target peptides in the context of HLA-A*02 and KDwindows were calculated compared to binding of the target peptide-HLA. Strongest TCER® binding to similar peptides was observed for GIMAP8-001 with KDwindows ranging from 26- to 168-fold. KDwindows of more than 25-fold already provide good therapeutic windows. TABLE 8KDvalues for binding of TCER ® Slot I variants to HLA-A*02/PRAME-004 and KDwindows for threeselected similar peptides serving as potential off-target peptides as measured via bio-layer interferometry.TCER ®PRAME-004KD(GIMAP8-001)/KD(SMARCD1-001)/KD(MYO1B-002)/variantRecruiterKD(M)KD(PRAME-004)KD(PRAME-004)KD(PRAME-004)TPP-108UCHT1-V171.03E−08168no bindingno bindingTPP-112UCHT1-V174.68E−0939380no bindingTPP-106UCHT1-V174.08E−0942272no bindingTPP-110BMA031(V36)1.33E−08Not analyzedNot analyzedNot analyzedTPP-111BMA031(V36)4.98E−09Not analyzedNot analyzedNot analyzedTPP-109BMA031(V36)4.45E−09Not analyzedNot analyzedNot analyzedTPP-113UCHT1-V175.24E−0933322no bindingTPP-114UCHT1-V175.68E−0937225no bindingTPP-115UCHT1-V175.06E−0938221no bindingTPP-116UCHT1-V175.18E−0931205no bindingTPP-117UCHT1-V173.42E−093441no bindingTPP-118UCHT1-V173.29E−094951no bindingTPP-119UCHT1-V174.57E−0930213no bindingTPP-120UCHT1-V175.49E−0928324no bindingTPP-121UCHT1-V175.41E−092698no bindingTPP-122UCHT1-V174.43E−0931174no bindingTPP-123UGHT1-V173.63E−092833no bindingTPP-124UCHT1-V173.43E−093032no bindingTPP-125UCHT1-V175.98E−0918248no bindingTPP-126UCHT1-V175.37E−0941221no bindingTPP-127UGHT1-V175.24E−0934195no bindingTPP-128UCHT1-V173.75E−094052no bindingTPP-129UCHT1-V173.05E−094047no binding Example 3.3: Slot II Further TCER® molecules were constructed utilizing VHand VLdomains derived from BMA031(V36) or ID4 as well as Valpha and Vbeta as described above (example 3.1). Production, purification and characterization of the respective TCER® molecules (Table 9, Table 18) was performed as outlined in example 2 whereby all ID4-based molecules were purified using MAbSelect SuRE columns (GE Lifesciences). TABLE 9Summary of productivity and stress stability data obtainedfor TCER ® molecules of slot II.Final productMonomer (%)yieldMonomerafter 14 daysProteinRecruiter(mg/L)(%)at 40° C.TPP-207BMA031(V36)31.898.9295.22TPP-208BMA031(V36)n/a96.9692.61TPP-209BMA031(V36)32.298.8794.79TPP-210BMA031(V36)19.698.1592.35TPP-211BMA031(V36)44.898.6096.35TPP-212BMA031(V36)34.497.6698.53TPP-213BMA031(V36)53.298.1292.45TPP-214BMA031(V36)45.298.2692.08TPP-215BMA031(V36)33.899.2195.15TPP-216BMA031(V36)4.596.5385.24TPP-217BMA031(V36)26.098.1693.87TPP-218BMA031(V36)19.898.2494.49TPP-219ID4>22.871.0736.49TPP-220ID421.898.3694.94TPP-221ID449.297.8096.51TPP-222ID445.498.2395.79TPP-227ID448.297.6093.67TPP-228ID412.197.5594.30TPP-229ID445.697.2296.99TPP-230ID447.497.2997.07 TCER® Slot II variants TPP-207-TPP-222 and TPP-227-TPP-230 were analyzed for their binding affinity to the target peptide-HLA complex (HLA-A*02/PRAME-004) via bio-layer interferometry. Measurements were performed on an Octet RED384 system as described above. Strong binding affinities were determined with KDvalues ranging from 1 nM to 7 nM (Table 10). Higher binding affinities were observed for the identical TCR variants (i.e. identical VAand VB) in combination with the ID4 recruiter when compared to combination with the BMA031(V36) recruiter (TPP-219-TPP-222 vs. TPP-211-TPP-214). As observed for the TCER® molecules from Slot I (example 3.2), the affinity-improving effects of TCR mutations bA84D and aN114Y could be confirmed for the TCER® variants generated in Slot II, while again no effects on affinity were found for the mutations bT115L/K, bP46M, bQ48R, aN20K. TCR binding motifs were assessed for selected TCER® molecules. To determine binding motifs, affinities were measured for the target peptide-HLA complex (HLA-A*02/PRAME-004) as well as for complexes with PRAME-004 variants carrying alanine-substitutions at peptide positions 1, 3, 4, 5, 6, 7 or 8. Affinity measurements were performed on an Octet RED384 or HTX system as described above. PRAME-004 positions were considered to be part of the TCR binding motif if an at least 2-fold reduction in binding affinity or signal (measured for the highest concentration analyzed) was detected for the alanine-substituted peptide variants. All TCER® variants showed broad binding motifs recognizing at least four peptide PRAME-004 positions (Table 10). TABLE 10KDvalues for binding of TCER ® Slot II variants to HLA-A*02/PRAME-004 andbinding motif determination according to KDwindows for Ala-substituted PRAME-004 peptidevariants as measured via bio-layer interferometry. For the A5 peptide, the KDwindowwas set to 100-fold since no to very low binding precluded affinity determination.PRAME-004Fold KDwindowTCER ®PRAME-004KD(M),Binding(Ala/PRAME-004)variantRecruiterKD(M)for motifmotifA1A3A4A5A6A7A8TPP-207BMA031(V36)4.33E−09TPP-208BMA031(V36)3.40E−09TPP-209BMA031(V36)3.29E−095.88E−09-x3-5678x1.116.01.2100.04.333.42.4TPP-210BMA031(V36)3.41E−09TPP-211BMA031(V36)4.53E−09TPP-212BMA031(V36)2.86E−09TPP-213BMA031(V36)4.55E−094.92E−09-x3-5678x1.113.41.3100.04.332.32.6TPP-214BMA031(V36)3.29E−092.76E−09-x3-5-78x1.33.01.2100.02.05.42.2TPP-215BMA031(V36)4.65E−09TPP-216BMA031(V36)3.38E−09TPP-217BMA031(V36)4.22E−09TPP-218BMA031(V36)2.51E−09TPP-219ID43.40E−09TPP-220ID41.85E−09TPP-221ID42.28E−092.61E−09-x3-5678x1.111.01.2100.04.124.23.2TPP-222ID41.47E−091.30E−09-x3-5678x1.42.91.2100.02.25.52.1TPP-227ID46.89E−09TPP-228ID43.46E−09TPP-229ID46.48E−09TPP-230ID42.93E−092.63E−09-x3-5678x1.313.01.9100.03.926.73.3 Example 3.4: Slot IIa Based on the data generated for the previous TCER® variants (example 3.3), new variants were generated carrying systematic substitutions of selected TCR amino acid positions for which a positive effect on protein properties or binding properties could be detected in previous experiments. Production, purification and characterization of the respective TCER® molecules (Table 11 and Table 18) was performed as outlined in example 3.3. Productivity and stress stability data are summarized in Table 11. TABLE 11Summary of productivity and stress stability data obtainedfor TCER ® molecules of slot IIa.Final productMonomer (%)TCER ®yieldMonomerafter 14 daysvariantRecruiter(mg/L)(%)at 40° C.TPP-235BMA031(V36)40.498.1296.16TPP-236BMA031(V36)48.598.3498.08TPP-237BMA031(V36)55.097.9898.21TPP-238BMA031(V36)37.898.2198.15TPP-239BMA031(V36)27.498.1997.22TPP-240BMA031(V36)44.298.6895.72TPP-241BMA031(V36)42.898.4598.02TPP-242BMA031(V36)23.698.8298.54TPP-243BMA031(V36)44.898.8198.10TPP-244BMA031(V36)22.698.2198.27TPP-245BMA031(V36)59.298.8198.32TPP-246BMA031(V36)4.792.2079.35TPP-247BMA031(V36)2.793.8082.82TPP-248BMA031(V36)2.492.0780.49TPP-249BMA031(V36)3.092.3881.45TPP-250BMA031(V36)3.893.1079.11TPP-252BMA031(V36)5.693.8680.14TPP-253BMA031(V36)3.794.8686.09TPP-254BMA031(V36)3.094.6681.85TPP-255BMA031(V36)12.092.4082.01TPP-256BMA031(V36)12.597.3492.67TPP-257BMA031(V36)8.295.2785.31TPP-258BMA031(V36)5.196.5084.32TPP-259BMA031(V36)2.497.3188.55TPP-260BMA031(V36)2.696.6986.45TPP-261BMA031(V36)7.997.3791.72TPP-262BMA031(V36)6.696.7191.53TPP-263BMA031(V36)3.693.7286.61TPP-264BMA031(V36)3.393.2582.35TPP-265BMA031(V36)9.991.8783.48TPP-266BMA031(V36)8.695.6790.72TPP-267BMA031(V36)6.094.5185.97TPP-266BMA031(V36)0.993.6487.21TPP-269BMA031(V36)0n/an/aTPP-270BMA031(V36)1.797.3091.83TPP-271BMA031(V36)2.295.1387.69TPP-272BMA031(V36)2.995.1687.63TPP-220ID45.997.3694.81TPP-273ID45.297.7792.43TPP-274ID42.697.1195.06TPP-275ID42.296.4794.08TPP-276ID41.897.0295.39TPP-277ID42.796.8494.89TPP-279ID45.498.0395.9 TCER® Slot IIa variants TPP-235-250, -252-268, -270-277, -279 were analyzed for their binding affinity to the target peptide-HLA complex (HLA-A*02/PRAME-004) via bio-layer interferometry. Measurements were performed on an Octet RED384 or HTX system as described above. Strong binding affinities were found with KDvalues ranging from 2 nM to 15 nM (Table 12). For position bA84, amino acid substitutions showed that bA84D is the most preferred substitution. At position aN114, alternative amino acid substitutions with affinities comparable to aN114Y were found, such as A, H, I and L. Alternatives to bT115K/L with comparable affinities were identified and included R, A, I and V. Introducing the mutation bA110S slightly reduced the affinities of the respective variants. Binding motifs were assessed for selected TCER® variants. To determine binding motifs, affinities were measured for the target peptide-HLA complex (HLA-A*02/PRAME-004) as well as for complexes with PRAME-004 variants carrying alanine substitutions at peptide positions 1, 3, 4, 5, 6, 7 or 8 as described above. PRAME-004 positions were considered to be part of the TCR binding motif if an at least 2-fold reduction in binding affinity or signal (measured for the highest concentration analyzed) was detected for the alanine-substituted peptide variants. All tested TCER® variants showed broad binding motifs recognizing at least three peptide positions (Table 12). In addition to binding motifs, the binding specificity of selected TCER® Slot II and IIa variants was further analyzed by bio-layer interferometry for binding to a set of 16 similar peptides potentially serving as off-target peptides. Measurements were performed on an Octet HTX system basically as described above. For the analysis, peptide-HLA complexes comprising the PRAME-004 target peptide, individual peptides out of a set of similar peptides or a control peptide were loaded onto HIS1K biosensors and binding of the TCER® variants was analyzed at a high TCER® concentration of 1 μM. The response signal at the end of a 5 min association phase was used to calculate the relative binding signal of the similar peptides in comparison to the PRAME-004 target peptide for selected TCER® variants (Table 13). Under these conditions, even binding events with very low affinity, which can be described as non-significant (e.g. binding with a KDthat is increased by a factor of ≥25, ≥30, ≥40, ≥50, ≥75, or ≥100, compared to the KDfor binding to the PRAME-004 peptide:MHC complex), will be detected. Among the 16 analyzed similar peptides, 11 peptides did not show any binding to any of the selected TCER® variants. Binding with lower signals compared to PRAME-004 was detected for five of the 16 similar peptides and four of these peptides were analyzed in more detail for TCER® Slot III variants such as measuring KDwindows compared to the PRAME-004 target peptide. TABLE 12KDvalues for binding of TCER ® Slot II variants to HLA-A*02/PRAME-004 andbinding motif determination according to KDwindows for Ala-substituted PRAME-004 peptidevariants as measured via bio-layer interferometry. For the A5 peptide, the KDwindowwas set to 100-fold since no to low binding precluded affinity determination.PRAME-004Fold KDwindowTCER ®PRAME-004KD(M)Binding(Ala/PRAME-004)variantRecruiterKD(M)for motifmotifA1A3A4A5A6A7A8TPP-246BMA031(V36)5.19E−09TPP-247BMA031(V36)8.94E−09TPP-248BMA031(V36)1.46E−08TPP-249BMA031(V36)6.69E−09TPP-250BMA031(V36)6.38E−09TPP-252BMA031(V36)6.30E−09TPP-220ID41.92E−09TPP-273ID42.78E−09TPP-274ID44.61E−09TPP-275ID47.21E−09TPP-276ID42.93E−09TPP-277ID43.71E−09TPP-279ID42.18E−09TPP-212BMA031(V36)3.38E−093.48E−09-x3-5-7-x1.12.51.0100.01.84.61.9TPP-235BMA031(V36)3.65E−09TPP-236BMA031(V36)6.01E−09TPP-237BMA031(V36)4.46E−09TPP-238BMA031(V36)4.74E−09TPP-239BMA031(V36)2.60E−093.44E−09-x3-5-7-x1.14.11.0100.02.07.81.9TPP-240BMA031(V36)3.48E−09TPP-241BMA031(V36)3.38E−093.84E−09-x3-567-x1.06.71.0100.02.113.82.0TPP-242BMA031(V36)5.23E−09TPP-243BMA031(V36)4.05E−09TPP-244BMA031(V36)4.90E−09TPP-245BMA031(V36)4.41E−09TPP-253BMA031(V36)3.43E−09TPP-254BMA031(V36)3.69E−09TPP-255BMA031(V36)6.13E−09TPP-256BMA031(V36)3.12E−094.08E−09-x3-5-7-x1.02.90.9100.01.86.41.8TPP-257BMA031(V36)3.52E−09TPP-258BMA031(V36)4.79E−09TPP-259BMA031(V36)4.80E−09TPP-260BMA031(V36)4.31E−09TPP-261BMA031(V36)3.45E−09TPP-262BMA031(V36)3.29E−094.18E−09-x3-5-7-x1.03.30.8100.01.86.51.7TPP-263BMA031(V36)3.87E−09TPP-264BMA031(V36)7.39E−09TPP-265BMA031(V36)6.72E−09TPP-266BMA031(V36)3.81E−094.57E−09-x3-5678x1.18.11.3100.02.710.32.3TPP-267BMA031(V36)4.78E−09TPP-268BMA031(V36)6.00E−09TPP-270BMA031(V36)5.74E−09TPP-271BMA031(V36)4.08E−09TPP-272BMA031(V36)4.11E−095.70E−09-x3-5678x1.26.91.1100.02.49.82.6 TABLE 13Relative binding signals for similar peptides (in percent of signals detected for PRAME-004 targetpeptide) of selected TCER ® Slot II and IIa variants as measured via bio-layer interferometry.TPP-214TPP-239TPP-241TPP-256TPP-266Recruiter:TPP-230Recruiter:Recruiter:Recruiter:Recruiter:BMA031Recruiter:BMA031BMA031BMA031BMA031Peptide(V36)ID4(V36)(V36)(V36)(V36)PRAME-004100100100100100100SMARCD1-001826065604919GIMAP8-0017046555638−3FARSA-0016935497217−5NOMAP-3-1408461124257−12VIM-009411028241011buffer control010010FAM114A2-002−11−7−5−4−3−6PDCD10-004−12−10−14−14−14−13NOMAP-5-0765−14−12−18−16−17−18IGHD-002−15−12−15−15−10−15TSN-001−16−12−17−18−17−18NOMAP-3-1587−16−14−16−17−18−18DDX5-001 (negative control)−17−13−16−17−17−16ALOX15B-003−18−15−15−19−14−17NOMAP-3-1768−18−16−19−19−21−19GPR56-002−18−14−19−19−17−19NOMAP-3-1265−18−13−16−20−15−20NOMAP-3-0972−22−17−22−23−20−23 Example 3.5: Slot III Further TCER® were constructed utilizing VHand VLdomains derived from BMA031(V36) or modified variants (A02 and D01) thereof, or ID4 as well as Valpha and Vbeta as described above (example 3.1). An additional TCER® molecule based on the UCHT1-V17 recruiting antibody (TPP-1109) was generated as reference. DNA constructs coding for the respective molecules were generated as outlined above. Resulting plasmids were used for transfection of CHO-S cells by electroporation (MaxCyte) for transient expression and production of TCER® variants (Table 14 and Table 18). Purification, formulation and initial characterization of molecules was performed as outlined above in example 3.3. TABLE 14Summary of productivity and stress stability data obtainedfor TCER ® molecules of slot III.Mono-Finalmer (%)Va, VbproductMono-after 14TCER ®(SEQyieldmerdays atvariantID NO)Recruiter(mg/L)(%)40° C.TPP-230132, 135ID473.898.8395.13TPP-871137, 135ID480.098.9297.33TPP-222132, 134ID470.698.8097.46TPP-872137, 134ID462.598.7797.87TPP-214132, 134BMA31(V36)36.297.9494.98TPP-876137, 134ID436.997.9492.28TPP-666132, 136BMA31(V36)A0249.797.5993.11TPP-879137, 134BMA31(V36)A0243.592.9890.42TPP-891137, 134BMA31(V36)D0140.098.1894.94TPP-669132, 136BMA31(V36)D0172.997.8394.66TPP-894132, 135BMA31(V36)D0140.297.4593.11TPP-1109(CDR6)UCHT1-V1713.698.1092.62 Potency of TCER® molecules with respect to killing of HLA-A*02-positive tumor cell lines presenting different levels of PRAME-004 target peptide on their cell surface, was assessed in LDH-release assays. In addition, an HLA-A*02-positive but PRAME-004-negative tumor cell line (e.g. T98G) was assessed to characterize unspecific or off-target activity of the TCER® variants. Tumor cell lines were co-incubated with PBMC effectors derived from healthy HLA-A*02-positive donors at a ratio of 1:10 and in the presence of increasing TCER® concentrations. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measurement of released LDH. EC50values of dose-response curves were calculated utilizing non-linear 4-point curve fitting. EC50values for two PRAME-004-positive tumor cell lines (Hs695T and U2OS) and a PRAME-004-negative tumor cell line (T98G) were determined in different experiments with different PBMC donors and are graphically summarized inFIG.9. TCER® Slot III variants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, -894 were analyzed for their binding affinity to the target peptide-HLA complex (HLA-A*02/PRAME-004) via bio-layer interferometry. Measurements were performed on an Octet HTX system at 30° C. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz on HIS1K biosensors in 16-channel mode using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following assay step sequence was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5)/neutralization (5 s, assay buffer; one regeneration cycle consists of four repeats of regeneration/neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg/ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, twofold serial dilution of TCER® ranging from 100 nM to 1.56 nM or 50 nM to 0.78 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was done using Octet Data Analysis HT Software. Reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded onto the biosensor (via a biosensor loaded with peptide-HLA measured in buffer). Data traces were aligned to baseline (average of the last 5 s), inter-step correction was done to the dissociation step, Savitzky-Golay filtering was applied and curves were fitted globally using a 1:1 binding model (with Rmax unlinked by sensor). Strong binding affinities were found with KDvalues ranging from 2 nM to 5 nM (Table 15). Furthermore, binding affinities were determined for four previously identified potential off-target peptides and KDwindows were calculated compared to binding of the target peptide-HLA. Measurements were performed on an Octet RED384 or HTX system at 30° C. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz on HIS1K biosensors in 16-channel mode using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following assay step sequence was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5)/neutralization (5 s, assay buffer; one regeneration cycle consists of four repeats of regeneration/neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg/ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, twofold serial dilution of TCER® ranging from 500 nM to 7.81 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was done using Octet Data Analysis HT Software. Reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded onto the biosensor (via a biosensor loaded with the respective peptide-HLA measured in buffer). Data traces were aligned to baseline (average of the last 5 s), inter-step correction was done to the dissociation step, Savitzky-Golay filtering was applied and curves were fitted globally using a 1:1 binding model (with Rmax unlinked by sensor). Overall, considerable weaker binding to the potential off-target peptides compared to target peptide was found for all variants showing windows of at least 60-fold to even no binding at all. NOMAP-3-1408 was not selected for KDdetermination, despite showing relative binding signals comparable to VIM-009 (Table 13). For VIM-009, the smallest measured KDwindows were >100-fold (Table 15). Thus, binding to VIM-009 is not relevant and affinity determination of NOMAP-3-1408 binding was not considered necessary based on its binding signals comparable to VIM-009. For one interaction, a KDwindow of 50-fold was calculated. However, for this interaction and also several others, the Rmax value calculated by the fitting algorithm was too low, so that the interaction is assumed to be weaker than calculated and thus the window larger. Respective interactions are indicated in Table 15. To further analyze specificity of the different variants, binding motifs were determined by measuring the affinities for the target peptide-HLA complex as well as for the alanine-substituted variants for positions 1, 3, 4, 5, 6, 7, 8. Measurements were performed on an Octet HTX system at 30° C. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz on HIS1K biosensors in 16- or 8-channel mode using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following assay step sequence was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5)/neutralization (5 s, assay buffer; one regeneration cycle consists of four repeats of regeneration/neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg/ml peptide-HLA), baseline (120 s, assay buffer), association (150 s, twofold serial dilution of TCER® ranging from 400 nM to 6.25 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was done using Octet Data Analysis HT Software. Reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded onto the biosensor (via a biosensor loaded with the respective peptide-HLA measured in buffer). Data traces were aligned to baseline (average of the last 5 s), inter-step correction was done to the dissociation step, Savitzky-Golay filtering was applied and curves were fitted globally using a 1:1 binding model (with Rmax unlinked by sensor). A position was considered part of the binding motif for an at least 2-fold reduction in affinity or binding signal (measured for the highest concentration analyzed). All tested TCER® variants showed broad binding motifs recognizing at least four and up to all analyzed peptide positions (Table 16). Positive effects on the binding motif were observed for bA84, aN114L and bA110S/bT115A, which is in accordance with previous data. For comparison, the binding motif of an alternative PRAME-004-targeting TCER® reference molecule (TPP-1109) was analyzed. This TCER® recognized positions 5-8 of the peptide and thus binding is limited to this peptide stretch, while positions recognized by TCER® Slot III variants are more evenly distributed throughout the whole peptide. TCER® Slot III variants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, -894 were additionally characterized for their ability to kill T2 cells loaded with varying levels of target peptide. After loading of the T2 cells with the respective concentrations of PRAME-004 for 2 h, peptide-loaded T2 cells were co-cultured with human PBMCs at an E:T ratio of 5:1 in the presence of increasing concentrations of TCER® variants for 48 h. Levels of LDH released into the supernatant were quantified using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). All TCER® variants showed potent killing of PRAME-004-loaded T2 cells with subpicomolar EC50values at a peptide loading concentration of 10 nM (FIG.10, Table 17). EC50values increased for decreasing PRAME-004 loading levels. However, even at a very low PRAME-004 loading concentration of 10 pM, killing was induced by all TCER® variants, except for TPP-214. TABLE 15KDvalues for binding to HLA-A*02/PRAME-004 and KDwindows of four selected off-targetpeptides measured via bio-layer interferometry for TCER ® Slot III variants.Va, VbTCER ®(SEQPRAME-004KDFARSA-001/KDGIMAP8-001/KDSMARCD1-001/KDVIM-009/variantRecruiterID NO)KD(M)KDPRAME-004KDPRAME-004KDPRAME-004KDPRAME-004TPP-230ID4132, 1353.05E−09—12011301—TPP-871ID4137, 1352.89E−09————TPP-222ID4132, 1341.56E−0911869741121TPP-872ID4137, 1341.60E−099510311912153TPP-214BMA031(V36)132, 1342.43E−092165966389TPP-876BMA031(V36)A02137, 1342.43E−0986802671601TPP-666BMA031(V36)A02132, 1363.37E−09507142121171TPP-879BMA031(V36)A02132, 1354.55E−09————TPP-891BMA031(V36)D01137, 1342.34E−0976852541461TPP-669BMA031(V36)D01132, 1363.65E−0983150184165TPP-894BMA031(V36)D01132, 1355.18E−09————1KDwindows are expected to be higher than the values given in the table (calculated Rmax values for these interactions are too low due to overall low binding signals). TABLE 16KDvalues for binding to HLA-A*02/PRAME-004 and KDwindows of Ala-substituted peptide variants for bindingmotif determination measured via bio-layer interferometry for TCER ® Slot III variants. For position5, a threshold of 100 is given for the KDwindow. Recognition of this position is at least 100-fold.Va, VbTCER ®(SEQPRAME-004BindingKDAla/targetvariantRecruiterID NO)KD, motif(M)motifA1A3A4A5A6A7A8TPP-230ID4132, 1353.03E−09-x3-5678x1.212.21.7100.03.925.53.0TPP-871ID4137, 1352.47E−091x345678x2.539.34.7100.016.589.98.3TPP-222ID4132, 1341.50E−09-x3-5-78x1.12.30.9100.01.84.31.8TPP-872ID4137, 1341.48E−09-x3-5678x1.17.61.1100.03.017.52.7TPP-214BMA031(V36)132, 1343.17E−09-x3-5-78x0.92.10.8100.01.64.61.7TPP-876BMA031(V36)A02137, 1342.87E−09-x3-567-x1.06.81.0100.02.313.92.0TPP-666BMA031(V36)A02132, 1363.84E−09-x3-5678x1.17.91.2100.02.69.72.1TPP-879BMA031V36)A02132, 1356.15E−09-x3-5678x1.112.51.6100.03.527.52.6TPP-891BMA031(V36)D01137, 1342.80E−09-x:3-5678x1.07.21.1100.02.614.72.3TPP-669BMA031(V36)D01132, 1363.28E−09-x3-5678x1.19.11.2100.02.511.02.4TPP-894BMA031(V36)D01132, 1356.04E−09-x3-5678x1.214.91.9100.03.826.42.8TPP-1109UCHT1-V17(CDR6)2.47E−09-x-5678x0.90.81.249.07.955.74.1 TABLE 17In vitro cytotoxicity of TCER ® Slot III variants on PRAME-004-loaded T2 cells. T2 cells were co-cultured with human PBMCs at an E:T ratio of 5:1 for 48 h. PRAME-004 loading concentrations are indicated.Ec50values and cytotoxicity levels in the plateau (Top) were calculated using non-linear 4-point curve fitting.Va, Vb10 nM1 nM100 pM10 pMTCER ®(SEQPRAME-004PRAME-004PRAME-004PRAME-004variantRecruiterID NO)EC50[pM]TopEC50[pM]TopEC50[pM]TopEC50[pM]TopTPP-230ID4132, 1350.091090.913923.2117914580TPP-871ID4137, 1350.131091.614376.519036176TPP-222ID4132, 134complete killing109complete killing782.811275890TPP-872ID4137, 134complete killing109complete killing1514.31844974TPP-876BMA031(V36)A02137, 1340.161112.011324.410053940TPP-666BMA031(V36)A02132, 1360.151132.411339.810018235TPP-879BMA031(V36)A02132, 1350.541066.210994.4117107039TPP-214BMA031(V36)132, 1340.221085.010992.8102no killing20TPP-891BMA031(V36)D01137, 1340.191202.211254.012561145TPP-669BMA031(V36)D01132, 1360.221243.210884.012624631TPP-894BMA031(V36)D01132, 1350.871089.9115226.0129108444TPP-214BMA031(V36)132, 1340.261215.4111105.499no killing231High variability within replicates do not allow for reliable EC50calculation. TABLE 18Bispecific moleculesα-β-chainchainSEQ IDSEQ IDIDNONOTPP-709394TPP-719395TPP-729396TPP-739397TPP-749398TPP-93100101TPP-79103102TPP-105105104TPP-106106107TPP-108106101TPP-109111110TPP-110111102TPP-111103110TPP-112100107TPP-113100119TPP-114100121TPP-115122121TPP-116106121TPP-117126121TPP-118128121TPP-119100131TPP-120100133TPP-121122133TPP-122106133TPP-123126133TPP-124128133TPP-125100143TPP-126122143TPP-127106143TPP-128126143TPP-129128143TPP-207103152TPP-208155152TPP-209157152TPP-210159152TPP-211103160TPP-212155162TPP-213157162TPP-214167160TPP-215169168TPP-216171168TPP-217173168TPP-218167168TPP-219177176TPP-220179176TPP-221181176TPP-222183176TPP-226159184TPP-227105186TPP-228189186TPP-229191186TPP-230193186TPP-235195160TPP-236197160TPP-237199160TPP-238201160TPP-239203160TPP-240205160TPP-241207160TPP-242209160TPP-243211160TPP-244213160TPP-245215160TPP-246217216TPP-247217218TPP-248217220TPP-249217222TPP-250217224TPP-252217228TPP-253217230TPP-254217232TPP-255217234TPP-256217236TPP-257217238TPP-258217240TPP-259217242TPP-260217244TPP-261217246TPP-262217248TPP-263217250TPP-264217252TPP-265217254TPP-266217256TPP-267217258TPP-268217260TPP-269217262TPP-270217264TPP-271217266TPP-272155268TPP-273189270TPP-274189272TPP-275189274TPP-276189276TPP-277189278TPP-279189282TPP-666285284TPP-669291284TPP-871295186TPP-872295296TPP-876299162TPP-879285300TPP-891303162TPP-892303284TPP-894291300TPP-1292151284TPP-1293156162TPP-1294158284TPP-1295158300TPP-1296303161TPP-1297303163TPP-1298291164TPP-1300291165TPP-1301166300TPP-1302291170TPP-1303291172TPP-1304291174TPP-1305166170TPP-1306166172TPP-1307166174TPP-1308291182TPP-1309291185TPP-1332175186TPP-1333178186TPP-1334180186 In table 18, except for TPP-70, TPP-71, TPP-72, TPP-73 and TPP74, the term “α-chain” refers to a polypeptide chain comprising a Vα, i.e. a variable domain derived from a TCR α-chain. The term “β-chain” refers to a polypeptide chain comprising a Vβ, i.e. a variable domain derived from a TCR β-chain. For TPP-70, TPP-71, TPP-72, TPP-73 and TPP74, the “α-chain” does not comprise any TCR derived variable domains, but the “β-chain” comprises two TCR-derived variable domains, one derived from a TCR α-chain and one derived from a TCR β-chain. Example 3.6: Safety Assessment for Selected TCER® Slot III Candidates The safety profile of the TCER® molecules TPP-230, TPP-666, TPP-871 and TPP-891 (Tables 14-18) was assessed in killing experiments with astrocytes and cardiomyocytes (derived from induced pluripotent stem cells) as well as aortic endothelial cells, mesenchymal stem cells and tracheal smooth muscle cells.FIG.11shows the results of co-cultures of above normal cell types (all expressing HLA-A*02) with PBMC effector cells from a healthy HLA-A*02+ donor at a ratio of 1:10 (target cells:effector cells) in presence of increasing TCER® concentrations. The cells were co-cultured in a 1:1 mixture of the respective normal tissue cell medium and T cell medium or in T cell medium alone (LDH-AM). After 48h of co-culture, supernatants were harvested and TCER®-induced normal tissue cell lysis was assessed by measuring LDH release with the LDH-Glo™ Kit (Promega). To determine a safety window, the TCER® molecules were co-incubated in an identical setup with the PRAME-004-positive tumor cell line Hs695T in the respective 1:1 mixture of normal tissue cell medium and T cell medium followed by the assessment of LDH release. As shown inFIG.11, no cytotoxicity against normal tissue cells was observed with TPP-230 and TPP-871 even at the highest TCER® concentration of 100 nM. For TPP-666 and TPP-891 some normal tissue cell lysis was observed at 100 nM TCER® concentration but no lysis was detected at 10 nM. When compared to Hs695T tumor cells that showed pronounced lysis at 100 pM for all tested TCER® molecules and for some molecules even lysis at 10 pM concentration, the normal tissue cell lysis at 100 nM concentration indicates a safety window of 1,000-fold (TPP-666 and TPP-891) or more (TPP-230 and TPP-871). Example 3.7: Slot IV Further TCER® were constructed utilizing VHand VLdomains derived from BMA031(V36) or modified variants (A02 and D01) thereof, or ID4 as well as Valpha and Vbeta as described above (example 3.1). DNA constructs coding for the respective molecules were generated as outlined above. Resulting plasmids were used for transfection of CHO-S cells by electroporation (MaxCyte) for transient expression and production of TCER® variants (Table 20 and Table 18). Purification, formulation and initial characterization of molecules was performed as outlined above in example 3.3. TABLE 20Summary of productivity and stress stability data obtainedfor TCER ® molecules of slot IV.α-chain,Finalβ-chainproductMonomer (%)TCER ®(SEQyieldMonomerafter 14 daysvariantID NO)Recruiter(mg/L)(%)at 40° C.TPP-1292151, 284BMA031(V36)A02_H90Y42.997.5393.46TPP-1294158, 284BMA031(V36)D01_H90Y39.897.7890.61TPP-1295158, 300BMA031(V36)D01_H90Y56.594.8991.49TPP-1296303, 161BMA031(V36)D0150.779.2175.17TPP-1297303, 163BMA031(V36)D0141.394.1286.77TPP-1298291, 164BMA031(V36)D0168.194.4189.7TPP-1300291, 165BMA031(V36)D0143.996.8187.5TPP-1301166, 300BMA031(V36)D0173.794.5790.89TPP-1302291, 170BMA031(V36)D0167.383.4879.58TPP-1303291, 172BMA031(V36)D0148.574.9571.03TPP-1304291, 174BMA031(V36)D0155.095.1388.87TPP-1305166, 170BMA031(V36)D0151.681.5577.75TPP-1306166, 172BMA031(V36)D0171.786.3781.18TPP-1307166, 174BMA031(V36)D0160.795.9388.16TPP-1308291, 182BMA031(V36)D0161.992.2887.98TPP-1309291, 185BMA031(V36)D0174.898.9891.11TPP-1332175, 186ID4 variant0n/an/aTPP-1333178, 186ID4 variant61.198.5295.51TPP-1334180, 186ID4 variant61.498.4295.94 In table 20, the term “α-chain” refers to a polypeptide chain comprising a Vα, i.e. a variable domain derived from a TCR α-chain. The term “β-chain” refers to a polypeptide chain comprising a Vβ, i.e. a variable domain derived from a TCR β-chain. Potency of TCER® molecules with respect to killing of HLA-A*02-positive tumor cell lines presenting different levels of PRAME-004 target peptide on their cell surface, was assessed in LDH-release assays. In addition, an HLA-A*02-positive but PRAME-004-negative tumor cell line (e.g. T98G) was assessed to characterize unspecific or off-target activity of the TCER® variants. Tumor cell lines were co-incubated with PBMC effectors derived from healthy HLA-A*02-positive donors at a ratio of 1:10 and in the presence of increasing TCER® concentrations. TCER®-induced cytotoxicity was quantified after 48 hours of co-culture by measurement of released LDH. EC50values of dose-response curves were calculated utilizing non-linear 4-point curve fitting. EC50values for a PRAME-004-positive tumor cell lines U2OS and a PRAME-004-negative tumor cell line (T98G) were determined in different experiments with different PBMC donors and are summarized in table 21. TABLE 24Summary of LDH-release assay data obtained for TCER ® molecules of slot IV.TCER ®EC50 [pM] forEC50 [pM] forEC50 [pM] forEC50 [pM] forvariantHBC-1005 vs U2OSHBC-1005 vs T98GHBC-848 vs U2OSHBC-848 vs T98GTPP-12926622,65954777,267TPP-12949969,150431>100,000TPP-1295150>100,000663>100,000TPP-12972,526>100,0004,096>100,000TPP-12984837,953249>100,000TPP-1300186>100,000811>100,000TPP-1301240>100,000979>100,000TPP-13047125>100,00013,686>100,000TPP-13078,056>100,000>100,000>100,000TPP-1333226>100,000719>100,000TPP-1334217>100,000829>100,000 TCER® Slot IV variants TPP-1292, -1294 to -1298, -1300 to -1309, -1333, -1334 were analyzed for their binding affinity to the target peptide-HLA complex (HLA-A*02/PRAME-004) via bio-layer interferometry. Measurements were performed on an Octet HTX system at 30° C. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz on HIS1K biosensors in 16-channel mode using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following assay step sequence was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5)/neutralization (5 s, assay buffer; one regeneration cycle consists of four repeats of regeneration/neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg/ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, twofold serial dilution of TCER® ranging from 100 nM to 1.56 nM or 50 nM to 0.78 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was done using Octet Data Analysis HT Software. Reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded onto the biosensor (via a biosensor loaded with peptide-HLA measured in buffer). Data traces were aligned to baseline (average of the last 5 s), inter-step correction was done to the dissociation step, Savitzky-Golay filtering was applied and curves were fitted globally using a 1:1 binding model (with Rmax unlinked by sensor). Strong binding affinities were found with KDvalues ranging from 2 nM to 15 nM (Table 22). Furthermore, binding affinities were determined for two previously identified potential off-target peptides and KDwindows were calculated compared to binding of the target peptide-HLA. Measurements were performed on an Octet RED384 or HTX system at 30° C. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz on HIS1K biosensors in 16-channel mode using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following assay step sequence was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5)/neutralization (5 s, assay buffer; one regeneration cycle consists of four repeats of regeneration/neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg/ml peptide-HLA), baseline (120 s, assay buffer), association (300 s, twofold serial dilution of TCER® ranging from 500 nM to 7.81 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was done using Octet Data Analysis HT Software. Reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded onto the biosensor (via a biosensor loaded with the respective peptide-HLA measured in buffer). Data traces were aligned to baseline (average of the last 5 s), inter-step correction was done to the dissociation step, Savitzky-Golay filtering was applied and curves were fitted globally using a 1:1 binding model (with Rmax unlinked by sensor). Overall, considerable weaker binding to the potential off-target peptides compared to target peptide was found for all variants showing windows of at least 10-fold to even no binding at all. Respective interactions are indicated in Table 22. To further analyze specificity of the variants TPP-1294, -1295, -1298, -1333, -1334, binding motifs were determined by measuring the affinities for the target peptide-HLA complex as well as for the alanine-substituted variants for positions 1, 3, 4, 5, 6, 7, 8. Measurements were performed on an Octet HTX system at 30° C. Assays were run at a sensor offset of 3 mm and an acquisition rate of 5 Hz on HIS1K biosensors in 16- or 8-channel mode using PBS, 0.05% Tween-20, 0.1% BSA as assay buffer. The following assay step sequence was repeated to measure all binding affinities: regeneration (5 s, 10 mM glycine pH 1.5)/neutralization (5 s, assay buffer; one regeneration cycle consists of four repeats of regeneration/neutralization), baseline (60 s, assay buffer), loading (120 s, 10 μg/ml peptide-HLA), baseline (120 s, assay buffer), association (150 s, twofold serial dilution of TCER® ranging from 400 nM to 6.25 nM, assay buffer as reference), dissociation (300 s, assay buffer). Data evaluation was done using Octet Data Analysis HT Software. Reference sensor subtraction was performed to subtract potential dissociation of peptide-HLA loaded onto the biosensor (via a biosensor loaded with the respective peptide-HLA measured in buffer). Data traces were aligned to baseline (average of the last 5 s), inter-step correction was done to the dissociation step, Savitzky-Golay filtering was applied and curves were fitted globally using a 1:1 binding model (with Rmax unlinked by sensor). A position was considered part of the binding motif for an at least 2-fold reduction in affinity or binding signal (measured for the highest concentration analyzed). All tested TCER® variants showed broad binding motifs recognizing at least five and up to all analyzed peptide positions (Table 23). TABLE 22KDvalues for binding to HLA-A*02/PRAME-004 and KDwindowsof two selected off-target peptides measured via bio-layer interferometry for TCER ® Slot IV variants.TCER ®KDIFIT-001/KDMCMB-002/variantPRAME-004 KD(M)KDPRAME-004KDPRAME-004TPP-12922.55E−0929.518.6TPP-12943.06E−0930.720.4TPP-12953.39E−0945.228.6TPP-12982.47E−0924.117.2TPP-13003.90E−0920.620.7TPP-13015.77E−0933.616.8TPP-13023.92E−0926.416.1TPP-13034.58E−0923.017.6TPP-13042.74E−08>100>100TPP-13055.19E−0923.813.7TPP-13065.20E−0947.223.3TPP-13073.97E−08>100>100TPP-13081.54E−0883.476.7TPP-13091.33E−0838.89.9TPP-13332.94E−0927.316.0TPP-13342.48E−0926.718.0 TABLE 23KDvalues for binding to HLA-A*02/PRAME-004 and KDwindows of Ala-substitutedpeptide variants for binding motif determination measured via bio-layer interferometryfor TCER ® Slot IV variants. For position 5, a threshold of 100 is givenfor the KDwindow. Recognition of this position is at least 100-fold.TCER ®PRAME-004BindingKDAla/targetvariantKD, motif(M)motifA1A3A4A5A6A7A8TPP-12944.35E−09-x3-5678x1.610.62.092.43.613.83.3TPP-12953.87E−091x345678x2.221.82.820.75.235.35.0TPP-12982.87E−09-x3-5678x1.410.31.6100.02.99.62.8TPP-13332.60E−09-x3-5678x1.412.82.0100.03.921.03.7TPP-13343.09E−09-x3-5678x1.19.21.6100.03.115.92.6 Example 3.8: Safety Assessment for Selected TCER® Slot IV Candidates The safety profile of the TCER® molecules TPP-1294, TPP-1295, TPP-1298, TPP-1333 and TPP-1334 (Tables 18 and 20-23) was assessed in killing experiments with astrocytes, GABAergic neurons and cardiomyocytes (derived from induced pluripotent stem cells; iHA, iHN and iHCM, respectively) as well as pulmonary fibroblasts (HPF), cardiac microvascular endothelial cells (HCMEC), dermal microvascular endothelial cells (HDMEC), aortic endothelial cells (HAoEC), coronary artery smooth muscle cells (HCASMC), renal cortical epithelial cells (HRCEpC) and tracheal smooth muscle cells (HTSMC). Furthermore, a bridging molecule TPP-891 was tested together with other molecules TPP-214 and TPP-669 from earlier slots.FIGS.12and13show the results of co-cultures of above normal cell types (all expressing HLA-A*02) with PBMC effector cells from a healthy HLA-A*02+ donor at a ratio of 1:10 (target cells:effector cells) in presence of increasing TCER® concentrations. The cells were co-cultured in a 1:1 mixture of the respective normal tissue cell medium and T cell medium or in T cell medium alone (LDH-AM). After 48h of co-culture, supernatants were harvested and TCER®-induced normal tissue cell lysis was assessed by measuring LDH release with the LDH-Glo™ Kit (Promega). To determine a safety window, the TCER® molecules were co-incubated in an identical setup with the PRAME-004-positive tumor cell line Hs695T in the respective 1:1 mixture of normal tissue cell medium and T cell medium followed by the assessment of LDH release. As shown inFIGS.12and13, no cytotoxicity against normal tissue cells was observed for any of the tested molecules until a concentration of 10 nM TCER®. At a concentration of 100 nM only the bridging and reference molecules TPP-891, TPP-669 and TPP-214 show a somehow increased cytotoxicity level above background. The only exception is TPP-1294 in iPSC-derived astrocytes with elevated cytotoxicity exclusively at 100 nM. When compared to Hs695T tumor cells that showed pronounced lysis at 100 pM for all tested TCER® molecules and for some molecules even lysis at 10 pM concentration, the normal tissue cell lysis at 100 nM concentration indicates a safety window of 1,000-fold (TPP-1294) or more (TPP-1295, TPP-1298, TPP-1334 and TPP-1335). Example 4: Detection of PRAME Peptide on Primary Tissues by Mass Spectrometry For the identification and relative quantitation of HLA ligands by mass spectrometry, HLA molecules from shock-frozen tissue samples were purified and HLA-associated peptides were isolated. The isolated peptides were separated, and sequences were identified by online nano-electrospray-ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments. Since the peptides were directly identified as ligands of HLA molecules of primary tumors, these results provide direct evidence for the natural processing and presentation of the identified peptides on the primary cancer tissue. The acquired LC-MS data are subsequently processed and quantified using a proprietary label-free quantitation data analysis pipeline, combining algorithms for sequence identification, spectral clustering, ion counting, retention time alignment, charge state deconvolution and normalization. Resulting target detection frequencies are depicted herein below in Table 19. TABLE 19Peptide detection frequency in tumor samples.The target detection frequency is indicatedas + (>0%), ++ (>10%), +++ (>30%), or ++++ (>50%).TargetdetectionEntityfrequencyacute myeloid leukemia (AML)+breast cancer (BRCA)++cholanglocellular carcinoma (CCC)+chronic lymphocytic leukemia (CLL)+colorectal carcinoma (CRC)+gallbladder cancer (GBC)++glioblastoma (GBM)+hepatocellular carcinoma (HCC)+head and neck squamous cell carcinoma (HNSCC)+melanoma (MEL)++++non-Hodgkin lymphoma (NHL)+non-small cell lung cancer adenocarcinoma+(NSCLCadeno)NSCLC samples that cannot unambiguously be assigned++to NSCLC adeno or NSCLCsquam (NSCLCother)squamous cell non-small cell lung cancer++(NSCLCsquam)ovarian cancer (OC)+++esophageal cancer (OSCAR)+renal cell carcinoma (RCC)++small cell lung cancer (SCLC)++urinary bladder carcinoma (UBC)+uterine and endometrial cancer (UEC)++++ ITEMS 1. An antigen binding protein specifically binding to a PRAME antigenic peptide that comprises or consists of the amino acid sequence SLLQHLIGL of SEQ ID NO: 50 and is in a complex with a major histocompatibility complex (MHC) protein, the antigen binding protein comprising(a) a first polypeptide comprising a variable domain VAcomprising complementarity determining regions (CDRs) CDRa1, CDRa2 and CDRa3, whereinthe CDRa1 comprises or consists of the amino acid sequence VKEFQD (SEQ ID NO: 16), or an amino acid sequence differing from SEQ ID NO: 16 by one, two or three amino acid mutations, preferably amino acid substitutions, and the CDRa3 comprises or consists of the amino acid sequence of ALYNNLDMR (SEQ ID NO: 33) or ALYNNYDMR (SEQ ID NO: 34), or an amino acid sequence differing from SEQ ID NO: 33 or SEQ ID NO: 34 by one, two or three, preferably one or two, amino acid mutations, preferably amino acid substitutions, and(b) a second polypeptide comprising a variable domain VBcomprising CDRb1, CDRb2 and CDRb3, whereinthe CDRb1 comprises or consists of the amino acid sequence SGHNS (SEQ ID NO: 10) or an amino acid sequence differing from SEQ ID NO: 10 by one or two amino acid mutations, preferably amino acid substitutions, andthe CDRb3 comprises or consists of the amino acid sequence ASSX1GX2X3DX4QY (SEQ ID NO: 327), wherein X1is P, A or T, X2is A or S, X3is T or I, and X4is K or A, or an amino acid sequence differing from SEQ ID NO: 327 by one, two or three amino acid mutations, preferably amino acid substitutions.2. The antigen binding protein of item 1, wherein(a) the CDRa2 comprises or consists of the amino acid sequence FGPYGKE (SEQ ID NO: 32), or an amino acid sequence differing from SEQ ID NO: 32 by one, two or three amino acid mutations, preferably amino acid substitutions, and/or(b) the CDRb2 comprises or consists of the amino acid sequence FQNTAV (SEQ ID NO: 36) or a CDRb2 amino acid sequence differing from SEQ ID NO: 36 by one, two, three, four, five or six amino acid mutations, preferably amino acid substitutions.3. The antigen binding protein of item 1 or 2, whereinPosition 27 of CDRa1 according to IMGT is V or is substituted by an amino acid selected from L, I, M, F, A, T, N, Q, H, E, D and S, particularly selected from T, N, S and I,Position 28 of CDRa1 according to IMGT is K or is substituted by an amino acid selected from R, Q, H, N, A, V, S, G, L, I and T, particularly selected from R, A and S,Position 38 of CDRa1 according to IMGT is D or is substituted by an amino acid selected from E, N, Q, H, K and R, particularly N,Position 64 of CDRa2 according to IMGT is K or is substituted by an amino acid selected from R, Q, H, N, T, V, A, L, I, M and F, particularly selected from R, T and V,Position 114 of CDRa3 according to IMGT is L or Y or is substituted by an amino acid selected from M, W, H, Q, A, I, K, R, V, D, E, F and N particularly selected from H, Q, A, I, K, R, V, D, E, F and N, more particularly selected from H, Q, A and I,Position 56 of CDRb2 according to IMGT is F or is substituted by an amino acid selected from Y, M, L, W, H, V, I and A, particularly selected from Y, M and L,Position 57 of CDRb2 according to IMGT is Q or is substituted by an amino acid selected from N, R, D, E, Q, H, K and K, particularly N, with the proviso that the amino acid at position 57 is not N when the amino acid at position 63 is T or S,Position 58 of CDRb2 according to IMGT is N or is substituted by an amino acid selected from Q, H, D, K, R, S and T, particularly S,Position 63 of CDRb2 according to IMGT is T or is substituted by an amino acid selected from S, V, A, D, Q and E, particularly selected from S and E, with the proviso that the amino acid at position 63 is not T or S when the amino acid at position 57 is N,Position 64 of CDRb2 according to IMGT is A or is substituted by an amino acid selected from V, L, I, S, G and T, particularly T,Position 65 of CDRb2 according to IMGT is V or is substituted by an amino acid selected from L, I, M, A, T, F and S, particularly selected from I, L and T,Position 108 of CDRb3 according to IMGT is P, A or T or is substituted by an amino acid selected from V, L, I, S, G, R, K, N and Q, particularly selected from R and S, with the proviso that the amino acid at position 108 is not N when the amino acid at position 110 is T or S,Position 110 of CDRb3 according to IMGT is A or S or is substituted by an amino acid selected from V, L, I, G, T and C, particularly T, with the proviso that the amino acid at position 110 is not T or S when the amino acid at position 108 is N,Position 113 of CDRb3 according to IMGT is T or I or is substituted by an amino acid selected from V, L, and G, andPosition 115 of CDRb3 according to IMGT is T, K or A or is substituted by an amino acid selected from G, L, I, V, R, Q, N, Y, H, E and F, particularly selected from L, I, V, R, Q, N, Y, H, E and F, more particularly from L, I, V and R.4. The antigen binding protein of any one of items 1 to 3, wherein said antigen binding protein specifically binds to the amino acid sequence of SEQ ID NO: 50 in a complex with a MHC protein, in particular a HLA protein, more particularly HLA-A, even more particularly HLA-A*02.5. The antigen binding protein of any one of items 1 to 4, wherein said antigen binding protein specifically binds to a functional epitope comprising or consisting of at least 3, 4 or 5 amino acid positions selected from the group consisting of positions 3, 5, 6, 7 and 8, in particular 3, 5 and 7, of SEQ ID NO: 50, preferably to a functional epitope consisting of amino acid positions 3, 5 and 7, or 3, 5, 6 and 7, or 3, 5, 7 and 8, or 3, 5, 6, 7 and 8 of SEQ ID NO: 50, but preferably not amino acid positions 1 and 4 of SEQ ID NO: 50.6. The antigen binding protein of any one of items 1 to 4, wherein said antigen binding protein specifically binds to a functional epitope comprising or consisting of at least 6 or 7 amino acid positions selected from the group consisting of positions 1, 3, 4, 5, 6, 7 and 8 of SEQ ID NO: 50.7. The antigen binding protein of any one of items 1 to 6, wherein said antigen binding protein binds to a complex of said PRAME antigenic peptide and a MHC protein, in particular a HLA protein, more particularly HLA-A, even more particularly HLA-A*02, with a KD of ≤100 nM, ≤50 nM, ≤10 nM, preferably ≤5 nM.8. The antigen binding protein of any one of items 1 to 7, wherein said antigen binding protein does not significantly bind to at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20 or all similar peptides selected from the group consisting of TMED9-001 (SEQ ID NO: 51), CAT-001 (SEQ ID NO: 52), DDX60L-001 (SEQ ID NO: 53), LRRC70-001 (SEQ ID NO: 54), PTPLB-001 (SEQ ID NO: 55), HDAC5-001 (SEQ ID NO: 56), VPS13B-002 (SEQ ID NO: 57), ZNF318-001 (SEQ ID NO: 58), CCDC51-001 (SEQ ID NO: 59), IFT17-003 (SEQ ID NO: 60), DIAPH1-004 (SEQ ID NO: 62), FADS2-001 (SEQ ID NO: 63), FRYL-003 (SEQ ID NO: 64), GIMAP8-001 (SEQ ID NO: 65), HSF1-001 (SEQ ID NO: 66), KNT-001 (SEQ ID NO: 67), MAU-001 (SEQ ID NO: 68), MCM4-001 (SEQ ID NO: 69), MPPE1-001 (SEQ ID NO: 71), MYO1B-002 (SEQ ID NO: 72), PRR12-001 (SEQ ID NO: 73), PTRF-003 (SEQ ID NO: 74), RASGRP1-001 (SEQ ID NO: 75), SMARCD1-001 (SEQ ID NO: 76), TGM2-001 (SEQ ID NO: 77), VAV1-001 (SEQ ID NO: 78), VIM-009 (SEQ ID NO: 317), FARSA-001 (SEQ ID NO: 306), ALOX15B-003 (SEQ ID NO: 304), FAM114A2-002 (SEQ ID NO: 305), GPR56-002 (SEQ ID NO: 307), IGHD-002 (SEQ ID NO: 308), NOMAP-3-0972 (SEQ ID NO: 309), NOMAP-3-1265 (SEQ ID NO: 310), NOMAP-3-1408 (SEQ ID NO: 311), NOMAP-3-1587 (SEQ ID NO: 312), NOMAP-3-1768 (SEQ ID NO: 313), NOMAP-5-0765 (SEQ ID NO: 314), PDCD10-004 (SEQ ID NO: 315), TSN-001 (SEQ ID NO: 316), ARMC9-002 (SEQ ID NO: 187), CLI-001 (SEQ ID NO: 188), COPG1-001 (SEQ ID NO: 190), COPS7A-001 (SEQ ID NO: 192), EIF-009 (SEQ ID NO: 194), EXT2-006 (SEQ ID NO: 196), LMNA-001 (SEQ ID NO: 198), PKM-005 (SEQ ID NO: 200), PSMB3-002 (SEQ ID NO: 202), RPL-007 (SEQ ID NO: 204), SPATS2L-003 (SEQ ID NO: 206), SYNE1-002 (SEQ ID NO: 208), TGM2-002 (SEQ ID NO: 210) and TPR-004 (SEQ ID NO: 212), in a complex with a MHC protein, preferably said antigen binding protein does not significantly bind to IFT17-003 (SEQ ID NO: 60) in a complex with a MHC protein.9. The antigen binding protein of any one of items 1 to 8, wherein the antigen binding protein is multispecific, e.g. tetra-, tri- or bispecific, preferably bispecific, in particular said antigen binding protein is a bispecific TCR, a bispecific antibody or a bispecific TCR-antibody molecule.10. The antigen binding protein of any one of items 1 to 9, wherein the first and the second polypeptide are comprised in a single polypeptide chain or two polypeptide chains, preferably wherein VAis comprised in a first polypeptide chain and VBis comprised in a second polypeptide chain.11. The antigen binding protein of any one of items 1 to 10, wherein VAfurther comprises one or more framework regions, preferably all framework regions, selected from the group consisting of FR1-a, FR2-a, FR3-a and FR4-a, whereinFR1-a comprises or consists of the amino acid sequence of SEQ ID NO: 345 or SEQ ID NO: 346, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 345, preferably comprising K or N, more preferably K, at position 20 and/or L or M more preferably L, at position 2;FR2-a comprises or consists of the amino acid sequence of SEQ ID NO: 347 or SEQ ID NO: 348, or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 347, preferably comprising L, I or M, more preferably L or I, at position 39, A or D, more preferably A, at position 47, K or W, preferably K, at position 44, F or A, preferably F, at position 52 and/or Y or V, preferably Y, at position 55;FR3-a comprises or consists of the amino acid sequence of SEQ ID NO: 349 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 349, preferably comprising T or K, more preferably T, at position 92 and/or D or G, preferably D, at position 93;FR4-a comprises or consists of the amino acid sequence of SEQ ID NO: 350 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 350; andVBfurther comprises one or more framework regions, preferably all framework regions, selected from the group consisting of FR1-b, FR2-b, FR3-b and FR4-b, whereinFR1-b comprises or consists of the amino acid sequence of SEQ ID NO: 351 or SEQ ID NO: 352 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 351, preferably comprising H or N, more preferably H, at position 10, E, L or K, preferably E, at position 11 and/or R or H, at position 22;FR2-b comprises or consists of the amino acid sequence of SEQ ID NO: 353 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 353, preferably comprising R or K, more preferably R, at position 43, E or Q, preferably E, at position 44, M or P, more preferably P, at position 46, and/or R or Q, more preferably Q, at position 48;FR3-b comprises or consists of the amino acid sequence of SEQ ID NO: 354 or SEQ ID NO: 355 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 354, preferably comprising D, A, E, R, K Q, N or S, more preferred D, A, E, Q, N or S, more preferably D or A, even more preferably D, at position 84; andFR4-b comprises or consists of the amino acid sequence of SEQ ID NO: 356 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 356.12. The antigen binding protein of any one of items 1 to 11, whereinVAcomprises or consists of the amino acid sequence of SEQ ID NO: 132 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 132, preferably comprising a CDRa1 of SEQ ID NO: 16, a CDRa2 of SEQ ID NO: 32 and a CDRa3 of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 9, and further K or N, preferably K, at position 20, L, M, or I, preferably L or I, at position 39, K or W, preferably K, at position 44, F or A, preferably F, at position 52, Y or V, preferably Y, at position 55, T or K, preferably T, at position 92 and/or D or G, preferably D, at position 93; andVBcomprises or consists of the amino acid sequence of SEQ ID NO: 134 or an amino acid sequence at least 85%, 90% or 95% identical to SEQ ID NO: 134, preferably comprising a CDRb1 of SEQ ID NO: 10, a CDRb2 of SEQ ID NO: 36, and a CDRb3 of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 47, SEQ ID NO: 281, SEQ ID NO: 292, SEQ ID NO: 294, SEQ ID NO: 297, SEQ ID NO: 298, SEQ ID NO: 301 or SEQ ID NO: 283, and further E, L or K, preferably E, at position 11, R or H at position 22, E or Q, preferably E, at position 44, P or M, preferably P, at position 46, Q or R, preferably Q, at position 48 and/or D, A, E, Q, N, or S, preferably D or A, at position 84.13. The antigen binding protein of any one of items 1 to 12, whereinVAcomprises or consists of the amino acid sequence of SEQ ID NO: 132, SEQ ID NO: 129, SEQ ID NO: 137 or SEQ ID NO: 142, andVBcomprises or consists of the amino acid sequence of SEQ ID NO: 134, SEQ ID NO: 130, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: 148.14. The antigen binding protein of any of items 1 to 13, further comprising one or more of the following:(i) one or more further antigen binding sites;(ii) a transmembrane region, optionally including a cytoplasmic signalling region;(iii) a diagnostic agent;(iv) a therapeutic agent; and(v) PK modifying moiety.15. The antigen binding protein of any one of items 1 to 14, further comprising an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).16. The antigen binding protein of item 15, wherein VLand VHbind to an antigen selected from the group consisting of CD2, CD3, in particular CD3γ, CD3δ, and/or CD3ε, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD90, CD94, CD95, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI, TCRα/β and TCRγ/β, HLA-DR and 4-1 BB, or combinations thereof and/or bind to an effector cell, in particular a T cell or natural killer cell (NK cell).17. The antigen binding protein of item 15 or 16, wherein the antigen binding protein comprises a first and a second polypeptide chain,whereinthe first polypeptide chain is represented by a formula [Ia]: V1-L1-D1-L2-V2-L3-D2[Ia],and the second polypeptide chain is represented by a formula [IIa] V3-L4-D3-L5-V4-L6-D4[IIa],whereinV1, V2, V3, and V4are variable domains, wherein one of V1to V4is VA, one is VB, one is VLand one is VH;D1, D2, D3, and D4are dimerization domains and may be present or absent, wherein D1and D3, and D2and D4, specifically bind to each other and at least one pair of D1and D3, or D2and D4is present; andL1, L2, L3, L4, L5, and L6are linkers, wherein L1and L4are present and L2, L3, L5, and L6may be present or absent.18. The antigen binding protein of any of items 15 to 17, wherein the antigen binding protein comprises a first and a second polypeptide chain,whereinthe first polypeptide chain is represented by a formula [Ib]: V1-L1-V2-L3-D2[Ib],and the second polypeptide chain is represented by a formula [IIb]: V3-L4-V4-L6-D4[IIb],whereinV1, V2, V3, V4, are variable domains, preferably wherein one of V1and V2is VA, one of V3and V4is VBand of the remaining two variable domains one is VLand the other is VH;D2and D4are dimerization domains, preferably Fc-domains; andL1, L3, L4and L6are linkers, wherein L3, and L6may be present or absent.19. The antigen binding protein of item 17 or 18, wherein(1) V1is VH, V2is VB, V3is VA, and V4is VL;(2) V1is VB, V2is VH, V3is VL, and V4is VA;(3) V1is VB, V2is VL, V3is VH, and V4is VA;(4) V1is VL, V2is VB, V3is VA, and V4is VH;(5) V1is VH, V2is VB, V3is VL, and V4is VA;(6) V1is VB, V2is VH, V3is VA, and V4is VL;(7) V1is VL, V2is VB, V3is VH, and V4is VA;(8) V1is VB, V2is VL, V3is VA, and V4is VH;(9) V1is VH, V2is VL, V3is VA, and V4is VB;(10) V1is VL, V2is VH, V3is VA, and V4is VB;(11) V1is VH, V2is VL, V3is VB, and V4is VA; or(12) V1is VL, V2is VH, V3is VB, and V4is VA.20. The antigen binding protein of any one of items 1 to 19, comprisinga first polypeptide chain selected from SEQ ID NO: 100, 103, 105, 106, 111, 122, 126, 128, 151, 155, 156, 157, 158, 159, 166, 167, 169, 171, 173, 175, 177, 178, 179, 180, 181, 183, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 285, 291, 295, 299 and 303, anda second polypeptide chain selected from SEQ ID NO: 101, 102, 104, 107, 110, 119, 121, 131, 133, 143, 152, 160, 161, 162, 163, 164, 165, 168, 170, 172, 174, 176, 182, 184, 185, 186, 216, 218, 220, 222, 224, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 282, 284, 296 or 300.21. The antigen binding protein of any one of items 1 to 13, wherein VAis comprised in a TCR α- or γ-chain; and/or VBis comprised in a TCR β- or δ-chain.22. An isolated nucleic acid comprising a sequence encoding the antigen binding protein of any one of items 1 to 21.23. A vector comprising the nucleic acid of item 22.24. A host cell comprising the antigen binding protein of any one of items 1 to 21, or the nucleic acid of item 22, or the vector of item 23.25. The host cell of item 24, wherein the host cell isa lymphocyte, preferably a T lymphocyte or T lymphocyte progenitor cell, for example a CD4 or CD8 positive T cell ora cell for recombinant expression, such as a Chinese Hamster Ovary (CHO) cell or a yeast cell.26. A pharmaceutical composition comprising the antigen binding protein of any one of items 1 to 21, the nucleic acid of item 22, the vector of item 23, or the host cell of item 24 or 25 and a pharmaceutically acceptable carrier.27. A method of producing the antigen binding protein according to any one of items 1 to 21, comprisinga. providing a host cell,b. providing a genetic construct comprising a coding sequence encoding the antigen binding protein of any of items 1 to 21,c. introducing said genetic construct into said host cell, andd. expressing said genetic construct by said host cell.28. The method of item 27, further comprising the isolation and purification of the antigen binding protein from the host cell and, optionally, reconstitution of the antigen binding protein in a T cell.29. The antigen binding protein of any one of items 1 to 21, the nucleic acid of item 22, the vector of item 23, the host cell of item 24 or 25, or the pharmaceutical composition of item 26 for use in medicine.29. The antigen binding protein of any one of items 1 to 21, the nucleic acid of item 22 or the vector of item 23, the host cell of item 24 or 25 or the pharmaceutical composition of item 26 for use in the diagnosis, prevention, and/or treatment of a proliferative disease, such as cancer, wherein said cancer is selected from the group of cancers consisting of acute myeloid leukemia, breast cancer, cholangiocellular carcinoma, gallbladder cancer, glioblastoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, amelanotic melanoma, non-Hodgkin lymphoma, non-small cell lung cancer adenocarcinoma, non-small cell lung cancer, squamous cell non-small cell lung cancer, ovarian cancer, esophageal cancer, renal cell carcinoma, small cell lung cancer, urinary bladder carcinoma, uterine and endometrial cancer, osteosarcoma, chronic lymphocytic leukemia, colorectal carcinoma, and synovial sarcoma. | 83,648 |
11859010 | DETAILED DESCRIPTION OF THE INVENTION Provided herein are antibodies, activatable antibodies (AAs), bispecific antibodies, and bispecific activatable antibodies (BAAs). In some embodiments, provided herein are humanized antibodies that specifically bind to the epsilon chain of CD3 (CD3ε; referred to herein interchangeably as CD3). In some embodiments, provided herein are IgG1 antibodies that specifically bind to Epidermal Growth Factor Receptor (EGFR), wherein the antibodies comprise point mutations in the Fc region, such that the antibody has reduced effector function. In some embodiments provided herein are AAs, for example AAs that specifically bind to EGFR or CD3. These AAs are optimized for affinity, effector function, masking, and cleavability. In some embodiments, provided herein are BAAs, for example BAAs that bind to a target antigen (e.g. tumor antigen, such as a target presented in Table 9) and a second antigen (e.g. immune effector antigen on an immune effector cell). In some embodiments, the immune effector cell is a leukocyte cell. In some embodiments, the immune effector cell is a T cell. In some embodiments, the immune effector cell is a natural killer (NK) cell. In some embodiments, the immune effector cell is a macrophage. In some embodiments, the immune effector cell is a mononuclear cell, such as a myeloid mononuclear cell. In some embodiments, the BAAs are immune effector cell-engaging BAAs. In some embodiments, the BAAs are leukocyte cell-engaging BAAs. In some embodiments, the BAAs are T cell engaging bispecific (TCB) AAs, also referred to herein as TCBAAs. In some embodiments, the BAAs are NK cell-engaging BAAs. In some embodiments, the BAAs are macrophage cell-engaging BAAs. In some embodiments, the BAAs are mononuclear cell-engaging BAAs, such as myeloid mononuclear cell-engaging BAAs. In some embodiments, the bispecific antibodies bind EGFR and CD3. These BAAs are optimized for affinity, effector function, masking, and cleavability. Also provided herein are methods of making and methods of use of these antibodies, AAs, and BAAs. AAs, including general production thereof and identification of masking moieties (MMs) and cleavable moieties (CMs) is described in International Publication Numbers WO 2009/025846 by Daugherty et al., published 26 Feb. 2009, and WO 2010/081173 by Stagliano et al., published 15 Jul. 2010, both of which are incorporated by reference in their entirety. BAAs, including general production thereof and identification of masking moieties (MMs) and cleavable moieties (CMs) is described in International Publication Numbers WO2015/013671 by Lowman et al., published 29 Jan. 2015 and WO2016/014974 by Irving et al., published 28 Jan. 2016, both of which are incorporated by reference in their entirety. Also incorporated by reference are International Publication WO2016/014974 by Irving et al., published 28 Jan. 2016, and International Publication WO2016/118629 by Moore et al., published 28 Jul. 2016 which provide AAs, general production, MMs, and CMs. As used herein, unless specified otherwise, the term “antibody” includes an antibody or antigen-binding fragment thereof that specifically binds its target and is a monoclonal antibody, domain antibody, single chain, Fab fragment, a F(ab′)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a scFv antibody. In some embodiments, such an antibody or immunologically active fragment thereof that binds its target is a mouse, chimeric, humanized or fully human monoclonal antibody. 1. CD3 Antibodies Provided herein is an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε, referred to herein throughout as CD3). Exemplary amino acid sequences of CD3-binding antibodies of the disclosure (variable domains) are provided in Table 1. (Predicted CDR sequences are underlined). As provided below, L3 is a linker, linking the light and heavy chain variable domains, in the exemplary CD3-binding antibodies. TABLE 1Anti-CD3 variant v12Light Chain Variable Domain LV12QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:1)Heavy Chain Variable Domain HV12, wherein L3 is SEQ ID NO: 98EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 2)LV12-L3-HV12QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 143)Anti-CD3 variant v16Light Chain Variable Domain LV12Sequence provided aboveHeavy Chain Variable Domain HV20EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 3)LV12-L3-HV20, wherein L3 is SEQ ID NO: 98QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 144)Anti-CD3 variant v19Light Chain Variable Domain LV19QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL (SEQ ID NO:4)Heavy Chain Variable Domain HV20Sequence provided aboveLV19-L3-HV20, wherein L3 is SEQ ID NO: 98QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 145)Anti-CD3 variant v26Light Chain Variable Domain LV19Sequence provided aboveHeavy Chain Variable Domain HV12Sequence provided aboveLV19-L3-HV12, wherein L3 is SEQ ID NO: 98QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 150) Exemplary scFv linkers (referred to herein as “L3” linking a VH and VL) are provided in Table 1-1. TABLE 1-1SEQ ID NO:Linker Amino Acid Sequence98GGGGSGGGGSGGGGS Exemplary CDR sequences of CD3-binding antibodies are provided in Table 2. TABLE 2NameCD3 Ab CDR SequencesSEQ ID NO:SP34L1RSSTGAVTTSNYANSEQ ID NO: 149SP34L2GTNKRAPSEQ ID NO: 5SP34L3ALWYSNLWVSEQ ID NO: 6SP34H1TYAMNSEQ ID NO: 7SP34H2RIRSKYNNYATYYADSVKDSEQ ID NO: 8SP34H3HGNFGNSYVSWFAYSEQ ID NO: 9 As provided herein, the CD3 antibody comprises at least one of the CDR sequences provided in Table 2. In some embodiments, the CD3 antibody comprises heavy chain variable domain as set forth in SEQ ID NO: 2. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 3. In some embodiments, the CD3 antibody comprises a light chain variable domain as set forth in SEQ ID NO: 1. In some embodiments, the CD3 antibody comprises a light chain variable domain as set forth in SEQ ID NO: 4. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 1. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 1. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 4. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 4. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or comprises a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4. In some embodiments, the CD3 antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 and comprises a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4. In some embodiments, the CD3 antibody is a scFv antibody. In some embodiments, the variable domains comprise the following structure from N terminus to C terminus: LV-HV. In some embodiments, the variable domains comprise the following structure from N terminus to C terminus: HV-LV. In some embodiments, the CD3 antibody is a scFv antibody comprising a heavy chain variable region (VH) linked to a light chain variable region (VL), wherein the VH is linked to the VL by a linker comprising amino acid sequence SEQ ID NO: 98. Exemplary sequences with such a linker are provided in Table 1. In exemplary embodiments, provided herein is an antibody that specifically binds to CD3 (AB), wherein the antibody is an IgG1 antibody or a scFv linked to an Fc domain, wherein the antibody comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises L234F, L235E, and P331S amino acid substitutions. In some embodiments, the antibody comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the antibody comprises L234F, L235E, P331S, and N297Q amino acid substitutions. In some embodiments, the heavy chain variable domain of the antibody comprises any one of SEQ ID NO: 2 or SEQ ID NO: 3 or wherein the light chain variable domain of the AB comprises any one of SEQ ID NO: 1 or SEQ ID NO: 4. 2. Activatable CD3 Antibodies In some embodiments, any one of the CD3 antibodies provided herein is in an activatable antibody (AA) format. As generally provided herein, the AAs of the invention comprise MM-CM constructs, also referred to herein as a prodomain. Accordingly, as used herein, the term “prodomain” refers to a polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM). In some embodiments, the MM and the CM are separated by a linker, referred to herein as L1. In some embodiments, the prodomain comprises a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, links the CM of the prodomain to the AB. In some embodiments, the prodomain comprises a linker between MM and CM and a linker after CM. In some embodiments, the MM and the CM are not separated by a linker. In certain embodiments a prodomain comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In exemplary embodiments, a prodomain comprises an EGFR MM and a CM cleavable by a matriptase or MMP; or a CD3ε MM and a CM cleavable by a matriptase or MMP. In some embodiments, a prodomain comprises an EGFR MM and a CM that is cleavable by a matriptase and an MMP. In some embodiments, a prodomain comprises a CD3ε MM and a CM that is cleavable by a matriptase and an MMP. Provided herein are activatable antibodies (AAs) comprising a prodomain. Also provided herein are nucleotides encoding a prodomain of the invention. Accordingly, provided herein is a CD3 AA comprising: (a) an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), wherein the antibody comprises a heavy chain domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or comprises a light chain domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3ε when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. As described above, (b) and (c) together are part of the prodomain. In some embodiments, the AB of the CD3 AA is any one of the CD3 antibodies described in the preceding section. In some embodiments, the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 2. In some embodiments, the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 3. In some embodiments, the AB of the CD3 AA comprises a light chain variable domain as set forth in SEQ ID NO: 1. In some embodiments, the AB of the CD3 AA comprises a light chain variable domain as set forth in SEQ ID NO: 4. In some embodiments, the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain domain as set forth in SEQ ID NO: 1. In some embodiments, the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain domain as set forth in SEQ ID NO: 1. In some embodiments, the AB of the CD3 AA comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain domain as set forth in SEQ ID NO: 4. In some embodiments, the AB is a scFv comprising a heavy chain variable region (VH) linked to a light chain variable region (VL), wherein the VH is linked to the VL by a linker L3 comprising amino acid sequence SEQ ID NO: 98. Exemplary sequences with such a linker are provided in Table 1. In some embodiments, the MM of the CD3 AA comprises any one of the sequences set forth in Table 3. Exemplary CD3 masking moieties (MMs) of the invention are provided in Table 3. In some embodiments, the MM of the CD3 AA comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the MM of the CD3 AA is the sequence set forth in SEQ ID NO: 10. In some embodiments, the MM of the CD3 AA is the sequence set forth in SEQ ID NO: 11. TABLE 3MMNameAA sequenceSEQ ID NO:CD3 MMJF15865MMYCGGNEVLCGPRVSEQ ID NO: 10CD3 MMJF15003GYRWGCEWNCGGITTSEQ ID NO: 11CD3 MMh20GGGYLWGCEWNCGGITTSEQ ID NO: 12 In some embodiments, the CM of the CD3 AA comprises any one of the sequences set forth in Table 4. Exemplary cleavable moieties (CMs) of the invention are provided in Table 4. In some embodiments, the CM of an AA of the disclosure comprises any one of the sequences set forth in Table 4-1. TABLE 4CM NameAA sequenceSEQ ID NO:0001LSGRSDNHSEQ ID NO: 130011LSGRSDDHSEQ ID NO: 142001ISSGLLSGRSDNHSEQ ID NO: 152008ISSGLLSGRSDQHSEQ ID NO: 162006ISSGLLSGRSDDHSEQ ID NO: 17 TABLE 4-1CM NameAA sequenceSEQ ID NO:0001LSGRSDNHSEQ ID NO: 180002LSGRSGNHSEQ ID NO: 190003TSTSGRSANPRGSEQ ID NO: 201001ISSGLLSSSEQ ID NO: 211002QNQALRMASEQ ID NO: 221003VHMPLGFLGPSEQ ID NO: 231004AVGLLAPPSEQ ID NO: 240011LSGRSDDHSEQ ID NO: 250021LSGRSDIHSEQ ID NO: 260031LSGRSDQHSEQ ID NO: 270041LSGRSDTHSEQ ID NO: 280051LSGRSDYHSEQ ID NO: 290061LSGRSDNPSEQ ID NO: 300071LSGRSANPSEQ ID NO: 310081LSGRSANISEQ ID NO: 320091LSGRSDNISEQ ID NO: 332001ISSGLLSGRSDNHSEQ ID NO: 342002ISSGLLSGRSGNHSEQ ID NO: 352003ISSGLLSGRSANPRGSEQ ID NO: 362005AVGLLAPPSGRSANPRGSEQ ID NO: 372006ISSGLLSGRSDDHSEQ ID NO: 382007ISSGLLSGRSDIHSEQ ID NO: 392008ISSGLLSGRSDQHSEQ ID NO: 402009ISSGLLSGRSDTHSEQ ID NO: 412010ISSGLLSGRSDYHSEQ ID NO: 422011ISSGLLSGRSDNPSEQ ID NO: 432012ISSGLLSGRSANPSEQ ID NO: 442013ISSGLLSGRSANISEQ ID NO: 452014ISSGLLSGRSDNISEQ ID NO: 463001AVGLLAPPGGLSGRSDNHSEQ ID NO: 473006AVGLLAPPGGLSGRSDDHSEQ ID NO: 483007AVGLLAPPGGLSGRSDIHSEQ ID NO: 493008AVGLLAPPGGLSGRSDQHSEQ ID NO: 503009AVGLLAPPGGLSGRSDTHSEQ ID NO: 513010AVGLLAPPGGLSGRSDYHSEQ ID NO: 523011AVGLLAPPGGLSGRSDNPSEQ ID NO: 533012AVGLLAPPGGLSGRSANPSEQ ID NO: 543013AVGLLAPPGGLSGRSANISEQ ID NO: 553014AVGLLAPPGGLSGRSDNISEQ ID NO: 56 3. Antibodies with Fc Mutations Provided herein are IgG1 antibodies that that have Fc mutations or antibody fragments containing antigen-binding domains (e.g. scFv, Fab, F(ab′)2) linked to a Fc domain, wherein the Fc exhibits reduced effector function (referred to herein as Fc variants). Any of the BAAs, AAs, and antibodies described herein may comprise any Fc variants disclosed herein. The antibodies that comprise these Fc mutations result in reduced effector function, while maintaining target binding affinity. Accordingly, provided herein are antibodies that bind to a target of interest, wherein the antibody is an IgG1 antibody or an antibody fragment linked to an Fc, wherein the Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, there is an additional mutation in N297. In some embodiments, the amino acid substitution is N297Q or N297A. In some embodiments, the Fc is selected from the Fc sequences presented in Table 4-2. In some embodiments, the Fc is selected from SEQ ID NO: 154, SEQ ID NO:156, SEQ ID NO: SEQ ID NO:158, and SEQ ID NO:160, wherein the X is selected from the group consisting of any naturally occurring amino acid (e.g. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine) or any non-naturally occurring amino acid (e.g. trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine). TABLE 4-2NameSEQ ID NO:AA SequenceFc-N297XASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP154)KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYXSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-N297QASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP155)KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-L234XASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP156)KSCDKTHTCPPCPAPEXLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-L234FQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG(SEQ ID NO:VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL157)TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-L235XQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG(SEQ ID NO:VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL158)TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELXGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-L235EASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP159)KSCDKTHTCPPCPAPELEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-P331XASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP160)KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAXIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-P331SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP161)KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFc-Fcmt3ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV(SEQ ID NO:HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP162)KSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5Fcmt4ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHCHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP(SEQ ID NO:KSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH163EDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibodies, AAs, bispecific antibodies, and BAAs comprising these Fc mutations are provided herein. In some embodiments, such Fc variant-containing AAs and BAAs can bind an immune effector cell. In some embodiments, they can bind a target selectively located on an immune effector cell. In some embodiments, they can bind CD3. In some embodiments, they can bind any target listed in Table 9. In some embodiments, they can bind EGFR. Accordingly, in some embodiments, provided herein is an activatable antibody (AA) comprising:a) an antibody (AB) that specifically binds a target, wherein the antibody is an IgG1 antibody, and wherein the Fc region of the antibody comprises an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function;b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the target when the AA is in an uncleaved state; andc) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, N297 and P331, as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function. In some embodiments, the target is selected from the group consisting of the targets presented in Table 9. In some embodiments, provided herein is a bispecific activatable antibody (BAA) comprising:a) an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises:i. two heavy chains (AB1 HCs) and two light chains (AB1 LCs); andii. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, whereinthe MM1 inhibits the binding of the AB1 to its target; andthe CM1 is a polypeptide that functions as a substrate for a first protease,b) two scFvs (AB2) that each specifically binds to a second target wherein each AB2 comprises:i. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; andii. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of a MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease, and wherein the AB1 comprises an Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. In some embodiments provided herein, the BAAs provided herein comprise:a) A bispecific activatable antibody (BAA) comprising:i) an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises two heavy chains (AB1 HCs) and two light chains (AB1 LCs); and wherein the AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, whereinthe MM1 inhibits the binding of the AB1 to its target; andthe CM1 is a polypeptide that functions as a substrate for a first protease,ii) two scFvs (each an AB2) that each specifically binds to a second target wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; and wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease, and wherein the AB1 comprises an Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. 4. EGFR Antibodies Provided herein are antibodies or antigen binding fragments thereof (AB) that specifically bind to EGFR. Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5. Provided herein are EGFR antibodies, bispecific antibodies with one arm targeting EGFR, AAs capable of binding EGFR upon activation, and BAAs capable of binding EGFR upon activation. In some embodiments, the EGFR antibody comprises the CDRs of Table 5. In some embodiments, e.g. in a BAA format, provided herein are IgG1 antibodies that specifically bind to the Epidermal Growth Factor Receptor (EGFR) and impart reduced effector function. The antibodies comprise Fc mutations that result in reduced effector function, while maintaining EGFR binding affinity. Accordingly, provided herein are antibodies that bind to EGFR, wherein the antibody is an IgG1 antibody, wherein the antibody comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the antibody has reduced effector function. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the antibody comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the antibody comprises L234F, L235E, and P331S amino acid substitutions. In some embodiments, the antibody comprises an Fc region comprising an amino acid substitution at N297 along with an amino acid substitution in at least one of amino acid positions L234, L235, and/or P331. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the Fc region comprises an N297A mutation. In some embodiments, the antibody comprises L234F, L235E, P331S and N297Q substitutions. In some embodiments, the antibody comprises L234F, L235E, P331S and N297A substitutions. Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5, set forth in Kabat. TABLE 5NameSequenceSEQ ID NO:C225L1RASQSIGTNIHSEQ ID NO: 57C225L2YASESISSEQ ID NO: 58C225L3QQNNNWPTTSEQ ID NO: 59C225H1NYGVHSEQ ID NO: 60C225H2VIWSGGNTDYNTPFTSSEQ ID NO: 61C225H3ALTYYDYEFAYSEQ ID NO: 62 Exemplary amino acid sequences of EGFR-binding antibodies are provided in Table 6. (VL and VH denote the variable light and variable heavy chains, respectively; LC and HC denote the light and heavy chains, respectively). In some embodiments, the EGFR antibodies comprise any one of the sequences provided in Table 6. In some embodiments, the heavy chain of the EGFR antibody comprises any one of the sequences set forth in SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, as set forth in Table 6. In some embodiments, the heavy chain EGFR antibody comprises any one of the sequences set forth in SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, and SEQ ID NO: 73, wherein in X is selected from any naturally occurring amino acid (e.g. alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine, valine) or any non-naturally occurring amino acid (e.g., trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine). The notation Fcmt3 comprises a triple point mutation, wherein the Fc region of the heavy chain of the EGFR antibody comprises the following three point mutations: L234F, L235E, and P331S. Accordingly, in some embodiments, the EGFR antibody comprises a heavy chain with an amino acid sequence set forth in SEQ ID NO: C225v5Fcmt3 HC. In some embodiments, the Fc region of the heavy chain of the EGFR antibody comprises a fourth point mutation, N297Q. The notation Fcmt4 comprises the Fcmt3 triple point mutation and the fourth point mutation, N297Q. Accordingly, in such embodiments, the EGFR antibody comprises a heavy chain with an amino acid sequence set forth in SEQ ID NO: 76. TABLE 6NameSEQ ID NO:AA SequenceC225v5-VLQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYAS(SEQ ID NO:ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKL63)ELKC225v5-VHQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG(SEQ ID NO:VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL64)TYYDYEFAYWGQGTLVTVS(S/A)C225v5 LCQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYAS(SEQ ID NO:ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKL65)ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECC225v5 HCQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG(SEQ ID NO:VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL66)TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5N297XQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGHCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC67)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYXSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5N297QQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGHCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC68)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGL234X HCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC69)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEXLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGL234F HCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC70)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGL235X HCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC71)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELXGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGL235E HCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC72)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGP331X HCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC73)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAXIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGP331S HCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC74)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5Fcmt3QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGHCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC75)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC225v5Fcmt4QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGHCVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL(SEQ ID NO:TYYDYEFAYWGQGTLVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAALGC76)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSynFcmt4QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLHCADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCA(SEQ ID NO:RSMITNWYFDVWGAGTTVTVS(S/A)ASTKGPSVFPLAPSSKSTSGGTAAL77)GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5. Activatable EGFR Antibodies In some embodiments, any one of the EGFR antibodies provided herein are in an AA format (EGFR AAs). As described above for the CD3 AAs, the EGFR AAs also comprise a prodomain. Accordingly provided herein are AAs comprising antibodies or antigen binding fragments thereof (AB) that specifically bind to EGFR. Exemplary CDR sequences of EGFR-binding antibodies are provided in Table 5. In some embodiments, the AA comprises: (a) any antibody or an antigen binding fragment thereof (AB) that specifically binds to Epidermal Growth Factor Receptor (EGFR); (b) and a prodomain, wherein the prodomain comprises (i) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the AA is in an uncleaved state, and wherein the MM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7; and (ii) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. Exemplary EGFR masking moieties (MMs) of the invention are provided in Table 7 and Table 8. TABLE 7MMNameAA sequenceSEQ ID NO:EGFR MMCF41LSCEGWAMNREQCRASEQ ID NO: 78EGFR MMCF08PPLECNTKSMCSKHDSEQ ID NO: 79EGFR MMCF13DRDCRGRRARCQQEGSEQ ID NO: 80EGFR MMCF19FTCEGWAMNREQCRTSEQ ID NO: 81EGFR MMCF22GRCPPSRDIRFCTYMSEQ ID NO: 82EGFR MMCF46FSCEGWAMNRSQCRTSEQ ID NO: 83EGFR MMCF48FTCEGWAMNRDQCRTSEQ ID NO: 84 TABLE 8MMNameAA sequenceSEQ ID NO:EGFR MM3954CISPRGCPDGPYVMYSEQ ID NO: 85EGFR MM3954aCISPRGCPDGPYVMSEQ ID NO: 86EGFR MM3960CISPRGCSEQ ID NO: 87 In some embodiments, the MM of the EGFR AA comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM of the EGFR AA comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CM of the EGFR AA comprises an amino acid sequence selected from the group consisting of sequences presented in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, provided herein is an activatable antibody (AA) comprising: (a) an antibody that specifically binds to Epidermal Growth Factor Receptor (EGFR), wherein the antibody is an IgG1 antibody, and wherein the Fc region of the antibody comprises an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. The EGFR IgG1 antibodies can be any of the IgG1 antibodies described in the immediately preceding section. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7. In an exemplary embodiment, provided herein is an activatable antibody (AA) comprising: (a) an antibody (AB) that specifically binds to Epidermal Growth Factor Receptor (EGFR), wherein the AB is an IgG1 antibody, and wherein the Fc region of the AB comprises an amino acid substitution in at least one of amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function; (b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the AA is in an uncleaved state; and (c) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. In some embodiments, the amino acid substitution is any one or more of L234F, L235E, and P331S. In some embodiments, the AB comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. In some embodiments, the AB comprises amino acid substitutions at amino acid positions L234, L235, and P331. In some embodiments, the AB comprises L234F, L235E, and P331S amino acid substitutions. In some embodiments, the AB comprises an Fc region comprising an amino acid substitution at N297. In some embodiments, the Fc region comprises an N297Q mutation. In some embodiments, the AB comprises L234F, L235E, P331S, and N297Q amino acid substitutions. In some embodiments, the MM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7 or Table 8. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the MM comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the CM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 4. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 14. In some embodiments, the CM comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the AA is part of a BAA. 6. Bispecific Activatable Antibodies (BAAs) Provided herein are BAAs (bispecific AAs, BAAs), wherein said BAA, when activated, specifically binds to two targets (e.g. binds two different targets, or binds two different epitopes on the same target) and can comprise and can comprise one of the exemplary structures provided inFIGS.17-19. In some embodiments, the first target is selected from the group consisting of the targets presented in Table 9 and the second target is selected from the group consisting of the target presented in Table 9. As generally provided herein, and as described above in the section describing AAs, the BAAs of the invention comprise MM-CM constructs, also referred to herein as a prodomain. Accordingly, as used herein, the term “prodomain” refers to a polypeptide comprising a masking moiety (MM) and a cleavable moiety (CM). In some embodiments, the MM and the CM are separated by a linker, referred to herein as L1. In some embodiments, the prodomain comprises a linker at the carboxyl terminus of the CM; this linker, referred to herein as L2, links the CM of the prodomain to the AB. In some embodiments, the prodomain comprises a linker between MM and CM and a linker after CM. In some embodiments, the MM and the CM are not separated by a linker. In certain embodiments a prodomain comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM)-L1-(CM), (MM)-(CM)-L2, (MM)-L1-(CM)-L2, or (MM)-(CM). In exemplary embodiments, a prodomain comprises an EGFR MM and a CM cleavable by a matriptase or MMP; or a CD3ε MM and a CM cleavable by a matriptase or MMP. In some embodiments, a prodomain comprises an EGFR MM and a CM that is cleavable by a matriptase and an MMP. In some embodiments, a prodomain comprises a CD3ε MM and a CM that is cleavable by a matriptase and an MMP. Provided herein are bispecific activatable antibodies (BAAs) comprising a prodomain. Also provided herein are nucleotides encoding a prodomain of the invention. In some embodiments, provided herein is a BAA, wherein said BAA, when activated, specifically binds to two targets (e.g. two different targets; or two different epitopes on the same target), and wherein said BAA, when not activated, comprises the following structure:a) an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises:i. two heavy chains (AB1 HCs) and two light chains (AB1 LCs); andii. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, wherein1. the MM1 inhibits the binding of the AB1 to its target; and2. the CM1 is a polypeptide that functions as a substrate for a first protease,b) two scFvs (AB2) that each specifically bind to a second target wherein each AB2 comprises:i. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; andii. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of a MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease,and wherein the BAA has at least one of the following characteristics:i. MM2 comprises amino acid sequence SEQ ID NO: 12;ii. MM1 comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7;iii. AB2 comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4; andiv. AB1 comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331 or L234, L235 and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. In some embodiments, the BAAs provided herein comprise:1. A bispecific activatable antibody (BAA), wherein said BAA, when activated, specifically binds to two targets and comprises the following structure:a. an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises two heavy chains (AB1 HCs) and two light chains (AB1 LCs); and wherein the AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, whereinthe MM1 inhibits the binding of the AB1 to its target; andthe CM1 is a polypeptide that functions as a substrate for a first protease,b. two scFvs (each an AB2) that each specifically binds to a second target wherein each AB2 comprises a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; and wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease,and wherein the BAA has at least one of the following characteristics:i. MM2 comprises amino acid sequence SEQ ID NO: 12;ii. MM1 comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7;iii. AB2 comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4; andiv. AB1 comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. In some embodiments, the BAAs provided herein comprise:a) an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises:a. two heavy chains (AB1 HCs) and two light chains (AB1 LCs); andb. a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, wherein1. the MM1 inhibits the binding of the AB1 to its target; and2. the CM1 is a polypeptide that functions as a substrate for a first protease,b) two scFvs (AB2) that each specifically binds to a second target wherein each AB2 comprises:a. a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; andb. a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of a MM2-CM2 construct is linked to the amino terminus of each AB2 wherein1. the MM2 inhibits the binding of the AB2 to its target; and2. the CM2 is a polypeptide that functions as a substrate for a second protease,and wherein the AB1 comprises an Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. In some embodiments, the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, N297 and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. In some embodiments, the BAAs provided herein comprise:(1) A bispecific activatable antibody (BAA) comprising:(a) an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises two heavy chains (AB1 HCs) and two light chains (AB1 LCs); and wherein the AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, whereinthe MM1 inhibits the binding of the AB1 to its target; andthe CM1 is a polypeptide that functions as a substrate for a first protease,(b) two scFvs (each an AB2) that each specifically binds to a second target wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; and wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease,and wherein the AB1 comprises an Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. As provided above, the BAAs of the invention comprise two scFvs (AB2) that each specifically binds to a second target. The VL and VH of the scFvs can be in any order, either VL-VH or VH-VL. In some embodiments, the Fc region of the AB1 comprises amino acid substitutions in at least amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. In some embodiments, the first target is selected from the group consisting of the targets presented in Table 9 and the second target is selected from the group consisting of the targets presented in Table 9. In some embodiments, AB1 binds a target antigen, e.g. a tumor antigen, and the AB2 binds an immune effector target. In some embodiments, AB2 binds a target antigen, e.g. a tumor antigen, and the AB1 binds an immune effector target. In some embodiments, the AB1 binds EGFR and the AB2 binds CD3. In some embodiments, the MM1 comprises SEQ ID NO: 78. In some embodiments, the MM2 comprises the amino acid sequence SEQ ID NO: 12. In some embodiments, the bispecific AA is CI106, as provided in Table 11, in Example 1. In some embodiments, the BAA is CI107, as provided in Table 11, in Example 1. In some embodiments, the BAA is CI011, as provided in Table 11, in Example 1. In some embodiments, the BAA is CI020, as provided in Table 11, in Example 1. In some embodiments, the BAA is CI040, as provided in Table 11, in Example 1. In some embodiments, the BAA is CI079, as provided in Table 11, in Example 1. In some embodiments, the BAA is CI090, as provided in Table 11, in Example 1. In an exemplary embodiment, AB1 comprises the amino acid sequence of C225v5Fcmt3 HC or C225v5Fcmt4 HC. In some embodiments, the first and second proteases are the same protease. In some embodiments, the first and second proteases are different proteases. In some embodiments, CM1 and CM2 comprise the same amino acid sequence. In some embodiments, CM1 and CM2 comprise different amino acid sequences. In some embodiments, CM1 and CM2 comprise different amino acid sequences that are cleavable by the same protease or proteases. In some embodiments, CM1 and CM2 are cleavable by more than one protease. In some embodiments, CM1 and/or CM2 is cleavable by a serine protease. In some embodiments, CM1 and/or CM2 is cleavable by a matrix metalloproteinase (MMP). In some embodiments, CM1 and/or CM2 is cleavable by a serine protease and an MMP. Exemplary BAAs of the disclosure include, for example, those shown in the Examples provided herein, and variants thereof. In some non-limiting embodiments, at least one of the AB in the BAA is specific for CD3 and at least one other AB is a binding partner for any target listed in Table 9. In an exemplary embodiment, AB2 of the BAA is specific for CD3 and AB1 is a binding partner for any target listed in Table 9. TABLE 9Exemplary Targets1-92-LFA-3CD52DL44HVEMLAG-3STEAP1Alpha-4CD56DLK1HyaluronidaseLIF-RSTEAP2integrinAlpha-VCD64DLL4ICOSLewis XTAG-72integrinalpha4beta1CD70DPP-4IFNalphaLIGHTTAPA1integrinalpha4beta7CD71DSG1IFNbetaLRP4TGFbetaintegrinAGR2CD74EGFRIFNgammaLRRC26TIGITAnti-Lewis-YEGFRviiiIgEMCSPTIM-3Apelin JCD80Endothelin BIgE ReceptorMesothelinTLR2receptorreceptor(FceRI)(ETBR)APRILCD81ENPP3IGFMRP4TLR4B7-H4CD86EpCAMIGF1RMUC1TLR6BAFFCD95EPHA2IL1BMucin-16TLR7(MUC16,CA-125)BTLACD117EPHB2IL1RNa/K ATPaseTLR8C5CD125ERBB3IL2NeutrophilTLR9complementelastaseC-242CD132F protein ofIL11NGFTMEM31(IL-2RG)RSVCA9CD133FAPIL12NicastrinTNFalphaCA19-9CD137FGF-2IL12p40NotchTNFR(Lewis a)ReceptorsCarbonicCD138FGF8IL-12R,Notch 1TNFRS12Aanhydrase 9IL-12Rbeta1CD2CD166FGFR1IL13Notch 2TRAIL-R1CD3CD172AFGFR2IL13RNotch 3TRAIL-R2CD6CD248FGFR3IL15Notch 4TransferrinCD9CDH6FGFR4IL17NOVTransferrinreceptorCD11aCEACAM5FolateIL18OSM-RTRK-A(CEA)receptorCD19CEACAM6GAL3ST1IL21OX-40TRK-B(NCA-90)CD20CLAUDIN-3G-CSFIL23PAR2uPARCD22CLAUDIN-4G-CSFRIL23RPDGF-AAVAP1CD24cMetGD2IL27/IL27RPDGF-BBVCAM-1(wsx1)CD25CollagenGITRIL29PDGFRalphaVEGFCD27CriptoGLUT1IL-31RPDGFRbetaVEGF-ACD28CSFRGLUT4IL31/IL31RPD-1VEGF-BCD30CSFR-1GM-CSFIL2RPD-L1VEGF-CCD33CTLA-4GM-CSFRIL4PD-L2VEGF-DCD38CTGFGP IIb/IIIaIL4RPhosphatidyl-VEGFR1receptorsserineCD40CXCL10Gp130IL6, IL6RP1GFVEGFR2CD40LCXCL13GPIIB/IIIAInsulinPSCAVEGFR3ReceptorCD41CXCR1GPNMBJaggedPSMAVISTALigandsCD44CXCR2GRP78Jagged 1RAAG12WISP-1CD44v6HER2/neuJagged 2RAGEWISP-2CD47CXCR4HGFSLC44A4WISP-3CD51CYR61hGHSphingosine 1Phosphate In some embodiments, the unmasked EGFR-CD3 bispecific antibody exhibits EGFR-dependent tumor cell killing, while the doubly-masked EGFR-CD3 BAA reduces target-dependent cytotoxicity by more than 100,000-fold. In established tumor models where tumor-resident proteases are expected to be active, it is shown that BAAs potently induce tumor regressions. In non-human primates, the maximum tolerated dose (MTD) of the EGFR-CD3 BAA is more than 60-fold higher than the MTD of the unmasked bispecific antibody, and the tolerated exposure (AUC) is more than 10,000-fold higher. Despite the 60-fold dose differential at the MTDs, transient serum cytokine and AST/ALT increases observed in non-human primates treated with the BAA are still lower than those induced by the bispecific antibody. By localizing activity to the tumor microenvironment, BAAs have the potential to expand clinical opportunities for T cell-engaging bispecific therapies that are limited by on target toxicities, especially in solid tumors. Moreover, an EGFR-CD3 BAA has the potential to address EGFR-expressing tumors that are poorly responsive to existing EGFR-directed therapies. 7. Cleavable Moieties (CM) Both the monospecific AAs and the BAAs of the disclosure comprise at least one CM, when masked and not activated. In some embodiments, the cleavable moiety (CM) of the AA or BAA includes an amino acid sequence that can serve as a substrate for at least one protease, usually an extracellular protease. In the case of a BAA, the CM may be selected based on a protease that is co-localized in tissue with the desired target of at least one AB of the BAA or AA. A CM can serve as a substrate for multiple proteases, e.g. a substrate for a serine protease and a second different protease, e.g. an MMP. In some embodiments, a CM can serve as a substrate for more than one serine protease, e.g., a matriptase and uPA. In some embodiments, a CM can serve as a substrate for more than one MMP, e.g., an MMP9 and an MMP14. A variety of different conditions are known in which a target of interest is co-localized with a protease, where the substrate of the protease is known in the art. In the example of cancer, the target tissue can be a cancerous tissue, particularly cancerous tissue of a solid tumor. There are reports in the literature of increased levels of proteases in a number of cancers, e.g., liquid tumors or solid tumors. See, e.g., La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Non-limiting examples of disease include: all types of cancers, (such as, but not limited to breast, lung, colorectal, gastric, glioblastoma, ovarian, endometrial, renal, sarcoma, skin cancer, cervical, liver, bladder, cholangiocarcinoma, prostate, melanomas, head and neck cancer (e.g. head and neck squamous cell cancer, pancreatic, etc.), rheumatoid arthritis, Crohn's disease, SLE, cardiovascular damage, ischemia, etc. For example, indications would include leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), lymphoblastic diseases including multiple myeloma, and solid tumors, including lung, colorectal, prostate, pancreatic and breast, including triple negative breast cancer. For example, indications include bone disease or metastasis in cancer, regardless of primary tumor origin; breast cancer, including by way of non-limiting example, ER/PR+ breast cancer, Her2+ breast cancer, triple-negative breast cancer; colorectal cancer; endometrial cancer; gastric cancer; glioblastoma; head and neck cancer, such as head and neck squamous cell cancer; esophageal cancer; lung cancer, such as by way of non-limiting example, non-small cell lung cancer; multiple myeloma ovarian cancer; pancreatic cancer; prostate cancer; sarcoma, such as osteosarcoma; renal cancer, such as by way of non-limiting example, renal cell carcinoma; and/or skin cancer, such as by way of non-limiting example, squamous cell cancer, basal cell carcinoma, or melanoma. In some embodiments, the cancer is a squamous cell cancer. In some embodiments, the cancer is a skin squamous cell carcinoma. In some embodiments, the cancer is an esophageal squamous cell carcinoma. In some embodiments, the cancer is a head and neck squamous cell carcinoma. In some embodiments, the cancer is a lung squamous cell carcinoma. The CM is specifically cleaved by an enzyme at a rate of about 0.001-1500×104M−1S−1or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×104M−1S−1. For specific cleavage by an enzyme, contact between the enzyme and CM is made. When the AA or BAA comprises at least a first AB coupled to a MM and a CM, e.g., the AA comprises an AB coupled to a MM via a CM, is in the presence of target and sufficient enzyme activity, the CM can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to make contact with the CM and effect cleavage. It can readily be envisioned that an enzyme may be in the vicinity of the CM but is unable to cleave because of other cellular factors or protein modification of the enzyme. Exemplary CMs of the disclosure are provided in Table 4 above. In some embodiments, the CM has a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a length in the range of 30-40 amino acids, a length in the range of 35-40 amino acids, a length in the range of 10-30 amino acids, a length in the range of 15-30 amino acids, a length in the range of 20-30 amino acids, a length in the range of 25-30 amino acids, a length in the range of 10-20 amino acids, or a length in the range of 10-15 amino acids. 8. Masking Moieties (MMs) In both the activatable monospecific CD3 and EGFR AAs and the BAAs described above, the AAs/BAAs contain a MM. As described herein, the AAs and BAAs of the invention comprise a prodomain, which comprises a MM. In some embodiments, the MM is selected for use with a specific antibody or antibody fragment. In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In other embodiments the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB. Exemplary MMs of the disclosure can have a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a length in the range of 30-40 amino acids, a length in the range of 35-40 amino acids, a length in the range of 10-30 amino acids, a length in the range of 15-30 amino acids, a length in the range of 20-30 amino acids, a length in the range of 25-30 amino acids, a length in the range of 10-20 amino acids, a length in the range of 10-15 amino acids, or a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. As provided herein, the MM inhibits the binding of the AB to the target. The MM binds the antigen binding domain of the AB and inhibits binding of the AB to the target. The MM can sterically inhibit the binding of the AB to the target. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments when the AB is modified by or coupled to a MM and in the presence of target there is no binding or substantially no binding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target, as compared to the binding of the AB not modified by or coupled to an MM, the parental AB, or the AB not coupled to an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay. When an AB is coupled to or modified with a MM, the MM ‘masks’ or reduces or otherwise inhibits the specific binding of the AB to the target. When an AB is coupled to or modified by a MM, such coupling or modification can effect a structural change that reduces or inhibits the ability of the AB to specifically bind its target. Exemplary MMs of the disclosure are provided in Tables 3, 7, and 8, above. In any of the AAs and BAAs provided herein, the masked AB has a lower binding affinity than unmasked AB. 9. Linkers In many embodiments, it may be desirable to insert one or more linkers, e.g., flexible linkers, into the AA/BAA constructs so as to provide for flexibility at one or more of the MM-CM junction, the CM-AB/CM-scFv junction, or both. For example, the AB, MM, and/or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser) to provide the desired flexibility. As such, the ability of such BAA constructs to remain intact (not activated) or be activated as disclosed herein may benefit from introduction of one or more amino acids to provide for a flexible linker. For example, in certain embodiments an AA comprises one of the following formulae (where the formula below represents an amino acid sequence in either N- to C-terminal direction or C- to N-terminal direction): (MM1)-L1-(CM1)-(AB1) (MM1)-(CM1)-L2-(AB1) (MM1)-L1-(CM1)-L2-(AB1) (MM2)-L1-(CM2)-(AB2) (MM2)-(CM2)-L2-(AB2) (MM2)-L1-(CM2)-L2-(AB2) wherein MM, CM, and AB are as defined above; wherein L1 and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g., Gly, Ser). In some embodiments, the BAA comprises 2 heavy chains, each comprising the structural arrangement from N-terminus to C-terminus of MM2-CM2-AB2-AB1 HC and two light chains each comprising the structural arrangement from N-terminus to C-terminus of MM1-CM1-AB1 LC. In some embodiments, the structure including with linkers is provided inFIG.17. In some embodiments, (MM2)-L1-(CM2)-L2-(AB2) is linked to the heavy chain of AB1 and AB2 is a scFv. Linkers suitable for use in compositions described herein are generally ones that provide flexibility of the modified AB or the AAs to facilitate the inhibition of the binding of the AB to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, a suitable linker can be from 4 to 25 amino acids in length. In some embodiments, a suitable linker can be from 5 to 25 amino acids in length. In some embodiments, a suitable linker can be from 4 to 20 amino acids in length. In some embodiments, a suitable linker can be from 5 to 20 amino acids in length. Exemplary linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 88) and (GGGS)n (SEQ ID NO: 89), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In some embodiments, n is from about 1 to about 10, or from about 1 to about 9, or from about 1 to about 8, or from about 1 to about 7, or from about 1 to about 6, or from about 1 to about 5, or from about 1 to about 4, or from about 1 to about 3, or from about 1 to about 2. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers are provided in Table 9-1. TABLE 9-1Exemplary L1 and L2 LinkersSEQ ID NO:Linker Amino Acid SequenceSEQ ID NO: 88GSGGSSEQ ID NO: 89GGGSSEQ ID NO: 90GGSGSEQ ID NO: 91GGSGGSEQ ID NO: 92GSGSGSEQ ID NO: 93GSGGGSEQ ID NO: 94GGGSGSEQ ID NO: 95GSSSGSEQ ID NO: 96GSSGGSGGSGGSEQ ID NO: 97GGGSSEQ ID NO: 99GGGGSSEQ ID NO: 100GSSGGSGGSGGSGSEQ ID NO: 101GSSGGSGGSGGGGGSGGGSGGGSSEQ ID NO: 102GSSGGSGGSGGSGGGSGGGSGGSSEQ ID NO: 103GSSGTSEQ ID NO: 104GGGSSGGS The ordinarily skilled artisan will recognize that design of an AA can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired AA structure. 10. Conjugation In some embodiments, any of the antibodies, or ABs of the AAs, and BAAs disclosed herein may be conjugated to an agent. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is a detectable moiety. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or fragment thereof. In some embodiments, the agent is conjugated to the AB via a linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is a microtubule inhibitor. In some embodiments, the agent is a nucleic acid damaging agent, such as a DNA alkylator or DNA intercalator, or other DNA damaging agent. In some embodiments, the linker is a cleavable linker. In some embodiments, the agent is an agent selected from the group listed in Table 10. TABLE 10Exemplary Pharmaceutical Agents for ConjugationCYTOTOXIC AGENTSAuristatinsAuristatin EMonomethyl auristatin D (MMAD)Monomethyl auristatin E (MMAE)Desmethyl auristatin E (DMAE)Auristatin FMonomethyl auristatin F (MMAF)Desmethyl auristatin F (DMAF)Auristatin derivatives, e.g., amides thereofAuristatin tyramineAuristatin quinolineDolastatinsDolastatin derivativesDolastatin 16 DmJDolastatin 16 DpvMaytansinoids, e.g. DM-1; DM-4Maytansinoid derivativesDuocarmycinDuocarmycin derivativesAlpha-amanitinAnthracyclinesDoxorubicinDaunorubicinBryostatinsCamptothecinCamptothecin derivatives7-substituted Camptothecin10,11-DifluoromethylenedioxycamptothecinCombretastatinsDebromoaplysiatoxinKahalalide-FDiscodermolideEcteinascidinsANTIVIRALSAcyclovirVira ASymmetrelANTIFUNGALSNystatinADDITIONAL ANTI-NEOPLASTICSAdriamycinCerubidineBleomycinAlkeranVelbanOncovinFluorouracilMethotrexateThiotepaBisantreneNovantroneThioguanineProcarabizineCytarabineANTI-BACTERIALSAminoglycosidesStreptomycinNeomycinKanamycinAmikacinGentamicinTobramycinStreptomycin BSpectinomycinAmpicillinSulfanilamidePolymyxinChloramphenicolTurbostatinPhenstatinsHydroxyphenstatinSpongistatin 5Spongistatin 7Halistatin 1Halistatin 2Halistatin 3Modified BryostatinsHalocomstatinsPyrrolobenzimidazolesCibrostatin6DoxaliformAnthracyclins analoguesCemadotin analogue (CemCH2-SH)Pseudomonas toxin A (PE38) variantPseudomonas toxin A (ZZ-PE38) variantZJ-101OSW-14-Nitrobenzyloxycarbonyl Derivatives ofO6-BenzylguanineTopoisomerase inhibitorsHemiasterlinCephalotaxineHomoharringtoninePyrrolobenzodiazepine dimers (PBDs)Functionalized pyrrolobenzodiazepenesCalicheamicinsPodophyllotoxinsTaxanesVinca alkaloidsCONJUGATABLE DETECTABLE MOIETIESFluorescein and derivatives thereofFluorescein isothiocyanate (FITC)RADIOPHARMACEUTICALS125I131I89Zr111In123I131I99mTc201Tl133Xe11C62Cu18F68Ga13N15O38K82Rb99mTc (Technetium)HEAVY METALSBariumGoldPlatinumANTI-MYCOPLASMALSTylosineSpectinomycin Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies, AAs, and BAAs of the disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). In some embodiments, the antibody, AA or BAA comprises a detectable moiety. In some embodiments, the detectable moiety is a diagnostic agent. In some embodiments, the antibody, AA or BAA contains one or more disulfide bonds. In some embodiments, the antibody, AA or BAA contains one or more lysines. In some embodiments, the antibody, AA or BAA can be engineered to include one or more disulfide bonds or can be otherwise engineered to enable site-specific conjugation. 11. Production The disclosure also provides an isolated nucleic acid molecule encoding an antibody, AA or BAA described herein, as well as vectors that include these isolated nucleic acid sequences. The disclosure provides methods of producing an antibody, AA or BAA by culturing a cell under conditions that lead to expression of the antibody, AA or BAA, wherein the cell comprises such a nucleic acid molecule. In some embodiments, the cell comprises such a vector. In some embodiments, the vector is pLW289. In some embodiments, the vector is pLW246. In some embodiments, the vector is pLW307. In some embodiments, the vector is pLW291. In some embodiments, the vector is pLW352. In some embodiments, the vector is pLW353. (these vectors and described and sequences provided below in Example 1) 12. Use of Antibodies, AAs, Bispecific Antibodies and BAAs In some embodiments, the antibodies/bispecific antibodies/AAs/BAAs thereof may be used as therapeutic agents. Such agents will generally be employed to treat, alleviate, and/or prevent a disease or pathology in a subject. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient or other mammal suffering from (or at risk of developing) a disorder using standard methods. Administration of the antibodies/bispecific antibodies/AAs/BAAs thereof may abrogate or inhibit or interfere with the signaling function of one or more of the targets. It will be appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists. Generally, alleviation or treatment of a disease or disorder involves the lessening of one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, the therapeutically effective amount of the drug can accomplish one or a combination of the following: reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., to decrease to some extent and/or stop) cancer cell infiltration into peripheral organs; inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. In some embodiments, a composition of this disclosure can be used to prevent the onset or reoccurrence of the disease or disorder in a subject, e.g., a human or other mammal, such as a non-human primate, companion animal (e.g., cat, dog, horse), farm animal, work animal, or zoo animal. The terms subject and patient are used interchangeably herein. A therapeutically effective amount of antibodies/bispecific antibodies/AAs/BAAs thereof of the disclosure relates generally to the amount needed to achieve a therapeutic objective. Common ranges for therapeutically effective dosing of an antibodies/bispecific antibodies/AAs/BAAs thereof of the disclosure may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week. Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disorder. Methods for the screening antibodies/bispecific antibodies/AAs/BAAs that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art. Other contemplated uses involve diagnostics, imaging, prognostics, and detection uses. In some embodiments, antibodies/bispecific antibodies/AAs/BAAs are used in methods known within the art relating to the localization and/or quantitation of the target (e.g., for use in measuring levels of one or more of the targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In some embodiments, antibodies/bispecific antibodies/AAs/BAAs are used to isolate one or more of the targets by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. An antibody, an AA, a bispecific antibody or a BAA can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include125I,131I,35S or3H. In yet another embodiment, an antibody, bispecific antibody, AA, BAA directed two or more targets can be used as an agent for detecting the presence of one or more of the targets (or a fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or in some embodiments, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab′)2) is used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of an antibody with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the disclosure can be used to detect a protein in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. The antibodies, bispecific antibodies, AAs, and bispecific antibodies of the disclosure are also useful in a variety of diagnostic and prophylactic formulations. In one embodiment, an antibody, AA, bispecific antibody, BAA is administered to patients that are at risk of developing one or more of the aforementioned disorders. A patient's or organ's predisposition to one or more of the disorders can be determined using genotypic, serological or biochemical markers. In another embodiment of the disclosure, an antibody, AA, bispecific antibody, BAA is administered to human individuals diagnosed with a clinical indication associated with one or more of the aforementioned disorders. Upon diagnosis, an antibody, AA, bispecific antibody, BAA is administered to mitigate or reverse the effects of the clinical indication. Antibodies, bispecific antibodies, AAs, and bispecific antibodies are also useful in the detection of one or more targets in patient samples and accordingly are useful as diagnostics. For example, the antibodies, bispecific antibodies, AAs, and bispecific antibodies of the disclosure are used in in vitro assays, e.g., ELISA, to detect one or more target levels in a patient sample. In one embodiment, an antibody, AA, bispecific antibody, BAA is immobilized on a solid support (e.g., the well(s) of a microtiter plate). The immobilized antibody and/or AA serves as a capture antibody for any target(s) that may be present in a test sample. Prior to contacting the immobilized antibody/AA with a patient sample, the solid support is rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte. Subsequently the wells are treated with a test sample suspected of containing the antigen, or with a solution containing a standard amount of the antigen. Such a sample is, e.g., a serum sample from a subject suspected of having levels of circulating antigen considered to be diagnostic of a pathology. After rinsing away the test sample or standard, the solid support is treated with a second antibody that is detectably labeled. The labeled second antibody serves as a detecting antibody. The level of detectable label is measured, and the concentration of target antigen(s) in the test sample is determined by comparison with a standard curve developed from the standard samples. It will be appreciated that based on the results obtained using the antibody, AA, bispecific antibody, BAA in an in vitro diagnostic assay, it is possible to stage a disease in a subject based on expression levels of the target antigen(s). For a given disease, samples of blood are taken from subjects diagnosed as being at various stages in the progression of the disease, and/or at various points in the therapeutic treatment of the disease. Using a population of samples that provides statistically significant results for each stage of progression or therapy, a range of concentrations of the antigen that may be considered characteristic of each stage is designated. Antibodies, bispecific antibodies, AAs, and BAAs can also be used in diagnostic and/or imaging methods. In some embodiments, such methods are in vitro methods. In some embodiments, such methods are in vivo methods. In some embodiments, such methods are in situ methods. In some embodiments, such methods are ex vivo methods. For example, AAs, and bispecific antibodies having an enzymatically cleavable CM can be used to detect the presence or absence of an enzyme that is capable of cleaving the CM. Such AAs, and bispecific antibodies can be used in diagnostics, which can include in vivo detection (e.g., qualitative or quantitative) of enzyme activity (or, in some embodiments, an environment of increased reduction potential such as that which can provide for reduction of a disulfide bond) through measured accumulation of activated or bispecific activated antibodies (i.e., antibodies or bispecific antibodies resulting from cleavage of an AA or a BAA) in a given cell or tissue of a given host organism. Such accumulation of activated bispecific antibodies indicates not only that the tissue expresses enzymatic activity (or an increased reduction potential depending on the nature of the CM) but also that the tissue expresses at least one target to which the activated bispecific antibody binds. For example, the CM can be selected to be a protease substrate for a protease found at the site of a tumor, at the site of a viral or bacterial infection at a biologically confined site (e.g., such as in an abscess, in an organ, and the like), and the like. At least one of the AB can be one that binds a target antigen. Using methods familiar to one skilled in the art, a detectable label (e.g., a fluorescent label or radioactive label or radiotracer) can be conjugated to an AB or other region of an antibody, AA, bispecific antibody, BAA. Suitable detectable labels are discussed in the context of the above screening methods and additional specific examples are provided below. Using at least one AB specific to a protein or peptide of the disease state, along with a protease whose activity is elevated in the disease tissue of interest, AAs will exhibit an increased rate of binding to disease tissue relative to tissues where the CM specific enzyme is not present at a detectable level or is present at a lower level than in disease tissue or is inactive (e.g., in zymogen form or in complex with an inhibitor). Since small proteins and peptides are rapidly cleared from the blood by the renal filtration system, and because the enzyme specific for the CM is not present at a detectable level (or is present at lower levels in non-disease tissues or is present in inactive conformation), accumulation of activated bispecific antibodies in the disease tissue is enhanced relative to non-disease tissues. In another example, antibodies, antibodies/bispecific antibodies/AAs/BAAs of the present disclosure can be used to detect the presence or absence of a cleaving agent in a sample. For example, where the antibodies/bispecific antibodies/AAs/BAAs contain a CM susceptible to cleavage by an enzyme, the BAAs can be used to detect (either qualitatively or quantitatively) the presence of an enzyme in the sample. In another example, where the antibodies/bispecific antibodies/AAs/BAAs contain a CM susceptible to cleavage by reducing agent, the antibodies/bispecific antibodies/AAs/BAAs can be used to detect (either qualitatively or quantitatively) the presence of reducing conditions in a sample. To facilitate analysis in these methods, the antibodies/bispecific antibodies/AAs/BAAs can be detectably labeled, and can be bound to a support (e.g., a solid support, such as a slide or bead). The detectable label can be positioned on a portion of the antibodies/bispecific antibodies/AAs/BAAs that is not released following cleavage, for example, the detectable label can be a quenched fluorescent label or other label that is not detectable until cleavage has occurred. The assay can be conducted by, for example, contacting the immobilized, detectably labeled antibodies/bispecific antibodies/AAs/BAAs with a sample suspected of containing an enzyme and/or reducing agent for a time sufficient for cleavage to occur, then washing to remove excess sample and contaminants. The presence or absence of the cleaving agent (e.g., enzyme or reducing agent) in the sample is then assessed by a change in detectable signal of the antibodies/bispecific antibodies/AAs/BAAs prior to contacting with the sample e.g., the presence of and/or an increase in detectable signal due to cleavage of the antibodies/bispecific antibodies/AAs/BAAs by the cleaving agent in the sample. Such detection methods can be adapted to also provide for detection of the presence or absence of a target that is capable of binding at least one AB of the antibodies/bispecific antibodies/AAs/BAAs of the present disclosure. Thus, the assays can be adapted to assess the presence or absence of a cleaving agent and the presence or absence of a target of interest. The presence or absence of the cleaving agent can be detected by the presence of and/or an increase in detectable label of the antibodies/bispecific antibodies/AAs/BAAs as described above, and the presence or absence of the target can be detected by detection of a target-AB complex e.g., by use of a detectably labeled anti-target antibody. AAs/BAAs of the present disclosure are also useful in in situ imaging for the validation of AA activation, e.g., by protease cleavage, and binding to a particular target. In situ imaging is a technique that enables localization of proteolytic activity and target in biological samples such as cell cultures or tissue sections. Using this technique, it is possible to confirm both binding to a given target and proteolytic activity based on the presence of a detectable label (e.g., a fluorescent label). These techniques are useful with any frozen cells or tissue derived from a disease site (e.g. tumor tissue) or healthy tissues. These techniques are also useful with fresh cell or tissue samples. In these techniques, an AA/BAA is labeled with a detectable label. The detectable label may be a fluorescent dye, (e.g. a fluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate (TRITC), an Alexa Fluor® label), a near infrared (NIR) dye (e.g., Qdot® nanocrystals), a colloidal metal, a hapten, a radioactive marker, biotin and an amplification reagent such as streptavidin, or an enzyme (e.g. horseradish peroxidase or alkaline phosphatase). Detection of the label in a sample that has been incubated with the labeled, AA or BAA indicates that the sample contains the target and contains a protease that is specific for the CM of the AAs or BAAs of the present disclosure. In some embodiments, the presence of the protease can be confirmed using broad spectrum protease inhibitors such as those described herein, and/or by using an agent that is specific for the protease, for example, an antibody such as A11, which is specific for the protease matriptase (MT-SP1) and inhibits the proteolytic activity of MT-SP1; see e.g., International Publication Number WO 2010/129609, published 11 Nov. 2010. The same approach of using broad spectrum protease inhibitors such as those described herein, and/or by using a more selective inhibitory agent can be used to identify a protease or class of proteases specific for the CM of the AAs or BAAs of the present disclosure. In some embodiments, the presence of the target can be confirmed using an agent that is specific for the target or the detectable label can be competed with unlabeled target. In some embodiments, unlabeled AA could be used, with detection by a labeled secondary antibody or more complex detection system. Similar techniques are also useful for in vivo imaging where detection of the fluorescent signal in a subject, e.g., a mammal, including a human, indicates that the disease site contains the target and contains a protease that is specific for the CM of the AAs or BAAs of the present disclosure. These techniques are also useful in kits and/or as reagents for the detection, identification or characterization of protease activity in a variety of cells, tissues, and organisms based on the protease-specific CM in the AAs or BAAs of the present disclosure. 13. Therapeutic Administration It will be appreciated that administration of therapeutic entities in accordance with the disclosure will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists. In some embodiments, the antibodies, bispecific antibodies, AAs, or BAAs (or conjugated compositions thereof) are administered in conjunction with one or more additional agents, or with a combination of additional agents. Suitable additional agents include current pharmaceutical and/or surgical therapies for an intended application. For example, they can be used in conjunction with an additional chemotherapeutic or anti-neoplastic agent. In some embodiments, the antibodies, bispecific antibodies, AAs, or BAAs (or conjugated compositions thereof) of the present disclosure are administered in conjunction with one or more additional agents selected from the group consisting of antibodies, conjugated antibodies, AAs, conjugated AAs, bispecific antibodies, conjugated bispecific antibodies, BAAs, or conjugated BAAs. In some embodiments, the antibody portion of any of the above-referenced additional agents is directed against a target such as one or more of the targets disclosed in Table 9. It is appreciated that in some embodiments the antibody portion of antibodies, bispecific antibodies, AAs, or BAAs (or conjugated compositions thereof) of the present disclosure and the antibody portion of the additional agent is directed against the same target (e.g. both may target EGFR). In some embodiments, they are directed against the same target, but target different epitopes. In some embodiments, they are directed against different targets entirely (e.g., an activatable antibody of the present disclosure that targets EGFR may be administered in conjunction with an AA targeting a different target; likewise e.g. a BAA of the present disclosure that targets EGFR and CD3 may be administered in conjunction with an AA targeting a different target. In some embodiments, antibodies, bispecific antibodies, AAs or BAAs (or conjugated compositions thereof) of the disclosure are administered in conjunction with an immunotherapeutic agent. In some embodiments, antibodies, bispecific antibodies, AAs or BAAs (or conjugated compositions thereof) of the disclosure are administered in conjunction with a chemotherapeutic agent. In some embodiments, antibodies, bispecific antibodies, AAs or BAAs (or conjugated compositions thereof) of the disclosure are administered in conjunction with both an immunotherapeutic agent and a chemotherapeutic agent. In some embodiments, one or more additional agents is administered with any of these combination embodiments. In some embodiments, they are formulated into a single therapeutic composition, and the antibodies/bispecific antibodies/AAs/BAAs thereof and the additional agent are administered simultaneously. Alternatively, the antibodies/bispecific antibodies/AAs/BAAs thereof are administered separate from each other, e.g., each is formulated into a separate therapeutic composition, and the antibodies/bispecific antibodies/AAs/BAAs thereof and the additional agent are administered simultaneously, or the antibodies/bispecific antibodies/AAs/BAAs thereof and the additional agent are administered at different times during a treatment regimen. The antibodies/bispecific antibodies/AAs/BAAs thereof and the additional agent can be administered in multiple doses. The antibodies/bispecific antibodies/AAs/BAAs thereof can be incorporated into pharmaceutical compositions suitable for administration. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York. Such compositions typically comprise the antibodies/bispecific antibodies/AAs/BAAs thereof and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be suitable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as sustained/controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. For example, the active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions. Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) and can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The formulation can also contain more than one active compound as necessary for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. In one embodiment, the active compounds are administered in combination therapy, i.e., combined with other agents, e.g., therapeutic agents, that are useful for treating pathological conditions or disorders, such as autoimmune disorders and inflammatory diseases. The term “in combination” in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially. If given sequentially, at the onset of administration of the second compound, the first of the two compounds is still detectable at effective concentrations at the site of treatment. For example, the combination therapy can include one or more antibodies/bispecific antibodies/AAs/BAAs thereof of the disclosure coformulated with, and/or coadministered with, one or more additional therapeutic agents, e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic or cytostatic agents, as described in more detail below. Furthermore, one or more antibodies/bispecific antibodies/AAs/BAAs thereof described herein may be used in combination with two or more of the therapeutic agents described herein (e.g. one BAA administered with another BAA or AA of the disclosure, and the like). Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies. In other embodiments, one or more antibodies of the disclosure can be coformulated with, and/or coadministered with, one or more anti-inflammatory drugs, immunosuppressants, or metabolic or enzymatic inhibitors. Nonlimiting examples of the drugs or inhibitors that can be used in combination with the antibodies described herein, include, but are not limited to, one or more of: nonsteroidal anti-inflammatory drug(s) (NSAIDs), e.g., ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, and indomethacin; sulfasalazine; corticosteroids such. as prednisolone; cytokine suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors of nucleotide biosynthesis, e.g., inhibitors of purine biosynthesis, folate antagonists (e.g., methotrexate (N-[4-[[(2,4-diamino-6-pteridinyl)methyl] methylamino] benzoyl]L-glutamic acid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotate dehydrogenase (DHODH) inhibitors. Suitable therapeutic agents for use in combination with the antibodies of the disclosure include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g., leflunomide), and folate antagonists (e.g., methotrexate). Examples of additional inhibitors include one or more of: corticosteroids (oral, inhaled and local injection); immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g., sirolimus (rapamycin—RAPAMUNE™ or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); agents that interfere with signaling by proinflammatory cytokines such as TNFα or IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, and variants thereof; phosphodiesterase inhibitors, e.g., R973401 (phosphodiesterase Type IV inhibitor); phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethyl ketone analogs); inhibitors of vascular endothelial cell growth factor or growth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor; and inhibitors of angiogenesis. Suitable therapeutic agents for use in combination with the antibodies of the disclosure are immunosuppressants, e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus (rapamycin) or rapamycin derivatives, e.g., soluble rapamycin derivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2 inhibitors, e.g., celecoxib and variants thereof; and phospholipase inhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g., trifluoromethyl ketone analogs. Additional examples of therapeutic agents that can be combined with an antibody of the disclosure include one or more of: 6-mercaptopurines (6-MP); azathioprine sulphasalazine; mesalazine; olsalazine; chloroquine/hydroxychloroquine)(PLAQUENIL®); pencillamine; aurothiornalate (intramuscular and oral); azathioprine; coichicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline, salmeteral); xanthines (theophylline, aminophylline); cromoglycate; nedocromil; ketotifen; ipratropium and oxitropium; mycophenolate mofetil; adenosine agonists; antithrombotic agents; complement inhibitors; and adrenergic agents. In some embodiments, antibodies/bispecific antibodies/AAs/BAAs thereof of the disclosure can be combined with one or more antibodies/bispecific antibodies/AAs/BAAs thereof. 14. Kits and Articles of Manufacture Provided herein are kits and articles of manufacture comprising any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein The kits and articles of manufacture may comprise any one or more of the antibodies, AAs, bispecific antibodies, and BAAs provided herein in a format suitable for storage or shipping. The kits and articles of manufacture may comprise at least a second component. The kits and articles of manufacture may comprise a vessel, a diluent, a solvent, a second composition, or any component useful for converting a composition in a format for storage into a composition suitable for use in a method disclosed herein, if such a conversion is required. The method may be, for instance, a therapeutic method disclosed herein. The kit may comprise instructions for use. The kits and articles of manufacture may comprise an agent as disclosed herein, for instance a cytotoxic agent or a detectable label, in a format suitable for conjugation to the antibodies, AAs, bispecific antibodies, and BAAs provided herein. The following examples are included for illustrative purposes and are not intended to limit the scope of the invention. ENUMERATED EMBODIMENTS The invention may be defined by reference to the following enumerated, illustrative embodiments. 1. A bispecific activatable antibody (BAA), wherein said BAA, when activated, specifically binds to two targets and comprises the following structure:a. an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises two heavy chains (AB1 HCs) and two light chains (AB1 LCs); and wherein the AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, whereinthe MM1 inhibits the binding of the AB1 to its target; andthe CM1 is a polypeptide that functions as a substrate for a first protease,b. two scFvs (each an AB2) that each specifically binds to a second target wherein each AB2 comprises a light chain variable region linked to a heavy chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; and wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease,and wherein the BAA has at least one of the following characteristics:i. MM2 comprises amino acid sequence SEQ ID NO: 12;ii. MM1 comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7;iii. AB2 comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4; andiv. AB1 comprises an Fc region comprising an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. 2. The BAA of embodiment 1, wherein AB2 comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4. 3. The BAA of embodiment 1, wherein the AB1 binds a tumor target and the AB2 binds an immune effector target. 4. The BAA of any one of embodiments 1 to 3, wherein the BAA is a T cell-engaging bispecific (TCB) AA (TCBAA). 5. The BAA of any one of embodiments 1 to 4, wherein the AB1 binds EGFR and the AB2 binds CD3E. 6. The BAA of any one of embodiments 1 to 5, wherein the MM1 comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7. 7. The BAA of any one of embodiments 1 to 5, wherein the MM1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 85 and SEQ ID NO: 78. 8. The BAA of any one of embodiments 1 to 5, wherein the MM1 comprises SEQ ID NO: 78. 9. The BAA of any one of embodiments 1 to 8, wherein the MM2 comprises the amino acid sequence SEQ ID NO: 12. 10. The BAA of any one of embodiments 1 to 9, wherein the CM comprises the amino acid sequence of SEQ ID NO: 14. 11. The BAA of any one of embodiments 1 to 9, wherein the CM comprises the amino acid sequence of SEQ ID NO: 17. 12. The BAA of any one of embodiments 1 to 9, wherein the CM the CM comprises the amino acid sequence of SEQ ID NO: 16. 13. The BAA of any one of embodiments 1 to 9, wherein CM1 comprises an amino acid sequence selected from the group comprising SEQ ID NO: 14 and SEQ ID NO: 16. 14. The BAA of any one of embodiments 1 to 9, wherein CM2 comprises an amino acid sequence selected from the group comprising SEQ ID NO: 14 and SEQ ID NO: 17. 15. The BAA of any one of embodiments 1 to 14, wherein AB1 comprises amino acid substitutions in at least two of amino acid positions L234, L235, and P331. 16. The BAA of embodiment 15, wherein AB1 comprises amino acid substitutions at amino acid positions L234, L235, and P331. 17. The BAA of embodiment 15, wherein AB1 comprises L234F, L235E, and P331S amino acid substitutions. 18. The BAA of embodiment 15, wherein the AB1 comprises an Fc region comprising an amino acid substitution at N297. 19. The BAA of any one of embodiments 1 to 14, wherein AB1 comprises amino acid substitutions at amino acid positions L234F, L235E, P331S, and N297Q. 20. The BAA of embodiment 1, wherein the heavy chain of the AB1 comprises any one of SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, as set forth in Table 6. 21. The BAA CI106, comprising the layout and sequence as provided in Table 11 and Example 1. 22. The BAA CI107, comprising the layout and sequence as provided in Table 11 and Example 1. 23. The BAA CI079, comprising the layout and sequence as provided in Table 11 and Example 1. 24. The BAA CI090, comprising the layout and sequence as provided in Table 11 and Example 1. 25. An activatable antibody (AA) comprising:a) an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε);b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3ε when the AA is in an uncleaved state, wherein the MM comprises amino acid sequence SEQ ID NO: 12; andc) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. 26. The AA of embodiment 25, wherein the CM comprises any one of the sequences set forth in Table 4. 27. The AA of embodiment 25, wherein the CM comprises a substrate cleavable by a serine protease or an MMP. 28. The AA of embodiment 25, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-56. 29. The AA of embodiment 25, wherein the protease is an MMP. 30. The AA of embodiment 25, wherein the protease is a serine protease. 31. The AA of embodiment 25, wherein the AB that specifically binds to CD3 is the antibody of any one of embodiments 38-47. 32. An activatable antibody (AA) comprising:a. an antibody or an antigen binding fragment thereof (AB) that specifically binds to Epidermal Growth Factor Receptor (EGFR);b. a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the EGFR when the AA is in an uncleaved state, and wherein the MM comprises an amino acid sequence selected from the group consisting of sequences presented in Table 7; andc. a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. 33. The AA of embodiment 32, wherein the MM comprises the amino acid sequence of SEQ ID NO: 78. 34. The AA of any one of embodiments 32 to 33, wherein the CM comprises a substrate cleavable by a serine protease or an MMP. 35. The AA of any one of embodiments 32 to 33, wherein the CM comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 18-56. 36. The AA of embodiment 32, wherein the CM comprises the amino acid sequence of SEQ ID NO: 14. 37. The AA of embodiment 32, wherein the CM comprises the amino acid sequence of SEQ ID NO: 16. 38. An antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), wherein the antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4. 39. The AB of embodiment 38, wherein, the antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4. 40. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO: 2. 41. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO: 3. 42. The AB of embodiment 38, wherein the antibody comprises a light chain variable domain as set forth in SEQ ID NO: 1. 43. The AB of embodiment 38, comprising a light chain variable domain as set forth in SEQ ID NO: 4. 44. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 1. 45. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 1. 46. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 4. 47. The AB of embodiment 38, comprising a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 4. 48. The AB of any one of embodiments 32 to 47, wherein the AB is a bispecific AB. 49. The AA of any one of embodiments 32 to 47, wherein the antibody is a scFv. 50. The AA of any one of embodiments 32 to 47, wherein the antibody is an IgG1 antibody. 51. An activatable antibody (AA) comprising:a) an antibody or antigen binding fragment thereof (AB) that specifically binds to the epsilon chain of CD3 (CD3ε), wherein the antibody comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or comprises a light chain variable domain as set forth in SEQ ID NO: 1 or SEQ ID NO: 4;b) a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the CD3ε when the AA is in an uncleaved state; andc) a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. 52. The AA of embodiment 51, wherein the AB comprises a heavy chain variable domain as set forth in SEQ ID NO: 2. 53. The AA of embodiment 51, wherein the AB comprises a heavy chain variable domain as set forth in SEQ ID NO: 3. 54. The AA of embodiment 51, wherein the AB comprises a light chain variable domain as set forth in SEQ ID NO: 1. 55. The AA of embodiment 51, wherein the AB comprises a light chain variable domain as set forth in SEQ ID NO: 4. 56. The AA of embodiment 51, wherein the AB comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 1. 57. The AA of embodiment 51, wherein the AB comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 1. 58. The AA of embodiment 51, wherein the AB comprises a heavy chain variable domain as set forth in SEQ ID NO: 2 and a light chain variable domain as set forth in SEQ ID NO: 4. 59. The AA of embodiment 51, wherein the AB comprises a heavy chain variable domain as set forth in SEQ ID NO: 3 and a light chain variable domain as set forth in SEQ ID NO: 4. 60. The AA of any one of embodiments 51 to 59, wherein the MM comprises any one of the sequences set forth in Table 3. 61. The AA of any one of embodiments 51 to 59, wherein the CM comprises any one of the sequences set forth in Table 4. 62. A bispecific activatable antibody (BAA) comprising any one of the AAs of embodiments 51 to 61. 63. An activatable antibody (AA) comprising:a. an antibody (AB) that specifically binds a target, wherein the antibody is an IgG1 antibody, and wherein the Fc region of the antibody comprises an amino acid substitution in amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function;b. a masking moiety (MM) coupled to the AB, wherein the MM reduces or inhibits the binding of the AB to the target when the AA is in an uncleaved state; andc. a cleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptide that functions as a substrate for a protease. 64. The AA of embodiment 63, wherein the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, N297 and P331, as numbered by the EU index as set forth in Kabat, such that the AA has reduced effector function. 65. The AA of embodiment 63 or 64, wherein the target is selected from the group consisting of the targets presented in Table 9. 66. A bispecific activatable antibody (BAA) comprising:a. an IgG antibody (AB1) that specifically binds to a first target wherein the AB1 comprises two heavy chains (AB1 HCs) and two light chains (AB1 LCs); and wherein the AB1 is linked to a first masking moiety (MM1) linked to a first cleavable moiety (CM1) to form a MM1-CM1 construct, wherein the carboxyl terminus of a MM1-CM1 construct is linked to each amino terminus of each light chain of the AB1, whereinthe MM1 inhibits the binding of the AB1 to its target; andthe CM1 is a polypeptide that functions as a substrate for a first protease,b. two scFvs (each an AB2) that each specifically binds to a second target wherein each AB2 comprises a heavy chain variable region linked to a light chain variable region, wherein the carboxyl terminus of each AB2 is linked to the amino terminus each of the AB1 heavy chains; and wherein each AB2 is linked to a second masking moiety (MM2) linked to a second cleavable moiety (CM2) to form a MM2-CM2 construct, wherein the carboxyl terminus of each MM2-CM2 construct is linked to the amino terminus of each AB2 whereinthe MM2 inhibits the binding of the AB2 to its target; andthe CM2 is a polypeptide that functions as a substrate for a second protease, and wherein the AB1 comprises an Fc region comprises an amino acid substitution in at least one of amino acid positions L234, L235, N297, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. 67. The BAA of embodiment 66, wherein the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, N297 and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. 68. The BAA of embodiment 66, wherein the Fc region comprises amino acid substitutions in at least amino acid positions L234, L235, and P331, as numbered by the EU index as set forth in Kabat, such that the BAA has reduced effector function. 69. The BAA of any one of embodiments 66 to 68, wherein the first target is selected from the group consisting of the targets presented in Table 9 and the second target is selected from the group consisting of the targets presented in Table 9. 70. The AA or BAA of any one of the above embodiments, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab′)2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. 71. The AA or BAA of any one of the above embodiments wherein the antibody is a rodent antibody, a chimeric antibody, a humanized antibody, or a fully human monoclonal antibody. 72. The AA of any one of embodiments 32-37 and 51-71, wherein the AA is a BAA. 73. A pharmaceutical composition comprising the antibody, AA, or BAA of any one of embodiments 1-72 and optionally a carrier. 74. The pharmaceutical composition of embodiment 73 comprising an additional agent. 75. The pharmaceutical composition of embodiment 74, wherein the additional agent is a therapeutic agent. 76. An isolated nucleic acid molecule encoding the antibody, AA, or BAA of any one of embodiments 1-72. 77. A vector comprising the isolated nucleic acid molecule of embodiment 76. 78. A vector comprising the nucleic acid sequence of pLW289. 79. A vector comprising the nucleic acid sequence of pLW246. 80. A vector comprising the nucleic acid sequence of pLW307. 81. A vector comprising the nucleic acid sequence of pLW291. 82. A cell comprising any one of the vectors of embodiments 77-81. 83. A cell comprising pLW289 and pLW246. 84. A cell comprising pLW307 and pLW291. 85. A method of producing the antibody, AA, or BAA of any one of embodiments 1-72 by culturing a cell under conditions that lead to expression of the antibody, AA, or BAA, wherein the cell comprises the nucleic acid molecule of embodiment 76 or the vector of any one of embodiments 78-81. 86. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease comprising administering a therapeutically effective amount of the antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75 to a subject in need thereof. 87. The method of embodiment 86, wherein the disorder or disease comprises disease cells expressing EGFR. 88. The method of embodiments 86 or 87, wherein the disorder or disease is cancer. 89. The method of embodiment 88, wherein the cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gall bladder cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, small bowel carcinoma, squamous cell cancer, skin cancer testicular cancer, thyroid cancer or uterine cancer. 90. A method of inhibiting angiogenesis in a subject comprising administering a therapeutically effective amount of the antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75 to a subject in need thereof. 91. The method of any one of embodiments 86-90, wherein the method comprises administering an additional agent. 92. The method of embodiment 91 wherein the additional agent is a therapeutic agent. 93. A method of reducing damage to healthy tissue caused by an antibody binding to its target on healthy tissue as well as on diseased tissue, the method comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA of any one of the embodiments provided herein. 94. A method to improve tolerability of an antibody treatment comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA of any one of the embodiments provided herein. 95. A method to recruit T cells to tumor tissue comprising administering to a subject in need thereof an AA or BAA or a pharmaceutical composition comprising an AA or BAA, wherein said AA or BAA is an AA or BAA of any one of the embodiments provided herein. 96. An antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75, for use as a medicament. 97. An antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75, for use in a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease, wherein the disorder or disease comprises disease cells expressing EGFR. 98. An antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75, for use in a method of treating cancer; optionally wherein the cancer is anal cancer, basal cell carcinoma, brain cancer, bladder cancer, breast cancer, bone cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gall bladder cancer, gastric cancer, glioblastoma, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, sarcoma, small bowel carcinoma, squamous cell cancer, skin cancer testicular cancer, thyroid cancer or uterine cancer. 99. An antibody, AA, or BAA of any one of embodiments 1-72, or the pharmaceutical composition of any one of embodiments 73-75, for use in a method of treatment, wherein the method comprises inhibiting angiogenesis. 100. The antibody, AA, or BAA, or the pharmaceutical composition, for use according to any of embodiments 96 to 99, wherein the use comprises administering an additional agent, optionally wherein the additional agent is a therapeutic agent. EXAMPLES Example 1. Sequences, Vector Construction and Expression of Antibodies, BAAs and Activated BAAs Antibodies of Interest The molecules as provided in Table 11 below were constructed and tested. As indicated, activated molecules were produced as masked and proteolytically cleaved to produce the activated forms. TABLE 11HeavyLightMoleculeChainChainNameMolecule Component PartsVectorVectorCI0113954-0001-C225v5N297Q-JF15865-0001-CD3LvHv-H-NpLW023OPP022CI0203954-Nsub-C225v5N297Q-JF15865-Nsub-hSP34LvHv-H-NpLW073pLW071CI0403954-2001-C225v5N297Q-JF15865-2001-hSP34LvHv-H-NpLW101CTX122CI048Activated CI011: 3954-0001-C225v5N297Q-JF15865-0001-pLW023OPP022CD3LvHv-H-NCI0793954-0001-C225v5Fcmt3-h20GG-0001-v16sc-H-NpLW225OPP022CI0903954-0001-C225v5Fcmt4-h20GG-0001-v16sc-H-NpLW233OPP022ActivatedActivated 3954-0001-C225v5Fcmt4-h20GG-0001-v16sc-H-NpLW233OPP022CI090ActivatedActivated 3954-0011-C225v5Fcmt4-h20GG-0011-v16sc-H-NpLW289pLW291CI104CI106CF41-2008-C225v5Fcmt4-h20GG-0011-v16sc-H-NpLW289pLW246CI1073954-0011-C225v5Fcmt4-h20GG-2006-v16sc-H-NpLW307pLW291CI127SynFcmt4-h20GG-0011-v16sc-H-NpLW334pLW139CI128SynFcmt4-h20GG-2006-v16sc-H-NpLW335pLW139CI135CF41-2008-C225v5Fcmt4-h20GG-0011-v12sc-H-NpLW352pLW246CI136CF41-2008-C225v5Fcmt4-h20GG-0011-v19sc-H-NpLW353pLW246CI0913954-1490DQH-C225v5Fcmt4-h20GG-2008-v16sc-H-NpLW242CX320CI064SynN297Q-JF15865-0001-hSP34LvHv-H-NpLW138pLW139v12Anti-CD3 variantHV12LV12v16Anti-CD3 variantHV20LV12v19Anti-CD3 variantHV20LV19v26Anti-CD3 variantHV12LV19 The sequences of the molecules and vectors are provided below. Brackets denote some of the component parts of the molecules presented. In some sequences, linkers are provided. Underlined amino acids denote predicted CDR sequences. CI011:3954-0001-C225v5N297Q-JF15865-0001-CD3LvHv-H-NpLW023: HC JF15865-0001-CD3LvHv-C225v5N297Q (H-N)Nucleotide Sequence[spacer SEQ ID NO: 176][pLW023 without spacer SEQ ID NO: 177]CAAGGCCAGTCTGGCCAAATGATGTATTGCGGTGGGAATGAGGTGTTGTGCGGGCCGCGGGTTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTCAGACCGTGGTCACACAGGAGCCCTCACTGACAGTGAGCCCTGGCGGGACCGTCACACTGACTTGTCGCAGTTCAACTGGCGCCGTGACTACCAGCAATTACGCTAACTGGGTCCAGCAGAAACCAGGACAGGCACCACGAGGACTGATCGGAGGAACTAATAAGAGAGCACCAGGAACCCCTGCAAGGTTCTCCGGATCTCTGCTGGGGGGAAAAGCCGCTCTGACACTGAGCGGCGTGCAGCCTGAGGACGAAGCTGAGTACTATTGCGCACTGTGGTACTCCAACCTGTGGGTGTTTGGCGGGGGAACTAAGCTGACCGTCCTGGGAGGAGGAGGAAGCGGAGGAGGAGGGAGCGGAGGAGGAGGATCCGAAGTGCAGCTGGTCGAGAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAAGCTGTCTTGTGCAGCCAGTGGCTTCACCTTCAACACTTACGCAATGAACTGGGTGCGGCAGGCACCTGGGAAGGGACTGGAATGGGTCGCCCGGATCAGATCTAAATACAATAACTATGCCACCTACTATGCTGACAGTGTGAAGGATAGGTTCACCATTTCACGCGACGATAGCAAAAACACAGCTTATCTGCAGATGAATAACCTGAAGACCGAGGATACAGCAGTGTACTATTGCGTCAGACACGGCAATTTCGGGAACTCTTACGTGAGTTGGTTTGCCTATTGGGGACAGGGGACACTGGTCACCGTCTCCTCAGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 105)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW023 without spacer SEQ ID NO: 179]QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGSGG][LSGRSDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)OPP022: LC 3954-0001-C225v5Nucleotide Sequence[spacer SEQ ID NO: 180][OPP022 without spacer SEQ ID NO: 181]TCCGATAATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 107)Amino Acid Sequence[spacer SEQ ID NO: 178][OPP022 without spacer SEQ ID NO: 182][SDNH][GSSGT][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 108)CI020: 3954-Nsub-C225v5N297O-JF15865-Nsub-hSP34LvHv-H-NpLW073: HC C225v5N297Q-JF15865-Nsub-hSP34LvHv (H-N)Nucleotide Sequence[spacer SEQ ID NO: 176][pLW073 without spacer SEQ ID NO: 183]CAAGGCCAGTCTGGCCAAATGATGTATTGCGGTGGGAATGAGGTGTTGTGCGGGCCGCGGGTTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTGGTGGAGGCTCGGGCGGTGGGAGCGGCGGCGGTTCTCAGACCGTGGTCACACAGGAGCCCTCACTGACAGTGAGCCCTGGCGGGACCGTCACACTGACTTGTCGCAGTTCAACTGGCGCCGTGACTACCAGCAATTACGCTAACTGGGTCCAGCAGAAACCAGGACAGGCACCACGAGGACTGATCGGAGGAACTAATAAGAGAGCACCAGGAACCCCTGCAAGGTTCTCCGGATCTCTGCTGGGGGGAAAAGCCGCTCTGACACTGAGCGGCGTGCAGCCTGAGGACGAAGCTGAGTACTATTGCGCACTGTGGTACTCCAACCTGTGGGTGTTTGGCGGGGGAACTAAGCTGACCGTCCTGGGAGGAGGAGGAAGCGGAGGAGGAGGGAGCGGAGGAGGAGGATCCGAAGTGCAGCTGGTCGAGAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAAGCTGTCTTGTGCAGCCAGTGGCTTCACCTTCAACACTTACGCAATGAACTGGGTGCGGCAGGCACCTGGGAAGGGACTGGAATGGGTCGCCCGGATCAGATCTAAATACAATAACTATGCCACCTACTATGCTGACAGTGTGAAGGATAGGTTCACCATTTCACGCGACGATAGCAAAAACACAGCTTATCTGCAGATGAATAACCTGAAGACCGAGGATACAGCAGTGTACTATTGCGTCAGACACGGCAATTTCGGGAACTCTTACGTGAGTTGGTTTGCCTATTGGGGACAGGGGACACTGGTCACCGTCTCCTCAGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 109)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW073 without spacer SEQ ID NO: 184]QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGSGGGGGSGGGSGGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 110)pLW071: LC 3954-Nsub-C225v5Nucleotide Sequence[spacer SEQ ID NO: 180][pLW071 without spacer SEQ ID NO: 185]CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGCTCAGGTGGAGGCTCGGGCGGTGGGAGCGGCGGTTCTGATATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 111)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW071 without spacer SEQ ID NO: 186]QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSGGGSGGGSGGS]DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 112)CI040: 3954-2001-C225v5N297Q-JF15865-2001-hSP34LvHv-H-NpLW101: HC JF15865-2001-CD3LvHv-C225v5N297Q (H-N)Nucleotide Sequence[spacer SEQ ID NO: 176][pLW101 without spacer SEQ ID NO: 187]CAAGGCCAGTCTGGCCAAATGATGTATTGCGGTGGGAATGAGGTGTTGTGCGGGCCGCGGGTTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACAATCACGGCGGCGGTTCTCAGACCGTGGTCACACAGGAGCCCTCACTGACAGTGAGCCCTGGCGGGACCGTCACACTGACTTGTCGCAGTTCAACTGGCGCCGTGACTACCAGCAATTACGCTAACTGGGTCCAGCAGAAACCAGGACAGGCACCACGAGGACTGATCGGAGGAACTAATAAGAGAGCACCAGGAACCCCTGCAAGGTTCTCCGGATCTCTGCTGGGGGGAAAAGCCGCTCTGACACTGAGCGGCGTGCAGCCTGAGGACGAAGCTGAGTACTATTGCGCACTGTGGTACTCCAACCTGTGGGTGTTTGGCGGGGGAACTAAGCTGACCGTCCTGGGAGGAGGAGGAAGCGGAGGAGGAGGGAGCGGAGGAGGAGGATCCGAAGTGCAGCTGGTCGAGAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAAGCTGTCTTGTGCAGCCAGTGGCTTCACCTTCAACACTTACGCAATGAACTGGGTGCGGCAGGCACCTGGGAAGGGACTGGAATGGGTCGCCCGGATCAGATCTAAATACAATAACTATGCCACCTACTATGCTGACAGTGTGAAGGATAGGTTCACCATTTCACGCGACGATAGCAAAAACACAGCTTATCTGCAGATGAATAACCTGAAGACCGAGGATACAGCAGTGTACTATTGCGTCAGACACGGCAATTTCGGGAACTCTTACGTGAGTTGGTTTGCCTATTGGGGACAGGGGACACTGGTCACCGTCTCCTCAGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO:113)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW101 without spacer SEQ ID NO: 188]QGQSGQ[MMYCGGNEVLCGPRV][GSSGGSGGSGG][ISSGLLSGRSDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114)CTX122: LC 3954-2001-C225v5Nucleotide Sequence[spacer SEQ ID NO: 180][CTX122 without spacer SEQ ID NO: 189]CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTATTAGCAGTGGTCTGTTAAGCGGTCGTAGCGATAATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 115)Amino Acid Sequence[spacer SEQ ID NO: 178][CTX122 without spacer SEQ ID NO: 190]QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSG][ISSGLLSGRSDNH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 116)CI048: Activated CI011: 3954-0001-C225v5N2970-JF15865-0001-CD3LvHv-H-NpLW023 and OPP022 sequences encoding corresponding masked antibodycomponents are provided herein as “pLW023” and “OPP022, respectively andare summarized in Table 11. Activated pLW023: HC JF15865-0001-CD3LvHv-C225v5N297Q (H-N)Nucleotide SequenceTCCGATAATCATGGCGGCGGTTCTCAGACCGTGGTCACACAGGAGCCCTCACTGACAGTGAGCCCTGGCGGGACCGTCACACTGACTTGTCGCAGTTCAACTGGCGCCGTGACTACCAGCAATTACGCTAACTGGGTCCAGCAGAAACCAGGACAGGCACCACGAGGACTGATCGGAGGAACTAATAAGAGAGCACCAGGAACCCCTGCAAGGTTCTCCGGATCTCTGCTGGGGGGAAAAGCCGCTCTGACACTGAGCGGCGTGCAGCCTGAGGACGAAGCTGAGTACTATTGCGCACTGTGGTACTCCAACCTGTGGGTGTTTGGCGGGGGAACTAAGCTGACCGTCCTGGGAGGAGGAGGAAGCGGAGGAGGAGGGAGCGGAGGAGGAGGATCCGAAGTGCAGCTGGTCGAGAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAAGCTGTCTTGTGCAGCCAGTGGCTTCACCTTCAACACTTACGCAATGAACTGGGTGCGGCAGGCACCTGGGAAGGGACTGGAATGGGTCGCCCGGATCAGATCTAAATACAATAACTATGCCACCTACTATGCTGACAGTGTGAAGGATAGGTTCACCATTTCACGCGACGATAGCAAAAACACAGCTTATCTGCAGATGAATAACCTGAAGACCGAGGATACAGCAGTGTACTATTGCGTCAGACACGGCAATTTCGGGAACTCTTACGTGAGTTGGTTTGCCTATTGGGGACAGGGGACACTGGTCACCGTCTCCTCAGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 117)Amino Acid Sequence[SDNH][GGGS]QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 118)Activated OPP022: LC 3954-0001-C225v5Nucleotide SequenceTCCGATAATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 119)Amino Acid Sequence[SDNH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 120)CI079: 3954-0001-C225v5Fcmt3-h20GG-0001-v16sc-H-NpLW225: HC h20GG-0001-v16sc-C225v5Fcmt3 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW225_without spacer SEQ ID NO: 192]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAATAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 121)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW225 without spacer SEQ ID NO: 193]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDNH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 122)OPP022: LC 3954-0001-C225v5Sequences provided aboveCI090: 3954-0001-C225v5Fcmt4-h20GG-0001-v16sc-H-NpLW233: HC h20GG-0001-v16sc-C225v5Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW233_without spacer SEQ ID NO: 194]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO: 123)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW233 without spacer SEQ ID NO: 195]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDNH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 124)OPP022: LC 3954-0001-C225v5Sequences provided aboveActivated CI090: Activated-3954-0001-C225v5Fcmt4-h20GG-0001-v16sc-H-NActivated pLW233: HC C225v5Fcmt4-h20GG-0001-v16sc (H-N)Nucleic Acid SequenceTCCGATAATCATGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 164Amino Acid Sequence[SDNH]GGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 165)Activated OPP022: 3954-0001-C225v5Nucleic Acid SequenceTCCGATAATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 166)Amino Acid Sequence[SDNH]GSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 167)CI104: 3954-0011-C225v5Fcmt4-h20GG-0011-v16sc-H-NpLW289 and pLW291 sequences encoding corresponding masked antibodycomponents are provided herein as “pLW289” and “pLW291”, respectively andare summarized in Table 11.Activated CI104: 3954-0011-C225v5Fcmt4-h20GG-0011-v16sc-H-NActivated pLW289: HC h20GG-0011-v16sc-C225v5Fcmt4 (H-N)Nucleotide SequenceTCCGATGATCATGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 125)Amino Acid Sequence[SDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL][GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 126)Activated pLW291: LC 3954-0011-C225v5Nucleotide SequenceTCCGATGATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 127)Amino Acid Sequence[SDDH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 128)CI106: CF41-2008-C225v5Fcmt4-h20GG-0011-v16sc-H-NpLW289: HC h20GG-0011-v16sc-C225v5Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW289 without spacer SEQ ID NO: 196]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATGATCATGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 129)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW289 without spacer SEQ ID NO: 197]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 130)pLW246: LC CF41-2008-C225v5Nucleotide Sequence[spacer SEQ ID NO: 176][pLW246 without spacer SEQ ID NO: 198]CAAGGCCAGTCTGGCCAAGGTCTTAGTTGTGAAGGTTGGGCGATGAATAGAGAACAATGTCGAGCCGGAGGTGGCTCGAGCGGCGGCTCTATCTCTTCCGGACTGCTGTCCGGCAGATCCGACCAGCACGGCGGAGGATCCCAAATCCTGCTGACACAGTCTCCTGTCATACTGAGTGTCTCCCCCGGCGAGAGAGTCTCTTTCTCATGTCGGGCCAGTCAGTCTATTGGGACTAACATACACTGGTACCAGCAACGCACCAACGGAAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 131)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW246 without spacer SEQ ID NO: 199]QGQSGQG[LSCEGWAMNREQCRA][GGGSSGGS][ISSGLLSGRSDQH][GGGS]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 132)CI107: 3954-0011-C225v5Fcmt4-h20GG-2006-v16sc-H-NpLW307: HC h20GG-2006-v16sc-C225v5Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW307 without spacer SEQ ID NO: 200]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATATCGAGTGGATTGCTGTCTGGCAGATCTGACGATCACGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 133)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW307 without spacer SEQ ID NO: 201]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][ISSGLLSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 134)pLW291: LC 3954-0011-C225v5Nucleotide Sequence[spacer SEQ ID NO: 180][pLW291 without spacer SEQ ID NO: 202]CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTCTGAGCGGCCGTTCCGATGATCATGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 135)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW291 without spacer SEQ ID NO: 203]QGQSGQ[CISPRGCPDGPYVMY][GSSGGSGGSGGSG][LSGRSDDH][GSSGT]QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136)CI127: SynFcmt4-h20GG-0011-v16sc-H-NpLW334: HC h20GG-0011-v16sc-Synagis ®Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW334 without spacer SEQ ID NO: 204]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATGATCATGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAAGTGACCCTGAGAGAGTCTGGCCCTGCCCTCGTGAAGCCTACCCAGACCCTGACACTGACCTGCACCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGTCTGTGGGCTGGATCAGACAGCCTCCTGGCAAGGCCCTGGAATGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCAGATCCATGATCACCAACTGGTACTTCGACGTGTGGGGAGCCGGCACCACCGTGACAGTGTCATCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 137)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW334 without spacer SEQ ID NO: 205]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 138)pLW139: LC Synagis ®Nucleotide SequenceGACATCCAGATGACCCAGAGCCCCAGCACACTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGTGCCAGCTGAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO: 139)Amino AcidSequenceDIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 140)CI128: SynFcmt4-h20GG-2006-v16sc-H-NpLW335: HC h20GG-2006-v16sc-Synagis ®Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW335 without spacer SEQ ID NO: 206]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATATCGAGTGGATTGCTGTCTGGCAGATCTGACGATCACGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAAGTGACCCTGAGAGAGTCTGGCCCTGCCCTCGTGAAGCCTACCCAGACCCTGACACTGACCTGCACCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGTCTGTGGGCTGGATCAGACAGCCTCCTGGCAAGGCCCTGGAATGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCAGATCCATGATCACCAACTGGTACTTCGACGTGTGGGGAGCCGGCACCACCGTGACAGTGTCATCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 141)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW335 without spacer SEQ ID NO: 207]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][ISSGLLSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 142)pLW139: LC Synagis ®Sequences provided aboveCI135: CF41-2008-C225v5Fcmt4-h20GG-0011-v12sc-H-NpLW352: HC h20GG-0011-v12sc-C225v5Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 208][pLW352 without spacer SEQ ID NO: 209]CAAGGCCAGTCTGGTTCTGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATGATCATGGCGGCGGATCCCAGACGGTAGTGACTCAGGAGCCATCATTTTCTGTCTCTCCTGGAGGTACTGTGACACTCACATGTAGAAGCTCAACTGGTGCAGTCACCACTTCAAATTACGCGAATTGGGTCCAGCAGACCCCTGGGCAGGCTCCGAGAGGGTTGATTGGAGGTACTAACAAACGGGCACCGGGAGTGCCTGATAGGTTTTCCGGTTCTATTCTCGGAAACAAGGCGGCTCTCACGATCACGGGTGCGCAGGCCGACGATGAATCAGACTATTACTGCGCTTTGTGGTACTCAAACCTGTGGGTATTCGGAGGGGGCACCAAGCTGACGGTGTTGGGTGGGGGGGGCTCTGGGGGAGGGGGAAGCGGAGGTGGGGGCAGCGAGGTTCAGCTTGTTGAAAGTGGTGGCGGACTCGTACAACCGGGTGGAAGTCTTAGACTCTCATGTGCAGCATCTGGATTTACTTTTTCTACTTATGCTATGAACTGGGTAAGACAGGCACCGGGGAAAGGGCTGGAATGGGTTGCACGCATTCGATCTAAATACAATAACTATGCTACATACTACGCCGATAGTGTTAAGGATCGATTCACTATATCTCGGGACGACAGTAAGAACTCACTTTACCTGCAGATGAATTCCTTGAAAACTGAGGACACGGCCGTTTATTATTGTGTACGGCACGGGAATTTCGGCAATTCTTACGTTTCCTGGTTCGCCTATTGGGGGCAAGGTACGCTGGTCACGGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 151)Amino Acid Sequence[spacer SEQ ID NO: 176][pLW352 without spacer SEQ ID NO: 210]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDDH][GGGS]QTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 146)pLW246: LC CF41-2008-C225v5Nucleotide SequenceSequences provided aboveAmino Acid SequenceSequences provided aboveCI136: CF41-2008-C225v5Fcmt4-h20GG-0011-v19sc-H-NpLW353: HC h20GG-0011-v19sc-C225v5Fcmt4 (H-N)Nucleotide Sequence[spacer SEQ ID NO: 191][pLW353 without spacer SEQ ID NO: 211]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATGATCATGGCGGCGGTTCTCAGGCCGTTGTTACACAAGAGCCTTCACTTACTGTGTCTCCAGGAGGCACTGTGACACTTACGTGCCGATCCTCTACGGGTGCCGTGACCACAAGCAACTATGCCAACTGGGTCCAGCAGAAGCCAGGTCAAGCGCCTCGAGGTCTGATCGGGGGCACGAATAAACGAGCTCCTGGAACTCCGGCCAGATTTTCTGGGAGTCTTATTGGTGGCAAGGCGGCGTTGACCCTGAGTGGAGCCCAACCGGAAGACGAGGCCGAGTACTACTGCGCCTTGTGGTATTCCAATTTGTGGGTCTTCGGAGGCGGAACAAAGCTCACAGTACTGGGAGGTGGAGGTAGCGGGGGCGGAGGCTCCGGGGGAGGTGGTTCCGAAGTCCAGCTTGTTGAATCAGGTGGGGGCTTGGTACAACCAGGTGGTTCACTGAAGTTGTCCTGTGCAGCGTCCGGATTTACATTTAGTACGTATGCTATGAACTGGGTCAGGCAGGCCAGTGGTAAAGGTCTCGAATGGGTTGGCCGGATAAGGTCAAAGTACAATAATTACGCAACCTACTACGCGGATTCCGTGAAAGACAGGTTCACTATTTCACGAGATGATAGCAAAAATACTGCGTATCTCCAAATGAATAGTCTTAAAACTGAAGACACTGCCGTATATTATTGCACTAGGCACGGCAACTTTGGTAACTCTTATGTTTCTTGGTTCGCATACTGGGGACAAGGAACTTTGGTCACTGTCTCATCTGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 152)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW353 without spacer SEQ ID NO: 212]QGQSGS[GYLWGCEWNCGGITT][GSSGGSGGSGG][LSGRSDDH][GGGS]QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLIGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL[GGGGSGGGGSGGGGS]EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS[GGGGS]QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 148)pLW246: LC CF41-2008-C225v5Sequences provided aboveCI091: 3954-1490DQH-C225v5Fcmt4-h20GG-2008-v16sc-H-NpLW242: HC C225v5Fcmt4-h20GG-2008-v16sc (H-N)Nucleic Acid Sequence[spacer SEQ ID NO: 191][pLW242 without spacer SEQ ID NO: 213]CAAGGCCAGTCTGGATCCGGTTATCTGTGGGGTTGCGAGTGGAATTGCGGAGGGATCACTACAGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTATATCGAGTGGATTGCTGTCTGGCAGATCTGACCAACACGGCGGCGGTTCTCAAACTGTAGTAACTCAAGAACCAAGCTTCTCCGTCTCCCCTGGGGGAACAGTCACACTTACCTGCCGAAGTAGTACAGGTGCTGTTACGACCAGTAACTATGCCAATTGGGTACAACAAACGCCTGGTCAGGCTCCGCGCGGATTGATAGGAGGCACGAATAAACGGGCACCCGGTGTCCCGGACAGATTCAGCGGAAGCATACTCGGTAATAAGGCAGCTCTTACTATCACTGGGGCCCAAGCTGATGATGAAAGTGATTATTATTGTGCGCTCTGGTACAGCAACCTCTGGGTGTTTGGGGGTGGCACGAAACTTACTGTCTTGGGCGGCGGCGGATCAGGGGGAGGTGGCTCTGGAGGAGGAGGCTCAGAAGTCCAACTGGTCGAATCCGGGGGAGGGCTCGTACAGCCGGGTGGGTCCCTCAAACTCTCTTGTGCGGCCTCAGGGTTTACCTTCAGTACATACGCGATGAATTGGGTCCGGCAGGCCAGTGGGAAAGGGCTCGAATGGGTAGGACGAATCCGATCAAAATACAACAACTACGCTACTTATTACGCTGATTCCGTGAAGGACAGATTCACAATATCCCGCGACGATAGCAAGAATACGGCATATCTTCAGATGAATTCTCTTAAAACTGAGGATACCGCTGTGTATTACTGCACAAGACATGGTAATTTTGGAAACTCATATGTCTCTTGGTTCGCTTATTGGGGACAGGGCACGTTGGTTACCGTGTCTAGCGGAGGTGGTGGATCCCAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAAAGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACCAGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGCAAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTTGAAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCAATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 168)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW242 without spacer SEQ ID NO: 214]QGQSGS[GYLWGCEWNCGGITT]GSSGGSGGSGG[ISSGLLSGRSDQH]GGGSQTVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAPRGLIGGTNKRAPGVPDRFSGSILGNKAALTITGAQADDESDYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 169)CX320: 3954-C225v5-2008Nucleic Acid Sequence[spacer SEQ ID NO: 180][CX320 without spacer SEQ ID NO: 215]CAAGGCCAGTCTGGCCAGTGCATCTCACCTCGTGGTTGTCCGGACGGCCCATACGTCATGTACGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGATCCGGTATATCGAGTGGATTGCTGTCTGGCAGATCTGACCAACACGGCAGTAGCGGTACCCAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTAGCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAGCCCGCGCCTGCTGATTAAATATGCGAGCGAAAGCATTAGCGGCATTCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATTATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO: 170)Amino Acid Sequence[spacer SEQ ID NO: 178][CX320 without spacer SEQ ID NO: 216]QGQSGQ[CISPRGCPDGPYVMY]GSSGGSGGSGGSG[ISSGLLSGRSDQH]GSSGTQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 171)CI064: SynN2970-JF15865-0001-hSP34LvHv-H-NpLW138: HC SynN297Q-JF15865-0001-hSP34LvHv-H-NNucleic Acid Sequence[spacer SEQ ID NO: 176][pLW138 without spacer SEQ ID NO: 147]CAAGGCCAGTCTGGCCAAATGATGTATTGCGGTGGGAATGAGGTGTTGTGCGGGCCGCGGGTTGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGAGCGGCCGTTCCGATAATCATGGCGGCGGTTCTCAGACCGTGGTCACACAGGAGCCCTCACTGACAGTGAGCCCTGGCGGGACCGTCACACTGACTTGTCGCAGTTCAACTGGCGCCGTGACTACCAGCAATTACGCTAACTGGGTCCAGCAGAAACCAGGACAGGCACCACGAGGACTGATCGGAGGAACTAATAAGAGAGCACCAGGAACCCCTGCAAGGTTCTCCGGATCTCTGCTGGGGGGAAAAGCCGCTCTGACACTGAGCGGCGTGCAGCCTGAGGACGAAGCTGAGTACTATTGCGCACTGTGGTACTCCAACCTGTGGGTGTTTGGCGGGGGAACTAAGCTGACCGTCCTGGGAGGAGGAGGAAGCGGAGGAGGAGGGAGCGGAGGAGGAGGATCCGAAGTGCAGCTGGTCGAGAGCGGAGGAGGACTGGTGCAGCCAGGAGGATCCCTGAAGCTGTCTTGTGCAGCCAGTGGCTTCACCTTCAACACTTACGCAATGAACTGGGTGCGGCAGGCACCTGGGAAGGGACTGGAATGGGTCGCCCGGATCAGATCTAAATACAATAACTATGCCACCTACTATGCTGACAGTGTGAAGGATAGGTTCACCATTTCACGCGACGATAGCAAAAACACAGCTTATCTGCAGATGAATAACCTGAAGACCGAGGATACAGCAGTGTACTATTGCGTCAGACACGGCAATTTCGGGAACTCTTACGTGAGTTGGTTTGCCTATTGGGGACAGGGGACACTGGTCACCGTCTCCTCAGGAGGTGGTGGATCCCAAGTGACCCTGAGAGAGTCTGGCCCTGCCCTCGTGAAGCCTACCCAGACCCTGACACTGACCTGCACCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGTCTGTGGGCTGGATCAGACAGCCTCCTGGCAAGGCCCTGGAATGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCAGATCCATGATCACCAACTGGTACTTCGACGTGTGGGGAGCCGGCACCACCGTGACAGTGTCATCTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO: 172)Amino Acid Sequence[spacer SEQ ID NO: 178][pLW138 without spacer SEQ ID NO: 153]QGQSGQ[MMYCGGNEVLCGPRV]GSSGGSGGSGG[LSGRSDNH]GGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGGGSQVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 173)pLW139: LC Syn kappaNucleic Acid SequenceGACATCCAGATGACCCAGAGCCCCAGCACACTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACATGCAAGTGCCAGCTGAGCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGAGTTCACCCTGACAATCAGCAGCCTGCAGCCCGACGACTTCGCCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT(SEQ ID NO: 174)Amino Acid SequenceDIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 175)Anti-CD3 scFv variant v12Light chain LV12Heavy chain HV12Sequences provided aboveAnti-CD3 scFv variant v16Light chain LV12Heavy chain HV20Sequences provided aboveAnti-CD3 scFv variant v19Light chain LV19Heavy chain HV20Sequences provided aboveAnti-CD3 scFv variant v26Light chain LV 19Heavy chain HV 12Sequences provided above Vector Construction The heavy and light chains were cloned separately into a mammalian expression vector using standard molecular biology techniques. Briefly, DNA fragments encoding the region of interest were amplified with primers binding to the terminal ends. Overlapping fragments were combined and amplified with flanking primers as needed to build the entire desired region. DNA fragments were subsequently cloned into the expression vector using a commercially available homologous recombination kit (MCLabs, South San Francisco, CA). The mammalian expression vector is a modified version of cDNA™3.1 (+) from Invitrogen with selection marker of G418 or hygromycin. Mutations were introduced using the QuikChange Kit (Agilent, Santa Clara, CA). Expression of AAs and Dually Masked BAAs (BAAs) AAs and BAAs were expressed in mammalian cells using a standard transfection kit (Life Technologies, Grand Island, NY). Briefly, 293 cells were transfected with nucleic acids using a lipid-based system, following the manufacturer's recommended protocol. AAs and dually masked BAAs were purified from cell-free supernatant using Protein A beads (GE, Piscataway, NJ) and concentrated using standard buffer exchange columns (Millipore, Temecula, CA). Example 2. Binding of Dually Masked, Bispecific, AAs to EGFR+ HT-29 Cells and CD3ε+ Jurkat Cells To determine if the described EGFR and CD3ε masking peptides and protease substrates could inhibit binding in the context of a dually masked, bispecific, AA, a flow cytometry-based binding assay was performed. HT-29-luc2 (Caliper) and Jurkat (Clone E6-1, ATCC, TIB-152) cells were cultured in RPMI-1640+glutamax (Life Technologies, Catalog 72400-047), 10% Heat Inactivated-Fetal Bovine Serum (HI-FBS, Life Technologies, Catalog 10438-026), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Catalog 15140-122) according to manufacturer guidelines. The following bispecific, activated antibodies CI048 and CI104 (act-104), and dually masked, bispecific, AAs CI011, CI106, and CI107 were tested. Two versions of SP34 scFv were utilized, namely the scFv in CI011 and CI048 versus the scFv in CI104, CI106, and CI107. Two versions of the EGFR mask were utilized, namely the EGFR mask in CI011 and CI107 versus the EGFR mask in CI106. Two versions of the CD3 mask were utilized, namely the CD3 mask in CI011 versus the CD3 mask in CI106 and CI107. HT29-luc2 cells were detached with Versene™ (Life Technologies, Catalog 15040-066), washed, plated in 96 well plates at 150,000 cells/well, and re-suspended in 50 μL of primary antibody. Titrations started at the concentrations indicated inFIGS.1A-1Bfollowed by 3-fold serial dilutions in FACS Stain Buffer+2% FBS (BD Pharmingen, Catalog 554656). Cells were incubated at 4° C. with shaking for about 1 hour, harvested, and washed with 2×200 μL of FACS Stain Buffer. Cells were resuspended in 50 μL of Alexa Fluor 647 conjugated anti-Human IgG Fc (10 μg/ml, Jackson ImmunoResearch, Product 109-606-008) and incubated at 4° C. with shaking for about 1 hour. HT29-luc2 were harvested, washed, and resuspended in a final volume of 60 μL of FACS Stain Buffer containing 2.5 μg/ml 7-AAD (BD Biosciences, Catalog 559925). Cells stained with secondary antibody alone were used as a negative control. Data was acquired on a MACSQuant® Analyzer 10 (Miltenyi) and the median fluorescence intensity (MFI) of viable cells was calculated using FlowJo® V10 (Treestar). Background subtracted MFI data was graphed in GraphPad Prism 6 using curve fit analysis. Jurkats growing in suspension were harvested, washed, plated in 96 well plates at 150000 cells/well and resuspended in 50 μl of primary antibody. Staining and data acquisition were carried out as described for HT29-luc2 cells above. FIG.1Ademonstrates that incorporation of the h20GG CD3ε masking peptide into the EGFR masked BAAs CI106 and CI107 significantly reduced binding to Jurkat cells relative to CI011. In some embodiments, the reduction in binding to Jurkat cells was more than 5,000-fold. In some embodiments, an scFv of the disclosure also led to reduced binding. A reduction in binding to EGFR+ HT29-luc2 cells was also evident for CI106 and CI107 relative to CI011 (FIG.1B). In some embodiments, the reduction in binding to EGFR+HT29-luc2 cells was more than 1,000-fold. InFIG.1AandFIG.1B, the dually masked, BAAs exhibit reduced binding relative to the activated bispecific antibodies. Example 3. EGFR-Dependent Cytotoxicity of Dually Masked BAAs To determine if the CD3ε and EGFR masks and the protease substrates in CI106 and CI107 could further attenuate cell killing relative to CI011 and CI040, a cytotoxicity assay was performed. Human PBMCs were purchased in frozen aliquots (HemaCare) and co-cultured with EGFR expressing HT29-luc2 cells at a ratio of 10:1 in RPMI-1640+glutamax supplemented with 5% heat inactivated human serum (Sigma, Catalog H3667). Titrations of the following bispecific, activated antibodies and dually masked BAAs were tested: CI011, CI040, activated CI104, CI106, and CI107. In addition, non-EGFR binding, masked bispecific, AAs CI127 and CI128 were used to demonstrate the EGFR dependence of cytotoxicity. After 48 hours, cytotoxicity was evaluated using the ONE-Glo™ Luciferase Assay System (Promega, Catalog E6130). Luminescence was measured on the Infinite M200 Pro (Tecan). Percent cytotoxicity was calculated and plotted in GraphPad PRISM with curve fit analysis. EGFR receptor number on a panel of cell lines was quantified by flow cytometry using QIFIKIT (Dako). FIG.2Ademonstrates that killing of EGFR+HT29-luc2 cells was further attenuated by CI106 and CI106 relative to CI011 and CI040.FIG.2Bshows that no cytotoxicity was observed when cells were treated with CI127 and CI128 demonstrating the dependence of EGFR targeting for cell killing. Additionally,FIG.2Bdepicts a more than 300,000 fold EC50 shift of the dually masked bispecific antibodies CI106 and CI107 relative to the protease activated bispecific antibody act-104.FIG.2Cdepicts the EGFR receptor number on a panel of cell lines that includes HT29. The approximate EGFR receptor number on HT29 cells was 75,000, indicating that high antigen density was not required for potent cytotoxicity of the tested antibodies. Example 4. Primary T Cell Activation by Dually Masked BAAs To determine if the CD3ε and EGFR masks in CI106 and CI107 could attenuate primary T cell activation relative to CI011 and CI040, a flow cytometry assay was performed. Human PBMCs and U266 cells were co-cultured according to the conditions described in Example 3. After a 48 hour incubation, cells were pelleted, media was removed, and cells were resuspended in 50 μl of a cocktail containing anti-CD45 VioBlue® (Miltenyi, Catalog 130-002-880), anti-CD8 APC-Vio770 (Miltenyi, Catalog 130-096-561) and anti-CD69 PE (BD Pharmingen, Catalog 555531) in FACS Stain Buffer+2% FBS. Cells were stained for 1 h at 4° C. with shaking, harvested, washed, and re-suspended in a final volume of 60 μL FACS Buffer. Data was acquired on a MACSQuant® Analyzer 10 (Miltenyi) and activation was quantified in FlowJo® V10 (Treestar) as the percentage of CD8+ T cells with expression of CD69 above the PE isotype control. Data was plotted in GraphPad PRISM 6 with curve fit analysis. FIG.3Ademonstrates that activation of primary CD8+ T cells was attenuated by CI106 and CI107, relative to CI011 and CI040.FIG.3Bdemonstrates that dually masked antibodies display a shifted dose response curve for T cell activation relative to protease activated bispecific antibody act-104 indicating that masking attenuates T cell activation. Example 5. Dually Masked, Bispecific, AAs of the Embodiments Induced Regression of Established HT29-Luc2 Tumors in Mice In this example, dually masked BAAs CI106 and CI107 targeting EGFR and CD3ε were analyzed for the ability to induce regression or reduce growth of established HT-29-Luc2 xenograft tumors in human T-cell engrafted NSG mice. The human colon cancer cell line HT29-luc2 was obtained from Perkin Elmer, Inc., Waltham, MA (formerly Caliper Life Sciences, Inc.) and cultured according to established procedures. Purified, frozen human PBMCs were obtained from Hemacare, Inc., Van Nuys, CA NSG™ (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were obtained from The Jackson Laboratories, Bar Harbor, ME On day 0, each mouse was inoculated subcutaneously at the right flank with 2×106HT29-luc2 cells in 100 μL RPMI+Glutamax, serum-free medium. Previously frozen PBMCs from a single donor were administered (i.p.) on day 3 at a CD3+ T cell to tumor cell ratio of 1:1. When tumor volumes reached 200 mm3(approximately day 12), mice were randomized, assigned to treatment groups and dosed i.v. according to Table 12. Tumor volume and body weights were measured twice weekly. TABLE 12Groups and doses for HT2-9luc2 xenograft study.GroupCountTreatmentDose (mg/kg)17PBSN/A27CI1060.537CI1061.547CI1070.557CI1071.5 FIG.4, which plots tumor volume versus days post initial treatment dose, demonstrates a dose-dependent effect of CI106 and CI107 dually masked, bispecific, AAs on the growth of HT29-luc2 xenograft tumors. The most efficacious dose tested was 1.5 mg/kg, resulting in tumor regression. Statistical analysis (RMANOVA with Dunnett's vs. PBS control) was carried out in GraphPad PRISM. *=p<0.05, **=p<0.01, ****=p<0.0001. Example 6. Dually Masked, Bispecific, AAs and Bispecific Antibodies of the Embodiments Reduce Growth of Established HCT116 Tumors in Mice In this example, bispecific antibody, activated CI104, and dually masked BAAs CI106 and CI107 targeting EGFR and CD3ε were analyzed for the ability to induce regression or reduce growth of established HCT116 xenograft tumors in human T-cell engrafted NSG mice. The human colon cancer cell line HCT116 was obtained from ATCC and was cultured in RPMI+Glutamax+10% FBS according to established procedures. The tumor model was carried out as described in Example 5. Mice were dosed according to Table 13. TABLE 13Groups and doses for HCT116 xenograft study.GroupCountTreatmentDose (mg/kg)18PBSN/A28CI1060.338CI1061.048CI1070.358CI1071.068act-1040.3 FIG.5which plots tumor volume versus days post initial treatment dose, demonstrated a dose-dependent effect of CI106 and CI107 dually masked bispecific, AAs on the growth of HCT116 xenograft tumors. The most efficacious dose tested was 1.0 mg/kg, resulting in tumor stasis. Act-104 dosed at 0.3 mg/kg also resulted in tumor stasis, demonstrating a 3 fold difference in efficacy between dually masked and protease activated bispecific antibodies. Statistical analysis (RMANOVA with Dunnett's vs. PBS control) was carried out in GraphPad PRISM. *=p<0.05, **=p<0.01, ****=p<0.0001. Example 7. Cross Reactivity of Dually Masked Bispecific, AAs to Cynomolgus Monkey T Cells To confirm that Cynomolgus monkey is a relevant toxicity species, protease activated CI104, CI106 and CI107 were used in a flow cytometry based cell binding assay and a HT29-luc2 cytotoxicity assay using Cynomolgus pan T cells (BioreclamationIVT) and the potency was compared to human PBMCs. Protocol was as described in Examples 2 and 3. FIG.6AandFIG.6Bdemonstrate that the EC50s of the tested dually masked and protease activated bispecific antibodies in a cytotoxicity assay are similar when either human (6A) or cyno (6B) effector cells are used.FIG.6CandFIG.6Ddemonstrate that binding of the protease-activated and dually masked antibodies to human (6C) and cyno (6D) T cells is similar. Therefore, cynomolgus monkey was determined to be a relevant species for tolerability studies. Example 8. Mutations in the Fc Region Affect Tolerability of Dually Masked, Bispecific, AAs in Cynomolgus Monkeys In this example, CI079 and CI090 were dosed at 600, μg/kg in naïve cynomolgus monkeys (n=1) to assess tolerability. The starting dose of 600 μg/kg was chosen based on the MTD of CI011 as previously established. The monkeys were of Chinese origin and ranged in weight from 2.5 to 4 kg. Each study animal was monitored for a minimum of 7 days. Tolerability was evaluated based on clinical signs, body weight, and food consumption. This study was conducted in compliance with standard operating procedures at SNBL USA, Ltd. (Everett, WA). Table 14 describes clinical observations following dosing of CI079 and CI090 dually masked, bispecific, AAs (BAAs) that differ only in their Fc regions (Table 15). CI079 contains Fc mutations L234F, L235E, and P331S. CI090 contains those Fc mutations and N297Q mutation. No clinical observations were noted following dosing of CI090 at 600 μg/kg, whereas, emesis was noted in the first 24 hours following dosing of CI079, demonstrating that mutations in the Fc region contribute to tolerability of these molecules. TABLE 14BAADose (μpk)ObservationsCI079600emesis during 1st24 hoursCI090600no clinical observations TABLE 15EGFR Mask &CD3 MaskBAASubstrate&SubstrateFcCI0793954 0001h20GG 0001Fcmt3CI0903954 0001h20GG 0001Fcmt4 Example 9. Tolerability of Dually Masked BAAs in Cynomolgus Monkeys In this example, CI106 and CI107 were dosed at 600, 2000, 4000 μg/kg (CI107 only), or 6000 μg/kg (CI107 only) to establish the maximum tolerated dose (MTD) following a single IV bolus administration to naïve cynomolgus monkeys (n=1). The starting dose of 600 μg/kg was chosen based on the MTD of CI011 as previously established. The monkeys were of Chinese origin and ranged in weight from 2.5 to 4 kg. Each study animal was monitored for a minimum of 7 days. Tolerability was evaluated based on clinical signs, body weight, food consumption and laboratory analyses that included serum chemistry, hematology, cytokine analysis, and flow cytometry to evaluate T cell activation. Blood was collected for standard serum chemistry and hematology analysis once during acclimation and at pre-dose, 48 h, 72 h (hematology only), and 7 days post dose. Blood was collected for cytokine analysis pre-dose and at 1 h, 4 h, 8 h, and 24 h post dose. Flow cytometry was performed on peripheral blood pre-dose, 72 h, and 7 days post dose. This study was conducted in compliance with standard operating procedures at SNBL USA, Ltd. (Everett, WA). CI107 dosed at 6000 μg/kg was fatal within 24 hours post dose. In the other groups, abnormal clinical signs including emesis and reduced food intake were observed in cynos treated with CI106 and CI107 at doses of 2000 μg/kg and above. These findings, when present, were transient and generally confined to the 48 h post-dose period. Serum chemistry findings at these doses included mild elevations of alanine transaminase (ALT) and aspartate aminotransferase (AST) at 48 h that did not exceed normal ranges. In CI107 treated animals at 2000 and 4000 μg/kg, total bilirubin increased outside of normal range at 48 hours and was fully reversed by day 8. In both CI106 and CI107 treated animals, transient increases in serum cytokines IL-2, IL-6, and IFNg were observed after dosing and were resolved by 24 h post dose. An increase in the percentage of T cells expressing CD69, Ki67, and PD-1 was observed at 72 h post-dose and, generally, the percentage of positive cells was greater for CI107 treated animals. FIGS.7A-7Cdepict pre-dose, 48 h, and 7 days post-dose serum concentration of ALT (7A), AST (7B), and total bilirubin (7C). With the exception of total bilirubin at 2000 and 4000 μg/kg, all values are within established normal ranges for cynomolgus monkeys. Only pre-dose data was available for CI107 at 6000 μg/kg, and was not included. FIGS.8A-8Cplot the increase in serum cytokine levels for IL-2 (8A), IL-6 (8B), and IFNg (8C). FIGS.9A-9Cdepict T cell activation as measured by CD69 (9A), Ki67 (9B), and PD-1 (9B) expression on CD4+ T cells. Example 10. Dually Masked, Bispecific, AAs are Safer in Cynomolgus Monkeys than Activated Bispecific Antibodies In this example, protease activated CI104 and dually masked CI106 and CI107 were dosed in cynomolgus monkeys (n=1) at 60, 180 (activated CI104 only), 600, 2000, 4000 μg/kg (CI107 only), or 6000 μg/kg (CI107 only) to compare the tolerability of masked and unmasked antibodies following a single IV bolus. Tolerability evaluation and blood collections were as described in Example 9. Dually masked, BAAs CI106 and CI107 were tolerated at 30-60 fold higher dose level than the protease activated, bispecific antibody. FIGS.10A-Eplot dose dependent increases in AST at 48 h post dose (10A), ALT at 48 h post dose (10B), IL-6 at 8 h post dose (10C), IFNg at 8 h post dose (10D), and Ki67 at 72 h post dose (10E). The dose response curve for all parameters was shifted for the dually masked antibodies indicating improved tolerability and decreased pharmacodynamics effects relative to the protease activated bispecific antibody. In some embodiments, the IL-6 dose response curve was shifted by more than 60-fold. Example 11. Tolerability of EGFR Binding, Dually Masked, Bispecific, AAs is Dependent on EGFR In this example, dually masked bispecific antibody, CI107, targeting EGFR and CD3ε and CI128, targeting RSV and CD3ε were dosed at 2000 μg/kg in cynomolgus monkeys (n=1). Tolerability evaluation and blood collections were as described in Example 9 above. There was no effect of CI128 on measures of acute organ toxicity (total bilirubin) and T cell activation (IL-6, PD-1) demonstrating that the toxicity observed in cyno was dependent on EGFR binding. These data also demonstrate that CD3ε binding alone was not sufficient to induce toxicity. FIGS.11A-11Ccompares the effects of EGFR binding CI107 and non EGFR binding CI128 on increases in total bilirubin (11A), IL-6 (11B), and PD-1 expressing CD4+ T cells (11C). Example 12. Humanization of Anti-CD3 Variants v12, v16, and v19 which have Different Affinities and Potencies This example describes anti-CD3 antibody variants v12, v16, and v19. These three variants were derived from the parent antibody hSP34 Humanization of the anti-human CD3 single-chain variable fragment (scFv) was performed by selectively mutating the framework. Briefly, CDRs were grafted into a series of light chain (LC) and heavy chain (HC) human IgG scaffolds and a number of amino acids in the variable region framework was selectively mutated. Immunoglobulins were expressed in all possible combinations of LC and HC, and then evaluated for expression level, percent monomer, and CD3 affinity using ELISA and on-cell binding to Jurkat cells. The variable regions of desirable combinations were expressed as scFv in the bispecific antibody (TCB) format and then evaluated for expression levels, percent monomer, CD3 affinity and function in cell cytotoxicity assays. The affinity of v12, v16, and v19 variant was measured using surface plasmon resonance (SPR). Surfaces were HC200m, carboxylated hydrogel based on a linear, synthetic polycarboxylate. Surface channels were activated with a standard EDC/NHS amine coupling protocols. Channels 1 and 2 were blank, Channels 3 and 4 were various anti-human CD3 antibodies. Surfaces were generated by diluting v12, v16, v19 and MM194 antibodies to 5 μg/ml in 1.0 mL 10 mM Sodium Acetate pH 4.5. Kinetic analysis was performed in PBST (10 mM Sodium Phosphates, pH 7.4, 150 mM Sodium Chloride, 0.05% TWEEN®-20) at 20° C. Regeneration was a series of three injections; a single 5 μl injection of 20 mM Sodium Hydroxide followed by two 5 μL injection of 10 mM Sodium Hydroxide freshly-made. The configuration was run with an inverse 3-fold serial dilution alternating with buffer blanks. Human CD3egFc was from Sino Biological Inc., (Beijing, China, Catalog #CT041-H0305H) reconstituted with sterile water from a lyophilized formulation based on PBS and stabilizers. Serial dilutions with the analyte in solution from concentrations starting at 300 nM or 100 nM human CD3. Processing was done with Scrubber software. These variants were also engineered using described methods into dually masked, bispecific, AAs targeting EGFR and CD3 and used in an in vitro cytotoxicity assay as described in example 3. FIG.12Adepicts the affinity measurements of v12, v16, and v19 relative to hSP34. V12 was the highest affinity at 12 nM while v16 was the lowest affinity at 70 nM. FIG.12Bdepicts the cytotoxicity of activated or dually masked, bispecific antibodies on HT29-luc2 cells. There were slight differences in the potency of cell killing of the activated molecules with v16 being the most potent. There are also slight differences in protection against cell killing for the dually masked molecules. Example 13. Dually Masked BAAs Enable Extended PK in Cynomolgus Monkeys In this example, protease activated, bispecific antibody act-104 and dually masked, bispecific antibody CI107 were dosed at 60 μg/kg, 180 μg/kg (act-104) or 2000 μg/kg (CI107) in cynomolgus monkey. Plasma samples were collected at 5 min (act-104 only), 30 min, 4 h (act-104 only), 24 h, 48 h (act-104 only), 96 h, and 168 h. Plasma concentration was measured by ELISA using an anti-idiotype antibody to capture, a horseradish peroxidase (HRP) labeled anti-human IgG (Fc) for detection, and visualized using 3,3′,5,5′-tetramethylbenzidine (TMB). Plasma concentration values were interpolated from a standard curve and plotted using GraphPad PRISM. Area under the curve (AUC) analysis was also performed. FIG.13depicts the extended PK of the dually masked molecule, CI107, relative to the protease activated molecule, act-104. Exposure (AUC) of CI107 was 448 day*nM and act-104 (60 μg/kg) was 0.04 day*nM representing a greater than 10,000 fold difference in plasma exposure. Example 14. Sensitivity to Protease Cleavage of Dually Masked, Bispecific, AAs Correlates to Tumor Efficacy and Tumor T Cell Infiltration This example describes anti-tumor efficacy and tumor T cell infiltration in a HT29-luc2 xenograft model. The model was carried out as described in example 5. In the tumor T cell infiltration study, mice received a single dose of test article and tumors were harvested 7 days post dose. Formalin fixed paraffin embedded (FFPE) blocks were created to use for histology. Test articles used are CI011, CI020 (a dually masked bispecific antibody devoid of a cleavable substrate), CI040, and CI048. Protease sensitivity and substrate cleavability of the test articles is as follows: CI040>CI011>CI020. Mice were dosed according to Table 16. TABLE 16Groups and doses for HT29-luc2 xenograft study.GroupCountTreatmentDose (mg/kg)Study18PBSN/AEfficacy28CI0110.3Efficacy38CI0200.3Efficacy48CI0400.3Efficacy58CI0480.3Efficacy65PBSN/AInfiltration75CI0111.0Infiltration85CI0201.0Infiltration95CI0401.0Infiltration105CI0481.0Infiltration FIG.14Adepicts efficacy in a HT29-luc2 tumor intervention model in PBMC engrafted NSG mice. Anti-tumor potency in this example correlates to protease sensitivity and substrate cleavability of the test articles, with the most efficacious test article being the fully protease activated CI048. FIG.14Bdepicts staining of tumor sections for CD3 (dark staining) as a measure of the degree of T cell infiltration into tumors. Tumor T cell infiltration correlates with protease sensitivity and substrate cleavability of the test articles. Example 15. Second Generation Dually Masked, Bispecific, AAs are Safer in Cynomolgus Monkeys than First Generation Molecules In this example, cynomolgus tolerability data was compared for CI011, CI040, CI048 (first generation molecules), act-104, CI106, and CI107 (second generation molecules). Data presented in this example was compiled from two cyno tolerability studies. Protease activated CI104 and CI048 were dosed in cynomolgus monkeys at 20 (CI048 only), 60 or 180 μg/kg (act-104 only). Dually masked CI011, CI040, CI106 and CI107 were dosed at 600, 2000, 4000 (CI107 only), or 6000 (CI107 only) μg/kg to compare the tolerability of dually masked and activated bispecific antibodies following a single IV bolus. Tolerability evaluation was as described in Example 8. Table 17 summarizes the clinical observations following a single dose of test article. Second generation, protease activated bispecific antibody act-104 was tolerated at 2-fold higher dose than first generation protease activated bispecific antibody CI048. CI106 and CI107 were tolerated at 30-60-fold higher dose than first generation antibodies CI011 and CI040. TABLE 17DoseBAA(μg/kg)Clinical ObservationsCI04820 (n = 2)1. Emesis and hunching in 1st24 hrs2. None60Emesis at 4 hr; hunching lasting 4 days and inappetencelasting 2 days; 10% weight lossCI011600 (n = 3)1. None2. Emesis at 4 hrs, inappetence on day 43. Hunching and inappetence through day 5.5% weight loss2000Emesis days 1-2, inappetence days 2-4CI040600 (n = 2)1. Emesis at 4 hr2. Emesis within 12 hrs, inappetence on day 42000Emesis days 1-2, moribund and euthanized day 2act-10460Emesis and hunching in 1st24 hrsCI104180Severe emesis; hunching, paleness, inappetence lasting3 daysCI106600none2000noneCI107600None2000Emesis, once in 1st24 hrs4000Emesis, multiple incidences in 1st24 hrs6000Severe emesis; bloody diarrhea Example 16. Evaluation of Masking Efficiencies of Activatable Anti-EGFR Antibodies Masking the ability of an antibody to bind to its antigen is an example of inhibition of binding and is enumerated herein as masking efficiency (ME). Masking efficiency can be calculated as the KDfor binding of the AA divided by the KDfor binding of the antibody measured under the same conditions. The extent of inhibition is dependent on the affinity of the antibody for its antigen, the affinity of the inhibitor (i.e., the masking moiety) for the antibody and the concentration of all reactants. Local concentrations of the tethered masking moiety peptide (inhibitor) is very high in the AA context, on the order of 10 mM, therefore moderate affinity peptides would effectively mask AA antigen binding. The general outline for this assay is as follows: Nunc, Maxisorp™ plates are coated overnight at 4° C. with 100 μl/well of a 1 μg/mL solution of human EGFR (R and D Systems) in PBS, pH 7.4. Plates are washed 3×PBST (PBS, pH 7.4, 0.05% TWEEN®-20), and wells are blocked with 200 μl/well, 10 mg/mL BSA in PBST for 2 hours at RT. Plates are washed 3×PBST (PBS, pH 7.4, 0.05% TWEEN®-20). Dilution curves can be prepared, in 10 mg/mL BSA in PBST, as illustrated below in Table 18. In this example the highest concentrations are 10 nM for the parental antibody and 400 nM for the AAs, however, the top concentrations can be increased or decreased to give full saturation binding curves for AAs with stronger or weaker masking. TABLE 18Plate layout for masking efficiency assay.[Antibody] = nM[AA 1] = nM[AA 2] = nM[AA 3] = nMColumns 1-3Columns 4-6Columns 7-9Columns 10-12A10400400400B2100100100C0.625252525D0.1566.256.256.25E0.0391.561.561.56F0.00970.390.390.39G0.00240.0980.0980.098H0.00060.0240.0240.024 The binding solutions are added to the plates, which are then are incubated for 1 hour at room temperature, and then washed 3×PBST (PBS, pH 7.4, 0.05% TWEEN®-20). 100 μl/well 1:4000 dilution goat-anti-human IgG (Fab specific, Sigma cat #A0293) in 10 mg/mL BSA in PBST is added, and the plate is incubated for 1 hour at room temperature. The plate is developed with TMB and IN HCL. Shown inFIG.15andFIG.16are plots of binding isotherms for activatable anti-EGFR C225v5 antibodies of the disclosure, for activatable anti-EGFR antibody 3954-2001-C225v5 described above, and for anti-EGFR antibody C225v5. Plots are generated in GraphPad PRISM and the data are fit to a model of single site saturation and a KDis determined. The KDand ME values are provided in Table 19. TABLE 19KDand ME values calculated from the bindingisotherms shown in FIG. 15 and FIG. 16.KD(nM)ME3954-2001-C225v513130CF08-2001-C225v50.22CF13-2001-C225v50.33CF19-2001-C225v527270CF22-2001-C225v578780CF41-2001-C225v552520CF46-2001-C225v5110 Example 17. Pharmacokinetics of Dually Masked BAAs in Cynomolgus Monkeys In this example, dually masked bispecific antibody CI107 was dosed at 600 μg/kg, 2000 μg/kg, or 4000 μg/kg in cynomolgus monkey. Plasma samples were collected at 30 min, 4 h (600 μg/kg only), 24 h, 48 h (600 and 4000 μg/kg only), 96 h, and 168 h. Plasma concentration was measured by ELISA as in example 13. FIG.20depicts the PK of the dually masked BAA CI107 following a single i.v. dose of either 600, 2000, or 4000 μg/kg. Example 18. EGFR-Dependent Cytotoxicity of Dually Masked, Bispecific, Activatable Antibodies To determine whether the anti-CD3ε, CD3 mask, and protease substrates in CI090 and CI091 could further attenuate cell killing relative to CI011, a cytotoxicity assay was performed using the method described in Example 3. Titrations of the following bispecific, activated antibodies and dually masked, bispecific, activatable antibodies were tested: CI011, CI090, CI091, activated CI090, and CI048. In addition, non-EGFR binding, bispecific, activatable antibody CI064 was used to demonstrate the EGFR dependence of cytotoxicity. FIG.21demonstrates that killing of EGFR+HT29-luc2 cells was further attenuated by CI090 and CI091 relative to CI011, however the potency of the activated CI090 is equivalent to CI048. The EC50 shift of CI090 and CI091 relative to activated bispecific antibody is increased representing increased masking efficiency of these molecules relative to CI011. No cytotoxicity was observed when cells were treated with CI064, demonstrating the dependence of EGFR targeting for cell killing. Example 19. Primary T Cell Activation by Dually Masked, Bispecific, Activatable Antibodies To determine if the anti-CD3ε, CD3 mask, and protease substrates in CI090 and CI091 could attenuate primary T cell activation relative to CI011, a flow cytometry assay was performed as described in Example 4. FIG.22demonstrates that activation of primary CD8+ T cells was further attenuated by CI090 and CI091, relative to CI011. Example 20. Dually Masked, Bispecific, Activatable Antibodies of the Embodiments Induced Regression of Established HT29-luc2 Tumors in Mice In this example, bispecific activatable antibodies CI011, CI090, and CI091 were analyzed for the ability to induce regression or reduce growth of established HT-29-Luc2 xenograft tumors in human PBMC engrafted NSG mice. The method is as described in Example 5. TABLE 20Groups and doses for HT-29-luc2 xenograft study.GroupCountTreatmentDose (mg/kg)17PBSN/A27CI0111.037CI0901.047CI0911.0 FIG.23, which plots tumor volume versus days post initial treatment dose, demonstrates that a 1 mg/kg, weekly dose induced tumor regression for all bispecific activatable antibodies tested. Example 21. Dually Masked, Bispecific, Activatable Antibodies Elicit Less Cytokine Release than Activated Bispecific Antibodies in Cynomolgus Monkey In this example, protease activated CI104, and dually masked CI011, CI090, and CI091 were dosed in cynomolgus monkeys (n=1) at 0.06, 0.18 (activated CI104), or 600 mg/kg (CI011, CI090, CI091). Blood was collected for cytokine analysis pre-dose and at 1 h, 4 h, 8 h, and 24 h post dose. Samples were analyzed using Life Technologies Monkey Magnetic 29-Plex Panel Kit (Product No. LCP0005M). Data was acquired on a BioRad BioPlex 200 instrument. This analysis was conducted in compliance with standard operating procedures at SNBL USA, Ltd. (Everett, WA). FIG.24plots IL-6 levels at 8 h post dose. The dually masked bispecific activatable antibody CI011 induces significantly less cytokine release than activated CI104 even when delivered at a higher dose, demonstrating the effect of masking on T cell activation. IL-6 is even further reduced in CI090 and CI091 treated animals reflecting the increased masking efficiency of these molecules relative to CI011. Other Embodiments While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following. | 237,755 |
11859011 | DETAILED DESCRIPTION OF THE INVENTION I. Definitions In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents. By “ablation” herein is meant a decrease or removal of activity. Thus for example, “ablating FcγR binding” means the Fc region amino acid variant has less than 50% starting binding as compared to an Fc region not containing the specific variant, with less than 70-80-90-95-98% loss of activity being preferred, and in general, with the activity being below the level of detectable binding in a Biacore assay. Of particular use in the ablation of FcγR binding are those shown inFIG.16. By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is correlated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to an increase in ADCC activity. By “ADCP” or antibody dependent cell-mediated phagocytosis as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. By “modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. By “amino acid modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. For clarity, unless otherwise noted, the amino acid modification is always to an amino acid coded for by DNA, e.g. the 20 amino acids that have codons in DNA and RNA. By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution. By “amino acid insertion” or “insertion” as used herein is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, −233E or 233E designates an insertion of glutamic acid after position 233 and before position 234. Additionally, −233ADE or A233ADE designates an insertion of AlaAspGlu after position 233 and before position 234. By “amino acid deletion” or “deletion” as used herein is meant the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233# or E233( ) designates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233# designates a deletion of the sequence GluAspAla that begins at position 233. By “variant protein” or “protein variant”, or “variant” as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification. Protein variant may refer to the protein itself, a composition comprising the protein, or the amino sequence that encodes it. Preferably, the protein variant has at least one amino acid modification compared to the parent protein, e.g. from about one to about seventy amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent. As described below, in some embodiments the parent polypeptide, for example an Fc parent polypeptide, is a human wild type sequence, such as the Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as “parent polypeptides”, for example the IgG1/2 hybrid ofFIG.19. The protein variant sequence herein will preferably possess at least about 80% identity with a parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. Variant protein can refer to the variant protein itself, compositions comprising the protein variant, or the DNA sequence that encodes it. Accordingly, by “antibody variant” or “variant antibody” as used herein is meant an antibody that differs from a parent antibody by virtue of at least one amino acid modification, “IgG variant” or “variant IgG” as used herein is meant an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by virtue of at least one amino acid modification, and “immunoglobulin variant” or “variant immunoglobulin” as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification. “Fc variant” or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain. The Fc variants of the present invention are defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S is an Fc variant with the substitution serine at position 434 relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. Likewise, M428L/N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, 428L/434S is the same Fc variant as M428L/N434S, and so on. For all positions discussed in the present invention that relate to antibodies, unless otherwise noted, amino acid position numbering is according to the EU index. The EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.) The modification can be an addition, deletion, or substitution. Substitutions can include naturally occurring amino acids and, in some cases, synthetic amino acids. Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated by reference. As used herein, “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The peptidyl group may comprise naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA 89(20):9367 (1992), entirely incorporated by reference). The amino acids may either be naturally occurring or synthetic (e.g. not an amino acid that is coded for by DNA); as will be appreciated by those in the art. For example, homo-phenylalanine, citrulline, ornithine and noreleucine are considered synthetic amino acids for the purposes of the invention, and both D- and L-(R or S) configured amino acids may be utilized. The variants of the present invention may comprise modifications that include the use of synthetic amino acids incorporated using, for example, the technologies developed by Schultz and colleagues, including but not limited to methods described by Cropp & Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101 (2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et at, 2003, Science 301(5635):964-7, all entirely incorporated by reference. In addition, polypeptides may include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels. By “residue” as used herein is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in the human antibody IgG1. By “Fab” or “Fab region” as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of a single antibody. As will be appreciated by those in the art, these generally are made up of two chains. By “IgG subclass modification” or “isotype modification” as used herein is meant an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, because IgG1 comprises a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an IgG subclass modification. By “non-naturally occurring modification” as used herein is meant an amino acid modification that is not isotypic. For example, because none of the IgGs comprise a serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification. By “amino acid” and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids that are coded for by DNA and RNA. By “effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC. By “IgG Fc ligand” as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include but are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirely incorporated by reference). Fc ligands may include undiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. By “Fc ligand” as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex. By “Fc gamma receptor”, “FcγR” or “FcqammaR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes. By “FcRn” or “neonatal Fc Receptor” as used herein is meant a protein that binds the IgG antibody Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, the functional FcRn protein comprises two polypeptides, often referred to as the heavy chain and light chain. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn protein refers to the complex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRn variants used to increase binding to the FcRn receptor, and in some cases, to increase serum half-life, are shown in the Figure Legend ofFIG.83. By “parent polypeptide” as used herein is meant a starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it. Accordingly, by “parent immunoglobulin” as used herein is meant an unmodified immunoglobulin polypeptide that is modified to generate a variant, and by “parent antibody” as used herein is meant an unmodified antibody that is modified to generate a variant antibody. It should be noted that “parent antibody” includes known commercial, recombinantly produced antibodies as outlined below. By “Fc” or “Fc region” or “Fc domain” as used herein is meant the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain and in some cases, part of the hinge. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcγR receptors or to the FcRn receptor. By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portion of an antibody. By “Fc fusion protein” or “immunoadhesin” herein is meant a protein comprising an Fc region, generally linked (optionally through a linker moiety, as described herein) to a different protein, such as a binding moiety to a target protein, as described herein. In some cases, one monomer of the heterodimeric antibody comprises an antibody heavy chain (either including an scFv or further including a light chain) and the other monomer is a Fc fusion, comprising a variant Fc domain and a ligand. In some embodiments, these “half antibody-half fusion proteins” are referred to as “Fusionbodies”. By “position” as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for antibody numbering. By “target antigen” as used herein is meant the molecule that is bound specifically by the variable region of a given antibody. A target antigen may be a protein, carbohydrate, lipid, or other chemical compound. A wide number of suitable target antigens are described below. By “strandedness” in the context of the monomers of the heterodimeric antibodies of the invention herein is meant that, similar to the two strands of DNA that “match”, heterodimerization variants are incorporated into each monomer so as to preserve the ability to “match” to form heterodimers. For example, if some pI variants are engineered into monomer A (e.g. making the pI higher) then steric variants that are “charge pairs” that can be utilized as well do not interfere with the pI variants, e.g. the charge variants that make a pI higher are put on the same “strand” or “monomer” to preserve both functionalities. Similarly, for “skew” variants that come in pairs of a set as more fully outlined below, the skilled artisan will consider pI in deciding into which strand or monomer that incorporates one set of the pair will go, such that pI separation is maximized using the pI of the skews as well. By “target cell” as used herein is meant a cell that expresses a target antigen. By “variable region” as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the V.kappa., V.lamda., and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively. By “wild type or WT” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified. The antibodies of the present invention are generally isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. “Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells. “Specific binding” or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10-4 M, at least about 10-5 M, at least about 10-6 M, at least about 10-7 M, at least about 10-8 M, at least about 10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, at least about 10-12 M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope. Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction. II. Overview Bispecific antibodies that co-engage CD3 and a tumor antigen target have been designed and used to redirect T cells to attack and lyse targeted tumor cells. Examples include the BiTE and DART formats, which monovalently engage CD3 and a tumor antigen. While the CD3-targeting approach has shown considerable promise, a common side effect of such therapies is the associated production of cytokines, often leading to toxic cytokine release syndrome. Because the anti-CD3 binding domain of the bispecific antibody engages all T cells, the high cytokine-producing CD4 T cell subset is recruited. Moreover, the CD4 T cell subset includes regulatory T cells, whose recruitment and expansion can potentially lead to immune suppression and have a negative impact on long-term tumor suppression. In addition, these formats do not contain Fc domains and show very short serum half-lives in patients. While the CD3-targeting approach has shown considerable promise, a common side effect of such therapies is the associated production of cytokines, often leading to toxic cytokine release syndrome. Because the anti-CD3 binding domain of the bispecific antibody engages all T cells, the high cytokine-producing CD4 T cell subset is recruited. Moreover, the CD4 T cell subset includes regulatory T cells, whose recruitment and expansion can potentially lead to immune suppression and have a negative impact on long-term tumor suppression. One such possible way to reduce cytokine production and possibly reduce the activation of CD4 T cells is by reducing the affinity of the anti-CD3 domain for CD3. Accordingly, in some embodiments the present invention provides antibody constructs comprising anti-CD3 antigen binding domains that are “strong” or “high affinity” binders to CD3 (e.g. one example are heavy and light variable domains depicted as H1.30_11.47 (optionally including a charged linker as appropriate)) and also bind to CD38. In other embodiments, the present invention provides antibody constructs comprising anti-CD3 antigen binding domains that are “lite” or “lower affinity” binders to CD3. Additional embodiments provides antibody constructs comprising anti-CD3 antigen binding domains that have intermediate or “medium” affinity to CD3 that also bind to CD38. It should be appreciated that the “high, medium, low” anti-CD3 sequences of the present invention can be used in a variety of heterodimerization formats. While the majority of the disclosure herein uses the “bottle opener” format of heterodimers, these variable heavy and light sequences, as well as the scFv sequences (and Fab sequences comprising these variable heavy and light sequences) can be used in other formats, such as those depicted in FIG. 2 of WO Publication No. 2014/145806, the Figures, formats and legend of which is expressly incorporated herein by reference. Accordingly, the present invention provides heterodimeric antibodies that bind to two different antigens, e.g the antibodies are “bispecific”, in that they bind two different target antigens, generally target tumor antigens (TTAs) as described below. These heterodimeric antibodies can bind these target antigens either monovalently (e.g. there is a single antigen binding domain such as a variable heavy and variable light domain pair) or bivalently (there are two antigen binding domains that each independently bind the antigen). The heterodimeric antibodies of the invention are based on the use different monomers which contain amino acid substitutions that “skew” formation of heterodimers over homodimers, as is more fully outlined below, coupled with “pI variants” that allow simple purification of the heterodimers away from the homodimers, as is similarly outlined below. For the heterodimeric bispecific antibodies of the invention, the present invention generally relies on the use of engineered or variant Fc domains that can self-assemble in production cells to produce heterodimeric proteins, and methods to generate and purify such heterodimeric proteins. III. Antibodies The present invention relates to the generation of bispecific antibodies that bind two different antigens, e.g. CD3 and a target tumor antigen such as CD20, CD38 and CD123, and are generally therapeutic antibodies. As is discussed below, the term “antibody” is used generally. Antibodies that find use in the present invention can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, described herein. Traditional antibody structural units typically comprise a tetramer. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as kappa and lambda light chains. The present invention is directed to the IgG class, which has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It should be understood that therapeutic antibodies can also comprise hybrids of isotypes and/or subclasses. For example, as shown in US Publication 2009/0163699, incorporated by reference, the present invention covers pI engineering of IgG1/G2 hybrids. The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, generally referred to in the art and herein as the “Fv domain” or “Fv region”. In the variable region, three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a “CDR”), in which the variation in the amino acid sequence is most significant. “Variable” refers to the fact that certain segments of the variable region differ extensively in sequence among antibodies. Variability within the variable region is not evenly distributed. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-15 amino acids long or longer. Each VH and VL is composed of three hypervariable regions (“complementary determining regions,” “CDRs”) and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described below. Throughout the present specification, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g, Kabat et al., supra (1991)). The present invention provides a large number of different CDR sets. In this case, a “full CDR set” comprises the three variable light and three variable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a larger variable light or variable heavy domain, respectfully. In addition, as more fully outlined herein, the variable heavy and variable light domains can be on separate polypeptide chains, when a heavy and light chain is used (for example when Fabs are used), or on a single polypeptide chain in the case of scFv sequences. The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning.” The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected numerous primary sequences of the variable regions of heavy chains and light chains. Based on the degree of conservation of the sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E. A. Kabat et al., entirely incorporated by reference). In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By “immunoglobulin (Ig) domain” herein is meant a region of an immunoglobulin having a distinct tertiary structure. Of interest in the present invention are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, “CH” domains in the context of IgG are as follows: “CH1” refers to positions 118-220 according to the EU index as in Kabat. “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat. As shown herein and described below, the pI variants can be in one or more of the CH regions, as well as the hinge region, discussed below. It should be noted that the sequences depicted herein start at the CH1 region, position 118; the variable regions are not included except as noted. For example, the first amino acid of SEQ ID NO: 2, while designated as position“1” in the sequence listing, corresponds to position 118 of the CH1 region, according to EU numbering. Another type of Ig domain of the heavy chain is the hinge region. By “hinge” or “hinge region” or “antibody hinge region” or “immunoglobulin hinge region” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 220, and the IgG CH2 domain begins at residue EU position 237. Thus for IgG the antibody hinge is herein defined to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein the numbering is according to the EU index as in Kabat. In some embodiments, for example in the context of an Fc region, the lower hinge is included, with the “lower hinge” generally referring to positions 226 or 230. As noted herein, pI variants can be made in the hinge region as well. The light chain generally comprises two domains, the variable light domain (containing the light chain CDRs and together with the variable heavy domains forming the Fv region), and a constant light chain region (often referred to as CL or Cκ). Another region of interest for additional substitutions, outlined below, is the Fc region. Thus, the present invention provides different antibody domains. As described herein and known in the art, the heterodimeric antibodies of the invention comprise different domains within the heavy and light chains, which can be overlapping as well. These domains include, but are not limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3 domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), the variable heavy domain, the variable light domain, the light constant domain, FAb domains and scFv domains. Thus, the “Fc domain” includes the—CH2-CH3 domain, and optionally a hinge domain. The heavy chain comprises a variable heavy domain and a constant domain, which includes a CH1-optional hinge-Fc domain comprising a CH2-CH3. The light chain comprises a variable light chain and the light constant domain. Some embodiments of the invention comprise at least one scFv domain, which, while not naturally occurring, generally includes a variable heavy domain and a variable light domain, linked together by a scFv linker. As shown herein, there are a number of suitable scFv linkers that can be used, including traditional peptide bonds, generated by recombinant techniques. The linker peptide may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. In one embodiment, the linker is from about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used, with from about 5 to about 10 amino acids finding use in some embodiments. Useful linkers include glycine-serine polymers, including for example (GS)n, (GSGGS)n (SEQ ID NO:449), (GGGGS)n (SEQ ID NO:450), and (GGGS)n (SEQ ID NO:451), where n is an integer of at least one (and generally from 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. Alternatively, a variety of nonproteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers, that is may find use as linkers. Other linker sequences may include any sequence of any length of CL/CH1 domain but not all residues of CL/CH1 domain; for example the first 5-12 amino acid residues of the CL/CH1 domains. Linkers can be derived from immunoglobulin light chain, for example Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins. In some embodiments, the linker is a “domain linker”, used to link any two domains as outlined herein together. While any suitable linker can be used, many embodiments utilize a glycine-serine polymer, including for example (GS)n, (GSGGS)n (SEQ ID NO:449), (GGGGS)n (SEQ ID NO:450), and (GGGS)n (SEQ ID NO:451), where n is an integer of at least one (and generally from 3 to 4 to 5) as well as any peptide sequence that allows for recombinant attachment of the two domains with sufficient length and flexibility to allow each domain to retain its biological function. In some cases, and with attention being paid to “strandedness”, as outlined below, charged domain linkers, as used in some embodiments of scFv linkers can be used. In some embodiments, the scFv linker is a charged scFv linker, a number of which are shown inFIG.33. Accordingly, the present invention further provides charged scFv linkers, to facilitate the separation in pI between a first and a second monomer. That is, by incorporating a charged scFv linker, either positive or negative (or both, in the case of scaffolds that use scFvs on different monomers), this allows the monomer comprising the charged linker to alter the pI without making further changes in the Fc domains. These charged linkers can be substituted into any scFv containing standard linkers. Again, as will be appreciated by those in the art, charged scFv linkers are used on the correct “strand” or monomer, according to the desired changes in pI. For example, as discussed herein, to make triple F format heterodimeric antibody, the original pI of the Fv region for each of the desired antigen binding domains are calculated, and one is chosen to make an scFv, and depending on the pI, either positive or negative linkers are chosen. Charged domain linkers can also be used to increase the pI separation of the monomers of the invention as well, and thus those included inFIG.33an be used in any embodiment herein where a linker is utilized. In some embodiments, the antibodies are full length. By “full length antibody” herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions, including one or more modifications as outlined herein, particularly in the Fc domains to allow either heterodimerization formation or the purification of heterodimers away from homodimers. Full length antibodies generally include Fab and Fc domains, and can additionally contain extra antigen binding domains such as scFvs, as is generally depicted in the Figures. In one embodiment, the antibody is an antibody fragment, as long as it contains at least one constant domain which can be engineered to produce heterodimers, such as pI engineering. Other antibody fragments that can be used include fragments that contain one or more of the CH1, CH2, CH3, hinge and CL domains of the invention that have been pI engineered. For example, Fc fusions are fusions of the Fc region (CH2 and CH3, optionally with the hinge region) fused to another protein. A number of Fc fusions are known the art and can be improved by the addition of the heterodimerization variants of the invention. In the present case, antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2; CH3; CH2 and CH3; CH1 and CH3, any or all of which can be made optionally with the hinge region, utilizing any combination of heterodimerization variants described herein. In particular, the formats depicted inFIG.1are antibodies, usually referred to as “heterodimeric antibodies”, meaning that the protein has at least two associated Fc sequences self-assembled into a heterodimeric Fc domain. Chimeric and Humanized Antibodies In some embodiments, the antibody can be a mixture from different species, e.g. a chimeric antibody and/or a humanized antibody. In general, both “chimeric antibodies” and “humanized antibodies” refer to antibodies that combine regions from more than one species. For example, “chimeric antibodies” traditionally comprise variable region(s) from a mouse (or rat, in some cases) and the constant region(s) from a human. “Humanized antibodies” generally refer to non-human antibodies that have had the variable-domain framework regions swapped for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody, except the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acids originating in a non-human organism, are grafted into the beta-sheet framework of a human antibody variable region to create an antibody, the specificity of which is determined by the engrafted CDRs. The creation of such antibodies is described in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporated by reference. “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; 5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirely incorporated by reference). The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region. Humanized antibodies can also be generated using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated by reference. A variety of techniques and methods for humanizing and reshaping non-human antibodies are well known in the art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA), and references cited therein, all entirely incorporated by reference). Humanization methods include but are not limited to methods described in Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8, all entirely incorporated by reference. Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods, as described for example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91:969-973, entirely incorporated by reference. In one embodiment, the parent antibody has been affinity matured, as is known in the art. Structure-based methods may be employed for humanization and affinity maturation, for example as described in U.S. Ser. No. 11/004,590. Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all entirely incorporated by reference. Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in U.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirely incorporated by reference. IV. Heterodimeric Antibodies Accordingly, in some embodiments the present invention provides heterodimeric antibodies that rely on the use of two different heavy chain variant Fc sequences, that will self-assemble to form heterodimeric Fc domains and heterodimeric antibodies. The present invention is directed to novel constructs to provide heterodimeric antibodies that allow binding to more than one antigen or ligand, e.g. to allow for bispecific binding. The heterodimeric antibody constructs are based on the self-assembling nature of the two Fc domains of the heavy chains of antibodies, e.g. two “monomers” that assemble into a “dimer”. Heterodimeric antibodies are made by altering the amino acid sequence of each monomer as more fully discussed below. Thus, the present invention is generally directed to the creation of heterodimeric antibodies which can co-engage antigens in several ways, relying on amino acid variants in the constant regions that are different on each chain to promote heterodimeric formation and/or allow for ease of purification of heterodimers over the homodimers. Thus, the present invention provides bispecific antibodies. An ongoing problem in antibody technologies is the desire for “bispecific” antibodies that bind to two different antigens simultaneously, in general thus allowing the different antigens to be brought into proximity and resulting in new functionalities and new therapies. In general, these antibodies are made by including genes for each heavy and light chain into the host cells. This generally results in the formation of the desired heterodimer (A-B), as well as the two homodimers (A-A and B-B (not including the light chain heterodimeric issues)). However, a major obstacle in the formation of bispecific antibodies is the difficulty in purifying the heterodimeric antibodies away from the homodimeric antibodies and/or biasing the formation of the heterodimer over the formation of the homodimers. There are a number of mechanisms that can be used to generate the heterodimers of the present invention. In addition, as will be appreciated by those in the art, these mechanisms can be combined to ensure high heterodimerization. Thus, amino acid variants that lead to the production of heterodimers are referred to as “heterodimerization variants”. As discussed below, heterodimerization variants can include steric variants (e.g. the “knobs and holes” or “skew” variants described below and the “charge pairs” variants described below) as well as “pI variants”, which allows purification of homodimers away from heterodimers. As is generally described in WO2014/145806, hereby incorporated by reference in its entirety and specifically as below for the discussion of “heterodimerization variants”, useful mechanisms for heterodimerization include “knobs and holes” (“KIH”; sometimes herein as “skew” variants (see discussion in WO2014/145806), “electrostatic steering” or “charge pairs” as described in WO2014/145806, pI variants as described in WO2014/145806, and general additional Fc variants as outlined in WO2014/145806 and below. In the present invention, there are several basic mechanisms that can lead to ease of purifying heterodimeric antibodies; one relies on the use of pI variants, such that each monomer has a different pI, thus allowing the isoelectric purification of A-A, A-B and B-B dimeric proteins. Alternatively, some scaffold formats, such as the “triple F” format, also allows separation on the basis of size. As is further outlined below, it is also possible to “skew” the formation of heterodimers over homodimers. Thus, a combination of steric heterodimerization variants and pI or charge pair variants find particular use in the invention. In general, embodiments of particular use in the present invention rely on sets of variants that include skew variants, that encourage heterodimerization formation over homodimerization formation, coupled with pI variants, which increase the pI difference between the two monomers. Additionally, as more fully outlined below, depending on the format of the heterodimer antibody, pI variants can be either contained within the constant and/or Fc domains of a monomer, or charged linkers, either domain linkers or scFv linkers, can be used. That is, scaffolds that utilize scFv(s) such as the Triple F format can include charged scFv linkers (either positive or negative), that give a further pI boost for purification purposes. As will be appreciated by those in the art, some Triple F formats are useful with just charged scFv linkers and no additional pI adjustments, although the invention does provide pI variants that are on one or both of the monomers, and/or charged domain linkers as well. In addition, additional amino acid engineering for alternative functionalities may also confer pI changes, such as Fc, FcRn and KO variants. In the present invention that utilizes pI as a separation mechanism to allow the purification of heterodimeric proteins, amino acid variants can be introduced into one or both of the monomer polypeptides; that is, the pI of one of the monomers (referred to herein for simplicity as “monomer A”) can be engineered away from monomer B, or both monomer A and B change be changed, with the pI of monomer A increasing and the pI of monomer B decreasing. As is outlined more fully below, the pI changes of either or both monomers can be done by removing or adding a charged residue (e.g. a neutral amino acid is replaced by a positively or negatively charged amino acid residue, e.g. glycine to glutamic acid), changing a charged residue from positive or negative to the opposite charge (aspartic acid to lysine) or changing a charged residue to a neutral residue (e.g. loss of a charge; lysine to serine.). A number of these variants are shown in the Figures. Accordingly, this embodiment of the present invention provides for creating a sufficient change in pI in at least one of the monomers such that heterodimers can be separated from homodimers. As will be appreciated by those in the art, and as discussed further below, this can be done by using a “wild type” heavy chain constant region and a variant region that has been engineered to either increase or decrease it's pI (wt A−+B or wt A−−B), or by increasing one region and decreasing the other region (A+−B− or A−B+). Thus, in general, a component of some embodiments of the present invention are amino acid variants in the constant regions of antibodies that are directed to altering the isoelectric point (pI) of at least one, if not both, of the monomers of a dimeric protein to form “pI antibodies”) by incorporating amino acid substitutions (“pI variants” or “pI substitutions”) into one or both of the monomers. As shown herein, the separation of the heterodimers from the two homodimers can be accomplished if the pIs of the two monomers differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in the present invention. As will be appreciated by those in the art, the number of pI variants to be included on each or both monomer(s) to get good separation will depend in part on the starting pI of the components, for example in the triple F format, the starting pI of the scFv and Fab of interest. That is, to determine which monomer to engineer or in which “direction” (e.g. more positive or more negative), the Fv sequences of the two target antigens are calculated and a decision is made from there. As is known in the art, different Fvs will have different starting pIs which are exploited in the present invention. In general, as outlined herein, the pIs are engineered to result in a total pI difference of each monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred as outlined herein. Furthermore, as will be appreciated by those in the art and outlined herein, in some embodiments, heterodimers can be separated from homodimers on the basis of size. As shown inFIG.1for example, several of the formats allow separation of heterodimers and homodimers on the basis of size. In the case where pI variants are used to achieve heterodimerization, by using the constant region(s) of the heavy chain(s), a more modular approach to designing and purifying bispecific proteins, including antibodies, is provided. Thus, in some embodiments, heterodimerization variants (including skew and purification heterodimerization variants) are not included in the variable regions, such that each individual antibody must be engineered. In addition, in some embodiments, the possibility of immunogenicity resulting from the pI variants is significantly reduced by importing pI variants from different IgG isotypes such that pI is changed without introducing significant immunogenicity. Thus, an additional problem to be solved is the elucidation of low pI constant domains with high human sequence content, e.g. the minimization or avoidance of non-human residues at any particular position. A side benefit that can occur with this pI engineering is also the extension of serum half-life and increased FcRn binding. That is, as described in U.S. Ser. No. 13/194,904 (incorporated by reference in its entirety), lowering the pI of antibody constant domains (including those found in antibodies and Fc fusions) can lead to longer serum retention in vivo. These pI variants for increased serum half life also facilitate pI changes for purification. In addition, it should be noted that the pI variants of the heterodimerization variants give an additional benefit for the analytics and quality control process of bispecific antibodies, as the ability to either eliminate, minimize and distinguish when homodimers are present is significant. Similarly, the ability to reliably test the reproducibility of the heterodimeric antibody production is important. Heterodimerization Variants The present invention provides heterodimeric proteins, including heterodimeric antibodies in a variety of formats, which utilize heterodimeric variants to allow for heterodimeric formation and/or purification away from homodimers. There are a number of suitable pairs of sets of heterodimerization skew variants. These variants come in “pairs” of “sets”. That is, one set of the pair is incorporated into the first monomer and the other set of the pair is incorporated into the second monomer. It should be noted that these sets do not necessarily behave as “knobs in holes” variants, with a one-to-one correspondence between a residue on one monomer and a residue on the other; that is, these pairs of sets form an interface between the two monomers that encourages heterodimer formation and discourages homodimer formation, allowing the percentage of heterodimers that spontaneously form under biological conditions to be over 90%, rather than the expected 50% (25% homodimer A/A:50% heterodimer A/B:25% homodimer B/B). Steric Variants In some embodiments, the formation of heterodimers can be facilitated by the addition of steric variants. That is, by changing amino acids in each heavy chain, different heavy chains are more likely to associate to form the heterodimeric structure than to form homodimers with the same Fc amino acid sequences. Suitable steric variants are included inFIG.29. One mechanism is generally referred to in the art as “knobs and holes”, referring to amino acid engineering that creates steric influences to favor heterodimeric formation and disfavor homodimeric formation can also optionally be used; this is sometimes referred to as “knobs and holes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated by reference in their entirety. The Figures identify a number of “monomer A—monomer B” pairs that rely on “knobs and holes”. In addition, as described in Merchant et al., Nature Biotech. 16:677 (1998), these “knobs and hole” mutations can be combined with disulfide bonds to skew formation to heterodimerization. An additional mechanism that finds use in the generation of heterodimers is sometimes referred to as “electrostatic steering” as described in Gunasekaran et at, J. Biol. Chem. 285(25):19637 (2010), hereby incorporated by reference in its entirety. This is sometimes referred to herein as “charge pairs”. In this embodiment, electrostatics are used to skew the formation towards heterodimerization. As those in the art will appreciate, these may also have have an effect on pI, and thus on purification, and thus could in some cases also be considered pI variants. However, as these were generated to force heterodimerization and were not used as purification tools, they are classified as “steric variants”. These include, but are not limited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g. these are “monomer corresponding sets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R. Additional monomer A and monomer B variants that can be combined with other variants, optionally and independently in any amount, such as pI variants outlined herein or other steric variants that are shown in FIG. 37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which are incorporated expressly by reference herein. In some embodiments, the steric variants outlined herein can be optionally and independently incorporated with any pI variant (or other variants such as Fc variants, FcRn variants, etc.) into one or both monomers, and can be independently and optionally included or excluded from the proteins of the invention. A list of suitable skew variants is found inFIG.29, withFIG.34showing some pairs of particular utility in many embodiments. Of particular use in many embodiments are the pairs of sets including, but not limited to, S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L and K370S: S364K/E357Q. In terms of nomenclature, the pair “S364K/E357Q: L368D/K370S” means that one of the monomers has the double variant set S364K/E357Q and the other has the double variant set L368D/K370S. pI (Isoelectric Point) Variants for Heterodimers In general, as will be appreciated by those in the art, there are two general categories of pI variants: those that increase the pI of the protein (basic changes) and those that decrease the pI of the protein (acidic changes). As described herein, all combinations of these variants can be done: one monomer may be wild type, or a variant that does not display a significantly different pI from wild-type, and the other can be either more basic or more acidic. Alternatively, each monomer is changed, one to more basic and one to more acidic. Preferred combinations of pI variants are shown inFIG.30. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be altered this way, as well as isotype hybrids. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used. Antibody Heterodimers Light Chain Variants In the case of antibody based heterodimers, e.g. where at least one of the monomers comprises a light chain in addition to the heavy chain domain, pI variants can also be made in the light chain. Amino acid substitutions for lowering the pI of the light chain include, but are not limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E, K207E and adding peptide DEDE (SEQ ID NO:452) at the c-terminus of the light chain. Changes in this category based on the constant lambda light chain include one or more substitutions at R108Q, Q124E, K126Q, N138D, K145T and Q199E. In addition, increasing the pI of the light chains can also be done. Isotypic Variants In addition, many embodiments of the invention rely on the “importation” of pI amino acids at particular positions from one IgG isotype into another, thus reducing or eliminating the possibility of unwanted immunogenicity being introduced into the variants. A number of these are shown in FIG. 21 of US Publ. 2014/0370013, hereby incorporated by reference. That is, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the heavy constant region of IgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). By introducing IgG2 residues at particular positions into the IgG1 backbone, the pI of the resulting monomer is lowered (or increased) and additionally exhibits longer serum half-life. For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI 3.22); importing the glutamic acid will affect the pI of the resulting protein. As is described below, a number of amino acid substitutions are generally required to significant affect the pI of the variant antibody. However, it should be noted as discussed below that even changes in IgG2 molecules allow for increased serum half-life. In other embodiments, non-isotypic amino acid changes are made, either to reduce the overall charge state of the resulting protein (e.g. by changing a higher pI amino acid to a lower pI amino acid), or to allow accommodations in structure for stability, etc. as is more further described below. In addition, by pI engineering both the heavy and light constant domains, significant changes in each monomer of the heterodimer can be seen. As discussed herein, having the pIs of the two monomers differ by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point. Calculating pI The pI of each monomer can depend on the pI of the variant heavy chain constant domain and the pI of the total monomer, including the variant heavy chain constant domain and the fusion partner. Thus, in some embodiments, the change in pI is calculated on the basis of the variant heavy chain constant domain, using the chart in the FIG. 19 of US Pub. 2014/0370013. As discussed herein, which monomer to engineer is generally decided by the inherent pI of the Fv and scaffold regions. Alternatively, the pI of each monomer can be compared. pI Variants that Also Confer Better FcRn In Vivo Binding In the case where the pI variant decreases the pI of the monomer, they can have the added benefit of improving serum retention in vivo. Although still under examination, Fc regions are believed to have longer half-lives in vivo, because binding to FcRn at pH 6 in an endosome sequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598, entirely incorporated by reference). The endosomal compartment then recycles the Fc to the cell surface. Once the compartment opens to the extracellular space, the higher pH, −7.4, induces the release of Fc back into the blood. In mice, Dall'Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half life as wild-type Fc (Dail′ Acqua et al. 2002, J. Immunol. 169:5171-5180, entirely incorporated by reference). The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid the release of the Fc back into the blood. Therefore, the Fc mutations that will increase Fc's half-life in vivo will ideally increase FcRn binding at the lower pH while still allowing release of Fc at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is not surprising to find His residues at important positions in the Fc/FcRn complex. Recently it has been suggested that antibodies with variable regions that have lower isoelectric points may also have longer serum half-lives (Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated by reference). However, the mechanism of this is still poorly understood. Moreover, variable regions differ from antibody to antibody. Constant region variants with reduced pI and extended half-life would provide a more modular approach to improving the pharmacokinetic properties of antibodies, as described herein. Additional Fc Variants for Additional Functionality In addition to pI amino acid variants, there are a number of useful Fc amino acid modification that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcγR receptors, altered binding to FcRn receptors, etc. Accordingly, the proteins of the invention can include amino acid modifications, including the heterodimerization variants outlined herein, which includes the pI variants and steric variants. Each set of variants can be independently and optionally included or excluded from any particular heterodimeric protein. FcγR Variants Accordingly, there are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcγR receptors. Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the present invention include those listed in U.S. Ser. No. 11/124,620 (particularlyFIG.41), Ser. Nos. 11/174,287, 11/396,495, 11/538,406, all of which are expressly incorporated herein by reference in their entirety and specifically for the variants disclosed therein. Particular variants that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and 299T. In addition, there are additional Fc substitutions that find use in increased binding to the FcRn receptor and increased serum half life, as specifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporated by reference in its entirety, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L. Ablation Variants Similarly, another category of functional variants are “FcγR ablation variants” or “Fc knock out (FcKO or KO)” variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or remove the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. That is, for example, in many embodiments, particularly in the use of bispecific antibodies that bind CD3 monovalently it is generally desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. wherein one of the Fc domains comprises one or more Fcγ receptor ablation variants. These ablation variants are depicted inFIG.31, and each can be independently and optionally included or excluded, with preferred aspects utilizing ablation variants selected from the group consisting of G236R/L328R, E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del. It should be noted that the ablation variants referenced herein ablate FcγR binding but generally not FcRn binding. Combination of Heterodimeric and Fc Variants As will be appreciated by those in the art, all of the recited heterodimerization variants (including skew and/or pI variants) can be optionally and independently combined in any way, as long as they retain their “strandedness” or “monomer partition”. In addition, all of these variants can be combined into any of the heterodimerization formats. In the case of pI variants, while embodiments finding particular use are shown in the Figures, other combinations can be generated, following the basic rule of altering the pI difference between two monomers to facilitate purification. In addition, any of the heterodimerization variants, skew and pI, are also independently and optionally combined with Fc ablation variants, Fc variants, FcRn variants, as generally outlined herein. Useful Formats of the Invention As will be appreciated by those in the art and discussed more fully below, the heterodimeric fusion proteins of the present invention can take on a wide variety of configurations, as are generally depicted inFIG.1. Some figures depict “single ended” configurations, where there is one type of specificity on one “arm” of the molecule and a different specificity on the other “arm”. Other figures depict “dual ended” configurations, where there is at least one type of specificity at the “top” of the molecule and one or more different specificities at the “bottom” of the molecule. Thus, the present invention is directed to novel immunoglobulin compositions that co-engage a different first and a second antigen. As will be appreciated by those in the art, the heterodimeric formats of the invention can have different valencies as well as be bispecific. That is, heterodimeric antibodies of the invention can be bivalent and bispecific, wherein one target tumor antigen (e.g. CD3) is bound by one binding domain and the other target tumor antigen (e.g. CD20, CD19, CD38, CD123, etc.) is bound by a second binding domain. The heterodimeric antibodies can also be trivalent and bispecific, wherein the first antigen is bound by two binding domains and the second antigen by a second binding domain. As is outlined herein, when CD3 is one of the target antigens, it is preferable that the CD3 is bound only monovalently, to reduce potential side effects. The present invention utilizes anti-CD3 antigen binding domains in combination with anti-target tumor antigen (TTA) antigen binding domains. As will be appreciated by those in the art, any collection of anti-CD3 CDRs, anti-CD3 variable light and variable heavy domains, Fabs and scFvs as depicted in any of the Figures (see particularlyFIGS.2through7, andFIGS.68A-68Z) can be used. Similarly, any of the anti-TTA antigen binding domains can be used, e.g. anti-CD38, anti-CD20, anti-CD19 and anti-CD123 antigen binding domains, whether CDRs, variable light and variable heavy domains, Fabs and scFvs as depicted in any of the Figures can be used, optionally and independently combined in any combination. Bottle Opener Format One heterodimeric scaffold that finds particular use in the present invention is the “triple F” or “bottle opener” scaffold format as shown inFIGS.1A, A and B. In this embodiment, one heavy chain of the antibody contains an single chain Fv (“scFv”, as defined below) and the other heavy chain is a “regular” FAb format, comprising a variable heavy chain and a light chain. This structure is sometimes referred to herein as “triple F” format (scFv-FAb-Fc) or the “bottle-opener” format, due to a rough visual similarity to a bottle-opener (seeFIG.1). The two chains are brought together by the use of amino acid variants in the constant regions (e.g. the Fc domain, the CH1 domain and/or the hinge region) that promote the formation of heterodimeric antibodies as is described more fully below. There are several distinct advantages to the present “triple F” format. As is known in the art, antibody analogs relying on two scFv constructs often have stability and aggregation problems, which can be alleviated in the present invention by the addition of a “regular” heavy and light chain pairing. In addition, as opposed to formats that rely on two heavy chains and two light chains, there is no issue with the incorrect pairing of heavy and light chains (e.g. heavy 1 pairing with light 2, etc.). Many of the embodiments outlined herein rely in general on the bottle opener format that comprises a first monomer comprising an scFv, comprising a variable heavy and a variable light domain, covalently attached using an scFv linker (charged, in many but not all instances), where the scFv is covalently attached to the N-terminus of a first Fc domain usually through a domain linker (which, as outlined herein can either be un-charged or charged). The second monomer of the bottle opener format is a heavy chain, and the composition further comprises a light chain. In general, in many preferred embodiments, the scFv is the domain that binds to the CD3, with the Fab of the heavy and light chains binding to the other TTA. In addition, the Fc domains of the invention generally comprise skew variants (e.g. a set of amino acid substitutions as shown inFIG.29andFIG.34, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L and K370S: S364K/E357Q), optionally ablation variants (including those shown inFIG.31), optionally charged scFv linkers (including those shown inFIG.33) and the heavy chain comprises pI variants (including those shown inFIG.30). The present invention provides bottle opener formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides bottle opener formats with CD38 antigen binding domains wherein the anti-CD38 sequences are as shown in the Figures, includingFIGS.8to10. The present invention provides bottle opener formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in the Figures. The present invention provides bottle opener formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in the Figures. The present invention provides bottle opener formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in the Figures. mAb-Fv Format One heterodimeric scaffold that finds particular use in the present invention is the mAb-Fv format shown inFIG.1. In this embodiment, the format relies on the use of a C-terminal attachment of an “extra” variable heavy domain to one monomer and the C-terminal attachment of an “extra” variable light domain to the other monomer, thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind a TTA and the “extra” scFv domain binds CD3. In this embodiment, the first monomer comprises a first heavy chain, comprising a first variable heavy domain and a first constant heavy domain comprising a first Fc domain, with a first variable light domain covalently attached to the C-terminus of the first Fc domain using a domain linker. The second monomer comprises a second variable heavy domain of the second constant heavy domain comprising a second Fc domain, and a third variable heavy domain covalently attached to the C-terminus of the second Fc domain using a domain linker. The two C-terminally attached variable domains make up a scFv that binds CD3. This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that bind a TTA. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The present invention provides mAb-Fv formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides mAb-Fv formats wherein the anti-CD38 sequences are as shown inFIGS.8to10. The present invention provides mAb-Fv formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in the Figures. The present invention provides mAb-Fv formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in in the Figures. The present invention provides mAb-Fv formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in in the Figures. The present invention provides mAb-Fv formats comprising ablation variants as shown inFIG.31. The present invention provides mAb-Fv formats comprising skew variants as shown inFIGS.29and34. mAb-scFv One heterodimeric scaffold that finds particular use in the present invention is the mAb-Fv format shown inFIG.1. In this embodiment, the format relies on the use of a C-terminal attachment of a scFv to one of the monomers, thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind a TTA and the “extra” scFv domain binds CD3. Thus, the first monomer comprises a first heavy chain (comprising a variable heavy domain and a constant domain), with a C-terminally covalently attached scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain. This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that bind a TTA. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The present invention provides mAb-Fv formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides mAb-Fv formats wherein the anti-CD38 sequences are as shown inFIGS.8to10. The present invention provides mAb-Fv formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in in the Figures. The present invention provides mAb-Fv formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in in the Figures. The present invention provides mAb-Fv formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in in the Figures. The present invention provides mAb-Fv formats comprising ablation variants as shown inFIG.31. The present invention provides mAb-Fv formats comprising skew variants as shown inFIGS.29and34. Central scFv One heterodimeric scaffold that finds particular use in the present invention is the Central-scFv format shown inFIG.1. In this embodiment, the format relies on the use of an inserted scFv domain thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind a TTA and the “extra” scFv domain binds CD3. The scFv domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers, thus providing a third antigen binding domain. In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain and Fc domain, with a scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain. The scFv is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers. This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that bind a TTA. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The present invention provides Central-scFv formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides Central-scFv formats wherein the anti-CD38 sequences are as shown inFIGS.8to10. The present invention provides Central-scFv formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in in the Figures. The present invention provides Central-scFv formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in in the Figures. The present invention provides Central-scFv formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in v The present invention provides Central-scFv formats comprising ablation variants as shown inFIG.31. The present invention provides Central-scFv formats comprising skew variants as shown inFIGS.29and34. Central-Fv Format One heterodimeric scaffold that finds particular use in the present invention is the Central-Fv format shown inFIG.1. In this embodiment, the format relies on the use of an inserted scFv domain thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind a TTA and the “extra” scFv domain binds CD3. The scFv domain is inserted between the Fc domain and the CH1-Fv region of the monomers, thus providing a third antigen binding domain, wherein each monomer contains a component of the scFv (e.g. one monomer comprises a variable heavy domain and the other a variable light domain). In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain and Fc domain and an additional variable light domain. The light domain is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers. The other monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain and Fc domain and an additional variable heavy domain. The light domain is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers. This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that bind a TTA. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The present invention provides Central-Fv formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides Central-Fv formats wherein the anti-CD38 sequences are as shown inFIGS.8to10. The present invention provides Central-Fv formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in the Figures. The present invention provides Central-Fv formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in the Figures. The present invention provides Central-Fv formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in the Figures. The present invention provides Central-Fv formats comprising ablation variants as shown inFIG.31. The present invention provides Central-Fv formats comprising skew variants as shown inFIGS.29and34. One Armed Central-scFv One heterodimeric scaffold that finds particular use in the present invention is the one armed central-scFv format shown inFIG.1. In this embodiment, one monomer comprises just an Fc domain, while the other monomer uses an inserted scFv domain thus forming the second antigen binding domain. In this format, either the Fab portion binds a TTA and the scFv binds CD3 or vice versa. The scFv domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers. In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain and Fc domain, with a scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain. The scFv is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers. The second monomer comprises an Fc domain. This embodiment further utilizes a light chain comprising a variable light domain and a constant light domain, that associates with the heavy chain to form a Fab. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The present invention provides one armed central-scFv formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides one armed central-scFv formats wherein the anti-CD38 sequences are as shown inFIGS.8to10. The present invention provides one armed central-scFv formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in the Figures. The present invention provides one armed central-scFv formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in the Figures. The present invention provides one armed central-scFv formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in the Figures. The present invention provides one armed central-scFv formats comprising ablation variants as shown inFIG.31. The present invention provides one armed central-scFv formats comprising skew variants as shown inFIGS.29and34. Dual scFv Formats The present invention also provides dual scFv formats as are known in the art and shown inFIG.1. The present invention provides dual scFv formats where the anti-CD3 scFv sequences are as shown inFIG.2toFIG.7andFIGS.68A-68Z. The present invention provides dual scFv formats wherein the anti-CD38 sequences are as shown inFIGS.8to10. The present invention provides dual scFv formats with CD20 antigen binding domains wherein the anti-CD20 sequences are as shown in the Figures. The present invention provides dual scFv formats with CD19 antigen binding domains wherein the anti-CD19 sequences are as shown in the Figures. The present invention provides dual scFv formats with CD123 antigen binding domains wherein the anti-CD123 sequences are as shown in the Figures. The present invention provides dual scFv formats comprising ablation variants as shown inFIG.31. The present invention provides dual scFv formats comprising skew variants as shown inFIGS.29and34. Target Antigens The bispecific antibodies of the invention have two different antigen binding domains: one that binds to CD3 (generally monovalently), and one that binds to a target tumor antigen (sometimes referred to herein as “TTA”). Suitable target tumor antigens include, but are not limited to, CD20, CD38, CD123; ROR1, ROR2, BCMA; PSMA; SSTR2; SSTR5, CD19, FLT3, CD33, PSCA, ADAM 17, CEA, Her2, EGFR, EGFR-vIII, CD30, FOLR1, GD-2, CA-IX, Trop-2, CD70, CD38, mesothelin, EphA2, CD22, CD79b, GPNMB, CD56, CD138, CD52, CD74, CD30, CD123, RON, ERBB2, and EGFR. The “triple F” format is particularly beneficial for targeting two (or more) distinct antigens. (As outlined herein, this targeting can be any combination of monovalent and divalent binding, depending on the format). Thus the immunoglobulins herein preferably co-engage two target antigens. Each monomer's specificity can be selected from the lists herein. Additional useful bispecific formats for use with an anti-CD3 binding domain are shown inFIG.1. Particular suitable applications of the heterodimeric antibodies herein are co-target pairs for which it is beneficial or critical to engage each target antigen monovalently. Such antigens may be, for example, immune receptors that are activated upon immune complexation. Cellular activation of many immune receptors occurs only by cross-linking, achieved typically by antibody/antigen immune complexes, or via effector cell to target cell engagement. For some immune receptors, for example the CD3 signaling receptor on T cells, activation only upon engagement with co-engaged target is critical, as nonspecific cross-linking in a clinical setting can elicit a cytokine storm and toxicity. Therapeutically, by engaging such antigens monovalently rather than multivalently, using the immunoglobulins herein, such activation occurs only in response to cross-linking only in the microenvironment of the primary target antigen. The ability to target two different antigens with different valencies is a novel and useful aspect of the present invention. Examples of target antigens for which it may be therapeutically beneficial or necessary to co-engage monovalently include but are not limited to immune activating receptors such as CD3, FcγRs, toll-like receptors (TLRs) such as TLR4 and TLR9, cytokine, chemokine, cytokine receptors, and chemokine receptors. In many embodiments, one of the antigen binding sites binds to CD3, and in some embodiments it is the scFv-containing monomer. Virtually any antigen may be targeted by the immunoglobulins herein, including but not limited to proteins, subunits, domains, motifs, and/or epitopes belonging to the following list of target antigens, which includes both soluble factors such as cytokines and membrane-bound factors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACε, ACε-2, Activin, Activin A, Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACε, ADAMS, ADAMS, ADAMTS, ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACε, BACε-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC,Clostridium botulinumtoxin,Clostridium perfringenstoxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, Cλ3CL1, Cλ3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, Factor Ha, Factor VII, Factor VIIIc, Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growth hormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, High molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICε, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain, Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5 (alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L L Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin Beta Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Mud.), MUC18, Muellerian-inhibitin substance, Mug, MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin, Neurotrophin-3,-4, or -6, Neurturin, Neuronal growth factor (NGF), NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin, respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACε, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT, TECK, TEM1, TEMS, TEM7, TEM8, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R), TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11 (TRANCε/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125, tumor-associated antigen expressing Lewis Y related carbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors. Exemplary antigens that may be targeted specifically by the immunoglobulins of the invention include but are not limited to: CD20, CD19, Her2, EGFR, EpCAM, CD3, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRI (CD64), Toll-like receptors (TLRs) such as TLR4 and TLR9, cytokines such as IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα, cytokine receptors such as IL-2R, chemokines, chemokine receptors, growth factors such as VEGF and HGF, and the like. To form the bispecific antibodies of the invention, antibodies to any combination of these antigens can be made; that is, each of these antigens can be optionally and independently included or excluded from a bispecific antibody according to the present invention. Particularly preferred combinations for bispecific antibodies are an antigen-binding domain to CD3 and an antigen binding domain selected from a domain that binds CD19, CD20, CD38 and CD123, the sequences of which are shown in the Figures. Nucleic Acids of the Invention The invention further provides nucleic acid compositions encoding the bispecific antibodies of the invention. As will be appreciated by those in the art, the nucleic acid compositions will depend on the format and scaffold of the heterodimeric protein. Thus, for example, when the format requires three amino acid sequences, such as for the triple F format (e.g. a first amino acid monomer comprising an Fc domain and a scFv, a second amino acid monomer comprising a heavy chain and a light chain), three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, some formats (e.g. dual scFv formats such as disclosed inFIG.1) only two nucleic acids are needed; again, they can be put into one or two expression vectors. As is known in the art, the nucleic acids encoding the components of the invention can be incorporated into expression vectors as is known in the art, and depending on the host cells used to produce the heterodimeric antibodies of the invention. Generally the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). The expression vectors can be extra-chromosomal or integrating vectors. The nucleic acids and/or expression vectors of the invention are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with mammalian cells (e.g. CHO cells), finding use in many embodiments. In some embodiments, nucleic acids encoding each monomer and the optional nucleic acid encoding a light chain, as applicable depending on the format, are each contained within a single expression vector, generally under different or the same promoter controls. In embodiments of particular use in the present invention, each of these two or three nucleic acids are contained on a different expression vector. As shown herein and in 62/025,931, hereby incorporated by reference, different vector ratios can be used to drive heterodimer formation. That is, surprisingly, while the proteins comprise first monomer:second monomer:light chains (in the case of many of the embodiments herein that have three polypeptides comprising the heterodimeric antibody) in a 1:1:2 ratio, these are not the ratios that give the best results. The heterodimeric antibodies of the invention are made by culturing host cells comprising the expression vector(s) as is well known in the art. Once produced, traditional antibody purification steps are done, including an ion exchange chromotography step. As discussed herein, having the pIs of the two monomers differ by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point. That is, the inclusion of pI substitutions that alter the isoelectric point (pI) of each monomer so that such that each monomer has a different pI and the heterodimer also has a distinct pI, thus facilitating isoelectric purification of the “triple F” heterodimer (e.g., anionic exchange columns, cationic exchange columns). These substitutions also aid in the determination and monitoring of any contaminating dual scFv-Fc and mAb homodimers post-purification (e.g., IEF gels, cIEF, and analytical IEX columns). Treatments Once made, the compositions of the invention find use in a number of applications. CD20, CD38 and CD123 are all unregulated in many hematopoeitic malignancies and in cell lines derived from various hematopoietic malignancies, accordingly, the heterodimeric antibodies of the invention find use in treating cancer, including but not limited to, all B cell lymphomas and leukemias, including but not limited to non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's Lymphoma (HL), chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma, and chronic myeloid leukemia (CML). Accordingly, the heterodimeric compositions of the invention find use in the treatment of these cancers. Antibody Compositions for In Vivo Administration Formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to provide antibodies with other specificities. Alternatively, or in addition, the composition may comprise a cytotoxic agent, cytokine, growth inhibitory agent and/or small molecule antagonist. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). The formulations to be used for in vivo administration should be sterile, or nearly so. This is readily accomplished by filtration through sterile filtration membranes. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. Administrative Modalities The antibodies and chemotherapeutic agents of the invention are administered to a subject, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred. Treatment Modalities In the methods of the invention, therapy is used to provide a positive therapeutic response with respect to a disease or condition. By “positive therapeutic response” is intended an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition. For example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (6) an increased patient survival rate; and (7) some relief from one or more symptoms associated with the disease or condition. Positive therapeutic responses in any given disease or condition can be determined by standardized response criteria specific to that disease or condition. Tumor response can be assessed for changes in tumor morphology (i.e., overall tumor burden, tumor size, and the like) using screening techniques such as magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, bone scan imaging, endoscopy, and tumor biopsy sampling including bone marrow aspiration (BMA) and counting of tumor cells in the circulation. In addition to these positive therapeutic responses, the subject undergoing therapy may experience the beneficial effect of an improvement in the symptoms associated with the disease. An improvement in the disease may be characterized as a complete response. By “complete response” is intended an absence of clinically detectable disease with normalization of any previously abnormal radiographic studies, bone marrow, and cerebrospinal fluid (CSF) or abnormal monoclonal protein in the case of myeloma. Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to 8 weeks, following treatment according to the methods of the invention. Alternatively, an improvement in the disease may be categorized as being a partial response. By “partial response” is intended at least about a 50% decrease in all measurable tumor burden (i.e., the number of malignant cells present in the subject, or the measured bulk of tumor masses or the quantity of abnormal monoclonal protein) in the absence of new lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks. Treatment according to the present invention includes a “therapeutically effective amount” of the medicaments used. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the medicaments to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A “therapeutically effective amount” for tumor therapy may also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer may be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to inhibit cell growth or to induce apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. The efficient dosages and the dosage regimens for the bispecific antibodies used in the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of an bispecific antibody used in the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, or about 3 mg/kg. In another embodiment, he antibody is administered in a dose of 1 mg/kg or more, such as a dose of from 1 to 20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg. A medical professional having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or a veterinarian could start doses of the medicament employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In one embodiment, the bispecific antibody is administered by infusion in a weekly dosage of from 10 to 500 mg/kg such as of from 200 to 400 mg/kg Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. In one embodiment, the bispecific antibody is administered by slow continuous infusion over a long period, such as more than 24 hours, if required to reduce side effects including toxicity. In one embodiment the bispecific antibody is administered in a weekly dosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4 to 6 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. The dosage may be determined or adjusted by measuring the amount of compound of the present invention in the blood upon administration by for instance taking out a biological sample and using anti-idiotypic antibodies which target the antigen binding region of the bispecific antibody. In a further embodiment, the bispecific antibody is administered once weekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8 weeks. In one embodiment, the bispecific antibody is administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more. In one embodiment, the bispecific antibody is administered by a regimen including one infusion of an bispecific antibody followed by an infusion of an bispecific antibody conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9 days later. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of an antibody in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof. In some embodiments the bispecific antibody molecule thereof is used in combination with one or more additional therapeutic agents, e.g. a chemotherapeutic agent. Non-limiting examples of DNA damaging chemotherapeutic agents include topoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecin and analogs or metabolites thereof, and doxorubicin); topoisomerase II inhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylating agents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine, streptozocin, decarbazine, methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators and free radical generators such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea). Chemotherapeutic agents that disrupt cell replication include: paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, and related analogs; thalidomide, lenalidomide, and related analogs (e.g., CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinib mesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κB inhibitors, including inhibitors of IκB kinase; antibodies which bind to proteins overexpressed in cancers and thereby downregulate cell replication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab); and other inhibitors of proteins or enzymes known to be upregulated, over-expressed or activated in cancers, the inhibition of which downregulates cell replication. In some embodiments, the antibodies of the invention can be used prior to, concurrent with, or after treatment with Velcade® (bortezomib). All cited references are herein expressly incorporated by reference in their entirety. Whereas particular embodiments of the invention have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims. EXAMPLES Examples are provided below to illustrate the present invention. These examples are not meant to constrain the present invention to any particular application or theory of operation. For all constant region positions discussed in the present invention, numbering is according to the EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, entirely incorporated by reference). Those skilled in the art of antibodies will appreciate that this convention consists of nonsequential numbering in specific regions of an immunoglobulin sequence, enabling a normalized reference to conserved positions in immunoglobulin families. Accordingly, the positions of any given immunoglobulin as defined by the EU index will not necessarily correspond to its sequential sequence. General and specific scientific techniques are outlined in US Publications 2015/0307629, 2014/0288275 and WO2014/145806, all of which are expressly incorporated by reference in their entirety and particularly for the techniques outlined therein. EXAMPLES Example 1: Alternate Formats Bispecifics Production Cartoon schematics of anti-CD38×anti-CD3 bispecifics are shown inFIG.1. Amino acid sequences for alternate format anti-CD38×anti-CD3 bispecifics are listed inFIG.39toFIG.43. DNA encoding the three chains needed for bispecific expression were generated by gene synthesis (Blue Heron Biotechnology, Bothell, Wash.) and were subcloned using standard molecular biology techniques into the expression vector pTT5. Substitutions were introduced using either site-directed mutagenesis (QuikChange, Stratagene, Cedar Creek, Tex.) or additional gene synthesis and subcloning. DNA was transfected into HEK293E cells for expression and resulting proteins were purified from the supernatant using protein A affinity (GE Healthcare) and cation exchange chromatography. Yields following protein A affinity purification are shown inFIG.35. Cation exchange chromatography purification was performed using a HiTrap SP HP column (GE Healthcare) with a wash/equilibration buffer of 50 mM MES, pH 6.0 and an elution buffer of 50 mM MES, pH 6.0+1 M NaCl linear gradient (seeFIG.36for chromatograms). Redirected T Cell CγTotoxicity Anti-CD38×anti-CD3 bispecifics were characterized in vitro for redirected T cell cytotoxicity (RTCC) of the CD38+RPMI8266 myeloma cell line. 10 k RPMI8266 cells were incubated for 24 h with 500 k human PBMCs. RTCC was measured by LDH fluorescence as indicated (seeFIG.37). Example 2 Redirected T Cell Cytotoxicity Anti-CD38×anti-CD3 Fab-scFv-Fc bispecifics were characterized in vitro for redirected T cell cytotoxicity (RTCC) of the CD38+ RPMI8266 myeloma cell line. 40 k RPMI8266 cells were incubated for 96 h with 400 k human PBMCs. RTCC was measured by flow cytometry as indicated (seeFIG.44). CD4+ and CD8+ T cell expression of CD69, Ki-67, and PI-9 were also characterized by flow cytometry and are shown inFIG.45. Mouse Model of Anti-Tumor Activity Four groups of five NOD scid gamma (NSG) mice each were engrafted with 5×106 RPMI8226TrS tumor cells (multiple myeloma, luciferase-expressing) by intravenous tail vein injection on Day −23. On Day 0, mice were engrafted intraperitoneally with 10×106 human PBMCs. After PBMC engraftment on Day 0, test articles are dosed weekly (Days 0, 7) by intraperitoneal injection at dose levels indicated inFIG.4. Study design is further summarized inFIG.46. Tumor growth was monitored by measuring total flux per mouse using an in vivo imaging system (IVIS®). Both XmAb13551 and XmAb15426 showed substantial anti-tumor effects (seeFIG.47andFIG.48). Studies in Cynomolgus Monkey Cynomolgus monkeys were given a single dose of anti-CD38×anti-CD3 bispecifics. An anti-RSV×anti-CD3 bispecific control was also included. Dose levels were: 20 μg/kg XmAb13551 (n=2), 0.5 mg/kg XmAb15426 (n=3), 3 mg/kg XmAb14702 (n=3), or 3 mg/kg XmAb13245 (anti-RSV×anti-CD3 control, n=3) (in 3 independent studies). Anti-CD38×anti-CD3 bispecifics rapidly depleted CD38+ cells in peripheral blood (seeFIG.49). Anti-CD38×anti-CD3 bispecifics resulted in T cell activation as measured by CD69 expression (seeFIG.50). Serum levels of IL-6 were also measured (seeFIG.51). Note that, compared to XmAb13551, XmAb15426 had an increased duration of CD38+ cell depletion and lower levels of T cell activation and IL-6 production. XmAb15426 and XmAb14702 were tested at single doeses of 0.5 mg/kg and 3 mg/kg respectively. Both antibodies were well-tolerated at these higher doeses, consistent with the moderate levels of IL6 observed in serum from the treated monkeys. Moreover, XmAb15426, with intermediate CD3 affinity, more effectively depleted CD38+ cells at 0.5 mg/kg compared to the original high-affinity XmAb13551 dosed at 2, 5 or 20 μg/kg. Depletion by XmAb15426 was more sustained compared to the highest dose of of XmAb13551 in the previous study (7 vs. 2 days, respectively). Notably, although target cell depletion was greater for XmAb15426, T cell activation (CD69, CD25 and PD1 induction) was much lower in monkeys treated with XmAb15426 even dosed 25-fold higher than the 20 μg/kg XmAb13551 group. XmAb14702, with very low CD3 affinity, had little effect on CD38+ cells and T cell activation. These results demonstrate that modulating T cell activation by attenuating CD3 affinity is a promising method to improve the therapeutic window of T cell-engaging bispecific antibodies. This strategy has potential to expand the set of antigens amenable to targeted T cell immunotherapy by improving tolerability and enabling higher dosing to overcome antigen sink clearance with targets such as CD38. We have shown that by reducing affinity for CD3, XmAb 15426 effectively depletes CD38+ cells while minimizing the CRS effects ween with comparable doses of its high-affinity counterpart XmAb13551. | 124,446 |
11859012 | DETAILED DESCRIPTION The present invention provides heterodimeric bispecific antibodies that bind to human CD3ε and human GPC3. A. Overview Anti-bispecific antibodies that co-engage CD3 and a tumor antigen target are used to redirect T cells to attack and lyse targeted tumor cells. Examples include the BiTE® and DART formats, which monovalently engage CD3 and a tumor antigen. While the CD3-targeting approach has shown considerable promise, a common side effect of such therapies is the associated production of cytokines, often leading to toxic cytokine release syndrome. Because the anti-CD3 binding domain of the bispecific antibody engages all T cells, the high cytokine-producing CD4 T cell subset is recruited. Moreover, the CD4 T cell subset includes regulatory T cells, whose recruitment and expansion can potentially lead to immune suppression and have a negative impact on long-term tumor suppression. In addition, these formats do not contain Fc domains and show very short serum half-lives in patients. Provided herein are novel anti-CD3×anti-GPC3 (also referred to as anti-GPC3×anti-CD3, αCD3×αGPC3, αGPC3×αCD3 or sometimes just GPC3×CD3) heterodimeric bispecific antibodies and methods of using such antibodies for the treatment of cancers. In particular, provided herein are anti-CD3, anti-GPC3 bispecific antibodies in a variety of formats. These bispecific antibodies are useful for the treatment of cancers, particularly those with increased GPC3 expression such as renal cell carcinoma. Such antibodies are used to direct CD3+ effector T cells to GPC3+ tumors, thereby allowing the CD3+ effector T cells to attack and lyse the GPC3+ tumors. Additionally, in some embodiments, the disclosure provides bispecific antibodies that have different binding affinities to human CD3 that can alter or reduce the potential side effects of anti-CD3 therapy. That is, in some embodiments the antibodies described herein provide antibody constructs comprising anti-CD3 antigen binding domains that are “strong” or “high affinity” binders to CD3 (e.g. one example are heavy and light variable domains depicted as H1.30_L1.47 (optionally including a charged linker as appropriate)) and also bind to GPC3. In other embodiments, the antibodies described herein provide antibody constructs comprising anti-CD3 antigen binding domains that are “lite” or “lower affinity” binders to CD3. Additional embodiments provides antibody constructs comprising anti-CD3 antigen binding domains that have intermediate or “medium” affinity to CD3 that also bind to GPC3. While a very large number of anti-CD3 antigen binding domains (ABDs) can be used, particularly useful embodiments use 6 different anti-CD3 ABDs, although they can be used in two scFv orientations as discussed herein. Affinity is generally measured using a Biacore assay. It should be appreciated that the “high, medium, low” anti-CD3 sequences provided herein can be used in a variety of heterodimerization formats as discussed herein. In general, due to the potential side effects of T cell recruitment, exemplary embodiments utilize formats that only bind CD3 monovalently, such as depicted inFIGS.15A and15B, and in the formats depicted herein, it is the CD3 ABD that is a scFv as more fully described herein. In contrast, the subject bispecific antibodies can bind GPC3 either monovalently (e.g.FIG.15A) or bivalently (e.g.FIG.15B). Provided herein are compositions that include GPC3 binding domains, including antibodies with such GPC3 binding domains (e.g., GPC3×CD3 bispecific antibodies). Subject antibodies that include such GPC3 binding domains advantageously elicit a range of different immune responses, depending on the particular GPC3 binding domain used. For example, the subject antibodies exhibit differences in selectivity for cells with different GPC3 expression, potencies for GPC3 expressing cells, ability to elicit cytokine release, and sensitivity to soluble GPC3. Such GPC3 binding domains and related antibodies find use, for example, in the treatment of GPC3 associated cancers. Accordingly, in one aspect, provided herein are heterodimeric antibodies that bind to two different antigens, e.g. the antibodies are “bispecific”, in that they bind two different target antigens, generally GPC3 and CD3 as described herein. These heterodimeric antibodies can bind these target antigens either monovalently (e.g. there is a single antigen binding domain such as a variable heavy and variable light domain pair) or bivalently (there are two antigen binding domains that each independently bind the antigen). In some embodiments, the heterodimeric antibody provided herein includes one CD3 binding domain and one GPC3 binding domain (e.g., heterodimeric antibodies in the “1+1 Fab-scFv-Fc” format described herein). In other embodiments, the heterodimeric antibody provided herein includes one CD3 binding domain and two GPC3 binding domains (e.g., heterodimeric antibodies in the “2+1 Fab2-scFv-Fc” formats described herein). The heterodimeric antibodies provided herein are based on the use different monomers which contain amino acid substitutions that “skew” formation of heterodimers over homodimers, as is more fully outlined below, coupled with “pI variants” that allow simple purification of the heterodimers away from the homodimers, as is similarly outlined below. The heterodimeric bispecific antibodies provided generally rely on the use of engineered or variant Fc domains that can self-assemble in production cells to produce heterodimeric proteins, and methods to generate and purify such heterodimeric proteins. B. Nomenclature The antibodies provided herein are listed in several different formats. In some instances, each monomer of a particular antibody is given a unique “XENP” number, although as will be appreciated in the art, a longer sequence might contain a shorter one. For example, a “scFv-Fc” monomer of a 1+1 Fab-scFv-Fc format antibody may have a first XENP number, while the scFv domain itself will have a different XENP number. Some molecules have three polypeptides, so the XENP number, with the components, is used as a name. Thus, the molecule XENP33744, which is in 2+1 Fab2-scFv-Fc format, comprises three sequences (seeFIG.18) a “Fab-Fc Heavy Chain” monomer; 2) a “Fab-scFv-Fc Heavy Chain” monomer; and 3) a “Light Chain” monomer or equivalents, although one of skill in the art would be able to identify these easily through sequence alignment. These XENP numbers are in the sequence listing as well as identifiers, and used in the Figures. In addition, one molecule, comprising the three components, gives rise to multiple sequence identifiers. For example, the listing of the Fab includes, the full heavy chain sequence, the variable heavy domain sequence and the three CDRs of the variable heavy domain sequence, the full light chain sequence, a variable light domain sequence and the three CDRs of the variable light domain sequence. A Fab-scFv-Fc monomer includes a full length sequence, a variable heavy domain sequence, 3 heavy CDR sequences, and an scFv sequence (include scFv variable heavy domain sequence, scFv variable light domain sequence and scFv linker). Note that some molecules herein with a scFv domain use a single charged scFv linker (+H), although others can be used. In addition, the naming nomenclature of particular antigen binding domains (e.g., GPC3 and CD3 binding domains) use a “Hx.xx_Ly.yy” type of format, with the numbers being unique identifiers to particular variable chain sequences. Thus, an Fv domain of the antigen binding domain is “H1 L1”, which indicates that the variable heavy domain, H1, was combined with the light domain L1. In the case that these sequences are used as scFvs, the designation “H1 L1”, indicates that the variable heavy domain, H1 is combined with the light domain, L1, and is in VH-linker-VL orientation, from N- to C-terminus. This molecule with the identical sequences of the heavy and light variable domains but in the reverse order (VL-linker-VH orientation, from N- to C-terminus) would be designated “L1_H1.1”. Similarly, different constructs may “mix and match” the heavy and light chains as will be evident from the sequence listing and the figures. Additionally, the bispecific antibodies of the invention are referred to herein as “anti-CD3×anti-GPC3”, “αCD3×αGPC3”, “αGPC3×αCD3” or sometimes just “GPC3×CD3”. The order of the antigens is not determinative as will be discussed below, although the majority of the formats that utilize as scFv have the an anti-CD3 ABD as the scFv. C. Definitions In order that the application may be more completely understood, several definitions are set forth below. Such definitions are meant to encompass grammatical equivalents. By “GPC3” herein is meant a protein belonging to the claudin family. GPC3 sequences are depicted, for example, inFIG.11. The ABDs of the invention bind to human GPC3. By “ablation” herein is meant a decrease or removal of activity. Thus for example, “ablating FcγR binding” means the Fc region amino acid variant has less than 50% starting binding as compared to an Fc region not containing the specific variant, with more than 70-80-90-95-98% loss of activity being preferred, and in general, with the activity being below the level of detectable binding in a Biacore, SPR or BLI assay. Of particular use in the ablation of FcγR binding are those shown inFIG.3, which generally are added to both monomers. By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as used herein is meant the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is correlated with binding to FcγRIIIa; increased binding to FcγRIIIa leads to an increase in ADCC activity. By “ADCP” or antibody dependent cell-mediated phagocytosis as used herein is meant the cell-mediated reaction wherein nonspecific phagocytic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. As used herein, term “antibody” is used generally. Antibodies described herein can take on a number of formats as described herein, including traditional antibodies as well as antibody derivatives, fragments and mimetics, including a number of bispecific formats described herein. Traditional immunoglobulin (Ig) antibodies are “Y” shaped tetramers. Each tetramer is typically composed of two identical pairs of polypeptide chains, each pair having one “light chain” monomer (typically having a molecular weight of about 25 kDa) and one “heavy chain” monomer (typically having a molecular weight of about 50-70 kDa). Other useful antibody formats include, but are not limited to, the 1+1 Fab-scFv-Fc format and 2+1 Fab-scFv-Fc antibody formats described herein and depicted inFIG.15, as well as “mAb-Fv,” “mAb-scFv,” “central-Fv”, “one-armed scFv-mAb,” “scFv-mAb,” “dual scFv,” and “trident” format antibodies, as discussed below and shown inFIG.44. Antibody heavy chains typically include a variable heavy (VH) domain, which includes vhCDR1-3, and an Fc domain, which includes a CH2-CH3 monomer. In some embodiments, antibody heavy chains include a hinge and CH1 domain. Traditional antibody heavy chains are monomers that are organized, from N- to C-terminus: VH-CH1-hinge-CH2-CH3. The CH1-hinge-CH2-CH3 is collectively referred to as the heavy chain “constant domain” or “constant region” of the antibody, of which there are five different categories or “isotypes”: IgA, IgD, IgG, IgE and IgM. Thus, “isotype” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. It should be understood that therapeutic antibodies can also comprise hybrids of isotypes and/or subclasses. For example, as shown in US Publication 2009/0163699, incorporated by reference, the antibodies described herein include the use of human IgG1/G2 hybrids. In some embodiments, the antibodies provided herein include IgG isotype constant domains, which has several subclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4. In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By “immunoglobulin (Ig) domain” herein is meant a region of an immunoglobulin having a distinct tertiary structure. Of interest in the antibodies described herein are the heavy chain domains, including, the constant heavy (CH) domains and the hinge domains. In the context of IgG antibodies, the IgG isotypes each have three CH regions. Accordingly, “CH” domains in the context of IgG are as follows: “CH1” refers to positions 118-220 according to the EU index as in Kabat. “CH2” refers to positions 237-340 according to the EU index as in Kabat, and “CH3” refers to positions 341-447 according to the EU index as in Kabat. As shown herein and described below, the pI variants can be in one or more of the CH regions, as well as the hinge region, discussed below. It should be noted that IgG1 has different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). The sequences depicted herein use the 356D/358M allotype, however the other allotype is included herein. That is, any sequence inclusive of an IgG1 Fc domain included herein can have 356E/358L replacing the 356D/358M allotype. It should be understood that therapeutic antibodies can also comprise hybrids of isotypes and/or subclasses. For example, as shown in US Publication 2009/0163699, incorporated by reference, the present antibodies, in some embodiments, include IgG1/IgG2 hybrids. By “Fc” or “Fc region” or “Fc domain” as used herein is meant the polypeptide comprising the constant region of an antibody, in some instances, excluding all of the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and in some cases, optionally including all or part of the hinge. For IgG, the Fc domain comprises immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all or a portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, in some cases, the Fc domain includes, from N- to C-terminal, CH2-CH3 and hinge-CH2-CH3. In some embodiments, the Fc domain is that from human IgG1, IgG2, IgG3 or IgG4, with human IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3 finding particular use in many embodiments. Additionally, in the case of human IgG1 Fc domains, frequently the hinge includes a C220S amino acid substitution. Furthermore, in the case of human IgG4 Fc domains, frequently the hinge includes a S228P amino acid substitution. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues E216, C226, or A231 to its carboxyl-terminal, wherein the numbering is according to the EU index as in Kabat. In some embodiments, as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcγR or to the FcRn. By “heavy chain constant region” herein is meant the CH1-hinge-CH2-CH3 portion of an antibody (or fragments thereof), excluding the variable heavy domain; in EU numbering of human IgG1 this is amino acids 118-447 By “heavy chain constant region fragment” herein is meant a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region. Another type of Ig domain of the heavy chain is the hinge region. By “hinge” or “hinge region” or “antibody hinge region” or “hinge domain” herein is meant the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain begins at residue EU position 231. Thus for IgG the antibody hinge is herein defined to include positions 216 (E216 in IgG1) to 230 (p230 in IgG1), wherein the numbering is according to the EU index as in Kabat. In some cases, a “hinge fragment” is used, which contains fewer amino acids at either or both of the N- and C-termini of the hinge domain. As noted herein, pI variants can be made in the hinge region as well. Many of the antibodies herein have at least one the cysteines at position 220 according to EU numbering (hinge region) replaced by a serine. Generally, this modification is on the “scFv monomer” side for most of the sequences depicted herein, although it can also be on the “Fab monomer” side, or both, to reduce disulfide formation. Specifically included within the sequences herein are one or both of these cysteines replaced (C220S). As will be appreciated by those in the art, the exact numbering and placement of the heavy constant region domains can be different among different numbering systems. A useful comparison of heavy constant region numbering according to EU and Kabat is as below, see Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, entirely incorporated by reference. TABLE 1EU NumberingKabat NumberingCH1118-215114-223Hinge216-230226-243CH2231-340244-360CH3341-447361-478 The antibody light chain generally comprises two domains: the variable light domain (VL), which includes light chain CDRs vlCDR1-3, and a constant light chain region (often referred to as CL or CIO. The antibody light chain is typically organized from N- to C-terminus: VL-CL. By “antigen binding domain” or “ABD” herein is meant a set of six Complementary Determining Regions (CDRs) that, when present as part of a polypeptide sequence, specifically binds a target antigen (e.g., GPC3 or CD3) as discussed herein. As is known in the art, these CDRs are generally present as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and a second set of variable light CDRs (vlCDRs or VLCDRs), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 variable heavy CDRs and vlCDR1, vlCDR2 and vlCDR3 vhCDR3 variable light CDRs. The CDRs are present in the variable heavy domain (vhCDR1-3) and variable light domain (vlCDR1-3). The variable heavy domain and variable light domain form an Fv region. The antibodies described herein provide a large number of different CDR sets. In this case, a “full CDR set” comprises the three variable light and three variable heavy CDRs, e.g., a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 and vhCDR3. These can be part of a larger variable light or variable heavy domain, respectfully. In addition, as more fully outlined herein, the variable heavy and variable light domains can be on separate polypeptide chains, when a heavy and light chain is used (for example when Fabs are used), or on a single polypeptide chain in the case of scFv sequences. As will be appreciated by those in the art, the exact numbering and placement of the CDRs can be different among different numbering systems. However, it should be understood that the disclosure of a variable heavy and/or variable light sequence includes the disclosure of the associated (inherent) CDRs. Accordingly, the disclosure of each variable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1, vhCDR2 and vhCDR3) and the disclosure of each variable light region is a disclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3). A useful comparison of CDR numbering is as below, see Lafranc et al.,Dev. Comp. Immunol.27(1):55-77 (2003): TABLE 2Kabat + ChothiaIMGTKabatAbMChothiaContactXencorvhCDR126-3527-3831-3526-3526-3230-3527-35vhCDR250-6556-6550-6550-5852-5647-5854-61vhCDR395-102105-11795-10295-10295-10293-101103-116vlCDR124-3427-3824-3424-3424-3430-3627-38vlCDR250-5656-6550-5650-5650-5646-5556-62vlCDR389-97105-11789-9789-9789-9789-9697-105 Throughout the present specification, the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately, residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) and the EU numbering system for Fc regions (e.g., Kabat et al., supra (1991)). The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of the antigen binding domains and antibodies. “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope. The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning.” As outlined below, the disclosure not only includes the enumerated antigen binding domains and antibodies herein, but those that compete for binding with the epitopes bound by the enumerated antigen binding domains. In some embodiments, the six CDRs of the antigen binding domain are contributed by a variable heavy and a variable light domain. In a “Fab” format, the set of 6 CDRs are contributed by two different polypeptide sequences, the variable heavy domain (vh or VH; containing the vhCDR1, vhCDR2 and vhCDR3) and the variable light domain (vl or VL; containing the vlCDR1, vlCDR2 and vlCDR3), with the C-terminus of the vh domain being attached to the N-terminus of the CH1 domain of the heavy chain and the C-terminus of the vl domain being attached to the N-terminus of the constant light domain (and thus forming the light chain). In a scFv format, the vh and vl domains are covalently attached, generally through the use of a linker (a “scFv linker”) as outlined herein, into a single polypeptide sequence, which can be either (starting from the N-terminus) vh-linker-vl or vl-linker-vh, with the former being generally preferred (including optional domain linkers on each side, depending on the format used (e.g., fromFIG.44). In general, the C-terminus of the scFv domain is attached to the N-terminus of the hinge in the second monomer. By “variable region” or “variable domain” as used herein is meant the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively, and contains the CDRs that confer antigen specificity. Thus, a “variable heavy domain” pairs with a “variable light domain” to form an antigen binding domain (“ABD”). In addition, each variable domain comprises three hypervariable regions (“complementary determining regions,” “CDRs”) (VHCDR1, VHCDR2 and VHCDR3 for the variable heavy domain and VLCDR1, VLCDR2 and VLCDR3 for the variable light domain) and four framework (FR) regions, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The hypervariable region generally encompasses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable region and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described in Table 2. By “Fab” or “Fab region” as used herein is meant the polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g. VH-CH1 on one chain and VL-CL on the other). Fab may refer to this region in isolation, or this region in the context of a bispecific antibody described herein. In the context of a Fab, the Fab comprises an Fv region in addition to the CH1 and CL domains. By “Fv” or “Fv fragment” or “Fv region” as used herein is meant a polypeptide that comprises the VL and VH domains of an ABD. Fv regions can be formatted as both Fabs (as discussed above, generally two different polypeptides that also include the constant regions as outlined above) and scFvs, where the VL and VH domains are combined (generally with a linker as discussed herein) to form an scFv. By “single chain Fv” or “scFv” herein is meant a variable heavy domain covalently attached to a variable light domain, generally using a scFv linker as discussed herein, to form a scFv or scFv domain. A scFv domain can be in either orientation from N- to C-terminus (VH-linker-VL or VL-linker-VH). In the sequences depicted in the sequence listing and in the figures, the order of the VH and VL domain is indicated in the name, e.g. H.X_L.Y means N- to C-terminal is VH-linker-VL, and L.Y_H.X is VL-linker-VH. However, the disclosure of any “H L” pairs is meant to include them in either order. Some embodiments of the subject antibodies provided herein comprise at least one scFv domain, which, while not naturally occurring, generally includes a variable heavy domain and a variable light domain, linked together by a scFv linker. As outlined herein, while the scFv domain is generally from N- to C-terminus oriented as VH-scFv linker-VL, this can be reversed for any of the scFv domains (or those constructed using vh and vl sequences from Fabs), to VL-scFv linker-VH, with optional linkers at one or both ends depending on the format. By “modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence or an alteration to a moiety chemically linked to a protein. For example, a modification may be an altered carbohydrate or PEG structure attached to a protein. By “amino acid modification” herein is meant an amino acid substitution, insertion, and/or deletion in a polypeptide sequence. For clarity, unless otherwise noted, the amino acid modification is always to an amino acid coded for by DNA, e.g. the 20 amino acids that have codons in DNA and RNA. By “amino acid substitution” or “substitution” herein is meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturally occurring at the particular position, either not naturally occurring within the organism or in any organism. For example, the substitution E272Y refers to a variant polypeptide, in this case an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein which has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels) is not an “amino acid substitution”; that is, despite the creation of a new gene encoding the same protein, if the protein has the same amino acid at the particular position that it started with, it is not an amino acid substitution. By “amino acid insertion” or “insertion” as used herein is meant the addition of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, −233E or 233E designates an insertion of glutamic acid after position 233 and before position 234. Additionally, −233ADE or A233ADE designates an insertion of AlaAspGlu after position 233 and before position 234. By “amino acid deletion” or “deletion” as used herein is meant the removal of an amino acid sequence at a particular position in a parent polypeptide sequence. For example, E233- or E233 #, E233( ) or E233del designates a deletion of glutamic acid at position 233. Additionally, EDA233- or EDA233 # designates a deletion of the sequence GluAspAla that begins at position 233. By “variant protein” or “protein variant”, or “variant” as used herein is meant a protein that differs from that of a parent protein by virtue of at least one amino acid modification. The protein variant has at least one amino acid modification compared to the parent protein, yet not so many that the variant protein will not align with the parental protein using an alignment program such as that described below. In general, variant proteins (such as variant Fc domains, etc., outlined herein, are generally at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the parent protein, using the alignment programs described below, such as BLAST. “Variant” as used herein also refers to particular amino acid modifications that confer particular function (e.g., a “heterodimerization variant,” “pI variant,” “ablation variant,” etc.). As described below, in some embodiments the parent polypeptide, for example an Fc parent polypeptide, is a human wild-type sequence, such as the heavy constant domain or Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequences with variants can also serve as “parent polypeptides”, for example the IgG1/2 hybrid of US Publication 2006/0134105 can be included. The protein variant sequence herein will preferably possess at least about 80% identity with a parent protein sequence, and most preferably at least about 90% identity, more preferably at least about 95-98-99% identity. Accordingly, by “antibody variant” or “variant antibody” as used herein is meant an antibody that differs from a parent antibody by virtue of at least one amino acid modification, “IgG variant” or “variant IgG” as used herein is meant an antibody that differs from a parent IgG (again, in many cases, from a human IgG sequence) by virtue of at least one amino acid modification, and “immunoglobulin variant” or “variant immunoglobulin” as used herein is meant an immunoglobulin sequence that differs from that of a parent immunoglobulin sequence by virtue of at least one amino acid modification. “Fc variant” or “variant Fc” as used herein is meant a protein comprising an amino acid modification in an Fc domain. The modification can be an addition, deletion, or substitution. The Fc variants are defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S is an Fc variant with the substitution for serine at position 434 relative to the parent Fc polypeptide, wherein the numbering is according to the EU index. Likewise, M428L/N434S defines an Fc variant with the substitutions M428L and N434S relative to the parent Fc polypeptide. The identity of the WT amino acid may be unspecified, in which case the aforementioned variant is referred to as 428L/434S. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, 428L/434S is the same Fc variant as 434S/428L, and so on. For all positions discussed herein that relate to antibodies or derivatives and fragments thereof (e.g., Fc domains), unless otherwise noted, amino acid position numbering is according to the EU index. The “EU index” or “EU index as in Kabat” or “EU numbering” scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference). In general, variant Fc domains have at least about 80, 85, 90, 95, 97, 98 or 99 percent identity to the corresponding parental human IgG Fc domain (using the identity algorithms discussed below, with one embodiment utilizing the BLAST algorithm as is known in the art, using default parameters). Alternatively, the variant Fc domains can have from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications as compared to the parental Fc domain. Alternatively, the variant Fc domains can have up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications as compared to the parental Fc domain. Additionally, as discussed herein, the variant Fc domains described herein still retain the ability to form a dimer with another Fc domain as measured using known techniques as described herein, such as non-denaturing gel electrophoresis. By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. In addition, polypeptides that make up the antibodies described herein may include synthetic derivatization of one or more side chains or termini, glycosylation, PEGylation, circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains, and addition of peptide tags or labels. By “residue” as used herein is meant a position in a protein and its associated amino acid identity. For example, Asparagine 297 (also referred to as Asn297 or N297) is a residue at position 297 in the human antibody IgG1. By “IgG subclass modification” or “isotype modification” as used herein is meant an amino acid modification that converts one amino acid of one IgG isotype to the corresponding amino acid in a different, aligned IgG isotype. For example, because IgG1 comprises a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y substitution in IgG2 is considered an IgG subclass modification. By “non-naturally occurring modification” as used herein is meant an amino acid modification that is not isotypic. For example, because none of the human IgGs comprise a serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered a non-naturally occurring modification. By “amino acid” and “amino acid identity” as used herein is meant one of the 20 naturally occurring amino acids that are coded for by DNA and RNA. By “effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC. By “IgG Fc ligand” as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an IgG antibody to form an Fc/Fc ligand complex. Fc ligands include but are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan binding lectin, mannose receptor, staphylococcal protein A, streptococcal protein G, and viral FcγR. Fc ligands also include Fc receptor homologs (FcRH), which are a family of Fc receptors that are homologous to the FcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirely incorporated by reference). Fc ligands may include undiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gamma receptors. By “Fc ligand” as used herein is meant a molecule, preferably a polypeptide, from any organism that binds to the Fc region of an antibody to form an Fc/Fc ligand complex. By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and is encoded by an FcγR gene. In humans this family includes but is not limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporated by reference), as well as any undiscovered human FcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRs or FcγR isoforms or allotypes. By “FcRn” or “neonatal Fc Receptor” as used herein is meant a protein that binds the IgG antibody Fc region and is encoded at least in part by an FcRn gene. The FcRn may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. As is known in the art, the functional FcRn protein comprises two polypeptides, often referred to as the heavy chain and light chain. The light chain is beta-2-microglobulin and the heavy chain is encoded by the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn protein refers to the complex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRn variants used to increase binding to the FcRn receptor, and in some cases, to increase serum half-life. An “FcRn variant” is one that increases binding to the FcRn receptor, and suitable FcRn variants are shown below. By “parent polypeptide” as used herein is meant a starting polypeptide that is subsequently modified to generate a variant. The parent polypeptide may be a naturally occurring polypeptide, or a variant or engineered version of a naturally occurring polypeptide. Accordingly, by “parent immunoglobulin” as used herein is meant an unmodified immunoglobulin polypeptide that is modified to generate a variant, and by “parent antibody” as used herein is meant an unmodified antibody that is modified to generate a variant antibody. It should be noted that “parent antibody” includes known commercial, recombinantly produced antibodies as outlined below. In this context, a “parent Fc domain” will be relative to the recited variant; thus, a “variant human IgG1 Fc domain” is compared to the parent Fc domain of human IgG1, a “variant human IgG4 Fc domain” is compared to the parent Fc domain human IgG4, etc. By “position” as used herein is meant a location in the sequence of a protein. Positions may be numbered sequentially, or according to an established format, for example the EU index for antibody numbering. By “target antigen” as used herein is meant the molecule that is bound specifically by the antigen binding domain comprising the variable regions of a given antibody. By “strandedness” in the context of the monomers of the heterodimeric antibodies described herein is meant that, similar to the two strands of DNA that “match”, heterodimerization variants are incorporated into each monomer so as to preserve the ability to “match” to form heterodimers. For example, if some pI variants are engineered into monomer A (e.g. making the pI higher) then steric variants that are “charge pairs” that can be utilized as well do not interfere with the pI variants, e.g. the charge variants that make a pI higher are put on the same “strand” or “monomer” to preserve both functionalities. Similarly, for “skew” variants that come in pairs of a set as more fully outlined below, the skilled artisan will consider pI in deciding into which strand or monomer one set of the pair will go, such that pI separation is maximized using the pI of the skews as well. By “target cell” as used herein is meant a cell that expresses a target antigen. By “host cell” in the context of producing a bispecific antibody according to the antibodies described herein is meant a cell that contains the exogeneous nucleic acids encoding the components of the bispecific antibody and is capable of expressing the bispecific antibody under suitable conditions. Suitable host cells are discussed below. By “wild type or WT” herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein has an amino acid sequence or a nucleotide sequence that has not been intentionally modified. Provided herein are a number of antibody domains that have sequence identity to human antibody domains. Sequence identity between two similar sequences (e.g., antibody variable domains) can be measured by algorithms such as that of Smith, T. F. & Waterman, M. S. (1981) “Comparison Of Biosequences,” Adv. Appl. Math. 2:482 [local homology algorithm]; Needleman, S. B. & Wunsch, C D. (1970) “A General Method Applicable To The Search For Similarities In The Amino Acid Sequence Of Two Proteins,” J. Mol. Biol. 48:443 [homology alignment algorithm], Pearson, W. R. & Lipman, D. J. (1988) “Improved Tools For Biological Sequence Comparison,” Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 [search for similarity method]; or Altschul, S. F. et al, (1990) “Basic Local Alignment Search Tool,” J. Mol. Biol. 215:403-10, the “BLAST” algorithm, see blast.ncbi.nlm.nih.gov. When using any of the aforementioned algorithms, the default parameters (for Window length, gap penalty, etc.) are used. In one embodiment, sequence identity is done using the BLAST algorithm, using default parameters The antibodies described herein are generally isolated or recombinant. “Isolated,” when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated and/or recovered from a cell or cell culture from which it was expressed. Ordinarily, an isolated polypeptide will be prepared by at least one purification step. An “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. “Recombinant” means the antibodies are generated using recombinant nucleic acid techniques in exogeneous host cells, and they can be isolated as well. “Specific binding” or “specifically binds to” or is “specific for” a particular antigen or an epitope means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target. Specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KD for an antigen or epitope of at least about 10−4M, at least about 10−5M, at least about 10−6M, at least about 10−7M, at least about 10−8M, at least about 10−9M, alternatively at least about 10−10M, at least about 10−11M, at least about 10−12M, or greater, where KD refers to a dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a control molecule relative to the antigen or epitope. Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relative to a control, where KA or Ka refers to an association rate of a particular antibody-antigen interaction. Binding affinity is generally measured using a Biacore, SPR or BLI assay. D. Antibodies of the Invention The present invention provides antibodies, including monoclonal antibodies and bispecific antibodies, that bind to human GPC3 (it should be noted that many, if not most, of the exemplified antibodies also bind to cyno GPC3 for ease of pre-clinical testing, but this is not required in all embodiments). In particularly, bispecific antibodies are provided that bind CD3 and GPC3 that make take on a variety of formats as more fully described below. 1. Antibodies The antibodies provided herein include different antibody domains as is more fully described below. As described herein and known in the art, the antibodies described herein include different domains within the heavy and light chains, which can be overlapping as well. These domains include, but are not limited to, the Fc domain, the CH1 domain, the CH2 domain, the CH3 domain, the hinge domain, the heavy constant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), the variable heavy domain, the variable light domain, the light constant domain, Fab domains and scFv domains. In particular, the formats depicted inFIGS.15A and15Bare antibodies, usually referred to as “heterodimeric antibodies”, meaning that the protein has at least two associated Fc sequences self-assembled into a heterodimeric Fc domain and at least two Fv regions, whether as Fabs or as scFvs. a. Chimeric and Humanized Antibodies In certain embodiments, the antibodies described herein comprise a heavy chain variable region from a particular germline heavy chain immunoglobulin gene and/or a light chain variable region from a particular germline light chain immunoglobulin gene. For example, such antibodies may comprise or consist of a human antibody comprising heavy or light chain variable regions that are “the product of” or “derived from” a particular germline sequence. A human antibody that is “the product of” or “derived from” a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence (i.e., greatest % identity) to the sequence of the human antibody (using the methods outlined herein). A human antibody that is “the product of” or “derived from” a particular human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence, due to, for example, naturally-occurring somatic mutations or intentional introduction of site-directed mutation. However, a humanized antibody typically is at least 90% identical in amino acids sequence to an amino acid sequence encoded by a human germline immunoglobulin gene and contains amino acid residues that identify the antibody as being derived from human sequences when compared to the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences). In certain cases, a humanized antibody may be at least 95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a humanized antibody derived from a particular human germline sequence will display no more than 10-20 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene (prior to the introduction of any skew, pI and ablation variants herein; that is, the number of variants is generally low, prior to the introduction of the variants described herein). In certain cases, the humanized antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene (again, prior to the introduction of any skew, pI and ablation variants herein; that is, the number of variants is generally low, prior to the introduction of the variants described herein). In one embodiment, the parent antibody has been affinity matured, as is known in the art. Structure-based methods may be employed for humanization and affinity maturation, for example as described in U.S. Ser. No. 11/004,590. Selection based methods may be employed to humanize and/or affinity mature antibody variable regions, including but not limited to methods described in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering 16(10):753-759, all entirely incorporated by reference. Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in U.S. Ser. No. 09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol. 169:3076-3084, all entirely incorporated by reference. 2. Heterodimeric Antibodies In exemplary embodiments, the bispecific antibodies provided herein are heterodimeric bispecific antibodies that include two variant Fc domain sequences. Such variant Fc domains include amino acid modifications to facilitate the self-assembly and/or purification of the heterodimeric antibodies. An ongoing problem in antibody technologies is the desire for “bispecific” antibodies that bind to two different antigens simultaneously, in general thus allowing the different antigens to be brought into proximity and resulting in new functionalities and new therapies. In general, these antibodies are made by including genes for each heavy and light chain into the host cells. This generally results in the formation of the desired heterodimer (A-B), as well as the two homodimers (A-A and B-B (not including the light chain heterodimeric issues)). However, a major obstacle in the formation of bispecific antibodies is the difficulty in biasing the formation of the desired heterodimeric antibody over the formation of the homodimers and/or purifying the heterodimeric antibody away from the homodimers. There are a number of mechanisms that can be used to generate the subject heterodimeric antibodies. In addition, as will be appreciated by those in the art, these different mechanisms can be combined to ensure high heterodimerization. Amino acid modifications that facilitate the production and purification of heterodimers are collectively referred to generally as “heterodimerization variants.” As discussed below, heterodimerization variants include “skew” variants (e.g., the “knobs and holes” and the “charge pairs” variants described below) as well as “pI variants,” which allow purification of heterodimers from homodimers. As is generally described in U.S. Pat. No. 9,605,084, hereby incorporated by reference in its entirety and specifically as below for the discussion of heterodimerization variants, useful mechanisms for heterodimerization include “knobs and holes” (“KIH”) as described in U.S. Pat. No. 9,605,084, “electrostatic steering” or “charge pairs” as described in U.S. Pat. No. 9,605,084, pI variants as described in U.S. Pat. No. 9,605,084, and general additional Fc variants as outlined in U.S. Pat. No. 9,605,084 and below. Heterodimerization variants that are useful for the formation and purification of the subject heterodimeric antibody (e.g., bispecific antibodies) are further discussed in detailed below. a. Skew Variants In some embodiments, the heterodimeric antibody includes skew variants which are one or more amino acid modifications in a first Fc domain (A) and/or a second Fc domain (B) that favor the formation of Fc heterodimers (Fc dimers that include the first and the second Fc domain; (A-B) over Fc homodimers (Fc dimers that include two of the first Fc domain or two of the second Fc domain; A-A or B-B). Suitable skew variants are included in the FIG. 29 of US Publ. App. No. 2016/0355608, hereby incorporated by reference in its entirety and specifically for its disclosure of skew variants, as well as inFIG.1. Thus, suitable Fc heterodimerization variant pairs that will permit the formation of heterodimeric Fc regions are shown inFIG.1. Thus a first Fc domain has first Fc heterodimerization variants and the second Fc domain has second Fc heterodimerization variants selected from the pairs inFIG.1. One mechanism is generally referred to in the art as “knobs and holes”, referring to amino acid engineering that creates steric influences to favor heterodimeric formation and disfavor homodimeric formation can also optionally be used; this is sometimes referred to as “knobs and holes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated by reference in their entirety. The Figures identify a number of “monomer A-monomer B” pairs that rely on “knobs and holes”. In addition, as described in Merchant et al., Nature Biotech. 16:677 (1998), these “knobs and hole” mutations can be combined with disulfide bonds to skew formation to heterodimerization. An additional mechanism that finds use in the generation of heterodimers is sometimes referred to as “electrostatic steering” as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), hereby incorporated by reference in its entirety. This is sometimes referred to herein as “charge pairs”. In this embodiment, electrostatics are used to skew the formation towards heterodimerization. As those in the art will appreciate, these may also have an effect on pI, and thus on purification, and thus could in some cases also be considered pI variants. However, as these were generated to force heterodimerization and were not used as purification tools, they are classified as “steric variants”. These include, but are not limited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g. these are “monomer corresponding sets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R. In some embodiments, the skew variants advantageously and simultaneously favor heterodimerization based on both the “knobs and holes” mechanism as well as the “electrostatic steering” mechanism. In some embodiments, the heterodimeric antibody includes one or more sets of such heterodimerization skew variants. These variants come in “pairs” of “sets”. That is, one set of the pair is incorporated into the first monomer and the other set of the pair is incorporated into the second monomer. It should be noted that these sets do not necessarily behave as “knobs in holes” variants, with a one-to-one correspondence between a residue on one monomer and a residue on the other. That is, these pairs of sets may instead form an interface between the two monomers that encourages heterodimer formation and discourages homodimer formation, allowing the percentage of heterodimers that spontaneously form under biological conditions to be over 90%, rather than the expected 50% (25 homodimer A/A:50% heterodimer A/B:25% homodimer B/B). Exemplary heterodimerization “skew” variants are depicted inFIG.1. In exemplary embodiments, the heterodimeric antibody includes Fc heterodimerization variants as sets: S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L; K370S: S364K/E357Q; or a T366S/L368A/Y407V: T366W (optionally including a bridging disulfide, T366S/L368A/Y407V/Y349C: T366W/S354C) are all “skew” variant amino acid substitution sets of Fc heterodimerization variants. In an exemplary embodiment, the heterodimeric antibody includes a “S364K/E357Q: L368D/K370S” amino acid substitution set. In terms of nomenclature, the pair “S364K/E357Q: L368D/K370S” means that one of the monomers includes an Fc domain that includes the amino acid substitutions S364K and E357Q and the other monomer includes an Fc domain that includes the amino acid substitutions L368D and K370S; as above, the “strandedness” of these pairs depends on the starting pI. In some embodiments, the skew variants provided herein can be optionally and independently incorporated with any other modifications, including, but not limited to, other skew variants (see, e.g., in FIG. 37 of US Publ. App. No. 2012/0149876, herein incorporated by reference, particularly for its disclosure of skew variants), pI variants, isotypic variants, FcRn variants, ablation variants, etc. into one or both of the first and second Fc domains of the heterodimeric antibody. Further, individual modifications can also independently and optionally be included or excluded from the subject the heterodimeric antibody. Additional monomer A and monomer B variants that can be combined with other variants, optionally and independently in any amount, such as pI variants outlined herein or other steric variants that are shown in FIG. 37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which are incorporated expressly by reference herein. In some embodiments, the steric variants outlined herein can be optionally and independently incorporated with any pI variant (or other variants such as Fc variants, FcRn variants, etc.) into one or both monomers, and can be independently and optionally included or excluded from the proteins of the antibodies described herein. A list of suitable skew variants is found inFIG.1, which shows some pairs of particular utility in many embodiments. Of particular use in many embodiments are the pairs of sets including, but not limited to, S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L and K370S: S364K/E357Q. In terms of nomenclature, the pair “S364K/E357Q: L368D/K370S” means that one of the monomers has the double variant set S364K/E357Q and the other has the double variant set L368D/K370S. b. pI (Isoelectric Point) Variants for Heterodimers In some embodiments, the heterodimeric antibody includes purification variants that advantageously allow for the separation of heterodimeric antibody (e.g., anti-GPC3×anti-CD3 bispecific antibody) from homodimeric proteins. There are several basic mechanisms that can lead to ease of purifying heterodimeric antibodies. For example, modifications to one or both of the antibody heavy chain monomers A and B such that each monomer has a different pI allows for the isoelectric purification of heterodimeric A-B antibody from monomeric A-A and B-B proteins. Alternatively, some scaffold formats, such as the “1+1 Fab-scFv-Fc” format and the “2+1 Fab2-scFv-Fc” format, also allows separation on the basis of size. As described above, it is also possible to “skew” the formation of heterodimers over homodimers using skew variants. Thus, a combination of heterodimerization skew variants and pI variants find particular use in the heterodimeric antibodies provided herein. Additionally, as more fully outlined below, depending on the format of the heterodimeric antibody, pI variants either contained within the constant region and/or Fc domains of a monomer, and/or domain linkers can be used. In some embodiments, the heterodimeric antibody includes additional modifications for alternative functionalities that can also create pI changes, such as Fc, FcRn and KO variants. In some embodiments, the subject heterodimeric antibodies provided herein include at least one monomer with one or more modifications that alter the pI of the monomer (i.e., a “pI variant”). In general, as will be appreciated by those in the art, there are two general categories of pI variants: those that increase the pI of the protein (basic changes) and those that decrease the pI of the protein (acidic changes). As described herein, all combinations of these variants can be done: one monomer may be wild type, or a variant that does not display a significantly different pI from wild-type, and the other can be either more basic or more acidic. Alternatively, each monomer is changed, one to more basic and one to more acidic. Depending on the format of the heterodimer antibody, pI variants can be either contained within the constant and/or Fc domains of a monomer, or charged linkers, either domain linkers or scFv linkers, can be used. That is, antibody formats that utilize scFv(s) such as “1+1 Fab-scFv-Fc”, format can include charged scFv linkers (either positive or negative), that give a further pI boost for purification purposes. As will be appreciated by those in the art, some 1+1 Fab-scFv-Fc formats are useful with just charged scFv linkers and no additional pI adjustments, although the antibodies described herein do provide pI variants that are on one or both of the monomers, and/or charged domain linkers as well. In addition, additional amino acid engineering for alternative functionalities may also confer pI changes, such as Fc, FcRn and KO variants. In subject heterodimeric antibodies that utilizes pI as a separation mechanism to allow the purification of heterodimeric proteins, amino acid variants are introduced into one or both of the monomer polypeptides. That is, the pI of one of the monomers (referred to herein for simplicity as “monomer A”) can be engineered away from monomer B, or both monomer A and B change be changed, with the pI of monomer A increasing and the pI of monomer B decreasing. As is outlined more fully below, the pI changes of either or both monomers can be done by removing or adding a charged residue (e.g., a neutral amino acid is replaced by a positively or negatively charged amino acid residue, e.g., glycine to glutamic acid), changing a charged residue from positive or negative to the opposite charge (aspartic acid to lysine) or changing a charged residue to a neutral residue (e.g., loss of a charge; lysine to serine). A number of these variants are shown in theFIGS.1and2. Thus, in some embodiments, the subject heterodimeric antibody includes amino acid modifications in the constant regions that alter the isoelectric point (pI) of at least one, if not both, of the monomers of a dimeric protein to form “pI antibodies”) by incorporating amino acid substitutions (“pI variants” or “pI substitutions”) into one or both of the monomers. As shown herein, the separation of the heterodimers from the two homodimers can be accomplished if the pIs of the two monomers differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use in the antibodies described herein. As will be appreciated by those in the art, the number of pI variants to be included on each or both monomer(s) to get good separation will depend in part on the starting pI of the components, for example in the 1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fc formats, the starting pI of the scFv and Fab(s) of interest. That is, to determine which monomer to engineer or in which “direction” (e.g., more positive or more negative), the Fv sequences of the two target antigens are calculated and a decision is made from there. As is known in the art, different Fvs will have different starting pIs which are exploited in the antibodies described herein. In general, as outlined herein, the pIs are engineered to result in a total pI difference of each monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferred as outlined herein. In the case where pI variants are used to achieve heterodimerization, by using the constant region(s) of the heavy chain(s), a more modular approach to designing and purifying bispecific proteins, including antibodies, is provided. Thus, in some embodiments, heterodimerization variants (including skew and pI heterodimerization variants) are not included in the variable regions, such that each individual antibody must be engineered. In addition, in some embodiments, the possibility of immunogenicity resulting from the pI variants is significantly reduced by importing pI variants from different IgG isotypes such that pI is changed without introducing significant immunogenicity. Thus, an additional problem to be solved is the elucidation of low pI constant domains with high human sequence content, e.g., the minimization or avoidance of non-human residues at any particular position. Alternatively or in addition to isotypic substitutions, the possibility of immunogenicity resulting from the pI variants is significantly reduced by utilizing isosteric substitutions (e.g. Asn to Asp; and Gln to Glu). As discussed below, a side benefit that can occur with this pI engineering is also the extension of serum half-life and increased FcRn binding. That is, as described in US Publ. App. No. US 2012/0028304 (incorporated by reference in its entirety), lowering the pI of antibody constant domains (including those found in antibodies and Fc fusions) can lead to longer serum retention in vivo. These pI variants for increased serum half-life also facilitate pI changes for purification. In addition, it should be noted that the pI variants give an additional benefit for the analytics and quality control process of bispecific antibodies, as the ability to either eliminate, minimize and distinguish when homodimers are present is significant. Similarly, the ability to reliably test the reproducibility of the heterodimeric antibody production is important. In general, embodiments of particular use rely on sets of variants that include skew variants, which encourage heterodimerization formation over homodimerization formation, coupled with pI variants, which increase the pI difference between the two monomers to facilitate purification of heterodimers away from homodimers. Exemplary combinations of pI variants are shown inFIGS.1and2, and FIG. 30 of US Publ. App. No. 2016/0355608, all of which are herein incorporated by reference in its entirety and specifically for the disclosure of pI variants. Preferred combinations of pI variants are shown inFIGS.1and2. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be altered this way, as well as isotype hybrids. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used. Accordingly, in some embodiments, one monomer has a set of substitutions fromFIG.2and the other monomer has a charged linker (either in the form of a charged scFv linker because that monomer comprises an scFv or a charged domain linker, as the format dictates, which can be selected from those depicted inFIGS.5and6). In some embodiments, modifications are made in the hinge of the Fc domain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, and 230 based on EU numbering. Thus, pI mutations and particularly substitutions can be made in one or more of positions 216-230, with 1, 2, 3, 4 or 5 mutations finding use. Again, all possible combinations are contemplated, alone or with other pI variants in other domains. Specific substitutions that find use in lowering the pI of hinge domains include, but are not limited to, a deletion at position 221, a non-native valine or threonine at position 222, a deletion at position 223, a non-native glutamic acid at position 224, a deletion at position 225, a deletion at position 235 and a deletion or a non-native alanine at position 236. In some cases, only pI substitutions are done in the hinge domain, and in others, these substitution(s) are added to other pI variants in other domains in any combination. In some embodiments, mutations can be made in the CH2 region, including positions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327, 334 and 339, based on EU numbering. It should be noted that changes in 233-236 can be made to increase effector function (along with 327A) in the IgG2 backbone. Again, all possible combinations of these 14 positions can be made; e.g., =may include a variant Fc domain with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pI substitutions. Specific substitutions that find use in lowering the pI of CH2 domains include, but are not limited to, a non-native glutamine or glutamic acid at position 274, a non-native phenylalanine at position 296, a non-native phenylalanine at position 300, a non-native valine at position 309, a non-native glutamic acid at position 320, a non-native glutamic acid at position 322, a non-native glutamic acid at position 326, a non-native glycine at position 327, a non-native glutamic acid at position 334, a non-native threonine at position 339, and all possible combinations within CH2 and with other domains. In this embodiment, the modifications can be independently and optionally selected from position 355, 359, 362, 384, 389,392, 397, 418, 419, 444 and 447 (EU numbering) of the CH3 region. Specific substitutions that find use in lowering the pI of CH3 domains include, but are not limited to, a non-native glutamine or glutamic acid at position 355, a non-native serine at position 384, a non-native asparagine or glutamic acid at position 392, a non-native methionine at position 397, a non-native glutamic acid at position 419, a non-native glutamic acid at position 359, a non-native glutamic acid at position 362, a non-native glutamic acid at position 389, a non-native glutamic acid at position 418, a non-native glutamic acid at position 444, and a deletion or non-native aspartic acid at position 447. In general, as will be appreciated by those in the art, there are two general categories of pI variants: those that increase the pI of the protein (basic changes) and those that decrease the pI of the protein (acidic changes). As described herein, all combinations of these variants can be done: one monomer may be wild type, or a variant that does not display a significantly different pI from wild-type, and the other can be either more basic or more acidic. Alternatively, each monomer is changed, one to more basic and one to more acidic. Preferred combinations of pI variants are shown inFIG.2. As outlined herein and shown in the figures, these changes are shown relative to IgG1, but all isotypes can be altered this way, as well as isotype hybrids. In the case where the heavy chain constant domain is from IgG2-4, R133E and R133Q can also be used. In one embodiment, for example in theFIGS.15and44formats, a preferred combination of pI variants has one monomer (the negative Fab side) comprising 208D/295E/384D/418E/421D variants (N208D/Q295E/N384D/Q418E/N421D when relative to human IgG1) and a second monomer (the positive scFv side) comprising a positively charged scFv linker, including (GKPGS)4(SEQ ID NO: 15). However, as will be appreciated by those in the art, the first monomer includes a CH1 domain, including position 208. Accordingly, in constructs that do not include a CH1 domain (for example for antibodies that do not utilize a CH1 domain on one of the domains, for example in a dual scFv format or a “one-armed” format such as those depicted inFIG.44Cor D), a preferred negative pI variant Fc set includes 295E/384D/418E/421D variants (Q295E/N384D/Q418E/N421D when relative to human IgG1). Accordingly, in some embodiments, one monomer has a set of substitutions fromFIG.4and the other monomer has a charged linker (either in the form of a charged scFv linker because that monomer comprises an scFv or a charged domain linker, as the format dictates, which can be selected from those depicted inFIGS.5and6). c. Isotypic Variants In addition, many embodiments of the antibodies described herein rely on the “importation” of pI amino acids at particular positions from one IgG isotype into another, thus reducing or eliminating the possibility of unwanted immunogenicity being introduced into the variants. A number of these are shown in FIG. 21 of US Publ. 2014/0370013, hereby incorporated by reference. That is, IgG1 is a common isotype for therapeutic antibodies for a variety of reasons, including high effector function. However, the heavy constant region of IgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). By introducing IgG2 residues at particular positions into the IgG1 backbone, the pI of the resulting monomer is lowered (or increased) and additionally exhibits longer serum half-life. For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI 3.22); importing the glutamic acid will affect the pI of the resulting protein. As is described below, a number of amino acid substitutions are generally required to significant affect the pI of the variant antibody. However, it should be noted as discussed below that even changes in IgG2 molecules allow for increased serum half-life. In other embodiments, non-isotypic amino acid changes are made, either to reduce the overall charge state of the resulting protein (e.g. by changing a higher pI amino acid to a lower pI amino acid), or to allow accommodations in structure for stability, etc. as is further described below. In addition, by pI engineering both the heavy and light constant domains, significant changes in each monomer of the heterodimer can be seen. As discussed herein, having the pIs of the two monomers differ by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point. d. Calculating pI The pI of each monomer can depend on the pI of the variant heavy chain constant domain and the pI of the total monomer, including the variant heavy chain constant domain and the fusion partner. Thus, in some embodiments, the change in pI is calculated on the basis of the variant heavy chain constant domain, using the chart in the FIG. 19 of US Pub. 2014/0370013. As discussed herein, which monomer to engineer is generally decided by the inherent pI of the Fv and scaffold regions. Alternatively, the pI of each monomer can be compared. e. pI Variants that Also Confer Better FcRn In Vivo Binding In the case where the pI variant decreases the pI of the monomer, they can have the added benefit of improving serum retention in vivo. Although still under examination, Fc regions are believed to have longer half-lives in vivo, because binding to FcRn at pH 6 in an endosome sequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598, entirely incorporated by reference). The endosomal compartment then recycles the Fc to the cell surface. Once the compartment opens to the extracellular space, the higher pH, —7.4, induces the release of Fc back into the blood. In mice, Dall'Acqua et al. showed that Fc mutants with increased FcRn binding at pH 6 and pH 7.4 actually had reduced serum concentrations and the same half-life as wild-type Fc (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180, entirely incorporated by reference). The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid the release of the Fc back into the blood. Therefore, the Fc mutations that will increase Fc's half-life in vivo will ideally increase FcRn binding at the lower pH while still allowing release of Fc at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is not surprising to find His residues at important positions in the Fc/FcRn complex. Recently it has been suggested that antibodies with variable regions that have lower isoelectric points may also have longer serum half-lives (Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated by reference). However, the mechanism of this is still poorly understood. Moreover, variable regions differ from antibody to antibody. Constant region variants with reduced pI and extended half-life would provide a more modular approach to improving the pharmacokinetic properties of antibodies, as described herein. f. Additional Fc Variants for Additional Functionality In addition to the heterodimerization variants discussed above, there are a number of useful Fc amino acid modification that can be made for a variety of reasons, including, but not limited to, altering binding to one or more FcγR receptors, altered binding to FcRn receptors, etc., as discussed below. Accordingly, the antibodies provided herein (heterodimeric, as well as homodimeric) can include such amino acid modifications with or without the heterodimerization variants outlined herein (e.g., the pI variants and steric variants). Each set of variants can be independently and optionally included or excluded from any particular heterodimeric protein. (i) FcγR Variants Accordingly, there are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcγR receptors. In certain embodiments, the subject antibody includes modifications that alter the binding to one or more FcγR receptors (i.e., “FcγR variants”). Substitutions that result in increased binding as well as decreased binding can be useful. For example, it is known that increased binding to FcγRIIIa generally results in increased ADCC (antibody dependent cell-mediated cytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell). Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can be beneficial as well in some circumstances. Amino acid substitutions that find use in the antibodies described herein include those listed in U.S. Pat. No. 8,188,321 (particularlyFIG.41) and U.S. Pat. No. 8,084,582, and US Publ. App. Nos. 20060235208 and 20070148170, all of which are expressly incorporated herein by reference in their entirety and specifically for the variants disclosed therein. Particular variants that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D/332E/330L, 243A, 243L, 264A, 264V and 299T. In addition, there are additional Fc substitutions that find use in increased binding to the FcRn receptor and increased serum half-life, as specifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporated by reference in its entirety, including, but not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 428L/434A, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L. Such modification may be included in one or both Fc domains of the subject antibody. (ii) Ablation Variants Similarly, another category of functional variants are “FcγR ablation variants” or “Fc knock out (FcKO or KO)” variants. In these embodiments, for some therapeutic applications, it is desirable to reduce or remove the normal binding of the Fc domain to one or more or all of the Fcγ receptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. That is, for example, in many embodiments, particularly in the use of bispecific antibodies that bind CD3 monovalently it is generally desirable to ablate FcγRIIIa binding to eliminate or significantly reduce ADCC activity. wherein one of the Fc domains comprises one or more Fcγ receptor ablation variants. These ablation variants are depicted inFIG.14, and each can be independently and optionally included or excluded, with preferred aspects utilizing ablation variants selected from the group consisting of G236R/L328R, E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G, E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del. It should be noted that the ablation variants referenced herein ablate FcγR binding but generally not FcRn binding. As is known in the art, the Fc domain of human IgG1 has the highest binding to the Fcγ receptors, and thus ablation variants can be used when the constant domain (or Fc domain) in the backbone of the heterodimeric antibody is IgG1. Alternatively, or in addition to ablation variants in an IgG1 background, mutations at the glycosylation position 297 (generally to A or S) can significantly ablate binding to FcγRIIIa, for example. Human IgG2 and IgG4 have naturally reduced binding to the Fcγ receptors, and thus those backbones can be used with or without the ablation variants. E. Combination of Heterodimeric and Fc Variants As will be appreciated by those in the art, all of the recited heterodimerization variants (including skew and/or pI variants) can be optionally and independently combined in any way, as long as they retain their “strandedness” or “monomer partition”. In some embodiments, the heterodimeric antibodies provided herein include the combination of heterodimerization skew variants, isosteric pI substitutions and FcKO variants as depicted inFIG.4. In addition, all of these variants can be combined into any of the heterodimerization formats. In the case of pI variants, while embodiments finding particular use are shown in the Figures, other combinations can be generated, following the basic rule of altering the pI difference between two monomers to facilitate purification. In addition, any of the heterodimerization variants, skew and pI, are also independently and optionally combined with Fc ablation variants, Fc variants, FcRn variants, as generally outlined herein. Exemplary combination of variants that are included in some embodiments of the heterodimeric 1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fc format antibodies are included inFIG.4. In certain embodiments, the antibody is a heterodimeric 1+1 Fab-scFv-Fc or 2+1 Fab2-scFv-Fc format antibody as shown inFIGS.15A and15B. F. Anti-GPC3×Anti-CD3 Bispecific Antibodies In another aspect, provided herein are anti-GPC3×anti-CD3 (also referred to herein as “αGPC3×αCD3” or sometimes just “GPC3×CD3”) bispecific antibodies. Such antibodies include at least one GPC3 binding domain and at least one CD3 binding domain. In some embodiments, bispecific αGPC3×αCD3 provided herein immune responses selectively in tumor sites that express GPC3. Note that unless specified herein, the order of the antigen list in the name does not confer structure; that is a GPC3×CD3 1+1 Fab-scFv-Fc antibody can have the scFv bind to GPC3 or CD3, although in some cases, the order specifies structure as indicated. As is more fully outlined herein, these combinations of ABDs can be in a variety of formats, as outlined below, generally in combinations where one ABD is in a Fab format and the other is in an scFv format. Exemplary formats that are used in the bispecific antibodies provided herein include the 1+1 Fab-scFv-Fc and 2+1 Fab2-scFv-Fv formats (see, e.g.,FIGS.15A and15B). Other useful antibody formats include, but are not limited to, “mAb-Fv,” “mAb-scFv,” “central-Fv”, “one-armed scFv-mAb,” “scFv-mAb,” “dual scFv,” and “trident” format antibodies, as depicted inFIG.44and more fully described below. In addition, in general, one of the ABDs comprises a scFv as outlined herein, in an orientation from N- to C-terminus of VH-scFv linker-VL or VL-scFv linker-VH. One or both of the other ABDs, according to the format, generally is a Fab, comprising a VH domain on one protein chain (generally as a component of a heavy chain) and a VL on another protein chain (generally as a component of a light chain). As will be appreciated by those in the art, any set of 6 CDRs or VH and VL domains can be in the scFv format or in the Fab format, which is then added to the heavy and light constant domains, where the heavy constant domains comprise variants (including within the CH1 domain as well as the Fc domain). The scFv sequences contained in the sequence listing utilize a particular charged linker, but as outlined herein, uncharged or other charged linkers can be used, including those depicted inFIG.5andFIG.6. In addition, as discussed above, the numbering used in the Sequence Listing for the identification of the CDRs is Kabat, however, different numbering can be used, which will change the amino acid sequences of the CDRs as shown in Table 2. For all of the variable heavy and light domains listed herein, further variants can be made. As outlined herein, in some embodiments the set of 6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (with amino acid substitutions finding particular use), as well as changes in the framework regions of the variable heavy and light domains, as long as the frameworks (excluding the CDRs) retain at least about 80, 85 or 90% identity to a human germline sequence selected from those listed in FIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend is incorporated by reference in its entirety herein. Thus, for example, the identical CDRs as described herein can be combined with different framework sequences from human germline sequences, as long as the framework regions retain at least 80, 85 or 90% identity to a human germline sequence selected from those listed in FIG. 1 of U.S. Pat. No. 7,657,380. Alternatively, the CDRs can have amino acid modifications (e.g., from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs (that is, the CDRs can be modified as long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications, with any combination of CDRs being changed; e.g., there may be one change in vlCDR1, two in vhCDR2, none in vhCDR3, etc.)), as well as having framework region changes, as long as the framework regions retain at least 80, 85 or 90% identity to a human germline sequence selected from those listed in FIG. 1 of U.S. Pat. No. 7,657,380. As discussed herein, the subject heterodimeric antibodies include two antigen binding domains (ABDs), each of which bind to GPC3 or CD3. As outlined herein, these heterodimeric antibodies can be bispecific and bivalent (each antigen is bound by a single ABD, for example, in the format depicted inFIG.15A), or bispecific and trivalent (one antigen is bound by a single ABD and the other is bound by two ABDs, for example as depicted inFIG.15B). In addition, in general, one of the ABDs comprises a scFv as outlined herein, in an orientation from N- to C-terminus of VH-scFv linker-VL or VL-scFv linker-VH. One or both of the other ABDs, according to the format, generally is a Fab, comprising a VH domain on one protein chain (generally as a component of a heavy chain) and a VL on another protein chain (generally as a component of a light chain). The disclosure provides a number of ABDs as outlined below. As will be appreciated by those in the art, any set of 6 CDRs or VH and VL domains can be in the scFv format or in the Fab format, which is then added to the heavy and light constant domains, where the heavy constant domains comprise variants (including within the CH1 domain as well as the Fc domain). The scFv sequences contained in the sequence listing utilize a particular charged linker, but as outlined herein, uncharged or other charged linkers can be used, including those depicted inFIG.5. In addition, as discussed above, the numbering used in the Sequence Listing for the identification of the CDRs is Kabat, however, different numbering can be used, which will change the amino acid sequences of the CDRs as shown in Table 2. For all of the variable heavy and light domains listed herein, further variants can be made. As outlined herein, in some embodiments the set of 6 CDRs can have from 0, 1, 2, 3, 4 or 5 amino acid modifications (with amino acid substitutions finding particular use), as well as changes in the framework regions of the variable heavy and light domains, as long as the frameworks (excluding the CDRs) retain at least about 80, 85 or 90% identity to a human germline sequence selected from those listed in FIG. 1 of U.S. Pat. No. 7,657,380, which Figure and Legend is incorporated by reference in its entirety herein. Thus, for example, the identical CDRs as described herein can be combined with different framework sequences from human germline sequences, as long as the framework regions retain at least 80, 85 or 90% identity to a human germline sequence selected from those listed in FIG. 1 of U.S. Pat. No. 7,657,380. Alternatively, the CDRs can have amino acid modifications (e.g. from 1, 2, 3, 4 or 5 amino acid modifications in the set of CDRs (that is, the CDRs can be modified as long as the total number of changes in the set of 6 CDRs is less than 6 amino acid modifications, with any combination of CDRs being changed; e.g. there may be one change in VLCDR1, two in VHCDR2, none in VHCDR3, etc.)), as well as having framework region changes, as long as the framework regions retain at least 80, 85 or 90% identity to a human germline sequence selected from those listed in FIG. 1 of U.S. Pat. No. 7,657,380. 1. GPC3 Antigen Binding Domains Herein is provided monoclonal and bispecific antibodies that contain antigen binding domains that bind to human GPC3. Suitable sets of 6 CDRs and/or VH and VL domains are depicted inFIGS.12and13. In some embodiments, the heterodimeric antibody is a 1+1 Fab-scFv-Fc or 2+1 Fab2-scFv-Fv format antibody (see, e.g.,FIGS.15A and15B) although any of the formats outlined below can be utilized. In some embodiments, the anti-GPC3 ABD has a set of vhCDRs selected from the vhCDR1, vhCDR2 and vhCDR3 sequences from a VH selected from the group consisting of H1, H1, H1.1, H1.2, H1.3 and H1.4 the sequences of which are shown inFIGS.12and13. In some embodiments, the VH domain of the anti-GPC3 ABD is selected from the group consisting of H1, H1, H1.1, H1.2, H1.3 and H1.4 the sequences of which are shown inFIGS.12and13. In some embodiments, the anti-GPC3 ABD has a set of vlCDRs selected from the vlCDR1, vlCDR2 and vlCDR3 sequences from a VL selected from the group consisting of L1, L1.1, L1.2, L1.3, L1.4, L1.5, L1.6, L1.7, L1.8, L1.9, L1.10, L1.16, L1.23, L1.29, L1.31. L1.65, L1.66, L1.68, L1.69, L1.70, L1.71, L1.72 and L1.73, the sequences of which are shown inFIGS.12and13. In some embodiments, the VL of the GPC3 ABD is selected from the group consisting of L1, L1.1, L1.2, L1.3, L1.4, L1.5, L1.6, L1.7, L1.8, L1.9, L1.10, L1.16, L1.23, L1.29, L1.31. L1.65, L1.66, L1.68, L1.69, L1.70, L1.71, L1.72 and L1.73, the sequences of which are shown inFIGS.12and13. Accordingly, included herein are GPC3 ABDs that have a set of 6 CDRs (vhCDR1, vhCDR2, vhCDR3, vlCDR1, vlCDR2 and vlCDR3) from VH/VLpairs selected from the group consisting of: [GPC3]H1_L1, [GPC3]H1_L1.1, [GPC3]H1_L1.2, [GPC3]H1_L1.3, [GPC3]H1_L1.4, [GPC3]H1_L1.5, [GPC3]H1_L1.6, [GPC3]H1_L1.7, [GPC3]H1_L1.8, [GPC3]H1_L1.9, [GPC3]H1_L1.10, [GPC3]H1_L1.16, [GPC3]H1_L1.23, [GPC3]H1_L1.29, [GPC3]H1_L1.31, [GPC3]H1_L1.65, [GPC3]H1_L1.66, [GPC3]H1_L1.67, [GPC3]H1_L1.68, [GPC3]H1_L1.70, [GPC3]H1_L1.71, [GPC3]H1_L1.72, [GPC3]H1_L1.73, [GPC3]H1.1_L1, [GPC3]H1.1_L1.1, [GPC3]H1.1_L1.2, [GPC3]H1.1_L1.3, [GPC3]H1.1_L1.4, [GPC3]H1.1_L1.5, [GPC3]H1.1_L1.6, [GPC3]H1.1_L1.7, [GPC3]H1.1_L1.8, [GPC3]H1.1_L1.9, [GPC3]H1.1_L1.10, [GPC3]H1.1_L1.16, [GPC3]H1.1_L1.23, [GPC3]H1.1_L1.29, [GPC3]H1.1_L1.31, [GPC3]H1.1_L1.65, [GPC3]H1.1_L1.66, L1.67, [GPC3]H1.1_L1.68, [GPC3]H1.1_L1.70, [GPC3]H1.1_L1.71, [GPC3]H1.1_L1.72, [GPC3]H1.1_L1.73, [GPC3]H1.2_L1, [GPC3]H1.2_L1.1, [GPC3]H1.2_L1.2, [GPC3]H1.2_L1.3, [GPC3]H1.2_L1.4, [GPC3]H1.2_L1.5, [GPC3]H1.2_L1.6, [GPC3]H1.2_L1.7, [GPC3]H1.2_L1.8, [GPC3]H1.2_L1.9, [GPC3]H1.2_L1.10, [GPC3]H1.2_L1.16, [GPC3]H1.2_L1.23, [GPC3]H1.2_L1.29, [GPC3]H1.2_L1.31, [GPC3]H1.2_L1.65, [GPC3]H1.2_L1.66, [GPC3]H1.2_L1.67, [GPC3]H1.2_L1.68, [GPC3]H1.2_L1.70, [GPC3]H1.2_L1.71, [GPC3]H1.2_L1.72, [GPC3]H1.2_L1.73, [GPC3]H1.3_L1, [GPC3]H1.3_L1.1, [GPC3]H1.3_L1.2, [GPC3]H1.3_L1.3, [GPC3]H1.3_L1.4, [GPC3]H1.3_L1.5, [GPC3]H1.3_L1.6, [GPC3]H1.3_L1.7, [GPC3]H1.3_L1.8, [GPC3]H1.3_L1.9, [GPC3]H1.3_L1.10, [GPC3]H1.3_L1.16, [GPC3]H1.3_L1.23, [GPC3]H1.3_L1.29, [GPC3]H1.3_L1.31, [GPC3]H1.3_L1.65, [GPC3]H1.3_L1.66, [GPC3]H1.3_L1.67, [GPC3]H1.3_L1.68, [GPC3]H1.3_L1.70, [GPC3]H1.3_L1.71, [GPC3]H1.3_L1.72, [GPC3]H1.3_L1.73, [GPC3]H1.4_L1, [GPC3]H1.4_L1.1, [GPC3]H1.4_L1.2, [GPC3]H1.4_L1.3, [GPC3]H1.4_L1.4, [GPC3]H1.4_L1.5, [GPC3]H1.4_L1.6, [GPC3]H1.4_L1.7, [GPC3]H1.4_L1.8, [GPC3]H1.4_L1.9, [GPC3]H1.4_L1.10, [GPC3]H1.4_L1.16, [GPC3]H1.4_L1.23, [GPC3]H1.4_L1.29, [GPC3]H1.4_L1.31, [GPC3]H1.4_L1.65, [GPC3]H1.4_L1.66, [GPC3]H1.4_L1.67, [GPC3]H1.4_L1.68, [GPC3]H1.4_L1.70, [GPC3]H1.4_L1.71, [GPC3]H1.4_L1.72 and [GPC3]H1.4_L1.73. Additionally, included herein are GPC3 ABDs that have VH/VL pairs selected from the group consisting of: [GPC3]H1_L1, [GPC3]H1_L1.1, [GPC3]H1_L1.2, [GPC3]H1_L1.3, [GPC3]H1_L1.4, [GPC3]H1_L1.5, [GPC3]H1_L1.6, [GPC3]H1_L1.7, [GPC3]H1_L1.8, [GPC3]H1_L1.9, [GPC3]H1_L1.10, [GPC3]H1_L1.16, [GPC3]H1_L1.23, [GPC3]H1_L1.29, [GPC3]H1_L1.31, [GPC3]H1_L1.65, [GPC3]H1_L1.66, [GPC3]H1_L1.67, [GPC3]H1_L1.68, [GPC3]H1_L1.70, [GPC3]H1_L1.71, [GPC3]H1_L1.72, [GPC3]H1_L1.73, [GPC3]H1.1_L1, [GPC3]H1.1_L1.1, [GPC3]H1.1_L1.2, [GPC3]H1.1_L1.3, [GPC3]H1.1_L1.4, [GPC3]H1.1_L1.5, [GPC3]H1.1_L1.6, [GPC3]H1.1_L1.7, [GPC3]H1.1_L1.8, [GPC3]H1.1_L1.9, [GPC3]H1.1_L1.10, [GPC3]H1.1_L1.16, [GPC3]H1.1_L1.23, [GPC3]H1.1_L1.29, [GPC3]H1.1_L1.31, [GPC3]H1.1_L1.65, [GPC3]H1.1_L1.66, L1.67, [GPC3]H1.1_L1.68, [GPC3]H1.1_L1.70, [GPC3]H1.1_L1.71, [GPC3]H1.1_L1.72, [GPC3]H1.1_L1.73, [GPC3]H1.2_L1, [GPC3]H1.2_L1.1, [GPC3]H1.2_L1.2, [GPC3]H1.2_L1.3, [GPC3]H1.2_L1.4, [GPC3]H1.2_L1.5, [GPC3]H1.2_L1.6, [GPC3]H1.2_L1.7, [GPC3]H1.2_L1.8, [GPC3]H1.2_L1.9, [GPC3]H1.2_L1.10, [GPC3]H1.2_L1.16, [GPC3]H1.2_L1.23, [GPC3]H1.2_L1.29, [GPC3]H1.2_L1.31, [GPC3]H1.2_L1.65, [GPC3]H1.2_L1.66, [GPC3]H1.2_L1.67, [GPC3]H1.2_L1.68, [GPC3]H1.2_L1.70, [GPC3]H1.2_L1.71, [GPC3]H1.2_L1.72, [GPC3]H1.2_L1.73, [GPC3]H1.3_L1, [GPC3]H1.3_L1.1, [GPC3]H1.3_L1.2, [GPC3]H1.3_L1.3, [GPC3]H1.3_L1.4, [GPC3]H1.3_L1.5, [GPC3]H1.3_L1.6, [GPC3]H1.3_L1.7, [GPC3]H1.3_L1.8, [GPC3]H1.3_L1.9, [GPC3]H1.3_L1.10, [GPC3]H1.3_L1.16, [GPC3]H1.3_L1.23, [GPC3]H1.3_L1.29, [GPC3]H1.3_L1.31, [GPC3]H1.3_L1.65, [GPC3]H1.3_L1.66, [GPC3]H1.3_L1.67, [GPC3]H1.3_L1.68, [GPC3]H1.3_L1.70, [GPC3]H1.3_L1.71, [GPC3]H1.3_L1.72, [GPC3]H1.3_L1.73, [GPC3]H1.4_L1, [GPC3]H1.4_L1.1, [GPC3]H1.4_L1.2, [GPC3]H1.4_L1.3, [GPC3]H1.4_L1.4, [GPC3]H1.4_L1.5, [GPC3]H1.4_L1.6, [GPC3]H1.4_L1.7, [GPC3]H1.4_L1.8, [GPC3]H1.4_L1.9, [GPC3]H1.4_L1.10, [GPC3]H1.4_L1.16, [GPC3]H1.4_L1.23, [GPC3]H1.4_L1.29, [GPC3]H1.4_L1.31, [GPC3]H1.4_L1.65, [GPC3]H1.4_L1.66, [GPC3]H1.4_L1.67, [GPC3]H1.4_L1.68, [GPC3]H1.4_L1.70, [GPC3]H1.4_L1.71, [GPC3]H1.4_L1.72 and [GPC3]H1.4_L1.73. In particular embodiments, the VH/VL pairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. In particular embodiments, the VH/VL pairs are Fabs and are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. As will be appreciated by those in the art, suitable GPC3 binding domains can comprise a set of 6 CDRs as depicted in the Figures, either as they are underlined or, in the case where a different numbering scheme is used as described herein and as shown in Table 2, as the CDRs that are identified using other alignments within the VH and VL sequences of those depicted inFIGS.12and13. Suitable ABDs can also include the entire VH and VL sequences as depicted in these sequences and Figures, used as scFvs or as Fabs. In many of the embodiments herein that contain an Fv to GPC3, it is the Fab monomer that binds GPC3. In addition to the parental CDR sets disclosed in the figures and sequence listing that form an ABD to GPC3, provided herein are variant GPC3 ABDs having CDRs that include at least one modification of the GPC3 ABD CDRs disclosed herein (e.g., (FIGS.12and13and the sequence listing). In one embodiment, the GPC3 ABD of the subject heterodimeric antibody includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of a GPC3 binding domain VH/VL pair as described herein, including the figures and sequence listing. In exemplary embodiments, the GPC3 ABD of the subject heterodimeric antibody includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of one of the following GPC3 binding domain [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. In certain embodiments, the GPC3 ABD of the subject antibody is capable of binding to GPC3, as measured at least one of a Biacore, surface plasmon resonance (SPR), BLI (biolayer interferometry, e.g., Octet assay) assay, and/or flow cytometry, with the latter finding particular use in many embodiments. In particular embodiments, the GPC3 ABD is capable of binding human GPC3 (seeFIG.11). In some cases, each variant CDR has no more than 1 or 2 amino acid changes, with no more than 1 per CDR being particularly useful. In some embodiments, the GPC3 ABD of the subject antibody includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of a GPC3 ABD as described herein, including the figures and sequence listing. In exemplary embodiments, the GPC3 ABD of the subject antibody includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of one of the following GPC3 binding domain VH/VL pairs: GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. In certain embodiments, the GPC3 ABD of the subject antibody is capable of binding to GPC3, as measured at least one of a Biacore, surface plasmon resonance (SPR), BLI (biolayer interferometry, e.g., Octet assay) assay, and/or flow cytometry. In particular embodiments, the GPC3 ABD is capable of binding human GPC3 antigen (seeFIG.11). In another exemplary embodiment, the GPC3 ABD of the subject antibody includes the variable heavy (VH) domain and variable light (VL) domain of any one of the GPC3 binding domain VH/VL pairs described herein, including the figures and sequence listing (e.g.,FIGS.12and13). In some embodiments, the subject antibody includes a GPC3 ABD that includes a variable heavy domain and/or a variable light domain that are variants of a GPC3 ABD VH and VL domain disclosed herein. In one embodiment, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of a GPC3 ABD described herein, including the figures and sequence listing. In exemplary embodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of one of the following GPC3 binding domain VH/VL pairs: GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. In some embodiments, the changes are in a VH domain depicted inFIGS.12and13. In some embodiments, the changes are in a VL domain are depicted inFIGS.12and13. In some embodiments, the changes are in a VH and VL domain are depicted inFIGS.12and13. In some embodiments, one or more amino acid changes are in the VH and/or VL framework regions (FR1, FR2, FR3, and/or FR4). In some embodiments, one or more amino acid changes are in one or more CDRs. In certain embodiments, the GPC3 ABD of the subject antibody is capable of binding to GPC3, as measured at least one of a Biacore, surface plasmon resonance (SPR), BLI (biolayer interferometry, e.g., Octet assay) assay, and/or flow cytometry. In particular embodiments, the GPC3 ABD is capable of binding human GPC3 antigen (seeFIG.11). In one embodiment, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of a GPC3 ABD as described herein, including the figures and sequence listing. In exemplary embodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of one of the following GPC3 binding domain VH/VL pairs: GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. In some embodiments, the GPC3 ABD includes a VH that is at least 90, 95, 97, 98 or 99% identical to VH domain depicted inFIGS.12and13. In some embodiments, the GPC3 ABD includes a VL that is at least 90, 95, 97, 98 or 99% identical to VL domain depicted inFIGS.12and13. In some embodiments, the GPC3 ABD includes a VH and a VL that is at least 90, 95, 97, 98 or 99% identical to a VH domain and a VL domain depicted inFIGS.12and13. In certain embodiments, the GPC3 ABD of the subject antibody is capable of binding to GPC3, as measured at least one of a Biacore, surface plasmon resonance (SPR), BLI (biolayer interferometry, e.g., Octet assay) assay, and/or flow cytometry. In particular embodiments, the GPC3 ABD is capable of binding human GPC3 antigen (seeFIG.11). 2. CD3 Antigen Binding Domains The heterodimeric bispecific of the invention (e.g., anti-GPC3×anti-CD3 antibodies) also include an ABD that binds to human episilon CD3 (CDR). Suitable sets of 6 CDRs and/or VH and VL domains, as well as scFv sequences, are depicted inFIG.10. CD3 binding domain sequences that are of particular use include, but are not limited to, anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3 H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3_L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31 as depicted inFIG.10. As shown inFIG.10, when the anti-CD3 ABD is a scFv domain, the VH and VL domains can be in either orientation. As will be appreciated by those in the art, suitable CD3 binding domains can comprise a set of 6 CDRs as depicted inFIG.10, either as they are underlined or, in the case where a different numbering scheme is used as described herein and as shown in Table 2, as the CDRs that are identified using other alignments within the VH and VL sequences of those depicted inFIGS.10A-10F. Suitable ABDs can also include the entire VH and VL sequences as depicted in these sequences and Figures, used as scFvs or as Fabs. In many of the embodiments herein that contain an Fv to CD3, it is the scFv monomer that binds CD3. In addition to the parental CDR sets disclosed in the figures and sequence listing that form an ABD to CD3, provided herein are variant CD3 ABDS having CDRs that include at least one modification of the CD3 ABD CDRs disclosed herein (e.g., (FIG.10and the sequence listing). In one embodiment, the CD3 ABD of the subject heterodimeric antibody (e.g., anti-GPC3×anti-CD3 antibody) includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of a CD3 ABD as described herein, including the figures and sequence listing. In exemplary embodiments, the CD3 ABD of the subject heterodimeric antibody includes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid modifications as compared to the 6 CDRs of one of the following CD3 binding domains: anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3 H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3_L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31 (FIG.10). In certain embodiments, the CD3 ABD of the subject antibody is capable of binding CD3 antigen, as measured by at least one of a Biacore, surface plasmon resonance (SPR), flow cytometry, and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the CD3ABD is capable of binding human CD3. In some embodiments, the CD3 ABD of the subject antibody includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of a CD3 ABD as described herein, including the figures and sequence listing. In exemplary embodiments, the CD3 ABD of the subject antibody includes 6 CDRs that are at least 90, 95, 97, 98 or 99% identical to the 6 CDRs of one of the following CD3 binding domains: anti-CD3_H1.30_L1.47, anti-CD3 H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3_L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3 L1.47_H1.31 (FIG.10). In certain embodiments, the CD3 ABD is capable of binding to the CD3, as measured by at least one of a Biacore, surface plasmon resonance (SPR), flow cytometry, and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the CD3 ABD is capable of binding human CD3 antigen. In another exemplary embodiment, the CD3 ABD of the subject antibody includes the variable heavy (VH) domain and variable light (VL) domain of any one of the CD3 binding domains described herein, including the figures and sequence listing. In some embodiments, the subject antibody includes a CD3 ABD that includes a variable heavy domain and/or a variable light domain that are variants of a CD3 ABD VH and VL domain disclosed herein. In one embodiment, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of a CD3 ABD described herein, including the figures and sequence listing. In exemplary embodiments, the variant VH domain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes from a VH and/or VL domain of one of the following CD3 binding domains: anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3 L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3_L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31 (FIG.10). In some embodiments, the changes are in a VH domain depicted inFIG.10. In some embodiments, the changes are in a VL domain are depicted inFIG.10. In some embodiments, the changes are in a VH and VL domain are depicted inFIG.10. In some embodiments, one or more amino acid changes are in the VH and/or VL framework regions (FR1, FR2, FR3, and/or FR4). In some embodiments, one or more amino acid changes are in one or more CDRs. In certain embodiments, the CD3 ABD of the subject antibody is capable of binding to CD3, as measured at least one of a Biacore, surface plasmon resonance (SPR), flow cytometry, and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the CD3 ABD is capable of binding human CD3 antigen. In one embodiment, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of a CD3 ABD as described herein, including the figures and sequence listing. In exemplary embodiments, the variant VH and/or VL domain is at least 90, 95, 97, 98 or 99% identical to the VH and/or VL of one of the following CD3 binding domains: anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3 L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3_L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31 (FIG.10). In some embodiments, the CD3 ABD includes a VH that is at least 90, 95, 97, 98 or 99% identical to VH domain depicted inFIG.10. In some embodiments, the CD3 ABD includes a VL that is at least 90, 95, 97, 98 or 99% identical to VL domain depicted inFIG.10. In some embodiments, the CD3 ABD includes a VH and a VL that is at least 90, 95, 97, 98 or 99% identical to a VH domain and a VL domain depicted inFIG.10. In certain embodiments, the CD3 ABD is capable of binding to CD3, as measured by at least one of a Biacore, surface plasmon resonance (SPR), flow cytometry, and/or BLI (biolayer interferometry, e.g., Octet assay) assay, with the latter finding particular use in many embodiments. In particular embodiments, the CD3 ABD is capable of binding human CD3 antigen. In addition to the αCD3 ABDs ofFIG.10, additional ABDs of use in the invention include those depicted in FIGS. 14 and 15 of WO2014/145806, hereby expressly incorporated herein in their entirety including the Figures and Legends therein. 3. Linkers As shown herein, there are a number of suitable linkers (for use as either domain linkers or scFv linkers) that can be used to covalently attach the recited domains (e.g., scFvs, Fabs, Fc domains, etc.), including traditional peptide bonds, generated by recombinant techniques. Exemplary linkers to attach domains of the subject antibody to each other are depicted inFIG.6. In some embodiments, the linker peptide may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity. In one embodiment, the linker is from about 1 to 50 amino acids in length, preferably about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used, with from about 5 to about 10 amino acids finding use in some embodiments. Useful linkers include glycine-serine polymers, including for example (GS)n, (GSGGS)n (SEQ ID NO: 3), (GGGGS)n (SEQ ID NO: 2), and (GGGS)n (SEQ ID NO: 4), where n is an integer of at least one (and generally from 3 to 4), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, some of which are shown inFIG.5andFIG.6. Alternatively, a variety of nonproteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers. Other linker sequences may include any sequence of any length of CL/CH1 domain but not all residues of CL/CH1 domain; for example the first 5-12 amino acid residues of the CL/CH1 domains. Linkers can be derived from immunoglobulin light chain, for example Cκ or Cλ. Linkers can be derived from immunoglobulin heavy chains of any isotype, including for example Cγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may also be derived from other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge region-derived sequences, and other natural sequences from other proteins. In some embodiments, the linker is a “domain linker”, used to link any two domains as outlined herein together. For example, inFIG.15B, there may be a domain linker that attaches the C-terminus of the CH1 domain of the Fab to the N-terminus of the scFv, with another optional domain linker attaching the C-terminus of the scFv to the CH2 domain (although in many embodiments the hinge is used as this domain linker). While any suitable linker can be used, many embodiments utilize a glycine-serine polymer as the domain linker, including for example (GS)n, (GSGGS)n (SEQ ID NO: 3), (GGGGS)n (SEQ ID NO: 2), and (GGGS)n (SEQ ID NO: 4), where n is an integer of at least one (and generally from 3 to 4 to 5) as well as any peptide sequence that allows for recombinant attachment of the two domains with sufficient length and flexibility to allow each domain to retain its biological function. In some cases, and with attention being paid to “strandedness”, as outlined below, charged domain linkers, as used in some embodiments of scFv linkers can be used. Exemplary useful domain linkers are depicted inFIG.6. With particular reference to the domain linker used to attach the scFv domain to the Fc domain in the “2+1” format, there are several domain linkers that find particular use, including “full hinge C220S variant,” “flex half hinge,” “charged half hinge 1,” and “charged half hinge 2” as shown inFIG.6. In some embodiments, the linker is a “scFv linker”, used to covalently attach the VH and VL domains as discussed herein. In many cases, the scFv linker is a charged scFv linker, a number of which are shown inFIG.5. Accordingly, in some embodiments, the antibodies described herein further provide charged scFv linkers, to facilitate the separation in pI between a first and a second monomer. That is, by incorporating a charged scFv linker, either positive or negative (or both, in the case of scaffolds that use scFvs on different monomers), this allows the monomer comprising the charged linker to alter the pI without making further changes in the Fc domains. These charged linkers can be substituted into any scFv containing standard linkers. Again, as will be appreciated by those in the art, charged scFv linkers are used on the correct “strand” or monomer, according to the desired changes in pI. For example, as discussed herein, to make 1+1 Fab-scFv-Fc format heterodimeric antibody, the original pI of the Fv region for each of the desired antigen binding domains are calculated, and one is chosen to make an scFv, and depending on the pI, either positive or negative linkers are chosen. Charged domain linkers can also be used to increase the pI separation of the monomers of the antibodies described herein as well, and thus those included inFIG.5can be used in any embodiment herein where a linker is utilized. G. Useful Formats of the Invention As will be appreciated by those in the art and discussed more fully below, the heterodimeric bispecific antibodies provided herein can take on a wide variety of configurations, as are generally depicted inFIG.15as well asFIG.44. Some figures depict “single ended” configurations, where there is one type of specificity on one “arm” of the molecule and a different specificity on the other “arm”. Other figures depict “dual ended” configurations, where there is at least one type of specificity at the “top” of the molecule and one or more different specificities at the “bottom” of the molecule. Thus, in some embodiments, the antibodies described herein are directed to novel immunoglobulin compositions that co-engage a different first and a second antigen. As will be appreciated by those in the art, the heterodimeric formats of the antibodies described herein can have different valencies as well as be bispecific. That is, heterodimeric antibodies of the antibodies described herein can be bivalent and bispecific, wherein one target tumor antigen (e.g. CD3) is bound by one binding domain and the other target tumor antigen (e.g. GPC3) is bound by a second binding domain. The heterodimeric antibodies can also be trivalent and bispecific, wherein the first antigen is bound by two binding domains and the second antigen by a second binding domain. As is outlined herein, when CD3 is one of the target antigens, it is preferable that the CD3 is bound only monovalently, to reduce potential side effects. The antibodies described herein utilize anti-CD3 antigen binding domains in combination with anti-GPC3 binding domains. As will be appreciated by those in the art, any collection of anti-CD3 CDRs, anti-CD3 variable light and variable heavy domains, Fabs and scFvs as depicted in any of the Figures can be used. Similarly, any of the anti-GPC3 antigen binding domains can be used, whether CDRs, variable light and variable heavy domains, Fabs and scFvs as depicted in any of the Figures can be used, optionally and independently combined in any combination. 1. 1+1 Fab-scFv-Fc Format One heterodimeric scaffold that finds particular use in the antibodies described herein is the “1+1 Fab-scFv-Fc” or “bottle-opener” format as shown inFIG.15Awith an exemplary combination of a CD3 binding domain and a tumor target antigen (GPC3) binding domain. In this embodiment, one heavy chain monomer of the antibody contains a single chain Fv (“scFv”, as defined below) and an Fc domain. The scFv includes a variable heavy domain (VH1) and a variable light domain (VL1), wherein the VH1 is attached to the VL1 using an scFv linker that can be charged (see, e.g.,FIG.5). The scFv is attached to the heavy chain using a domain linker (see, e.g.,FIG.6). The other heavy chain monomer is a “regular” heavy chain (VH-CH1-hinge-CH2-CH3). The 1+1 Fab-scFv-Fc also includes a light chain that interacts with the VH-CH1 to form a Fab. This structure is sometimes referred to herein as the “bottle-opener” format, due to a rough visual similarity to a bottle-opener. The two heavy chain monomers are brought together by the use of amino acid variants (e.g., heterodimerization variants, discussed above) in the constant regions (e.g., the Fc domain, the CH1 domain and/or the hinge region) that promote the formation of heterodimeric antibodies as is described more fully below. There are several distinct advantages to the present “1+1 Fab-scFv-Fc” format. As is known in the art, antibody analogs relying on two scFv constructs often have stability and aggregation problems, which can be alleviated in the antibodies described herein by the addition of a “regular” heavy and light chain pairing. In addition, as opposed to formats that rely on two heavy chains and two light chains, there is no issue with the incorrect pairing of heavy and light chains (e.g. heavy 1 pairing with light 2, etc.). Many of the embodiments outlined herein rely in general on the 1+1 Fab-scFv-Fc or “bottle opener” format antibody that comprises a first monomer comprising an scFv, comprising a variable heavy and a variable light domain, covalently attached using an scFv linker (charged, in many but not all instances), where the scFv is covalently attached to the N-terminus of a first Fc domain usually through a domain linker The domain linker can be either charged or uncharged and exogenous or endogenous (e.g., all or part of the native hinge domain). Any suitable linker can be used to attach the scFv to the N-terminus of the first Fc domain. In some embodiments, the domain linker is chosen from the domain linkers inFIG.6. The second monomer of the 1+1 Fab-scFv-Fc format or “bottle opener” format is a heavy chain, and the composition further comprises a light chain. In general, in many preferred embodiments, the scFv is the domain that binds to the CD3, and the Fab forms an GPC3 binding domain. An exemplary anti-GPC3×anti-CD3 bispecific antibody in the 1+1 Fab-scFv-Fc format is depicted inFIG.15A. Exemplary anti-GPC3×anti-CD3 bispecific antibodies in the 1+1 Fab-scFv-Fc format are depicted inFIGS.16and17. In addition, the Fc domains of the antibodies described herein generally include skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L; K370S: S364K/E357Q; T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In certain embodiments, the 1+1 Fab-scFv-Fc scaffold format includes a first monomer that includes a scFv-domain linker-CH2-CH3 monomer, a second monomer that includes a first variable heavy domain-CH1-hinge-CH2-CH3 monomer and a third monomer that includes a first variable light domain. In some embodiments, the CH2-CH3 of the first monomer is a first variant Fc domain and the CH2-CH3 of the second monomer is a second variant Fc domain. In some embodiments, the scFv includes a scFv variable heavy domain and a scFv variable light domain that form a CD3 binding moiety. In certain embodiments, the scFv variable heavy domain and scFv variable light domain are covalently attached using an scFv linker (charged, in many but not all instances. See, e.g.,FIG.5). In some embodiments, the first variable heavy domain and first variable light domain form a GPC3 binding domain. In some embodiments, the 1+1 Fab-scFv-Fc format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 1+1 Fab-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence ofFIG.5being preferred in some embodiments), the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and an scFv that binds to CD3 as outlined herein; b) a second monomer (the “Fab monomer”) that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain; and c) a light chain that includes a variable light domain light domain (VL) and a constant light domain (CL), wherein numbering is according to EU numbering. The variable heavy domain and variable light domain make up an GPC3 binding moiety. Any suitable CD3 ABD can be included in the 1+1 Fab-scFv-Fc format antibody, included those provided herein. CD3 binding domain sequences finding particular use in these embodiments include, but are not limited to, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 or a variant thereof, as well as those depicted inFIG.10and those depicted in FIGS. 14 and 15 of WO2014/145806, hereby incorporated by reference including the Legends. Any suitable GPC3 ABD can be included in the 1+1 Fab-scFv-Fc format antibody, included those provided herein. GPC3 ABDs that are of particular use in these embodiments include, but are not limited to, VH and VL domains selected from have VH/VL pairs selected from the group consisting of: [GPC3]H1_L1, [GPC3]H1_L1.1, [GPC3]H1_L1.2, [GPC3]H1_L1.3, [GPC3]H1_L1.4, [GPC3]H1_L1.5, [GPC3]H1_L1.6, [GPC3]H1_L1.7, [GPC3]H1_L1.8, [GPC3]H1_L1.9, [GPC3]H1_L1.10, [GPC3]H1_L1.16, [GPC3]H1_L1.23, [GPC3]H1_L1.29, [GPC3]H1_L1.31, [GPC3]H1_L1.65, [GPC3]H1_L1.66, [GPC3]H1_L1.67, [GPC3]H1_L1.68, [GPC3]H1_L1.70, [GPC3]H1_L1.71, [GPC3]H1_L1.72, [GPC3]H1_L1.73, [GPC3]H1.1_L1, [GPC3]H1.1_L1.1, [GPC3]H1.1_L1.2, [GPC3]H1.1_L1.3, [GPC3]H1.1_L1.4, [GPC3]H1.1_L1.5, [GPC3]H1.1_L1.6, [GPC3]H1.1_L1.7, [GPC3]H1.1_L1.8, [GPC3]H1.1_L1.9, [GPC3]H1.1_L1.10, [GPC3]H1.1_L1.16, [GPC3]H1.1_L1.23, [GPC3]H1.1_L1.29, [GPC3]H1.1_L1.31, [GPC3]H1.1_L1.65, [GPC3]H1.1_L1.66, L1.67, [GPC3]H1.1_L1.68, [GPC3]H1.1_L1.70, [GPC3]H1.1_L1.71, [GPC3]H1.1_L1.72, [GPC3]H1.1_L1.73, [GPC3]H1.2_L1, [GPC3]H1.2_L1.1, [GPC3]H1.2_L1.2, [GPC3]H1.2_L1.3, [GPC3]H1.2_L1.4, [GPC3]H1.2_L1.5, [GPC3]H1.2_L1.6, [GPC3]H1.2_L1.7, [GPC3]H1.2_L1.8, [GPC3]H1.2_L1.9, [GPC3]H1.2_L1.10, [GPC3]H1.2_L1.16, [GPC3]H1.2_L1.23, [GPC3]H1.2_L1.29, [GPC3]H1.2_L1.31, [GPC3]H1.2_L1.65, [GPC3]H1.2_L1.66, [GPC3]H1.2_L1.67, [GPC3]H1.2_L1.68, [GPC3]H1.2_L1.70, [GPC3]H1.2_L1.71, [GPC3]H1.2_L1.72, [GPC3]H1.2_L1.73, [GPC3]H1.3_L1, [GPC3]H1.3_L1.1, [GPC3]H1.3_L1.2, [GPC3]H1.3_L1.3, [GPC3]H1.3_L1.4, [GPC3]H1.3_L1.5, [GPC3]H1.3_L1.6, [GPC3]H1.3_L1.7, [GPC3]H1.3_L1.8, [GPC3]H1.3_L1.9, [GPC3]H1.3_L1.10, [GPC3]H1.3_L1.16, [GPC3]H1.3_L1.23, [GPC3]H1.3_L1.29, [GPC3]H1.3_L1.31, [GPC3]H1.3_L1.65, [GPC3]H1.3_L1.66, [GPC3]H1.3_L1.67, [GPC3]H1.3_L1.68, [GPC3]H1.3_L1.70, [GPC3]H1.3_L1.71, [GPC3]H1.3_L1.72, [GPC3]H1.3_L1.73, [GPC3]H1.4_L1, [GPC3]H1.4_L1.1, [GPC3]H1.4_L1.2, [GPC3]H1.4_L1.3, [GPC3]H1.4_L1.4, [GPC3]H1.4_L1.5, [GPC3]H1.4_L1.6, [GPC3]H1.4_L1.7, [GPC3]H1.4_L1.8, [GPC3]H1.4_L1.9, [GPC3]H1.4_L1.10, [GPC3]H1.4_L1.16, [GPC3]H1.4_L1.23, [GPC3]H1.4_L1.29, [GPC3]H1.4_L1.31, [GPC3]H1.4_L1.65, [GPC3]H1.4_L1.66, [GPC3]H1.4_L1.67, [GPC3]H1.4_L1.68, [GPC3]H1.4_L1.70, [GPC3]H1.4_L1.71, [GPC3]H1.4_L1.72 and [GPC3]H1.4_L1.73 or a variant thereof. In particular embodiments, the VH/VL pairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69 or a variant thereof. In some embodiments, the 1+1 Fab-scFv-Fc format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 1+1 Fab-scFv-Fc formats that comprise: a) a first monomer (the “scFv monomer”) that comprises a charged scFv linker (with the +H sequence ofFIG.5being preferred in some embodiments), the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and an scFv that binds to CD3 as outlined herein; b) a second monomer (the “Fab monomer”) that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S, and a variable heavy domain; and c) a light chain that includes a variable light domain (VL) and a constant light domain (CL), wherein numbering is according to EU numbering. The variable heavy domain and variable light domain make up a GPC3 binding domain. CD3 binding domain sequences finding particular use in these embodiments include, but are not limited to, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 or a variant thereof, as well as those depicted inFIG.10. GPC3 binding domain sequences that are of particular use in these embodiments include, but are not limited to, the αGPC3 ABD VH/VL pairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. Particularly useful GPC3 and CD3 sequence combinations for use with the 1+1 format antibody include, for example, are disclosed inFIGS.16and17. FIGS.7A-7Dshow some exemplary Fc domain sequences that are useful in the 1+1 Fab-scFv-Fc format antibodies. The “monomer 1” sequences depicted inFIGS.7A-7Dtypically refer to the Fc domain of the “Fab-Fc heavy chain” and the “monomer 2” sequences refer to the Fc domain of the “scFv-Fc heavy chain.” Further,FIG.9provides useful CL sequences that can be used with this format. In some embodiments, any of the VH and VL sequences depicted herein (including all VH and VL sequences depicted in the Figures and Sequence Listings, including those directed to GPC3) can be added to the bottle opener backbone formats ofFIG.7A-7Das the “Fab side”, using any of the anti-CD3 scFv sequences shown in the Figures and Sequence Listings. For bottle opener backbone 1 fromFIG.7A, (optionally including the 428L/434S variants), CD binding domain sequences finding particular use in these embodiments include, but are not limited to, CD3 binding domain anti-CD3 H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3 H1.33_L1.47 and anti-CD3_H1.31_L1.47 attached as the scFv side of the backbones shown inFIG.7. 2. mAb-Fv One heterodimeric scaffold that finds particular use in the antibodies described herein is the mAb-Fv format (FIG.44G). In this embodiment, the format relies on the use of a C-terminal attachment of an “extra” variable heavy domain to one monomer and the C-terminal attachment of an “extra” variable light domain to the other monomer, thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind a GPC3 and the “extra” scFv domain binds CD3. In this embodiment, the first monomer comprises a first heavy chain, comprising a first variable heavy domain and a first constant heavy domain comprising a first Fc domain, with a first variable light domain covalently attached to the C-terminus of the first Fc domain using a domain linker (VH1-CH1-hinge-CH2-CH3-[optional linker]-VL2). The second monomer comprises a second variable heavy domain of the second constant heavy domain comprising a second Fc domain, and a third variable heavy domain covalently attached to the C-terminus of the second Fc domain using a domain linker (vh1-CH1-hinge-CH2-CH3-[optional linker]-VH2. The two C-terminally attached variable domains make up a Fv that binds CD3 (as it is less preferred to have bivalent CD3 binding). This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain that associates with the heavy chains to form two identical Fabs that bind a GPC3. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The antibodies described herein provide mAb-Fv formats where the CD3 binding domain sequences are as shown inFIG.10or a variant thereof. The antibodies described herein provide mAb-Fv formats wherein the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. In addition, the Fc domains of the mAb-Fv format comprise skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In some embodiments, the mAb-Fv format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include mAb-Fv formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that, with the first variable light domain of the light chain, makes up an Fv that binds to GPC3, and a second variable heavy domain; b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and a first variable heavy domain that, with the first variable light domain, makes up the Fv that binds to GPC3 as outlined herein, and a second variable light chain, that together with the second variable heavy domain forms an Fv (ABD) that binds to CD3; and c) a light chain comprising a first variable light domain and a constant light domain. In some embodiments, the mAb-Fv format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include mAb-Fv formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a first variable heavy domain that, with the first variable light domain of the light chain, makes up an Fv that binds to GPC3, and a second variable heavy domain; b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a first variable heavy domain that, with the first variable light domain, makes up the Fv that binds to GPC3 as outlined herein, and a second variable light chain, that together with the second variable heavy domain of the first monomer forms an Fv (ABD) that binds CD3; and c) a light chain comprising a first variable light domain and a constant light domain. 3. mAb-scFv One heterodimeric scaffold that finds particular use in the antibodies described herein is the mAb-scFv format (FIG.44H). In this embodiment, the format relies on the use of a C-terminal attachment of a scFv to one of the monomers, thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind GPC3 and the “extra” scFv domain binds CD3. Thus, the first monomer comprises a first heavy chain (comprising a variable heavy domain and a constant domain), with a C-terminally covalently attached scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain in either orientation (VH1-CH1-hinge-CH2-CH3-[optional linker]-VH2-scFv linker-VL2 or VH1-CH1-hinge-CH2-CH3-[optional linker]-VL2-scFv linker-VH2). This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that bind GPC3. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The antibodies described herein provide mAb-scFv formats where the CD binding domain sequences are as shown inFIG.10A-10For a variant thereof, and the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. In addition, the Fc domains of the mAb-scFv format comprise skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In some embodiments, the mAb-scFv format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include mAb-scFv formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein; and c) a common light chain comprising a variable light domain and a constant light domain. In some embodiments, the mAb-scFv format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include mAb-scFv formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein; and c) a common light chain comprising a variable light domain and a constant light domain. 4. 2+1 Fab2-scFv-Fc Format One heterodimeric scaffold that finds particular use in the antibodies described herein is the “2+1 Fab2-scFv-Fc” format (also referred to in previous related filings as “Central-scFv format”) shown inFIG.15Bwith an exemplary combination of a CD3 binding domain and two tumor target antigen (GPC3) binding domains. In this embodiment, the format relies on the use of an inserted scFv domain thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind GPC3 and the “extra” scFv domain binds CD3. The scFv domain is inserted between the Fc domain and the CH1-Fv region of one of the monomers, thus providing a third antigen binding domain. As described, GPC3×CD3 bispecific antibodies having the 2+1 Fab2-scFv-Fc format are potent in inducing redirected T cell cytotoxicity in cellular environments that express low levels of GPC3. Moreover, as shown in the examples, GPC3×CD3 bispecific antibodies having the 2+1 Fab2-scFv-Fc format allow for the “fine tuning” of immune responses as such antibodies exhibit a wide variety of different properties, depending on the GPC3 and/or CD3 binding domains used. For example, such antibodies exhibit differences in selectivity for cells with different GPC3 expression, potencies for GPC3 expressing cells, ability to elicit cytokine release, and sensitivity to soluble GPC3. These GPC3 antibodies find use, for example, in the treatment of GPC3 associated cancers. In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain (and optional hinge) and Fc domain, with a scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain. The scFv is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using optional domain linkers (VH1-CH1-[optional linker]-VH2-scFv linker-VL2-[optional linker including the hinge]-CH2-CH3, or the opposite orientation for the scFv, VH1-CH1-[optional linker]-VL2-scFv linker-VH2-[optional linker including the hinge]-CH2-CH3). The optional linkers can be any suitable peptide linkers, including, for example, the domain linkers included inFIG.6. In some embodiments, the optional linker is a hinge or a fragment thereof. The other monomer is a standard Fab side (i.e., VH1-CH1-hinge-CH2-CH3). This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that bind GPC3. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. In one embodiment, the 2+1 Fab2-scFv-Fc format antibody includes an scFv with the VH and VL of a CD3 binding domain sequence depicted inFIG.10. In one embodiment, the 2+1 Fab2-scFv-Fc format antibody includes two Fabs having the VH and VL of a GPC3 binding domain as shown inFIGS.12and13. In exemplary embodiments, the GPC3 binding domain of the 2+1 Fab2-scFv-Fc GPC3×CD3 bispecific antibody includes the VH and VL CD3 binding domain sequences finding particular use in these embodiments include, but are not limited to, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 or a variant thereof, as well as those depicted inFIG.10and those depicted in FIGS. 14 and 15 of WO2014/145806, hereby incorporated by reference including the Legends. Any suitable GPC3 ABD can be included in the 2+1 Fab2-scFv-Fc format antibody, included those provided herein. GPC3 ABDs that are of particular use in these embodiments include, but are not limited to, VH and VL domains selected from the group consisting of: [GPC3]H1_L1, [GPC3]H1_L1.1, [GPC3]H1_L1.2, [GPC3]H1_L1.3, [GPC3]H1_L1.4, [GPC3]H1_L1.5, [GPC3]H1_L1.6, [GPC3]H1_L1.7, [GPC3]H1_L1.8, [GPC3]H1_L1.9, [GPC3]H1_L1.10, [GPC3]H1_L1.16, [GPC3]H1_L1.23, [GPC3]H1_L1.29, [GPC3]H1_L1.31, [GPC3]H1_L1.65, [GPC3]H1_L1.66, [GPC3]H1_L1.67, [GPC3]H1_L1.68, [GPC3]H1_L1.70, [GPC3]H1_L1.71, [GPC3]H1_L1.72, [GPC3]H1_L1.73, [GPC3]H1.1_L1, [GPC3]H1.1_L1.1, [GPC3]H1.1_L1.2, [GPC3]H1.1_L1.3, [GPC3]H1.1_L1.4, [GPC3]H1.1_L1.5, [GPC3]H1.1_L1.6, [GPC3]H1.1_L1.7, [GPC3]H1.1_L1.8, [GPC3]H1.1_L1.9, [GPC3]H1.1_L1.10, [GPC3]H1.1_L1.16, [GPC3]H1.1_L1.23, [GPC3]H1.1_L1.29, [GPC3]H1.1_L1.31, [GPC3]H1.1_L1.65, [GPC3]H1.1_L1.66, L1.67, [GPC3]H1.1_L1.68, [GPC3]H1.1_L1.70, [GPC3]H1.1_L1.71, [GPC3]H1.1_L1.72, [GPC3]H1.1_L1.73, [GPC3]H1.2_L1, [GPC3]H1.2_L1.1, [GPC3]H1.2_L1.2, [GPC3]H1.2_L1.3, [GPC3]H1.2_L1.4, [GPC3]H1.2_L1.5, [GPC3]H1.2_L1.6, [GPC3]H1.2_L1.7, [GPC3]H1.2_L1.8, [GPC3]H1.2_L1.9, [GPC3]H1.2_L1.10, [GPC3]H1.2_L1.16, [GPC3]H1.2_L1.23, [GPC3]H1.2_L1.29, [GPC3]H1.2_L1.31, [GPC3]H1.2_L1.65, [GPC3]H1.2_L1.66, [GPC3]H1.2_L1.67, [GPC3]H1.2_L1.68, [GPC3]H1.2_L1.70, [GPC3]H1.2_L1.71, [GPC3]H1.2_L1.72, [GPC3]H1.2_L1.73, [GPC3]H1.3_L1, [GPC3]H1.3_L1.1, [GPC3]H1.3_L1.2, [GPC3]H1.3_L1.3, [GPC3]H1.3_L1.4, [GPC3]H1.3_L1.5, [GPC3]H1.3_L1.6, [GPC3]H1.3_L1.7, [GPC3]H1.3_L1.8, [GPC3]H1.3_L1.9, [GPC3]H1.3_L1.10, [GPC3]H1.3_L1.16, [GPC3]H1.3_L1.23, [GPC3]H1.3_L1.29, [GPC3]H1.3_L1.31, [GPC3]H1.3_L1.65, [GPC3]H1.3_L1.66, [GPC3]H1.3_L1.67, [GPC3]H1.3_L1.68, [GPC3]H1.3_L1.70, [GPC3]H1.3_L1.71, [GPC3]H1.3_L1.72, [GPC3]H1.3_L1.73, [GPC3]H1.4_L1, [GPC3]H1.4_L1.1, [GPC3]H1.4_L1.2, [GPC3]H1.4_L1.3, [GPC3]H1.4_L1.4, [GPC3]H1.4_L1.5, [GPC3]H1.4_L1.6, [GPC3]H1.4_L1.7, [GPC3]H1.4_L1.8, [GPC3]H1.4_L1.9, [GPC3]H1.4_L1.10, [GPC3]H1.4_L1.16, [GPC3]H1.4_L1.23, [GPC3]H1.4_L1.29, [GPC3]H1.4_L1.31, [GPC3]H1.4_L1.65, [GPC3]H1.4_L1.66, [GPC3]H1.4_L1.67, [GPC3]H1.4_L1.68, [GPC3]H1.4_L1.70, [GPC3]H1.4_L1.71, [GPC3]H1.4_L1.72 and [GPC3]H1.4_L1.73 or a variant thereof. In particular embodiments, the VH/VL pairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69 or a variant thereof. In addition, the Fc domains of the 2+1 Fab2-scFv-Fc format comprise skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In some embodiments, the 2+1 Fab2-scFv-Fc format antibody includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include 2+1 Fab2-scFv-Fc formats that comprise: a) a first monomer (the Fab-scFv-Fc side) that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein, and an scFv domain that binds to CD3; b) a second monomer (the Fab-Fc side) that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein; and c) a common light chain comprising the variable light domain and a constant light domain, where numbering is according to EU numbering. In some embodiments, the αGPC3 VH/VL pairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. In some embodiments, the 2+1 Fab2-scFv-Fc format antibody includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include 2+1 Fab2-scFv-Fc formats that comprise: a) a first monomer (the Fab-scFv-Fc side) that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein, and an scFv domain that binds to CD3; b) a second monomer (the Fab-Fc side) that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein; and c) a common light chain comprising a variable light domain and a constant light domain, where numbering is according to EU numbering. In some embodiments, the αGPC3 VH/VLpairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69. CD3 binding domain sequences finding particular use in these embodiments include, but are not limited to, H1.30_L1.47, H1.32_L1.47, H1.89_L1.47, H1.90_L1.47, H1.33_L1.47, H1.31_L1.47, L1.47_H1.30, L1.47_H1.30, L1.47_H1.32, L1.47_H1.89, L1.47_H1.90, L1.47_H1.33, and L1.47_H1.31 or a variant thereof. FIGS.8A-8Cshows some exemplary Fc domain sequences that are useful with the 2+1 Fab2-scFv-Fc format. The “monomer 1” sequences depicted inFIGS.8A-8Ctypically refer to the Fc domain of the “Fab-Fc heavy chain” and the “monomer 2” sequences refer to the Fc domain of the “Fab-scFv-Fc heavy chain.” Further,FIG.9provides useful CL sequences that can be used with this format. Exemplary anti-GPC3×anti-CD3 2+1 Fab2-scFv-Fc format antibodies are depicted inFIGS.18-21. 5. Central-Fv One heterodimeric scaffold that finds particular use in the antibodies described herein is the Central-Fv format (FIG.44I). In this embodiment, the format relies on the use of an inserted Fv domain (i.e., the central Fv domain) thus forming an “extra” third antigen binding domain, wherein the Fab portions of the two monomers bind a GPC3 and the “extra” central Fv domain binds CD3. The “extra” central Fv domain is inserted between the Fc domain and the CH1-Fv region of the monomers, thus providing a third antigen binding domain (i.e., the “extra” central Fv domain), wherein each monomer contains a component of the “extra” central Fv domain (i.e., one monomer comprises the variable heavy domain and the other a variable light domain of the “extra” central Fv domain). In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain, and Fc domain and an additional variable light domain. The light domain is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers (VH1-CH1-[optional linker]-VL2-hinge-CH2-CH3). The other monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain and Fc domain and an additional variable heavy domain (VH1-CH1-[optional linker]-VH2-hinge-CH2-CH3). The light domain is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers. This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain, that associates with the heavy chains to form two identical Fabs that each bind an GPC3. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The antibodies described herein provide central-Fv formats where the CD3 binding domain sequences are as shown inFIG.10or a variant thereof, and the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. 6. One-Armed Central-scFv One heterodimeric scaffold that finds particular use in the antibodies described herein is the one-armed central-scFv format (FIG.44C). In this embodiment, one monomer comprises just an Fc domain, while the other monomer includes a Fab domain (a first antigen binding domain), a scFv domain (a second antigen binding domain) and an Fc domain, where the scFv domain is inserted between the Fc domain and the Fc domain. In this format, the Fab portion binds one receptor target and the scFv binds another. In this format, either the Fab portion binds a GPC3 and the scFv binds CD3 or vice versa. In this embodiment, one monomer comprises a first heavy chain comprising a first variable heavy domain, a CH1 domain and Fc domain, with a scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain. The scFv is covalently attached between the C-terminus of the CH1 domain of the heavy constant domain and the N-terminus of the first Fc domain using domain linkers, in either orientation, VH1-CH1-[optional domain linker]-VH2-scFv linker-VL2-[optional domain linker]-CH2-CH3 or VH1-CH1-[optional domain linker]-VL2-scFv linker-VH2-[optional domain linker]-CH2-CH3. The second monomer comprises an Fc domain (CH2-CH3). This embodiment further utilizes a light chain comprising a variable light domain and a constant light domain that associates with the heavy chain to form a Fab. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The antibodies described herein provide central-Fv formats where the CD3 binding domain sequences are as shown inFIG.10or a variant thereof, and the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. In addition, the Fc domains of the one-armed central-scFv format generally include skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In some embodiments, the one-armed central-scFv format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments of the one-armed central-scFv formats comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that includes an Fc domain having the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K; and c) a light chain comprising a variable light domain and a constant light domain. In some embodiments, the one-armed central-scFv format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments of the one-armed central-scFv formats comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that includes an Fc domain having the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and the FcRn variants M428L/N434S; and c) a light chain comprising a variable light domain and a constant light domain. 7. One-Armed scFv-mAb One heterodimeric scaffold that finds particular use in the antibodies described herein is the one-armed scFv-mAb format (FIG.44D). In this embodiment, one monomer comprises just an Fc domain, while the other monomer uses a scFv domain attached at the N-terminus of the heavy chain, generally through the use of a linker: VH-scFv linker-VL-[optional domain linker]-CH1-hinge-CH2-CH3 or (in the opposite orientation) VL-scFv linker-VH-[optional domain linker]-CH1-hinge-CH2-CH3. In this format, the Fab portions each bind GPC3 and the scFv binds CD3. This embodiment further utilizes a light chain comprising a variable light domain and a constant light domain, that associates with the heavy chain to form a Fab. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The antibodies described herein provide one-armed scFv-mAb formats where the CD3 binding domain sequences are as shown inFIG.10or a variant thereof, and wherein the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. In addition, the Fc domains of the one-armed scFv-mAb format generally include skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In some embodiments, the one-armed scFv-mAb format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments of the one-armed scFv-mAb formats comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that includes an Fc domain having the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K; and c) a light chain comprising a variable light domain and a constant light domain. In some embodiments, the one-armed scFv-mAb format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments one-armed scFv-mAb formats comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that includes an Fc domain having the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and the FcRn variants M428L/N434S; and c) a light chain comprising a variable light domain and a constant light domain. 8. scFv-mAb One heterodimeric scaffold that finds particular use in the antibodies described herein is the mAb-scFv format (FIG.44E). In this embodiment, the format relies on the use of a N-terminal attachment of a scFv to one of the monomers, thus forming a third antigen binding domain, wherein the Fab portions of the two monomers bind GPC3 and the “extra” scFv domain binds CD3. In this embodiment, the first monomer comprises a first heavy chain (comprising a variable heavy domain and a constant domain), with a N-terminally covalently attached scFv comprising a scFv variable light domain, an scFv linker and a scFv variable heavy domain in either orientation ((VH1-scFv linker-VL1-[optional domain linker]-VH2-CH1-hinge-CH2-CH3) or (with the scFv in the opposite orientation) ((VL1-scFv linker-VH1-[optional domain linker]-VH2-CH1-hinge-CH2-CH3)). This embodiment further utilizes a common light chain comprising a variable light domain and a constant light domain that associates with the heavy chains to form two identical Fabs that bind GPC3. As for many of the embodiments herein, these constructs include skew variants, pI variants, ablation variants, additional Fc variants, etc. as desired and described herein. The antibodies described herein provide scFv-mAb formats where the CD3 binding domain sequences are as shown inFIG.10or a variant thereof, and wherein the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. In addition, the Fc domains of the scFv-mAb format generally include skew variants (e.g. a set of amino acid substitutions as shown inFIG.1, with particularly useful skew variants being selected from the group consisting of S364K/E357Q: L368D/K370S; L368D/K370S: S364K; L368E/K370S: S364K; T411T/E360E/Q362E: D401K; L368D/K370S: S364K/E357L, K370S: S364K/E357Q, T366S/L368A/Y407V: T366W and T366S/L368A/Y407V/Y349C: T366W/S354C), optionally ablation variants (including those shown inFIG.3), optionally charged scFv linkers (including those shown inFIG.5) and the heavy chain comprises pI variants (including those shown inFIG.2). In some embodiments, the scFv-mAb format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include scFv-mAb formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein; and c) a common light chain comprising a variable light domain and a constant light domain. In some embodiments, the scFv-mAb format includes skew variants, pI variants, ablation variants and FcRn variants. Accordingly, some embodiments include scFv-mAb formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein, and a scFv domain that binds to CD3; b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a variable heavy domain that, with the variable light domain of the common light chain, makes up an Fv that binds to GPC3 as outlined herein; and c) a common light chain comprising a variable light domain and a constant light domain. 9. Dual scFv Formats The antibodies described herein also provide dual scFv formats as are known in the art (FIG.44B). In this embodiment, the GPC3×CD3 heterodimeric bispecific antibody is made up of two scFv-Fc monomers (both in either (VH-scFv linker-VL-[optional domain linker]-CH2-CH3) format or (VL-scFv linker-VH-[optional domain linker]-CH2-CH3) format, or with one monomer in one orientation and the other in the other orientation. The antibodies described herein provide dual scFv formats where the CD3 binding domain sequences are as shown inFIG.10or a variant thereof, and wherein the GPC3 binding domain sequences are as shown inFIGS.12and13or a variant thereof. In some embodiments, the dual scFv format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include dual scFv formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, and a first scFv that binds either CD3 or GPC3; and b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, and a second scFv that binds either CD3 or GPC3. In some embodiments, the dual scFv format includes skew variants, pI variants, ablation variants and FcRn variants. In some embodiments, the dual scFv format includes skew variants, pI variants, and ablation variants. Accordingly, some embodiments include dual scFv formats that comprise: a) a first monomer that comprises the skew variants S364K/E357Q, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a first scFv that binds either CD3 or GPC3; and b) a second monomer that comprises the skew variants L368D/K370S, the pI variants N208D/Q295E/N384D/Q418E/N421D, the ablation variants E233P/L234V/L235A/G236del/S267K, the FcRn variants M428L/N434S and a second scFv that binds either CD3 or GPC3. 10. Non-Heterodimeric Bispecific Antibodies As will be appreciated by those in the art, the anti-GPC3×anti-CD3 antibodies provided herein can also be included in non-heterodimeric bispecific formats (seeFIG.44J). In this format, the anti-GPC3×anti-CD3 includes: 1) a first monomer comprising a VH1-CH1-hinge-CH2-CH3; 2) a second monomer comprising a VH2-CH1-hinge-CH2-CH3; 3) a first light chain comprising a VL1-CL; and 4) a second light chain comprising a VL2-CL. In such embodiments, the VH1 and VL1 form a first antigen binding domain and VH2 and VL2 form a second antigen binding domain. One of the first or second antigen binding domains binds GPC3 and the other antigen binding domain binds CD3. Any suitable GPC3 binding domain and CD3 binding domain can be included in the anti-GPC3×anti-CD3 antibody in the non-heterodimeric bispecific antibody format, including any of the GPC3 binding domains and CD3 binding domains and related VHs and VLs provided herein or a variant thereof (see, e.g.,FIGS.10,12and13). 11. Trident Format In some embodiments, the bispecific antibodies described herein are in the “Trident” format as generally described in WO2015/184203, hereby expressly incorporated by reference in its entirety and in particular for the Figures, Legends, definitions and sequences of “Heterodimer-Promoting Domains” or “HPDs”, including “K-coil” and “E-coil” sequences. Tridents rely on using two different HPDs that associate to form a heterodimeric structure as a component of the structure. In this embodiment, the Trident format include a “traditional” heavy and light chain (e.g., VH1-CH1-hinge-CH2-CH3 and VL1-CL), a third chain comprising a first “diabody-type binding domain” or “DART®”, VH2-(linker)-VL3-HPD1 and a fourth chain comprising a second DART®, VH3-(linker)-(linker)-VL2-HPD2. The VH1 and VL1 form a first ABD, the VH2 and VL2 form a second ABD, and the VH3 and VL3 form a third ABD. In some cases, as is shown inFIG.1K, the second and third ABDs bind the same antigen, in this instance generally GPC3, e.g., bivalently, with the first ABD binding a CD3 monovalently. Any suitable GPC3 binding domain and CD3 binding domain can be included in the anti-GPC3×anti-CD3 antibody in the Trident bispecific antibody format, including any of the GPC3 binding domains and CD3 binding domains and related VHs and VLs provided herein or a variant thereof (see, e.g.,FIGS.10,12and13). 12. Monospecific, Monoclonal antibodies As will be appreciated by those in the art, the novel Fv sequences outlined herein can also be used in both monospecific antibodies (e.g., “traditional monoclonal antibodies”) or non-heterodimeric bispecific formats. Accordingly, in some embodiments, the antibodies described herein provide monoclonal (monospecific) antibodies comprising the 6 CDRs and/or the vh and vl sequences from the figures, generally with IgG1, IgG2, IgG3 or IgG4 constant regions, with IgG1, IgG2 and IgG4 (including IgG4 constant regions comprising a S228P amino acid substitution) finding particular use in some embodiments. That is, any sequence herein with a “H L” designation can be linked to the constant region of a human IgG1 antibody. In some embodiments, the monospecific antibody is an GPC3 monospecific antibody that has a VH/VLpairs selected from the group consisting of: [GPC3]H1_L1, [GPC3]H1_L1.1, [GPC3]H1_L1.2, [GPC3]H1_L1.3, [GPC3]H1_L1.4, [GPC3]H1_L1.5, [GPC3]H1_L1.6, [GPC3]H1_L1.7, [GPC3]H1_L1.8, [GPC3]H1_L1.9, [GPC3]H1_L1.10, [GPC3]H1_L1.16, [GPC3]H1_L1.23, [GPC3]H1_L1.29, [GPC3]H1_L1.31, [GPC3]H1_L1.65, [GPC3]H1_L1.66, [GPC3]H1_L1.67, [GPC3]H1_L1.68, [GPC3]H1_L1.70, [GPC3]H1_L1.71, [GPC3]H1_L1.72, [GPC3]H1_L1.73, [GPC3]H1.1_L1, [GPC3]H1.1_L1.1, [GPC3]H1.1_L1.2, [GPC3]H1.1_L1.3, [GPC3]H1.1_L1.4, [GPC3]H1.1_L1.5, [GPC3]H1.1_L1.6, [GPC3]H1.1_L1.7, [GPC3]H1.1_L1.8, [GPC3]H1.1_L1.9, [GPC3]H1.1_L1.10, [GPC3]H1.1_L1.16, [GPC3]H1.1_L1.23, [GPC3]H1.1_L1.29, [GPC3]H1.1_L1.31, [GPC3]H1.1_L1.65, [GPC3]H1.1_L1.66, L1.67, [GPC3]H1.1_L1.68, [GPC3]H1.1_L1.70, [GPC3]H1.1_L1.71, [GPC3]H1.1_L1.72, [GPC3]H1.1_L1.73, [GPC3]H1.2_L1, [GPC3]H1.2_L1.1, [GPC3]H1.2_L1.2, [GPC3]H1.2_L1.3, [GPC3]H1.2_L1.4, [GPC3]H1.2_L1.5, [GPC3]H1.2_L1.6, [GPC3]H1.2_L1.7, [GPC3]H1.2_L1.8, [GPC3]H1.2_L1.9, [GPC3]H1.2_L1.10, [GPC3]H1.2_L1.16, [GPC3]H1.2_L1.23, [GPC3]H1.2_L1.29, [GPC3]H1.2_L1.31, [GPC3]H1.2_L1.65, [GPC3]H1.2_L1.66, [GPC3]H1.2_L1.67, [GPC3]H1.2_L1.68, [GPC3]H1.2_L1.70, [GPC3]H1.2_L1.71, [GPC3]H1.2_L1.72, [GPC3]H1.2_L1.73, [GPC3]H1.3_L1, [GPC3]H1.3_L1.1, [GPC3]H1.3_L1.2, [GPC3]H1.3_L1.3, [GPC3]H1.3_L1.4, [GPC3]H1.3_L1.5, [GPC3]H1.3_L1.6, [GPC3]H1.3_L1.7, [GPC3]H1.3_L1.8, [GPC3]H1.3_L1.9, [GPC3]H1.3_L1.10, [GPC3]H1.3_L1.16, [GPC3]H1.3_L1.23, [GPC3]H1.3_L1.29, [GPC3]H1.3_L1.31, [GPC3]H1.3_L1.65, [GPC3]H1.3_L1.66, [GPC3]H1.3_L1.67, [GPC3]H1.3_L1.68, [GPC3]H1.3_L1.70, [GPC3]H1.3_L1.71, [GPC3]H1.3_L1.72, [GPC3]H1.3_L1.73, [GPC3]H1.4_L1, [GPC3]H1.4_L1.1, [GPC3]H1.4_L1.2, [GPC3]H1.4_L1.3, [GPC3]H1.4_L1.4, [GPC3]H1.4_L1.5, [GPC3]H1.4_L1.6, [GPC3]H1.4_L1.7, [GPC3]H1.4_L1.8, [GPC3]H1.4_L1.9, [GPC3]H1.4_L1.10, [GPC3]H1.4_L1.16, [GPC3]H1.4_L1.23, [GPC3]H1.4_L1.29, [GPC3]H1.4_L1.31, [GPC3]H1.4_L1.65, [GPC3]H1.4_L1.66, [GPC3]H1.4_L1.67, [GPC3]H1.4_L1.68, [GPC3]H1.4_L1.70, [GPC3]H1.4_L1.71, [GPC3]H1.4_L1.72 and [GPC3]H1.4_L1.73 or a variant thereof. In particular embodiments, the VH/VL pairs are selected from the group consisting of [GPC3]H1.1_L1.16 and [GPC3]H1.1_L1.69 or a variant thereof. H. Particular Embodiments of the Invention The invention specifically provides 1+1 and 2+1 formats that bind CD3 and GPC3. Certain embodiments include XENP38086 (Xtend analog to XENP34920, meaning they have identical sequences except that the Xtend analog includes 428L/434S on each Fc domain), XENP38087 (Xtend analog to XENP36935, ditto), and XENP38232 (Xtend analog to XENP37625, ditto.) 1. 1+1 Format In particular 1+1 format embodiments, the αGPC3 ABD is the Fab and has the VH/VL pair [GPC3]H1.1_L1.16 and the αCD3 ABD is a scFv selected from the group consisting of anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3 L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31. In particular 1+1 format embodiments, the αGPC3 ABD is the Fab and has the VH/VL pair [GPC3]H1.1_L1.69, and the αCD3 ABD is a scFv selected from the group consisting of anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3 L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31. Particular embodiments that find use in a number of applications include those listed inFIGS.16and17, including, but not limited to, XENP21971, XENP34364, XENP34365, XENP34367, XENP34368, XENP35843, XENP36140, XENP36931, XENP36932 XENP36933, XENP36934, XENP36935, XENP36936, XENP36937, XENP36938, XENP36939, XENP36941 and XENP38087. 2. 2+1 Format In particular 2+1 format embodiments, the αGPC3 ABD is the Fab and has the VH/VL pair [GPC3]H1.1_L1.16 and the αCD3 ABD is a scFv selected from the group consisting of anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3 L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31. In particular 2+1 format embodiments, the αGPC3 ABD is the Fab and has the VH/VL pair [GPC3]H1.1_L1.69, and the αCD3 ABD is a scFv selected from the group consisting of anti-CD3_H1.30_L1.47, anti-CD3_H1.32_L1.47, anti-CD3_H1.89_L1.47, anti-CD3_H1.90_L1.47, anti-CD3_H1.33_L1.47, anti-CD3_H1.31_L1.47, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.30, anti-CD3_L1.47_H1.32, anti-CD3_L1.47_H1.89, anti-CD3 L1.47_H1.90, anti-CD3_L1.47_H1.33, and anti-CD3_L1.47_H1.31. Particular embodiments that find use in a number of applications include those listed inFIGS.18,19,20and21, including, but not limited to, XENP33744, XENP33745, XENP27259, XENP33746, XENP34919, XENP34920, XENP34921, XENP34922, XENP34923, XENP35840, XENP35840, XENP37246, XENP37247, XENP38086, XENP33747, XENP35841, XENP37624, XENP37625, XENP37626, XENP38232, XENP37430, XENP37433 and XENP33748. II. Nucleic Acids of the Invention The disclosure further provides nucleic acid compositions encoding the anti-GPC3 antibodies provided herein, including, but not limited to, anti-GPC3×anti-CD3 bispecific antibodies and GPC3 monospecific antibodies. As will be appreciated by those in the art, the nucleic acid compositions will depend on the format and scaffold of the heterodimeric protein. Thus, for example, when the format requires three amino acid sequences, such as for the 1+1 Fab-scFv-Fc format (e.g. a first amino acid monomer comprising an Fc domain and a scFv, a second amino acid monomer comprising a heavy chain and a light chain), three nucleic acid sequences can be incorporated into one or more expression vectors for expression. Similarly, some formats (e.g. dual scFv formats such as disclosed inFIG.44) only two nucleic acids are needed; again, they can be put into one or two expression vectors. As is known in the art, the nucleic acids encoding the components of the antibodies described herein can be incorporated into expression vectors as is known in the art, and depending on the host cells used to produce the heterodimeric antibodies described herein. Generally the nucleic acids are operably linked to any number of regulatory elements (promoters, origin of replication, selectable markers, ribosomal binding sites, inducers, etc.). The expression vectors can be extra-chromosomal or integrating vectors. The nucleic acids and/or expression vectors of the antibodies described herein are then transformed into any number of different types of host cells as is well known in the art, including mammalian, bacterial, yeast, insect and/or fungal cells, with mammalian cells (e.g. CHO cells), finding use in many embodiments. In some embodiments, nucleic acids encoding each monomer and the optional nucleic acid encoding a light chain, as applicable depending on the format, are each contained within a single expression vector, generally under different or the same promoter controls. In embodiments of particular use in the antibodies described herein, each of these two or three nucleic acids are contained on a different expression vector. As shown herein and in 62/025,931, hereby incorporated by reference, different vector ratios can be used to drive heterodimer formation. That is, surprisingly, while the proteins comprise first monomer:second monomer:light chains (in the case of many of the embodiments herein that have three polypeptides comprising the heterodimeric antibody) in a 1:1:2 ratio, these are not the ratios that give the best results. The heterodimeric antibodies described herein are made by culturing host cells comprising the expression vector(s) as is well known in the art. Once produced, traditional antibody purification steps are done, including an ion exchange chromatography step. As discussed herein, having the pIs of the two monomers differ by at least 0.5 can allow separation by ion exchange chromatography or isoelectric focusing, or other methods sensitive to isoelectric point. That is, the inclusion of pI substitutions that alter the isoelectric point (pI) of each monomer so that such that each monomer has a different pI and the heterodimer also has a distinct pI, thus facilitating isoelectric purification of the “1+1 Fab-scFv-Fc” and “2+1” heterodimers (e.g., anionic exchange columns, cationic exchange columns). These substitutions also aid in the determination and monitoring of any contaminating dual scFv-Fc and mAb homodimers post-purification (e.g., IEF gels, cIEF, and analytical IEX columns). III. Biological and Biochemical Functionality of the Heterodimeric Bispecific Antibodies Generally the bispecific GPC3×CD3 antibodies described herein are administered to patients with cancer, and efficacy is assessed, in a number of ways as described herein. Thus, while standard assays of efficacy can be run, such as cancer load, size of tumor, evaluation of presence or extent of metastasis, etc., immuno-oncology treatments can be assessed on the basis of immune status evaluations as well. This can be done in a number of ways, including both in vitro and in vivo assays. IV. Treatments Once made, the compositions of the antibodies described herein find use in a number of applications including cancer such as liver cancer, such that the heterodimeric compositions of the antibodies described herein find use in the treatment of such GPC3 positive cancers. V. Antibody Compositions for In Vivo Administration Formulations of the antibodies used in accordance with the antibodies described herein are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the form of lyophilized formulations or aqueous solutions. VI. Administrative Modalities The antibodies and chemotherapeutic agents described herein are administered to a subject, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time. VII. Treatment Modalities In the methods described herein, therapy is used to provide a positive therapeutic response with respect to a disease or condition. By “positive therapeutic response” is intended an improvement in the disease or condition, and/or an improvement in the symptoms associated with the disease or condition. For example, a positive therapeutic response would refer to one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (5) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth; (6) an increased patient survival rate; and (7) some relief from one or more symptoms associated with the disease or condition. Positive therapeutic responses in any given disease or condition can be determined by standardized response criteria specific to that disease or condition. Tumor response can be assessed for changes in tumor morphology (i.e., overall tumor burden, tumor size, and the like) using screening techniques such as magnetic resonance imaging (MM) scan, x-radiographic imaging, computed tomographic (CT) scan, bone scan imaging, endoscopy, and tumor biopsy sampling including bone marrow aspiration (BMA) and counting of tumor cells in the circulation. In addition to these positive therapeutic responses, the subject undergoing therapy may experience the beneficial effect of an improvement in the symptoms associated with the disease. Treatment according to the disclosure includes a “therapeutically effective amount” of the medicaments used. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the medicaments to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. A “therapeutically effective amount” for tumor therapy may also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer may be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to inhibit cell growth or to induce apoptosis by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. The efficient dosages and the dosage regimens for the bispecific antibodies described herein depend on the disease or condition to be treated and may be determined by the persons skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of an bispecific antibody used in the antibodies described herein is about 0.1-100 mg/kg. All cited references are herein expressly incorporated by reference in their entirety. Whereas particular embodiments of the disclosure have been described above for purposes of illustration, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims. EXAMPLES A. Example 1: Engineering αGPC3×αCD3 Bispecific Antibodies A number of formats for αGPC3×αCD3 bispecific antibodies (bsAbs) were conceived, illustrative formats for which are outlined below and inFIG.15. One such format is the 1+1 Fab-scFv-Fc format which comprises a single-chain Fv (“scFv”) covalently attached to a first heterodimeric Fc domain, a heavy chain variable region (VH) covalently attached to a complementary second heterodimeric Fc domain, and a light chain (LC) transfected separately so that a Fab domain is formed with the variable heavy domain. Another format is the 2+1 Fab2-scFv-Fc format which comprises a VH domain covalently attached to a CH1 domain covalently attached to an scFv covalently attached to a first heterodimeric Fc domain (VH-CH1-scFv-Fc), a VH domain covalently attached to a complementary second heterodimeric Fc domain, and a LC transfected separately so that Fab domains are formed with the VH domains. DNA encoding chains of the αGPC3×αCD3 bsAbs were generated by standard gene synthesis followed by isothermal cloning (Gibson assembly) or subcloning into a pTT5 expression vector containing fusion partners (e.g. domain linkers as depicted inFIG.6and/or backbones as depicted inFIGS.7-8). DNA was transfected into HEK293E cells for expression. Sequences for illustrative αGPC3×αCD3 bsAbs in the 1+1 Fab-scFv-Fc format and in the 2+1 Fab2-scFv-Fc format are depicted respectively inFIGS.16-21. B. Example 2: Engineering GPC3 Binding Domains 2A: Humanization of a Murine GPC3 Binding Domain A murine clone that binds that C terminal peptide of GPC3 (amino acids 524-563) of the GPC3 protein, located close to the cell membrane was humanized using string content optimization (see, e.g., U.S. Pat. No. 7,657,380, issued Feb. 2, 2010). Sequences for the humanized variant are herein referred to as GPC3-A and are depicted inFIG.12. Variants reduced degradation (e.g. aspartic acid isomerization and deamidation) liability, modulated GPC3 binding affinity, and/or selectivity for high GPC3 expression cell lines. Sequences for illustrative such variants are depicted inFIG.13. 2B: Tuning the GPC3 Binding Affinity After humanization, GPC3 binding arms were engineered with single point mutations in the variable light domain with the aim to create a ladder of GPC3 binding affinity, illustrative sequences for which are depicted inFIG.13. 73 variable light domain variants were engineered and paired with GPC3-A H1.1 (see Example 3C for description of GPC3-A H1.1 variant). The GPC3-A variants were engineered in the 1+1 Fab-scFv-Fc bsAb format and binding for GPC3 antigen was measured using Octet, a BioLayer Interferometry (BLI)-based method. Experimental steps for Octet generally include the following: Immobilization (capture of ligand to a biosensor); Association (dipping of ligand-coated biosensors into wells containing the analyte); and Dissociation (returning of biosensors to well containing buffer). For ease of clinical development (e.g. by investigating the therapeutics in model animals), it is useful for the binding domains to be cross-reactive for cynomolgus antigen; therefore, binding to both human and cynomolgus GPC3 antigen were investigated. His-tagged human and cynomolgus GPC3 were captured on HIS1K sensors then dipped into multiple concentrations of the bispecific antibodies. The resulting dissociation constant (KD) are depicted inFIG.23. A broad range of KD values were obtained from high affinity variant H1.1_L1.6 (4 nM), to medium affinity variants H1.1_L1.16 and H1.1_L1.29 (100 nM and 70 nM respectively), to low affinity variant H1.1_L1.31 (400 nM). Next, binding of the variants to cell surface GPC3 was investigated. Illustrative affinity variants H1.1_L1.29, H1.1_L1.16, and H1.1_L1.31 were engineered in the 1+1 Fab-scFv-Fc format. BsAbs XENP36941 (L1.29, 70 nM GPC3 affinity), XENP35843 (L1.16, 100 nM GPC3 affinity), and XENP36140 (L1.31, 400 nM GPC3 affinity) were incubated with GPC3highHepG2 cells at indicated concentrations for 1 hr at 4° C. Cells were then washed and stained with a secondary antibody (typically anti-human Fc AlexaFlour647) for 1 hr at 4° C. After 2 more washes, cells were analyzed by flow cytometry. The resulting binding curves portrayed inFIG.24show that cell binding correlates with the GPC3 affinity, with XENP36941 (70 nM variable light domain) showing the tightest binding, and the XENP36140 (400 nM variable light domain) showing the weakest. 2C: Engineering to Remove Degradation Liable Residues The sequences for humanized GPC3-A antibodies were investigated for degradation liable residues. The GPC3-A H1 heavy chain CDR2 included D52/P52a (Kabat numbering) as an aspartic acid isomerization motif. Further, the L1 variable light domain contained an N28/G29 (Kabat numbering) as a deamidation motif. Accordingly, a library was made with mutations at these residues to investigate whether the liability could be removed without impacting on GPC3 binding, binding data for which are depicted inFIG.25. D52E mutation in the variable heavy variant H1.1 removed the aspartic acid isomerization liability (relative to H1) while maintaining similar GPC3 binding (D52N resulted in reduced binding, while D52Q and D52S were acceptable). G29T in the variable light variant L1.6 removed the deamidation liability (relative to L1) while maintaining similar GPC3 binding (N28Q, N28Y, N28S, and N28H drastically reduced binding, while G29A and G29K were acceptable). It should be noted that different mutations may be required to remove the deamidation liability from the affinity variants, binding data for illustrative variants as depicted inFIG.26. G29A mutation was utilized in the L1.69 variant to remove the deamidation liability from the L1.29 variant (N28Y mutation resulted in decreased binding, while G29K was also acceptable); and G29K mutation was utilized in the L1.73 variant to remove the deamidation liability from the L1.31 variant (N28Y and G29A mutations resulted in low or aberrant response). Sequences for the variants described here are depicted inFIG.13. C. Example 3: Tuning and Optimizing αGPC3×αCD3 bsAbs αGPC3×αCD3 bsAbs were engineered with various affinity-tuned GPC3 and CD3 binding domains and with different GPC3 binding valency and produced as generally described in Example 1 to optimize redirected T cell cytotoxicity (RTCC), selectivity, and potential therapeutic index. RTCC assays were used to investigate the potential of αGPC3×αCD3 bispecific antibodies (bsAbs) to redirect CD3+ effector T cells to destroy GPC3-expressing cell lines. RTCC assays were generally performed using HepG2 cells (a liver hepatocellular carcinoma line) as GPC3hightarget cells, Huh7 cells (also a liver hepatocellular carcinoma line) as a GPC3medtarget cells, and/or HEK293 cells as GPC3lowtarget cells (a surrogate for cells outside of the tumor environment). Two methods of redirected T cell cytotoxicity (RTCC) assays were used: flow cytometry based, and lactate dehydrogenase (LDH) based. For flow cytometry based RTCC assays, target cells were incubated with human PBMCs and test articles at indicated effector to target cell ratios at 37° C. After incubation, cells were stained with Aqua Zombie stain for 15 minutes at room temperature. Cells were then washed and stained with antibodies for cell surface markers and analyzed by flow cytometry. Induction of RTCC was determined using Zombie Aqua staining on CSFE+target cells; and activation and degranulation of T cells were determined by CD107a, CD25, and CD69 expression on lymphocytes. For LDH-based RTCC assays, cells and indicated concentrations of test articles would be incubated in a total volume of 200 ul in a flat bottom 96 well plate for 48 or 72 hours at 37° C. Then the cells were lysed and mixed with substrate using the Promega CytoTox-one kit according to its protocol. A Wallac machine was used to read the plate. A V-PLEX proinflammatory panel 1 human kit was used to measure cytokines. It should also be noted that some of the data sets are from the same experiment, as several engineering approaches were simultaneously explored. Throughout this section, a comparator bsAb XENP31308, which is based on the αGPC3×αCD3 bsAb as disclosed in WO 2016/047722 and sequences for which are depicted inFIG.22, was also utilized to benchmark the novel bsAbs of the invention. XENP31308 was characterized in RTCC assays as generally described above. Data depicted inFIG.27show that at 10:1 effector:target ratio, XENP31308 induced potent RTCC and cytokine secretion on both high and low GPC3 expressing cell lines. In another experiment utilizing 1:1 effector:target ratio data for which are depicted inFIG.28, XENP31308 induces little RTCC in the presence of low GPC3 expressing cell line HEK293 but still potently induces cytokine secretion. As the GPC3lowcell line is a surrogate for healthy tissues, maintaining very little to no killing and cytokine release in the presence of a low GPC3 expressing cell line is important as it may translate to less toxicity and cytokine storm in a clinical setting. Strong killing of a GPC3lowcell line would indicate likelihood of on-target off-tissue killing in vivo, which could cause undesirable toxicity. 3A: Investigating the Effect of GPC3 Binding Domain Binding Different GPC3 Epitopes GPC3 is membrane bound and consists of an alpha subunit and a beta subunit. The alpha subunit (residues 25-358) may be cleaved and released as soluble GPC3, the while the beta subunit remains attached to the cellular membrane. As described in Example 2A, GPC3 binding domain GPC3-A binds C-terminal residues 524-563 of GPC3 which is part of the beta subunit. Additional GPC3 binding domains have been described to bind the N-terminal residues 359-524 of the beta subunit or the alpha subunit. To investigate the effect of binding to different GPC3 epitopes, αGPC3×αCD3 bsAbs in the 1+1 format with CD3 High or 2+1 format with CD3 High-Int #1 scFv were engineered with different GPC3 binding domains binding different GPC3 epitopes. An RTCC assay was performed using the bsAbs and HepG2 cells (10:1 effector:target ratio). The data inFIG.29(depicting activity of the bsAbs as indicated by induction of IFNγ release) show that each of the additional epitopes were less potent at inducing T cell activity than GPC3-A. While some of the difference may be due to differences in binding affinity (between clones) or avidity (between formats), it is likely that the GPC3 epitope contributes to the reduced potency. 3B: Investigating the Effect of GPC3 Binding Avidity It was hypothesized that bsAbs in the 2+1 Fab2-scFv-Fc format having bivalent binding for GPC3 may be useful in achieving higher potency with a lower affinity binding domain due to the improved avidity from two binding domains. The avidity benefit of this format may confer selectivity to cell lines with higher target expression, reducing the potential for on-target off-tissue (e.g. healthy tissues expressing low levels of GPC3) effects in vivo. Additionally, high levels of soluble GPC3 may be present in vivo, potentially acting as an antigen sink for GPC3-targeting drugs. Lower GPC3 binding affinity coupled with bivalent GPC3 binding may help overcome the sink by preferably binding cells with high antigen density over soluble antigen. Accordingly, the cell binding of αGPC3×αCD3 bispecific antibodies in the 1+1 Fab-scFv-Fc format, the 2+1 Fab2-scFv-Fc format, and the traditional monospecific bivalent format were investigated. The GPC3-A variable light domains L1.16 (having a GPC3 binding affinity of 100 nM) and L1.31 (having a GPC3 binding affinity of 400 nM) were engineered into the 1+1 Fab-scFv-Fc format, the 2+1 Fab2-scFv-Fc (VL/VH), and bivalent monospecific IgG1 format. Test articles were incubated with GPC3highHepG2 cells at indicated concentrations, washed, stained with a secondary antibody, washed again, and analyzed by flow cytometry as described above. As seen inFIG.30, the avidity conferred by the 2+1 Fab2-scFv-Fc (VL/VH) format improves binding compared to the 1+1 Fab-scFv-Fc format, reaching a level similar to that of the standard bivalent IgG. Additionally, the lower affinity L1.31 clone more drastically shows this improvement over the 1+1 Fab-scFv-Fc indicating that the lower binding affinity creates more of a dependence on avidity and binding valency. 3C: Investigating the Effect of Variant CD3 Binding Domains on RTCC To investigate the effect of CD3 binding affinity, αGPC3×αCD3 bsAbs were produced in the 1+1 Fab-scFv-Fc and the 2+1 Fab2-scFv-Fc formats with CD3 High scFv, CD3 High-Int #1 scFv, CD3 High-Int #2 scFv, and CD3 Intermediate scFvs and GPC3-A variants having 100 nM or 400 nM binding affinity for GPC3 and investigated in a flow-based RTCC performed as described above using a 10:1 E:T ratio on GPC3highHepG2 target cells with a 48 hour incubation time. The data plotted inFIGS.31A and32A(respectively for bsAbs in the 1+1 format and in the 2+1 format) showed that affinity-engineered αGPC3×αCD3 bsAbs demonstrated a range of potencies to GPC3highHepG2 cells, from high (XENP33744 having a 5 nM CD3 arm and 2+1 format, with an EC50 value of 62.63 pg/ml) to low (XENP27259 having a 30 nM CD3 arm in a 1+1 format, with an EC50 value of 19920 pg/ml). The bispecific antibodies also induced release of IFNγ (as depicted inFIGS.31B and32B) and T cell activation (as indicated by expression of activation markers such as CD69 and 107a; data not shown) in a manner correlated to their potency. Notably, de-tuning the CD3 binding affinity from CD3 High to CD3 High-Int #1 in the 1+1 Fab-scFv-Fc format provided a much greater potency reduction in induction of cytokine release in comparison to in the 2+1 Fab2-scFv-Fc format. However, in the 2+1 Fab2-scFv-Fc format, de-tuning the CD3 binding affinity with CD3 High-Int #2 and CD3 Intermediate still resulted in potency reduction in induction of cytokine release. 3D: Investigating the Effect of Affinity Detuned GPC3 Binding Domains on RTCC In another experiment, the effect of engineering affinity reductions in the GPC3 binding domains of a set of αGPC3×αCD3 bsAbs as described in Example 2 on the ability to redirect CD3+ effector T cells was investigated. An LDH-based RTCC assay was performed as described above, using GPC3medHuh7 as target cells, mixed with indicated concentrations of test articles and PBMC effector cells at a 10:1 E:T ratio, for a 48 hour incubation time.FIG.33shows that XENP37625 (having a variable light domain L1.69, 70 nM GPC3 affinity), XENP37624 (having a variable light domain L1.16, 100 nm GPC3 affinity), and XENP37626 (having a variable light domain L1.73, 400 nM GPC3 affinity) each demonstrated reduced potency of killing and IFNγ secretion roughly in accordance with their respective affinities. 3E: Investigating the Ability of αGPC3×αCD3 bsAbs to Induce RTCC on Cell Lines with Different GPC3 Expression Levels On-target, off-tumor toxicity and cytokine release syndrome can result in severe adverse effects in patients; therefore, it is important to tune the bsAbs of the invention to avoid killing and induction of cytokine release in the presence of healthy tissues which express low levels of GPC3. Accordingly, based on the various observations described above, additional αGPC3×αCD3 bsAbs were engineered, produced, and investigated to identify bsAbs with maximal therapeutic potential with minimal potential for toxicity. Towards this, the effects of modulating GPC3 binding affinity, GPC3 binding valency, and CD3 binding affinity on selectivity of the bispecific antibodies for cell lines with high and low expression were investigated. HepG2 (GPC3high) and HEK293 (GPC3low) cells were each incubated with human PBMCs (1:1 effector to target cell ratio) and indicated concentrations of the test articles for 72 hours at 37° C. Data depicting induction of RTCC and cytokine secretion are depicted inFIGS.34and35. Consistent with the above, the bispecific antibodies demonstrated a range of potencies in inducing RTCC and cytokine release in the presence of GPC3highHepG2 cell. Notably, in the presence of GPC3lowHEK293 cells, each of the bispecific antibodies demonstrated little to no induction of RTCC and cytokine release. 3F: Further Characterization of αGPC3×αCD3 bsAbs Using 2D RTCC on Incucyte The novel αGPC3×αCD3 bsAbs and the comparator molecule XENP31308 were investigated utilizing a different system (2D RTCC on Incucyte). Indicated concentrations of indicated test articles were incubated with HepG2 or Huh7 cells and T cells at 10:1 or 1:1 effector:target ratio. Data depicting RTCC are depicted inFIGS.36-38. At a 10:1 effector:target (HepG2) ratio, bsAbs XENP34920, XENP37430, XENP37625, and XENP37624 demonstrated similar potency (less than EC50 of 100 ng/ml) as comparator XENP31308; other bsAbs XENP35843, XENP36935, XENP37433, and XENP37626 were much less potent but were able to achieve efficacious killing at higher concentrations, and XENP36939 did not demonstrate any killing. Notably on lower density Huh7 and at lower effector:target ratio, several of the bsAbs of the invention (e.g. XENP34920, XENP36935, and XENP37625) demonstrate much greater difference in EC50 in comparison to the XENP31308 comparator indicating potential for enhanced therapeutic index. Additionally, when GPC3high HepG2 and GPC3med Huh7 target cells were treated with test articles at a concentration of 10 pg/ml at a 10:1 E:T ratio and observed over different time points ranging from 24 to 144 hours, similar results were seen. As depicted inFIGS.39-40, the affinity detuned αGPC3×αCD3 bsAbs including XENP34920, XENP37430, XENP36935, XENP37624, and XENP37625 were effective at inhibiting tumor growth over time in both HepG2 and Huh7 target cells, and their efficacy was very similar to that of XENP31308. 3G: Further Characterization of αGPC3×αCD3 bsAbs Using a 3D Spheroid RTCC Model An additional RTCC system was used to further investigate the affinity detuned αGPC3×αCD3 bsAbs. In this system, target tumor cells grow in a 3D spheroid format, which is physiologically more similar to an in vivo tumor compared to cells growing in a monolayer on a flat surface. In this experiment, 1,000 HepG2 cells (which have a doubling time of 24 hours) were seeded and given 72-96 hours to grow before adding 40,000 PBMCs (resulting in an E:T ratio of approximately 3:1) along with the indicated bsAb at a concentration of 10 ug/ml. The intensity of each signal (tumor or immune cell) was integrated over each well across different time points, and this data is depicted inFIGS.41-42.FIG.43additionally shows photographic images taken of the tumor cells and PBMCs. With PBS treatment only, the tumor spheroids and the PBMCs clustered around them remain unchanged over time, whereas when treated with the αGPC3×αCD3 bsAbs PBMCs proliferate dramatically (as depicted quantitatively inFIG.42), and the tumor cells are destroyed (as depicted quantitatively inFIG.41). All αGPC3×αCD3 bsAbs except XENP36939 show 100% efficacy after 144 hours. The 3D spheroid model also produced an unexpected result in test articles with the lower affinity High-Int #2 CD3 binding domain. Generally, for 2+1 bsAbs in a 2D model, the CD3 affinity difference between the High-Int #1 binding domain and the High-Int #2 binding domain results in roughly a 10-fold potency difference, such as what can be seen inFIG.38. However, in this 3D model, XENP34920 and XENP37624, both having with the same 100 nM GPC3 binding domain but XENP34920 having the High-Int #1 CD3 binding domain and XENP37624 having the High-Int #2 binding domain, showed potency curves that were very similar. Depicted inFIG.41, the unexpected result of this 3D model, which should more closely mimic an in vivo model, provides useful insight on the potency of a weaker CD3 binder in this context. D. Example 4: Identifying αGPC3×αCD3 bsAbs with Optimal Selectivity and Therapeutic Index Based on the above in vitro experiments, several bsAbs were selected for further analysis in vivo. These antibodies were further engineered with Xtend Fc (M428L/N434S) to enhance serum half-life, illustrative sequences which are depicted inFIGS.17,19, and20as XENP38086 (Xtend analog to XENP34920), XENP38087 (Xtend analog to XENP36935), and XENP38232 (Xtend analog to XENP37625). 1. 4A: In Vivo Investigation for Toxicity in Cynomolgus Studies in cynomolgus are planned to investigate whether the improvement in vitro (i.e. selectivity for GPChighover GPClowtarget cells, and reduced cytokine release in the presence of GPClowcells) translates to improved safety in an in vivo setting. In a Phase 1 dose escalation study, animals (n=1) are intravenously dosed with 1×, 3×, 10×, 30×, and 60× dose of XENP38086, XENP38087, or XENP38232. Blood is drawn to determine IL-6 concentrations as an indicator of cytokine release syndrome. Animals may be sacrificed to investigate additional signs of toxicity. | 199,527 |
11859014 | DETAILED DESCRIPTION In one embodiment, compounds and compositions useful in treating, preventing, or curing norovirus (NoV) infection are disclosed. Methods for treating, preventing, or curing NoV infection are also disclosed. In other embodiments, compounds for treating, preventing, or curing infections caused by Sapporo virus (human), Gastroenteritis, Jena virus (cattle), Murine norovirus (mouse), Fulminant organ dysfunction, Pistoia virus (lion), Hemorrhagic enteritis, Canine norovirus (dog), Swine43 (pig), Porcine enteric calicivirus (pig), Mink enteric calicivirus (mink), Rabbit hemorrhagic disease virus (rabbit), European brown hare syndrome virus (hare), Bovine enteric calicivirus/Newbury-1 virus (cattle), Bovine enteric calicivirus/Nebraska virus (cattle), Feline calicivirus (cat), Feline calicivirus-VS (cat), San Miguel sea lion virus (sea lion), Canine calicivirus No. 48 (dog), Tulane virus (monkey), St. Valerian virus/AB90 (pig), and Bayern virus (chicken) are disclosed. The compounds described herein show inhibitory activity against NoV in cell-based assays. Therefore, the compounds can be used to treat or prevent a NoV in a host, or reduce the biological activity of the virus. The host can be a mammal, and in particular, a human, infected with NoV. The methods involve administering an effective amount of one or more of the compounds described herein. Pharmaceutical formulations including one or more compounds described herein, in combination with a pharmaceutically acceptable carrier or excipient, are also disclosed. In one embodiment, the formulations include at least one compound described herein and at least one further therapeutic agent. The present invention will be better understood with reference to the following definitions: I. Definitions The term “independently” is used herein to indicate that the variable, which is independently applied, varies independently from application to application. Thus, in a compound such as R″XYR″, wherein R″ is “independently carbon or nitrogen,” both R″ can be carbon, both R″ can be nitrogen, or one R″ can be carbon and the other R″ nitrogen. As used herein, the term “enantiomerically pure” refers to a compound composition that comprises at least approximately 95%, and, preferably, approximately 97%, 98%, 99% or 100% of a single enantiomer of that compound. As used herein, the term “substantially free of” or “substantially in the absence of” refers to a compound composition that includes at least 85 to 90% by weight, preferably 95% to 98% by weight, and, even more preferably, 99% to 100% by weight, of the designated enantiomer of that compound. In a preferred embodiment, the compounds described herein are substantially free of enantiomers. Similarly, the term “isolated” refers to a compound composition that includes at least 85 to 90% by weight, preferably 95% to 98% by weight, and, even more preferably, 99% to 100% by weight, of the compound, the remainder comprising other chemical species or enantiomers. The term “alkyl,” as used herein, unless otherwise specified, refers to a saturated straight, branched, or cyclic, primary, secondary, or tertiary hydrocarbons, including both substituted and unsubstituted alkyl groups. The alkyl group can be optionally substituted with any moiety that does not otherwise interfere with the reaction or that provides an improvement in the process, including but not limited to but limited to halo, haloalkyl, hydroxyl, carboxyl, acyl, aryl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acid halide, anhydride, oxime, hydrozine, carbamate, phosphonic acid, phosphonate, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al.,Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991, hereby incorporated by reference. Specifically included are CF3and CH2CF3. In the text, whenever the term C(alkyl range) is used, the term independently includes each member of that class as if specifically and separately set out. The term “alkyl” includes C1-22alkyl moieties, and the term “lower alkyl” includes C1-6alkyl moieties. It is understood to those of ordinary skill in the art that the relevant alkyl radical is named by replacing the suffix “-ane” with the suffix “-yl”. As used herein, a “bridged alkyl” refers to a bicyclo- or tricyclo alkane, for example, a 2:1:1 bicyclohexane. As used herein, a “spiro alkyl” refers to two rings that are attached at a single (quaternary) carbon atom. The term “alkenyl” refers to an unsaturated, hydrocarbon radical, linear or branched, in so much as it contains one or more double bonds. The alkenyl group disclosed herein can be optionally substituted with any moiety that does not adversely affect the reaction process, including but not limited to but not limited to those described for substituents on alkyl moieties. Non-limiting examples of alkenyl groups include ethylene, methylethylene, isopropylidene, 1,2-ethane-diyl, 1,1-ethane-diyl, 1,3-propane-diyl, 1,2-propane-diyl, 1,3-butane-diyl, and 1,4-butane-diyl. The term “alkynyl” refers to an unsaturated, acyclic hydrocarbon radical, linear or branched, in so much as it contains one or more triple bonds. The alkynyl group can be optionally substituted with any moiety that does not adversely affect the reaction process, including but not limited to those described above for alkyl moieties. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl, butyn-2-yl, pentyn-1-yl, pentyn-2-yl, 4-methoxypentyn-2-yl, 3-methylbutyn-1-yl, hexyn-1-yl, hexyn-2-yl, and hexyn-3-yl, 3,3-dimethylbutyn-1-yl radicals. The term “alkylamino” or “arylamino” refers to an amino group that has one or two alkyl or aryl substituents, respectively. The term “protected” as used herein and unless otherwise defined refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction or for other purposes. A wide variety of oxygen and nitrogen protecting groups, including protecting groups for amines, are known to those skilled in the art of organic synthesis, and are described, for example, in Greene et al., Protective Groups in Organic Synthesis, supra. Specific examples include Carbobenzyloxy (Cbz), tosylate (Ts), nosylate, brosylate, mesylate, -tert-butoxycarbonyl (t-boc or boc), p-Methoxybenzyl carbonyl (Moz or MeOZ), 9-Fluorenylmethyloxycarbonyl (FMOC), Acetyl (Ac), Benzoyl (Bz), Benzyl (Bn), Carbamate, p-Methoxybenzyl (PMB), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), Tosyl (Ts), Troc (trichloroethyl chloroformate), and other sulfonamides (such as Nosyl, mesyl, triflyl, and Nps). The term “aryl”, alone or in combination, means a carbocyclic aromatic system containing one, two or three rings wherein such rings can be attached together in a pendent manner or can be fused. Non-limiting examples of aryl include phenyl, biphenyl, or naphthyl, or other aromatic groups that remain after the removal of a hydrogen from an aromatic ring. The term aryl includes both substituted and unsubstituted moieties. The aryl group can be optionally substituted with any moiety that does not adversely affect the process, including but not limited to but not limited to those described above for alkyl moieties. Non-limiting examples of substituted aryl include heteroarylamino, N-aryl-N-alkylamino, N-heteroarylamino-N-alkylamino, heteroaralkoxy, arylamino, aralkylamino, arylthio, monoarylamidosulfonyl, arylsulfonamido, diarylamidosulfonyl, monoaryl amidosulfonyl, arylsulfinyl, arylsulfonyl, heteroarylthio, heteroarylsulfinyl, heteroarylsulfonyl, aroyl, heteroaroyl, aralkanoyl, heteroaralkanoyl, hydroxyaralkyl, hydoxyheteroaralkyl, haloalkoxyalkyl, aryl, aralkyl, aryloxy, aralkoxy, aryloxyalkyl, saturated heterocyclyl, partially saturated heterocyclyl, heteroaryl, heteroaryloxy, heteroaryloxyalkyl, arylalkyl, heteroarylalkyl, arylalkenyl, and heteroarylalkenyl, carboaralkoxy. The terms “alkaryl” or “alkylaryl” refer to an alkyl group with an aryl substituent. The terms “aralkyl” or “arylalkyl” refer to an aryl group with an alkyl substituent. The term “halo,” as used herein, includes chloro, bromo, iodo and fluoro. The term “acyl” refers to a carboxylic acid ester in which the non-carbonyl moiety of the ester group is selected from the group consisting of straight, branched, or cyclic alkyl or lower alkyl, alkoxyalkyl, including, but not limited to methoxymethyl, aralkyl, including, but not limited to, benzyl, aryloxyalkyl, such as phenoxymethyl, aryl, including, but not limited to, phenyl, optionally substituted with halogen (F, Cl, Br, or I), alkyl (including but not limited to C1, C2, C3, and C4) or alkoxy (including but not limited to C1, C2, C3, and C4), sulfonate esters such as alkyl or aralkyl sulphonyl including but not limited to methanesulfonyl, the mono, di or triphosphate ester, trityl or monomethoxytrityl, substituted benzyl, trialkylsilyl (e.g., dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the esters optimally comprise a phenyl group. The term “lower acyl” refers to an acyl group in which the non-carbonyl moiety is lower alkyl. The terms “alkoxy” and “alkoxyalkyl” embrace linear or branched oxy-containing radicals having alkyl moieties, such as methoxy radical. The term “alkoxyalkyl” also embraces alkyl radicals having one or more alkoxy radicals attached to the alkyl radical, that is, to form monoalkoxyalkyl and dialkoxyalkyl radicals. The “alkoxy” radicals can be further substituted with one or more halo atoms, such as fluoro, chloro or bromo, to provide “haloalkoxy” radicals. Examples of such radicals include fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy, trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, and fluoropropoxy. The term “alkylamino” denotes “monoalkylamino” and “dialkylamino” containing one or two alkyl radicals, respectively, attached to an amino radical. The terms arylamino denotes “monoarylamino” and “diarylamino” containing one or two aryl radicals, respectively, attached to an amino radical. The term “aralkylamino”, embraces aralkyl radicals attached to an amino radical. The term aralkylamino denotes “monoaralkylamino” and “diaralkylamino” containing one or two aralkyl radicals, respectively, attached to an amino radical. The term aralkylamino further denotes “monoaralkyl monoalkylamino” containing one aralkyl radical and one alkyl radical attached to an amino radical. The term “heteroatom,” as used herein, refers to oxygen, sulfur, nitrogen and phosphorus. The terms “heteroaryl” or “heteroaromatic,” as used herein, refer to an aromatic that includes at least one sulfur, oxygen, nitrogen or phosphorus in the aromatic ring. The term “heterocyclic,” “heterocyclyl,” and cycloheteroalkyl refer to a nonaromatic cyclic group wherein there is at least one heteroatom, such as oxygen, sulfur, nitrogen, or phosphorus in the ring. Nonlimiting examples of heteroaryl and heterocyclic groups include furyl, furanyl, pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl, benzimidazolyl, purinyl, carbazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl, quinazolinyl, cinnolinyl, phthalazinyl, xanthinyl, hypoxanthinyl, thiophene, furan, pyrrole, isopyrrole, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, oxazole, isoxazole, thiazole, isothiazole, pyrimidine or pyridazine, and pteridinyl, aziridines, thiazole, isothiazole, 1,2,3-oxadiazole, thiazine, pyridine, pyrazine, piperazine, pyrrolidine, oxaziranes, phenazine, phenothiazine, morpholinyl, pyrazolyl, pyridazinyl, pyrazinyl, quinoxalinyl, xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl, 5-azauracilyl, triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, adenine, N6-alkylpurines, N6-benzylpurine, N6-halopurine, N6-vinypurine, N6-acetylenic purine, N6-acyl purine, N6-hydroxyalkyl purine, N6-thioalkyl purine, thymine, cytosine, 6-azapyrimidine, 2-mercaptopyrmidine, uracil, N5-alkylpyrimidines, N5-benzylpyrimidines, N5-halopyrimidines, N5-vinylpyrimidine, N5-acetylenic pyrimidine, N5-acyl pyrimidine, N5-hydroxyalkyl purine, and N6-thioalkyl purine, and isoxazolyl. The heteroaromatic group can be optionally substituted as described above for aryl. The heterocyclic or heteroaromatic group can be optionally substituted with one or more substituents selected from the group consisting of halogen, haloalkyl, alkyl, alkoxy, hydroxy, carboxyl derivatives, amido, amino, alkylamino, and dialkylamino. The heteroaromatic can be partially or totally hydrogenated as desired. As a nonlimiting example, dihydropyridine can be used in place of pyridine. Functional oxygen and nitrogen groups on the heterocyclic or heteroaryl group can be protected as necessary or desired. Suitable protecting groups are well known to those skilled in the art, and include trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl or substituted trityl, alkyl groups, acyl groups such as acetyl and propionyl, methanesulfonyl, and p-toluenelsulfonyl. The heterocyclic or heteroaromatic group can be substituted with any moiety that does not adversely affect the reaction, including but not limited to but not limited to those described above for aryl. The term “host,” as used herein, refers to a unicellular or multicellular organism in which the virus can replicate, including but not limited to cell lines and animals, and, preferably, humans. Alternatively, the host can be carrying a part of the viral genome, whose replication or function can be altered by the compounds of the present invention. The term host specifically refers to infected cells, cells transfected with all or part of the viral genome and animals, in particular, primates (including but not limited to chimpanzees) and humans. In most animal applications of the present invention, the host is a human being. Veterinary applications, in certain indications, however, are clearly contemplated by the present invention (such as for use in treating chimpanzees). The term “peptide” refers to a natural or synthetic compound containing two to one hundred amino acids linked by the carboxyl group of one amino acid to the amino group of another. The term “pharmaceutically acceptable salt or prodrug” is used throughout the specification to describe any pharmaceutically acceptable form (such as an ester) compound which, upon administration to a patient, provides the compound. Pharmaceutically-acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. The term “pharmaceutically acceptable salt or prodrug” is used throughout the specification to describe any pharmaceutically acceptable form (such as an ester) compound which, upon administration to a patient, provides the compound. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art. Pharmaceutically acceptable prodrugs refer to a compound that is metabolized, for example hydrolyzed or oxidized, in the host to form the compound of the present invention. Typical examples of prodrugs include compounds that have biologically labile protecting groups on functional moieties of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce the active compound. The prodrug forms of the compounds of this invention can possess antiviral activity, can be metabolized to form a compound that exhibits such activity, or both. II. Active Compounds NoV is composed of small, icosahedral, non-enveloped viruses, from the Caliciviridae family, that have a linear, positive-sense, single-stranded RNA genome. The virus particles are thought to attach to protein receptors via carbohydrate attachment factors (histo-blood group antigens). After entry and uncoating, translation occurs using cellular translation factors and the viral protease (PR) cleaves the synthesized polyprotein. The replication complex is then formed and the genome is replicated by the RNA-dependent RNA-polymerase (RdRp). The newly synthesized genomes are finally translated or are packaged into new virions to exit the cell. The compounds described herein are active as NoV protease inhibitors. The NoV protease is also known as cysteine protease or thiol protease, containing a catalytic triad consisting of His30, Glu54, and Cys139. In one embodiment, the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:R5is selected from R2, R2′, R10, R10′, R11and R11′ are, independently, hydrogen, CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl,R12and R12′ are, independently, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl,R12and R12′can come together to form an optionally substituted C3-7ring, optionally containing an N, O, or S;R8is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, C2-6alkenyl, aryl, or arylalkyl;m, n, p and r are independently 0, 1, 2, 3, 4 or 5;q is 1, 2, 3, 4 or 5X is independently selected from a bond, O or NH,Y is independently Cl, F, I or Br,R12is hydrogen, CF3, CO2R′, S(O)2R′, S(O)2N(R′)2, P(O)(OR′)2, C2-6alkenyl, C2-6alkynyl, C3-6alkoxyalkyl, C1-6alkyl, arylalkoxycarbonyl, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl;R6and R6′are, independently, hydrogen, halogen, CF3, hydroxy, N(R′)S(O)2R′, S(O)2R′, S(O)2N(R′)2, C1-6alkoxy, C2-6alkenyl, cyano, C2-6alkynyl, C3-6alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, C1-6alkyl, arylalkoxycarbonyl, carboxy, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl, or R6and R6′, together with the carbon to which they are attached, form a carbonyl;Each R′ is, independently, H, C1-6alkyl, C1-6haloalkyl, C1-6alkoxy, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, aryl, heteroaryl, alkylaryl, or arylalkyl,the R′ groups, and other optionally substituted groups, can optionally be substituted with one or more substituents, which substituents are, independently, halo, C1-6haloalkyl, C1-6hydroxyalkyl, hydroxyl, carboxyl, acyl, aryl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, alkoxyalkyl, aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acid halide, anhydride, oxime, hydrozine, carbamate, phosphonic acid, or phosphonate;two R′ residing on the same carbon or nitrogen atom can come together to form a C3-6ring optionally containing an N, O, or S;R6and R6′can come together to form an optionally substituted double bond, a C3-6ring optionally containing an N, O, or S;R7and R7′are, independently, hydrogen, CF3, N(R′)S(O)2R′, S(O)2R′, S(O)2N(R′)2, C1-6alkoxy, C2-6alkenyl, cyano, C2-6alkynyl, C3-6alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, C1-6alkyl, arylalkoxycarbonyl, carboxy, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl;R7and R7′can come together to form an optionally substituted double bond or a C3-6ring optionally containing an N, O, or S;R4and R3are, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, C2-8alkoxyalkyl, arylalkyl, heteroarylalkyl, or —CH2—R4′.R4′is a six-membered ring or a six-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are independently N, O, or S, a seven-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, a five-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S; a four-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, or a three membered ring; andR1is optionally substituted aryl, heteroaryl, aryloxy, heteroaryloxy, arylalkoxy, or heteroarlalkoxy. In another embodiment, the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:R5is ketoamides, bisulfite salts, R9is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,R4and R3are, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, C2-8alkoxyalkyl, arylalkyl, heteroarylalkyl, or —CH2—R4′,R4′is a six-membered ring or a six-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are independently N, O, or S, a seven-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, a five-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S; a four-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, or a three membered ring;R1is optionally substituted aryl, heteroaryl, aryloxy, heteroaryloxy, arylalkoxy, or heteroarlalkoxy,R2, R2′, R10and R10′ are, independently, hydrogen, CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl,X is, independently, a bond, O or NH,m, n, and p are, independently, 0, 1, 2, 3, 4 or 5;when n and m are not 1: R6and R6′are, independently, hydrogen, halogen, CF3, hydroxy, N(R′)S(O)2R′, S(O)2R′, S(O)2N(R′)2, C1-6alkoxy, C2-6alkenyl, cyano, C2-6alkynyl, C3-6alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, C1-6alkyl, arylalkoxycarbonyl, carboxy, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl; or R6and R6′, together with the carbon to which they are attached, form a carbonyl, Each R′ is, independently, H, C1-6alkyl, C1-6haloalkyl, C1-6alkoxy, C2-6alkenyl, C2-6alkynyl, C3-6cycloalkyl, aryl, heteroaryl, alkylaryl, or arylalkyl, the R′ groups can optionally be substituted with one or more substituents, which substituents are, independently, halo, C1-6haloalkyl, C1-6hydroxyalkyl, hydroxyl, carboxyl, acyl, aryl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, alkoxyalkyl, aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acid halide, anhydride, oxime, hydrozine, carbamate, phosphonic acid, or phosphonate;two R′ residing on the same carbon or nitrogen atom can come together to form a C3-6ring optionally containing a N, O, or S heteroatom;R6and R6′can come together to form an optionally substituted double bond or a C3-6ring optionally containing a N, O, or S heteroatom;R7and R7′are, independently, hydrogen, CF3, N(R′)S(O)2R′, S(O)2R′, S(O)2N(R′)2, C1-6alkoxy, C2-6alkenyl, cyano, C2-6alkynyl, C3-6alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, C1-6alkyl, arylalkoxycarbonyl, carboxy, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl; R7and R7can come together to form an optionally substituted double bond or a C3-6ring optionally containing a N, O, or S heteroatom; andwhen n and m are 1, at least one or R2, R6, R6′, R7and R7′is not hydrogen. In yet another embodiment, the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:R5is ketoamides, bisulfite salts, R9is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,R2and R2′ are, independently, hydrogen, CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl,R3is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, C2-8alkoxyalkyl, arylalkyl, heteroarylalkyl, or —CH2—R4′.R4is, independently, optionally substituted C1-6haloalkyl, C2-8alkoxyalkyl, heteroarylalkyl, or —CH2—R4′,R4′is a six-membered ring or a six-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are independently N, O, or S, a seven-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, a five-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S; a four-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, or a three membered ring;R1is optionally substituted aryl, heteroaryl, aryloxy, heteroaryloxy, arylalkoxy, or heteroarlalkoxy,p is 0, 1, 2, 3, 4 or 5; andX is a bond, O or NH. In still another embodiment, the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:R5is ketoamides, bisulfite salts, wherein a ketoamide has the formula —C(O)C(O)NHRx, where Rxis a branched or unbranched alkyl, cycloalkyl, or arylalkyl, and an α-hydroxyphosphonate of the formula —CH(O)(P═O)(ORy)2, where each Ryis H, a substituted or unsubstituted alkyl, aryl, or arylalkyl, and a bisulfite has the formula —H(OH)SO3−, and the salt is any pharmaceutically acceptable salt,R9is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,R4is optionally substituted C1-6alkyl, cycloalkyl, aryl, arylakyl, alkenyl, alkynyl, or a natural amino acid side chain,R4′is a six-membered ring or a six-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are independently N, O, or S, a seven-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, a five-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S; a four-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, or a three membered ring;R1is optionally substituted aryl, heteroaryl, aryloxy, heteroaryloxy, arylalkoxy, or heteroarlalkoxy,p is 0, 1, 2, 3, 4 or 5;X is, independently, a bond, O or NH,R3is, independently, optionally substituted C1-6haloalkyl, C2-8alkoxyalkyl, heteroarylalkyl, or —CH2—R4*, andR2and R2′ are, independently, hydrogen, CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl. In still another embodiment, the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:R5is ketoamides, bisulfite salts, R9is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,R4is optionally substituted C1-6alkyl, cycloalkyl, aryl, arylakyl, alkenyl, alkynyl, or a natural amino acid side chain,R4′is a six-membered ring or a six-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are independently N, O, or S, a seven-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, a five-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S; a four-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, or a three membered ring;R1is optionally substituted aryl, heteroaryl, aryloxy, heteroaryloxy, arylalkoxy, or heteroarlalkoxy,p is 0, 1, 2, 3, 4 or 5;X is, independently, a bond, O or NH,R3is, independently, optionally substituted C1-6haloalkyl, C2-8alkoxyalkyl, heteroarylalkyl, or —CH2—R4′,R2and R2′ are, independently, CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl. In still another embodiment, the compounds have the following formula: or a pharmaceutically acceptable salt or prodrug thereof, wherein:R1is optionally substituted aryl, heteroaryl, aryloxy, heteroaryloxy, arylalkoxy, or heteroarylalkoxy,R2and R2′ are, independently, H, —NH2, —NH-carboxybenzyl (i.e., —NHCBz), CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl, where the —NH2can optionally be protected with an amine protecting group.R3is, independently, optionally substituted C1-6haloalkyl, C2-8alkoxyalkyl, heteroarylalkyl, or —CH2—R4′,R4is optionally substituted C1-6alkyl, cycloalkyl, aryl, arylakyl, alkylaryl, alkenyl, alkynyl, or a natural amino acid side chain,R4′is a six-membered ring or a six-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are independently N, O, or S, a seven-membered bridged or spiro-fused ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, a five-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S; a four-membered ring containing zero, one, or two heteroatoms, which are, independently, N, O, or S, or a three membered ring;R5is an acrylamide (—C(R2)═C(R2)—, C1-6-haloalkyl, C1-6hydroxyalkyl, C1-6-alkyl sulfonate, aryl sulfonate, heteroaryl sulfonate, C1-6-alkyl sulfoxide, or C1-6-ketoalkyl, wherein the alkyl moiety on any of these groups can be substituted with an epoxide (on two adjacent carbons), CN, OH, halo, keto, —CF3,R6and R6′are, independently, hydrogen, halogen, CF3, hydroxy, N(R′)S(O)2R′, S(O)2R′, S(O)2N(R′)2, C1-6alkoxy, C2-6alkenyl, cyano, C2-6alkynyl, C3-6alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, C1-6alkyl, arylalkoxycarbonyl, carboxy, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl, or R6and R6′, together with the carbon to which they are attached, form a carbonyl;R6and R6′can come together to form an optionally substituted double bond, a C3-6ring optionally containing an N, O, or S;R7and R7′are, independently, hydrogen, CF3, N(R′)S(O)2R′, S(O)2R′, S(O)2N(R′)2, C1-6alkoxy, C2-6alkenyl, cyano, C2-6alkynyl, C3-6alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, C1-6alkyl, arylalkoxycarbonyl, carboxy, C1-6haloalkyl, heterocyclylalkyl, or C1-6hydroxyalkyl;R9is, independently, optionally substituted C1-6alkyl, C1-6haloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,R10and R10′ are, independently, hydrogen, CF3, C1-6alkyl, C1-6haloalkyl, or C2-6alkenyl,m, n and p are independently 0, 1, 2, 3, 4 or 5; andX is, independently, a bond, O or NH, and pharmaceutically acceptable salts and prodrugs thereof. A subset of these compounds has the following formula: Individual compounds include the following: and pharmaceutically acceptable salts or prodrugs thereof. Additional compounds include the following: Preferred compounds include the following: and pharmaceutically acceptable salts or prodrugs thereof. III Stereoisomerism and Polymorphism The compounds described herein can have asymmetric centers and occur as racemates, racemic mixtures, individual diastereomers or enantiomers, with all isomeric forms being included in the present invention. Compounds of the present invention having a chiral center can exist in and be isolated in optically active and racemic forms. Some compounds can exhibit polymorphism. The present invention encompasses racemic, optically-active, polymorphic, or stereoisomeric forms, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein. The optically active forms can be prepared by, for example, resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution. One can either purify the respective compound, then derivatize the compound to form the compounds described herein, or purify the compound themselves. Optically active forms of the compounds can be prepared using any method known in the art, including but not limited to by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase. Examples of methods to obtain optically active materials include at least the following.i) physical separation of crystals: a technique whereby macroscopic crystals of the individual enantiomers are manually separated. This technique can be used if crystals of the separate enantiomers exist, i.e., the material is a conglomerate, and the crystals are visually distinct;ii) simultaneous crystallization: a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, possible only if the latter is a conglomerate in the solid state;iii) enzymatic resolutions: a technique whereby partial or complete separation of a racemate by virtue of differing rates of reaction for the enantiomers with an enzyme;iv) enzymatic asymmetric synthesis: a synthetic technique whereby at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer;v) chemical asymmetric synthesis: a synthetic technique whereby the desired enantiomer is synthesized from an achiral precursor under conditions that produce asymmetry (i.e., chirality) in the product, which can be achieved using chiral catalysts or chiral auxiliaries;vi) diastereomer separations: a technique whereby a racemic compound is reacted with an enantiomerically pure reagent (the chiral auxiliary) that converts the individual enantiomers to diastereomers. The resulting diastereomers are then separated by chromatography or crystallization by virtue of their now more distinct structural differences and the chiral auxiliary later removed to obtain the desired enantiomer;vii) first- and second-order asymmetric transformations: a technique whereby diastereomers from the racemate equilibrate to yield a preponderance in solution of the diastereomer from the desired enantiomer or where preferential crystallization of the diastereomer from the desired enantiomer perturbs the equilibrium such that eventually in principle all the material is converted to the crystalline diastereomer from the desired enantiomer. The desired enantiomer is then released from the diastereomer;viii) kinetic resolutions: this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral, non-racemic reagent or catalyst under kinetic conditions;ix) enantiospecific synthesis from non-racemic precursors: a synthetic technique whereby the desired enantiomer is obtained from non-chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis;x) chiral liquid chromatography: a technique whereby the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their differing interactions with a stationary phase (including but not limited to via chiral HPLC). The stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the differing interactions;xi) chiral gas chromatography: a technique whereby the racemate is volatilized and enantiomers are separated by virtue of their differing interactions in the gaseous mobile phase with a column containing a fixed non-racemic chiral adsorbent phase;xii) extraction with chiral solvents: a technique whereby the enantiomers are separated by virtue of preferential dissolution of one enantiomer into a particular chiral solvent;xiii) transport across chiral membranes: a technique whereby a racemate is placed in contact with a thin membrane barrier. The barrier typically separates two miscible fluids, one containing the racemate, and a driving force such as concentration or pressure differential causes preferential transport across the membrane barrier. Separation occurs as a result of the non-racemic chiral nature of the membrane that allows only one enantiomer of the racemate to pass through. Chiral chromatography, including but not limited to simulated moving bed chromatography, is used in one embodiment. A wide variety of chiral stationary phases are commercially available. IV. Salt or Prodrug Formulations In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compound as a pharmaceutically acceptable salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate and α-glycerophosphate. Suitable inorganic salts can also be formed, including but not limited to, sulfate, nitrate, bicarbonate and carbonate salts. For certain transdermal applications, it can be preferred to use fatty acid salts of the compounds described herein. The fatty acid salts can help penetrate the stratum corneum. Examples of suitable salts include salts of the compounds with stearic acid, oleic acid, lineoleic acid, palmitic acid, caprylic acid, and capric acid. Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid, affording a physiologically acceptable anion. In those cases where a compound includes multiple amine groups, the salts can be formed with any number of the amine groups. Alkali metal (e.g., sodium, potassium or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be made. A prodrug is a pharmacological substance that is administered in an inactive (or significantly less active) form and subsequently metabolized in vivo to an active metabolite. Getting more drug to the desired target at a lower dose is often the rationale behind the use of a prodrug and is generally attributed to better absorption, distribution, metabolism, and/or excretion (ADME) properties. Prodrugs are usually designed to improve oral bioavailability, with poor absorption from the gastrointestinal tract usually being the limiting factor. Additionally, the use of a prodrug strategy can increase the selectivity of the drug for its intended target thus reducing the potential for off target effects. V. Isotopes Compounds described herein include isotopically-labeled compounds, which are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. In other embodiments are examples of isotopes that are incorporated into the present compounds including isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as, for example,2H,3H,13C,14C,15N,18O,17O,35S,18F,36Cl, respectively. Certain isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as2H are incorporated, are useful in drug and/or substrate tissue distribution assays. Further, in some embodiments, substitution with isotopes such as deuterium, i.e.,2H, can affords certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. VI. Methods of Treatment The compounds described herein can be used to prevent, treat or cure Norovirus (NoV) infections. Hosts, including but not limited to humans infected with NoV, or a gene fragment thereof, can be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, transdermally, subcutaneously, or topically, in liquid or solid form. VII. Combination of Alternation Therapy In one embodiment, the compounds of the invention can be employed together with at least one other antiviral agent, selected from the group consisting of polymerase inhibitors, anti-NoV nucleosides and their prodrugs, viral entry inhibitor, viral maturation inhibitor, and agents of distinct or unknown mechanism. For example, when used to treat or prevent NoV infection, the active compound or its prodrug or pharmaceutically acceptable salt can be administered in combination or alternation with another anti-NoV agent including, but not limited to, those of the formulae above. In general, in combination therapy, effective dosages of two or more agents are administered together, whereas during alternation therapy, an effective dosage of each agent is administered serially. The dosage will depend on absorption, inactivation and excretion rates of the drug, as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens and schedules should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Nonlimiting examples of antiviral agents that can be used in combination with the compounds disclosed herein include those in the tables below. Norovirus TherapiesFAMILY/DRUG NAMEMECHANISMCOMPANYCC-1845Polymerase inhibitorCocrystal PharmaZofranAnti-emeticNovartisImmodiumAnti-diarrhealJohnson and JohnsonFavipiravirViral polymerase,Toyama Chemicalinduces lethalmutagenesisRibavirinViral polymerase,Kadmoninduces lethalPharmaceuticals, LLCmutagenesis(RibaPak ®)rupintrivirViral proteaseAgouroninhibitor; irreversiblePharmaceuticals, Inc.inhibitor of active siteWP1130Small-moleculeCayman Chemicalinhibitor of cellulardeubiquitinases,Indirect activation ofthe unfolded proteinresponse2′-C-methylcytidineViral polymeraseCocrystal PharmainhibitorIFN-λ specificInduces an antiviralinhibitorsstate in the host cellsSuramin (Germanin)Non-nucleosideBayerpolymerase inhibitorNF203Non-nucleosidepolymerase inhibitorPPNDSNon-nucleosideSanta Cruzpolymerase inhibitorBiotechnologyStructures for PPNDS and WP1130 are provided below: VI. Pharmaceutical Compositions Hosts, including but not limited to humans, infected with NoV can be treated by administering to the patient an effective amount of the active compound or a pharmaceutically acceptable prodrug or salt thereof in the presence of a pharmaceutically acceptable carrier or diluent. The active materials can be administered by any appropriate route, for example, orally, parenterally, intravenously, intradermally, subcutaneously, or topically, in liquid or solid form. A preferred dose of the compound for will be in the range of between about 0.01 and about 10 mg/kg, more generally, between about 0.1 and 5 mg/kg, and, preferably, between about 0.5 and about 2 mg/kg, of body weight of the recipient per day. The effective dosage range of the pharmaceutically acceptable salts and prodrugs can be calculated based on the weight of the parent compound to be delivered. If the salt or prodrug exhibits activity in itself, the effective dosage can be estimated as above using the weight of the salt or prodrug, or by other means known to those skilled in the art. The compound is conveniently administered in unit any suitable dosage form, including but not limited to but not limited to one containing 7 to 600 mg, preferably 70 to 600 mg of active ingredient per unit dosage form. An oral dosage of 1-400 mg is usually convenient. The concentration of active compound in the drug composition will depend on absorption, inactivation and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient can be administered at once, or can be divided into a number of smaller doses to be administered at varying intervals of time. A preferred mode of administration of the active compound is oral, although for certain patients a sterile injectable form can be given sc, ip or iv. Oral compositions will generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, unit dosage forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup can contain, in addition to the active compound(s), sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. The compound or a pharmaceutically acceptable prodrug or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, antifungals, anti-inflammatories or other antiviral compounds. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS). Transdermal Formulations In some embodiments, the compositions are present in the form of transdermal formulations, such as that used in the FDA-approved agonist rotigitine transdermal (Neupro patch). Another suitable formulation is that described in U.S. Publication No. 20080050424, entitled “Transdermal Therapeutic System for Treating Parkinsonism.” This formulation includes a silicone or acrylate-based adhesive, and can include an additive having increased solubility for the active substance, in an amount effective to increase dissolving capacity of the matrix for the active substance. The transdermal formulations can be single-phase matrices that include a backing layer, an active substance-containing self-adhesive matrix, and a protective film to be removed prior to use. More complicated embodiments contain multiple-layer matrices that may also contain non-adhesive layers and control membranes. If a polyacrylate adhesive is used, it can be crosslinked with multivalent metal ions such as zinc, calcium, aluminum, or titanium ions, such as aluminum acetylacetonate and titanium acetylacetonate. When silicone adhesives are used, they are typically polydimethylsiloxanes. However, other organic residues such as, for example, ethyl groups or phenyl groups may in principle be present instead of the methyl groups. Because the active compounds are amines, it may be advantageous to use amine-resistant adhesives. Representative amine-resistant adhesives are described, for example, in EP 0 180 377. Representative acrylate-based polymer adhesives include acrylic acid, acrylamide, hexylacrylate, 2-ethylhexylacrylate, hydroxyethylacrylate, octylacrylate, butylacrylate, methylacrylate, glycidylacrylate, methacrylic acid, methacrylamide, hexylmethacrylate, 2-ethylhexylmethacrylate, octylmethacrylate, methylmethacrylate, glycidylmethacrylate, vinylacetate, vinylpyrrolidone, and combinations thereof. The adhesive must have a suitable dissolving capacity for the active substance, and the active substance most be able to move within the matrix, and be able to cross through the contact surface to the skin. Those of skill in the art can readily formulate a transdermal formulation with appropriate transdermal transport of the active substance. Certain pharmaceutically acceptable salts tend to be more preferred for use in transdermal formulations, because they can help the active substance pass the barrier of the stratum corneum. Examples include fatty acid salts, such as stearic acid and oleic acid salts. Oleate and stearate salts are relatively lipophilic, and can even act as a permeation enhancer in the skin. Permeation enhancers can also be used. Representative permeation enhancers include fatty alcohols, fatty acids, fatty acid esters, fatty acid amides, glycerol or its fatty acid esters, N-methylpyrrolidone, terpenes such as limonene, alpha-pinene, alpha-terpineol, carvone, carveol, limonene oxide, pinene oxide, and 1,8-eucalyptol. The patches can generally be prepared by dissolving or suspending the active agent in ethanol or in another suitable organic solvent, then adding the adhesive solution with stirring. Additional auxiliary substances can be added either to the adhesive solution, the active substance solution or to the active substance-containing adhesive solution. The solution can then be coated onto a suitable sheet, the solvents removed, a backing layer laminated onto the matrix layer, and patches punched out of the total laminate. Nanoparticulate Compositions The compounds described herein can also be administered in the form of nanoparticulate compositions. In one embodiment, the controlled release nanoparticulate formulations comprise a nanoparticulate active agent to be administered and a rate-controlling polymer which functions to prolong the release of the agent following administration. In this embodiment, the compositions can release the active agent, following administration, for a time period ranging from about 2 to about 24 hours or up to 30 days or longer. Representative controlled release formulations including a nanoparticulate form of the active agent are described, for example, in U.S. Pat. No. 8,293,277. Nanoparticulate compositions comprise particles of the active agents described herein, having a non-crosslinked surface stabilizer adsorbed onto, or associated with, their surface. The average particle size of the nanoparticulates is typically less than about 800 nm, more typically less than about 600 nm, still more typically less than about 400 nm, less than about 300 nm, less than about 250 nm, less than about 100 nm, or less than about 50 nm. In one aspect of this embodiment, at least 50% of the particles of active agent have an average particle size of less than about 800, 600, 400, 300, 250, 100, or 50 nm, respectively, when measured by light scattering techniques. A variety of surface stabilizers are typically used with nanoparticulate compositions to prevent the particles from clumping or aggregating. Representative surface stabilizers are selected from the group consisting of gelatin, lecithin, dextran, gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glycerol monostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, polyvinylpyrrolidone, tyloxapol, poloxamers, poloxamines, poloxamine 908, dialkylesters of sodium sulfosuccinic acid, sodium lauryl sulfate, an alkyl aryl polyether sulfonate, a mixture of sucrose stearate and sucrose distearate, p-isononylphenoxypoly-(glycidol), SA9OHCO, decanoyl-N-methylglucamide, n-decyl-D-glucopyranoside, n-decyl-D-maltopyranoside, n-dodecyl-D-glucopyranoside, n-dodecyl-D-maltoside, heptanoyl-N-methylglucamide, n-heptyl-D-glucopyranoside, n-heptyl-D-thioglucoside, n-hexyl-D-glucopyranoside, nonanoyl-N-methylglucamide, n-nonyl-D-glucopyranoside, octanoyl-N-methylglucamide, n-octyl-D-glucopyranoside, and octyl-D-thioglucopyranoside. Lysozymes can also be used as surface stabilizers for nanoparticulate compositions. Certain nanoparticles such as poly(lactic-co-glycolic acid) (PLGA)-nanoparticles are known to target the liver when given by intravenous (IV) or subcutaneously (SQ). In one embodiment, the nanoparticles or other drug delivery vehicles are targeted to the liver. One such type of liver-targeted drug delivery vehicle is described in Park, et al., Mol Imaging. February 2011; 10(1): 69-77, and uses Glypican-3 (GPC3) as a molecular target. Park taught using this target for hepatocellular carcinoma (HCC), a primary liver cancer frequently caused by chronic persistent hepatitis. In one aspect of this embodiment, this drug delivery vehicle is also used to target therapeutics to the liver to treat viral infections. Further, since the compounds described herein have anti-cancer uses, this type of system can target the compounds to the liver and treat liver cancers. GPC3 is a heparan sulfate proteoglycan that is not expressed in normal adult tissues, but significantly over-expressed in up to 80% of human HCC's. GPC3 can be targeted, for example, using antibody-mediated targeting and binding (See Hsu, et al., Cancer Res. 1997; 57:5179-84). Another type of drug delivery system for targeting the liver is described in U.S. Pat. No. 7,304,045. The '045 patent discloses a dual-particle tumor or cancer targeting system that includes a first ligand-mediated targeting nanoparticle conjugated with galactosamine, with the ligand being on a target cell. The first nanoparticle includes poly(γ-glutamic acid)/poly(lactide) block copolymers and n antiviral compound, which in this case is a compound described herein, and in the '045 patent, was gancyclovir. A second nanoparticle includes poly(γ-glutamic acid)/poly(lactide) block copolymers, an endothelial cell-specific promoter, and a (herpes-simplex-virus)-(thymidine kinase) gene constructed plasmid, and provides enhanced permeability and retention-mediated targeting. The first and said second nanoparticles are mixed in a solution configured for delivering to the liver. When the disorder to be treated is a liver tumor or cancer, the delivery can be directly to, or adjacent to, the liver tumor or cancer. Representative rate controlling polymers into which the nanoparticles can be formulated include chitosan, polyethylene oxide (PEO), polyvinyl acetate phthalate, gum arabic, agar, guar gum, cereal gums, dextran, casein, gelatin, pectin, carrageenan, waxes, shellac, hydrogenated vegetable oils, polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcelluose (HPMC), sodium carboxymethylcellulose (CMC), poly(ethylene) oxide, alkyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, hydrophilic cellulose derivatives, polyethylene glycol, polyvinylpyrrolidone, cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose acetate trimellitate, polyvinyl acetate phthalate, hydroxypropylmethyl cellulose phthalate, hydroxypropylmethyl cellulose acetate succinate, polyvinyl acetaldiethylamino acetate, poly(alkylmethacrylate), poly(vinyl acetate), polymers derived from acrylic or methacrylic acid and their respective esters, and copolymers derived from acrylic or methacrylic acid and their respective esters. Methods of making nanoparticulate compositions are described, for example, in U.S. Pat. Nos. 5,518,187 and 5,862,999, both for “Method of Grinding Pharmaceutical Substances;” U.S. Pat. No. 5,718,388, for “Continuous Method of Grinding Pharmaceutical Substances;” and U.S. Pat. No. 5,510,118 for “Process of Preparing Therapeutic Compositions Containing Nanoparticles.” Nanoparticulate compositions are also described, for example, in U.S. Pat. No. 5,298,262 for “Use of Ionic Cloud Point Modifiers to Prevent Particle Aggregation During Sterilization;” U.S. Pat. No. 5,302,401 for “Method to Reduce Particle Size Growth During Lyophilization;” U.S. Pat. No. 5,318,767 for “X-Ray Contrast Compositions Useful in Medical Imaging;” U.S. Pat. No. 5,326,552 for “Novel Formulation For Nanoparticulate X-Ray Blood Pool Contrast Agents Using High Molecular Weight Non-ionic Surfactants;” U.S. Pat. No. 5,328,404 for “Method of X-Ray Imaging Using lodinated Aromatic Propanedioates;” U.S. Pat. No. 5,336,507 for “Use of Charged Phospholipids to Reduce Nanoparticle Aggregation;” U.S. Pat. No. 5,340,564 for Formulations Comprising Olin 10-G to Prevent Particle Aggregation and Increase Stability;” U.S. Pat. No. 5,346,702 for “Use of Non-Ionic Cloud Point Modifiers to Minimize Nanoparticulate Aggregation During Sterilization;” U.S. Pat. No. 5,349,957 for “Preparation and Magnetic Properties of Very Small Magnetic-Dextran Particles;” U.S. Pat. No. 5,352,459 for “Use of Purified Surface Modifiers to Prevent Particle Aggregation During Sterilization;” U.S. Pat. Nos. 5,399,363 and 5,494,683, both for “Surface Modified Anticancer Nanoparticles;” U.S. Pat. No. 5,401,492 for “Water Insoluble Non-Magnetic Manganese Particles as Magnetic Resonance Enhancement Agents;” U.S. Pat. No. 5,429,824 for “Use of Tyloxapol as a Nanoparticulate Stabilizer;” U.S. Pat. No. 5,447,710 for “Method for Making Nanoparticulate X-Ray Blood Pool Contrast Agents Using High Molecular Weight Non-ionic Surfactants;” U.S. Pat. No. 5,451,393 for “X-Ray Contrast Compositions Useful in Medical Imaging;” U.S. Pat. No. 5,466,440 for “Formulations of Oral Gastrointestinal Diagnostic X-Ray Contrast Agents in Combination with Pharmaceutically Acceptable Clays;” U.S. Pat. No. 5,470,583 for “Method of Preparing Nanoparticle Compositions Containing Charged Phospholipids to Reduce Aggregation;” U.S. Pat. No. 5,472,683 for “Nanoparticulate Diagnostic Mixed Carbamic Anhydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;” U.S. Pat. No. 5,500,204 for “Nanoparticulate Diagnostic Dimers as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;” U.S. Pat. No. 5,518,738 for “Nanoparticulate NSAID Formulations;” U.S. Pat. No. 5,521,218 for “Nanoparticulate Tododipamide Derivatives for Use as X-Ray Contrast Agents;” U.S. Pat. No. 5,525,328 for “Nanoparticulate Diagnostic Diatrizoxy Ester X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;” U.S. Pat. No. 5,543,133 for “Process of Preparing X-Ray Contrast Compositions Containing Nanoparticles;” U.S. Pat. No. 5,552,160 for “Surface Modified NSAID Nanoparticles;” U.S. Pat. No. 5,560,931 for “Formulations of Compounds as Nanoparticulate Dispersions in Digestible Oils or Fatty Acids;” U.S. Pat. No. 5,565,188 for “Polyalkylene Block Copolymers as Surface Modifiers for Nanoparticles;” U.S. Pat. No. 5,569,448 for “Sulfated Non-ionic Block Copolymer Surfactant as Stabilizer Coatings for Nanoparticle Compositions;” U.S. Pat. No. 5,571,536 for “Formulations of Compounds as Nanoparticulate Dispersions in Digestible Oils or Fatty Acids;” U.S. Pat. No. 5,573,749 for “Nanoparticulate Diagnostic Mixed Carboxylic Anydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;” U.S. Pat. No. 5,573,750 for “Diagnostic Imaging X-Ray Contrast Agents;” U.S. Pat. No. 5,573,783 for “Redispersible Nanoparticulate Film Matrices With Protective Overcoats;” U.S. Pat. No. 5,580,579 for “Site-specific Adhesion Within the GI Tract Using Nanoparticles Stabilized by High Molecular Weight, Linear Poly(ethylene Oxide) Polymers;” U.S. Pat. No. 5,585,108 for “Formulations of Oral Gastrointestinal Therapeutic Agents in Combination with Pharmaceutically Acceptable Clays;” U.S. Pat. No. 5,587,143 for “Butylene Oxide-Ethylene Oxide Block Copolymers Surfactants as Stabilizer Coatings for Nanoparticulate Compositions;” U.S. Pat. No. 5,591,456 for “Milled Naproxen with Hydroxypropyl Cellulose as Dispersion Stabilizer;” U.S. Pat. No. 5,593,657 for “Novel Barium Salt Formulations Stabilized by Non-ionic and Anionic Stabilizers;” U.S. Pat. No. 5,622,938 for “Sugar Based Surfactant for Nanocrystals;” U.S. Pat. No. 5,628,981 for “Improved Formulations of Oral Gastrointestinal Diagnostic X-Ray Contrast Agents and Oral Gastrointestinal Therapeutic Agents;” U.S. Pat. No. 5,643,552 for “Nanoparticulate Diagnostic Mixed Carbonic Anhydrides as X-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;” U.S. Pat. No. 5,718,388 for “Continuous Method of Grinding Pharmaceutical Substances;” U.S. Pat. No. 5,718,919 for “Nanoparticles Containing the R(−)Enantiomer of Ibuprofen;” U.S. Pat. No. 5,747,001 for “Aerosols Containing Beclomethasone Nanoparticle Dispersions;” U.S. Pat. No. 5,834,025 for “Reduction of Intravenously Administered Nanoparticulate Formulation Induced Adverse Physiological Reactions;” U.S. Pat. No. 6,045,829 “Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors Using Cellulosic Surface Stabilizers;” U.S. Pat. No. 6,068,858 for “Methods of Making Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors Using Cellulosic Surface Stabilizers;” U.S. Pat. No. 6,153,225 for “Injectable Formulations of Nanoparticulate Naproxen;” U.S. Pat. No. 6,165,506 for “New Solid Dose Form of Nanoparticulate Naproxen;” U.S. Pat. No. 6,221,400 for “Methods of Treating Mammals Using Nanocrystalline Formulations of Human Immunodeficiency Virus (HIV) Protease Inhibitors;” U.S. Pat. No. 6,264,922 for “Nebulized Aerosols Containing Nanoparticle Dispersions;” U.S. Pat. No. 6,267,989 for “Methods for Preventing Crystal Growth and Particle Aggregation in Nanoparticle Compositions;” U.S. Pat. No. 6,270,806 for “Use of PEG-Derivatized Lipids as Surface Stabilizers for Nanoparticulate Compositions;” U.S. Pat. No. 6,316,029 for “Rapidly Disintegrating Solid Oral Dosage Form,” U.S. Pat. No. 6,375,986 for “Solid Dose Nanoparticulate Compositions Comprising a Synergistic Combination of a Polymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate;” U.S. Pat. No. 6,428,814 for “Bioadhesive nanoparticulate compositions having cationic surface stabilizers;” U.S. Pat. No. 6,431,478 for “Small Scale Mill;” and U.S. Pat. No. 6,432,381 for “Methods for targeting drug delivery to the upper and/or lower gastrointestinal tract,” all of which are specifically incorporated by reference. In addition, U.S. Patent Application No. 20020012675 A1, published on Jan. 31, 2002, for “Controlled Release Nanoparticulate Compositions,” describes nanoparticulate compositions, and is specifically incorporated by reference. The nanoparticle formulations including the compounds described herein, and also in the form of a prodrug or a salt, can be used to treat or prevent infections by hepatitis B virus. Amorphous small particle compositions are described, for example, in U.S. Pat. No. 4,783,484 for “Particulate Composition and Use Thereof as Antimicrobial Agent;” U.S. Pat. No. 4,826,689 for “Method for Making Uniformly Sized Particles from Water-Insoluble Organic Compounds;” U.S. Pat. No. 4,997,454 for “Method for Making Uniformly-Sized Particles From Insoluble Compounds;” U.S. Pat. No. 5,741,522 for “Ultrasmall, Non-aggregated Porous Particles of Uniform Size for Entrapping Gas Bubbles Within and Methods;” and U.S. Pat. No. 5,776,496, for “Ultrasmall Porous Particles for Enhancing Ultrasound Back Scatter.” Controlled Release Formulations In a preferred embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including but not limited to implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. For example, enterically coated compounds can be used to protect cleavage by stomach acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Suitable materials can also be obtained commercially. Liposomal suspensions (including but not limited to liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 (incorporated by reference). For example, liposome formulations can be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the active compound is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension. The terms used in describing the invention are commonly used and known to those skilled in the art. As used herein, the following abbreviations have the indicated meanings:Boc2O Di-tert-butyl dicarbonateCbzCl Benzyl chloroformateCDI N,N′-CarbonyldiimidazoleDCE dichloroethaneDCM DichloromethaneDIPEA diisopropyl ethyl amine (Hünig's base)DMSO dimethylsulfoxideEDC 1-ethyl-3-(3-dimethyl)aminopropyl)carbodiimide hydrochlorideEt3N TriethylamineEtOAc ethyl acetateEtOH ethanolh hourHOBt HydroxybenzotriazoleKOAC Potassium acetateLiHMDS Lithium bis(trimethylsilyl)amideM molarmCPBA meta-Chloroperoxybenzoic acidMeOH MethanolMePPh3Br Methyltriphenylphosphonium bromideMsCl Methanesulfonyl chloridemin minutePy·SO3Sulfur trioxide pyridine complexrt or RT room temperatureTFA trifluoroacetic acidTHF tetrahydrofuranTLC Thin layer chromatographyTMSCF3trimethyl(trifluoromethyl)silane IX. General Methods for Preparing Active Compounds Methods for the facile preparation of active compounds are known in the art and result from the selective combination of known methods. The compounds disclosed herein can be prepared as described in detail below, or by other methods known to those skilled in the art. It will be understood by one of ordinary skill in the art that variations of detail can be made without departing from the spirit and in no way limiting the scope of the present invention. The various reaction schemes are summarized below.Scheme 1 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, a synthetic approach to compound A.Scheme 2 is a non-limiting example of the synthesis of intermediates of the present invention, and in particular, a synthetic approach to compound XVI, XVIII, XIX and XXI.Scheme 3 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, a synthetic approach to compound B-D.Scheme 4 is a non-limiting example of the synthesis of active compounds of the present invention, and in particular, a synthetic approach to compound E. Compounds of formula A can be prepared by first reaction of an amino acid derivative of general formula I with an alcohol. Intermediate II can be then N-protected for example, by treatment with Boc2O in the presence of a base such as Et3N and then reacted with a compound of general formula IV in presence of a base such as LiHMDS. Cyano derivative of general formula V can be then reduced and finally cyclized to give VII. Intermediate VII can be deprotected for example, in the presence of TFA when Boc was used as a protecting group, and reacted with an amino acid of general formula VIII in the presence of peptide coupling reagents like EDC and HOBt. After deprotection for example, in the presence of TFA when Boc was used as a protecting group, compound of general formula IX can be reacted in presence peptide coupling reagents like EDC and HOBt with compound XII, prepared by reaction of amino acid of general formula X and halogenated reagent XI in the presence of a base such as NaHCO3. Esters of general formula XIII can then be reduced with, for instance, LiAlH4to give compounds of general formula A. Intermediates of formula XVI, XVIII, XIX and XXI can be prepared by first reduction of compound of general formula VI, with a reducing agent such as for instance LiBH4followed by oxidation to form aldehyde of general formula XIV and then reaction with either compounds XV, XVII, XX or MePPh3Br. Compounds of formula B-D can be prepared by first deprotection of compound of general formula XVI, XVIII or XXI, for example, in the presence of TFA when Boc was used as a protecting group, and reaction with a carboxylic acid of general formula XII in the presence of peptide coupling reagents like EDC and HOBt. Compounds of formula E can be prepared by first deprotection of compound of general formula XIX, for example, in the presence of TFA when Boc was used as a protecting group; reaction with a carboxylic acid of general formula XII in the presence of peptide coupling reagents like EDC and HOBt and epoxidation using for instance mCPBA. SPECIFIC EXAMPLES Specific compounds which are representative of this invention were prepared as per the following examples and reaction sequences; the examples and the diagrams depicting the reaction sequences are offered by way of illustration, to aid in the understanding of the invention and should not be construed to limit in any way the invention set forth in the claims which follow thereafter. The present compounds can also be used as intermediates in subsequent examples to produce additional compounds of the present invention. No attempt has necessarily been made to optimize the yields obtained in any of the reactions. One skilled in the art would know how to increase such yields through routine variations in reaction times, temperatures, solvents and/or reagents. Anhydrous solvents were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI) and EMD Chemicals Inc. (Gibbstown, NJ). Reagents were purchased from commercial sources. Unless noted otherwise, the materials used in the examples were obtained from readily available commercial suppliers or synthesized by standard methods known to one skilled in the art of chemical synthesis.1H and13C NMR spectra were taken on a Bruker Ascend™ 400 MHz Fourier transform spectrometer at room temperature and reported in ppm downfield from internal tetranmethylsilane. Deuterium exchange, decoupling experiments or 2D-COSY were performed to confirm proton assignments. Signal multiplicities are represented by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quadruplet), br (broad), bs (broad singlet), m (multiplet). All J-values are in Hz. Mass spectra were determined on a Micromass Platform LC spectrometer using electrospray techniques. Analytic TLC were performed on Sigma-Aldrich® aluminum supported silica gel (25 μm) plates. Column chromatography was carried out on Silica Gel or via reverse-phase high performance liquid chromatography. Example 1 Dimethyl (2S,4S)-2-((tert-butoxycarbonyl)amino)-4-(2-cyanoethyl)pentanedioate (2) To a solution of N-Boc-L-glutamic acid dimethyl ester (1, 16.5 g, 60.0 mmol) in THF (180 mL) was added dropwise a solution of lithium bis(trimethylsilyl)amide in THF (130 mL, 1 M, 130 mmol) at −78° C. under an argon atmosphere. The resulting mixture was stirred at −78° C. for 1.5 h. At the same time, 3-bromopropionitrile (9.63 g, 71.9 mmol) was added dropwise to the dianion solution over a period of 1 h while maintaining the temperature below −70° C. The reaction mixture was stirred at −78° C. for an additional 3 h. The reaction was quenched with aqueous NH4Cl (80 mL). The reaction mixture was allowed to warm up to room temperature and then EtOAc (140 mL) was added. The layers were separated, and the aqueous layer was extracted with EtOAc (100 mL×2). The combined organic layers were washed with brine, dried over Na2SO4, and filtered. The filtrate was evaporated to dryness. The crude residue was purified by flash column chromatography (hexanes/ethyl acetate=4/1) to give product 2 (5.25 g, 27%) as a colorless oil.1H NMR (CDCl3, 400 MHz) δ: 5.08 (1H, d, J=8.0 Hz), 4.38 (1H, m), 3.74 (3H, s), 3.71 (3H, s), 2.62-2.65 (1H, m), 2.35-2.42 (2H, m), 1.97-2.04 (4H, m), 1.44 (9H, s).13C-NMR (CDCl3, 100 MHz) δ: 15.16, 27.31, 28.26, 34.47, 40.77, 51.55, 52.20, 52.60, 80.37, 118.70, 115.38, 172.36, 174.42. ESI-MS (m/z): 329.4 (M+H)+. Methyl (S)-2-((tert-butoxycarbonyl)amino)-3-((S)-2-oxopiperidin-3-yl)propanoate (4) In a hydrogenation flask was placed compound 2 (2.15 g, 6.55 mmol), 5 mL of chloroform and 60 mL of methanol before addition of PtO2(160 mg, 0.65 mmol). The resulting mixture was mechanically stirred at room temperature for 2 days under hydrogen pressure (50 Psi). The mixture was then filtered over a pad of silica gel. KOAc (1.27 g, 13 mmol) was added to the filtrate and the resulting mixture was stirred at 60° C. for 12 h. After removal of the solvents, the crude residue was purified by silica gel column chromatography (DCM/MeOH=50:1 to 20:1) to give the product 4 as a colorless oil (1.21 g, 62%, over two steps).1H NMR (CDCl3, 400 MHz) δ: 6.41 (1H, s), 5.64 (1H, d, J=8.0 Hz), 4.30-4.36 (1H, m), 3.31-3.33 (1H, m), 2.38-2.42 (1H, m), 2.25-2.34 (1H, m), 2.13-2.16 (1H, m), 1.81-1.93 (3H, m), 1.71-1.79 (1H, m), 1.52-1.61 (1H, m), 1.47 (9H, s).13C-NMR (CDCl3, 100 MHz) δ: 21.54, 26.53, 28.29, 34.28, 37.97, 42.35, 51.70, 52.30, 79.81, 155.92, 173.18, 174.58. ESI-MS (m/z): 301.4 (M+H)+. Methyl (5S, 8S, 11S)-5-(4-hydroxybenzyl)-8-isobutyl-3, 6, 9-trioxo-11-(((S)-2-oxopiperidin-3-yl)methyl)-1-phenyl-2-oxa-4, 7, 10-triazadodecan-12-oate (9) To a solution of 4 (300 mg, 1.0 mmol) in dioxane was added a solution of 4 M HCl in dioxane. The reaction was stirred for 2 h at room temperature and then concentrated. The crude HCl salt was suspended in DCM (10 mL) and (tert-butoxycarbonyl)-L-leucine (254 mg, 1.1 mmol), 1-hydroxybenzotriazole (169 mg, 1.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (240 mg, 1.25 mmol) and N,N-diisopropylethyl amine (0.7 mL, 4.0 mmol) were added at 0° C. The ice bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with EtOAc (100 mL) and washed with 1N HCl, NaHCO3(5%) and brine, dried over Na2SO4and concentrated in vacuo. The residue was purified by flash chromatography (DCM/MeOH 30:1 to 10:1) to give compound 6 (262 mg, 63%). Compound 6 (200 mg, 0.48 mmol) was dissolved in a 1:2 TFA-DCM solution (10 mL) and stirred 2 h at room temperature and then concentrated under vacuum. The crude HCl salt was suspended in DCM (10 mL) and ((benzyloxy)carbonyl)-L-tyrosine 8 (166 mg, 0.53 mmol), 1-hydroxybenzotriazole (82 mg, 0.61 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (161 mg, 0.61 mmol) and N,N-diisopropylethyl amine (0.33 mL, 1.92 mmol) at were added at 0° C. The ice bath was removed and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc (100 mL) and washed with 1N HCl, NaHCO3(5%) and brine. The organic layer was dried over Na2SO4and concentrated in vacuo. The residue was purified by flash chromatography (DCM/MeOH 30:1 to 10:1) to give compound 9 (170 mg, 58%).1H NMR (400 MHz, Methanol-d4) δ 7.39-7.20 (m, 5H), 7.12-7.03 (m, 2H), 6.80-6.65 (m, 2H), 5.13-4.94 (m, 2H), 4.64 (s, 1H), 4.54 (dd, J=11.5, 4.2 Hz, 1H), 4.40 (ddd, J=17.8, 9.2, 5.5 Hz, 2H), 3.72 (s, 3H), 3.31-3.20 (m, 2H), 3.05 (dd, J=14.0, 4.9 Hz, 1H), 2.76 (dd, J=14.0, 9.4 Hz, 1H), 2.41 (dt, J=9.9, 5.0 Hz, 1H), 2.30 (ddd, J=15.5, 11.6, 4.1 Hz, 1H), 2.04-1.88 (m, 2H), 1.88-1.80 (m, 1H), 1.79-1.58 (m, 4H), 1.52 (dtt, J=13.3, 10.3, 4.4 Hz, 2H), 0.95 (dd, J=13.9, 6.1 Hz, 6H).13C NMR (101 MHz, MeOD) δ 175.04, 173.48, 172.75, 172.45, 156.89, 155.80, 136.77, 130.01, 128.05, 127.22, 114.83, 66.16, 56.45, 51.83, 51.43, 49.83, 41.56, 40.48, 37.32, 36.82, 32.76, 25.52, 24.31, 21.99, 20.85, 20.81. ESI-MS (m/z): 611.4 (M+H)+. Benzyl ((S)-1-(((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)carbamate (10) To a solution of 9 (120 mg, 0.2 mmol) in THF (3 mL) was added LiBH4(4M in THF, 0.3 mL, 1.2 mmol) dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 h and then quenched with 1N HCl (15 mL). After being stirred for 1 h at room temperature, the suspension was extracted with ethyl acetate, and washed with NaHCO3and brine. The organic layers was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (DCM/MeOH 30:1 to 10:1) to give compound, to afford 10 (83 mg, 73%).1H NMR (400 MHz, Methanol-d4) δ 7.44-7.23 (m, 5H), 7.08 (d, J=8.5 Hz, 2H), 6.71 (d, J=8.5 Hz, 2H), 5.18-4.97 (m, 3H), 4.62 (s, 1H), 4.43-4.28 (m, 2H), 4.09-3.91 (m, 1H), 3.51 (qd, J=11.0, 5.7 Hz, 2H), 3.33 (t, J=1.7 Hz, 1H), 3.06 (dd, J=14.1, 5.0 Hz, 1H), 2.80 (dd, J=14.1, 9.1 Hz, 1H), 2.30 (d, J=9.6 Hz, 1H), 2.16-1.94 (m, 2H), 1.79 (d, J=9.4 Hz, 1H), 1.75-1.56 (m, 5H), 1.56-1.46 (m, 1H), 1.01-0.88 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.01, 173.42, 172.89, 157.15, 155.87, 136.70, 129.99, 128.05, 127.62, 127.55, 127.34, 114.88, 66.33, 64.15, 56.75, 52.22, 41.63, 40.51, 37.29, 36.60, 32.74, 25.66, 24.38, 22.11, 20.64, 20.56. EST-MS (m/z): 583.5 (M+H)+. Benzyl ((S)-3-(4-hydroxyphenyl)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-v)amino)-1-oxopropan-2-yl)carbamate (11) To a solution of 10 (50 mg, 0.086 mmol) in dichloromethane-DMSO (4:1, 1 mL) were added sulfur trioxide pyridine complex (55 mg, 0.34 mmol) and N,N-diisopropylethyl amine (0.06 mL, 0.34 mmol). The resulting mixture was stirred at room temperature for 12 h and then quenched with 1N HCl (5 mL). The suspension was extracted with ethyl acetate washed with a saturated solution of NaHCO3and brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by preparative TLC (DCM/MeOH=12/1) to give product 11 as a white solid (28 mg, 56%).1H NMR (400 MHz, MeOD) δ 7.39-7.23 (m, 5H), 7.08 (d, J=8.4 Hz, 2H), 6.71 (d, J=8.4 Hz, 2H), 5.01 (dd, J=25.6, 12.8 Hz, 1H), 4.65 (s, 1H), 4.53-4.45 (m, 1H), 4.37 (dd, J=9.5, 4.3 Hz, 2H), 4.01 (d, J=2.8 Hz, 1H), 3.36 (s, 1H), 3.23 (d, J=4.1 Hz, 2H), 3.06 (dd, J=14.1, 4.3 Hz, 1H), 2.77 (dd, J=13.4, 10.4 Hz, 1H), 2.27 (d, J=6.4 Hz, 1H), 2.15 (t, J=13.1 Hz, 1H), 2.01 (dd, J=6.9, 3.5 Hz, 1H), 1.84-1.43 (m, 6H), 1.01-0.87 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.10, 173.55, 173.46, 172.89, 172.80, 157.02, 155.82, 136.75, 129.98, 128.05, 127.74, 127.51, 127.25, 114.84, 98.40, 98.32, 66.21, 56.57, 54.07, 53.77, 52.22, 50.72, 50.62, 41.62, 40.66, 40.59, 37.05, 37.02, 36.77, 30.50, 29.93, 25.43, 24.38, 24.32, 21.97, 20.85, 20.52. ESI-MS (m/z): 581.4 (M+H)+. Example 2 Methyl (5R, 8S, 11S)-8-isobutyl-5-(naphthalen-1-ylmethyl)-3, 6, 9-trioxo-11-(((S)-2-oxopiperidin-3-yl)methyl)-1-phenyl-2-oxa-4, 7, 10-triazadodecan-12-oate (13) Compound 13 was prepared from (R)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanoic acid using a similar procedure as that used in the synthesis of compound 9. White solid 188 mg (83% yield).1H NMR (400 MHz, MeOD) δ 8.17 (d, J=8.3 Hz, 1H), 7.89 (d, J=8.1 Hz, 1H), 7.79 (d, J=7.4 Hz, 1H), 7.56 (t, J=7.1 Hz, 1H), 7.50 (t, J=7.4 Hz, 1H), 7.44-7.24 (m, 7H), 5.08 (d, J=3.0 Hz, 2H), 4.63 (s, 2H), 4.52 (dd, J=15.4, 6.8 Hz, 3H), 4.21 (dd, J=10.9, 3.8 Hz, 2H), 3.66 (s, 3H), 3.50 (t, J=7.3 Hz, 2H), 3.25-3.16 (m, 2H), 2.50-2.28 (m, 3H), 1.99-1.85 (m, 3H), 1.80 (d, J=13.8 Hz, 2H), 1.69 (d, J=13.5 Hz, 2H), 1.54-1.37 (m, 3H), 1.15 (t, J=11.4 Hz, 1H), 0.99 (dd, J=16.3, 5.6 Hz, 1H), 0.63-0.67 (m, 6H).13C NMR (101 MHz, MeOD) δ 175.11, 173.65, 172.72, 172.29, 156.89, 136.72, 134.10, 132.56, 131.94, 128.48, 128.09, 127.63, 127.58, 127.37, 125.99, 125.37, 125.15, 123.33, 66.42, 56.53, 51.56, 51.36, 49.68, 41.51, 39.70, 37.24, 34.36, 32.53, 25.23, 23.64, 22.03, 20.66, 20.20. ESI-MS (m/z): 645.4 (M+H)+. Benzyl ((R)-1-(((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl))propan-2-yl))amino)-4-methyl-1-oxopentan-2-yl))amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl))carbamate (14) Prepared from 13 using a similar procedure as that used in the synthesis of compound 10. White solid 83 mg (75% yield).1H NMR (400 MHz, MeOD) δ 8.13 (d, J=8.4 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.79 (d, J=7.9 Hz, 1H), 7.65 (d, J=9.2 Hz, 1H), 7.52 (dt, J=14.7, 6.9 Hz, 2H), 7.43-7.26 (m, 6H), 5.12 (dd, J=29.6, 12.4 Hz, 2H), 5.07 (s, 1H), 4.48 (dd, J=9.6, 6.0 Hz, 1H), 4.13 (dd, J=11.4, 3.3 Hz, 1H), 4.02-3.93 (m, 1H), 3.71 (dt, J=12.2, 6.1 Hz, 1H), 3.57-3.37 (m, 5H), 3.19 (t, J=5.8 Hz, 2H), 2.26-2.02 (m, 2H), 1.93 (dd, J=6.7, 3.8 Hz, 1H), 1.77 (dd, J=12.7, 4.6 Hz, 1H), 1.63 (t, J=11.3 Hz, 3H), 1.52-1.36 (m, 3H), 1.13 (d, J=6.1 Hz, 7H), 0.62-0.57 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.05, 173.35, 173.26, 156.99, 136.61, 134.11, 132.37, 131.90, 128.53, 128.15, 127.71, 127.67, 127.59, 127.46, 126.08, 125.45, 125.22, 123.32, 68.72, 66.53, 64.10, 56.79, 51.82, 41.58, 39.88, 37.18, 34.07, 32.70, 25.30, 23.46, 22.19, 21.74, 20.26, 20.04. ESI-MS (m/z): 617.4 (M+H)+. Benzyl ((R)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (15) Prepared from compound 14 using a similar procedure as that used in the synthesis of compound 11. White solid 13 mg (41% yield).1H NMR (400 MHz, MeOD) δ 8.22 (d, J=8.1 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.77 (d, J=7.4 Hz, 1H), 7.51 (tt, J=18.1, 9.0 Hz, 2H), 7.37 (dd, J=14.9, 7.7 Hz, 2H), 7.29 (s, 2H), 7.22 (d, J=7.2 Hz, 2H), 5.05-4.92 (m, 2H), 4.59 (dt, J=21.6, 10.7 Hz, 1H), 4.48 (t, J=3.9 Hz, 1H), 4.43 (dd, J=9.9, 4.6 Hz, 1H), 4.10 (dt, J=19.3, 9.6 Hz, 1H), 4.02 (ddd, J=11.3, 7.7, 3.5 Hz, 1H), 3.73 (dd, J=14.5, 4.4 Hz, 1H), 3.34 (dd, J=18.8, 7.1 Hz, 4H), 3.26-3.11 (m, 3H), 2.28 (s, 1H), 2.17 (t, J=12.4 Hz, 1H), 2.08-1.96 (m, 1H), 1.85-1.44 (m, 8H), 1.47 (d, J=9.7 Hz, 1H), 1.25 (t, J=7.1 Hz, 1H), 1.03-0.85 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.08, 173.45, 173.43, 172.71, 172.61, 156.86, 136.69, 134.03, 133.09, 131.99, 128.45, 128.01, 127.53, 127.49, 127.27, 125.86, 125.26, 125.00, 123.28, 98.40, 98.32, 98.25, 66.26, 66.17, 55.83, 55.61, 52.36, 52.28, 52.22, 50.72, 50.54, 41.61, 40.64, 40.57, 40.29, 38.11, 37.08, 37.04, 34.53, 34.36, 30.44, 29.89, 26.44, 25.49, 25.46, 24.44, 24.38, 22.14, 21.92, 21.13, 20.90, 20.56. ESI-MS (m/z): 615.4 (M+H)+. Example 3 Methyl (5S, 8S, 11S)-8-isobutyl-5-(naphthalen-1-ylmethyl)-3, 6, 9-trioxo-11-(((S)-2-oxopiperidin-3-yl)methyl)-1-phenyl-2-oxa-4, 7, 10-triazadodecan-12-oate (17) Compound 17 was prepared from (S)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanoic acid 16 using a similar procedure as that used in the synthesis of compound 9. White solid 120 mg (80% yield).1H NMR (400 MHz, MeOD) δ 8.21 (d, J=8.1 Hz, 1H), 7.88 (d, J=7.9 Hz, 1H), 7.77 (d, J=7.1 Hz, 1H), 7.52 (dt, J=20.4, 7.1 Hz, 2H), 7.43-7.34 (m, 2H), 7.30 (d, J=7.1 Hz, 2H), 7.22 (d, J=7.2 Hz, 2H), 5.01 (2H, overlapped with water peak), 4.71-4.59 (m, 2H), 4.54 (dt, J=26.7, 11.3 Hz, 1H), 4.51-4.39 (m, 1H), 3.79-3.67 (m, 4H), 3.32 (t, J=5.8 Hz, 2H), 3.29-3.17 (m, 2H), 2.47-2.38 (m, 1H), 2.35-2.31 (m, 1H), 2.32 (dd, J=18.3, 7.5 Hz, 1H), 2.06-1.96 (m, 1H), 1.95-1.86 (m, 1H), 1.77 (d, 0.1=16.4 Hz, 1H), 1.75-1.65 (m, 2H), 1.66-1.53 (m, 2H), 1.03-0.85 (m, 6H).13C NMR (101 MHz, MeOD) δ 175.04, 173.57, 173.48, 172.62, 172.48, 156.80, 136.69, 134.02, 133.05, 132.01, 128.45, 128.03, 127.51, 127.25, 125.86, 125.27, 125.01, 123.29, 66.16, 55.53, 51.98, 51.42, 49.92, 49.82, 41.55, 40.43, 37.33, 34.51, 32.78, 25.54, 24.36, 21.95, 20.87, 20.83. ESI-MS (m/z): 645.5 (M+H)+. Benzyl ((S)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl))-1-oxopropan-2-yl)carbamate (19) Compound 19 was synthesized from compound 17 using a similar procedure as that used in the synthesis of compound 11. White solid 11 mg (41% yield).1H NMR (400 MHz, MeOD) δ 8.22 (d, J=8.1 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.77 (d, 0.1=7.4 Hz, 1H), 7.51 (tt, J=18.1, 9.0 Hz, 2H), 7.37 (dd, J=15.0, 7.8 Hz, 2H), 7.27 (d, J=15.0 Hz, 2H), 7.23 (t, J=7.0 Hz, 2H), 4.97 (d, J=9.8 Hz, 2H), 4.60 (dd, J=9.5, 4.4 Hz, 1H), 4.47 (dd, J=10.1, 6.1 Hz, 1H), 4.44-4.35 (m, 1H), 4.02 (ddd, J=11.3, 7.7, 3.5 Hz, 1H), 3.73 (dd, J=14.5, 4.4 Hz, 1H), 3.40-3.32 (m, 3H), 3.26-3.12 (m, 3H), 2.46-2.35 (m, 1H), 2.28 (s, 1H), 2.17 (t, J=12.4 Hz, 1H), 1.84-1.55 (m, 7H), 1.47 (d, J=9.7 Hz, 1H), 1.02-0.83 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.08, 173.45, 173.43, 172.71, 172.61, 156.86, 136.69, 134.03, 133.09, 131.99, 128.45, 128.01, 127.53, 127.49, 127.27, 125.86, 125.26, 125.00, 123.28, 98.32, 98.25, 66.26, 66.17, 55.61, 52.36, 52.28, 52.22, 50.72, 50.54, 41.61, 40.64, 40.57, 40.29, 38.11, 37.08, 37.04, 34.53, 34.36, 30.44, 29.89, 26.44, 25.49, 25.46, 24.44, 24.38, 22.14, 21.92, 21.13, 20.90, 20.56. ESI-MS (m/z): 615.5 (M+H)+. N—((S)-1-(((S)-4-methyl-1l-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl))-1-oxopropan-2-yl)pyrazine-2-carboxamide Compound 23 was synthesized from compound 6 using a similar procedure as that used in the synthesis of compound 11.1H NMR (400 MHz, Methanol-d4) δ 9.09 (dd, J=16.5, 1.4 Hz, 1H), 8.75 (dd, J=4.9, 2.4 Hz, 1H), 8.63 (td, J=2.5, 1.4 Hz, 1H), 8.30 (d, J=8.5 Hz, 1H), 7.85 (dd, J=8.3, 5.0 Hz, 1H), 7.76 (t, J=7.3 Hz, 1H), 7.60-7.43 (m, 3H), 7.37 (dt, J=9.7, 7.5 Hz, 1H), 5.14-5.00 (m, 1H), 4.59-4.37 (m, 2H), 4.25-4.12 (m, 1H), 4.08-3.99 (m, 1H), 3.89 (ddd, J=19.4, 9.9, 3.8 Hz, 1H), 3.62-3.46 (m, 1H), 3.25 (td, J=9.3, 8.4, 3.7 Hz, 2H), 2.41 (dt, J=10.3, 5.7 Hz, 1H1), 2.31 (d, J=8.8 Hz, 1H1), 2.18 (ddd, J=14.7, 8.8, 3.3 Hz, 1H), 2.04 (tt, J=9.8, 5.0 Hz, 1H), 1.93-1.58 (m, 4H), 1.07-0.82 (m, 6H). ESI-MS (m/z): 587.5 (M+H)+. N—((S)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl))propan-2-yl)amino)pentan-2-yl))amino)-3-(naphthalen-1-yl))-1-oxopropan-2-yl))pyrazine-2-carboxamide (29) Compound 29 was synthesized from compound 24 using a similar procedure as that used in the synthesis of compound 11.1H NMR (400 MHz, Methanol-d4) δ 9.09 (dd, J=20.6, 1.4 Hz, 1H), 8.75 (dd, J=6.2, 2.5 Hz, 1H), 8.62 (tt, J=2.6, 1.3 Hz, 1H), 8.29 (d, J=8.5 Hz, 1H), 7.85 (t, J=7.8 Hz, 1H), 7.75 (t, J=9.2 Hz, 1H), 7.51 (dq, J=27.0, 7.3 Hz, 3H), 7.37 (dt, J=15.0, 7.6 Hz, 1H), 5.16-4.97 (m, 1H), 4.58-4.36 (m, 2H), 4.09 (dq, J=8.5, 4.3 Hz, 1H), 4.03-3.93 (m, 1H), 3.87 (td, J=15.6, 14.5, 5.2 Hz, 1H), 3.53 (ddd, J=19.6, 14.2, 9.0 Hz, 1H), 2.60-2.43 (m, 1H), 2.41-2.29 (m, 1H), 2.22 (dt, J=14.0, 4.7 Hz, 1H), 2.11-1.98 (m, 1H), 1.89 (q, J=10.7, 9.9 Hz, 1H), 1.83-1.54 (m, 4H), 0.97-0.91 (m, 6H). ESI-MS (m/z): 573.5 (M+H)+. (S)-2-(((Benzyloxy)carbonyl)amino)-3-(4-fluorophenyl)propanoic acid (31) To a solution of p-fluoro-L-phenylalanine (2.56 g, 13.98 mmol), NaHCO3(1.76 g, 21 mmol), K2CO3(2.90 g, 21 mmol) in THF—H2O (v/v=1:1, 50 mL) was added CbzCl (2.2 mL, 15.4 mmol). The reaction mixture was stirred overnight at room temperature. After evaporation of the volatils, the reaction mixture was washed with ethyl acetate (10 mL) and then the pH of the water phase was adjusted to pH=1 by addition of 1N HCl. The water layer was finally extracted with ethyl acetate (30 mL×4) and the combined organic layers dried over Na2SO4, to give, after evaporation, compound 31 (4.2 g, 95%).1H NMR (400 MHz, Methanol-d4) δ 7.37-7.12 (m, 7H), 6.96 (t, J=8.8 Hz, 2H), 5.11-4.93 (m, 2H), 4.47 (dd, J=9.3, 5.0 Hz, 1H), 3.19 (dd, J=14.0, 5.0 Hz, 1H), 2.92 (dd, J=14.0, 9.3 Hz, 1H).13C NMR (101 MHz, MeOD) δ 173.61, 163.03, 160.62, 156.94, 136.74, 133.08, 133.05, 130.72, 130.64, 128.08, 127.61, 127.34, 114.78, 114.57, 66.20, 55.37, 36.45.19F NMR (377 MHz, Methanol-d4) δ −119.46. LC-MS: m/z [M+H]+calcd. for C17H17FNO4: 318.1, found: 318.2. Methyl (5S,8S,11S)-5-(4-fluorobenzyl)-8-isobutyl-3,6,9-trioxo-11-(((S)-2-oxopiperidin-3-yl)methyl)-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (33) To a solution of compound 32 (230 mg, 0.66 mmol) and amino acid 31 (250 mg, 0.79 mmol) in DCM (6.0 mL) was added 1-hydroxybenzotriazole (135 mg, 1.0 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (190 mg, 1.0 mmol) and N,N-diisopropylethyl amine (0.7 mL, 4.0 mmol) at 0° C. After being stirred at room temperature overnight, the reaction mixture was diluted with EtOAc (50 mL) and washed with 1N HCl, NaHCO3(5%) and a saturated solution of NaCl. The organic layer was dried over Na2SO4. The solvent was concentrated in vacua and was purified by flash chromatography (DCM/MeOH 20:1) to afford compound 33 (280 mg, 69%).1H NMR (400 MHz, Methanol-d4) δ 8.64 (d, J=8.0 Hz, 1H), 8.19 (d, J=7.5 Hz, 1H), 7.28 (m, 4H), 7.08-6.90 (m, 3H), 5.16-4.94 (m, 2H), 4.42 (ddd, J=8.3, 5.7, 3.1 Hz, 2H), 3.72 (s, 4H), 3.29-3.20 (m, 2H), 3.14 (dd, J=14.0, 4.7 Hz, 1H), 2.82 (dd, J=14.0, 9.7 Hz, 1H), 2.47-2.18 (m, 2H), 2.08-1.78 (m, 2H), 1.73-1.51 (m, J=4H), 0.93 and 0.98 (2s, 6H).13C NMR (101 MHz, CD3OD) δ 175.01, 173.60, 173.51, 172.52, 172.45, 172.43, 163.01, 160.59, 156.90, 156.85, 136.79, 133.19, 133.15, 130.77, 130.69, 130.61, 128.03, 127.55, 127.31, 127.28, 114.69, 114.48, 66.16, 66.07, 56.19, 52.00, 51.97, 51.91, 51.87, 51.37, 49.94, 49.84, 41.56, 40.55, 40.51, 37.35, 36.77, 32.83, 32.79, 25.58, 24.34, 21.97, 20.85, 20.82.19F NMR (377 MHz, Methanol-d4) δ −119.88-−119.94 (m). LC-MS: m/z [M+H]+calcd. for C32H42FN4O7: 613.3, found: 613.5. Benzyl ((S)-3-(4-fluorophenyl)-1-(((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-1-oxopropan-2-yl)carbamate (34) To a solution of 33 (230 mg, 0.38 mmol) in THF (2.0 mL) was added LiBH4(4M in THF, 0.25 mL, 1.0 mmol) dropwise at 0° C. The reaction mixture was stirred at room temperature for 2 h. Then the reaction was quenched with 1N HCl (5 mL) and stirred for 1 h at room temperature. Ethyl acetate (30 mL) was added to the mixture, and the organic layer was washed with 1N HCl, NaHCO3and brine. The organic layer was dried over Na2SO4, and the filtrate evaporated to dryness. The residue was purified by was purified by flash chromatography (DCM/MeOH 30:1 to 10:1) to afford product 34 (174 mg, 80%).1H NMR (400 MHz, Methanol-d4) δ 7.88 (d, J=8.9 Hz, 1H), 7.38-7.21 (m, 6H), 6.98 (t, J=8.8 Hz, 2H), 5.16-4.96 (m, 2H), 4.39 (dt, J=12.9, 6.2 Hz, 3H), 4.02 (ddd, J=8.9, 5.8, 2.9 Hz, 1H), 3.60-3.42 (m, 2H), 3.25 (t, J=4.7 Hz, 2H), 3.15 (dd, J=14.1, 4.7 Hz, 1H), 2.85 (dd, J=14.1, 9.5 Hz, 1H), 2.31 (d, J=8.7 Hz, 1H), 2.18-1.96 (m, 2H), 1.90-1.77 (m, 1H), 1.72-1.57 (m, 5H), 0.95 (d, J=5.7 Hz, 3H), 0.92 (d, J=5.2 Hz, 3H).13C NMR (101 MHz, MeOD) δ 175.98, 173.48, 173.40, 172.53, 163.02, 160.60, 157.06, 136.71, 133.11, 130.75, 130.67, 128.02, 127.56, 127.38, 127.29, 114.72, 114.50, 66.29, 64.16, 56.42, 52.26, 52.22, 41.62, 40.52, 37.29, 36.57, 32.76, 25.71, 24.42, 22.07, 20.62.19F NMR (377 MHz, Methanol-d4) δ −119.74-−119.82 (m). LC-MS: m/z [M+H]+calcd. for C31H42FN4O6: 585.3, found: 585.5. Benzyl ((S)-3-(4-fluorophenyl)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxopropan-2-yl)carbamate (35) To a solution of compound 34 (123 mg, 0.21 mmol) in dichloromethane (2.0 mL) was added Dess-Martin periodinane (43 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 2 h then filtered through a silica gel pad, washed with ethyl acetate. The filtrate was evaporated to dryness and the residue was purified by flash chromatography (DCM/MeOH 30:1 to 12:1) to afford product 35 (70 mg, 57%).1H NMR (400 MHz, Methanol-d4) δ 8.18 (dd, J=7.5, 4.1 Hz, 1H), 7.86 (d, J=9.3 Hz, 1H), 7.42-7.15 (m, 12H), 6.98 (t, J=8.8 Hz, 3H), 5.16-4.95 (m, 3H), 4.41 (qd, J=6.2, 3.8, 3.3 Hz, 3H), 4.02 (ddt, J=9.1, 5.8, 2.7 Hz, 1H), 3.23 (t, J=4.9 Hz, 3H), 3.15 (dd, J=14.1, 4.9 Hz, 2H), 2.83 (dd, J=13.7, 10.0 Hz, 1H), 2.35-2.24 (m, 1H), 2.23-2.11 (m, 2H), 2.02 (dt, J=10.5, 3.5 Hz, 1H), 1.75-1.57 (m, 7H), 0.94 (dd, J=13.6, 6.1 Hz, 10H).13C NMR (101 MHz, MeOD) δ 176.05, 173.53, 173.50, 172.51, 172.42, 163.00, 160.59, 156.92, 136.75, 133.17, 130.75, 130.67, 128.04, 128.02, 127.54, 127.35, 127.32, 114.75, 114.71, 114.54, 114.49, 98.39, 98.32, 66.19, 56.26, 54.03, 53.80, 53.73, 52.28, 52.24, 50.80, 50.62, 41.62, 40.71, 40.64, 40.40, 37.07, 37.03, 36.77, 30.45, 29.92, 26.46, 25.50, 25.47, 24.41, 24.35, 22.15, 21.97, 21.94, 21.14, 20.89, 20.85, 20.57, 20.54.19F NMR (377 MHz, Chloroform-d) δ −117.12-−117.37 (m). LC-MS: m/z [M+H]+calcd. for C31H40FN4O6: 583.3, found: 583.5. Benzyl ((S)-1-(((S)-1-(((S,E)-5-amino-4-cyano-5-oxo-1-((S)-2-oxopiperidin-3-yl)pent-3-en-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (36) To a solution of 2-cyanoacetamide (6.5 mg, 0.08 mmol) and 19 (50 mg, 0.08 mmol) in ethanol (0.2 mL) was added piperidine (0.66 M in ethanol, 12 μL, 0.008 mmol). The reaction vessel was then placed into a microwave reactor (CEM Discover), and irradiated for 25 minutes at 80° C. After removal of the volatils under vacuo, the reaction mixture was purified by preparative TLC (EtOAc/MeOH 20/1) to give 2-cyano-3-(substituted phenyl)acrylamide product 36 as a white solid (8 mg, 15%).1H NMR (400 MHz, Methanol-d4) δ 8.21 (q, J=12.1, 10.0 Hz, 1H), 7.89 (d, J=8.1 Hz, 1H), 7.84-7.72 (m, 1H), 7.53 (dt, J=22.7, 7.3 Hz, 2H), 7.45-7.36 (m, 1H), 7.36-7.17 (m, 6H), 5.00 (d, J=8.2 Hz, 1H), 4.62 (d, J=10.2 Hz, 1H), 4.45-4.26 (m, 1H), 3.78-3.65 (m, 1H), 3.28-3.16 (m, 4H), 2.50-2.16 (m, 1H), 2.04 (s, 1H), 1.85 (d, J=15.2 Hz, 1H), 1.64 (dd, J=8.4, 4.7 Hz, 1H), 1.06-0.73 (m, 6H).13C NMR (101 MHz, CD3OD) δ 175.97, 175.49, 173.37, 172.76, 157.28, 156.97, 136.65, 134.03, 132.99, 131.98, 128.45, 128.03, 128.00, 127.51, 127.31, 127.26, 125.88, 125.27, 125.00, 123.26, 114.64, 113.42, 66.27, 64.16, 55.77, 52.35, 52.19, 41.61, 41.56, 40.09, 37.34, 37.30, 34.30, 24.56, 24.46, 22.06, 20.81, 20.66, 20.51. LC-MS: m/z [M+H]+calcd. for C38H44N6O6: 680.3, found: 680.5. Sodium (5S,8S,11S)-12-hydroxy-8-isobutyl-5-(naphthalen-1-ylmethyl)-3,6,9-trioxo-11-(((S)-2-oxopiperidin-3-yl)methyl)-1-phenyl-2-oxa-4,7,10-triazadodecane-12-sulfonate (37) A solution of 19 (19 mg, 0.03 mmol) and sodium bisulfite (4.5 mg, 0.04 mmol) in a mixture of EtOAc/EtOH/H2O (1:0.6:0.25, 0.2 mL) was stirred for 3 h at 55° C. and then allowed to cool down to room temperature. The precipitate formed was vacuum filtered and the solid was thoroughly washed with absolute ethanol. The filtrate was then dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to yield a yellowish oil which was treated with ethyl ether to form a white solid. Careful removal of the solvent using a pipette yielded compound 37 (15 mg, 67%).1H NMR (400 MHz, DMSO-d6) δ 8.23 (d, J=8.3 Hz, 2H), 7.92 (dd, J=7.7, 3.6 Hz, 1H), 7.85-7.74 (m, 2H), 7.70-7.49 (m, 2H), 7.30 (dt, J=9.1, 5.6 Hz, 3H), 7.24-7.13 (m, 2H), 4.45 (q, J=10.8, 9.2 Hz, 1H), 4.39-4.18 (m, 1H), 3.87 (d, J=4.8 Hz, 0H), 3.21-2.97 (m, 3H), 2.17 (ddd, J=21.8, 11.5, 4.6 Hz, 1H), 2.01 (d, J=14.7 Hz, 1H), 1.96-1.79 (m, 1H), 1.76-1.63 (m, 2H), 1.57-1.41 (m, 4H), 0.92 (d, J=4.8 Hz, 3H), 0.88 (d, J=4.3 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 201.42, 173.16, 173.00, 172.94, 171.82, 171.72, 156.25, 137.40, 134.19, 133.83, 132.07, 129.03, 128.72, 128.08, 127.87, 127.75, 127.70, 127.48, 126.52, 126.00, 125.77, 124.19, 65.63, 61.62, 56.05, 55.81, 55.69, 51.70, 41.68, 41.55, 41.40, 34.98, 26.18, 24.66, 23.54, 23.37, 22.27, 22.12, 21.85, 21.77, 15.60. LC-MS: m/z [M+H]+calcd. for C35H45N4O9S: 697.3, found: 697.5. Benzyl ((2S)-1-(((2S)-4-methyl-1-oxo-1-(((2S)-4,4,4-trifluoro-3-hydroxy-1-((S)-2-oxopiperidin-3-yl)butan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (38) To a solution of compound 19 (61 mg, 0.1 mmol) and cesium fluoride in THF (76 mg, 0.5 mmol) was added Me3SiCF3(2M in THF, 0.1 mL, 0.2 mmol) at −78° C. dropwise over 5 minutes. The reaction mixture was then stirred at room temperature for 2 h and quenched with 1N HCl (0.5 mL). After 1 h, EtOAc (10 mL) was added to the reaction mixture and the organic layer was washed with 1N HCl, NaHCO3and water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by preparative TLC (DCM/MeOH 20:1) to provide compound 38 (19 mg, 28%).1H NMR (400 MHz, Chloroform-d) δ 8.21 (s, 1H), 8.08 (s, 1H), 7.98-7.80 (m, 1H), 7.78 (d, J=8.0 Hz, 1H), 7.65-7.45 (m, 1H), 7.36 (d, J=15.8 Hz, 5H), 6.70-6.38 (m, 2H), 5.99 (d, J=33.3 Hz, 1H), 5.66-5.40 (m, 1H), 5.04 (s, 1H), 4.66 (d, J=8.5 Hz, 1H), 4.46 (d, J=8.9 Hz, 1H), 4.33 (t, J=7.4 Hz, 1H), 3.76-3.62 (m, 1H), 3.46-3.36 (m, 1H), 3.24 (s, 2H), 2.22 (s, 1H), 2.10-1.86 (m, 3H), 1.75 (s, 2H), 0.98-0.78 (m, 6H).13C NMR (101 MHz, CD3OD) δ 175.83, 174.65, 173.21, 172.79, 172.58, 156.79, 136.72, 134.04, 133.08, 132.87, 131.99, 128.45, 128.03, 128.00, 127.51, 127.36, 127.28, 125.86, 125.26, 125.00, 123.25, 66.22, 55.64, 52.55, 41.68, 41.57, 39.75, 38.56, 37.11, 36.84, 34.59, 26.45, 24.52, 24.42, 22.22, 22.09, 21.54, 20.53, 20.30.19F NMR (377 MHz, Chloroform-d) δ −75.94, −77.58. LC-MS: m/z [M+H]+calcd. for C36H44F3N4O6: 685.3, found: 685.5. Benzyl ((S)-1-(((S)-4-methyl-1-oxo-1-(((S)-4,4,4-trifluoro-3-oxo-1-((S)-2-oxopiperidin-3-yl)butan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (39) A solution of compound 38 (19 mg, 0.03 mmol) and Dess-Martin periodinane (45 mg, 0.11 mmol) in dichloromethane (0.8 mL) was stirred at room temperature for 1 h. The reaction mixture was then filtered through silica gel pad, washed with ethyl acetate and concentrated under vacuum. The residue was purified by two successive preparative TLCs (DCM/methanol=20/1 then 100% ethyl acetate) to give compound 39 as a white solid (10 mg, 53%).1H NMR (400 MHz, Methanol-d4) δ 8.22 (d, J=8.5 Hz, 1H), 7.88 (d, J=8.3 Hz, 1H), 7.77 (d, J=7.7 Hz, 1H), 7.52 (dt, J=15.2, 7.6 Hz, 2H), 7.38 (d, J=9.6 Hz, 2H), 7.30 (d, J=6.3 Hz, 3H), 7.21 (d, J=7.2 Hz, 2H), 4.95 (s, 1H), 4.69-4.55 (m, 1H), 4.47 (dd, J=10.2, 6.1 Hz, 1H), 4.38 (d, J=11.6 Hz, 1H), 3.73 (d, J=14.7 Hz, 1H), 3.28-3.16 (m, 2H), 2.40-2.18 (m, 2H), 2.04 (s, 1H), 1.85-1.67 (m, 1H), 1.60 (dt, J=13.3, 7.3 Hz, 2H), 1.44 (d, J=11.1 Hz, 1H), 1.03-0.81 (m, 6H).13C NMR (101 MHz, MeOD) δ 176.01, 175.84, 173.66, 173.39, 172.79, 172.66, 156.79, 136.70, 134.03, 133.09, 131.99, 128.45, 128.01, 127.48, 127.30, 127.22, 125.85, 125.25, 125.00, 123.25, 66.13, 66.09, 55.49, 52.29, 52.05, 49.71, 49.43, 41.57, 40.32, 37.00, 36.87, 34.58, 30.12, 29.99, 29.36, 25.62, 25.43, 24.35, 24.32, 22.06, 21.98, 20.76, 20.72, 20.62.19F NMR (377 MHz, CD3OD) δ −79.48, −79.97. LC-MS: m/z [M+H]+calcd. for C36H42F3N4O6: 683.3, found: 683.5 and 701.5 [M+H2O]+. tert-Butyl ((S)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)carbamate (41) To a solution of compound 40 (600 mg, 2.0 mmol) in MeOH (10 mL) was added NaBH4(152 mg, 4.0 mmol) at 0° C. The reaction mixture was stirred at room temperature for 3 h then quenched with 1N HCl (5 mL) and finally stirred for 1 h at room temperature. The suspension was extracted with ethyl acetate (3×30 mL), and washed with NaHCO3and brine. The organic layer was dried over Na2SO4, and then evaporated to dryness. The residue was purified by flash chromatography on silica gel (DCM/MeOH 50:1 to 10:1) to afford compound 41 (480 mg, 88%).1H NMR (400 MHz, Chloroform-d) δ 6.42 (s, 1H), 5.57 (d, J=8.1 Hz, 1H), 3.70 (dt, J=24.3, 5.3 Hz, 2H), 3.63-3.51 (m, 2H), 3.48 (s, OH), 3.32 (qd, J=4.8, 2.2 Hz, 2H), 2.38 (dt, J=11.0, 5.5 Hz, 1H), 2.17 (s, 1H), 2.01-2.1 (m, 1H), 1.96-1.81 (m, 1H), 1.80-1.66 (m, 2H), 1.56 (dtd, J=13.5, 10.5, 3.0 Hz, 1H), 1.44 (s, 9H).13C NMR (101 MHz, CDCl3) δ 175.78, 156.55, 79.31, 65.64, 53.43, 50.66, 50.47, 42.45, 38.10, 32.80, 28.39, 26.90, 21.64. LC-MS: m/z [M+H]+calcd. for C13H25N2O4: 273.2, found: 273.5. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)carbamate (42) A solution of compound 41 (400 mg, 1.47 mmol) and Dess-Martin periodinane (750 mg, 1.77 mmol) in dichloromethane (10 mL) was stirred at room temperature for 2 h. The reaction mixture was filtered through a celite pad and washed with ethyl acetate (50 mL). The organic layer was washed with a solution of sodium thiosulfate (0.4 N, 10 mL) and a solution of NaHCO3(5%, 10 mL). The organic layer was dried over Na2SO4, and filtered. The filtrate was evaporated under reduced pressure to give the crude product 42 (364 mg, 92%).1H NMR (400 MHz, Chloroform-d) δ 9.55 (s, 1H), 6.33-6.14 (m, 1H), 4.28-4.15 (m, 1H), 3.43-3.20 (m, 4H), 2.47-2.25 (m, 1H), 2.19 (ddd, J=14.2, 8.6, 7.1 Hz, 1H), 1.88 (tt, J=8.5, 4.4 Hz, 2H), 1.75 (dtd, J=13.8, 7.3, 3.3 Hz, 1H), 1.64-1.51 (m, 1H), 1.45 (d, J=12.1 Hz, 9H).13C NMR (101 MHz, CDCl3) δ 200.77, 174.84, 156.18, 79.94, 60.40, 58.30, 42.39, 37.34, 31.50, 28.40, 28.32, 27.38, 21.31. LC-MS: m/z [M+H]+calcd. for C13H23N2O4: 271.2, found: 271.5. tert-Butyl ((2S)-4,4,4-trifluoro-3-hydroxy-1-((S)-2-oxopiperidin-3-yl)butan-2-yl)carbamate (44) To a solution of crude product 42 (270 mg, 1.0 mmol) and CsF (180 mg, 1.18 mmol) in THF (3.0 mL) was added at −78° C., TMSCF3(2.0M in THF, 0.7 mL, 1.4 mmol) dropwise over 10 minutes. After addition, the reaction mixture was then stirred at room temperature for 1 h, quenched by addition of a 1 N HCl solution (10 mL) and stirred for another 30 minutes. The reaction mixture was extracted with ethyl acetate (30 ml×3), washed with a saturated solution of NaHCO3and water, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel column (DCM/MeOH 30:1 to 10:1) to afford compound 44. LC-MS: m/z [M+H]+calcd. for C14H24F3N2O4: 341.2, found: 341.5. Benzyl ((2S)-1-(((2S)-4-methyl-1-oxo-1-(((2S)-4,4,4-trifluoro-3-hydroxy-1-((S)-2-oxopiperidin-3-yl)butan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (38) To a solution of crude product 44 (50 mg, 0.18 mmol) in DCM (1.5 mL) was added 4N HCl in dioxane (0.6 mL, 2.4 mmol). The reaction mixture was stirred for 2 h at room temperature and then the volatils were remove under reduced pressure. The residue was dissolved in DCM (1.0 mL) and compound 46 (46 mg, 0.1 mmol), 1-hydroxybenzotriazole (28 mg, 0.2 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (38 mg, 0.2 mmol) and N,N-diisopropylethyl amine (0.14 mL, 0.8 mmol) were added at 0° C. The reaction mixture was stirred at room temperature overnight before being diluted with EtOAc (20 mL). The organic layer was washed with a 1N HCl solution, a solution of NaHCO3(5%) and brine. The organic layer was dried over Na2SO4. The solvent was evaporated and the residue was purified by flash chromatography on silica gel (DCM/MeOH 30:1 to 15:1) to afford product 38. LC-MS: m/z [M+H]+calcd. for C36H44F3N4O6: 685.3, found: 685.5. tert-Butyl ((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)carbamate (49) To a solution of compound 4 (1.0 g, 3.49 mmol) in methanol (40 mL) was added NaBH4(0.53 g, 14 mmol) at room temperature. The reaction mixture was stirred at this temperature for 2 h, then quenched with water (30 mL). The suspension was extracted with EtOAc (50 mL×3) and the combined organic layers were dried over Na2SO4and finally evaporated under vacuum. The residue was then dissolved in dichloromethane (20 mL) and Dess-Martin periodinane (1.48 g, 3.49 mmol) and NaHCO3(0.37 g, 3.49 mmol) were added. The resulting mixture was stirred at room temperature for 5 h. The mixture was diluted with EtOAc (150 mL) and the organic layer was washed with an aqueous solution of 10% Na2S2O4, a saturated solution of NaHCO3, a solution of 1N HCl, and brine successively. The organic layer was dried over Na2SO4and then concentrated to give product 49 as a white solid which was used in the next step without further purification.1H NMR (CDCl3) δ 9.73 (1H, s), 6.02 (1H, br), 5.48 (1H, d, J=7.8 Hz), 4.36-4.25 (1H, m), 3.38-3.32 (2H, m), 2.50-2.44 (2H, m), 2.11-2.03 (1H, m), 1.88-1.76 (2H, m), 1.43 (9H, s). LCMS-ESI (m/z): 257 (M+H)+. tert-Butyl((S)-1-((S)-2-oxopyrrolidin-3-yl)but-3-en-2-yl)carbamate (50) To a suspension of methyltriphenylphosphonium bromide (3.29 g, 9.29 mmol) in THF (10 mL) at −78° C. was added LiHMDS (30.3 g, 152 mmol). The resulting yellow suspension was warmed up to room temperature and stirred at the same temperature for 1 hour. After the reaction mixture was cooled down to −78° C., a solution of aldehyde 49 (1.07 g, 4.4 mmol) in THF (5 mL) was added dropwise. The mixture was stirred at 0° C. overnight. The reaction was quenched with MeOH (0.5 mL) and the resulting mixture was poured into 1 N HCl solution (20 mL). Extraction with Et2O (3×20 mL), drying over Na2SO4and evaporation of the solvent in vacuo afforded an orange semi-solid that was purified by silica gel chromatography (DCM/MeOH=20/1) to afford 50 as a white solid (0.36 g, 32%).1H NMR (400 MHz, Chloroform-d) δ 6.89 (s, 1H), 5.72-5.80 (m, 1H), 5.03-5.20 (m, 3H), 4.14 (s, br, 1H), 3.23-3.31 (m, 2H), 2.40-2.46 (m, 2H), 1.72-1.77 (m, 1H), 1.45-1.52 (m, 1H), 1.40 (s, 9H). tert-Butyl ((S)-4-methyl-1-oxo-1-(((S)-1-((S)-2-oxopyrrolidin-3-yl)but-3-en-2-yl)amino)pentan-2-yl)carbamate (51) To a solution of 50 (250 mg, 1.04 mmol) in dioxane (5 mL) was added a solution of 4 M HCl in dioxane (2 mL). The reaction was stirred at room temperature for 3 h and then the volatils were removed under vacuum. The residue was finally coevaporated with toluene the deprotected deprotected product as a colorless oil. To a solution of this amino derivative in DCM (20 mL) was added EDC (250 mg, 1.3 mmol), HOBt (176 mg, 1.3 mmol), Boc-L-Leu-OH (280 mg, 1.2 mmol) and DIPEA (0.84 mL, 4.8 mmol). The solution was stirred at room temperature overnight before being diluted with ethyl acetate (80 mL). The organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine, dried over Na2SO4. After removal of the volatils under vacuum, the title compound 51 was obtained as a colorless oil (250 mg, 67%). LCMS-ESI (m/z): 368 (M+H)+. Benzyl ((S)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-((S)-2-oxopyrrolidin-3-yl)but-3-en-2-yl)amino) pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (52) Compound 51 (370 mg, 1.0 mmol) was dissolved in DCM (6 mL) and treated with trifluoroacetic acid (2 mL). The solution was stirred at room temperature for 3 h and the solvent was removed under vacuum. The crude compound was dried under vacuum for 5 h and used in the next step without further purification. To a solution of the deprotected amino acid in DCM (20 mL) was added EDCI (230 mg, 1.2 mmol), HOBt (160 mg, 1.2 mmol), Z-L-Ala(−1-naphthyl)-OH (350 mg, 1.0 mmol) and DIPEA (0.7 mL, 4.0 mmol). After being stirred at room temperature overnight, the reaction mixture was diluted with ethyl acetate (80 mL). The organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine. The organic layer was then dried over Na2SO4and the solvent removed under vacuum. The residue was purified by column chromatography (DCM:MeOH=20:1) to give title compound 52 as a white solid (320 mg, 54%).1H NMR (400 MHz, MeOH-d4) δ 8.18 (t, J=10.1 Hz, 2H), 7.87 (d, J=7.6 Hz, 1H), 7.76 (d, J=7.3 Hz, 1H), 7.48-7.54 (m, 2H), 7.21-7.40 (m, 7H), 5.80-5.85 (m, 1H), 5.21 (dt, J1=1.4 Hz, J2=17.2 Hz, 1H), 5.17 (dt, J1=1.4 Hz, J2=10.4 Hz, 1H), 4.95-4.97 (m, 2H), 4.59-4.65 (m, 1H), 4.40-4.52 (m, 2H), 3.67-3.73 (m, 1H), 3.16-3.28 (m, 2H), 2.47-2.49 (m, 1H), 2.24-2.27 (m, 1H), 1.47-1.77 (m, 4H), 0.91-0.96 (m, 6H);13C NMR (100 MHz, MeOH-d4) δ 181.02, 172.93, 171.58, 156.92, 138.32, 136.69, 134.04, 132.98, 131.98, 128.48, 128.03, 127.52, 127.29, 125.90, 125.30, 125.02, 123.25, 113.62, 66.23, 60.14, 55.81, 52.43, 49.33, 40.68, 40.10, 38.28, 35.58, 34.53, 27.51, 24.40, 21.97, 20.84, 19.49, 13.09; LCMS-ESI (m/z): 599 (M+H)+. Benzyl ((2S)-1-(((2S)-4-methyl-1-(((1S)-1-(oxiran-2-yl)-2-((S)-2-oxopyrrolidin-3-yl)ethyl)amino)-1-oxopentan-2-yl))amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl))carbamate To a solution of compound 2 (50 mg, 0.08 mmol) in DCM (5 mL) containing aq. Na2HP3O4(6 M, 40 μL, 0.24 mmol) was added mCPBA (70%, 62 mg, 0.25 mmol). The reaction mixture was stirred at room temperature for 24 h. EtOAc (30 mL) was added and the solution was washed with a saturated solution of NaHCO3, 1 N HCl, brine, and dried over Na2SO4. After concentration under vacuum, the residue was purified on preparative TLC to afford compound 53 as a white solid (20 mg, 39%).1H NMR (400 MHz, MeOH-d4) δ 8.11-8.23 (m, 1H), 7.94-8.01 (m, 1H), 7.89 (d, J=7.7 Hz, 1H), 7.78 (d, J=7.2 Hz, 1H), 7.23-7.61 (m, 8H), 4.98 (s, 2H), 4.59-4.62 (m, 1H), 4.35-4.40 (m, 1H), 4.02-4.10 (m, 1H), 3.69-3.74 (m, 1H), 3.21-3.28 (m, 2H), 3.04-3.07 (m, 1H), 2.75-2.77 (m, 1H), 2.58-2.60 (m, 1H), 2.45-2.52 (m, 1H), 2.09-2.33 (m, 2H), 1.53-1.81 (m, 4H), 1.30-1.37 (m, 1H), 0.88-0.98 (m, 6H);13C NMR (100 MHz, MeOH-d4) δ 180.88, 173.64, 173.56, 172.75, 156.87, 136.69, 134.03, 133.00, 132.19, 131.98, 129.67, 128.44, 128.01, 127.50, 127.26, 127.20, 125.87, 125.28, 124.99, 123.24, 66.20, 55.57, 53.29, 52.40, 44.06, 40.51, 40.06, 38.05, 34.38, 32.54, 27.43, 24.50, 21.95, 20.66; LCMS-ESI (m/z): 615 (M+H)+, 633 (M+H+H2O)+ Methyl ((S)-2-(((benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanoyl)-L-leucinate (56) To a solution of Cbz-L-Ala(1-Naphthyl)-OH 55 (0.78 g, 2.25 mmol) and L-Leu-OMe (0.45 g, 2.48 mmol) in DCM (50 mL) was added EDCI (560 mg, 2.9 mmol), HOBt (400 mg, 2.9 mmol), and DIPEA (1.6 mL, 9 mmol). The reaction mixture was stirred overnight at room temperature at which time H2O (100 mL) and EtOAc (200 mL) were added. The organic layer was washed successively with aq. HCl (1 M, 50 mL), sat. aq. NaHCO3(200 mL) and brine (100 mL), and then dried over Na2SO4. The solvent was removed under vacuum to give the title compound as a yellow solid after crystallization from ethyl acetate (0.9 g, 85%).1H NMR (400 MHz, MeOH-d4) δ 7.89 (d, J=9.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.49-7.58 (m, 2H), 7.36-7.41 (m, 8H), 5.82 (d, J=8.0 Hz, 1H), 5.51-5.53 (m, 1H), 5.08-5.16 (m, 2 H), 4.60-4.65 (m, 1H), 4.44-4.59 (m, 1H), 4.09-4.23 (m, 2H), 3.21-3.27 (m, 2H), 2.48-2.52 (m, 1H), 1.57-1.80 (m, 4H), 1.21-1.40 (m, LCMS-ESI (m/z): 477 (M+H)+. ((S)-2-(((Benzyloxy)carbonyl)amino)-3-(naphthalen-1-yl)propanoyl)-L-leucine (57) To a solution of methyl ester 56 (2.0 g, 4.2 mmol) in MeOH (19 mL) was added a solution of LiOH H2O (270 mg, 6.3 mmol) in H2O (1 mL). The reaction mixture was stirred overnight at room temperature. The volatiles were removed under vacuum to give a colourless residue that was partitioned between EtOAc and aq. HCl (1M). The organic layer was separated, washed successively with aq. HCl (1M) and brine, then dried over MgSO4, and the solvent removed under vacuum to give a glassy solid. Recrystallization from EtOAc gave the title compound 57 as a white solid (1.86 g, 96%).1H NMR (400 MHz, Methanol-d4) δ 8.13-8.21 (m, 1H), 7.89 (s, br, 1H), 7.79 (s, 1H), 7.25-7.57 (m, 9H), 5.02-5.17 (m, 1H), 4.33-4.59 (m, 1H), 3.34-3.56 (m, 1H), 1.34-1.54 (m, 3H), 0.84-0.88 (m, 6H); LCMS-ESI (m/z): 463 (M+H)+. Methyl (5S,8S,11S,E)-8-isobutyl-5-(naphthalen-1-ylmethyl)-3,6,9-trioxo-11-(((S)-2-oxopyrrolidin-3-yl)methyl)-1-phenyl-2-oxa-4,7,10-triazatetradec-12-en-14-oate (60) To a solution of compound 59 (330 mg, 1.0 mmol) in dioxane (5 ML) was added HCl (4 M in dioxane, 2 mL). The reaction was stirred at room temperature for 3 h and then the volatils were removed under vacuum to give the crude deprotected amine which was used directly in the next step. This compound was thus dissolved in DCM (20 mL) and EDC (250 mg, 1.3 mmol), HOBt (176 mg, 1.3 mmol), dipeptide 57 (460 mg, 1.0 mmol) and DIPEA (0.84 mL, 4.8 mmol) were added. The solution was stirred at room temperature overnight and then diluted with ethyl acetate (80 mL). The organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine, dried over Na2SO4and the solvent removed under vacuum. The residue was purified by column chromatography to give 60 as a white solid (460 mg, 70%).1H NMR (400 MHz, Methanol-d4) δ 8.18-8.21 (m, 1H), 7.74-7.94 (m, 3H), 7.23-7.68 (m, 8H), 6.91 (dd, J=15.5, 5.3 Hz, 1H), 5.90-6.02 (m, 1H), 4.95-5.01 (m, 1H), 4.60-4.65 (m, 1H), 4.32-4.86 (m, 1H), 4.01-4.23 (m, 2H), 3.70-3.75 (m, 1H), 3.21-3.27 (m, 2H), 2.48-2.52 (m, 1H), 2.40-2.66 (m, 1H), 1.58-1.80 (m, 4H), 1.20-1.40 (m, 6H), 0.89-1.10 (m, 6H); LCMS-ESI (m/z): 657 (M+H)+. Benzyl ((S)-1-(((S)-1-(((R)-5-hydroxy-1-((S)-2-oxopyrrolidin-3-yl)pentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (61) To a solution of methyl ester 60 (34 mg, 0.05 mmol) in dry THF (3 mL) was added LiBH4(2M in THF, 0.03 mL, 0.06 mmol) at 0° C. The resulting mixture was stirred at room temperature for 3 h and then quenched with aq. HCl (1M). Ethyl acetate (20 ML) was added and the organic layer was further washed with H2O, dried over Na2SO4, and the solvent removed under vacuum to give a white solid. Recrystallization from EtOAc gave 61 as a white solid (9 mg, 29%).1H NMR (400 MHz, Methanol-d4) δ 8.21 (d, J=8.3 Hz, 1H), 7.97-7.71 (m, 3H), 7.52 (dt, J=14.6, 7.5 Hz, 2H), 7.45-7.20 (m, 9H), 5.10-4.94 (m, 2H), 4.58 (dd, J=9.5, 5.3 Hz, 1H), 4.46-4.29 (m, 1H), 4.12 (q, J=7.1 Hz, 2H), 3.97 (d, J=10.6 Hz, 1H), 3.70 (dd, J=14.0, 5.0 Hz, 1H), 3.30-3.17 (m, 2H), 2.59-2.20 (m, 5H), 2.05-1.38 (m, 7H), 1.37-1.16 (m, 4H), 0.93 (td, J=12.7, 10.8, 5.7 Hz, 6H); LCMS-ESI (m/z): 631 (M+H)+. Benzyl ((S)-1-(((S)-4-methyl-1-oxo-1-(((R)-5-oxo-1-((S)-2-oxopyrrolidin-3-yl)pentan-2-yl)amino) pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (62) To a solution of j (47 mg, 0.075 mmol) in DCM (4 mL) and DMSO (1 mL) was added DIPEA (50 μL, 0.3 mmol) at 0° C. The solution was stirred for 30 min before addition of SO3pyridine complex (47 mg, 0.3 mmol). The reaction mixture was stirred for 3 h at room temperature, and then diluted with EtOAc (50 mL). The organic phase was separated and then washed successively with aq. HCl (1M), sat. aq. NaHCO3, and brine, dried over Na2SO4. After removal of the voaltils under vacuum, the residue was purified by preparative TLC (DCM:MeOH 20:1) to give 62 as a white solid. LCMS-ESI (m/z): 629 (M+H)+. Benzyl ((S)-1-(((S)-1-(((S,E)-5-hydroxy-1-((S)-2-oxopyrrolidin-3-yl)pent-3-en-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (63) To a solution of 60 (67 mg, 0.1 mmol) in DCM (3 mL) was added DIBAL (1 M, 0.2 mL, 0.3 mmol) at 0° C. The reaction was stirred for 2 h at 0° C. and then quenched with 1 M HCl (0.1 mL). The reaction mixture was warmed up to room temperature and diluted with EtOAc (15 mL). The organic layer was washed successively with 1 M HCl (5 mL), sat. aq. NaHCO3(5 mL), and brine, dried over Na2SO4and the solvent was removed under vacuum. The residue was purified by silica gel chromatography (DCM:MeOH=20:1) to give 63.1H NMR (400 MHz, Methanol-d4) δ 8.22 (d, J=8.4 Hz, 1H), 7.84 (dd, J=41.9, 7.7 Hz, 2H), 7.53 (dt, J=14.5, 7.6 Hz, 1H), 7.44-7.13 (m, 7H), 5.81-5.63 (m, 1H), 4.68-4.27 (m, 4H), 4.07 (d, J=5.0 Hz, 1H), 3.80-3.62 (m, 1H), 2.60-2.17 (m, 3H), 2.03 (dd, J=24.1, 7.1 Hz, 2H), 1.82-1.45 (m, 6H), 1.31 (s, H), 1.04-0.85 (m, 6H); LCMS-ESI (m/z): 629 (M+H)+. Benzyl ((S)-1-(((S)-4-methyl-1-oxo-1-(((S,E)-5-oxo-1-((S)-2-oxopyrrolidin-3-yl)pent-3-en-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (64) To a solution of alcohol 63 (56 mg, 0.09 mmol) in DCM (4 mL) and DMSO (1 mL) was added DIPEA (0.13 mL, 0.36 mmol) at 0° C. The solution was stirred for 30 min before addition of SO3·pyridine complex (60 mg, 0.36 mmol). The reaction mixture was then stirred at 0° C. overnight and then diluted with EtOAc (25 mL). The organic phase was separated and then washed successively with aq. HCl (1M, 10 mL), sat. aq. NaHCO3(10 mL), and brine, dried over Na2SO4. After removal of the volatils under vacuum, the residue was purified by preparative TLC to give 64 as a white solid (mg, 75%).1H NMR (400 MHz, Methanol-d4) δ 9.55 (dd, J=1.6, 7.8 Hz, 1H), 8.21 (d, J=8.4 Hz, 1H), 7.88 (d, J=7.4 Hz, 1H), 7.78 (d, J=6.6 Hz, 1H), 7.48-7.57 (m, 2H), 7.23-7.41 (m, 7H), 6.91-6.97 (m, 1H), 6.14-6.20 (m, 1H), 4.99 (s, 2H), 4.74-4.85 (m, 1H), 4.59-4.63 (m, 1H), 4.37-4.41 (m, 1H), 3.68-3.74 (m, 1H), 3.24-3.29 (m, 2H), 2.45-2.61 (m, 1H), 2.24-2.32 (m, 1H), 1.62-1.84 (m, 5H), 0.89-0.98 (m, 6H); LCMS-ESI (m/z): 627 (M+H)+. tert-Butyl ((S,E)-4-(methylsulfonyl)-1-((S)-2-oxopyrrolidin-3-yl)but-3-en-2-yl)carbamate (65) To a solution of diethyl ((methylsulfonyl)methyl)phosphonate (1.46 g, 6.3 mmol) in THF (60 mL) was added BuLi (1 M, 6.5 mL, 6.5 mmol) dropwise at −78° C. After stirred for 30 min, aldehyde 49 (1.35 g, 5.3 mmol) in THF (10 mL) was added over 30 min. The reaction mixture was warmed up to it over 1 h and stirred for further 3 h. Quenched the reaction by addition of MeOH (1 mL), and the solvent was removed in vacuum. The residue was partitioned between EtOAc (150 mL) and aq. 1N HCl (80 mL), and the organic phase was washed respectively with aq. NaHCO3and brine, dried (Na2SO4), concentrated, and the residue was purified by silica gel chromatography (DCM:MeOH=20:1) to afford 65 (25%). LCMS-ESI (m/z): 333 [M+H]. Benzyl ((S)-1-(((S)-4-methyl-1-(((S,E)-4-(methylsulfonyl)-1-((S)-2-oxopyrrolidin-3-yl)but-3-en-2-yl)amino)-1-oxopentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (66) A solution of compound 65 (330 mg, 1 mmol) and trifluoroacetic acid (2 mL) in DCM (6 mL) was stirred at room temperature for 3 h. The solvent was removed under vacuum and the residue coevaporated three times with toluene. The residue was then dissolved in DCM (30 mL) and dipeptide 57 (480 mg, 1.05 mmol), EDCI (250 mg, 1.3 mmol), HOBt (180 mg, 1.3 mmol), and DIPEA (0.7 mL, 4 mmol) were added. The solution was stirred at room temperature overnight before being diluted with EtOAc (80 mL). The organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine, dried over Na2SO4. Removal of the volatils under vacuum and recrystallization of the residue from EtOAc gave 66 as a white solid (27 mg, 31%).1H NMR (400 MHz, Methanol-d4) δ 8.21 (d, J=8.72 Hz, 1H), 7.89 (d, J=7.76 Hz, 1H), 7.79 (d, J=4.84 Hz, 1H), 7.48-7.59 (m, 2H), 7.25-7.40 (m, 7H), 6.86 (dd, J=4.8, 15.32 Hz, 1H), 6.7 (d, J=15.16 Hz, 1H), 5.01-5.05 (m, 2H), 4.70-4.72 (m, 1H), 4.56-4.60 (m, 1H), 4.31-4.34 (m, 1H), 3.66-3.73 (m, 1H), 2.99 (s, 3H), 2.51-2.54 (m, 1H), 2.27-2.29 (m, 1H), 1.61-1.84 (m, 4H), 0.91-0/98 (m, 6H); LCMS-ESI (m/z): 677 (M+H)+. Benzyl ((2S)-1-(((2S)-4-methyl-1-(((1S)-1-(3-(methylsulfonyl)oxiran-2-yl)-2-((S)-2-oxopyrrolidin-3-yl)ethyl)amino)-1-oxopentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (67) To a solution of TBHP (5.5 M, 50 μL, 0.27 mmol) in THF (5 mL) was added MeLi (2.5 M, 0.1 mL, 0.25 mmol) at −78° C. The resulting mixture was stirred at −78° C. for 15 min and then a solution of compound 66 (123 mg, 0.18 mmol) in THF (1 mL) was added dropwise. The resulting mixture was stirred at 0° C. overnight. Solid Na2SO3(200 mg) was added and the suspension was stirred for 15 min. After dilution with sat. aq. NH4Cl solution, extraction with EtOAc (30 mL×3), the combined organic layers were washed with brine, dried over Na2SO4, and concentrated under vacuum. The crude oil was purified by column chromatography (DCM:MeOH=20:1) to give 67 as a pale yellow solid.1H NMR (400 MHz, Methanol-d4) δ 8.23 (d, J=8.6 Hz, 1H), 7.88 (d, J=8.2 Hz, 1H), 7.78 (d, J=7.3 Hz, 1H), 7.53 (dd, J=17.7, 7.7 Hz, 1H), 7.45-7.18 (m, 6H), 4.98 (d, J=3.6 Hz, 1H), 4.72 (q, J=7.2, 6.7 Hz, 1H), 4.60 (q, J=3.8 Hz, 1H), 4.47 (dd, J=26.2, 3.5 Hz, 1H), 4.19-4.05 (m, 2H), 3.69 (dt, J=13.1, 6.2 Hz, 1H), 3.38 (d, J=6.7 Hz, 1H), 2.58 (d, J=10.3 Hz, 1H), 2.43-2.30 (m, 1H), 2.05-1.92 (m, 1H), 1.82 (ddd, J=13.2, 8.8, 4.4 Hz, 1H), 1.75-1.44 (m, 2H), 1.40-1.21 (m, 2H), 0.94 (hept, J=6.7 Hz, 6H); LCMS-ESI (m/z): 693 (M+H)+. S—((S)-2-((tert-Butoxycarbonyl)amino)-3-((S)-2-oxopyrrolidin-3-yl)propyl) ethanethioate (32) To a solution of N-[1-(hydroxymethyl)cyclopropyl]carbamic acid-t-butyl ester 2 (3.74 g, 20.0 mmol) and NEt3(3.4 mL, 24.0 mmol) in CH2Cl2(100 mL), methanesulfonyl chloride (1.9 mL, 24.0 mmol) was added dropwise at 0° C. The reaction mixture was stirred for 20 h at 0° C. and after removal of the volatile components under reduced pressure, the residue was diluted with H2O (60 mL). The aqueous phase was extracted with EtOAc (3×60 mL) and the combined organic layers were dried over Na2SO4and concentrated under vacuum. To a solution of the residue in DMF (90 mL) was added K2CO3(6.78 g, 20.8 mmol) and thioacetic acid (1.5 mL, 20.8 mmol). The reaction mixture was stirred for 24 h at room temperature and then the volatile components were removed under reduced pressure. 1N HCl (90 mL) was added to the residue and the aqueous phase was extracted with EtOAc (3×100 mL). The combined organic phases were dried over Na2SO4and concentrated under vacuum. Recrystallization of the residue from hexane/Et2O yielded 68 (2.97 g, 12.1 mmol, 61%) as pale yellow solid. LCMS-EST (m/z): 317 [M+H]. S-((5S,8S,11S)-8-Isobutyl-5-(naphthalen-1-ylmethyl)-3,6,9-trioxo-11-(((S)-2-oxopyrrolidin-3-yl)methyl)-1-phenyl-2-oxa-4,7,10-triazadodecan-12-yl) ethanethioate (69) A solution of compound 68 (130 mg, 0.41 mmol) in DCM (6 mL) and trifluoroacetic acid (2 mL) was stirred at room temperature for 2 h. The solvent was removed under vacuum, the residue coevaporated three times with toluene. To a solution of the dry residue in DCM (20 mL) was added dipeptide 57 (200 mg, 0.43 mmol), EDCI (103 mg, 0.54 mmol), HOBt (73 mg, 0.54 mmol), and DIPEA (0.3 mL, 1.72 mmol). The solution was stirred at room temperature overnight before being diluted with EtOAc (80 mL). The organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine, dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, the thioacetate 69 was obtained as a white solid (227 mg, 50%).1H NMR (400 MHz, Methanol-d4) δ 8.13 (d, J=8.4 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.48-7.59 (m, 2H), 7.30-7.44 (m, 7H), 5.32 (d, J=12.4 Hz, 1H), 5.13 (d, J=12.4 Hz, 1H), 4.42-4.47 (m, 1H), 3.97-4.07 (m, 2H), 3.45-3.51 (m, 1H), 3.24-3.30 (m, 1H), 3.16-3.20 (m, 1H), 2.87-2.93 (m, 1H), 2.29 (s, 3H), 1.67-1.73 (m, 1H), 1.48-1.52 (m, 1H), 1.35-1.42 (m, 1H), 1.09-1.16 (m, 1H), 0.92-0.97 (m 1H), 0.60 (d, J=6.4 Hz, 3H), 0.52 (d, J=6.4 Hz, 3H); LCMS-EST (m/z): 661 [M+H]. Benzyl ((S)-1-(((S)-1-(((S)-1-mercapto-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (70) A solution of thioacetate 60 (21 mg, 0.03 mmol) and LiOH (2.4 mg, 0.1 mmol) in methanol (1 ml), and the mixture was stirred for 3 days in room temperature. The solvent was removed under vacuum and the residue was purified by preparative TLC (DCM:MeOH=20:1) to give solid thiol 70 in quantitative yield.1H NMR (400 MHz, Methanol-d4) δ 8.13 (d, J=8.4 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.48-7.59 (m, 2H), 7.30-7.44 (m, 7H), 5.32 (d, J=12.4 Hz, 1H), 5.13 (d, J=12.4 Hz, 1H), 4.42-4.47 (m, 1H), 3.97-4.07 (m, 2H), 3.45-3.51 (m, 1H), 3.24-3.30 (m, 1H), 3.16-3.20 (m, 1H), 2.87-2.93 (m, 1H), 1.67-1.73 (m, 1H), 1.48-1.52 (m, 1H), 1.35-1.42 (m, 1H), 1.09-1.16 (m, 1H), 0.92-0.97 (m 1H), 0.60 (d, J=6.4 Hz, 3H), 0.52 (d, J=6.4 Hz, 3H); LCMS-ESI (m/z): 619 [M+H]. (S)-3-(naphthalen-1-yl)-2-((phenethoxycarbonyl)amino)propanoic acid (71) To a mixture of amino acid a (545 mg, 2.53 mmol), NaHCO3(320 mg, 3.8 mmol) in THF—H2O (2:3, 20 mL) was added PhCH2CH2OCOCl (0.33 mL, 2.78 mmol) at 0° C. The reaction mixture stirred at rt for 5 h and the acidified with 1 N HCl (8-10 mL) to pH 2.0, organic solvents were then removed under vacuum and the remaining aqueous phase extracted with EtOAc (20 mL×3). The combined organic layers were washed with brine, dried, and concentrated under vacuum to give 71 as a white solid after recrystallization from EtOAc.1H NMR (400 MHz, Chloroform-d) δ 8.12 (d, J=8.4 Hz, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.82 (d, J=8.2 Hz, 1H), 7.54 (q, J=8.1, 7.1 Hz, 2H), 7.15-7.48 (m, 7H), 5.13 (m, 1H), 4.79 (m, 1H), 4.27 (m, 2H), 3.76 (d, J=13.7 Hz, 1H), 3.50 (m, 1H), 2.89 (m, 2H). LCMS-ESI (m/z): 364 [M+H]. Methyl (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (24) To a solution of compound 47 (4.14 g, 14.47 mmol) in dioxane (30 mL) was added HCl (4 M in dioxane, 20 mL). The reaction mixture was stirred at room temperature for 2 h and the solvent removed under vacuum. The residue was carefully dried in vacuo for 5 h and then used directly in the next step without further purification. The residue was dissolved in DCM (100 mL) and Boc-L-Leu-OH (4.02 g, 17.4 mmol), EDCI (3.61 g, 18.8 mmol), HOBt (2.54 g, 18.8 mmol), and DIPEA (10.4 mL, 60 mmol) were added. The solution was stirred at room temperature overnight before solvents were removed under vacuum. EtOAc (200 mL) was then added and the organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine and finally dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 24 was obtained as a white solid (72%). LCMS-ESI (m/z): 400 [M+H]. Methyl (6S,9S,12S)-9-isobutyl-6-(naphthalen-1-ylmethyl)-4,7,10-trioxo-12-(((S)-2-oxopyrrolidin-3-yl)methyl)-1-phenyl-3-oxa-5,8,11-triazatridecan-13-oate (72) A solution of compound 24 (520 mg, 1.3 mmol) and TFA (5 mL) in DCM (20 mL) was stirred at room temperature for 3 h. The solvent was removed under vacuum and the residue used in the next step without further purification. The residue was dissolved in DCM (40 mL) and compound 21 (494 mg, 1.2 mmol mmol), EDCI (310 mg, 1.62 mmol), HOBt (220 mg, 1.62 mmol), and DIPEA (0.88 mL, 5 mmol) were added The solution was stirred at room temperature overnight before solvents were removed under vacuum. EtOAc (100 mL) was then added and the organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3and brine and finally dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 72 was obtained as a white solid (62%).1H NMR (400 MHz, Methanol-d4) δ 8.16 (d, J=8.4 Hz, 1H), 7.89 (d, J=8.1 Hz, 1H), 7.80 (d, J=7.9 Hz, 1H), 7.54 (dt, J=27.5, 7.3 Hz, 2H), 7.44-7.20 (m, 7H), 4.64 (m, 1H), 4.52-4.40 (m, 1H), 4.34-4.11 (m, 3H), 3.72 (d, J=15.2 Hz, 1H), 3.64 (s, 2H), 3.47 (d, J=7.8 Hz, 1H), 3.30-3.18 (m, 1H), 2.91 (t, J=6.9 Hz, 1H), 2.50 (d, J=10.7 Hz, 1H), 2.28 (ddd, J=34.9, 17.0, 7.3 Hz, 2H), 1.92-1.37 (m, 2H), 1.14 (d, J=6.0 Hz, 1H), 0.64 (d, J=12.8 Hz, 6H); LCMS-ESI (m/z): 645 [M+H]. Phenethyl ((S)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (74) To a solution of methyl ester 72 (420 mg, 0.65 mmol) in THF-EtOH (2:3, 10 mL) were added LiBH4(4M, 250 μL, 1 mmol) at 0° C. The resulting mixture was stirred at room temperature for 3 h and then quenched with aq. HCl (1 M). Ethyl acetate (50 mL) was added and the organic phase was further washed with brine and dried over Na2SO4. After removal of the solvent under vacuum compound 73 was obtained as a white solid. To a solution of alcohol 73 in a mixture of DCM (16 mL) and DMSO (4 mL) was added at 0° C. DIPEA (360 μL, 2.07 mmol). The resulting solution was stirred for 30 min before addition of SO3pyridine complex (330 mg, 2.06 mmol). The reaction mixture was stirred overnight at 0° C. and then diluted with EtOAc (100 mL). The organic phase was then washed successively with aq. HCl (1M), sat. aq. NaHCO3, and brine and dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 74 was obtained as a white solid (68%).1H NMR (400 MHz, Methanol-d4) δ 8.16 (d, J=8.4 Hz, 1H), 7.89 (d, J=7.8 Hz, 1H), 7.79 (d, J=8.0 Hz, 1H), 7.64-7.44 (m, 2H), 7.15-7.46 (m, 7H), 4.53-4.41 (m, 1H), 4.31-4.14 (m, 3H), 3.69 (s, 1H), 3.64 (s, 2H), 3.51-3.44 (m, 1H), 3.29-3.14 (m, 2H), 2.90 (q, J=7.2 Hz, 2H), 2.59-2.44 (m, 1H), 2.39-2.14 (m, 1H), 1.91-1.64 (m, 1H), 1.64-1.37 (m, 1H), 0.72-0.51 (m, 6H); LCMS-ESI (m/z): 615 [M+H]. (S)-2-Dodecanamido-3-(naphthalen-1-yl)propanoic acid (75) To a mixture of amino acid 54 (215 mg, 1 mmol), NaHCO3(125 mg, 1.5 mmol) in THF—H2O (2:3, 10 mL) was added C11H23COCl (0.33 mL, 2.78 mmol) at 0° C. The reaction mixture was stirred at it for 2 h and then acidified with 1 N HCl (8-10 mL) to reach pH 2.0. After removal of the solvent the aqueous layer was extracted with EtOAc (20 mL×3), and the combined organic phases were washed with brine, dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 75 (73%) was obtained as a white solid. LCMS-ESI (m/z): 398 [M+H]. Methyl ((S)-2-dodecanamido-3-(naphthalen-1-yl)propanoyl)-L-leucinate (76) To a solution of compound 75 (380 mg, 1.05 mmol) and L-Leu-OMe (230 mg, 1.27 mmol) in DCM (30 mL) were added EDCI (260 mg, 1.35 mmol), HOBt (185 mg, 1.34 mmol), and DIPEA (0.73 mL, 4.2 mmol). The reaction mixture was stirred overnight at room temperature and then H2O (800 mL) and EtOAc (100 mL) were added. The organic phase was washed successively with aq. HCl (1 M, 50 mL), sat. aq. NaHCO3(50 mL) and brine (500 mL), and then dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 76 (350 mg, 65%) was obtained. LCMS-ESI (m/z): 525 [M+H]. ((S)-2-Dodecanamido-3-(naphthalen-1-yl)propanoyl)-L-leucine (77) To a solution of methyl ester 76 (345 mg, 0.66 mmol) in THF-MeOH—H2O (3:1:1, 10 mL) was added a solution of LiOH H2O (32 mg, 1.33 mmol) in H2O (1 mL). The reaction mixture was stirred overnight at room temperature. The volatiles were removed under vacuum to give a colourless residue that was partitioned between EtOAc and aq. HCl (1M). The organic layer was separated, washed successively with aq. HCl (1M) and brine, then dried over Na2SO4, After removal of the solvent under vacuum and recrystallization from EtOAc, compound 77 was obtained as a white solid (430 mg, 96%). LCMS-ESI (m/z): 692 [M+H]. N—((S)-1-(((S)-4-methyl-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxocyclohexyl)propan-2-yl)amino)pentan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)dodecanamide (79) To a solution of methyl ester 4 (466 mg, 0.67 mmol) in THF-EtOH (2:3, 20 mL) was added LiBH4(2 M, 440 μL, 0.88 mmol) at 0° C. The resulting mixture was stirred at room temperature for 3 h and then quenched with aq. HCl (1 M). After addition of ethyl acetate (50 mL) the organic phase was separated and further washed with brine and dried over Na2SO4. Removal of the solvent under vacuum afforded compound 78 as a white solid which was used directly in the next without further purification. To a solution of alcohol 78 (70 mg, 0.11 mmol) in a mixture of DCM (3 mL) and DMSO (1 mL) was added at 0° C. DIPEA (90 μL, 0.5 mmol). The solution was stirred at this temperature for 10 min before addition of SO3pyridine complex (70 mg, 0.43 mmol). The reaction mixture was stirred overnight at 0° C. and then diluted with EtOAc (50 mL). The organic phase was separated and then washed successively with aq. HCl (1M), sat. aq. NaHCO3, and brine, dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 79 was obtained as a white solid (48%).1H NMR (400 MHz, Methanol-d4) δ 8.23 (d, 0.1=6.4 Hz, 1H), 7.87 (d, J=6.8 Hz, 1H), 7.75-7.78 (m, 1H), 7.48-7.59 (m, 2H), 7.37-7.42 (m, 2H), 4.37-4.44 (m, 1H), 3.95-4.04 (m, 1H), 3.67-3.75 (m, 1H), 3.45-3.55 (m, 2H), 3.21-3.28 (m, 2H), 2.27-2.35 (m, 1H), 2.08-2.18 (m, 4H), 1.60-1.82 (m, 7H), 1.19-1.43 (m, 18H), 0.90-0.98 (m, 9H); LCMS-ESI (m/z): 662 [M+H]. Methyl (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-3-((S)-2-oxocyclohexyl) propanoate (80) To a solution of compound 4 (630 mg, 2.1 mmol) in dioxane (8 mL) was added HCl (4M in dioxane, 8 mL). The solution was stirred at room temperature for 3 h and the solvent was removed under reduced pressure. The obtained residue was used in the next step without further purification. To a solution of the residue in DCM (80 mL) was added L-Phe-OH (725 mg, 2.73 mmol), EDCI (564 mg, 2.94 mmol), HOBt (400 mg, 2.94 mmol), and DIPEA (1.54 mL, 8.83 mmol). The reaction mixture was stirred at room temperature overnight before evaporation of the solvent under vacuum. EtOAc (200 mL) was added to the residue and the organic layer was washed successively with aq. HCl (1 M), sat. aq. NaHCO3, brine and dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 80 was obtained as a white solid (72%).1H NMR (400 MHz, Chloroform-d) δ 7.69 (di, J=7.5 Hz, 1H), 7.34-7.16 (m, 7H), 6.36 (s, 1H), 5.19 (di, J=8.4 Hz, 1H), 4.56 (dd, J=19.2, 10.7 Hz, 2H), 3.71 (s, 3H), 3.16 (dd, J=13.9, 5.5 Hz, 1H), 3.03 (dd, J=14.1, 7.0 Hz, 1H), 2.35 (ddd, J=14.0, 11.3, 5.1 Hz, 1H), 2.24 (dq, J=14.9, 5.7 Hz, 1H), 2.15-2.00 (m, 2H), 1.88 (dtd, J=14.3, 7.9, 3.9 Hz, 2H), 1.80-1.61 (m, 1H), 1.60-1.46 (m, 1H), 1.37 (d, J=17.7 Hz, 9H); LCMS-ESI (nm/z): 413 [M+H]. Methyl (5S,8S,11S)-8-benzyl-5-(naphthalen-1-ylmethyl)-3,6,9-trioxo-11-(((S)-2-oxopiperidin-3-yl)methyl)-1-phenyl-2-oxa-4,7,10-triazadodecan-12-oate (81) A solution of compound 80 (592 mg, 1.28 mmol) and TFA (5 mL) in DCM (15 mL) was stirred at room temperature for 2 h. The solvent was removed under vacuum and the residue used in the next step without further purification. The residue was dissolved in DCM (50 mL) and compound 9 (540 mg, 1.54 mmol mmol), EDCI (320 mg, 1.68 mmol), HOBt (225 mg, 1.67 mmol), and DIPEA (0.9 mL, 5.2 mmol) were added. The solution was stirred at room temperature overnight before removal of the solvent under vacuum and addition of EtOAc (150 mL). The organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3, brine and dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 81 was obtained as a white solid (62%). LCMS-ESI (m/z): 679 [M+H]. Benzyl ((S)-1-(((S)-1-(((S)-1-hydroxy-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (82) To a solution of methyl ester 81 (870 mg, 1.28 mmol) in THF-EtOH (2:3, 30 mL) was added LiBH4(2 M, 1 mL, 2 mmol) at 0° C. The resulting mixture was stirred at room temperature for 3 h and then quenched with aq. HCl (1 M). Ethyl acetate (50 mL) was added and the organic phase was washed with brine and dried over Na2SO4. Removal of the solvent under vacuum gave 82 as a white solid which was used in the next step without further purification.1H NMR (400 MHz, Methanol-d4) δ 8.14 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.3 Hz, 2H), 7.74 (d, J=8.1 Hz, 1H), 7.49 (dq, J=14.6, 7.2 Hz, 2H), 7.27 (tdd, J=16.9, 11.8, 7.9 Hz, 12H), 4.96 (d, J=4.2 Hz, 2H), 4.58 (dt, J=19.6, 7.1 Hz, 2H), 4.02-3.89 (m, 1H), 3.55 (dd, J=14.3, 5.4 Hz, 1H), 3.48-3.06 (m, 4H), 2.98 (dd, J=13.6, 8.0 Hz, 1H), 2.24 (h, J=5.7, 4.5 Hz, 1H), 1.98 (dtq, J=12.7, 6.3, 3.5 Hz, 3H), 1.79-1.53 (m, 4H), 1.50-1.35 (m, 1H); LCMS-ESI (m/z): 651 [M+H]. Benzyl ((S)-3-(naphthalen-1-yl)-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-yl)amino)propan-2-yl)carbamate (83) To a solution of 82 (70 mg, 0.11 mmol) in a mixture of DCM (3 mL) and DMSO (1 mL) was added at 0° C. DIPEA (90 μL, 0.5 mmol). After 10 minutes at this temperature, SO3pyridine complex (70 mg, 0.43 mmol) was added and the reaction mixture was stirred overnight at 0° C. After addition of EtOAc (50 mL), the organic layer was washed successively with aq. HCl (1M), sat. aq. NaHCO3, brine and dried over Na2SO4. After removal of the solvent under vacuum and recrystallization from EtOAc, compound 83 as a white solid (56%).1H NMR (400 MHz, Methanol-d4) δ 9.22 (s, 0.3H), 8.22-8.07 (m, 1H), 7.87 (d, J=8.1 Hz, 1H), 7.76 (d, J=8.1 Hz, 1H), 7.51 (dq, J=14.6, 7.1 Hz, 2H), 7.39-7.12 (m, 12H), 5.06-4.94 (m, 2H), 4.69-4.46 (m, 1H), 4.35-4.21 (m, 1H), 3.98 (d, J=12.3 Hz, 1H), 3.57 (dt, J=14.2, 7.7 Hz, 1H), 3.28-2.95 (m, 5H), 2.29-1.93 (m, 1H), 1.81 (d, J=14.4 Hz, 1H), 1.66 (s, 1H), 1.46 (q, J=11.5, 10.3 Hz, 1H), 1.35-1.18 (m, 1H); LCMS-ESI (m/z): 649 [M+H]. Benzyl ((S)-1-(((S)-1-(((S)-1-chloro-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl)carbamate (84) A solution of 82 (32 mg, 0.05 mmol), Ph3P (50 mg, 0.19 mmol), CCl4(0.1 mL) in 1,2-dichloroethane (0.5 mL) was heated at 80° C. for 3 min under microwave irradiation. After removal of the solvent under vacuum, the residue was purified by preparative TLC to give 84 (78%) as a white solid.1H NMR (400 MHz, Methanol-d4) δ 8.15 (d, J=8.1 Hz, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.76 (d, J=8.1 Hz, 1H), 7.51 (dq, J=15.3, 7.5 Hz, 2H), 7.27 (tdd, J=20.7, 9.9, 5.4 Hz, 13H), 4.96 (d, J=19.5 Hz, 2H), 4.57 (dq, J=21.7, 6.2, 5.1 Hz, 2H), 4.19-4.09 (m, 1H), 3.67-3.37 (m, 2H), 3.27-2.96 (m, 5H), 2.27 (s, 1H), 2.19-1.88 (m, 2H), 1.85-1.52 (m, 3H); LCMS-ESI (m/z): 669 [M+H]. Benzyl ((S)-3-(naphthalen-1-yl)-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxo-3-((S)-2-oxopiperidin-3-yl)propan-2-yl)amino)-3-phenylpropan-2-yl)amino)propan-2-yl)carbamate (85) To a solution of 82 (41 mg, 0.06 mmol) and NEt3(30 μL, 0.2 mmol) in CH2Cl2(5 mL) was added methanesulfonyl chloride (9 μL, 0.09 mmol) at 0° C. The reaction mixture was stirred for 1 h at this temperature. After removal of the volatile components under reduced pressure, the residue was subjected to preparative TLC to give 85 as a white solid.1H NMR (400 MHz, Methanol-d4) δ 8.20-8.05 (m, 2H), 7.87 (d, J=7.9 Hz, 1H), 7.77 (t, J=8.2 Hz, 1H), 7.51 (dd, J=13.4, 7.6 Hz, 2H), 7.27 (tt, J=17.2, 7.0 Hz, 6H), 5.06-4.96 (m, 2H), 4.64-4.44 (m, 1H), 4.07 (q, 0.1=7.7, 5.7 Hz, 1H), 3.65-3.49 (m, 1H), 3.23 (d, 0.1=5.0 Hz, 3H), 3.04 (s, 3H), 2.37-2.21 (m, 2H), 2.22-1.94 (m, 1H). Example 2 Cellular Toxicity Assays The toxicity of the compounds was assessed in Vero, human PBM, CEM (human lymphoblastoid), MT-2, and HepG2 cells, as described previously (see Schinazi R. F., Sommadossi J.-P., Saalmann V., Cannon D. L., Xie M.-Y., Hart G. C., Smith G. A. & Hahn E. F.Antimicrob. Agents Chemother.1990, 34, 1061-67). Cycloheximide was included as positive cytotoxic control, and untreated cells exposed to solvent were included as negative controls. The cytotoxicity CC50was obtained from the concentration-response curve using the median effective method described previously (see Chou T.-C. & Talalay P.Adv. Enzyme Regul.1984, 22, 27-55; Belen'kii M. S. & Schinazi R. F. Antiviral Res. 1994, 25, 1-11). The results are shown in Table 1 below: TABLE 1Cytotoxicity, CC50, μM (% inhibition)Cytotoxicity; CC50(μM)CompoundPBMCEMVEROHuh711>100>100>100>1019243220>1023>10039>100>1029>10038>100>10355235>1006036459>1008374418>1001138186221339392111467>10064>100ND8348.717.041.2ND8475.026.651.7ND In the table, Compounds 11, 19, 28 and 29 have the following structures: Compounds 35, 36, 37, 38, 39, 67, 83, and 84 have the following structures: Example 3 Mitochondrial Toxicity Assays in HepG2 Cells: i) Effect of Compounds on Cell Growth and Lactic Acid Production: The effect on the growth of HepG2 cells was determined by incubating cells in the presence of 0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM drug. Cells (5×104 per well) were plated into 12-well cell culture clusters in minimum essential medium with nonessential amino acids supplemented with 10% fetal bovine serum, 1% sodium pyruvate, and 1% penicillin/streptomycin and incubated for 4 days at 37° C. At the end of the incubation period the cell number was determined using a hemocytometer. Also taught by Pan-Zhou X-R, Cui L, Zhou X-J, Sommadossi J-P, Darley-Usmer VM. “Differential effects of antiretroviral nucleoside analogs on mitochondrial function in HepG2 cells,” Antimicrob. Agents Chemother. 2000; 44:496-503. To measure the effects of the compounds on lactic acid production, HepG2 cells from a stock culture were diluted and plated in 12-well culture plates at 2.5×104 cells per well. Various concentrations (0 μM, 0.1 μM, 1 μM, 10 μM and 100 μM) of compound were added, and the cultures were incubated at 37° C. in a humidified 5% CO2atmosphere for 4 days. At day 4, the number of cells in each well was determined and the culture medium collected. The culture medium was then filtered, and the lactic acid content in the medium was determined using a colorimetric lactic acid assay (Sigma-Aldrich). Since lactic acid product can be considered a marker for impaired mitochondrial function, elevated levels of lactic acid production detected in cells grown in the presence of test compounds would indicate a drug-induced cytotoxic effect. ii) Effect of Compounds on Mitochondrial DNA Synthesis: A real-time PCR assay to accurately quantify mitochondrial DNA content has been developed (see Stuyver L J, Lostia S, Adams M, Mathew J S, Pai B S, Grier J, Tharnish P M, Choi Y, Chong Y, Choo H, Chu C K, Otto M J, Schinazi R F. Antiviral activities and cellular toxicities of modified 2′,3′-dideoxy-2′,3′-didehydrocytidine analogs. Antimicrob. Agents Chemother. 2002, 46: 3854-60). This assay was used in all studies described in this application that determine the effect of compounds on mitochondrial DNA content. In this assay, low-passage-number HepG2 cells were seeded at 5,000 cells/well in collagen-coated 96-well plates. Test compounds were added to the medium to obtain final concentrations of 0 μM, 0.1 μM, 10 μM and 100 μM. On culture day 7, cellular nucleic acids were prepared by using commercially available columns (RNeasy 96 kit; Qiagen). These kits co-purify RNA and DNA, and hence, total nucleic acids are eluted from the columns. The mitochondrial cytochrome c oxidase subunit II (COXII) gene and the β-actin or rRNA gene were amplified from 5 μl of the eluted nucleic acids using a multiplex Q-PCR protocol with suitable primers and probes for both target and reference amplifications. For COXII the following sense, probe and antisense primers were used, respectively: 5′-TGCCCGCCATCATCCTA-3′,5′-tetrachloro-6-carboxyfluorescein-(SEQ ID No. 1) TCCTCATCGCCCT-CCCATCCC-TAMRA-3′ (SEQ ID No. 2) and 5′-CGTCTGTTTATGTAAAGGATGCGT-3′ (SEQ ID No. 3). For exon 3 of the β-actin gene (GenBank accession number E01094) the sense, probe, and antisense primers are 5′-GCGCGGCTACAGCTTCA-(SEQ ID No. 4) 3′,5′-6-FAMCACCACGGCCGAGCCGGGATAMRA-3′ (SEQ ID No. 5) and 5′-TCTCCTTAATGTCACGCACGAT-3′ (SEQ ID No. 6), respectively. The primers and probes for the rRNA gene are commercially available from Applied Biosystems. Since equal amplification efficiencies are obtained for all genes, the comparative CT method was used to investigate potential inhibition of mitochondrial DNA synthesis. The comparative CT method uses arithmetic formulas in which the amount of target (COXII gene) is normalized to the amount of an endogenous reference (the β-actin or rRNA gene) and is relative to a calibrator (a control with no drug at day 7). The arithmetic formula for this approach is given by 2-ΔΔCT, where ΔΔCT is (CT for average target test sample-CT for target control)-(CT for average reference test-CT for reference control) (see Johnson M R, K Wang, J B Smith, M J Heslin, R B Diasio. Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal. Biochem. 2000; 278:175-184). A decrease in mitochondrial DNA content in cells grown in the presence of drug indicated mitochondrial toxicity. Example 4 Mitochondrial Toxicity Assays in Neuro2A Cells To estimate the potential of the compounds of this invention to cause neuronal toxicity, mouse Neuro2A cells (American Type Culture Collection 131) can be used as a model system (see Ray A S, Hernandez-Santiago B I, Mathew J S, Murakami E, Bozeman C, Xie M Y, Dutschman G E, Gullen E, Yang Z, Hurwitz S, Cheng Y C, Chu C K, McClure H, Schinazi R F, Anderson K S. Mechanism of anti-human immunodeficiency virus activity of beta-D-6-cyclopropylamino-2′,3′-didehydro-2′,3′-dideoxyguanosine.Antimicrob. Agents Chemother.2005, 49, 1994-2001). The concentrations necessary to inhibit cell growth by 50% (CCso) can be measured using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide dye-based assay, as described. Perturbations in cellular lactic acid and mitochondrial DNA levels at defined concentrations of drug can be carried out as described above. ddC and AZT can be used as control nucleoside analogs. Example 5 Assay for Bone Marrow Cytotoxicity Primary human bone marrow mononuclear cells can be obtained commercially from Cambrex Bioscience (Walkersville, MD). CFU-GM assays is carried out using a bilayer soft agar in the presence of 50 units/mL human recombinant granulocyte/macrophage colony-stimulating factor, while BFU-E assays used a ethylcellulose matrix containing 1 unit/mL erythropoietin (see Sommadossi J P, Carlisle R. Toxicity of 3′-azido-3′-deoxythymidine and 9-(1,3-dihydroxy-2-propoxymethyl) guanine for normal human hepatopoietic progenitor cells in vitro. Antimicrob. Agents Chemother. 1987; 31: 452-454; Sommadossi, J P, Schinazi, R F, Chu, C K, and Xie, M Y. Comparison of cytotoxicity of the (−) and (+) enantiomer of 2′,3′-dideoxy-3′-thiacytidine in normal human bone marrow progenitor cells. Biochem. Pharmacol. 1992; 44:1921-1925). Each experiment can be performed in duplicate in cells from three different donors. AZT is used as a positive control. Cells can be incubated in the presence of the compound for 14-18 days at 37° C. with 5% CO2, and colonies of greater than 50 cells can be counted using an inverted microscope to determine the IC50. The 50% inhibitory concentration (IC50) can be obtained by least-squares linear regression analysis of the logarithm of drug concentration versus BFU-E survival fractions. Statistical analysis can be performed with Student's t test for independent non-paired samples. Example 6 Anti-Norovirus Activity Norwalk virus replicon assays were performed as reported by Constantini et al. (Antivir Ther2012, 17, 981-991). HG23 cells (derived from Huh-7 cells) containing NoV replicon RNA are seeded at a density of 3,000 cells/well in 96-well plates and incubated at 37° C. and 5% CO2overnight. Compounds were tested at concentrations ranging from 0.1 to 100 μM. Compounds were added in triplicate to 80 to 90% confluent monolayers and incubated at 37° C. and 5% CO2. Untreated cells were included in each plate. Following five days incubation (37° C., 5% CO2), total cellular RNA was isolated with RNeasy96 extraction kit from Qiagen. Replicon RNA and an internal control (TaqMan rRNA control reagents, Applied Biosystems) were amplified in a single step. The median effective concentrations (EC50) ranges of several of the compounds described herein against NoV are shown in Table 3: TABLE 3Anti-NoV activity (μM)CompoundEC50EC90110.72.4190.070.27230.070.33290.080.46674.33>20830.330.9184>10ND Example 12 The ability of these compounds to inhibit the NoV, specifically Minerva virus protease catalytic Cys139 covalently (IC50and Ki) was determined with an enzyme kinetic assay. NoV strains, specifically GII.4 such as the Minerva virus are responsible for causing the majority (˜80%) of infections in humans. The activity of the inhibitors was evaluated by monitoring the cleavage of a FRET substrate every one minute for 20 minutes (excitation/emission: 488/520 nm) using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale CA). Serial dilutions of each inhibitor were incubated with enzyme for 90 minutes at 37° C. before addition of the FRET substrate to ensure complete inactivation. Commercially available protease inhibitors chymostatin and rupintrivir were used as controls. TABLE 4CompoundIC50(μM)Ki(μM)110.112 ± 0.0250.427 ± 0.109190.150 ± 0.0021.19 ± 0.444230.204 ± 0.0091.59 ± 0.050290.140 ± 0.0170.670 ± 0.019350.167 ± 0.0050.858 ± 0.032361.17 ± 0.3333.60 ± 0.501372.63 ± 1.0414.03 ± 5.5538>10ND39>10ND64>100ND6730.0 ± 1.8>100830.482 ± 0.077.095 ± 5.58384>100NDchymostatin13.71.6 ± 1.0rupintrivir23.68.2 ± 2.3 Example 13 Norovirus GI.1 (Norwalk virus) protease were tested for enzymatic activity using a fluorescence resonance energy transfer (FRET) based enzyme assay. Norovirus GI.1 represents 5 to 10% of the clinical isolates. The FRET kinetic enzyme assays were performed as follows. The purified viral protease was diluted in reaction buffer (50 mM HEPES, pH 8.0, 120 mM NaCl, 0.4 mM EDTA, 20% glycerol, and 4 mM DTT) to a final concentration of 128 nM. Each reaction was initiated by addition of FRET substrate [(HiLyte Fluor 488) -DFELQGPK-(QXL520)]. To determine kinetic parameters, the FRET substrate was serially diluted to final concentrations of 100 μM to 49 nM and added to the reaction. The final reaction volume was 100 μL. The fluorescence emitted by substrate cleavage was monitored by a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA) at a 488 nm excitation wavelength with an emission wavelength of 520 nm. Readings were taken every minute for 20 minutes, and the reactions were performed at 37° C. In order to convert RFU into μM, a standard curve was created by measuring fluorescence of free HiLyte Fluor 488, which was serially diluted from 250 nM to 3.9 nM. All data were plotted and analyzed with GraphPad Prism v. 6.07. TABLE 5CompoundIC50(μM)Ki(μM)110.044 ± 0.0080.123 ± 0.006190.080 ± 0.0230.155 ± 0.008230.096 ± 0.0170.528 ± 0.167290.112 ± 0.0130.350 ± 0.140350.096 ± 0.0110.465 ± 0.201360.593 ± 0.1241.697 ± 0.332370.654 ± 0.2852.241 ± 0.43838>10ND39>10ND6410ND67>10ND830.084 ± 0.0190.256 ± 0.05784>100ND Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties for all purposes. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying FIGURES. Such modifications are intended to fall within the scope of the appended claims. | 152,930 |
11859015 | DETAILED DESCRIPTION OF THE INVENTION This invention provides immunogenic peptides, and compositions and vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering one or more immunogenic peptides. This invention provides synthetic peptides and methods of treating, reducing the incidence of, and inducing immune responses against a WT1-expressing cancer, comprising immunogenic peptides. The WT1 molecule from which the peptides of the present invention are derived has, in another embodiment, the sequence: (SEQ ID NO: 51)1SRQRPHPGAL RNPTACPLPH FPPSLPPTHS PTHPPRAGTA AQAPGPRRLL51AAILDFLLLQ DPASTCVPEP ASQHTLRSGP GCLQQPEQQG VRDPGGIWAK101LGAAEASAER LQGRRSRGAS GSEPQQMGSD VRDLNALLPA VPSLGGGGGC151ALPVSGAAQW APVLDFAPPG ASAYGSLGGP APPPAPPPPP PPPPHSFIKQ201EPSWGGAEPH EEQCLSAFTV HFSGQFTGTA GACRYGPFGP PPPSQASSGQ251ARMFPNAPYL PSCLESQPAI RNQGYSTVTF DGTPSYGHTP SHHAAQFPNH301SFKHEDPMGQ QGSLGEQQYS VPPPVYGCHT PTDSCTGSQA LLLRTPYSSD351NLYQMTSQLE CMTWNQMNLG ATLKGVAAGS SSSVKWTEGQ SNHSTGYESD401NHTTPILCGA QYRIHTHGVF RGIQDVRRVP GVAPTLVRSA SETSEKRPFM451CAYPGCNKRY FKLSHLQMHS RKHTGEKPYQ CDFKDCERRF SRSDQLKRHQ501RRHTGVKPFQ CKTCQRKFSR SDHLKTHTRT HTGKTSEKPF SCRWPSCQKK551FARSDELVRH HNMHQRNMTK LQLAL. The foregoing sequence of the WT-1 protein is that published by Gessler et al. (Gessler M, Poustka A, Cavenee W, Neve R L, Orkin S H, Bruns G A. Homozygous deletion in Wilms tumours of a zinc-finger gene identified by chromosome jumping.Nature.1990; 343(6260):774-778. Prepublished on 1990 Feb. 22 as DOI 10.1038/343774a0.) which comprises 575 amino acids and includes the first 126 amino acids in the N-terminus missing in the (Exon 5+, KTS+) isoform of WT-116. In another embodiment, the WT1 sequence is (GenBank Accession number AY245105; SEQ ID NO: 52)MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMQGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL. In another embodiment, the WT1 molecule has the sequence: (GenBank Accession number NM_000378;SEQ ID NO: 53)AAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL. In another embodiment, the WT1 molecule has the sequence: (GenBank Accession number NP_077742;SEQ ID No: 54)MQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL. In another embodiment, the WT1 protein has the sequence set forth in GenBank Accession #NM_024426. In other embodiments, the WT1 protein has or comprises one of the sequences set forth in one of the following sequence entries: NM_024425, NM_024424, NM_000378, 595530, D13624, D12496, D 12497, or X77549. In another embodiment, the WT1 protein has any other WT1 sequence known in the art. This invention provides peptides, compositions, and immunogenic compositions such as vaccines comprising immunogenic peptides, and methods of treating, reducing the incidence of, and inducing immune responses to a WT1-expressing cancer, comprising administering immunogenic peptides. In some cases, the peptides described herein are derived from peptides that are native sequences of WT1, and may be referred to herein as WT1-derived peptides or as a WT1 peptide. In one embodiment, the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In one embodiment, the present invention provides an isolated HLA class I binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence consisting of any one of the sequences SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In one embodiment, the present invention provides an isolated WT1 peptide having an amino acid (AA) sequence comprising any one of the sequences SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55, or a fragment thereof. In one embodiment, the present invention provides an isolated HLA class I binding WT1-derived peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In one embodiment, the present invention provides an isolated HLA class II binding WT1 peptide having an amino acid (AA) sequence comprising of any one of the sequences SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) a peptide selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class I binding peptide selected from SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In another embodiment, the present invention provides a composition comprising (a) an antigen-presenting cell and (b) an HLA class II binding peptide selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a vaccine comprising one or more peptides of SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48. In another embodiment, the present invention provides a vaccine comprising one or more HLA class II binding peptides selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a vaccine comprising one or more HLA class I binding peptides selected from SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and one or more HLA class II binding peptides selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer. In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject. In another embodiment, the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In one embodiment, the fragment of a WT1 protein consists of a peptide or comprises a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer. In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a WT1 peptide or vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject. In another embodiment, the present invention provides a method of inducing an anticancer immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment the fragment consists of a peptide or comprises a peptide from among SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55. In another embodiment, the present invention provides a method of treating a subject with a cancer, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, the present invention provides a method of reducing an incidence of a cancer, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; (b) a fragment of a WT protein; (c) a nucleotide molecule encoding a WT1 protein; or (d) a nucleotide molecule encoding a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In one embodiment, the fragment of a WT1 protein is a peptide from among SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, the cancer is a WT1-expressing cancer. In one embodiment, the WT1-expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms' tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematological cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is a gastric cancer. In another embodiment, the WT1-expressing cancer is a colon cancer. In another embodiment, the WT1-expressing cancer is a lung cancer. In another embodiment, the WT1-expressing cancer is a breast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is an ovarian cancer. In another embodiment, the WT1-expressing cancer is a uterine cancer. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma. In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma. In another embodiment, the present invention provides a composition comprising an isolated peptide of the invention in combination with at least 1 additional WT1-derived peptide. In certain embodiments, a composition comprising at least 2 different isolated peptides of the present invention is provided. In certain embodiments, a composition comprising at least 3 or at least 4 different isolated peptides of the present invention is provided. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the composition of the present invention is a vaccine. In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a peptide or composition of the present invention, thereby treating a subject with a WT1-expressing cancer. In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject, the method comprising administering to the subject a peptide or composition of the present invention, thereby reducing the incidence of a WT1-expressing cancer, or its relapse, in a subject. In another embodiment, the present invention provides a method of inducing formation and proliferation of a WT1 protein-specific CTL, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of a WT1 protein-specific CTL. This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect. In another embodiment, the present invention provides a method of inducing formation and proliferation of (a) a WT1 protein-specific CD8+lymphocyte; or (b) a CD4+lymphocyte specific for the WT1 protein, or the combination thereof, the method comprising contacting a lymphocyte population with a peptide or composition of the present invention, thereby inducing formation and proliferation of (a) a WT1 protein-specific CD8+lymphocyte; or (b) a CD4+lymphocyte specific for the WT1 protein; or a combination thereof. This method can be conducted in vitro, ex vivo or in vivo. When conducted in vitro or ex vivo, these CTL can then be infused into a patient for therapeutic effect. “Peptide,” in another embodiment of methods and compositions of the present invention, refers to a compound of subunit AA connected by peptide bonds. In another embodiment, the peptide comprises an AA analogue. In another embodiment, the peptide comprises a peptidomimetic. The different AA analogues and peptidomimetics that can be included in the peptides of methods and compositions of the present invention are enumerated hereinbelow. The subunits are, in another embodiment, linked by peptide bonds. In another embodiment, the subunit is linked by another type of bond, e.g. ester, ether, etc. Each possibility represents a separate embodiment of the present invention. The unaltered peptides of the present invention (as described both above and below) are referred to collectively herein as “WT1 peptides.” Each of the embodiments enumerated below for “WT1 peptides” applies to unaltered WT1 peptides and HLA class I and class II heteroclitic peptides of the present invention. Each possibility represents a separate embodiment of the present invention. In another embodiment, a WT1 peptide of the present invention binds to an HLA class I molecule or a class II molecule. In another embodiment the peptide binds to both a class I and a class II molecule. In another embodiment, the HLA class II molecule is an HLA-DRB molecule. In another embodiment, the HLA class II-molecule is an HLA-DRA molecule. In another embodiment, the HLA molecule is an HLA-DQA1 molecule. In another embodiment, the HLA molecule is an HLA-DQB1 molecule. In another embodiment, the HLA molecule is an HLA-DPA1 molecule. In another embodiment, the HLA molecule is an HLA-DPB 1 molecule. In another embodiment, the HLA molecule is an HLA-DMA molecule. In another embodiment, the HLA molecule is an HLA-DMB molecule. In another embodiment, the HLA molecule is an HLA-DOA molecule. In another embodiment, the HLA molecule is an HLA-DOB molecule. In another embodiment, the HLA molecule is any other HLA class Il-molecule known in the art. Each possibility represents a separate embodiment of the present invention. In another embodiment, the HLA class I molecule whose binding motif is contained in or comprising a peptide of the present invention is, in another embodiment, an HLA-A molecule. In another embodiment, the HLA class I molecule is an HLA-B molecule. In another embodiment, the HLA class I molecule is an HLA-C molecule. In another embodiment, the HLA class I molecule is an HLA-A0201 molecule. In another embodiment, the molecule is HLA A1. In another embodiment, the HLA class I molecule is HLA A2. In another embodiment, the HLA class I molecule is HLA A2.1. In another embodiment, the HLA class I molecule is HLA A3. In another embodiment, the HLA class I molecule is HLA A3.2. In another embodiment, the HLA class I molecule is HLA A11. In another embodiment, the HLA class I molecule is HLA A24. In another embodiment, the HLA class I molecule is HLA B7. In another embodiment, the HLA class I molecule is HLA B27. In another embodiment, the HLA class I molecule is HLA B8. Each possibility represents a separate embodiment of the present invention. In another embodiment, the HLA class I molecule-binding WT1-derived peptide of methods and compositions of the present invention binds to a superfamily of HLA class I molecules. In another embodiment, the superfamily is the A2 superfamily. In another embodiment, the superfamily is the A3 superfamily. In another embodiment, the superfamily is the A24 superfamily. In another embodiment, the superfamily is the B7 superfamily. In another embodiment, the superfamily is the B27 superfamily. In another embodiment, the superfamily is the B44 superfamily. In another embodiment, the superfamily is the C1 superfamily. In another embodiment, the superfamily is the C4 superfamily. In another embodiment, the superfamily is any other superfamily known in the art. Each possibility represents a separate embodiment of the present invention. In another embodiment, the HLA molecule is a A0101, A0201, A0203, A2402, A6901, B0702, A3101, B3501, B3503, B3508, B3802, B3801, B3901, B4001, B4402, B4701, B5701, C0401, C1701, DRB10101, DRB10402, DRB10402, DRB10401 or DRB11104 molecule. In another embodiment, the peptides of SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55, bind to the HLA class I or class II molecules described for each peptide in the Tables below. In another embodiment the HLA class I peptides consist of or comprise SEQ ID NO:6, 7, 30, 31, 32, 33, 34, 35, 36, 37, 38, 41, 42, 47 and 48, and the HLA class II peptide consists of or comprises SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 39, 43, 44, 46, 49, 50 and 55, and bind to the corresponding HLA molecule or molecules indicated for each peptide in the tables below. In one embodiment, certain peptides can bind to more than one HLA allele. In another embodiment, a modification of a peptide of the invention is provided. In one embodiment the modification comprises at least one heteroclitic amino acid change, also referred to as a mutation or mutated, or an anchor residue mutation (see below). An HLA class I molecule binding motif of a modified peptide of the present invention exhibits an increased affinity for the HLA class I molecule, relative to the unmutated counterpart of the peptide. In another embodiment, the point mutation increases the affinity of the isolated, mutated WT1-derived peptide for the HLA class I molecule. In another embodiment, the increase in affinity is relative to the affinity (for the same HLA class I molecule) of the isolated, unmutated WT1-derived peptide wherefrom the isolated, mutated WT1-derived peptide was derived. Each possibility represents a separate embodiment of the present invention. In another embodiment, a WT1 peptide of methods and compositions of the present invention is so designed as to exhibit affinity for an HLA molecule. In another embodiment, the affinity is a high affinity, as described herein. HLA molecules, known in another embodiment as major histocompatibility complex (MHC) molecules, bind peptides and present them to immune cells. Thus, in another embodiment, the immunogenicity of a peptide is partially determined by its affinity for HLA molecules. HLA class I molecules interact with CD8 molecules, which are generally present on cytotoxic T lymphocytes (CTL). HLA class II molecules interact with CD4 molecules, which are generally present on helper T lymphocytes. In another embodiment, a peptide of the present invention is immunogenic. In another embodiment, “immunogenic” refers to an ability to stimulate, elicit or participate in an immune response. In another embodiment, the immune response elicited is a cell-mediated immune response. In another embodiment, the immune response is a combination of cell-mediated and humoral responses. In another embodiment, T cells that bind to the MHC molecule-peptide complex become activated and induced to proliferate and lyse cells expressing a protein comprising the peptide. T cells are typically initially activated by “professional” antigen presenting cells (“APC”; e.g. dendritic cells, monocytes, and macrophages), which present costimulatory molecules that encourage T cell activation as opposed to anergy or apoptosis. In another embodiment, the response is heteroclitic, as described herein, such that the CTL lyses a neoplastic cell expressing a protein which has an AA sequence homologous to a peptide of this invention, or a different peptide than that used to first stimulate the T cell. In another embodiment, an encounter of a T cell with a peptide of this invention induces its differentiation into an effector and/or memory T cell. Subsequent encounters between the effector or memory T cell and the same peptide, or, in another embodiment, with a related peptide of this invention, leads to a faster and more intense immune response. Such responses are gauged, in another embodiment, by measuring the degree of proliferation of the T cell population exposed to the peptide. In another embodiment, such responses are gauged by any of the methods enumerated hereinbelow. In another embodiment, the peptides of methods and compositions of the present invention bind an HLA class II molecule with high affinity. In other embodiments, the HLA class II molecule is any HLA class II molecule enumerated herein. Each possibility represents a separate embodiment of the present invention. In another embodiment, derivatives of peptides of methods and compositions of the present invention bind an HLA class I molecule with high affinity. In other embodiments, the MHC class I molecule is any MHC class I molecule enumerated herein. Each possibility represents a separate embodiment of the present invention. In another embodiment, a peptide of methods and compositions of the present invention binds an HLA class II molecule with significant affinity, while a peptide derived from the original peptide binds an HLA class I molecule with significant affinity. In another embodiment, “affinity” refers to the concentration of peptide necessary for inhibiting binding of a standard peptide to the indicated MHC molecule by 50%. In another embodiment, “high affinity” refers to an affinity is such that a concentration of about 500 nanomolar (nM) or less of the peptide is required for 50% inhibition of binding of a standard peptide. In another embodiment, a concentration of about 400 nM or less of the peptide is required. In another embodiment, the binding affinity is 300 nM. In another embodiment, the binding affinity is 200 nM. In another embodiment, the binding affinity is 150 nM. In another embodiment, the binding affinity is 100 nM. In another embodiment, the binding affinity is 80 nM. In another embodiment, the binding affinity is 60 nM. In another embodiment, the binding affinity is 40 nM. In another embodiment, the binding affinity is 30 nM. In another embodiment, the binding affinity is 20 nM. In another embodiment, the binding affinity is 15 nM. In another embodiment, the binding affinity is 10 nM. In another embodiment, the binding affinity is 8 nM. In another embodiment, the binding affinity is 6 nM. In another embodiment, the binding affinity is 4 nM. In another embodiment, the binding affinity is 3 nM. In another embodiment, the binding affinity is 2 nM. In another embodiment, the binding affinity is 1.5 nM. In another embodiment, the binding affinity is 1 nM. In another embodiment, the binding affinity is 0.8 nM. In another embodiment, the binding affinity is 0.6 nM. In another embodiment, the binding affinity is 0.5 nM. In another embodiment, the binding affinity is 0.4 nM. In another embodiment, the binding affinity is 0.3 nM. In another embodiment, the binding affinity is less than 0.3 nM. In another embodiment, “affinity” refers to a measure of binding strength to the MHC molecule. In another embodiment, affinity is measured using a method known in the art to measure competitive binding affinities. In another embodiment, affinity is measured using a method known in the art to measure relative binding affinities. In another embodiment, the method is a competitive binding assay. In another embodiment, the method is radioimmunoassay or RIA. In another embodiment, the method is BiaCore analyses. In another embodiment, the method is any other method known in the art. In another embodiment, the method yields an IC50 in relation to an IC50 of a reference peptide of known affinity. Each type of affinity and method of measuring affinity represents a separate embodiment of the present invention. In another embodiment, “high affinity” refers to an IC50 of 0.5-500 nM. In another embodiment, the IC50 is 1-300 nM. In another embodiment, the IC50 is 1.5-200 nM. In another embodiment, the IC50 is 2-100 nM. In another embodiment, the IC50 is 3-100 nM. In another embodiment, the IC50 is 4-100 nM. In another embodiment, the IC50 is 6-100 nM. In another embodiment, the IC50 is 10-100 nM. In another embodiment, the IC50 is 30-100 nM. In another embodiment, the IC50 is 3-80 nM. In another embodiment, the IC50 is 4-60 nM. In another embodiment, the IC50 is 5-50 nM. In another embodiment, the IC50 is 6-50 nM. In another embodiment, the IC50 is 8-50 nM. In another embodiment, the IC50 is 10-50 nM. In another embodiment, the IC50 is 20-50 nM. In another embodiment, the IC50 is 6-40 nM. In another embodiment, the IC50 is 8-30 nM. In another embodiment, the IC50 is 10-25 nM. In another embodiment, the IC50 is 15-25 nM. Each affinity and range of affinities represents a separate embodiment of the present invention. In another embodiment, a peptide of methods and compositions of the present invention binds to a superfamily of HLA molecules. Superfamilies of HLA molecules share very similar or identical binding motifs. In another embodiment, the superfamily is a HLA class I superfamily. In another embodiment, the superfamily is a HLA class II superfamily. Each possibility represents a separate embodiment of the present invention. The terms “HLA-binding peptide,” “HLA class I molecule-binding peptide,” and “HLA class II molecule-binding peptide” refer, in another embodiment, to a peptide that binds an HLA molecule with measurable affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with high affinity. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to activate a T cell precursor. In another embodiment, the terms refer to a peptide that binds an HLA molecule with sufficient affinity to mediate recognition by a T cell. The HLA molecule is, in other embodiments, any of the HLA molecules enumerated herein. Each possibility represents a separate embodiment of the present invention. “Heteroclitic” refers, in another embodiment, to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived (e.g. the peptide not containing the anchor residue or other residue mutations). In another embodiment, “original peptide” refers to a peptide of the present invention. In another embodiment, “heteroclitic” refers to a peptide that generates an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the improved peptide was derived (e.g. the peptide not containing the anchor residue mutations). In another embodiment, a “heteroclitic” immune response refers to an immune response that recognizes the original peptide from which the heteroclitic peptide was derived, wherein the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response generated by vaccination with the original peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response substantially equal to the response to vaccination with the original peptide. In another embodiment, the magnitude of the immune response generated by vaccination with the heteroclitic peptide is greater than the immune response less than the response to vaccination with the original peptide. In another embodiment, a heteroclitic peptide of the present invention is an HLA class I heteroclitic peptide. Methods for identifying HLA class I and class II residues, and for improving HLA binding by mutating the residues, are well known in the art, as described below. Each possibility represents a separate embodiment of the present invention. In another embodiment, a heteroclitic peptide of the present invention induces an immune response that is increased at least 2-fold relative to the WT1 peptide from which the heteroclitic peptide was derived (“native peptide”). In another embodiment, the increase is 3-fold relative to the native peptide. In another embodiment, the increase is 5-fold relative to the native peptide. In another embodiment, the increase is 7-fold relative to the native peptide. In another embodiment, the increase is 10-fold relative to the native peptide. In another embodiment, the increase is 15-fold relative to the native peptide. In another embodiment, the increase is 20-fold relative to the native peptide. In another embodiment, the increase is 30-fold relative to the native peptide. In another embodiment, the increase is 50-fold relative to the native peptide. In another embodiment, the increase is 100-fold relative to the native peptide. In another embodiment, the increase is 150-fold relative to the native peptide. In another embodiment, the increase is 200-fold relative to the native peptide. In another embodiment, the increase is 300-fold relative to the native peptide. In another embodiment, the increase is 500-fold relative to the native peptide. In another embodiment, the increase is 1000-fold relative to the native peptide. In another embodiment, the increase is more than 1000-fold relative to the native peptide. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a HLA class II heteroclitic peptide derived from an isolated WT1 peptide of the present invention. In another embodiment, the process of deriving comprises introducing a mutation that enhances a binding of the peptide to an HLA class II molecule. In another embodiment, the process of deriving consists of introducing a mutation that enhances a binding of the peptide to an HLA class I molecule. In another embodiment, the mutation is in an HLA class II anchor residue. In another embodiment, a heteroclitic class II peptide of the present invention is identified and tested in a manner analogous to identification and testing of HLA class I heteroclitic peptides, as exemplified herein. Each possibility represents a separate embodiment of the present invention. In another embodiment, the HLA class II binding site in a peptide of the present invention is created or improved by mutation of an HLA class II motif anchor residue. In another embodiment, the anchor residue that is modified is in the P1 position. In another embodiment, the anchor residue is at the P2 position. In another embodiment, the anchor residue is at the P6 position. In another embodiment, the anchor residue is at the P9 position. In another embodiment, the anchor residue is selected from the P1, P2, P6, and P9 positions. In another embodiment, the anchor residue is at the P3 position. In another embodiment, the anchor residue is at the P4 position. In another embodiment, the anchor residue is at the P5 position. In another embodiment, the anchor residue is at the P6 position. In another embodiment, the anchor residue is at the P8 position. In another embodiment, the anchor residue is at the P10 position. In another embodiment, the anchor residue is at the P11 position. In another embodiment, the anchor residue is at the P12 position. In another embodiment, the anchor residue is at the P13 position. In another embodiment, the anchor residue is at any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. Each possibility represents a separate embodiment of the present invention. In another embodiment, a heteroclitic peptide is generated by introduction of a mutation that creates an anchor motif. “Anchor motifs” or “anchor residues” refers, in another embodiment, to 1 or a set of preferred residues at particular positions in an HLA-binding sequence. In another embodiment, the HLA-binding sequence is an HLA class II-binding sequence. In another embodiment, the HLA-binding sequence is an HLA class I-binding sequence. In another embodiment, the positions corresponding to the anchor motifs are those that play a significant role in binding the HLA molecule. In another embodiment, the anchor residue is a primary anchor motif. In another embodiment, the anchor residue is a secondary anchor motif. Each possibility represents a separate embodiment of the present invention. Methods for predicting MHC class I and II epitopes are well known in the art. In one embodiment, the software of the Bioinformatics & Molecular Analysis Section (National Institutes of Health, Washington, DC) available at http://bimas.dcrt.nih.gov/cgi-bin/molbio/ken parker comboform is useful. This software ranks 9-mer or 10-mer peptides on a predicted half-time dissociation coefficient from HLA class I molecules (Pinilla, et al. Curr Opin Immunol, 11 (2): p. 193-202 (1999)). In another embodiment, MHC class II epitope is predicted using TEPITOPE (Meister G E, Roberts C G et al, Vaccine 1995 13: 581-91). In another embodiment, the MHC class II epitope is predicted using EpiMatrix (De Groot A S, Jesdale B M et al, AIDS Res Hum Retroviruses 1997 13: 529-31). In another embodiment, the MHC class II epitope is predicted using the Predict Method (Yu K, Petrovsky N et al, Mol Med. 2002 8: 137-48). In another embodiment, the MHC class II epitope is predicted using the SYFPEITHI epitope prediction algorithm (Examples). In another embodiment, the MHC class II epitope is predicted using Rankpep. In another embodiment, the MHC class II epitope is predicted using any other method known in the art. Each possibility represents a separate embodiment of the present invention. In another embodiment, in the case of HLA class II-binding peptides (e.g. HLA-DR-binding peptides), the anchor residue that is modified is in the P1 position. In another embodiment, the anchor residue is in the P2 position. In another embodiment, the anchor residue is in the P6 position. In another embodiment, the anchor residue is in the P9 position. In other embodiments, the anchor residue is the P3, P4, P5, P6, P8, P10, P11, P12, or P13 position. In another embodiment, the anchor residue is any other anchor residue of an HLA class II molecule that is known in the art. In another embodiment, residues other than P1, P2, P6, and P9 serve as secondary anchor residues; therefore, mutating them can improve HLA class II binding. In another embodiment, any combination of the above residues is mutated. Each possibility represents a separate embodiment of the present invention. In another embodiment, a WT1 peptide of the present invention binds to 2 distinct HLA class II molecules. In another embodiment, the peptide binds to three distinct HLA class II molecules. In another embodiment, the peptide binds to four distinct HLA class II molecules. In another embodiment, the peptide binds to five distinct HLA class II molecules. In another embodiment, the peptide binds to six distinct HLA class II molecules. In another embodiment, the peptide binds to more than six distinct HLA class II molecules. In another embodiment, the HLA class II molecules that are bound by a WT1 peptide of the present invention are encoded by two or more distinct alleles at a given HLA class II locus. In another embodiment, the HLA class II molecules are encoded by 3 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 4 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 5 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by 6 distinct alleles at a locus. In another embodiment, the HLA class II molecules are encoded by more than six distinct alleles at a locus. In another embodiment, the HLA class II molecules bound by the WT1 peptide are encoded by HLA class II genes at 2 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 2 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 3 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at 4 or more distinct loci. In another embodiment, the HLA molecules bound are encoded by HLA class II genes at more than 4 distinct loci. In other embodiments, the loci are selected from HLA-DRB loci. In another embodiment, the HLA class II-binding peptide is an HLA-DRA binding peptide. In another embodiment, the peptide is an HLA-DQA1 binding peptide. In another embodiment, the peptide is an HLA-DQB 1 binding peptide. In another embodiment, the peptide is an HLA-DPA1 binding peptide. In another embodiment, the peptide is an HLA-DPB 1 binding peptide. In another embodiment, the peptide is an HLA-DMA binding peptide. In another embodiment, the peptide is an HLA-DMB binding peptide. In another embodiment, the peptide is an HLA-DOA binding peptide. In another embodiment, the peptide is an HLA-DOB binding peptide. In another embodiment, the peptide binds to any other HLA class II molecule known in the art. Each possibility represents a separate embodiment of the present invention. In another embodiment, a WT1 peptide of the present invention binds to 2 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 3 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 4 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 5 distinct HLA-DRB molecules. In another embodiment, the peptide binds to 6 distinct HLA-DRB molecules. In another embodiment, the peptide binds to more than 6 distinct HLA-DRB molecules. In another embodiment, a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 3 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 4 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 5 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by 6 distinct HLA-DRB alleles. In another embodiment, the HLA-DRB molecules are encoded by more than 6 distinct HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention. In another embodiment, a WT1 peptide of the present invention binds to HLA-DRB molecules that are encoded by 2 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 3 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 4 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by 5 distinct HLA-DRB alleles selected from DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, DRB 1104 and DRB 1501. In another embodiment, the WT1 peptide binds to HLA-DRB molecules encoded by each of the following HLA-DRB alleles: DRB 101, DRB 301, DRB 401, DRB 701, DRB 1101, and DRB 1501. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a composition comprising 2 distinct WT1 peptides of the present invention. In another embodiment, the 2 distinct WT1 peptides are both unaltered. In another embodiment, 1 of the WT1 peptides is unaltered, while the other is heteroclitic. In another embodiment, both of the WT1 peptides are heteroclitic. In another embodiment, the composition comprises 3 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises 4 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises 5 distinct WT1 peptides of the present invention. In another embodiment, the composition comprises more than 5 distinct isolated WT1 peptides of the present invention. In another embodiment, 2 of the WT1 peptides in the composition are unaltered. In another embodiment, 2 of the WT1 peptides in the composition are heteroclitic. In another embodiment, 2 of the WT1 peptides in the composition are unaltered, and 2 are heteroclitic. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered. In another embodiment, more than 2 of the WT1 peptides in the composition are heteroclitic. In another embodiment, more than 2 of the WT1 peptides in the composition are unaltered, and more than 2 are heteroclitic. Each possibility represents a separate embodiment of the present invention. In another embodiment, 1 of the additional WT1 peptides in a composition of the present invention has a sequence selected from the sequences set forth in SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, 2 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, 3 of the additional WT1 peptides have a sequence selected from the sequences set forth in SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, any other immunogenic WT1 peptide known in the art is utilized as an additional WT1 peptide. In another embodiment, any combination of immunogenic WT1 peptides known in the art is utilized. Non-limiting sources of other WT1 peptides include WO2005053618, WO2007047764 and WO2007120673. Each additional WT1 peptide, and each combination thereof, represents a separate embodiment of the present invention. In another embodiment, a composition of the present invention contains 2 HLA class II heteroclitic peptides that are derived from the same isolated WT1 peptide of the present invention. In another embodiment, the 2 HLA class II heteroclitic peptides contain mutations in different HLA class II molecule anchor residues. In another embodiment, the 2 HLA class II heteroclitic peptides contain different mutations in the same anchor residues. In another embodiment, 2 of the HLA class II heteroclitic peptides are derived from different isolated WT1 peptides of the present invention. Each possibility represents a separate embodiment of the present invention. In another embodiment, 2 WT1 peptides of the present invention, or the WT1 peptides that correspond to two HLA class II heteroclitic peptides of the present invention, overlap with one another. In another embodiment, the overlap between the peptides is at least 7 amino acids (AA). In another embodiment, the overlap is at least 8 AA. In another embodiment, the overlap is at least 9 AA. In another embodiment, the overlap is 7 AA. In another embodiment, the overlap is 8 AA. In another embodiment, the overlap is 9 AA. In another embodiment, the overlap is 10 AA. In another embodiment, the overlap is 11 AA. In another embodiment, the overlap is 12 AA. In another embodiment, the overlap is 13 AA. In another embodiment, the overlap is 14 AA. In another embodiment, the overlap is 15 AA. In another embodiment, the overlap is 16 AA. In another embodiment, the overlap is more than 16 AA. Each possibility represents a separate embodiment of the present invention. In another embodiment, the peptides in a composition of the present invention bind to 2 distinct HLA class II molecules. In another embodiment, the peptides bind to 3 distinct HLA class II molecules. In another embodiment, the peptides bind to 4 distinct HLA class II molecules. In another embodiment, the peptides bind to 5 distinct HLA class II molecules. In another embodiment, the peptides bind to more than 5 distinct HLA class II molecules. In another embodiment, the peptides in the composition bind to the same HLA class II molecules. In another embodiment, each of the WT 1 peptides in a composition of the present invention binds to a set of HLA class II molecules. In another embodiment, each of the WT1 peptides binds to a distinct set of HLA class II molecules. In another embodiment, the WT1 peptides in the composition bind to the same set of HLA class II molecules. In another embodiment, 2 of the WT1 peptides bind to a distinct but overlapping set of HLA class II molecules. In another embodiment, 2 or more of the WT1 peptides bind to the same set of HLA class II molecules, while another of the WT1 peptides binds to a distinct set. In another embodiment, 2 or more of the WT1 peptides bind to an overlapping set of HLA class II molecules, while another of the WT1 peptides binds to a distinct set. In another embodiment, 2 or more of the WT1 peptides in a composition of the present invention each binds to more than 1 HLA-DRB molecule. In another embodiment, the 4 or more HLA-DRB molecules bound by the peptides in the composition are distinct from one another. In another embodiment, the HLA-DRB molecules are encoded by different HLA-DRB alleles. Each possibility represents a separate embodiment of the present invention. In another embodiment, 2 or more of the HLA class II molecules bound by WT1 peptides in a composition of the present invention are HLA-DRB molecules. In another embodiment, 3 or more of the HLA class II molecules that are bound are HLA-DRB molecules. In other embodiments, the HLA class II molecules that are bound can be any of the HLA class II molecules enumerated herein. In another embodiment, the HLA class II molecules that are bound are encoded by 2 or more distinct HLA class II alleles at a given locus. In another embodiment, the HLA class II molecules that are bound are encoded by HLA class II genes at 2 or more distinct loci. Each of the above compositions represents a separate embodiment of the present invention. In another embodiment, a “set of HLA class II molecules” refers to the HLA class II molecules encoded by different alleles at a particular locus. In another embodiment, the term refers to HLA class II molecules with a particular binding specificity. In another embodiment, the term refers to HLA class II molecules with a particular peptide consensus sequence. In another embodiment, the term refers to a superfamily of HLA class II molecules. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a composition comprising an unaltered HLA class II molecule-binding WT1 peptide of the present invention and a second, HLA class I molecule-binding WT1 peptide. In another embodiment, the composition comprises more than 1 HLA class II molecule-binding WT1 peptide of the present invention, in addition to the HLA class I molecule-binding WT1 peptide. In another embodiment, the composition comprises more than 1 HLA class I molecule-binding WT1 peptide, in addition to the HLA class II molecule-binding WT1 peptide. Each possibility represents a separate embodiment of the present invention. In another embodiment, the AA sequence of the HLA class I molecule-binding WT1 peptide comprises a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. In another embodiment, the AA sequence of the HLA class I molecule-binding WT1 peptide is selected from the sequences set forth in SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55. Each possibility represents a separate embodiment of the present invention. In another embodiment, the HLA class I molecule-binding WT1 peptide is an HLA class I heteroclitic peptide. In another embodiment, the HLA class I molecule-binding WT1 peptide contains a mutation in an HLA class I molecule anchor residue thereof, as described further herein. As provided herein, WT1-derived peptides were modified in HLA anchor residues to generate heteroclitic peptides with increased predicted binding to HLA-A0201 and HLA-A0301. Peptides with increased predicted binding also exhibited enhanced ability to bind HLA class I molecules and increased immunogenicity. In another embodiment, the mutation that enhances MHC binding is in the residue at position 1 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to tyrosine. In another embodiment, the residue is changed to glycine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to phenylalanine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, a substitution in position 1 (e.g. to tyrosine) stabilizes the binding of the position 2 anchor residue. In another embodiment, the mutation is in position 2 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to methionine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, the mutation is in position 6 of the HLA class I heteroclitic peptide. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to glutamine. In another embodiment, the residue is changed to histidine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, the mutation is in position 9 of the HLA class I heteroclitic peptide. In another embodiment, the mutation changes the residue at the C-terminal position thereof. In another embodiment, the residue is changed to valine. In another embodiment, the residue is changed to threonine. In another embodiment, the residue is changed to isoleucine. In another embodiment, the residue is changed to leucine. In another embodiment, the residue is changed to alanine. In another embodiment, the residue is changed to cysteine. In another embodiment, the residue is changed to any other residue known in the art. In another embodiment, the point mutation is in a primary anchor residue. In another embodiment, the HLA class I primary anchor residues are positions 2 and 9. In another embodiment, the point mutation is in a secondary anchor residue. In another embodiment, the HLA class I secondary anchor residues are positions 1 and 8. In another embodiment, the HLA class I secondary anchor residues are positions 1, 3, 6, 7, and 8. In another embodiment, the point mutation is in a position selected from positions 4, 5, and 8. Each possibility represents a separate embodiment of the present invention. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, 8, and 9 of the HLA class I binding motif. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 6, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 2, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 1, 3, and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 2 and 9. In another embodiment, the point mutation is in 1 or more residues in positions selected from positions 6 and 9. Each possibility represents a separate embodiment of the present invention. Each of the above anchor residues and substitutions represents a separate embodiment of the present invention. In another embodiment, the HLA class I molecule-binding WT peptide has length of 9 AA. In another embodiment, the peptide has length of 10 AA. As provided herein, native and heteroclitic peptides of 9-10 AA exhibited substantial binding to HLA class I molecules and ability to elicit cytokine secretion and cytolysis by CTL. In another embodiment, the HLA class I molecule that is bound by the HLA class I molecule-binding WT1 peptide is an HLA-A molecule. In another embodiment, the HLA class I-molecule is an HLA-A2 molecule. In another embodiment, the HLA class I-molecule is an HLA-A3 molecule. In another embodiment, the HLA class I-molecule is an HLA-A11 molecule. In another embodiment, the HLA class I-molecule is an HLA-B 8 molecule. In another embodiment, the HLA class I-molecule is an HLA-0201 molecule. In another embodiment, the HLA class I-molecule binds any other HLA class I molecule known in the art. Each possibility represents a separate embodiment of the present invention. In another embodiment, a WT1 peptide of methods and compositions of the present invention has a length of 8-30 amino acids. In another embodiment, the peptide has a length of 9-11 AA. In another embodiment, the peptide ranges in size from 7-25 AA, or in another embodiment, 8-11, or in another embodiment, 8-15, or in another embodiment, 9-20, or in another embodiment, 9-18, or in another embodiment, 9-15, or in another embodiment, 8-12, or in another embodiment, 9-11 AA in length. In another embodiment, the peptide is 8 AA in length, or in another embodiment, 9 AA or in another embodiment, 10 AA or in another embodiment, 12 AA or in another embodiment, 25 AA in length, or in another embodiment, any length therebetween. In another embodiment, the peptide is of greater length, for example 50, or 100, or more. In this embodiment, the cell processes the peptide to a length of 7 and 25 AA in length. In this embodiment, the cell processes the peptide to a length of 9-11 AA Each possibility represents a separate embodiment of the present invention. In another embodiment, the peptide is 15-23 AA in length. In another embodiment, the length is 15-24 AA. In another embodiment, the length is 15-25 AA. In another embodiment, the length is 15-26 AA. In another embodiment, the length is 15-27 AA. In another embodiment, the length is 15-28 AA. In another embodiment, the length is 14-30 AA. In another embodiment, the length is 14-29 AA. In another embodiment, the length is 14-28 AA. In another embodiment, the length is 14-26 AA. In another embodiment, the length is 14-24 AA. In another embodiment, the length is 14-22 AA. In another embodiment, the length is 14-20 AA. In another embodiment, the length is 16-30 AA. In another embodiment, the length is 16-28 AA. In another embodiment, the length is 16-26 AA. In another embodiment, the length is 16-24 AA. In another embodiment, the length is 16-22 AA. In another embodiment, the length is 18-30 AA. In another embodiment, the length is 18-28 AA. In another embodiment, the length is 18-26 AA. In another embodiment, the length is 18-24 AA. In another embodiment, the length is 18-22 AA. In another embodiment, the length is 18-20 AA. In another embodiment, the length is 20-30 AA. In another embodiment, the length is 20-28 AA. In another embodiment, the length is 20-26 AA. In another embodiment, the length is 20-24 AA. In another embodiment, the length is 22-30 AA. In another embodiment, the length is 22-28 AA. In another embodiment, the length is 22-26 AA. In another embodiment, the length is 24-30 AA. In another embodiment, the length is 24-28 AA. In another embodiment, the length is 24-26 AA. Each of the above peptides, peptide lengths, and types of peptides represents a separate embodiment of the present invention. In another embodiment, minor modifications are made to peptides of the present invention without decreasing their affinity for HLA molecules or changing their TCR specificity, utilizing principles well known in the art. In the case of HLA class I-binding peptides, “minor modifications” refers, in another embodiment, to e.g. insertion, deletion, or substitution of one AA, inclusive, or deletion or addition of 1-3 AA outside of the residues between 2 and 9, inclusive. While the computer algorithms described herein are useful for predicting the MHC class I-binding potential of peptides, they have 60-80% predictive accuracy; and thus, the peptides should be evaluated empirically before a final determination of MHC class I-binding affinity is made. Thus, peptides of the present invention are not limited to peptides predicated by the algorithms to exhibit strong MHC class I-binding affinity. The types are modifications that can be made are listed below. Each modification represents a separate embodiment of the present invention. In another embodiment, a peptide enumerated in the Examples of the present invention is further modified by mutating an anchor residue to an MHC class I preferred anchor residue, which can be, in other embodiments, any of the anchor residues enumerated herein. In another embodiment, a peptide of the present invention containing an MHC class I preferred anchor residue is further modified by mutating the anchor residue to a different MHC class I preferred residue for that location. The different preferred residue can be, in other embodiments, any of the preferred residues enumerated herein. In another embodiment, the anchor residue that is further modified is in the 1 position. In another embodiment, the anchor residue is in the 2 position. In another embodiment, the anchor residue is in the 3 position. In another embodiment, the anchor residue is in the 4 position. In another embodiment, the anchor residue is in the 5 position. In another embodiment, the anchor residue is in the 6 position. In another embodiment, the anchor residue is in the 7 position. In another embodiment, the anchor residue is in the 8 position. In another embodiment, the anchor residue is in the 9 position. In the case of HLA class I-binding peptides, residues other than 2 and 9 can serve as secondary anchor residues; therefore, mutating them can improve MHC class I binding. Each possibility represents a separate embodiment of the present invention. In another embodiment, a peptide of methods and compositions of the present invention is a length variant of a peptide enumerated in the Examples. In another embodiment, the length variant is one amino acid (AA) shorter than the peptide from the Examples. In another embodiment, the length variant is two AA shorter than the peptide from the Examples. In another embodiment, the length variant is more than two AA shorter than the peptide from the Examples. In another embodiment, the shorter peptide is truncated on the N-terminal end. In another embodiment, the shorter peptide is truncated on the C-terminal end. In another embodiment, the truncated peptide is truncated on both the N-terminal and C-terminal ends. Peptides are, in another embodiment, amenable to truncation without changing affinity for HLA molecules, as is well known in the art. Each of the above truncated peptides represents a separate embodiment of the present invention. In another embodiment, the length variant is longer than a peptide enumerated in the Examples of the present invention. In another embodiment, the longer peptide is extended on the N-terminal end in accordance with the surrounding WT1 sequence. Peptides are, in another embodiment, amenable to extension on the N-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples. In another embodiment, the N-terminal extended peptide is extended by one residue. In another embodiment, the N-terminal extended peptide is extended by two residues. In another embodiment, the N-terminal extended peptide is extended by three residues. In another embodiment, the N-terminal extended peptide is extended by more than three residues. In another embodiment, the longer peptide is extended on the C terminal end in accordance with the surrounding WT1 sequence. Peptides are, in another embodiment, amenable to extension on the C-terminal end without changing affinity for HLA molecules, as is well known in the art. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the C-terminal extended peptide is extended by one residue. In another embodiment, the C-terminal extended peptide is extended by two residues. In another embodiment, the C-terminal extended peptide is extended by three residues. In another embodiment, the C-terminal extended peptide is extended by more than three residues. In another embodiment, the extended peptide is extended on both the N-terminal and C-terminal ends in accordance with the surrounding WT1 sequence. Each of the above extended peptides represents a separate embodiment of the present invention. In another embodiment, a truncated peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a smaller number of intervening residues (e.g., 5) than a peptide enumerated in the Examples of the present invention. Peptides are, in another embodiment, amenable to such mutation without changing affinity for HLA molecules. In another embodiment, such a truncated peptide is designed by removing one of the intervening residues of one of the above sequences. In another embodiment, the HLA anchor residues are retained on the second and eighth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. Each possibility represents a separate embodiment of the present invention. In another embodiment, an extended peptide of the present invention retains the HLA anchor residues (e.g. the HLA class I anchor residues) on the second residue and the C-terminal residue, with a larger number of intervening residues (e.g. 7 or 8) than a peptide enumerated in the Examples of the present invention. In another embodiment, such an extended peptide is designed by adding one or more residues between two of the intervening residues of one of the above sequences. It is well known in the art that residues can be removed from or added between the intervening sequences of HLA-binding peptides without changing affinity for HLA. Such peptides are thus equivalents of the peptides enumerated in the Examples of the present invention. In another embodiment, the HLA anchor residues are retained on the second and ninth residues. In another embodiment, the HLA anchor residues are retained on the first and eighth residues. In another embodiment, the HLA anchor residues are retained on the two residues separated by six intervening residues. Each possibility represents a separate embodiment of the present invention. “Fragment,” in another embodiment, refers to a peptide of 11 or more AA in length. In another embodiment, a peptide fragment of the present invention is 16 or more AA long. In another embodiment, the fragment is 12 or more AA long. In another embodiment, the fragment is 13 or more AA. In another embodiment, the fragment is 14 or more AA. In another embodiment, the fragment is 15 or more AA. In another embodiment, the fragment is 17 or more AA. In another embodiment, the fragment is 18 or more AA. In another embodiment, the fragment is 19 or more AA. In another embodiment, the fragment is 22 or more AA. In another embodiment, the fragment is 8-12 AA. In another embodiment, the fragment is about 8-12 AA. In another embodiment, the fragment is 16-19 AA. In another embodiment, the fragment is about 16-19 AA. In another embodiment, the fragment 10-25 AA. In another embodiment, the fragment is about 10-25 AA. In another embodiment, the fragment has any other length. Each possibility represents a separate embodiment of the present invention. “Fragment of a WT1 protein,” in another embodiment, refers to any of the definitions of “fragment” found herein. Each definition represents a separate embodiment of the present invention. In another embodiment, a peptide of the present invention is homologous to a peptide enumerated in the Examples. The terms “homology,” “homologous,” etc., when in reference to any protein or peptide, refer, in another embodiment, to a percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Methods and computer programs for the alignment are well known in the art. In another embodiment, the term “homology,” when in reference to any nucleic acid sequence similarly indicates a percentage of nucleotides in a candidate sequence that are identical with the nucleotides of a corresponding native nucleic acid sequence. Homology is, in another embodiment, determined by computer algorithm for sequence alignment, by methods well described in the art. In other embodiments, computer algorithm analysis of nucleic acid sequence homology includes the utilization of any number of software packages available, such as, for example, the BLAST, DOMAIN, BEAUTY (BLAST Enhanced Alignment Utility), GENPEPT and TREMBL packages. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 70%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 72%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 75%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 78%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 80%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 82%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 83%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 85%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 87%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than [0128] 88%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 90%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 92%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 93%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 95%. In another embodiment, “homology” refers to identity to a sequence selected from SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 96%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 97%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 98%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of greater than 99%. In another embodiment, “homology” refers to identity to one of SEQ ID No: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 30, 31, 32, 33, 35, 36, 37, 38, 39, 41, 42, 43, 44, 46, 47, 48, 49, 50 and 55 of 100%. Each possibility represents a separate embodiment of the present invention. [00114] In another embodiment, homology is determined via determination of candidate sequence hybridization, methods of which are well described in the art (See, for example, “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y). In another embodiments, methods of hybridization are carried out under moderate to stringent conditions, to the complement of a DNA encoding a native caspase peptide. Hybridization conditions being, for example, overnight incubation at 42<0>C in a solution comprising: 10-20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 [mu]g/ml denatured, sheared salmon sperm DNA. Each of the above homologues and variants of peptides enumerated in the Examples represents a separate embodiment of the present invention. In another embodiment, the present invention provides a composition comprising a peptide of this invention. In another embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the composition further comprises an adjuvant. In another embodiment, the composition comprises 2 or more peptides of the present invention. In another embodiment, the composition further comprises any of the additives, compounds, or excipients set forth hereinbelow. In another embodiment, the adjuvant is KLH, QS21, Freund's complete or incomplete adjuvant, aluminum phosphate, aluminum hydroxide, BCG or alum. In other embodiments, the carrier is any carrier enumerated herein. In other embodiments, the adjuvant is any adjuvant enumerated herein. Each possibility represents a separate embodiment of the present invention. In another embodiment, this invention provides a vaccine comprising a peptide of this invention. In another embodiment, this invention provides a vaccine comprising an antigen-presenting cell (APC) and a peptide of this invention. In another embodiment, the vaccine further comprises a carrier. In another embodiment, the vaccine further comprises an adjuvant. In another embodiment, the vaccine further comprises an APC. In another embodiment, the vaccine further comprises a combination of more than 1 of an antigen, carrier, and/or APC. In another embodiment, the vaccine is a cell-based composition. Each possibility represents a separate embodiment of the present invention. In another embodiment, the term “vaccine” refers to a material or composition that, when introduced into a subject, provides a prophylactic or therapeutic response for a particular disease, condition, or symptom of same. In another embodiment, this invention comprises peptide-based vaccines, wherein the peptide comprises any embodiment listed herein, including immunomodulating compounds such as cytokines, adjuvants, etc. In another embodiment, a vaccine of methods and compositions of the present invention further comprises an adjuvant. In another embodiment, the adjuvant is Montanide ISA 51. Montanide ISA 51 contains a natural metabolizable oil and a refined emulsifier. In another embodiment, the adjuvant is GM-CSF. Recombinant GM-CSF is a human protein grown, in another embodiment, in a yeast (S. cerevisiae) vector. GM-CSF promotes clonal expansion and differentiation of hematopoietic progenitor cells, APC, and dendritic cells and T cells. In another embodiment, the adjuvant is a cytokine. In another embodiment, the adjuvant is a growth factor. In another embodiment, the adjuvant is a cell population. In another embodiment, the adjuvant is QS21. In another embodiment, the adjuvant is Freund's incomplete adjuvant. In another embodiment, the adjuvant is aluminum phosphate. In another embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is BCG. In another embodiment, the adjuvant is alum. In another embodiment, the adjuvant is an interleukin. In another embodiment, the adjuvant is a chemokine. In another embodiment, the adjuvant is any other type of adjuvant known in the art. In another embodiment, the WT1 vaccine comprises two the above adjuvants. In another embodiment, the WT1 vaccine comprises more than two the above adjuvants. Each possibility represents a separate embodiment of the present invention. In other embodiments, a vaccine or composition of the present invention can comprise any of the embodiments of WT1 peptides of the present invention and combinations thereof. Each possibility represents a separate embodiment of the present invention. It is to be understood that any embodiments described herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention. Each combination of peptide, vaccine, or composition with a method represents an embodiment thereof. In another embodiment, the present invention provides a method of treating a subject with a WT1-expressing cancer, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby treating a subject with a WT1-expressing cancer. In another embodiment, the present invention provides a method of treating a subject with an MDS, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby treating a subject with an MDS. In another embodiment, the present invention provides a method of suppressing or halting the progression of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby suppressing or halting the progression of a WT1-expressing cancer. In another embodiment, the present invention provides a method of reducing the incidence of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of a WT1-expressing cancer in a subject. In another embodiment, the present invention provides a method of reducing the incidence of an AML in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of an AML. In another embodiment, the present invention provides a method of reducing the incidence of relapse of a WT1-expressing cancer in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of relapse of a WT1-expressing cancer in a subject. In another embodiment, the present invention provides a method of reducing the incidence of relapse of an AML in a subject, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby reducing the incidence of relapse of an AML in a subject. In another embodiment, the present invention provides a method of breaking a T cell tolerance of a subject to a WT1-expressing cancer, the method comprising administering to the subject a WT1 vaccine of the present invention, thereby breaking a T cell tolerance to a WT1-expressing cancer. In another embodiment, the present invention provides a method of treating a subject having a WT1-expressing cancer, comprising (a) inducing in a donor formation and proliferation of human cytotoxic T lymphocytes (CTL) that recognize a malignant cell of the cancer by a method of the present invention; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer. In another embodiment, the present invention provides a method of treating a subject having a WT 1-expressing cancer, comprising (a) inducing ex vivo formation and proliferation of human CTL that recognize a malignant cell of the cancer by a method of the present invention, wherein the human immune cells are obtained from a donor; and (b) infusing the human CTL into the subject, thereby treating a subject having a cancer. Methods for ex vivo immunotherapy are well known in the art and are described, for example, in United States Patent Application Serial Numbers 2006/0057130, 2005/0221481, 2005/0214268, 2003/0175272, 2002/0127718, and U.S. Pat. No. 5,229,115, which are incorporated herein by reference. Additional methods are well known in the art and are described, for example, in Davis I D et al (Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients. J Immunother. 2006 September-October; 29(5):499-511) and Mitchell M S et al (The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2. Cancer Immunol Immunother. 2006 Jul. 28). Each method represents a separate embodiment of the present invention. In another embodiment, the present invention provides a method of inducing the formation and proliferation of CTL specific for cells of a WT1-expressing cancer, the method comprising contacting a lymphocyte population with a vaccine of the present invention. In another embodiment, the vaccine is an APC associated with a peptide of the present invention. In another embodiment, the vaccine is an APC associated with a mixture of peptides of the present invention. Each possibility represents a separate embodiment of the present invention. In another embodiment, this invention provides a method of generating a heteroclitic immune response in a subject, wherein the heteroclitic immune response is directed against a WT1-expressing cancer, the method comprising administering to the subject a vaccine of the present invention, thereby generating a heteroclitic immune response. In another embodiment, the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby inducing an anti-mesothelioma immune response in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a method of inducing an anti-mesothelioma immune response in a subject, the method comprising the step of contacting the subject with an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby inducing an anti-mesothelioma immune response in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby treating a subject with a mesothelioma. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a method of treating a subject with a mesothelioma, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby treating a subject with a mesothelioma. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising (a) a WT1 protein; or (b) a fragment of a WT protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. In another embodiment, the present invention provides a method of reducing an incidence of a mesothelioma, or its relapse, in a subject, the method comprising the step of administering to the subject an immunogenic composition comprising a nucleotide molecule encoding (a) a WT1 protein; or (b) a fragment of a WT1 protein, thereby reducing an incidence of a mesothelioma, or its relapse, in a subject. In another embodiment, the mesothelioma is a malignant mesothelioma. Each possibility represents a separate embodiment of the present invention. In another embodiment, a target cell of an immune response elicited by a method of the present invention presents the WT1 peptide of the present invention, or a corresponding WT1 fragment, on an HLA molecule. In another embodiment, the HLA molecule is an HLA class I molecule. In other embodiments, the HLA molecule is any HLA class I subtype or HLA class I molecule known in the art. In another embodiment, the immune response against the WT1 peptide or fragment is a heteroclitic immune response. Each possibility represents a separate embodiment of the present invention. In another embodiment, the WT1-expressing cancer is an acute myelogenous leukemia (AML). In another embodiment, the WT1-expressing cancer is associated with a myelodysplastic syndrome (MDS). In another embodiment, the WT1-expressing cancer is an MDS. In another embodiment, the WT1-expressing cancer is a non-small cell lung cancer (NSCLC). In another embodiment, the WT1-expressing cancer is a Wilms' tumor. In another embodiment, the WT1-expressing cancer is a leukemia. In another embodiment, the WT1-expressing cancer is a hematological cancer. In another embodiment, the WT1-expressing cancer is a lymphoma. In another embodiment, the WT1-expressing cancer is a desmoplastic small round cell tumor. In another embodiment, the WT1-expressing cancer is a mesothelioma. In another embodiment, the WT1-expressing cancer is a malignant mesothelioma. In another embodiment, the WT1-expressing cancer is a gastric cancer. In another embodiment, the WT1-expressing cancer is a colon cancer. In another embodiment, the WT1-expressing cancer is a lung cancer. In another embodiment, the WT1-expressing cancer is a breast cancer. In another embodiment, the WT1-expressing cancer is a germ cell tumor. In another embodiment, the WT1-expressing cancer is an ovarian cancer. In another embodiment, the WT 1-expressing cancer is a uterine cancer. In another embodiment, the WT 1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a hepatocellular carcinoma. In another embodiment, the WT1-expressing cancer is a thyroid cancer. In another embodiment, the WT1-expressing cancer is a liver cancer. In another embodiment, the WT1-expressing cancer is a renal cancer. In another embodiment, the WT1-expressing cancer is a Kaposi's sarcoma. In another embodiment, the WT1-expressing cancer is a sarcoma. In another embodiment, the WT1-expressing cancer is any other carcinoma or sarcoma. In another embodiment, the WT1-expressing cancer is a solid tumor. In another embodiment, the solid tumor is associated with a WT1-expressing cancer. In another embodiment, the solid tumor is associated with a myelodysplastic syndrome (MDS). In another embodiment, the solid tumor is associated with a non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is associated with a lung cancer. In another embodiment, the solid tumor is associated with a breast cancer. In another embodiment, the solid tumor is associated with a colorectal cancer. In another embodiment, the solid tumor is associated with a prostate cancer. In another embodiment, the solid tumor is associated with an ovarian cancer. In another embodiment, the solid tumor is associated with a renal cancer. In another embodiment, the solid tumor is associated with a pancreatic cancer. In another embodiment, the solid tumor is associated with a brain cancer. In another embodiment, the solid tumor is associated with a gastrointestinal cancer. In another embodiment, the solid tumor is associated with a skin cancer. In another embodiment, the solid tumor is associated with a melanoma. In another embodiment, a cancer or tumor treated by a method of the present invention is suspected to express WT1. In another embodiment, WT1 expression has not been verified by testing of the actual tumor sample. In another embodiment, the cancer or tumor is of a type known to express WT1 in many cases. In another embodiment, the type expresses WT1 in the majority of cases. Each type of WT1-expressing cancer or tumor, and cancer or tumor suspected to express WT1, represents a separate embodiment of the present invention. Any embodiments enumerated herein, regarding peptides, vaccines and compositions of this invention can be employed in any of the methods of this invention, and each represents an embodiment thereof. In another embodiment, multiple peptides of this invention are used to stimulate an immune response in methods of the present invention. The methods disclosed herein will be understood by those in the art to enable design of other WT1-derived peptides. The methods further enable design of peptides binding to other HLA molecules. The methods further enable design of vaccines combining WT1-derived peptides of the present invention. Each possibility represents a separate embodiment of the present invention. In another embodiment, vaccines of the present invention have the advantage of activating or eliciting WT1-specific CD4<+> T cells containing a variety of different HLA class II alleles. In another embodiment, the vaccines have the advantage of activating or eliciting WT1-specific CD4<+> T cells in a substantial proportion of the population (e.g. in different embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%. 85%, 90%, 95%, or greater than 95%). In another embodiment, the vaccines activate or elicit WT1-specific CD4<+> T cells in a substantial proportion of a particular population (e.g. American Caucasians). Each possibility represents a separate embodiment of the present invention. In another embodiment, methods of the present invention provide for an improvement in an immune response that has already been mounted by a subject. In another embodiment, methods of the present invention comprise administering the peptide, composition, or vaccine 2 or more times. In another embodiment, the peptides are varied in their composition, concentration, or a combination thereof. In another embodiment, the peptides provide for the initiation of an immune response against an antigen of interest in a subject who has not yet initiated an immune response against the antigen. In another embodiment, the CTL that are induced proliferate in response to presentation of the peptide on the APC or cancer cell. In other embodiments, reference to modulation of the immune response involves, either or both the humoral and cell-mediated arms of the immune system, which is accompanied by the presence of Th2 and ThI T helper cells, respectively, or in another embodiment, each arm individually. In other embodiments, the methods affecting the growth of a tumor result in (1) the direct inhibition of tumor cell division, or (2) immune cell mediated tumor cell lysis, or both, which leads to a suppression in the net expansion of tumor cells. Inhibition of tumor growth by either of these two mechanisms can be readily determined by one of ordinary skill in the art based upon a number of well-known methods. In another embodiment, tumor inhibition is determined by measuring the actual tumor size over a period of time. In another embodiment, tumor inhibition can be determined by estimating the size of a tumor (over a period of time) utilizing methods well known to those of skill in the art. More specifically, a variety of radiologic imaging methods (e.g., single photon and positron emission computerized tomography; see generally, “Nuclear Medicine in Clinical Oncology,” Winkler, C. (ed.) Springer-Verlag, New York, 1986), can be utilized to estimate tumor size. Such methods can also utilize a variety of imaging agents, including for example, conventional imaging agents (e.g., Gallium-67 citrate), as well as specialized reagents for metabolite imaging, receptor imaging, or immunologic imaging (e.g., radiolabeled monoclonal antibody specific tumor markers). In addition, non-radioactive methods such as ultrasound (see, “Ultrasonic Differential Diagnosis of Tumors”, Kossoff and Fukuda, (eds.), Igaku-Shoin, New York, 1984), can also be utilized to estimate the size of a tumor. In addition to the in vivo methods for determining tumor inhibition discussed above, a variety of in vitro methods can be utilized in order to predict in vivo tumor inhibition. Representative examples include lymphocyte mediated anti-tumor cytolytic activity determined for example, by a <51>Cr release assay (Examples), tumor dependent lymphocyte proliferation (Ioannides, et al., J. Immunol. 146(5):1700-1707, 1991), in vitro generation of tumor specific antibodies (Herlyn, et al., J. Immunol. Meth. 73:157-167, 1984), cell (e.g., CTL, helper T-cell) or humoral (e.g., antibody) mediated inhibition of cell growth in vitro (Gazit, et al., Cancer Immunol Immunother 35:135-144, 1992), and, for any of these assays, determination of cell precursor frequency (Vose, Int. J. Cancer 30:135-142 (1982), and others. In another embodiment, methods of suppressing tumor growth indicate a growth state that is curtailed compared to growth without contact with, or exposure to a peptide of this invention. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a <3>H-thymidine incorporation assay, or counting tumor cells. “Suppressing” tumor cell growth refers, in other embodiments, to slowing, delaying, or stopping tumor growth, or to tumor shrinkage. Each possibility represents a separate embodiment of the present invention. In another embodiment of methods and compositions of the present invention, WT1 expression is measured. In another embodiment, WT1 transcript expression is measured. In another embodiment, WT1 protein levels in the tumor are measured. Each possibility represents a separate embodiment of the present invention. Methods of determining the presence and magnitude of an immune response are well known in the art. In another embodiment, lymphocyte proliferation assays, wherein T cell uptake of a radioactive substance, e.g. <3>H-thymidine is measured as a function of cell proliferation. In other embodiments, detection of T cell proliferation is accomplished by measuring increases in interleukin-2 (IL-2) production, Ca<2+> flux, or dye uptake, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium. Each possibility represents a separate embodiment of the present invention. In another embodiment, CTL stimulation is determined by means known to those skilled in the art, including, detection of cell proliferation, cytokine production and others. Analysis of the types and quantities of cytokines secreted by T cells upon contacting ligand-pulsed targets can be a measure of functional activity. Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. (Fujihashi K. et al. (1993) J. Immunol. Meth. 160: 181; Tanguay S. and Killion J. J. (1994) Lymphokine Cytokine Res. 13:259). In another embodiment, CTL activity is determined by <51>Cr-release lysis assay. Lysis of peptide-pulsed <51>Cr-labeled targets by antigen-specific T cells can be compared for target cells pulsed with control peptide. In another embodiment, T cells are stimulated with a peptide of this invention, and lysis of target cells expressing the native peptide in the context of MHC can be determined. The kinetics of lysis as well as overall target lysis at a fixed timepoint (e.g., 4 hours) are used, in another embodiment, to evaluate ligand performance. (Ware C. F. et al. (1983) J Immunol 131: 1312). Methods of determining affinity of a peptide for an HLA molecule are well known in the art. In another embodiment, affinity is determined by TAP stabilization assays. In another embodiment, affinity is determined by competition radioimmunoassay. In another embodiment, the following protocol is utilized: Target cells are washed two times in PBS with 1% bovine serum albumin (BSA; Fisher Chemicals, Fairlawn, NJ). Cells are resuspended at 10<7>/ml on ice, and the native cell surface bound peptides are stripped for 2 minutes at 0 [deg.] C using citrate-phosphate buffer in the presence of 3 mg/ml beta2 microglobulin. The pellet is resuspended at 5×10<6>cells/ml in PBS/1% BSA in the presence of 3 mg/ml beta2 microglobulin and 30 mg/ml deoxyribonuclease, and 200 ml aliquots are incubated in the presence or absence of HLA-specific peptides for 10 min at 20<0>C, then with <125>I-labeled peptide for 30 min at 20<0>C. Total bound <125>I is determined after two washes with PBS/2% BSA and one wash with PBS. Relative affinities are determined by comparison of escalating concentrations of the test peptide versus a known binding peptide. In another embodiment, a specificity analysis of the binding of peptide to HLA on surface of live cells (e.g. SKLY-16 cells) is conducted to confirm that the binding is to the appropriate HLA molecule and to characterize its restriction. This includes, in another embodiment, competition with excess unlabeled peptides known to bind to the same or disparate HLA molecules and use of target cells which express the same or disparate HLA types. This assay is performed, in another embodiment, on live fresh or 0.25% paraformaldehyde-fixed human PBMC, leukemia cell lines and EBV-transformed T-cell lines of specific HLA types. The relative avidity of the peptides found to bind MHC molecules on the specific cells are assayed by competition assays as described above against <125>I-labeled peptides of known high affinity for the relevant HLA molecule, e.g., tyrosinase or HBV peptide sequence. [00165] In another embodiment, an HLA class II-binding peptide of methods and compositions of the present invention is longer than the minimum length for binding to an HLA class II molecule, which is, in another embodiment, about 12 AA. In another embodiment, increasing the length of the HLA class II-binding peptide enables binding to more than one HLA class II molecule. In another embodiment, increasing the length enables binding to an HLA class II molecule whose binding motif is not known. In another embodiment, increasing the length enables binding to an HLA class I molecule. In another embodiment, the binding motif of the HLA class I molecule is known. In another embodiment, the binding motif of the HLA class I molecule is not known. Each possibility represents a separate embodiment of the present invention. In another embodiment, the peptides utilized in methods and compositions of the present invention comprise a non-classical amino acid such as: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al. (1991) J. Am Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41): 5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D. Thesis, University of Arizona); hydroxy-1,2,3, 4-tetrahydroisoquinoline-3-carboxylate (Miyake et al. (1984) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylic acid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); and HIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep. Protein Res. 42(1):68-77) and ((1992) Acta. Crst., Crystal Struc. Comm 48(IV): 1239-124). In another embodiment, a peptide of this invention comprises an AA analog or peptidomimetic, which, in other embodiments, induces or favors specific secondary structures. Such peptides comprise, in other embodiments, the following: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a [beta]-turn inducing dipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); [beta]-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); [beta]-turn inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5057-5060); alpha-helix inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:4935-4938); gamma-turn inducing analogs (Kemp et al. (1989) J. Org. Chem. 54:109:115); analogs provided by the following references: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans, p. 1687; a GIy-Ala turn analog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bond isostere (Jones et al. (1988) Tetrahedron Lett. 29(31):3853-3856); tretrazol (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al. (1990) J. Org. Chem. 55(3):936-940. Conformationally restricted mimetics of beta turns and beta bulges, and peptides containing them, are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn. In other embodiments, a peptide of this invention is conjugated to one of various other molecules, as described hereinbelow, which can be via covalent or non-covalent linkage (complexed), the nature of which varies, in another embodiment, depending on the particular purpose. In another embodiment, the peptide is covalently or non-covalently complexed to a macromolecular carrier, (e.g. an immunogenic carrier), including, but not limited to, natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. In another embodiment, a peptide of this invention is linked to a substrate. In another embodiment, the peptide is conjugated to a fatty acid, for introduction into a liposome (U.S. Pat. No. 5,837,249). In another embodiment, a peptide of the invention is complexed covalently or non-covalently with a solid support, a variety of which are known in the art. In another embodiment, linkage of the peptide to the carrier, substrate, fatty acid, or solid support serves to increase an elicited an immune response. In other embodiments, the carrier is thyroglobulin, an albumin (e.g. human serum albumin), tetanus toxoid, polyamino acids such as poly (lysine: glutamic acid), an influenza protein, hepatitis B virus core protein, keyhole limpet hemocyanin, an albumin, or another carrier protein or carrier peptide; hepatitis B virus recombinant vaccine, or an APC. Each possibility represents a separate embodiment of the present invention. In another embodiment, the term “amino acid” (AA) refers to a natural or, in another embodiment, an unnatural or synthetic AA, and can include, in other embodiments, glycine, D- or L optical isomers, AA analogs, peptidomimetics, or combinations thereof. In another embodiment, the terms “cancer,” “neoplasm,” “neoplastic” or “tumor,” are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. In another embodiment, a tumor is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation, and in another embodiment, is identified by biochemical or immunologic findings, the latter which is used to identify cancerous cells, as well, in other embodiments. Methods for synthesizing peptides are well known in the art. In another embodiment, the peptides of this invention are synthesized using an appropriate solid-state synthetic procedure (see for example, Steward and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco, Calif. (1968); Merrifield (1967) Recent Progress in Hormone Res 23: 451). The activity of these peptides is tested, in other embodiments, using assays as described herein. In another embodiment, the peptides of this invention are purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. In another embodiment, immuno-affinity chromatography is used, whereby an epitope is isolated by binding it to an affinity column comprising antibodies that were raised against that peptide, or a related peptide of the invention, and were affixed to a stationary support. In another embodiment, affinity tags such as hexa-His (Invitrogen), Maltose binding domain (New England Biolabs), influenza coat sequence (Kolodziej et al. (1991) Meth. Enzymol. 194:508-509), glutathione-S-transferase, or others, are attached to the peptides of this invention to allow easy purification by passage over an appropriate affinity column. Isolated peptides can also be physically characterized, in other embodiments, using such techniques as proteolysis, nuclear magnetic resonance, and x-ray crystallography. In another embodiment, the peptides of this invention are produced by in vitro translation, through known techniques, as will be evident to one skilled in the art. In another embodiment, the peptides are differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule or other ligand, (Ferguson et al. (1988) Ann. Rev. Biochem. 57:285-320). In another embodiment, the peptides of this invention further comprise a detectable label, which in another embodiment, is fluorescent, or in another embodiment, luminescent, or in another embodiment, radioactive, or in another embodiment, electron dense. In other embodiments, the detectable label comprises, for example, green fluorescent protein (GFP), DS-Red (red fluorescent protein), secreted alkaline phosphatase (SEAP), beta-galactosidase, luciferase, <32>P, <125>I, <3>H and <14>C, fluorescein and its derivatives, rhodamine and its derivatives, dansyl and umbelliferone, luciferin or any number of other such labels known to one skilled in the art. The particular label used will depend upon the type of immunoassay used. In another embodiment, a peptide of this invention is linked to a substrate, which, in another embodiment, serves as a carrier. In another embodiment, linkage of the peptide to a substrate serves to increase an elicited an immune response. In another embodiment, peptides of this invention are linked to other molecules, as described herein, using conventional cross-linking agents such as carbodiimides. Examples of carbodiimides are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC), 1-ethyl-3-(3-dimethyaminopropyl) carbodiimide (EDC) and 1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide. In other embodiments, the cross-linking agents comprise cyanogen bromide, glutaraldehyde and succinic anhydride. In general, any of a number of homo-bifunctional agents including a homo-bifunctional aldehyde, a homo-bifunctional epoxide, a homo-bifunctional imido-ester, a homo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctional maleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyl disulfide, a homo-bifunctional aryl halide, a homo-bifunctional hydrazide, a homo-bifunctional diazonium derivative and a homo-bifunctional photoreactive compound can be used. Also envisioned, in other embodiments, are hetero-bifunctional compounds, for example, compounds having an amine-reactive and a sulfhydryl-reactive group, compounds with an amine-reactive and a photoreactive group and compounds with a carbonyl-reactive and a sulfhydryl-reactive group. In other embodiments, the homo-bifunctional cross-linking agents include the bifunctional N-hydroxysuccinimide esters dithiobis(succinimidylpropionate), disuccinimidyl suberate, and disuccinimidyl tartarate; the bifunctional imido-esters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1,4-di-[3′-(2′-pyridyldithio)propionamido]butane, bismaleimidohexane, and bis-N-maleimido-1, 8-octane; the bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactive agents such as bis-[b-(4-azidosalicylamido)ethyl] di sulfide; the bifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as 1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipic acid dihydrazide, carbohydrazide, and succinic acid dihydrazide; the bifunctional diazoniums o-tolidine, diazotized and bis-diazotized benzidine; the bifunctional alkylhalides N1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide), N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides and halomustards, such as ala′-diiodo-p-xylene sulfonic acid and tri(2-chloroethyl)amine, respectively, In other embodiments, hetero-bifunctional cross-linking agents used to link the peptides to other molecules, as described herein, include, but are not limited to, SMCC (succinimidyl-4-(N-rnaleimidomethyl)cyclohexane-1-carboxylate), MB S (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS (N-(.gamma.-maleimidobutyryloxy)succmimide ester), MPBH (4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl) cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate). In another embodiment, the peptides of the invention are formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules can be accomplished, in another embodiment, through salt bridge formation under low ionic strength environments, such as in deionized water. Large complexes can be created, in another embodiment, using charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine), which contain numerous negative and positive charges, respectively. In another embodiment, peptides are adsorbed to surfaces such as microparticle latex beads or to other hydrophobic polymers, forming non-covalently associated peptide-superantigen complexes effectively mimicking cross-linked or chemically polymerized protein, in other embodiments. In another embodiment, peptides are non-covalently linked through the use of biospecific interactions between other molecules. For instance, utilization of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives could be used to form peptide complexes. The peptides, according to this aspect, and in another embodiment, can be modified to possess biotin groups using common biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups. In another embodiment, a peptide of the present invention is linked to a carrier. In another embodiment, the carrier is KLH. In other embodiments, the carrier is any other carrier known in the art, including, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly (lysine:glutamic acid), influenza, hepatitis B virus core protein, hepatitis B virus recombinant vaccine and the like. Each possibility represents a separate embodiment of the present invention. In another embodiment, the peptides of this invention are conjugated to a lipid, such as P3 CSS. In another embodiment, the peptides of this invention are conjugated to a bead. In another embodiment, the compositions of this invention further comprise immunomodulating compounds. In other embodiments, the immunomodulating compound is a cytokine, chemokine, or complement component that enhances expression of immune system accessory or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-Ia and MIP-Ib, complement components, or combinations thereof. In other embodiments, the immunomodulating compound stimulate expression, or enhanced expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors, or combination thereof. In another embodiment, the immunomodulatory compound induces or enhances expression of co-stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule. In another embodiment, the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175:437-445), chondroitin sulfate-modified MHC invariant chain (Ii-CS) (Naujokas M. F. et al (1993) Cell 74:257-268), or an intracellular adhesion molecule 1 (ICAM-I) (Van R. H. (1992) Cell 71: 1065-1068), which assists, in another embodiment, co-stimulation by interacting with their cognate ligands on the T cells. In another embodiment, the composition comprises a solvent, including water, dispersion media, cell culture media, isotonic agents and the like. In another embodiment, the solvent is an aqueous isotonic buffered solution with a pH of around 7.0. In another embodiment, the composition comprises a diluent such as water, phosphate buffered saline, or saline. In another embodiment, the composition comprises a solvent, which is non-aqueous, such as propyl ethylene glycol, polyethylene glycol and vegetable oils. In another embodiment, the composition is formulated for administration by any of the many techniques known to those of skill in the art. For example, this invention provides for administration of the pharmaceutical composition parenterally, intravenously, subcutaneously, intradermally, intramucosally, topically, orally, or by inhalation. In another embodiment, the vaccine comprising a peptide of this invention further comprises a cell population, which, in another embodiment, comprises lymphocytes, monocytes, macrophages, dendritic cells, endothelial cells, stem cells or combinations thereof, which, in another embodiment are autologous, syngeneic or allogeneic, with respect to each other. In another embodiment, the cell population comprises a peptide of the present invention. In another embodiment, the cell population takes up the peptide. Each possibility represents a separate embodiment of the present invention. In another embodiment, the cell populations of this invention are obtained from in vivo sources, such as, for example, peripheral blood, leukopheresis blood product, apheresis blood product, peripheral lymph nodes, gut associated lymphoid tissue, spleen, thymus, cord blood, mesenteric lymph nodes, liver, sites of immunologic lesions, e.g. synovial fluid, pancreas, cerebrospinal fluid, tumor samples, granulomatous tissue, or any other source where such cells can be obtained. In another embodiment, the cell populations are obtained from human sources, which are, in other embodiments, from human fetal, neonatal, child, or adult sources. In another embodiment, the cell populations of this invention are obtained from animal sources, such as, for example, porcine or simian, or any other animal of interest. In another embodiment, the cell populations of this invention are obtained from subjects that are normal, or in another embodiment, diseased, or in another embodiment, susceptible to a disease of interest. In another embodiment, the cell populations of this invention are separated via affinity-based separation methods. Techniques for affinity separation include, in other embodiments, magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or use in conjunction with a monoclonal antibody, for example, complement and cytotoxins, and “panning” with an antibody attached to a solid matrix, such as a plate, or any other convenient technique. In other embodiment, separation techniques include the use of fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. In other embodiments, any technique that enables separation of the cell populations of this invention can be employed, and is to be considered as part of this invention. In another embodiment, the dendritic cells are from the diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, qualified as such (Steinman (1991) Ann. Rev. Immunol. 9:271-296). In another embodiment, the dendritic cells used in this invention are isolated from bone marrow, or in another embodiment, derived from bone marrow progenitor cells, or, in another embodiment, from isolated from/derived from peripheral blood, or in another embodiment, derived from, or are a cell line. In another embodiment, the cell populations described herein are isolated from the white blood cell fraction of a mammal, such as a murine, simian or a human (See, e.g., WO 96/23060). The white blood cell fraction can be, in another embodiment, isolated from the peripheral blood of the mammal. Methods of isolating dendritic cells are well known in the art. In another embodiment, the DC are isolated via a method which includes the following steps: (a) providing a white blood cell fraction obtained from a mammalian source by methods known in the art such as leukophoresis; (b) separating the white blood cell fraction of step (a) into four or more subfractions by countercurrent centrifugal elutriation; (c) stimulating conversion of monocytes in one or more fractions from step (b) to dendritic cells by contacting the cells with calcium ionophore, GM-CSF and IL-13 or GM-CSF and IL-4, (d) identifying the dendritic cell-enriched fraction from step (c); and (e) collecting the enriched fraction of step (d), preferably at about 4[deg.] C. In another embodiment, the dendritic cell-enriched fraction is identified by fluorescence-activated cell sorting, which identifies at least one of the following markers: HLA-DR, HLA-DQ, or B7.2, and the simultaneous absence of the following markers: CD3, CD14, CD16, 56, 57, and CD 19, 20. In another embodiment, the cell population comprises lymphocytes, which are, in another embodiment, T cells, or in another embodiment, B cells. The T cells are, in other embodiments, characterized as NK cells, helper T cells, cytotoxic T lymphocytes (CTL), TBLs, naive T cells, or combinations thereof. It is to be understood that T cells which are primary, or cell lines, clones, etc. are to be considered as part of this invention. In another embodiment, the T cells are CTL, or CTL lines, CTL clones, or CTLs isolated from tumor, inflammatory, or other infiltrates. In another embodiment, hematopoietic stem or early progenitor cells comprise the cell populations used in this invention. In another embodiment, such populations are isolated or derived, by leukaphoresis. In another embodiment, the leukapheresis follows cytokine administration, from bone marrow, peripheral blood (PB) or neonatal umbilical cord blood. In another embodiment, the stem or progenitor cells are characterized by their surface expression of the surface antigen marker known as CD34<+>, and exclusion of expression of the surface lineage antigen markers, Lin−. In another embodiment, the subject is administered a peptide, composition or vaccine of this invention, in conjunction with bone marrow cells. In another embodiment, the administration together with bone marrow cells embodiment follows previous irradiation of the subject, as part of the course of therapy, in order to suppress, inhibit or treat cancer in the subject. In another embodiment, the phrase “contacting a cell” or “contacting a population” refers to a method of exposure, which can be, in other embodiments, direct or indirect. In another embodiment, such contact comprises direct injection of the cell through any means well known in the art, such as microinjection. It is also envisaged, in another embodiment, that supply to the cell is indirect, such as via provision in a culture medium that surrounds the cell, or administration to a subject, via any route well known in the art, and as described herein. In another embodiment, CTL generation of methods of the present invention is accomplished in vivo, and is effected by introducing into a subject an antigen presenting cell contacted in vitro with a peptide of this invention (See for example Paglia et al. (1996) J. Exp. Med. 183:317-322). In another embodiment, the peptides of methods and compositions of the present invention are delivered to APC. In another embodiment, the peptide-pulsed APC are administered to a subject to elicit and immune response or treat or inhibit growth or recurrence of a tumor. Each possibility represents a separate embodiment of the present invention. In another embodiment, the peptides are delivered to APC in the form of cDNA encoding the peptides. In another embodiment, the term “antigen-presenting cells” (APC) refers to dendritic cells (DC), monocytes/macrophages, B lymphocytes or other cell type(s) expressing the necessary MHC/co-stimulatory molecules, which effectively allow for T cell recognition of the presented peptide. In another embodiment, the APC is a cancer cell. Each possibility represents a separate embodiment of the present invention. In another embodiment, the CTL are contacted with 2 or more APC populations. In another embodiment, the 2 or more APC populations present different peptides. Each possibility represents a separate embodiment of the present invention. In another embodiment, techniques that lead to the expression of antigen in the cytosol of APC (e.g. DC) are used to deliver the peptides to the APC. Methods for expressing antigens on APC are well known in the art. In another embodiment, the techniques include (1) the introduction into the APC of naked DNA encoding a peptide of this invention, (2) infection of APC with recombinant vectors expressing a peptide of this invention, and (3) introduction of a peptide of this invention into the cytosol of an APC using liposomes. (See Boczkowski D. et al. (1996) J. Exp. Med. 184:465-472; Rouse et al. (1994) J. Virol. 68:5685-5689; and Nair et al. (1992) J. Exp. Med. 175:609-612). In another embodiment, foster APC such as those derived from the human cell line 174xCEM.T2, referred to as T2, which contains a mutation in its antigen processing pathway that restricts the association of endogenous peptides with cell surface MHC class I molecules (Zweerink et al. (1993) J. Immunol. 150:1763-1771), are used, as exemplified herein. In another embodiment, as described herein, the subject is exposed to a peptide, or a composition/cell population comprising a peptide of this invention, which differs from the native protein expressed, wherein subsequently a host immune cross-reactive with the native protein/antigen develops. In another embodiment, the subject, as referred to in any of the methods or embodiments of this invention is a human. In other embodiments, the subject is a mammal, which can be a mouse, rat, rabbit, hamster, guinea pig, horse, cow, sheep, goat, pig, cat, dog, monkey, or ape. Each possibility represents a separate embodiment of the present invention. In another embodiment, peptides, vaccines, and compositions of this invention stimulate an immune response that results in tumor cell lysis. In another embodiment, any of the methods described herein is used to elicit CTL, which are elicited in vitro. In another embodiment, the CTL are elicited ex-vivo. In another embodiment, the CTL are elicited in vitro. The resulting CTL, are, in another embodiment, administered to the subject, thereby treating the condition associated with the peptide, an expression product comprising the peptide, or a homologue thereof. Each possibility represents a separate embodiment of the present invention. In another embodiment, the method entails introduction of the genetic sequence that encodes the peptides of this invention using, e.g., one or more nucleic acid delivery techniques. Nucleic acids of the invention include, in another embodiment, DNA, RNA and mixtures of DNA and RNA, alone or in conjunction with non-nucleic acid components. In another embodiment, the method comprises administering to the subject a vector comprising a nucleotide sequence, which encodes a peptide of the present invention (Tindle, R. W. et al. Virology (1994) 200:54). In another embodiment, the method comprises administering to the subject naked DNA which encodes a peptide, or in another embodiment, two or more peptides of this invention (Nabel, et al. PNAS-USA (1990) 90: 11307). In another embodiment, multi-epitope, analogue-based cancer vaccines are utilized (Fikes et al, Design of multi-epitope, analogue-based cancer vaccines. Expert Opin Biol Ther. 2003 September; 3(6):985-93). Each possibility represents a separate embodiment of the present invention. Nucleic acids can be administered to a subject via any means as is known in the art, including parenteral or intravenous administration, or in another embodiment, by means of a gene gun. In another embodiment, the nucleic acids are administered in a composition, which correspond, in other embodiments, to any embodiment listed herein. Vectors for use according to methods of this invention can comprise any vector that facilitates or allows for the expression of a peptide of this invention. Vectors comprises, in some embodiments, attenuated viruses, such as vaccinia or fowlpox, such as described in, e.g., U.S. Pat. No. 4,722,848, incorporated herein by reference. In another embodiment, the vector is BCG (Bacille Calmette Guerin), such as described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g.,Salmonella typhivectors and the like, will be apparent to those skilled in the art from the description herein. In another embodiment, the vector further encodes for an immunomodulatory compound, as described herein. In another embodiment, the subject is administered an additional vector encoding same, concurrent, prior to or following administration of the vector encoding a peptide of this invention to the subject. In another embodiment, the peptides, compositions and vaccines of this invention are administered to a subject, or utilized in the methods of this invention, in combination with other anticancer compounds and chemotherapeutics, including monoclonal antibodies directed against alternate cancer antigens, or, in another embodiment, epitopes that consist of an AA sequence which corresponds to, or in part to, that from which the peptides of this invention are derived. Various embodiments of dosage ranges are contemplated by this invention. [mu] refers to micro; [mu]g referring to microgram or micrograms. In another embodiment, the dosage is 20 [mu]g per peptide per day. In another embodiment, the dosage is 10 [mu]g/peptide/day. In another embodiment, the dosage is 30 [mu]g/peptide/day. In another embodiment, the dosage is 40 [mu]g/peptide/day. In another embodiment, the dosage is 60 [mu]g/peptide/day. In another embodiment, the dosage is 80 [mu]g/peptide/day. In another embodiment, the dosage is 100 [mu]g/peptide/day. In another embodiment, the dosage is 150 [mu]g/peptide/day. In another embodiment, the dosage is 200 [mu]g/peptide/day. In another embodiment, the dosage is 300 [mu]g/peptide/day. In another embodiment, the dosage is 400 [mu]g/peptide/day. In another embodiment, the dosage is 600 [mu]g/peptide/day. In another embodiment, the dosage is 800 [mu]g/peptide/day. In another embodiment, the dosage is 1000 [mu]g/peptide/day. In another embodiment, the dosage is 1500 [mu]g/peptide/day. In another embodiment, the dosage is 2000 [mu]g/peptide/day. In another embodiment, the dosage is 10 [mu]g/peptide/dose. In another embodiment, the dosage is 30 [mu]g/peptide/dose. In another embodiment, the dosage is 40 [mu]g/peptide/dose. In another embodiment, the dosage is 60 [mu]g/peptide/dose. In another embodiment, the dosage is 80 [mu]g/peptide/dose. In another embodiment, the dosage is 100 [mu]g/peptide/dose. In another embodiment, the dosage is 150 [mu]g/peptide/dose. In another embodiment, the dosage is 200 [mu]g/peptide/dose. In another embodiment, the dosage is 300 [mu]g/peptide/dose. In another embodiment, the dosage is 400 [mu]g/peptide/dose. In another embodiment, the dosage is 600 [mu]g/peptide/dose. In another embodiment, the dosage is 800 [mu]g/peptide/dose. In another embodiment, the dosage is 1000 [mu]g/peptide/dose. In another embodiment, the dosage is 1500 [mu]g/peptide/dose. In another embodiment, the dosage is 2000 [mu]g/peptide/dose. In another embodiment, the dosage is 10-20 [mu]g/peptide/dose. In another embodiment, the dosage is 20-30 [mu]g/peptide/dose. In another embodiment, the dosage is 20-40 [mu]g/peptide/dose. In another embodiment, the dosage is 30-60 [mu]g/peptide/dose. In another embodiment, the dosage is 40-80 [mu]g/peptide/dose. In another embodiment, the dosage is 50-100 [mu]g/peptide/dose. In another embodiment, the dosage is 50-150 [mu]g/peptide/dose. In another embodiment, the dosage is 100-200 [mu]g/peptide/dose. In another embodiment, the dosage is 200-300 [mu]g/peptide/dose. In another embodiment, the dosage is 300-400 [mu]g/peptide/dose. In another embodiment, the dosage is 400-600 [mu]g/peptide/dose. In another embodiment, the dosage is 500-800 [mu]g/peptide/dose. In another embodiment, the dosage is 800-1000 [mu]g/peptide/dose. In another embodiment, the dosage is 1000-1500 [mu]g/peptide/dose. In another embodiment, the dosage is 1500-2000 [mu]g/peptide/dose. In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts. Each of the above doses represents a separate embodiment of the present invention. Various embodiments of dosage ranges are contemplated by this invention. In another embodiment, the dosage is 20 mg per peptide per day. In another embodiment, the dosage is 10 mg/peptide/day. In another embodiment, the dosage is 30 mg/peptide/day. In another embodiment, the dosage is 40 mg/peptide/day. In another embodiment, the dosage is 60 mg/peptide/day. In another embodiment, the dosage is 80 mg/peptide/day. In another embodiment, the dosage is 100 mg/peptide/day. In another embodiment, the dosage is 150 mg/peptide/day. In another embodiment, the dosage is 200 mg/peptide/day. In another embodiment, the dosage is 300 mg/peptide/day. In another embodiment, the dosage is 400 mg/peptide/day. In another embodiment, the dosage is 600 mg/peptide/day. In another embodiment, the dosage is 800 mg/peptide/day. In another embodiment, the dosage is 1000 mg/peptide/day. In another embodiment, the dosage is 10 mg/peptide/dose. In another embodiment, the dosage is 30 mg/peptide/dose. In another embodiment, the dosage is 40 mg/peptide/dose. In another embodiment, the dosage is 60 mg/peptide/dose. In another embodiment, the dosage is 80 mg/peptide/dose. In another embodiment, the dosage is 100 mg/peptide/dose. In another embodiment, the dosage is 150 mg/peptide/dose. In another embodiment, the dosage is 200 mg/peptide/dose. In another embodiment, the dosage is 300 mg/peptide/dose. In another embodiment, the dosage is 400 mg/peptide/dose. In another embodiment, the dosage is 600 mg/peptide/dose. In another embodiment, the dosage is 800 mg/peptide/dose. In another embodiment, the dosage is 1000 mg/peptide/dose. In another embodiment, the dosage is 10-20 mg/peptide/dose. In another embodiment, the dosage is 20-30 mg/peptide/dose. In another embodiment, the dosage is 20-40 mg/peptide/dose. In another embodiment, the dosage is 30-60 mg/peptide/dose. In another embodiment, the dosage is 40-80 mg/peptide/dose. In another embodiment, the dosage is 50-100 mg/peptide/dose. In another embodiment, the dosage is 50-150 mg/peptide/dose. In another embodiment, the dosage is 100-200 mg/peptide/dose. In another embodiment, the dosage is 200-300 mg/peptide/dose. In another embodiment, the dosage is 300-400 mg/peptide/dose. In another embodiment, the dosage is 400-600 mg/peptide/dose. In another embodiment, the dosage is 500-800 mg/peptide/dose. In another embodiment, the dosage is 800-1000 mg/peptide/dose. In another embodiment, the total amount of peptide per dose or per day is one of the above amounts. In another embodiment, the total peptide dose per dose is one of the above amounts. Each of the above doses represents a separate embodiment of the present invention. In another embodiment, the present invention provides a kit comprising a peptide, composition or vaccine of the present invention. In another embodiment, the kit further comprises a label or packaging insert. In another embodiment, the kit is used for detecting a WT1-specific CD4 response through the use of a delayed-type hypersensitivity test. In another embodiment, the kit is used for any other method enumerated herein. In another embodiment, the kit is used for any other method known in the art. Each possibility represents a separate embodiment of the present invention. Example 1. Materials and Methods Peptide Design. Using three computer-based predictive algorisms BIMAS (http://www-bimas.cit.nih.gov/cgi-bin/molbio/ken_parker_comboform), SYFPEITHI (http://www.syfpeithi.de/) and RANKPEP (http://bio.dfci.harvard.edu/Tools/rankpep.html), epitopes were selected for both CD8 and CD4 T cells by starting with the native WT1 protein sequences that are capable of inducing immune response in normal donors. Heteroclitic peptides were designed by altering a single amino acid in the anchor residues of the native peptides for class I, which resulted in a higher predicted binding than its native sequences. The class II peptides were designed by adding flanking residues to the class I peptides, in order to simultaneously stimulate both CD4 and CD8 T cells. While many sequences can be predicted by the algorithms, these models do not predict binding to MHC when tested on live cells in 30% of cases (Gomez-Nunez et al. Leuk Res. 2006; 30(10): 1293-8), therefore in vitro testing is necessary. In addition, even if binding is demonstrated, a cytotoxic T cell response may not occur, requiring additional in vitro study. Peptide Synthesis. All peptides were purchased and synthesized by Genemed Synthesis, Inc. (San Antonio, TX). Peptides were sterile with purity of 70% to 90%. The peptides were dissolved in DMSO and diluted in saline at 5 mg/mL and stored at −80° C. Control peptides used are: for HLA-DR.B1: JAK-2-derived DR.B1-binding peptide JAK2-DR (GVCVCGDENILVQEF; SEQ ID NO:59) or BCR.ABL-derived peptide (IVHSATGFKQSSKALQRPVASDFEP; SEQ ID NO:60); for HLA-A0201: ewing sarcoma-derived peptide EW (QLQNPSYDK; SEQ ID NO:61) and for HLA-A2402: prostate-specific membrane antigen (PMSA)-derived peptide 624-632 (TYSVSFDSL; SEQ ID NO:62). Cells Lines, Cytokines and Antibodies. Human leukemia cell lines BA25 and HL-60 were used as a targets for measuring cytotoxicity of T cells. Human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1beta, IL-4, IL-6, IL-15, tumor necrosis factor (TNF)-alpha and prostaglandin E2 (PGE2) were purchased from R&D Systems (Minneapolis, MN). Beta 2-microglobulin (b2-m) was purchased from Sigma (St. Louis, MO). The antibodies used for immunofluorescence assays including mAbs to human CD3, CD4, CD8, HLA-A2 (clone BB7.2) and isotype controls were obtained from BD Biosciences (San Diego, CA). Cell isolation kits for CD14 and CD3 were purchased from Miltenyi Biotec. (Bergisch Gladbach, Germany). T2 Assay for Peptide Binding. T2 cells (TAP-, HLA-A0201+) were incubated overnight at 37° C. at 1×106cells/ml in FCS-free RPMI medium supplemented with 10 ug/ml human beta-2m (Sigma, St Louis, MO, USA) in the absence (negative control) or presence peptides at various final concentrations (50, 10 and 2 ug/ml). Brefeldin A (Sigma) at 5 ug/ml was added to the cultures for the final two hrs of incubation. Then T2 cells were washed and stained with anti-HLA-A2.1 (BB7.2) mAb conjugated to FITC for 30 min at 4° C. and followed by washing with staining buffer (PBS plus 1% FBS and 0.02% azide). The expression of the HLA-A2 on the cell surface was measured by flow cytometry on a FACScalibur (Becton Dickinson) and analyzed with FlowJo 9.6.3 software. In Vitro Stimulation and Human T-Cell Cultures. Peripheral blood mononuclear cells (PBMCs) from HLA-typed healthy donors were obtained by Ficoll density centrifugation. CD14+ monocytes were isolated by positive selection using mAb to human CD14 coupled with magnetic beads (Miltenyi Biotec) and were used for the first stimulation of T cells. The CD14− fraction of PBMC were used for isolation of CD3, by negative immunomagnetic cell separation using a pan T cell isolation kit (Miltenyi Biotec). The purity of the cells was always more than 98%. T cells were stimulated for 7 days in the presence of RPMI 1640 supplemented with 5% autologous plasma (AP), 20 ug/mL synthetic peptides, 1 ug/mL B2-m, and 10 ng/mL IL-15. Monocyte-derived dendritic cells (DCs) were generated from CD14+ cells, by culturing the cells in RPMI 1640 medium supplemented with 1% AP, 500 units/mL recombinant IL-4, and 1,000 units/mL GM-CSF. On days 2 and 4 of incubation, fresh medium with IL-4 and GM-CSF was either added or replaced half of the culture medium. On day 5, 20 ug/mL class II peptide was added to the immature DCs, for the processing. On day 6, maturation cytokine cocktail was added (Dao et al. Plos One 2009; 4(8):e6730). On day 7 or 8, T cells were re-stimulated with mature DCs, with IL-15. In most cases, T cells were stimulated 3 times in the same manner, using either DCs or CD14+ cells as antigen-presenting cells (APCs). A week after final stimulation, the peptide-specific T cell response was examined by IFN-g enzyme-linked immunospot (ELISPOT) assay and the cytotoxicity was tested, by51chromium (Cr)-release assay. IFN-g ELISPOT. HA-Multiscreen plates (Millipore) were coated with 100 uL of mouse anti-human IFN-g antibody (10 Ag/mL; clone 1-D1K; Mabtech) in PBS, incubated overnight at 4 C, washed with PBS to remove unbound antibody, and blocked with RPMI 1640/10% autologous plasma (AP) for 2 h at 37° C. Purified CD3+ T cells (>98% pure) were plated with either autologous CD14+ (10:1 E:APC ratio) or autologous DCs (30:1 E:APC ratio). Various test peptides were added to the wells at 20 ug/mL. Negative control wells contained APCs and T cells without peptides or with irrelevant peptides. Positive control wells contained T cells plus APCs plus 20 ug/mL phytohemagglutinin (PHA, Sigma). All conditions were done in triplicates. Microtiter plates were incubated for 20 h at 37° C. and then extensively washed with PBS/0.05% Tween and 100 ul/well biotinylated detection antibody against human IFN-g (2 ug/mL; clone 7-B6-1; Mabtech) was added. Plates were incubated for an additional 2 h at 37° C. and spot development was done as described (Dao et al., op. cit.). Spot numbers were automatically determined with the use of a computer-assisted video image analyzer with KS ELISPOT 4.0 software (Carl Zeiss Vision). 51Chromium Release Assay. The presence of specific CTLs was measured in a standard chromium release assay as described (Dao et al., op. cit.). Briefly, target cells alone, or pulsed with 50 ug/mL of synthetic peptides for 2 hours (in some cases for over night) at 37° C., are labeled with 50 uCi/million cells of Na251CrO4(NEN Life Science Products, Inc.). After extensive washing, target cells are incubated with T cells at E:T ratios ranging from 100:1 to 10:1. All conditions were done in triplicate. Plates were incubated for 4-5 hrs at 37° C. in 5% CO2. Supernatant fluids were harvested and radioactivity was measured in a gamma counter. Percentage specific lysis was determined from the following formula: [(experimental release−spontaneous release)/(maximum release−spontaneous release)]×100%. Maximum release was determined by lysis of radiolabeled targets in 1% SDS. Example 2. Binding of the Native and its Analogue Peptides to HLA-A0201 and HLA-A2402 Using a pool of 15 mer overlapping peptides spanning human WT1 protein to sensitize human T cells in vitro, the sequence 239-248 (NQMNLGATL; SEQ ID NO:5; herein abbreviated NQM or) has recently been identified as an immunogenic CD8 T cell epitope in the context of HLA-A2402 (Doubrovina et al., Blood 2012; 123(8):1633-46). In order to generate analog peptides with stronger immunogenicity, the prediction scores of the native peptide and possible analogs with various amino acid substitutions in the position 2 and 9 (class I anchor residues) was screened, using three online available databases (BIMAS, RANKPEP and SYFPEITHI). The predicted binding scores from all three databases showed better binding of the native NQMNLGATL (SEQ ID NO:5) peptide to HLA-A0201 than HLA-A2402 molecule (Table I). When the glutamine at the position 2 was substituted by leucine, the binding score to HLA-A2402 remained at the similar level by all 3 prediction programs. However, a significantly stronger binding score was predicted for HLA-A0201. On the other hand, when the glutamine at the position 2 was substituted by tyrosine, binding score to HLA-A2402 was dramatically improved, showing about 90-fold increased binding by BIMAS prediction. All three peptides were predicted to be cleaved at c-terminal by RANKPEP algorithm, suggesting the processing of the peptide fragment. The binding score was checked by substitution with various amino acids at position 9 but none of them showed a significant improved binding compared to the substitution at the position 2. Therefore, the two analogue peptides NLMNLGATL (SEQ ID NO:6; herein abbreviated NLM or A24-het-1) and NYMNLGATL (SEQ ID NO:7; herein abbreviated NYM or A24-het-2) were selected for further studies. TABLE 1Predictive binding scores of the peptides toHLA-A0201 and A2402RANKPEPBIMASSYFPETHI(score; % opt)SequencesHLA-HLA-HLA-HLA-HLA-HLA-(p 239-247)A0201A2402A0201A24A0201A2402NQMNLGATL8.0147.200171034; 26.56%10.482;(SEQ ID NO: 5)Cleaved27.23%,CleavedNLMNLGATL79.0417.2261078; 60.94%8.948;(SEQ ID NO: 6)Cleaved23.24%,CleavedNYMNLGATL0.011360.00092041; 32.03%23.573;(SEQ ID NO: 7)Cleaved61.22%,Cleaved Example 3. Binding of the Peptides to HLA-A0201 and HLA-A2402 Molecules The immunogenicity of MHC class I-restricted peptides requires the capacity to bind and stabilize MHC class I molecules on the live cell surface. Moreover, the computer prediction has only up to 70% accuracy; therefore, direct measurement was sought of the strength of the interaction between the peptides and the HLA-A0201 molecules using a conventional binding and stabilization assay that uses the antigen-transporting-deficient (TAP2 negative) HLA-A0201 human T2 cells. T2 cells lack TAP function and consequently are defective in properly loading class I molecules with antigenic peptides generated in the cytosol. The association of exogenously added peptides with thermolabile, empty HLA-A0201 molecules stabilizes them and results in an increase in the level of surface HLA-A0201 recognizable by specific anti-HLA-A0201 mAb such as BB7.2. The T2 binding assay showed that native NQMNLGATL (SEQ ID NO:5) peptide did not increase the HLA-A2 expression on T2 cells (FIG.1, upper panel). However, the NLMNLGATL (SEQ ID NO:6) analogue peptide stabilized the HLA-A2 molecule by showing a dose-dependent increase in HLA-A2 expression, compared to the T2 cells without peptide pulsing (FIG.1middle panel). Similar to the native peptide NQMNLGATL, NYMNLGATL (SEQ ID NO:7) peptide did not increase the HLA-A2 expression (FIG.1lower panel). These data confirmed the HLA-A2 biding scores, predicted by the computer-based algorithm. Example 4. Induction of a Peptide-Specific of CD8 T Cell Response the Context of HLA-A0201 and A2402 Molecules Although affinity for MHC molecules is necessary for the peptide presentation, T cell recognition of the peptide presented by HLA molecules is another important requirement for eliciting the peptide-specific response. Therefore, using an in vitro stimulation protocol, the new synthetic WT1 peptide analogs were evaluated for their ability to stimulate peptide-specific T cell response in both HLA-A0201 and A2402 donors. To expand the peptide-specific T cell precursors, three to five in vitro stimulation were performed and the specific T cell response was measured by IFN-g production, when challenged with individual peptide. NLMNLGATL peptide induced strong IFN-g secretion which crossed reacted with the native NQMNLGATL peptide. Five stimulations of T cells enhanced the response showing by more IFN-g spots (FIG.2B) than 3 stimulation (FIG.2A). T cells after 5 stimulation with NLMNLGATL peptide were also tested for the cytotoxicity using51Cr release assay. No killing was observed against HL-60 cells that were WT1 positive but HLA-A2 negative. However, the T cells killed the WT1+ and HLA-A0201+ AML cell line SET-2 and primary leukemia blasts derived from a patient who is HLA-A0201 positive (FIG.3). Whether both NLMNLGATL and NYMNLGATL heteroclitic peptides could induce a better CD8 T cell responses in HLA-A2402 donors was determined. NLMNLGATL peptide could induce T cell responses against both NLMNLGATL and the native NQMNLGATL peptides, but there was no significant enhancement compared to the T cell response induced by the native NQMNLGATL peptide. In the contrast, NYMNLGATL peptide induced a strong T cell response against itself and the native peptide after 3 stimulation (FIG.4A) but the response was demised after 5 round stimulation (FIG.4B), which also showed a weak cross reactivity with native sequence. These data demonstrated that NLMNLGATL heteroclitic peptide is a strong epitope for CD8 T cells in the context of HLA-A0201 molecule. NYMNLGATL peptide, on the other hand, induced CD8 T cell response in HLA-A0201 positive donors, but the response was not significantly better than the NQMNLGATL peptide. Example 5. Induction of T Cell Response by HLA-DR.B1 Peptides that Recognizes NQMNLGATL CD8 T Cell Epitope It has been shown that a peptide combining both CD4 and CD8 epitopes is more effective than the single class I epitope in eliciting effective immune response for vaccine design, because CD4 T cells can help CD8 CTL by fully activating DCs through the CD40/CD40L signaling as well as by producing IL-2 and IFN-g. In addition, if T cells stimulated with longer peptides, in which CD8 T cell epitopes are imbedded in, could recognize the short peptides, it would confirm the processing of the CD8 T cell epitopes. Therefore, four HLA-DR.B1-binding peptides that span the NQMNLGATL and NLMNLGATL epitopes, respectively, were designed: (SEQ ID NO: 8)DR-Native-1: cmtwNQMNLGATLkg(SEQ ID NO: 9)DR-Native-2: wNQMNLGATLkgvaa(SEQ ID NO: 14)DR-het-1: cmtwNLMNLGATLkg(SEQ ID NO: 17)DR-het-2: wNLMNLGATLkgvaa Since there is no definitive method to predict the class II peptide cleavage, two different versions of the class II peptides were designed using the BIMAS, SYFPEITHI and RANKPEP algorithms (Table 2). TABLE 2Predictive binding scores of HLA-DRB bindingpeptides.DR.B1-DR.B1-DR.B1-DR.B1-DR.B1-DR.B1-SYFPEITHI010103010401070111011501Native-1DR-Native 11711610164cmtwNQMNLGATLkgSEQ ID NO: 8Het-1DR-het-11821610164cmtwNLMNLGATLkgSEQ ID NO: 14Native-2DR-Native-2171314161324wNQMNLGATLkgvaaSEQ ID NO: 9Het-2DR-het-2171314161324wNLMNLGATLkgvaaSEQ ID NO: 17 When T cells were stimulated with two “heteroclitic” DR.B1 peptides spanning the NLMNLGATL epitope, they induced T cell responses that were specific for both short and long peptides, showing by IFN-g secretion. Since CD4 peptides induce more potent response due to their massive production of cytokines, the background is usually higher than CD8 T cell peptide stimulation. Therefore, although both DR-heteroclitic peptides induced specific responses, DR-het-2 peptide showed a more clear response than the DR-het-1 peptide in a donor shown inFIG.5A. It was evident that DR-het-2 peptide induced responses were specific for both short peptides NQMNGATL and NLMNGATL, and DR-native 2 and het-2 peptides. More importantly, the responses were directed against irradiated tumor cell line BA-25 (WT1+A2+), but not for the HL-60 cells that were WT1+ but A0201 negative. Similarly, when T cells were stimulated with short peptides (NQMNLGATL or NLMNGATL) or long peptides as indicated inFIG.5B, only BA-25 but not HL-60 cells were killed. Example 6. Other HLA-DR.B1 Binding Peptides that Recognize NQMNLGATL CD8 T Cell Epitope In addition to those DR peptides described above, additional HLA-DR.B1-binding peptides that span the NQMNLGATL, NLMNLGATL and NLMNLGATL epitopes were designed and evaluated (Table 3): TABLE 3Predictive binding scores of the peptides to HLA-DR.B1SYFPEITHIDR.B1-DR.B1-DR.B1-DR.B1-DR.B1-DR.B1-010103010401070111011501NativecmtwNQMNLGATLkg1711610164(SEQ ID NO: 8)mtwNQMNLGATLkgv17116808(SEQ ID NO: 12)twNQMNLGATLkgva18212078(SEQ ID NO: 13)wNQMNLGATLkgvaa171314161324(SEQ ID NO: 9)Het24-1cmtwNLMNLGATLkg1821610164(SEQ ID NO: 14)mtwNLMNLGATLkgv17136808(SEQ ID NO: 15)twNLMNLGATLkgva26122081318(SEQ ID NO: 16)wNLMNLGATLkgvaa171314161324(SEQ ID NO: 17)Het24-2cmtwNYMNLGATLkg1711610164(SEQ ID NO: 10)mtwNYMNLGATLkgv17116808(SEQ ID NO: 19)twNYMNLGATLkgva2822210178(SEQ ID NO: 20)wNYMNLGATLkgvaa171314161324(SEQ ID NO: 11)DR.B1-DR.B1-DR.B1-DR.B1-DR.B1-DR.B1-RANKPEP010103010401070111011501cmtwNQMNLGATLkgva10.188;3.577;13.521;7.85;21.138;1.731;Native21.12%8.78%30.67%15.27%32.2%4.14%(SEQ ID NO:21)Binder:Binder:Binder:wNQMNtwNQMmtwNQLGATNLGAMNLG(CMT-(CM-(C-ATL-LKG-TLK-13aa)15aa)14aa)(SEQ ID(SEQ ID(SEQ IDNO: 28)NO: 24)NO: 26)cmtwNLMNLGATLkgva9.377; 12.728;11.145;7.85;22.089;6.209;Het24-19.44%6.7%25.28%15.27%33.65%14.84%(SEQ ID NO: 22)Binder:Binder:Binder:wNLMNMNLGAmtwNQLGATTLkgMNLG(CMT-(WNL-(C-ATL-LKG-VA-13aa)15aa)14aa)(SEQ ID(SEQ ID(SEQ IDNO:28)NO: 25)NO: 27)cmtwNYMNLGATLkgva7.184;4.061;11.145;7.85;18.539;8.439;Het24-214.89%9.97%25.28%15.27%28.23%20.17%(SEQ ID NO: 23)Binder:Binder:MNLGAmtwNQTLkgMNLG(WNY-(C-ATL-VA-13aa)14aa)(SEQ ID(SEQ IDNO: 28)NO: 18) Example 7. Generation of Peptides Derived from WTI Oncoprotein that Bind to Human HLA-B7 Class I and HLA-Dr Class II Molecules Peptides were also designed that that bind to HLA-B0702 (Table 4). The following peptide sequences were designed: RQRPHPGAL (B7-Native 1; SEQ ID NO:34), RLRPHPGAL (B7-het-1; SEQ ID NO:37), RIRPHPGAL (B7-het-2; SEQ ID NO:38), GALRNPTAC (Native 2; SEQ ID NO:29), and GALRNPTAL (B7-het-3; SEQ ID NO:31). The predictive binding scores of these and other variants are shown in Table 4. These peptides were tested in vitro and stimulate heteroclitic T cell responses (FIG.6). CD3 T cells from a HLA-B0702-positive donor were stimulated with 2 sets of peptides (total five) for 5 times in vitro. The peptide-specific response was measured by IFN-gamma ELISPOT assay, against individual peptide. For the first set of peptides, both heteroclitic-1 and 2, induced the peptide-specific responses, but the cross reactivity to the native 1 (N1) peptide was stronger for the het-2 than the het-1 peptide. For the second set of the peptides, heteroclitic peptide induced strong IFN-g production, when challenged with the stimulating peptide, but no cross-reactivity to the native sequence was found. TABLE 4Predictive binding scores of B7 peptides to HLA-B7 and other haplotypes.RANKPEPSYFPEITHI-SYFPEITHI-B0702SYFPEITHI-SYFPEITHI-SYFPEITHI-SYFPEITHI-SYFPEITHI-B-B-Score; % OptB0702A0201A0301A0101B-08270539021. GALRNPTAC−18.084−44.75%214(p -118 to -110)(B5101)SEQ ID NO: 29GYLRNPTAC−20.632−51.06%AllSEQ ID NO: 30below8GALRNPTAL−9.401−23.27%12188161720SEQ ID NO: 31(B5101)YALRNPTAC−14.528−35.95%10AllSEQ ID NO: 32below10GLLRNPTAC−20.18−49.94%2141814SEQ ID NO: 332. RQRPHPGAL−3.687−9.12%151313171423(p -125 to -117)(1501)SEQ ID NO: 34RYRPHPGAL−4.517−11.18%15131317SEQ ID NO: 35YQRPHPGAL−5.618−13.90%15SEQ ID NO: 36RLRPHPGAL−4.065−10.06%1523232317SEQ ID NO: 37(B37)RIRPHPGAL−2.674−6.62%15212121SEQ ID NO: 38BIMAS-B7GALRNPTAC0.3SEQ ID NO: 30GALRNPTAL12SEQ ID NO: 31RQRPHPGAL40SEQ ID NO: 34RLRPHPGAL40SEQ ID NO: 37RIRPHPGAL40SEQ ID NO: 38 Based in the finding that the native peptides RQRPHPGAL (p-125 to -117; SEQ ID NO:34) and GALRNPTAC (p-118 to -110; SEQ ID NO:29) induce T cells responses in the context of HLA-B7 molecule, using HLA-binding prediction algorithms, one heteroclitic peptide for the GALRNPTAC peptide was designed (SEQ ID NO:31), and two heteroclitic peptides for RQRPHPGAL (SEQ ID NOS:37 and 38). Based on the binding prediction, these peptides may also be able to stimulate T cells in the context of other HLA haplotypes, such as: A0201, A0301, B8, B1501, B37 and B5101 (Table 4). Example 8. Generation of Peptides Derived from WTI Oncoprotein that Bind to Human HLA-B35, A0101, A0301, A1101 Class I and HLA-DR Class II Molecules Peptide QFPNHSFKHEDPMGQ (p170-182) (SEQ ID NO:39) induces T cells response in the context of HLA-DR.B1 0301 and 0401. The short sequences imbedded within the long peptide, HSFKHEDPM, induces T cell response in the context of B3501. Based on the predictions by the HLA-binding prediction algorithms, one heteroclitic long peptide was designed, which is the extension of the het-B35-1 short peptide. The sequences of the peptides are: Class II peptide: DR.B1-03/04-Native: QFPNHSFKHEDPM (SEQ ID NO:42), DR.B1-03/04-Het: QFPNHSFKHEDPY (SEQ ID NO:43; Class I peptides: 1. Native: HSFKHEDPM (SEQ ID NO:40), 2. Het-01/03-1: HSFKHEDPY (for A0101 and A0301) (SEQ ID NO:41), and 3. Het-03/11-1 HSFKHEDPK (for A0301 and A1101) (SEQ ID NO:42). Heteroclitic peptides for the HLA-B3501 haplotype were tested in silico (Table 5). TABLE 5Predictive binding scores of the natural peptides to HLA-DR.B1-0301,0402 and B3501, A0101, A0301 and A1101.Class IIDR.B1-DR.B1-DR.B1-DR.B1-DR.B1-DR.B1-SYFPEITHI (15 mer)010103010401070111011501QFPNHSFKHEDPMGQ821201414SEQ ID NO: 39RANKPEPQFPNHSFKHEDPMGQ−1.949;−2.786;6.717; 15.23%−4.04;3.393;8.864;SEQ ID NO: 39−4.04%−6.84%(0401)−8.56%5.17%21.18%6.165; 13.82%(0402)Class ISYFPEITHIBIMASRANKPEPHSFKHEDPM (B35-native)SEQ ID NO: 40B3501N/A10−3.568;−8.95%A010140.002−16.2;−26.61%A030100.005−3.832;−10.86%A1101110−12.047;−30.69%HSFKHEDPY (B35-het1)SEQ ID NO: 41B3501N/A10−2.86;−7.18%CleavedA0101190.075−5.164;−8.48% CleavedA030160.17.127;20.20% CleavedA11011100.433;1.1% CleavedHSFKHEDPK (B35-het-2)SEQ ID NO: 42B3501N/A0.05−11.223;−28.16%A010140.03−15.83;−26%A0301100.5−8.783;24.77%A1101210.044.316;11% Example 9. Generation of Peptides Derived from WT1 Oncoprotein that Bind to Human HLA-A1, A3, A11 Class I and HLA-DR.B1-0401 Class II Molecules Peptide KRPFMCAYPGCNK (320-332) (SEQ ID NO:44) was shown to induce T cell response in the context of HLA-DR.B1 0401. The short sequence imbedded within the long peptide, FMCAYPGCN (SEQ ID NO:45), induces T cell response in the context of B35, B7 and A0101 (Table 6). The binding scores were investigated of the peptides to multiple HLA haplotypes using prediction algorithms. One heteroclitic long peptide was designed, which is the extension of the het-1 short peptide. Two short heteroclitic peptides were designed that bind better to HLA-A0101, 0301 and 1101. The sequences of the peptides are: Class II peptide: DR.B1-04 Native: KRPFMCAYPGCNK (SEQ ID NO:44), DR.B1-04 het: KRPFMCAYPGCYK (SEQ ID NO:46); Class I peptides: 1. Native: FMCAYPGCN (SEQ ID NO:45), 2. DR.B1-04-Het-1 short: FMCAYPGCY (for A0101) (SEQ ID NO:47), 3. DR.B1-04-Het-2-short: FMCAYPGCK (for A0301 and A1101) (SEQ ID NO:48). KRPFMCAYPGCYK (SEQ ID NO:46) is the extension of DR.B1-04-het 1 short, FMCAYPGCN (SEQ ID NO:45), in which the end of the sequences CN becomes CY. TABLE 6Predictive binding scores of the peptides to HLA-DR.B1-0401 andB35, B7, A0101, A0301 and A1101.HLA-DR.B1DR.B1-SYFPEITHI (15 mer)010103010401070111011501KRPFMCAYPGCNKRY16822161016SEQ ID NO: 49KRPFMCAYPGCYKRY161616221012SEQ ID NO: 55SEKRPFMCAYPGCNK15000128SEQ ID NO: 50RANKPEPKRPFMCAYPGCNK5.381;−9.13;3.131;−0.486;0.756;4.199;SEQ ID NO: 4411.15%−22.35%7.1%−0.95%1.15%10.04%Class ISYFPEITHIBIMASRANKPEPFMCAYPGCN (native)SEQ ID NO: 45A010100.005-4.165; -6.84%B710.02-21.654;53.59%B35N/AN/A-23.926;-60.03%A030140.018-2.503;-7.09%A110180-2.509;5.25%FMCAYPGCY (DR.B1-04-hetl-short)SEQ ID NO: 47A0101150.256.613; 10.86%CleavedB710.02-13.887;34.37%,cleavedB35N/A0.02-11.078;27.8%A0301103.67.557;21.42%,cleavedA110180.0049.516;24.24%,cleavedFMCAYPGCK (DR.B1-04-Het-2 short)SEQ ID NO: 48A010100.14.053; −6.66%B710.01−20.85,51.71%B35N/A0.01−21.886;47.27%A030114189.168;25.99%A1101180.48.883;22.09% Example 10. Additional Cross-Reactivity Studies An ELISPOT assay was conducted using donor SA after 5 stimulations for the Het24-1 (SEQ ID NO:6) and Het24-2 (SEQ ID NO:7) A24 peptides, in comparison to the native sequence (SEQ ID NO:5). As shown inFIG.7, the heteroclitic peptides generate cross-reactive responses. | 167,869 |
11859016 | BEST MODE The present disclosure relates to a peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3. Mode of Disclosure Hereinafter, the present disclosure will be described in more detail with reference to the following examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. Preparation Example. Synthesis of Sequence List 70 g of chloro trityl chloride resin (CTC resin, Nova biochem Cat No. 01-64-0021) was added to a reactor and 490 ml of methylene chloride (MC) was added thereto, followed by stirring for 3 minutes. Subsequently, after the solution was removed therefrom, 490 ml of dimethyl formamide (DMF) was added thereto, the mixture was stirred for 3 minutes, and the solvent was removed therefrom. 700 ml of dichloromethane solution was added to the reactor, and then 200 mmole of Fmoc-Tyr(tBu)-OH (Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA) were added thereto. The mixture was dissolved by stirring and maintained for 1 hour while stirring. After the resultant was washed, methanol and DIEA (2:1) were dissolved in dichloromethane (DCM) and maintained for 10 minutes, and then the resultant was washed with an excess of DCM/DMF (1:1). Then, after the solution was removed, 490 ml of dimethyl formamide (DMF) was added thereto, and the mixture was stirred for 3 minutes, and then the solvent was removed therefrom. 700 ml of a deprotection solution (20% piperidine/DMF) was added to the reactor and stirred for 10 minutes at room temperature, and then the solution was removed. After the same amount of the deprotection solution was added thereto and maintained for 10 minutes, the solution was removed and the resultant was washed twice with DMF, once with MC, and once with DMF, each for 3 minutes to prepare a Tyr(tBu)-CTL resin. 700 ml of a DMF solution was added to a new reactor, and 200 mmole of Fmoc-Arg(Pbf)-OH (Bachem, Swiss), 200 mmole of HoBt, and 200 mmole of HBTu were added thereto and dissolved by stirring. 400 mmole of DIEA was added to the reactor in twice and the mixture was stirred for at least 5 minutes to completely dissolve all solids. The dissolved amino acid mixture solution was added to the reactor including the deprotected resin and maintained for 1 hour at room temperature while stirring. After the reaction solution was removed, the resultant was stirred with a DMF solution three times each for 5 minutes and then the DMF solution was removed therefrom. A small amount of the reaction resin was taken and the degree of reaction was checked by Kaiser test (Nihydrin Test). Deprotection reaction was conducted twice in the same manner using the deprotection solution to prepare an Arg(Pbf)-Tyr(tBu)-CTL resin. The resin was sufficiently washed with DMF and MC and subjected to the Kaiser test again, followed by amino acid binding test as describe above. According to a selected amino acid sequence, chain reactions were conducted in the order of Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Trp-OH, and Fmoc-Lys(Boc)-OH. After the Fmoc-protective group was reacted with the deprotection solution twice each for 10 minutes, the deprotection solution was removed by washing. The peptidyl resin was washed with DMF, MC, and methanol three times each, and dried while slowly flowing nitrogen gas, and then completely dried under the P2O5atmosphere in a vacuum. Then, 1,900 ml of a leaving solution [81.5% of trifluroacetic acid, 5.0% of distilled water, 5.0% of thioanisole, 5.0% of phenol, 2.5% of ethanedithiol (EDT), and 1.0 5 of triisopropylsilane (TIS)] was added thereto, and reactions of the mixture were maintained at room temperature for 2 hours while shaking. The resin was filtered, washed with a small amount of a TFA solution, and mixed with a mother solution. Cold ether was added to 2,090 ml of the mother solution to induce precipitation, and the mixture was centrifuged to collect precipitates, and then washed twice with cold ether. After removing the mother solution, the resultant was sufficiently dried under a nitrogen atmosphere to synthesize 70.8 g of a peptide consisting of an amino acid sequence of SEQ ID NO: 1 before purification (Yield: 97.0%). A molecular weight of 822.9 (Theoretical value: 822.9) was obtained using a molecular weight measurer. Peptides having amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 3 are synthesized in the same manner as the above method. TABLE 1Analysis value (massspectrometer)SEQ IDSequenceObtainedTheoreticalNO:listvaluevalue1KWGGGRY822.9822.92ILGRWCG803.9803.93DVENTS663.6663.6 Example 1. In Vitro Binding Assay Each of the peptides having amino acid sequences of SEQ ID NOS: 1, 2, and 3 mixed with a coating buffer (20 mM sodium phosphate, pH 9.6) at a concentration of 1.8 mM was seeded on a plate for an enzyme-linked immunosorbent assay (ELISA) and cultured at 4° C. overnight. Subsequently, the peptide was washed with phosphate buffered saline with Tween-20 (PBST) and blocked with 3% of bovine serum albumin (BSA) for 2 hours at room temperature. After washing with PBST, 2 μM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (hereinafter, referred to as TCDD) was added to each well and cultured at room temperature for 2 hours. Subsequently, after washing with PBST, treatment with anti-TCDD antibody conjugated with fluorescein isothiocyanate (FITC) was conducted at a ratio of antibody:PBST=1:100 and the resultant was cultured for 2 hours at room temperature. Then, after washing with PBST, an excitation 488 nm/emission 520 nm value was measured using a fluorescence meter, and the results are shown inFIGS.1A to1C, and Table 2. TABLE 2SEQ ID NO:Control50 μM500 μM1000 μM2000 μM1100%193%360%394%575%2100%128%264%358%405%3100%159%253%400%420% As shown inFIGS.1A to1Cand Table 2, it was confirmed that the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 directly binds to TCDD. Example 2. AhR Nuclear Translocation Test HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×105cells/well and cultured overnight. Subsequently, 10 nM of TCDD and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 1 hour and collected to obtain nuclei and cytoplasmic proteins separated from each other. Westin blotting was performed using an aryl hydrocarbon receptor (AhR) antibody (Santa Cruz Biotechnology, U.S.A.) to identify activated nuclear translocation of AhR, and the results are shown inFIGS.2A to2Fand Table 3. TABLE 3TCDD + PeptideSEQ ID NO:ControlTCDD5 μM50 μM11 times5.8 times1.9 times1.9 times21 times5.7 times2.5 times0.9 times31 times5.9 times3 times1.5 times As shown inFIGS.2A to2Fand Table 3, it was confirmed that the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 inhibits nuclear translocation of AhR by TCDD. Example 3. TCDD ICC HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×105cells/well and cultured overnight. Subsequently, 50 nM of TCDD and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 5 minutes and immobilized with 4% paraformaldehyde for 30 minutes. Then, after washing three times, the cells were reacted with 0.5% Triton X-100 for 15 minutes and washed three times. Subsequently, the cells were blocked with 3% BSA for 1 hour and reacted with a primary antibody against TCDD conjugated with fluorescein isothiocyanate (FITC) (1:100) at 4° C. overnight. The cells were stained and mounted with 4,6-diamidino-2-phenylindole (DAPI) and observed with a fluorescence microscope. The results are shown inFIGS.3A to3C. As shown inFIGS.3A to3C, the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 inhibited introduction of TCDD into cells. Example 4. ROS Analysis in Cell HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×105cells/well and cultured overnight. Subsequently, 10 nM of TCDD and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 24 hours and further treated with DCFH-DA for 30 minutes. Then, the cells were collected and subjected to FACS analysis to observe changes of average FL1 values, and the results are shown inFIGS.4A to4C. As shown inFIGS.4A to4C, the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 reduced ROS levels increased by TCDD in cells. Example 5. RT-PCR of CYP1A1 and Inflammatory Molecules HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×105cells/well and cultured overnight. Subsequently, 10 nM of TCDD and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 6 hours or 24 hours and collected to separate RNA therefrom. After quantifying RNA, cDNA was synthesized using a cDNA synthesis kit (Intron, Korea). Then, polymerase chain reaction (PCR) was performed using a PCR PreMix kit (Intron, Korea) and a primer for each of CYP1A1, TNF-a, IL-6, IL-1 b, and COX-2 shown in Table 4. Then, by running the resultant on a 5% agarose gel, the expression levels of mRNA of the growth factors were compared under the conditions of treating the respective samples, and the results are shown inFIGS.5A to5C. TABLE 4SEQIDNO:PrimerSequence (5′-3′)4CYP1A1_FGGATCTTTCTCTGTACCCTGG5CYP1A1_RAGCATGTCCTTCAGCCCAGA6TNF-a_FCGTCAGCCGATTRTGCTATCT7TNF-a_RCGGACTCCGCAAAGTCTAAG8IL-6_FAAAGAGGCACTGCCAGAAAA9IL-6_RATCTGAGGTGCCCATGCTAC10IL-1b_FTTCGACACATGGGATAACGA11IL-1b_RTCTTTCAACACGCAGGACAG12COX-2_FATCATTCACCAGGCAAATTGC13COX-2_RGGCTTCAGCATAAAGCGTTTG As shown inFIGS.5A to5C, the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 had the effect of inhibiting expressions of CYP1A1 and various inflammatory factors induced by TCDD. Example 6. AhR Nuclear Translocation Test HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×105cells/well and cultured overnight. 10 nM of urban particulate matter (PM, Sigma Aldrich, USA) and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 1 hour and collected to obtain nuclei and cytoplasmic proteins separated from each other. Then, Westin blotting was performed using aryl hydrocarbon receptor (AhR) antibody (Santa Cruz Biotechnology, USA) to identify activated nuclear translocation of AhR, and the results are shown inFIGS.6A to6C. As shown inFIGS.6A to6C, the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 inhibited nuclear translocation of AhR by particulate matter. Example 7. RT-PCR of CYP1A1 and Inflammatory Molecules HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×105cells/well and cultured overnight. Subsequently, 10 nM of particulate matter (PM) and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 6 hours or 24 hours and collected to separate RNA therefrom. After quantifying RNA, cDNA was synthesized using a cDNA synthesis kit (Intron, Korea). Then, PCR was performed using a PCR PreMix kit (Intron, Korea) and a primer for each of CYP1A1, TNF-a, IL-6, IL-1b, and COX-2 shown in Table 5. Then, by running the resultant on a 5% agarose gel, the expression levels of mRNA of the growth factors were compared under the conditions of treating the respective samples, and the results are shown inFIGS.7A to7C. TABLE 5SEQIDNO:PrimerSequence (5′-3′)4CYP1A1_FGGATCTTTCTCTGTACCCTGG5CYP1A1_RAGCATGTCCTTCAGCCCAGA6TNF-a_FCGTCAGCCGATTRTGCTATCT7TNF-a_RCGGACTCCGCAAAGTCTAAG8IL-6_FAAAGAGGCACTGCCAGAAAA9IL-6_RATCTGAGGTGCCCATGCTAC10IL-1b_FTTCGACACATGGGATAACGA11IL-1b_RTCTTTCAACACGCAGGACAG12COX-2_FATCATTCACCAGGCAAATTGC13COX-2_RGGCTTCAGCATAAAGCGTTTG As identified inFIGS.7A to7C, the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 showed the effects of inhibiting expressions of CYP1 A1 and various inflammatory factors induced by particulate matter. INDUSTRIAL AVAILABILITY The present disclosure relates to a peptide with a cytoprotective effect against environmental pollutants and a use thereof. | 12,645 |
11859017 | EXAMPLES Example 1 Identification and Quantitation of Tumor Associated Peptides Presented on the Cell Surface Tissue Samples Patients' tumor tissues were provided by University of Heidelberg, Heidelberg, Germany. Written informed consents of all patients had been given before surgery. Tissues were shock-frozen in liquid nitrogen immediately after surgery and stored until isolation of TUMAPs at −80° C. Isolation of HLA Peptides from Tissue Samples HLA peptide pools from shock-frozen tissue samples were obtained by immune precipitation from solid tissues according to a slightly modified protocol (Falk, K., 1991; Seeger, F. H. T., 1999) using the HLA-A*02-specific antibody BB7.2, the HLA-A, -B, -C-specific antibody W6/32, CNBr-activated sepharose, acid treatment, and ultrafiltration. Methods The HLA peptide pools as obtained were separated according to their hydrophobicity by reversed-phase chromatography (Acquity UPLC system, Waters) and the eluting peptides were analyzed in an LTQ-Orbitrap hybrid mass spectrometer (ThermoElectron) equipped with an ESI source. Peptide pools were loaded directly onto the analytical fused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) applying a flow rate of 400 nL per minute. Subsequently, the peptides were separated using a two-step 180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient was composed of Solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in the data-dependent mode using a TOPS strategy. In brief, a scan cycle was initiated with a full scan of high mass accuracy in the orbitrap (R=30 000), which was followed by MS/MS scans also in the orbitrap (R=7500) on the 5 most abundant precursor ions with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST and additional manual control. The identified peptide sequence was assured by comparison of the generated natural peptide fragmentation pattern with the fragmentation pattern of a synthetic sequence-identical reference peptide.FIGS.1A through1Dshow an exemplary spectrum obtained from tumor tissue for the MHC class I associated peptide ABCA13-001 and its elution profile on the UPLC system. Label-free relative LC-MS quantitation was performed by ion counting i.e. by extraction and analysis of LC-MS features (Mueller et al. 2007a). The method assumes that the peptide's LC-MS signal area correlates with its abundance in the sample. Extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al. 2007b; Sturm et al. 2008). Finally, all LC-MS features were cross-referenced with the sequence identification results to combine quantitative data of different samples and tissues to peptide presentation profiles. The quantitative data were normalized in a two-tier fashion according to central tendency to account for variation within technical and biological replicates. Thus each identified peptide can be associated with quantitative data allowing relative quantification between samples and tissues. In addition, all quantitative data acquired for peptide candidates was inspected manually to assure data consistency and to verify the accuracy of the automated analysis. For each peptide a presentation profile was calculated showing the mean sample presentation as well as replicate variations. The profile juxtaposes NSCLC samples to a baseline of normal tissue samples. Presentation profiles of exemplary over-presented peptides are shown inFIGS.3A through3C. Example 2 Expression Profiling of Genes Encoding the Peptides of the Invention Not all peptides identified as being presented on the surface of tumor cells by MHC molecules are suitable for immunotherapy, because the majority of these peptides are derived from normal cellular proteins expressed by many cell types. Only few of these peptides are tumor-associated and likely able to induce T cells with a high specificity of recognition for the tumor from which they were derived. In order to identify such peptides and minimize the risk for autoimmunity induced by vaccination the inventors focused on those peptides that are derived from proteins that are over-expressed on tumor cells compared to the majority of normal tissues. The ideal peptide will be derived from a protein that is unique to the tumor and not present in any other tissue. To identify peptides that are derived from genes with an expression profile similar to the ideal one the identified peptides were assigned to the proteins and genes, respectively, from which they were derived and expression profiles of these genes were generated. RNA Sources and Preparation Surgically removed tissue specimens were provided by University of Heidelberg, Heidelberg, Germany (see Example 1) after written informed consent had been obtained from each patient. Tumor tissue specimens were snap-frozen in liquid nitrogen immediately after surgery and later homogenized with mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TM Reagent (Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol. Total RNA from healthy human tissues was obtained commercially (Ambion, Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam, Netherlands; BioChain, Hayward, CA, USA). The RNA from several individuals (between 2 and 123 individuals) was mixed such that RNA from each individual was equally weighted. Quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent). Microarray Experiments Gene expression analysis of all tumor and normal tissue RNA samples was performed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA). All steps were carried out according to the Affymetrix manual. Briefly, double-stranded cDNA was synthesized from 5-8 μg of total RNA, using SuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech, Ebersberg, Germany) as described in the manual. In vitro transcription was performed with the BioArray High Yield RNA Transcript Labelling Kit (ENZO Diagnostics, Inc., Farmingdale, NY, USA) for the U133A arrays or with the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0 arrays, followed by cRNA fragmentation, hybridization, and staining with streptavidin-phycoerythrin and biotinylated anti-streptavidin antibody (Molecular Probes, Leiden, Netherlands). Images were scanned with the Agilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-Chip Scanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOS software (Affymetrix), using default settings for all parameters. For normalisation, 100 housekeeping genes provided by Affymetrix were used. Relative expression values were calculated from the signal log ratios given by the software and the normal kidney sample was arbitrarily set to 1.0. Exemplary expression profiles of source genes of the present invention that are highly over-expressed or exclusively expressed in non-small-cell lung carcinoma are shown inFIGS.2A and2B. Example 4 In Vitro Immunogenicity for NSCLC MHC Class I Presented Peptides In order to obtain information regarding the immunogenicity of the TUMAPs of the present invention, we performed investigations using an in vitro T-cell priming assay based on repeated stimulations of CD8+ T cells with artificial antigen presenting cells (aAPCs) loaded with peptide/MHC complexes and anti-CD28 antibody. This way we could show immunogenicity for 9 HLA-A*0201 restricted TUMAPs of the invention so far, demonstrating that these peptides are T-cell epitopes against which CD8+ precursor T cells exist in humans (Table 4). In Vitro Priming of CD8+ T Cells In order to perform in vitro stimulations by artificial antigen presenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, we first isolated CD8+ T cells from fresh HLA-A*02 leukapheresis products via positive selection using CD8 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtained from the Transfusion Medicine Tuebingen, Germany, after informed consent. Isolated CD8+ lymphocytes or PBMCs were incubated until use in T-cell medium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat inactivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/ml Streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nurnberg, Germany) were also added to the TCM at this step. Generation of pMHC/anti-CD28 coated beads, T-cell stimulations and readout was performed in a highly defined in vitro system using four different pMHC molecules per stimulation condition and 8 different pMHC molecules per readout condition. All pMHC complexes used for aAPC loading and cytometric readout were derived from UV-induced MHC ligand exchange (Rodenko et al., 2006) with minor modifications. In order to determine the amount of pMHC monomer obtained by exchange we performed streptavidin-based sandwich ELISAs according to (Rodenko et al., 2006). The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung et al., 1987) was chemically biotinylated using Sulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer (Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidin coated polystyrene particles (Bangs Laboratories, Illinois, USA). pMHC used for positive and negative control stimulations were A*0201/MLA-001 (peptide ELAGIGILTV from modified Melan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5), respectively. 800.000 beads/200 μl were coated in 96-well plates in the presence of 4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 were added subsequently in a volume of 200 μl. Stimulations were initiated in 96-well plates by co-incubating 1×106CD8+ T cells with 2×105washed coated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell) for 3-4 days at 37° C. Half of the medium was then exchanged by fresh TCM supplemented with 80 U/ml IL-2 and incubating was continued for 3-4 days at 37° C. This stimulation cycle was performed for a total of three times. For the pMHC multimer readout using 8 different pMHC molecules per condition, a two-dimensional combinatorial coding approach was used as previously described (Andersen et al., 2012) with minor modifications encompassing coupling to 5 different fluorochromes. Finally, multimeric analyses were performed by staining the cells with Live/dead near IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BD LSRII SORP cytometer equipped with appropriate lasers and filters was used. Peptide specific cells were calculated as percentage of total CD8+ cells. Evaluation of multimeric analysis was done using the FlowJo software (Tree Star, Oregon, USA). In vitro priming of specific multimer+ CD8+ lymphocytes was detected by by comparing to negative control stimulations. Immunogenicity for a given antigen was detected if at least one evaluable in vitro stimulated well of one healthy donor was found to contain a specific CD8+ T-cell line after in vitro stimulation (i.e. this well contained at least 1% of specific multimer+ among CD8+ T-cells and the percentage of specific multimer+ cells was at least 10× the median of the negative control stimulations). In Vitro Immunogenicity for NSCLC Peptides For tested HLA class I peptides, in vitro immunogenicity could be demonstrated by generation of peptide specific T-cell lines. Exemplary flow cytometry results after TUMAP-specific multimer staining for two peptides of the invention are shown inFIG.4together with corresponding negative controls. Results for 25 peptides from the invention are summarized in Table 5. TABLE 5In vitro immunogenicity of HLA class I peptides of the inventionExemplary results of in vitro immunogenicity experimentsconducted by the applicant for the peptides of the invention.SEQ ID NO:WellsDonors1+++2+++3+++4+++7++++++8+++9++10+++11++++++ (100%)15++++16+++19+++18++++21++++22++++24+++30+++31++++32++++33++++35+++37+++++ (100%)38+++39+++40+++42++++++ (100%)43++++44+++45++46++++47+++48++52++53++++54+++55+++56++++++ (100%)62++++++57+++59++++60+++++++ (100%)61++++63+++64++++65+++++66++++67+++68++69+++++70++++71++++72++++73+++74++++75+++78++++79+++++80+++81+++85++++++86+++87++++88+++92+++<20% = +;20%-49% = ++;50%-70%= +++;and >70% = ++++ Example 5 Syntheses of Peptides All peptides were synthesized using standard and well-established solid phase peptide synthesis using the Fmoc-strategy. After purification by preparative RP-HPLC, ion-exchange procedure was performed to incorporate physiological compatible counter ions (for example trifluoro-acetate, acetate, ammonium or chloride). Identity and purity of each individual peptide have been determined by mass spectrometry and analytical RP-HPLC. After ion-exchange procedure the peptides were obtained as white to off-white lyophilizates in purities of 90% to 99.7%. All TUMAPs are preferably administered as trifluoro-acetate salts or acetate salts, other salt-forms are also possible. For the measurements of example 4, trifluoro-acetate salts of the peptides were used. Example 6 UV-Ligand Exchange Candidate peptides for the vaccines according to the present invention were further tested for immunogenicity by in vitro priming assays. The individual peptide-MHC complexes required for these assays were produced by UV-ligand exchange, where a UV-sensitive peptide is cleaved upon UV-irradiation, and exchanged with the peptide of interest as analyzed. Only peptide candidates that can effectively bind and stabilize the peptide-receptive MHC molecules prevent dissociation of the MHC complexes. To determine the yield of the exchange reaction, an ELISA was performed based on the detection of the light chain (β2m) of stabilized MHC complexes. The assay was performed as generally described in Rodenko et al. (Rodenko B, Toebes M, Hadrup S R, van Esch W J, Molenaar A M, Schumacher T N, Ovaa H. Generation of peptide-MHC class I complexes through UV-mediated ligand exchange. Nat Protoc. 2006; 1(3):1120-32). 96 well MAXISorp plates (NUNC) were coated over night with 2 ug/ml streptavidin in PBS at room temperature, washed 4× and blocked for 30 min at 37° C. in 2% BSA containing blocking buffer. Refolded HLA-A*0201/MLA-001 monomers served as standards, covering the range of 8-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction were diluted 100 fold in blocking buffer. Samples were incubated for 1 h at 37° C., washed four times, incubated with 2 ug/ml HRP conjugated anti-β2m for 1 h at 37° C., washed again and detected with TMB solution that is stopped with NH2SO4. Absorption was measured at 450 nm. TABLE 6UV-Ligand exchangeAverage exchangeSEQ ID NO.Peptide nameyield in %Exchange yield81ANKS1A-00178++++87AURKB-00154+++85BUB1B-00159+++48SNRNP20-00154+++80CEP192-00156+++90COG4-00157+++89IFT81-00157+++83MDN1-00167+++82CEP250-00270+++91NCBP1-00165+++92NEFH-00150++84OLFM1-00148++86PI4KA-00151+++11SLC3A2-00156+++78SLI-00147++79TLX3-00170+++2MMP12-00357+++68FAP-00331++66IGF2BP3-00146++4DST-00150++5MXRA5-00157+++31GFPT2-00143++1ABCA13-00193++++6DST-00259+++40MXRA5-00256+++49SAMSN1-00147++8HNRNPH-00126++69WNT5A-00137++15IL8-00141++50STAT2-00169+++72ADAM8-00167+++73COL6A3-00281++++18VCAN-00141++12SMYD3-00150++3ABCA13-00236++35BNC1-00143++7CDK4-00145++19DROSHA-00168+++33GALNT2-00173++++13AKR-00113+39LAMC2-00161+++56RAD54B-00148++24COL12A1-00255+++43CSE1-00155+++45SEC61G-00118+47PCNXL3-00187++++9TANC2-00171++++70TPX2-00156+++17HUWE1-00145++54TACC3-00154+++32CERC-00162+++26SERPINB3-00147++58CCNA2-00154+++44DPYSL4-00177++++27KIF26B-00168+++51CNOT1-00157+++11SEC34A2-00151+++30RGS4-00149++20VCAN-00249++67CDC6-00148++74THY1-00165+++10RNF213-00184++++61RCN1-00175++++37FZD-00152+++71HMMR-00149++60C11orf24-00147++53JUNB-00151+++25ELANE-00162+++61RCC1-00177++++62MAGEF1-00183++++22ACACA-00161+++21PLEKHA8-00147++57EEF2-00231++41HSP-00247++38ATP-00119+46ORMDL1-00261+++59NET1-00182++++63NCAPD2-00176++++42VPS13B-00163+++64C12orf44-00134++23ITGA11-00153+++75DIO2-00150++28ANKH-00152+++65HERC4-00161+++16P2RY6-00191++++ Candidate peptides that show a high exchange yield (i.e. higher than 40%, preferably higher than 50%, more preferred higher than 70%, and most preferred higher than 80%) are generally preferred for a generation and production of antibodies or fragments thereof, and/or T cell receptors or fragments thereof, as they show sufficient avidity to the MHC molecules and prevent dissociation of the MHC complexes. Example 7 Binding and Immunogenicity of Selected MHC Class II Peptides HLA class II proteins are divided into 3 major isotypes HLA-DR, -DP, DQ which are encoded by numerous haplotypes. The combination of various α- and β-chains increases the diversity of the HLA class II proteins found in an arbitrary population. Thus, the selected HLA class II TUMAPs have to bind to several different HLA-DR molecules (i.e. show promiscuous binding ability) in order to be able to contribute to an effective T-cell response in a significant percentage of patients. The promiscuous binding of POSTN-002 and MMP12-002 to various HLA-DR haplotypes and the stability of the formed complexes was assessed in an in vitro binding assay by an external service provider as follows. Materials and Methods List of Peptides Sequence NoPeptide IDSequenceOriginSize76MMP12-002INNYTPDMNREDVDYAIRIMA-9421877POSTN-002TNGVIHVVDKLLYPADTIMA-94217 List of Investigated HLA-DR Haplotypes The 7 investigated HLA-DR haplotypes are selected according to their frequencies in HLA-A*02 and HLA-A*24 positive North Americans population (Table 7.1 and 7.2) Data are derived from the analysis of 1.35 million HLA-typed volunteers registered in the National Marrow Donor Program (Mori et al., 1997). The analyzed population was subdivided in the following ethnic groups: Caucasian Americans (N=997,193), African Americans (N=110,057), Asian Americans (N=81,139), Latin Americans (N=100,128), and Native Americans (N=19,203). TABLE 7.1Haplotype frequencies in HLA-A*02 positive North Americans: Theanalyzed haplotypes are indicated in the rightmost column.Haplotype Frequency [% of HLA-A*02 positive individuals]Serological haplotypeNativeHLA-AHLA-DRCaucasianAfricanAsianLatinAmericanAnalyzed218.87.83.06.16.8Yes2214.913.817.69.713.8Yes236.111.11.85.35.5Yes2421.39.415.723.624.9Yes251.22.31.01.31.8No2615.220.011.517.715.9Yes2713.010.52.57.89.0Yes284.25.710.216.28.7No291.22.816.01.02.9No2101.42.41.21.30.8No2118.710.65.26.44.8Yes2122.62.812.31.81.9No2901.40.82.01.73.3NoSUM100.0100.0100.0100.0100.0 TABLE 7.2Haplotype frequencies in HLA-A*24 positive North Americans: Theanalyzed haplotypes are indicated in the rightmost column.Haplotype Frequency [% of HLA-A*24 positive individuals]Serological haplotypeNativeHLA-AHLA-DRCaucasianAfricanAsianLatinAmericanAnalyzed2418.27.95.44.14.6Yes24215.718.824.610.714.8Yes2436.07.51.43.74.0Yes24414.914.419.825.821.6Yes2452.01.61.42.71.0No24617.018.79.620.520.7Yes2479.27.92.54.84.3Yes2484.03.85.712.411.3No2491.41.79.90.75.8No24101.61.20.82.00.6No241116.58.05.29.05.4Yes24121.87.511.52.22.4No24901.61.02.21.33.3NoSUM100.0100.0100.0100.0100.0 Principle of Test The ProImmune REVEAL® MHC-peptide binding assay determines the ability of each candidate peptide to bind to the selected HLA class II haplotype and stabilize the HLA-peptide complex. Thereby the candidate peptides are assembled in vitro with a particular HLA class II protein. The level of peptide incorporation into HLA molecules is measured by presence or absence of the native conformation of the assembled HLA-peptide complex at time 0 after completed refolding procedure (so called on-rate). The binding capacity of candidate peptide to a particular HLA molecule is compared to the one with known very strong binding properties (positive control) resulting in the corresponding REVEAL® MHC-peptide binding score. The positive control peptide is selected and provided by ProImmune based on their experience individually for each HLA haplotype. Besides the affinity of a peptide to a particular HLA molecule, the enduring stability of the formed HLA-peptide complex is crucial for the occurrence of an immune response. Accordingly presence of the formed HLA-peptide complex is measured after its incubation for 24 h at 37° C. Consequently the stability of the formed MHC-peptide complex is calculated as a ration of the binding scores at 24 h and the binding scores which are received right after the refolding (accordingly at time 0) in percent. Results The analysis of POSTN-002 and MMP12-002 in REVEAL® MHC-peptide binding assay showed that both peptides bind to various HLA haplotypes. POSTN-002 was shown to form a complex with 5 and MMP12-002 with 4 of 7 investigated HLA haplotypes (FIG.5). Both peptides did not bind to HLA-DR3 and HLA-DR6. The detected binding scores were within the range of 0.02 to about 2.5% compared to the positive control, and clearly above scores of non-binding peptides. The stability analysis of the formed HLA-POSTN-002 and HLA-MMP12-002 complexes revealed that 3 and 2 of 6 investigated HLA-peptide complexes were stable after 24 h at 37° C., respectively (FIG.6). A conclusion on the immugenicity of a peptide based on its binding capacity to a HLA molecule can be made by comparing the binding score of this peptide to the one with known immunogenicity. Therefore, five well investigated peptides with determined immunogenicity were selected for this comparison. The immunogenicity of these peptides was determined ex vivo in blood samples of vaccinated patients using intracellular cytokine staining (ICS) CD4 T-cells. In principle, ICS assays analyze the quality of specific T cells in terms of effector functions. Therefore, the peripheral mononuclear cells (PBMCs) were cultivated in vitro and subsequently restimulated by the peptide of interest, a reference peptide and a negative control (here MOCK). Following the restimulated cells were stained for FN-gamma, TNF-alpha, IL-2 and IL-10 production, as well as expression of the co-stimulatory molecule CD154. The counting of affected cells was performed on a flow cytometer (FIG.7). The immunogenicity analysis revealed 100% immune response by vaccination with IMA950 peptides (BIR-002 and MET-005) in 16 patients and 44% to 86% immune response by vaccination with IMA910 peptides (CEA-006, TGFBI-004 and MMP-001) in 71 patients. To compare the binding scores of POSTN-002 and MMP12-002 to the binding scores of IMA910 and IMA950 peptides, all peptides were arranged in a table for each investigated HLA-DR haplotype according to the detected binding score (Tables 8.1 to 8.5). TABLE 8.1Binding scores of POSTN-002 and MMP12-002 to HLA-DR1 compared to the binding scores of class IIpeptides with known immunogenicity: POSTN-002and MMP12-002 are ranked 4 and 6, respectively.Relative BindingPeptide RankPeptide CodeOriginScore HLA-DR11BIR-002IMA95040.062CEA-006IMA9101.313MET-005IMA9500.874POSTN-002IMA-9420.245MMP-001IMA9010.196MMP12-002IMA-9420.047TGFBI-004IMA9100.03 TABLE 8.2Binding scores of POSTN-002 and MMP12-002 to HLA-DR2 compared to the binding scores of class IIpeptides with known immunogenicity: POSTN-002and MMP12-002 are ranked 3 and 1, respectively.Relative BindingPeptide RankPeptide CodeOriginScore HLA-DR21MMP12-002IMA-9422.432MMP-001IMA9010.73POSTN-002IMA-9420.684MET-005IMA9500.285TGFBI-004IMA9100.286BIR-002IMA9500.057CEA-006IMA9100.03 TABLE 8.3Binding scores of POSTN-002 and MMP12-002 to HLA-DR4 compared to the binding scores of class IIpeptides with known immunogenicity: POSTN-002and MMP12-002 are ranked 6 and 4, respectively.Relative BindingPeptide RankPeptide CodeOriginScore HLA-DR41CEA-006IMA91039.652BIR-002IMA9506.123MET-005IMA9505.894MMP12-002IMA-9420.745MMP-001IMA9010.066POSTN-002IMA-9420.027TGFBI-004IMA9100.02 TABLE 8.4Binding scores of POSTN-002 and MMP12-002 to HLA-DR5 compared to the binding scores of class IIpeptides with known immunogenicity: POSTN-002and MMP12-002 are ranked 5 and 6, respectively.Relative BindingPeptide RankPeptide CodeOriginScore HLA-DR51BIR-002IMA950103.92MMP-001IMA90147.823CEA-006IMA91024.274MET-005IMA9500.125POSTN-002IMA-9420.086MMP12-002IMA-9420.047TGFBI-004IMA9100.04 TABLE 8.5Binding scores of POSTN-002 and MMP12-002 to HLA-DR7 compared to the binding scores of class IIpeptides with known immunogenicity: POSTN-002and MMP12-002 are ranked 3 and 7, respectively.Relative BindingPeptide RankPeptide CodeOriginScore HLA-DR71MET-005IMA9503.692CEA-006IMA9100.633POSTN-002IMA-9420.474BIR-002IMA9500.275TGFBI-004IMA9100.016MMP-001IMA90107MMP12-002IMA-9420 The comparison of the binding scores of POSTN-002 and MMP12-002 to the binding scores of the other class II peptides with known immunogenicity showed that the binding capacities of both peptides are mostly located in the middle till the lower half of the tables with exception of HLA-DR2. 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11859018 | DETAILED DESCRIPTION Cyclic Peptoid-Based Chelating Ligands The present disclosure provides cyclic peptoid-based chelating ligands. A cyclic peptoid-based chelating ligand of the present disclosure may be produced by macrocyclization of linear peptoid sequences. A peptoid is an N-alky or N-aryl glycine polymer having side chains appended to the nitrogen atom of the peptide backbone. This is in contrast to peptides, in which side chains are appended to α-carbons. The following are structures of generic monomers of α-peptide and α-peptoid backbones: Peptoids, unlike peptides, are resistant to hydrolysis under physiological conditions, thereby making peptoid-based chelating ligands of the present disclosure suitable candidates for treating metal poisoning in vivo. A ligand is a charged or neutral functional group that binds to a central atom to form a coordination complex via a Lewis base-Lewis acid type interaction, where the ligand acts as the Lewis base, and the central atom is the Lewis acid. Chelation is a type of bonding of ions and molecules to metal ions. More specifically, chelation involves the formation of at least two separation coordinate bonds between a polydentate ligand and a single central atom. In chelation therapy, chelating agents convert metal ions into a chemically and biochemically inert form that can be excreted. In view of the foregoing, as used herein, a “cyclic peptoid-based chelating ligand” may refer to a cyclic N-alky or N-aryl glycine polymer having multiple side chains appended to nitrogen atoms of the peptide backbone, with the cyclic peptoid-based chelating ligand being a charged or neutral molecule that binds to a metal ion to form a coordination complex via a Lewis base-Lewis acid type interaction, whereby each of the side chains form a separate coordinate bond with the metal ion, such that the cyclic peptoid-based chelating ligand converts the metal ion into a chemically and biochemically inert form that can be excreted from a subject. As used herein, the term “subject” may refer to a vertebrate mammal including but not limited to a human, non-human primate (e.g., monkey), mouse, rate, guinea pig, rabbit, cow, dog, cat, horse, goat, bird, reptile, or fish. A subject may be a domesticated animal, a wild animal, or an agricultural animal. Accordingly, teachings of the present disclosure may be used with respect to human and non-human subjects. For instance, teachings of the present disclosure can be used in veterinary applications (e.g., in zoos, reserves, farms, in the wild, etc.) as well as in human prevention and treatment regimens. Peptoid Backbones In at least some examples, a cyclic peptoid-based chelating ligand of the present disclosure may be produced from an α-peptoid (having a generic monomer as illustrated above). In at least some examples, a cyclic peptoid-based chelating ligand of the present disclosure may be produced from a β-peptoid. The following structure represents a generic monomer of a β-peptoid backbone: In at least some examples, a single cyclic peptoid-based chelating ligand may be produced from a hybrid system including at least one α-peptoid and at least one β-peptoid. One skilled in the art will appreciate that achievable ring sizes and conformations may depend on the α-peptoid(s) and/or β-peptoid(s) used. Solid phase peptoid synthesis can be used to produce linear peptoids from suitably produced monomers that have variable side chains with variable functional groups. Solid phase peptoid synthesis may include acylating a secondary amine, on a resin, by an activated haloacetic acid, such as bromoacetic acid, with N.N-diisopropylcarbodimide (DIC). One skilled in the art will appreciate that multiple resins with multiple coupling chemistries and protecting groups can be used for synthesis of a linear peptoid to achieve the optimal yield of desired peptoid. For example, the solid phase synthesis of a linear hexa-peptoid can be accomplished using a bromoacetic acid functionalized 2-Chlorotrityl resin [loading 200-400 mesh polystyrene 1% DVB cross linking]. A Biotage Alstra microwave-assisted peptide synthesizer or other similar peptide synthesizer can be used for automated synthesis. Initial displacement of the bromide of the functionalized resin adds the first peptoid and then the peptoid chain is elaborated by iterative coupling with successive functionalized monomers using an activation catalyst to accelerate the coupling reaction. Once the linear peptoid has been constructed, protecting groups on the functional groups of the peptoid side chains are removed, and then the completed linear peptoid can be removed from the resin. The peptoid can be purified by chromatography and analyzed prior to cyclization into the cyclic peptoid. Peptoid Cyclization In at least some examples, a string of peptoid monomers (that have yet to be modified to include side chains having hard chelating groups) may be cyclized to produce a cyclic peptoid scaffold upon which side chains (having hard chelating groups) may be appended. Such cyclic peptoid scaffolds may be produced via macrocyclization with peptoid coupling reagents, or cyclized on a resin solid support. Cyclization of a typical linear hexamer to cyclic hexamer can be accomplished using a variety of coupling reagents under conditions of high dilution (10−3M). Typically, cyclizations using PyBrOP were found to be low yielding and the resulting product is very difficult to separate from reaction by products. Just switching solvents from CH2Cl2to DMF still resulted in extremely low yields. Both PyClock and MSNT mediated cyclizations showed improved yields; however, cyclizations using HATU were found to be high yielding and could be scaled to 100 mg quantities of starting material to give the cyclic product in about 75% yield. FIG.1illustrates an example of a cyclic peptoid hexamer scaffold and hard chelating groups in accordance with the present disclosure. One skilled in the art will appreciate that cyclic peptoid scaffolds of the present disclosure may be of various ring sizes, such as detailed elsewhere herein. As illustrated inFIG.1, a cyclic peptoid hexamer scaffold (chemical structure on the left side ofFIG.1) of the present disclosure may include one or more side chains functionalized with chelating, anchoring, and/or polarity modifying groups bearing hard chelating groups favoring hard cation binding. Such substitution is described elsewhere herein. In at least some other examples, a string of peptoid monomers (that have already been modified to include side chains having hard chelating groups) may be synthesized with appropriate protecting groups on the hard chelating groups using either solid phase or solution synthesis to produce variously functionalized peptoids that can be cyclized as described herein to produce a cyclic peptoid-based chelating ligand after removal of the protecting groups. Alternatively, as one skilled in the art will appreciate, a peptoid with a protected functional group, such as an amine, alcohol, thiol, etc. or such as a halogen, acid, ester, etc., can be synthesized and cyclized. After removal of the protecting group, the desired binding functionality, such as a catecholate, phosphine, phosphate, amine, imine, amidine, guanidine, or aromatic hard ligand can be attached to the cyclic peptoid scaffold to tune desired binding properties. Ring Size Cyclic peptoid-based chelating ligands of the present disclosure may be of various ring sizes. Such various ring sizes enables selective chelation of various metal cations. A ring size of a cyclic peptoid-based chelating ligand of the present disclosure is determined by the number of glycine monomers incorporated into a linear peptoid scaffold. Ring sizes for the peptoid scaffold can vary from 4 to 9. However, synthesis yields may be affected as the ring contracts below 5 and expands to 8 or more. The nitrogens in the cyclic peptoid ring can also participate in the binding of metals in concert with the hard ligand binders that are located on the peptoid side chains, and by altering the ring size and the ligands one skilled in the art will appreciate that different size metal complexes can be preferentially bound with high affinity allowing the cyclic peptoid to select one metal complex in preference to another. A smaller ring can bind sodium in preference to potassium or cesium or iron while a larger ring can bind iron or copper in preference to ruthenium or iridium, which would require an even larger ring and/or modified ligand groups. Lanthanides and actinides, since they are larger, may require hexameric or larger ring systems for enhanced binding. Side Chains Cyclic peptoid-based chelating ligands of the present disclosure may be designed to be selective for various metal cations. As such, cyclic peptoid-based chelating ligands of the present disclosure may be useful in chelation therapy of various metals. Generally, selective binding of specific metal cations may be achieved by careful choice of the hardness of the Lewis basic site(s), linker length, ring size, and conformation dictated by the identity of all incorporated side chains. As described above, a string of peptoid monomers (that have yet to be modified to include side chains having hard chelating groups) may be cyclized to produce a cyclic peptoid scaffold. Post-cyclization, the cyclic peptoid scaffold may include one or more nitrogen atoms having side chains (e.g., benzyl (Bn) side chains) bound thereto. The side chains of the cyclic peptoid scaffold may be deprotected and modified with one or more different side chains having the same or different hard chelating groups. A cyclic hexamer with a protecting group, such as a CBZ group, can be deprotected by reduction over Pd/C by heating to reflux until the reduction is complete (about 1 hr or determined to be complete by mass spectrometric analysis). After addition of formic acid dropwise by syringe, the reaction mixture may be refluxed with stirring for another hour until mass spectroscopy indicates complete conversion to the triamine. Thereafter, the mixture may be filtered. The solid may be dissolved in aqueous HCl, frozen in liquid nitrogen, and lyophilized to produce a deprotected cyclic peptoid amine as an HCl salt in 95% yields. One skilled in the art will appreciate that the method of deprotection may be varied depending on the type of protecting group. For example deprotection to the linear peptoid side chains and removal from the resin support can be accomplished by reaction with trifluoroacetic acid and reagents specific to the chosen protecting group. Side chains may be chosen with respect to chain linker length, ligand type (e.g., degree of hardness), desired solubility of the cyclic peptoid-based chelating ligand, desired bioavailibliity of the cyclic peptoid-based chelating ligand, etc. In at least some examples, one or more side chains may be selected to serve as an anchor(s) to a solid support. In at least some examples, a side chain may include one or more hard chelating groups selective for one or more f-block elements. F-block elements consist of lanthanides and actinides, which are also known as the inner transition elements. Lanthanides are a series of fifteen metallic chemical elements from atomic number 57 to atomic number 71 in the periodic table. The fifteen lanthanides are lanthanum (La, atomic number 57), cerium (Ce, atomic number 58), praseodymium (Pr, atomic number 59), neodymium (Nd, atomic number 60), promethium (Pm, atomic number 61), samarium (Sm, atomic number 62), europium (Eu, atomic number 63), gadolinium (Gd, atomic number 64), terbium (Tb, atomic number 65), dysprosium (Dy, atomic number 66), holmium (Ho, atomic number 67), erbium (Er, atomic number 68), thulium (Tm, atomic number 69), ytterbium (Yb, atomic number 70), and lutetium (Lu, atomic number 71). Actinides are a series of fifteen metallic elements from atomic number 89 to atomic number 103 in the periodic table. The fifteen actinides are actinium (Ac, atomic number 89), thorium (Th, atomic number 90), protactinium (Pa, atomic number 91), uranium (U, atomic number 92), neptunium (Np, atomic number 93), plutonium (Pu, atonic number 94), americium (Am, atomic number 95), curium (Cm, atomic number 96), berkelium (Bk, atomic number 97), californium (Cf, atomic number 98), einsteinium (Es, atomic number 99), fermium (Fm, atomic number 100), mendelevium (Md, atomic number 101), nobelium (No, atomic number 102), and lawrencium (Lr, atomic number 103). In at least some examples, one or more hard chelating groups may be selected based on one or more of high affinity for a lanthanide(s), actinide(s), or other metal cation(s); selectivity for a lanthanide(s), actinide(s), or other metal cation(s); an ability to recover a metal cation once the metal cation is bound to a cyclic peptoid-based chelating ligand; and/or whether the overall cyclic peptoid-based chelating ligand should be hydrophobic or hydrophilic. Typical hard chelating ligands that are specific for lanthanide and actinide binding include catecholates, phosphoramidates, amidines, and guanidines. Since the size of the metal ion affects it's binding to the cyclic peptoid, the ring size and length of the tether between the ring and the hard chelating ligand can be tuned to provide optimal binding to the specific metal that is targeted. If a cyclic peptoid is to be used for treatment of metal poisoning, the goal is to remove the metal from the biological regime as rapidly as possible; consequently, the binding to the target metal must be as tight as possible so the metal is not released during the removal process. However, if the purpose is to separate a metal from other metals then, after it has been sequestered, the cyclic peptoid binding must be selective but not high enough that release after removal is prevented. Replacement of some of the side chains on the cyclic peptoid can be used to adjust the hydrophobicity or hydrophilicity of the cyclic peptoid complex. For example, cadmium and some radionuclides can be accumulated into lipid structures in the body, rendering normal chelation therapy inaffective in removing metal ions. By adding lipid like hydrocarbon side chains and aromatic groups, the cyclic peptoid will be better able to remove lipid soluble metal complexes. Contrarily, a side chain on the cyclic peptoid can be constructed with hydroxyl, amine, or carboxylic groups to make the overall complex more hydrophilic and water soluble to achieve a better separation of aqueous soluble actinides. Various hard chelating groups may be incorporated into a cyclic peptoid-based chelating ligand that is selective for one or more actinides and/or one or more lanthanides. Example actinide(s) and/or lanthanide(s) selective hard chelating groups include, but are not limited to, phosphonate (C—PO(OH)2or C—PO(OR)2, where R is alkyl or aryl), catecholate (C6H4O2), amine (either a primary amine, secondary amine, or tertiary amine), guanidinium (CH6N3+), phosphoramidate (H2NO3P−2), n-acyl derivatives of hydroxyamines (H3NO), n-hydroxypyridones (C5H5NO2being the molecular formula for hydroxypyridone), and carbamoylmethylphosphine oxide (CMPO). A cyclic peptoid may be tuned to be selective for one or more particular actinides and/or lanthanides by changing the size of the cyclic peptoid and/or the ligand groups. For example, instead of having three of the same hard ligands (such as tricatacholate), a cyclic peptoid complex can be constructed with one, two, or three catecholates and one or two amidines or guanidines to change the selectivity for the various lanthanides and actinides. For example, gadolinium is bound tightly by the carboxylic acids of DOTA and other lanthanides can be selected by altering the number of carboxylic acids used to bind the metal. Cyclic peptoid-based chelating ligands of the present disclosure may be of various hardnesses to enable chelation of various elements. In at least some examples, one or more hard atoms [e.g., one or more hard electron donor atoms such as nitrogen (N, atomic number 7), oxygen (O, atomic number 8), and fluorine (F, atomic number 9)] may be positioned between a peptoid backbone and a hard chelating group of a side chain. An electron-donating hard atom may increase the affinity of a hard chelating group for a hard cation, such as a trivalent lanthanide (having a +3 charge), trivalent actinide (having a +3 charge), and/or cations of transition metals and main group elements. In general, the harder the chelating group, the tighter the binding to the metal cation can be. Consequently, by reducing the hardness of the chelator (e.g., by adding additional functional groups that are soft electron donors), the extend of binding can be tuned to the target metal complex. In at least some examples, a side chain may be modulated with one or more soft electron donors. Examples of soft electron donors include thiolates and thioureas. A thiolate (RS−) is a derivative of a thiol (an organic compound containing the group —SH) in which a metal atom replaces the hydrogen attached to sulfur. A thiourea is an organosulfur compound with the formula SC(NH2)2. Modulating one or more side chains of a cyclic peptoid-based chelating ligand may enable the cyclic peptoid-based chelating ligand to be used as a lead scavenger in medical applications, as well as be used for precious metal recovery. A cyclic peptoid-based chelating ligand of the present disclosure may be configured such that the cyclic peptoid-based chelating ligand has a number of hard chelating groups corresponding to a charge of a metal cation to be bound by the cyclic peptoid-based chelating ligand. For example, a cyclic peptoid-based chelating ligand for binding a +3 metal cation may have three hard chelating groups, a cyclic peptoid-based chelating ligand for binding a +4 metal cation may have four hard chelating groups, a cyclic peptoid-based chelating ligand for binding a +5 metal cation may have five hard chelating groups, etc. Side chain length may be varied to select for certain sizes of metal cations. As the size of the metal cation increases, so may the cavity of the cyclic peptoid that will accommodate the metal cation. Consequently, small metal cations may be accommodated best with side chain lengths of n=2-4, and larger metal cations may be accommodated best with side chain lengths of n=4-8 or more. Administration of Peptoid-Based Chelating Ligand In connection with the treatment of metal poisoning, in vivo, the peptoid-based chelating ligands of the present disclosure may be introduced systemically to a patient having, or suspected of having, metal poisoning. The peptoid-based chelating ligands may preferably be introduced systemically, although localized administration may be appropriate in some circumstances (e.g., in the case of localized metal poisoning). The peptoid-based chelating ligands may be formulated for oral, topical, or rectal administration using well-known formulation methodologies. Additionally, when formulated in a physiologically acceptable buffer solution, the peptoid-based chelating ligands may be introduced parenterally (e.g., intravenously or by injection). The determination of effective therapeutic levels, and the formulations required to deliver such effective therapeutic levels, are determined on a case-by-case basis which is dependent, for example, on the extent of the metal poisoning to be treated. Such determinations are readily made by one skilled in the art using no more than routine experimentation. While biological model system data is not provided herein, one skilled in the art will appreciate that siderophores, like enterobactin, show extremely high binding to iron under physiological conditions. Unfortunately, enterobactin is readily hydrolyzed and rendered inactive. The peptoid backbone of the present disclosure, in at least some examples, is similar to the peptide backbone of enterobactin, but is resistant to hydrolysis and has been successfully used in construction of successful drugs. Consequently, there is a high degree of certainty that cyclic peptoids of the present disclosure will be physiologically effective and safe for chelation therapy. Additionally, cyclic peptoid of the present disclosure, in addition to being effective in chelation therapies, also have the potential as contrast agents for medical imaging, as targeted delivery mechanisms for radiotherapy applications, and as highly specific tools for separating actinides and other metals. EXAMPLES The following reagents were used as supplied by the manufacturer unless otherwise noted. Reactions, except for TFA deprotections of t-butyl esters were performed under argon (Ar). Flash chromatography was performed on a Biotage Isolera instrument. Products were typically dried under high vacuum (about 1×10−2mm Hg) overnight. Silica TLC plates were visualized via ceric ammonium molybdate, phosphomolybdic acid or I2stain. t-BuO2CCH2NHCH2Ph FIG.2illustrates the chemical t-BuO2CCH2NHCH2Ph, which may be used as a starting peptoid monomer for a backbone of a cyclic peptoid-based chelating ligand of the present disclosure. One skilled in the art will appreciate that t-BuO2CCH2NHCH2Ph is merely illustrative, and that other peptoid monomers may be used in accordance with the present disclosure. To produce t-BuO2CCH2NHCH2Ph, triethylamine (about 13 mL, about 93.3 mmol, 1.0 eq.) was added to a solution of benzylamine (about 10.2 mL, about 93.3 mmol, 1.0 eq.) in N,N-dimethylformamide (DMF) (about 60 mL) contained in a round bottomed flask (RBF) (e.g., a 250 mL RBF). After chilling to 0° C. in an ice bath, a solution of t-butyl bromoacetate (about 12.4 mL, about 84.0 mmol, 0.9 eq.) in DMF (about 33 mL) was added dropwise (e.g., through an addition funnel). The initial concentration of t-butyl bromoacetate after the addition was about 0.5 M. The reaction mixture was warmed to room temperature (rt) after stirring for about 0.5 hr at about 0° C. The resulting reaction mixture was stirred for about 3 hours at room temperature (rt), diluted with water (H2O) (about 200 mL) and extracted with ethyl acetate (EtOAc) (about 100 mL, 2×50 mL). The combined organic phase was washed with brine (about 100 mL), dried (e.g., over sodium sulfate (Na2SO4)), filtered, and concentrated. The resulting oil was dried under high vacuum overnight. The crude product was purified by EtOAc/hexane flash chromatography on silica and dried under high vacuum, yielding about 13.7 g t-BuO2CCH2NHCH2Ph as an oil (about 74% yield). t-BuO2CCH2NFMOC(CH2Ph) FIG.3illustrates the peptoid monomer t-BuO2CCH2NFMOC(CH2PH) synthesized from t-BuO2CCH2NHCH2Ph. One skilled in the art will appreciate that t-BuO2CCH2NFMOC(CH2Ph) is merely an illustrative peptoid monomer, and that other peptoid monomers may be synthesized in accordance with the present disclosure. 9-fluorenylmethyl chloroformate (FMOC-Cl) (about 17.2 g, about 67 mmol, 1.0 eq.) was added, in small portions, to a solution of BuO2CCH2NHCH2Ph (about 17.6 g, about 80 mmol, 1.2 eq.) in dichloromethane (CH2Cl2) (about 250 mL) contained in a RBF (e.g., a 500 mL RBF). CH2Cl2, (about 70 mL) was used to complete the transfer of FMOC-Cl, resulting in an about 0.25 M initial concentration of FMOC-Cl. After stirring for about 4.5 hrs under Ar, the solution was extracted with about 0.1 M aqueous hydrochloric acid (HCl) (about 500 mL, 2×250 mL) and brine (about 250 mL). The resulting organic phase was dried over Na2SO4, filtered, and concentrated to an oil. The resulting oil was dissolved in minimal EtOAc, and a seed crystal was added. The resulting solution was allowed to crystallize at about 4° C. overnight. The resulting slurry was filtered, washed with minimal EtOAc, and dried under high vacuum. The filtrate was evaporated and treated in the same manner twice to obtain two more crops of product, about 2.26 g and about 2.33 g respectively, resulting in about 20.2 g t-BuO2CCH2NFMOC(CH2PH) (about 57% yield). HO2CCH2NFMOC(CH2Ph) FIG.4illustrates the peptoid monomer HO2CCH2NFMOC(CH2Ph) synthesized from t-BuO2CCH2NFMOC(CH2PH). One skilled in the art will appreciate that HO2CCH2NFMOC(CH2Ph) is merely an illustrative peptoid monomer, and that other peptoid monomers may be synthesized in accordance with the present disclosure. t-BuO2CCH2NFMOC(CH2PH) (about 15 g, about 34 mmol, 1.0 eq.) was added to a RBF (e.g., a 1 L RBF), followed by addition of triisopropylsilane (i-Pr3SiH) (about 21 mL, about 102 mmol, 3.0 eq.). Trifluoroacetic acid (TFA) (about 68 mL, about 0.5 M) was added to the resulting slurry, followed by addition of CH2Cl2(about 5 mL). The resulting mixture was stirred at rt for about 1.5 hrs, then evaporated under vacuum and evaporated with toluene (about 150 mL, 3×50 mL). The resulting crude product was dried under high vacuum overnight. The resulting solid was triturated with hexanes (about 100 mL), washed with hexanes (about 100 mL, 2×50 mL), and dried under high vacuum, resulting in about 12.9 g HO2CCH2NFMOC(CH2Ph) (about 99% yield). BOCNHCH2CH2CH2CH2NHCBz FIG.5illustrates the side chain, BOCNHCH2CH2CH2CH2NHCBz. One skilled in the art will appreciate that BOCNHCH2CH2CH2CH2NHCBz is merely an illustrative side chain, and that other side chains may be synthesized in accordance with the present disclosure. Triethylamine (about 16 mL, about 116 mmol, 2.5 eq.) was added to a stirring slurry of BOCNHCH2CH2CH2CH2CH2NH3Cl (about 10.4 g, about 46 mmol, 1.0 eq.) in tetrahydrofuran (THF) (about 92 mL, about 0.5 M), contained in a RBF (e.g., a 250 mL RBF). The resulting slurry was chilled in an ice bath and N-(benzyloxycarbonyloxy)succinimide (CBz-OSu) (about 11.5 g, about 46 mmol, 1.0 eq.) was then added in portions. The reaction mixture was then stirred under Ar at about 0° C. for about 5 minutes, warmed to rt, then stirred at rt overnight. The solvent was then removed from the reaction mixture via rotary evaporation. H2O (about 100 mL) was added and the mixture was extracted with EtOAc (about 1502 mL, 3×50 mL), washed with aqueous 0.1 M HCl (about 100 mL) and brine (about 100 mL), and dried over Na2SO4. The solution was filtered and concentrated to dryness yielding a white colored solid produce (BOCNHCH2CH2CH2CH2NHCBz). The product was spectroscopically pure after drying overnight under high vacuum. The product weighed about 14.2 g (about 95% yield). CBzNHCH2CH2CH2CH2NH3Cl FIG.6illustrates the side chain CBzNHCH2CH2CH2CH2NH3Cl synthesized from BOCNHCH2CH2CH2CH2NHCBz. One skilled in the art will appreciate that CBzNHCH2CH2CH2CH2NH3Cl is merely an illustrative side chain, and that other side chains may be synthesized in accordance with the present disclosure. BOCNHCH2CH2CH2CH2NHCBz (about 14.2 g, about 44 mmol, 1.0 eq.) was added to a RBF (e.g., a 500 mL RBF), followed by addition of 1,4-dioxane (about 90 mL). To the resulting solution, 4M HCl-Dioxane (about 55 mL, about 220 mmol, 5.0 eq.) was added (e.g., via syringe). The resulting mixture was stirred under Ar overnight, resulting in the precipitation of a white colored solid. The resulting mixture was filtered and the solid was washed with THF (about 100 mL, 2×50 mL) and dried under high vacuum, yielding about 10.9 g of a white colored powder (about 95% yield). t-BuO2CCH2NH(C4H8NHCBz) FIG.7illustrates the peptoid monomer t-BuO2CCH2NH(C4H8NHCBz) synthesized from CBzNHCH2CH2CH2CH2NH3Cl. One skilled in the art will appreciate that t-BuO2CCH2NH(C4H8NHCBz) is merely an illustrative peptoid monomer, and that other peptoid monomers may be synthesized in accordance with the present disclosure. Triethylamine (about 12.3 mL, about 88 mmol, 2.0 eq.) was added to a stirring solution of CBzNHCH2CH2CH2CH2NH3Cl (about 11.4 g, about 44 mmol, 1.0 eq.) in DMF (about 70 mL) contained in a RBF (e.g., a 250 mL RBF) at about 0° C. The reaction mixture was stirred for about 5 minutes and a solution of tert-butylbromoacetate (t-BuO2CCH2Br) (about 5.9 mL, about 40 mmol, 0.9 eq.) in DMF (about 18 mL) was added dropwise (e.g., from an addition funnel), achieving an initial concentration of t-BuO2CCH2Br of about 0.5 M. After the addition was complete (after about 0.5 hours), the reaction mixture was allowed to warm to rt. After stirring overnight, the reaction mixture was diluted with H2O (about 250 mL), extracted with EtOAc (about 100 mL, 2×50 mL). The organic phase was then washed with brine (about 100 mL, 2×50 mL), dried over Na2SO4, filtered, and concentrated to an oil. The crude product was purified via flash chromatography on silica with a gradient of EtOAc/hexanes, yielding about 9.0 g of an oil product (about 67% yield). t-BuO2CCH2NFMOC(C4H8NHCBz) FIG.8illustrates the peptoid monomer t-BuO2CCH2NFMOC(C4H8NHCBz) synthesized from t-BuO2CCH2NH(C4H8NHCBz). One skilled in the art will appreciate that t-BuO2CCH2NFMOC(C4H8NHCBz) is merely an illustrative peptoid monomer, and that other peptoid monomers may be synthesized in accordance with the present disclosure. FMOC-Cl (about 4.3 g, about 16.8 mmol, 1.0 eq.) was added, in small portions, to a stirring solution of t-BuO2CCH2NH(C4H8NHCBz) (about 6.8 g, about 20.2 mmol, 1.2 eq.) in 0.25M CH2Cl2(about 68 mL). The reaction mixture was then stirred overnight at rt. The resulting solution was then diluted with aqueous 0.1 M HCl (about 100 mL) and the aqueous layer extracted with CH2Cl2(about 50 mL, 2×25 mL). The combined organic phase was washed with brine (about 50 mL), dried over Na2SO4, filtered, and concentrated to dryness. The resulting oil was dissolved in methanol (MeOH) and chilled in a freezer overnight, resulting in the product crystallizing. The product was filtered and washed with a small amount of MeOH. Thin-layer chromatograph (TLC) indicated non-polar FMOC containing by-products. The resulting solid was stirred with hexanes (about 25 mL), filtered, and washed with hexanes (about 50 mL, 2×25 mL). The resulting solid was dried under high vacuum. A second crop of crystals was obtained in the same manner from the initial filtrate, producing about 6.4 g (about a 68% combined yield). HO2CCH2NFMOC(C4H8NHCBz) FIG.9illustrates the peptoid monomer HO2CCH2NFMOC(C4H8NHCBz) synthesized from t-BuO2CCH2NFMOC(C4H8NHCBz). One skilled in the art will appreciate that HO2CCH2NFMOC(C4H8NHCBz) is merely an illustrative peptoid monomer, and that other peptoid monomers may be synthesized in accordance with the present disclosure. i-Pr3SiH, (about 11.7 mL, about 57.3 mmol, 5.0 eq.) was added to a RBF (e.g., a 500 mL RBF) containing t-BuO2CCH2NFMOC(C4H8NHCBz) (about 6.4 g, about 11.5 mmol, 1.0 eq.), resulting in the formation of a slurry. TFA (about 58 mL, about 0.2M) was then added slowly. The resulting mixture was stirred at rt for about 1 hr, after which time TLC indicated complete consumption of starting material. Volatiles were then removed with a rotary evaporator, leaving an oil that was evaporated with toluene (about 75 mL, 3×25 mL), and then dried under high vacuum for about 4 hrs. The resulting oil was triturated with hexanes (about 100 mL), the solvent decanted, and the resultant material dissolved in minimal EtOAc. Hexane was slowly added until the solution turned cloudy. This was then heated in an about 40° C. water bath until the solution cleared. A seed crystal was added, and the mixture was allowed to crystallize overnight at rt. The resulting solid was filtered and washed with 50% EtOAc/hexanes (about 70 mL, 2×35 mL), and then dried under high vacuum, to yield about 4.1 g (about 70% yield) of HO2CCH2NFMOC(C4H8NHCBz) as a white colored powder. t-BuO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) FIG.10illustrates the peptoid t-BuO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) synthesized from HO2CCH2NFMOC(C4H8NHCBz). One skilled in the art will appreciate that t-BuO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. Ethyl (hydroxyamino) cyanoacetate (oxyma) (about 0.85 g, about 6.0 mmol, 1.0 eq.) was added to a solution of HO2CCH2NFMOC(C4H8NHCBz) (about 3.0 g, about 6.0 mmol, 1.0 eq.) in DMF (about 20 mL). After stirring for about 5 minutes, N,N-diisopropylcarbodiimide (DIC) (about 0.93 mL, 1.0 eq.) was added. After stirring for an additional about 5 minutes, t-BuO2CCH2NHBn (about 1.32 g, about 6.0 mmol, 1.0 eq.) was added in DMF (about 10 mL) and the reaction mixture was stirred overnight at rt. The reaction mixture was then diluted with H2O (about 100 mL), and extracted with EtOAc (about 75 mL, 1×50 mL, 2×25 mL). The organic phase was then washed with H2O (about 50 mL, 2×25 mL), saturated aqueous NaHCO3(about 75 mL, 3×25 mL), and brine (about 50 mL). The organic phase was then dried over Na2SO4, filtered, and concentrated to yield an oil. The product was purified by flash chromatography on silica using EtOAc/hexanes, yielding about 3.9 g of a white colored foam after drying under high vacuum (about 92% yield). HO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) FIG.11illustrates the peptoid HO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) synthesized from the peptoid t-BuO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz). One skilled in the art will appreciate that HO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. i-Pr3SiH (about 1.45 mL, about 7.1 mmol, 5.0 eq.) was added to a RBF (e.g., a 50 mL RBF) containing t-BuO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) (about 1.0 g, about 1.4 mmol, 1.0 eq.), followed by addition of TFA (about 5.8 mL, about 0.25 M). CH2Cl2(about 1.0 mL) was then added. The resulting mixture was stirred at rt for about 35 min, after which time TLC indicated consumption of starting material. The resulting solution was evaporated to an oil and evaporated with CH2Cl2(about 30 mL, 3×10 mL). The crude product was loaded onto silica gel from CH2Cl2and purified via flash chromatography on silica using a gradient of EtOAc/hexanes. About 0.82 g of the product was obtained as a white colored foam after evaporation of the product containing fractions (about 89% yield). t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NFMOC(C4H8NHCBz) FIG.12illustrates the peptoid t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NFMOC(C4H8NHCBz) synthesized from the peptoid HO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz). One skilled in the art will appreciate that t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NFMOC(C4H8NHCBz) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. Bromotripyrrolidinophosphonium hexafluorphosphate (PyBrOP) (about 0.58 g, 1.2 eq.) was added to a solution of HO2CCH2N(Bn)COCH2NFMOC(C4H8NHCBz) (about 0.73 g, about 1.1 mmol, 1.0 eq.) in CH2Cl2(about 6 mL), followed by addition of N,N-diisopropylethylamine (i-Pr2NEt) (about 0.39 mL, about 2.2 mmol, 2.0 eq.). The resulting solution was stirred for about 5 min and a solution of t-BuO2CCH2NH(C4H8NHCBz) (about 0.38 g, about 1.1 mmol, 1.0 eq.) in CH2Cl2(about 6 mL) was added. Limiting reagent concentration was about 0.1 M. The reaction mixture was stirred overnight and poured into a saturated aqueous NaHCO3solution (about 100 mL). The mixture was extracted with EtOAc (about 100 mL, 1×75 mL, 1×25 mL) and the organic phase was washed with brine (about 50 mL). The resulting solution was dried over Na2SO4, filtered, and concentrated to an oil. The oil was dissolved in CH2Cl2, loaded onto silica, and purified via flash chromatography on silica using EtOAc/hexanes, resulting in about 0.76 g of product (about 70% yield). t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NH(C4H8NHCBz) FIG.13illustrates the peptoid t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NH(C4H8NHCBz) synthesized from the peptoid t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NFMOC(C4H8NHCBz). One skilled in the art will appreciate that t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NH(C4H8NHCBz) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. 20% piperidine in tetrahydrofuran (THF) (about 22 mL, about 0.1 M) was added to a RBF (e.g., a 250 mL RBF) containing t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NFMOC(C4H8NHCBz) (about 2.2 g, about 2.2 mmol, 1.0 eq.). The walls of the RBF were washed down with THF. The mixture was stirred vigorously and, after about 20 minutes, TLC indicated complete consumption of starting material. The resulting mixture was evaporated to dryness and evaporated with several small portions of toluene to remove excess piperidine. The resulting product was loaded onto silica gel using CH2Cl2and evaporated, and purified via flash chromatography on silica using a gradient of EtOAc/hexanes, then switching to a gradient of MeOH/EtOAc. The resulting product containing fractions were collected, concentrated, and dried under high vacuum, yielding about 1.62 g of the product as a white colored foam (about 98% yield). t-BuO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) FIG.14illustrates the peptoid t-BuO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) synthesized from HO2CCH2NFMOC(Bn). One skilled in the art will appreciate that t-BuO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. Oxyma (about 1.1 g, about 7.7 mmol, 1.0 eq.) was added to a solution of HO2CCH2NFMOC(Bn) (about 3.0 g, about 7.7 mmol, 1.0 eq.) in DMF (about 30 mL) contained in a RBF (e.g., a 100 mL RBF). The resulting solution was stirred for about 5 minutes. DIC (about 1.2 mL, about 7.7 mmol, 1.0 eq.) was added to the reaction mixture, which was then stirred for about 10 minutes. A solution of t-BuO2CCH2NH(C4H8NHCBz) (about 2.6 g, about 7.7 mmol, 1.0 eq.) in DMF (about 9.0 mL) was then added, achieving an initial limiting reactant concentration of about 0.2 M. The reaction mixture was stirred overnight and subsequently diluted with H2O (about 100 mL). The mixture was then extracted with EtOAc (about 100 mL, 1×50 mL, 2×25 mL). The organic phase was then washed with H2O (about 50 mL, 2×25 mL), and saturated with aqueous NaHCO3(about 75 mL, 3×25 mL) and brine (about 50 mL). The organic phase was then dried over Na2SO4, filtered, and concentrated. The resulting material was dissolved in CH2Cl2, loaded onto silica gel, and purified via flash chromatography on silica, using a gradient of EtOAc/hexanes to elute the product. Product containing fractions were concentrated and dried under high vacuum, yielding about 4.3 g of a white colored foam (about 80% yield). HO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) FIG.15illustrates the peptoid HO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) synthesized from t-BuO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn). One skilled in the art will appreciate that HO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. i-Pr3SiH (about 5.8 mL, about 28.3 mmol, 5.0 eq.) was added to a RBF (e.g., a 250 mL RBF) containing t-BuO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) (about 4.0 g, about 5.7 mmol, 1.0 eq.), followed by addition of TFA (about 23 mL, about 0.25 M) and CH2Cl2(about 5.0 mL). The reaction mixture was stirred at rt, with complete disappearance of starting material observed via TLC after about 40 minutes. The reaction mixture was evaporated to dryness and evaporated with CH2Cl2(about 20 mL, 2×10 mL). After drying under vacuum, the resulting oil was dissolved in CH2Cl2and loaded onto silica gel. The product was then purified via flash chromatography on silica, eluting with a gradient of EtOAc/hexanes. The resulting product containing fractions were collected, concentrated, and dried under high vacuum, yielding about 3.0 g of a white colored foam (about 82% yield). t-BuO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) FIG.16illustrates the peptoid t-BuO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) synthesized from HO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn). One skilled in the art will appreciate that t-BuO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. i-Pr2NEt (about 0.83 mL, about 4.8 mmol, 1.1 eq.) was added to a solution of HO2CCH2N(C4H8NHCBz)COCH2NFMOC(Bn) (about 2.8 g, about 4.3 mmol, 1.0 eq.) in CH2Cl2(about 10 mL) contained in a RBF (e.g., a 50 mL RBF), followed by addition of PyBrOP (about 2.0 g, about 4.3 mmol, 1.0 eq.). The resulting solution was stirred for about 10 minutes. Then, a solution of t-BuO2CCH2NHBn (about 1.25 g, about 5.6 mmol, 1.3 eq.) in CH2Cl2(about 7.0 mL) was added, resulting in a limiting reactant concentration of about 0.25 M. The resulting reaction mixture was stirred overnight under argon. The solvent was then evaporated and the resulting residue was diluted with saturated aqueous NaHCO3solution (about 50 mL). The resulting mixture was extracted with EtOAc (about 150 mL, 3×50 mL), and the organic phase was washed with brine (about 50 mL), dried over Na2SO4, then filtered and concentrated into an oil. The resulting oil was dissolved in CH2Cl2and loaded onto silica gel. The product was then purified by flash chromatography on silica gel, eluting with a gradient of EtOAc/hexanes. The resulting product containing fractions were concentrated and dried under high vacuum, yielding the product as a foam (about 3.3 g, about 88% yield). HO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) FIG.17illustrates the peptoid HO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) synthesized from t-BuO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn). One skilled in the art will appreciate that HO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. i-Pr3SiH (about 3.9 mL, about 19 mmol, 5.0 eq.) was added to a RBF (e.g., a100 mL RBF) containing t-BuO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) (about 3.3 g, about 3.8 mmol, 1.0 eq.), followed by addition of TFA (about 15 mL, about 0.25 M) and CH2Cl2(about 5 mL). The resulting mixture was stirred for about 1 hr and 20 mins and then concentrated to dryness. The crude product was dissolved in CH2Cl2and loaded onto silica gel. The product was then purified by flash chromatography on silica, eluting with EtOAc/hexanes. The product containing fractions were collected, concentrated, and dried under high vacuum, yielding the product as a foam (about 1.8 g, about 58% yield). t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NFMOC(Bn) FIG.18illustrates the peptoid t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NFMOC(Bn) synthesized from HO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn). One skilled in the art will appreciate that t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NFMOC(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. i-Pr2NEt (about 0.46 mL, about 2.7 mmol, 1.2 eq.) and PyBrOP (about 1.1 g, about 2.4 mmol, 1.1 eq.) were added to a solution of HO2CCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) (about 1.76 g, about 2.2 mmol, 1.0 eq.) in CH2Cl2(about 10 mL) contained in a RBF (e.g., a 50 mL RBF) under argon. The resulting solution was stirred for about 10 minutes. A solution of t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2NH(C4H8NHCBz) (about 1.6 g, about 2.2 mmol, 1.0 eq.) in CH2Cl2(about 12 mL) was then added, producing an initial limiting reactant concentration of about 0.1 M. The resulting solution was stirred at rt overnight and the solvent was then evaporated. The residue was then dissolved in EtOAc (about 50 mL), and washed with saturated aqueous NaHCO3solution (about 50 mL). The aqueous phase was then extracted with EtOAc (about 100 mL, 2×50 mL). The combined organic phase was then washed with brine (about 50 mL), dried over Na2SO4, filtered, and concentrated. The resulting material was then dissolved in CH2Cl2, loaded onto silica gel and purified via flash chromatography on silica using a gradient of EtOAc/hexanes. The resulting product containing fractions were concentrated, and dried under high vacuum, yielding a foam (about 2.96 g, about 89% yield). t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NH(Bn) FIG.19illustrates the peptoid t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NH(Bn) synthesized from t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NFMOC(Bn). One skilled in the art will appreciate that t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NH(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. 20% piperidine in THF (about 40 mL) was added to a solution of t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NFMOC(Bn) (about 2.96 g, about 1.9 mmol, 1.0 eq.) in THF (about 10 mL). A white colored solid precipitated from the reaction mixture. After about 0.5 hr, the reaction appeared to be complete via TLC. The reaction mixture was then concentrated to dryness and evaporated with several small portions of toluene. The resulting product was dissolved in wet acetonitrile (MeCN) and stirred with several portions of hexanes. The MeCN layer was concentrated to dryness, dissolved in CH2Cl2and loaded onto silica gel. The product was then purified via flash chromatography on silica, eluting first with a gradient of EtOAc/hexanes, then a gradient of MeOH/EtOAc. The resulting product containing fractions were collected, concentrated, and dried under high vacuum, resulting in about 2.3 g of product (about 91% yield). HO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NH(Bn) FIG.20illustrates the peptoid HO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NH(Bn) synthesized from t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NH(Bn). One skilled in the art will appreciate that HO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NH(Bn) is merely an illustrative peptoid, and that other peptoids may be synthesized in accordance with the present disclosure. i-Pr3SiH (about 0.39 mL, about 1.9 mmol, 5.0 eq.) was added to a solution of t-BuO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2NH(Bn) (about 0.50 g, about 0.38 mmol, 1.0 eq.) in CH2Cl2(about 2.0 mL), followed by addition of TFA (about 1.5 mL, about 0.25 M). The resulting solution was stirred at rt for about 2 hrs and then concentrated to dryness. The resulting residue was evaporated several times with toluene to remove residual TFA. The resulting material was dried under high vacuum overnight. The product was purified via reverse phase Biotage flash chromatography, running a gradient of 10-80% MeOH/H2O on a C18 column. Product containing fractions were collected, concentrated, and dried under high vacuum, yielding about 0.40 g of product as a solid (about 83% yield). Cyclic Hexamer FIG.21illustrates an example cyclic hexamer according to the present disclosure. To a solution of 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole (MSNT) (about 0.122 g, about 0.41 mmol, 5.0 eq.) in CH2Cl2(about 71 mL) contained in a RBF (e.g., a 100 mL RBF) under Ar, was added a solution of HO2CCH2N(C4H8NHCBz)COCH2N(Bn)COCH2N(C4H8NHCBz)COCH2N(Bn)CO CH2N(C4H8NHCBz)COCH2NH(Bn) (about 0.101 g, about 0.081 mmol, 1.0 eq.), i-Pr2NEt (about 0.14 mL, about 0.81 mmol, 10 eq.) in CH2Cl2(about 10 mL) via syringe pump configured to release 0.5 mL/hr. Final reactant concentration was about 0.001 M. The reaction mixture was stirred for about 4 days and diluted with about 50 mL H2O. The aqueous phase was extracted with CH2Cl2(about 50 mL, 2×25 mL). The combined organic phase was washed with H2O (about 50 mL), then evaporated to dryness. The resulting product was then dried under high vacuum. Purification was achieved by reverse phase Biotage chromatography on a C18column, eluting with a gradient of 70-100% MeCN/H2O. The product containing fractions were collected, concentrated, and dried under high vacuum, but still were impure via TLC. The product was then loaded onto silica from CH2Cl2, and purified via flash chromatography on silica using a gradient of 0-10% MeOH/CH2Cl2. The product containing fractions were then collected, concentrated, and dried under high vacuum, yielding about 21 mg of product as a colorless glass (about 21% yield). While in solution cyclization has been described, one skilled in the art will appreciate that other types of cyclization (e.g., solid stage synthesis or on resin) may be used. CBZ Deprotected Cyclic Hexamer FIG.22illustrates an example CBz deprotected cyclic hexamer (i.e., an example cyclic peptoid-based chelating ligand) according to the present disclosure. To a solution of the foregoing cyclic hexamer (about 18 mg, about 0.015 mmol, 1.0 eq.) in EtOH (about 5 mL) contained in a RBF (e.g., a 15 mL RBF) fitted with a reflux condenser was added 10% Pd/C. The solution was heated to reflux and an about 0.3 M formic acid solution in EtOH (about 2.5 mL, about 0.74 mmol, 50 eq.) was added. After refluxing for about 0.5 hr, the reaction was determined to be complete via mass spectrometry. The mixture was filtered to remove Pd/C and evaporated to dryness. Purification was achieved via reverse phase Biotage chromatography on a C18 column, and eluted with a gradient of 20-100% MeCN/H2O. Product containing fractions were concentrated, yielding the product as a white colored solid after drying under high vacuum (about 12 mg, about 56% yield). Protected Catechol Ester FIG.23illustrates a chemical structure of a protected catechol ester. The following is an example for preparing the protected catechol ester ofFIG.23. To a 500 mL RBF was added acetone (200 mL). The acetone was then degassed by sparging with Ar using a needle for 0.5 hr. 2,3-dihydroxybenzoic acid (1.0 g, 6.5 mmol, 1.0 eq.) was added, followed by K2CO3(3.64 g, 26.4 mmol, 4.1 eq.) and benzyl bromide (4.7 mL, 39.5 mmol, 6.1 eq.). A reflux condenser was attached, and the RBF was flushed with Ar and heated to reflux with stirring. The solution was refluxed for 24 hrs, cooled to rt, and filtered from solids. The resulting solids were washed with acetone. The solution was concentrated to dryness and excess benzyl bromide removed under high vacuum overnight. The resulting oil was purified via Biotage flash chromatography on a silica gel column, eluting with a gradient of EtOAc/hexanes. The product containing fractions were concentrated, yielding a colorless oil that was dried under high vacuum. The oil gradually crystallized (2.59 g, 94%). Protected Catechol Acid FIG.24illustrates a chemical structure of a protected catechol acid produced from the protected catechol ester ofFIG.23. The following is an example for preparing the protected catechol acid ofFIG.24from the protected catechol ester ofFIG.23. To a solution of the protected catechol ester ofFIG.23(2.54 g, 5.98 mmol, 1.0 eq.) in MeOH (360 mL) contained in a 1000 mL RBF was added aqueous NaOH, 5N (90 mL, 75 eq.). A reflux condenser was attached and the reaction apparatus was flushed with Ar. The reaction mixture was refluxed for 4 hrs, cooled to rt, and excess MeOH was evaporated. H2O (100 mL) was then added and the mixture was extracted with Et2O (2×100 mL). The aqueous layer was acidified with 12 N aqueous HCl until a white precipitate formed, pH 4.0. EtOAc was added, the aqueous layer was saturated with NaCl, and extracted with EtOAc (2×50 mL). The combined organic phase was dried over Na2SO4, filtered, and concentrated to dryness, yielding a white solid. Product was dissolved in CH2Cl2/hexanes, CH2Cl2was evaporated, and the resulting solution was chilled in a freezer overnight, then filtered and washed with hexanes, and dried under vacuum (1.88 g, 94%). Protected Catechol Acid Chloride FIG.25illustrates a chemical structure of a protected catechol acid chloride produced from the protected catechol acid ofFIG.24. The following is an example for preparing the protected catechol acid chloride ofFIG.25from the protected catechol acid ofFIG.24. Thionyl chloride was freshly distilled under Ar prior to use. To a 15 mL RBF containing the protected catechol acid ofFIG.24(0.25 g, 0.75 mmol, 1.0 eq.) was attached to a reflux condenser. The RBF was then flushed with Ar. SOCl2(4 mL, 55 mmol, 74 eq.) was added. The reaction mixture was heated with stirring under Ar. After refluxing for 3 hrs, the reaction mixture was cooled to rt and the excess SOCl2was evaporated under high vacuum, yielding a pink colored oil that solidified after standing (229 mg, 88%). Cyclic Hexamer Including Protected Catecholate Functionality In at least some examples, catecholate may be substituted for a hydrogen on a primary amine forming a terminal functional group of a side chain of a deprotected cyclic hexamer. For example, the oxygen, of a hydroxyl group of a carboxylic acid of catecholate, may be bonded to the nitrogen of a primary amine. The following is an example for preparing the cyclic hexamer ofFIG.26from the cyclic hexamer ofFIG.22and the protected catechol acid chloride ofFIG.25. To a solution of the protected catechol acid chloride ofFIG.25(161 mg, 0.48 mmol, 18.0 eq.) and oxyma (104 mg 0.73 mmol, 27.0 eq.) in DMF (1.0 mL) contained in a 5 mL RBF was added DIC (114 uL, 0.73 mmol, 27.0 eq.) dropwise. The resulting yellow colored solution was stirred under Ar for 15 min. To a 5 mL Biotage microwave vial containing a stir bar was added the triamine cyclic hexamer ofFIG.22(25 mg, 0.027 mmol, 1.0 eq.). Et3N (67 uL, 0.48 mmol, 18 eq.) was added to the microwave vial, followed by the solution of activated catechol acid. DMF (1.0 mL) was used to complete the transfer of the activated acid. The microwave vial was sealed and the resulting orange colored reaction mixture was heated in a Biotage Initiator microwave reactor (75° C., 1 hr). Volatiles were then removed by azeotropic distillation with toluene using a rotary evaporator. The resulting residue was dissolved in CH2Cl2(50 mL) and washed with saturated aqueous NaHCO3solution (50 mL). The aqueous phase was then extracted with CH2Cl2(2×25 mL). The combined organic phases were then washed with saturated aqueous NaHCO3solution (50 mL), brine (50 mL) and dried over Na2SO4. The solution was then filtered and evaporated to dryness. The product was purified 2× by RP C-18 Biotage chromatography on a 30 g column, 50-100% acetonitrile/H2O 0.5% formic acid additive in the acetonitrile. The product was isolated as an oil (43 mg, 92%). RP HPLC indicated some impurities. A partial separation by analytical TLC was obtained using silica gel plates, 10% MeOH/CH2Cl21% AcOH additive. Cyclic Hexamer Including Protected Catecholate Functionality In at least some examples, protected catecholate functionality of a cyclic hexamer may be deprotected by substituting benzyl groups (Bn) with hydrogen, resulting in the formation of hydroxyl groups. The following is an example for preparing the cyclic hexamer ofFIG.27from the cyclic hexamer ofFIG.26. To a solution of the cyclic hexamer ofFIG.26(including protected catecholate functionality) (38 mg, 0.021 mmol, 1.0 eq.) in ethanol (20 mL) in a 100 mL Teflon RBF was added Pd/C 10% Pd (15 mg, 40% by weight). The reaction apparatus was flushed with Ar and then flushed with H2. The reaction mixture was vigorously stirred under an atmosphere of H2provided by a balloon. After stirring for 26 hrs, the mixture was filtered through a 0.2 um Teflon syringe filter and the filter was washed with ethanol (3×5 mL). The combined ethanol phase was evaporated to dryness in a Teflon RBF. Purification by RP C-18 Biotage chromatography was attempted using a gradient of 40-100% MeCN/H2O, MeCN contained 0.5% formic acid as an additive. Some of the product ran off the column initially within the 1stcolumn volume, while the remainder eluted as a broad hump during the gradient run. Iron Affinity Ability of the cyclic hexamer ofFIG.27to bind Fe(III) was demonstrated by UV-visible spectroscopy. Solutions of the cyclic hexamer, Fe(III) triflate, and a solution of equal parts cyclic hexamer and Fe(III) triflate in methanol were prepared and examined by UV-visible spectroscopy. The solution of Fe(III) cyclic hexamer showed a broad absorbance band centered around 521 nm (seeFIG.28). Overview of Terms and Abbreviations The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the foregoing detailed description and the claims. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims, are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and/or limits of detection under standard test conditions/methods. While the present disclosure has been particularly described in conjunction with specific embodiments, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. It is therefore contemplated that the appended claims will embrace any such alternatives, modifications, and variations as falling within the true spirit and scope of the present disclosure. | 58,675 |
11859019 | DETAILED DESCRIPTION The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein. Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation. General Definitions As used herein, nomenclature for compounds, including organic compounds, can be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, Cahn-Ingold-Prelog rules for stereochemistry can be employed to designate stereochemical priority, E/Z specification, and the like. One of skill in the art can readily ascertain the structure of a compound if given a name, either by systemic reduction of the compound structure using naming conventions, or by commercially available software, such as CHEMDRAW™ (Cambridgesoft Corporation, U.S.A.). As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound. A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included. As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. As used herein, the term “subject” refers to the target of administration, e.g. a subject. Thus the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, fish, bird, rodent, or fruit fly. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In some examples, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some examples of the disclosed methods, the subject has been diagnosed with a need for treatment of cancer, autoimmune disease, and/or inflammation prior to the administering step. In some examples of the disclosed method, the subject has been diagnosed with cancer prior to the administering step. The term subject also includes a cell, such as an animal, for example human, cell. As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, or stabilize a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In some examples, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, “diagnosed with cancer” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can treat or prevent cancer. As a further example, “diagnosed with a need for treating or preventing cancer” refers to having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition characterized by cancer or other disease wherein treating or preventing cancer would be beneficial to the subject. As used herein, the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. For example, a subject can be identified as having a need for treatment of a disorder (e.g., a disorder related to cancer) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder. It is contemplated that the identification can, in some examples, be performed by a person different from the person making the diagnosis. It is also contemplated, in some examples, that the administration can be performed by one who subsequently performed the administration. As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In some examples, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In some examples, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition. The term “contacting” as used herein refers to bringing a disclosed compound and a target (e.g., a cell, target receptor, transcription factor, or other biological entity) together in such a manner that the compound can affect the activity of the target either directly, i.e., by interacting with the target itself, or indirectly, i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent. As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In some examples, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition. As used herein, “EC50,” is intended to refer to the concentration or dose of a substance (e.g., a compound or a drug) that is required for 50% enhancement or activation of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. EC50also refers to the concentration or dose of a substance that is required for 50% enhancement or activation in vivo, as further defined elsewhere herein. Alternatively, EC50can refer to the concentration or dose of compound that provokes a response halfway between the baseline and maximum response. The response can be measured in an in vitro or in vivo system as is convenient and appropriate for the biological response of interest. For example, the response can be measured in vitro using cultured cells or in an ex vivo organ culture system with isolated cells. Alternatively, the response can be measured in vivo using an appropriate research model such as rodent, including mice and rats. The mouse or rat can be an inbred strain with phenotypic characteristics of interest such as cancer or inflammation. As appropriate, the response can be measured in a transgenic or knockout mouse or rat wherein the gene or genes has been introduced or knocked-out, as appropriate, to replicate a disease process. As used herein, “IC50,” is intended to refer to the concentration or dose of a substance (e.g., a compound or a drug) that is required for 50% inhibition or diminuation of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. IC50also refers to the concentration or dose of a substance that is required for 50% inhibition or diminuation in vivo, as further defined elsewhere herein. Alternatively, IC50also refers to the half maximal (50%) inhibitory concentration (IC) or inhibitory dose of a substance. The response can be measured in an in vitro or in vivo system as is convenient and appropriate for the biological response of interest. For example, the response can be measured in vitro using cultured cells or in an ex vivo organ culture system with isolated cells. Alternatively, the response can be measured in vivo using an appropriate research model such as rodent, including mice and rats. The mouse or rat can be an inbred strain with phenotypic characteristics of interest such as cancer or inflammation. As appropriate, the response can be measured in a transgenic or knockout mouse or rat wherein a gene or genes has been introduced or knocked-out, as appropriate, to replicate a disease process. The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner. As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound. As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more —OCH2CH2O— units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more —CO(CH2)8CO— moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester. Chemical Definitions As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In some examples, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain examples, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted). In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents. The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like. This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term. The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “polyalkylene group” as used herein is a group having two or more CH2groups linked to one another. The polyalkylene group can be represented by the formula —(CH2)a—, where “a” is an integer of from 2 to 500. The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as —OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as —OA1-OA2or —OA1-(OA2)a-OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and/or cycloalkyl groups. The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A1A2)C═C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C═C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein. The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. “Carbocyclyl,” “carbocyclic ring” or “carbocycle” refers to a ring structure, wherein the atoms which form the ring are each carbon. Carbocyclic rings can comprise from 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryls and cycloalkyl, cycloalkenyl and cycloalkynyl as defined herein. The carbocyclic group can be substituted or unsubstituted. The carbocyclic group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl. The term “aldehyde” as used herein is represented by the formula —C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C═O. The terms “amine” or “amino” as used herein are represented by the formula —NA1A2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “alkylamino” as used herein is represented by the formula —NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like. The term “dialkylamino” as used herein is represented by the formula —N(-alkyl)2where alkyl is a described herein. Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like. The term “carboxylic acid” as used herein is represented by the formula —C(O)OH. The term “ester” as used herein is represented by the formula —OC(O)A1or —C(O)OA1, where A1can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula —(A1O(O)C-A2-C(O)O)a— or —(A1O(O)C-A2-OC(O))a—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups. The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula —(A1O-A2O)n—, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide. The term “halide” as used herein refers to the halogens fluorine, chlorine, bromine, and iodine. The term “heterocycle,” as used herein refers to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Heterocycle includes azetidine, dioxane, furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran, tetrazine, including 1,2,4,5-tetrazine, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole, thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine, triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like. The term “hydroxyl” as used herein is represented by the formula —OH. The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “azide” as used herein is represented by the formula —N3. The term “nitro” as used herein is represented by the formula —NO2. The term “nitrile” as used herein is represented by the formula —CN. The term “silyl” as used herein is represented by the formula —SiA1A2A3, where A1, A2, and A3can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfo-oxo” as used herein is represented by the formulas —S(O)A1, —S(O)2A1, —OS(O)2A1, or —OS(O)2OA1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S═O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula —S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “thiol” as used herein is represented by the formula —SH. “R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group. As described herein, compounds may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned herein are preferably those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in some examples, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted). The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in some examples, their recovery, purification, and use for one or more of the purposes disclosed herein. Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH2)0-4R∘; —O(CH2)0-4R∘, —O—(CH2)0-4C(O)OR∘; —(CH2)0-4CH(OR∘)2; —(CH2)0-4SR∘; —(CH2)0-4Ph, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1Ph which may be substituted with R∘; —CH═CHPh, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1-pyridyl which may be substituted with R∘; —NO2; —CN; —N3; —(CH2)0-4N(R∘)2; —(CH2)0-4N(R∘)C(O)R∘; —N(R∘) C(S)R∘; —(CH2)0- 4N(R∘)C(O)NR∘2; —N(R∘)C(S)NR∘2; —(CH2)0-4N(R∘) C(O)OR∘; —N(R∘)N(R∘)C(O)R∘; —N(R∘)N(R∘)C(O)NR∘2; —N(R∘)N(R∘)C(O)OR∘; —(CH2)0-4C(O)R∘; —C(S)R∘; —(CH2)0-4C(O)OR∘; —(CH2)0-4C(O)SR∘; —(CH2)0-4C(O)OSiR∘3; —(CH2)0-4OC(O)R∘; —OC(O)(CH2)0-4SR—, SC(S)SR∘; —(CH2)0-4SC(O)R∘; —(CH2)0-4C(O)NR∘2; —C(S)NR∘2; —C(S)SR∘; —SC(S)SR∘, —(CH2)0-4OC(O)NR∘2; —C(O)N(OR∘)R∘; —C(O)C(O)R∘; —C(O)CH2C(O)R∘; C(NOR∘)R∘; —(CH2)0-4SSR∘; —(CH2)0-4S(O)2R∘; —(CH2)0-4S(O)2OR∘; —(CH2)0-4OS(O)2R∘; —S(O)2NR∘2; —(CH2)0-4S(O)R∘; —N(R∘)S(O)2NR∘2; —N(R∘)S(O)2R∘; —N(OR∘)R∘; —C(NH)NR∘2; —P(O)2R∘; —P(O)R∘2; —OP(O)R∘2; —OP(O)(OR∘)2; SiR∘3; —(C1-4straight or branched)alkylene)O—N(R∘)2; or —(C1-4straight or branched) alkylene)C(O)O—N(R∘)2, wherein each R∘may be substituted as defined below and is independently hydrogen, C1-6aliphatic, —CH2Ph, —O(CH2)0-1Ph, —CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R∘, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below. Suitable monovalent substituents on R∘(or the ring formed by taking two independent occurrences of R∘together with their intervening atoms), are independently halogen, —(CH2)0-2R●, -(haloR●), —(CH2)0-2OH, —(CH2)O2OR●, —(CH2)0-2CH(OR●)2; —O(haloR●), —CN, —N3, —(CH2)0-2C(O)R●, —(CH2)0-2C(O)OH, —(CH2)0-2C(O)OR●, —(CH2)0-2SR●, —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR●, —(CH2)0-2NR●2, —NO2, —SiR●3, —OSiR●3, —C(O)SR, —(C1-4straight or branched alkylene)C(O)OR, or —SSR●wherein each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R∘include ═O and ═S. Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2))2-3O—, or —S(C(R*2))2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*2)2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on the aliphatic group of R* include halogen, —R●, -(halonR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R†, —NR†2, —C(O)R†, —C(O)OR†, —C(O)C(O)R†, —C(O)CH2C(O)R†, —S(O)2R†, —S(O)2NR†2, —C(S)NR†2, —C(NH)NR†2, or —N(R†)S(O)2R†; wherein each R†is independently hydrogen, C1-6aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on the aliphatic group of R†are independently halogen, —R●, -(halonR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate. The terms “hydrolysable group” and “hydrolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999). The term “organic residue” defines a carbon containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In some examples, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms. A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4-thiazolidinedione radical in a particular compound has the structure regardless of whether thiazolidinedione is used to prepare the compound. In some embodiments the radical (for example an alkyl) can be further modified (i.e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” The number of atoms in a given radical is not critical to the compounds and compositions disclosed herein unless it is indicated to the contrary elsewhere herein. “Organic radicals,” as the term is defined and used herein, contain one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In some examples, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bound to at least some of the carbon atoms of the organic radical. One example, of an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthyl radical. In some embodiments, an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, wherein the terms are defined elsewhere herein. A few non-limiting examples of organic radicals that include heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals and the like. “Inorganic radicals,” as the term is defined and used herein, contain no carbon atoms and therefore comprise only atoms other than carbon. Inorganic radicals comprise bonded combinations of atoms selected from hydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, and halogens such as fluorine, chlorine, bromine, and iodine, which can be present individually or bonded together in their chemically stable combinations. Inorganic radicals have 10 or fewer, or preferably one to six or one to four inorganic atoms as listed above bonded together. Examples of inorganic radicals include, but not limited to, amino, hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonly known inorganic radicals. The inorganic radicals do not have bonded therein the metallic elements of the periodic table (such as the alkali metals, alkaline earth metals, transition metals, lanthanide metals, or actinide metals), although such metal ions can sometimes serve as a pharmaceutically acceptable cation for anionic inorganic radicals such as a sulfate, phosphate, or like anionic inorganic radical. Inorganic radicals do not comprise metalloids elements such as boron, aluminum, gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gas elements, unless otherwise specifically indicated elsewhere herein. Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis/trans (E/Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the compounds and compositions disclosed herein include all such possible isomers, as well as mixtures of such isomers. As used herein, the symbol (hereinafter can be referred to as “a point of attachment bond”) denotes a bond that is a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment bond and the other of which is not depicted as being attached to the point of attachment bond. For example, indicates that the chemical entity “XY” is bonded to another chemical entity via the point of attachment bond. Furthermore, the specific point of attachment to the non-depicted chemical entity can be specified by inference. For example, the compound CH3—R3, wherein R3is H or infers that when R3is “XY”, the point of attachment bond is the same bond as the bond by which R3is depicted as being bonded to CH3. Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the compounds and compositions disclosed herein include all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers. Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon. Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically-labelled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds disclosed herein include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,35S,18F and36Cl respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e.,3H, and carbon-14, i.e.,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. The compounds described herein can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvate or water molecules can combine with the compounds disclosed herein to form solvates and hydrates. Unless stated to the contrary, all such possible solvates are included in the discussion herein. The term “co-crystal” means a physical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework in the crystalline lattice. In certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et. al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid. It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an α-hydrogen can exist in an equilibrium of the keto form and the enol form. Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, all such possible tautomers are included herein. It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, all such possible polymorphic forms are included. In some examples, a structure of a compound can be represented by a formula: which is understood to be equivalent to a formula: wherein n is typically an integer. That is, Rnis understood to represent five independent substituents, Rn(a), Rn(b), Rn(c), Rn(d), Rn(e), By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance Rn(a)is halogen, then Rn(b)is not necessarily halogen in that instance. Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification. Disclosed are the components to be used to prepare the compositions disclosed herein as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions disclosed herein. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods disclosed herein. It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result. Abbreviations used herein are as follows: Alloc, allyloxycarbonyl; Cpa, L-4-chlorophenylalanine; dap, D-2,3-diaminopropionic acid; Dap, L-2,3-diaminopropionic acid; FA, fluorescence anisotropy; FITC, fluorescein isothiocyanate; Fpa, L-4-fluorophenylalanine; F2pa, L-3,4-difluorophenylalanine; fpa, D-2-fluorophenylalanine; f2pa, D-3,4-difluorophenylalanine; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; miniPEG, 8-amino-3,6-dioxaoctanoic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Nal, L-2-naphthylalanine; Nip, (R)-nipecotic acid; Nle, norleucine; Orn, ornithine; Phg, L-α-phenylglycine; PPI, protein-protein interaction; Sar, sarcosine; TNFα, tumor necrosis factor-alpha; TNFR, TNFα receptor. Compounds In previous studies, screening of a naïve bicyclic peptide against TNFα identified anticachexin C1 as a moderately potent TNFα antagonist, which blocks the TNFα-TNFRI interaction with an IC50value of 3.1 μM (Lian, W.; et al., supra). Cyclic and bicyclic peptide hits derived from other screening campaigns against protein-protein interaction (PPI) targets (which generally do not contain major binding pockets) typically exhibit a similar level of potencies (i.e., IC50/KDvalues in the high nM to low μM range) (Liu, T., et al., “Synthesis and screening of a cyclic peptide library: Discovery of small-molecule ligands against human prolactin receptor.”Bioorg. Med. Chem.2009, 17:1026-1033; Liu, T.; et al., “High-Throughput Screening of One-Bead-One-Compound Libraries: Identification of Cyclic Peptidyl Inhibitors against Calcineurin/NFAT Interaction.”ACS Comb. Sci.2011, 13:537-546; Dewan, V.; et al., “Cyclic peptide inhibitors of HIV-1 capsid-human lysyl-tRNA synthetase interaction.”ACS Chem. Biol.2012, 7:761-769; Desimmie, B. A.; et al., “Phage Display-directed Discovery of LEDGF/p75 Binding Cyclic Peptide Inhibitors of HIV Replication.”Mol. Therapy2012, 20:2064-2075; Birts, C. N.; et al., “A cyclic peptide inhibitor of C-terminal binding protein dimerization links metabolism with mitotic fidelity in breast cancer cells.”Chem. Sci.2013, 4:3046-3057; Miranda, E.; et al., “A Cyclic Peptide Inhibitor of HIF-1 Heterodimerization That Inhibits Hypoxia Signaling in Cancer Cells.”J. Am. Chem. Soc.2013, 135:10418-10425). These hits require substantial improvement in potency (and specificity) before becoming useful as therapeutic agents or chemical probes. Disclosed herein, SAR analysis and optimization was performed on all nine residues within the bicyclic structure of anticachexin C1, which in certain examples improved the potency by 44-fold (IC50=70 nM). Further improvement of the potency was achieved by constructing and screening a second-generation library, in which a degenerate tripeptide sequence was appended to the side chain of a noncritical residue, to engage in additional interactions with the TNFα surface. The resulting TNFα inhibitor (IC50=12 nM) can be used as a potent TNFα inhibitor or can serve as a useful lead for further development into therapeutic agents. The combination of ring residue optimization and exocyclic structural extension can offer a general strategy for optimization of cyclic/bicyclic peptide hits derived from combinatorial libraries. More specifically, a planar scaffold, trimesic acid, can be used in order to maximize the surface area of the resulting molecules and therefore their ability to interact with flat protein surfaces such as the PPI interfaces. A bicyclic peptide library was generated by “wrapping” a peptide sequence of up to 10 random residues around the trimesoyl group. Peptide cyclization was mediated by the formation three amide bonds between the trimesoyl scaffold and the N-terminal amine, the side chain of a C-terminal L-2,3-diaminopropionic acid (Dap), and the side chain of a fixed lysine within the random region. In a particular aspect, disclosed herein are bicyclic peptides of Formula I. wherein AA1-AA3and AA5-AA10are amino acid residues, R is null, carboxylic acid, amide, or C1-20keto, ester, amino acid residue, or functionalized peptide moiety of from 2 to 10 amino acid residues in length, and n is an integer of from 1 to 6. Each amino acid residue can be a natural or non-natural amino acid residue. The term “non-natural amino acid” refers to an organic compound that is a congener of a natural amino acid in that it has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid can be a modified amino acid, and/or amino acid analog, that is not one of the 20 common naturally occurring amino acids or the rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be the D-isomer of the natural amino acids. In some examples, one or more amino acid residues is the D-isomer. Examples of suitable amino acids include, but are not limited to, alanine, allosoleucine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, a derivative, or combinations thereof. These, and others, are listed in the Table A along with their abbreviations used herein. TABLE AAmino Acid AbbreviationsAmino AcidAbbreviations*AlanineAla (A)AllosoleucineAIleArginineArg (R)asparagineAsn (N)aspartic acidAsp (D)cysteineCys (C)cyclohexylalanineCha4-chlorophenylalanineCpa2,3-diaminopropionic acidDap3,4,-difluorophenylalaniceF2pa4-fluorophenylalanineFpa (Σ)glutamic acidGlu (E)glutamineGln (Q)glycineGly (G)histidineHis (H)homoprolinePip (Θ)isoleucineIle (I)leucineLeu (L)lysineLys (K)methionineMet (M)naphthylalanineNal (Φ)norleucineNle (Ω)phenylalaninePhe (F)phenylglycinePhg (Ψ)4-(phosphonodifluoromethyl)phenylalanineF2Pmp (Λ)pipecolic acidPp (ϑ)prolinePro (P)sarcosineSar (Ξ)selenocysteineSec (U)serineSer (S)threonineThr (T)tyrosineTyr (Y)tryptophanTrp (W)valineVal (V)*single letter abbreviations: when shown in capital letters herein it indicates the L-amino acid form, when shown in lower case herein it indicates the D-amino acid form The amino acids AA1-AA3and AA5-AA10can be coupled to one another by a peptide bond or a modified peptide bond, such as by —N(alkyl)C(O)—. In certain examples, disclosed are a subset of compounds of Formula I where n is 3; this is labeled as Formula I-A. wherein AA1-AA3and AA5-AA10and R are as defined above for Formula I. In still further examples, disclosed herein are compounds of Formula II: wherein R1, R2, R3, R5, R6, R7, R8, R9, are side chains of amino acid residues AA1-3and AA5-9respectively. R11is OH, NH2, R12, or NHR12, where R12is an amino acid residue, or substituted or unsubstituted, branched or straight chain C1-20alkyl, substituted or unsubstituted, branched or straight chain OC1-20alkyl, or a functionalized peptide side moiety of from 2 to 10 amino acid residues in length, any of which is optionally coupled to a detectable moiety or therapeutic moiety. In specific examples, disclosed herein are compounds of Formula II, wherein R1can be a hydrophobic moiety. For example, R1can be phenyl, benzyl, or substituted or unsubstituted, branched or straight chain C1-20alkyl. In specific examples, R1can be methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pentyl, or hexyl. In preferred examples, R1can be phenyl or n-butyl. In specific examples, disclosed herein are compounds of Formula II, wherein R2can be an aryl moiety. For example, R2can be unsubstituted phenyl or benzyl, or phenyl or benzyl substituted with one or more halo, OH, SH, CO2H, or NH2groups. In specific examples, R2can be benzyl (CH2C6H6), 4-hydroxybenzyl, 4-fluorobenzyl, 4-chlorobenzyl, or 3,4-difluorobenzyl. In preferred examples, R2can be 4-hydroxybenzyl. In specific examples, disclosed herein are compounds of Formula II, wherein R3can be a small (e.g., less than 5 atoms in length) neutral or hydrophilic moiety. For example, R3can be H, unsubstituted C1-6alkyl or C1-6alkyl substituted with one or more halo, OH, SH, CO2H, or NH2groups. In specific examples, R3can be —CH2OH, —CHOHCH3, —CH2SH, —CH2CO2, —(CH2)2CO2H, —(CH2)4NH2, or —CH2CONH2. In preferred examples, R3can be —CH3or —CH2OH. In other examples, R3is a peptide of from 2 to 8 amino acids in length, e.g., from 3 to 6, from 4 to 5, from 2 to 4, or from 3 to 5 amino acids in length. The amino acids in the peptide of R3in these examples can be natural or unnatural amino acids. In specific examples R3is a tripeptide comprising natural and/or unnatural amino acids. In further specific examples, R3is a tripeptide X1-X2-X3where X1and X3are nonatural amino acids, e.g., D-α-amino acids (4-iodo-D-phenylalanine, 4-cyano-D-phenylalanine, 2-fluoro-D-phenylalanine (fpa), 3,4-difluoro-D-phenylalanine (f2pa), D-Trp, D-Asp, D-Val, D-Thr, D-Pro, D-Ser, D-Leu, D-Phe, D-Ala, D-Tyr, D-Nal, D-Glu, D-Asn, D-Lys, D-Arg, D-His). In further examples, X2is an unnatural amino acid, e.g., 3-amino benzoic acid (Abz), L-β-homoisoleucine, (R)-nipecotic acid (Nip), 4-amino-1-methylpyrrole-3-carboxylic acid, γ-aminobutyric acid, β-Ala, D-homophenylalanine, D-Thr, D-Pro, D-Ser, D-Leu, D-Phe, (S)-3-amino-5-phenylpentanoic acid (apa), D-Tyr, D-Nal, D-Glu, D-Asn, D-Lys, D-Arg, and D-His. In specific examples, disclosed herein are compounds of Formula II, wherein R5can be an aryl moiety. For example, R5can be unsubstituted phenyl, benzyl, heteroaryl or CH2heteroaryl, or phenyl, benzyl, heteroaryl or —CH2heteroaryl substituted with one or more halo, OH, SH, CO2H, or NH2groups. In specific examples, R5can be benzyl 4-hydroxybenzyl, 4-fluorobenzyl, 4-chlorobenzyl, 3,4-difluorobenzyl, —CH2Imidazole, or —CH2Indole. In a preferred examples, R5can be —CH2Indole. In specific examples, disclosed herein are compounds of Formula II, wherein R6can be a hydrophilic moiety. For example, R6can be CH2Imidazole, or C1-6alkyl, benzyl, or phenyl substituted with one or more halo, OH, SH, CO2H, or NH2groups. In a preferred example, R6can be CH2Indole. In specific examples, disclosed herein are compounds of Formula II, wherein R7can be a small (e.g., less than 5 atoms in length) moiety such as H, unsubstituted C1-2alkyl, or C1-2alkyl substituted with OH, SH, NH2, or CO2H. In a preferred example, R7is H. In specific examples, disclosed herein are compounds of Formula II, wherein R8can be a hydrophilic moiety. For example, R8can be C1-6alkyl substituted with one or more halo, OH, SH, CO2H, or NH2groups. In specific examples, R8can be —CH2OH, —CHOHCH3, —CH2SH, —CH2CO2, —(CH2)2CO2H, —(CH2)4NH2, or —CH2CONH2. In a preferred example, R8can be —(CH2)4NH2. In specific examples, disclosed herein are compounds of Formula II, wherein R9can be can be CH2Imidazole, or C1-6alkyl, benzyl, or phenyl substituted with one or more halo, OH, SH, CO2H, or NH2groups. In a preferred example, R9can be CH2Imidazole. In specific examples, R11and R12can be, independently, arginine, lysine, aspartic acid, norleucine, phenylalanine, beta alanine or any of the sequences for R1shown in Table 5 or 6, any of which can be coupled to a detectable moiety or therapeutic moiety. Disclosed herein is a bicyclic peptide having Formula II-A. wherein R11is H or R12as defined in Formula II. In some examples, the compounds disclosed herein can linked to label moiety at R11or R12. The label moiety can comprise any detectable label. Examples of suitable detectable labels include, but are not limited to, a UV-Vis label, a near-infrared label, a luminescent group, a phosphorescent group, a magnetic spin resonance label, a photosensitizer, a photocleavable moiety, a chelating center, a heavy atom, a radioactive isotope, a isotope detectable spin resonance label, a paramagnetic moiety, a chromophore, or any combination thereof. In some embodiments, the label is detectable without the addition of further reagents. In some embodiments, the label moiety is a biocompatible label moiety, such that the compounds can be suitable for use in a variety of biological applications. “Biocompatible” and “biologically compatible”, as used herein, generally refer to compounds that are, along with any metabolites or degradation products thereof, generally non-toxic to cells and tissues, and which do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in their presence. The label moiety can contain a luminophore such as a fluorescent label or near-infrared label. Examples of suitable luminophores include, but are not limited to, metal porphyrins; benzoporphyrins; azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatic hydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes; xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyanine dyes, metal-ligand complex such as bipyridine, bipyridyls, phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium; acridine, oxazine derivatives such as benzophenoxazine; aza-annulene, squaraine; 8-hydroxyquinoline, polymethines, luminescent producing nanoparticle, such as quantum dots, nanocrystals; carbostyril; terbium complex; inorganic phosphor; ionophore such as crown ethers affiliated or derivatized dyes; or combinations thereof. Specific examples of suitable luminophores include, but are not limited to, Pd (II) octaethylporphyrin; Pt (II)-octaethylporphyrin; Pd (II) tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II) meso-tetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetrapheny metrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II) octaethylporphyrin ketone; Pd (II) meso-tetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra (pentafluorophenyl) porphyrin; Ru (II) tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp)3); Ru (II) tris(1,10-phenanthroline) (Ru(phen)3), tris(2,2′-bipyridine)ruthenium (II) chloride hexahydrate (Ru(bpy)3); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; iridium (III) ((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III) ((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7; 4-nitirophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein; eosin; 2′,7′-dichlorofluorescein; 5(6)-carboxy-fluorecsein; carboxynaphtofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid; semi-naphthorhodafluor; semi-naphthofluorescein; tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride; (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron; platinum (II) octaethylporphyin; dialkylcarbocyanine; dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride; 7-amino-4-methylcourmarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof. In some examples, the label moiety is a fluorescence label. In some examples, the fluorescence label is a small molecule. Such small molecules are known in the art. In some examples, the label moiety has a structure represented by a formula: In the disclosed compounds R11or R12can be a substituted or unsubstituted, branched or straight chain C1-20alkyl, substituted or unsubstituted, branched or straight chain OC1-20alkyl, or a functionalized peptide side moiety of from two to 10 amino acid residues in length. As is shown herein, when R11or R12is a functionalized peptide, the potency of the disclosed cyclic peptides can be further boosted, generating a “lollipop” shaped molecule. Optimization of the tripeptidyl branch structure by medicinal chemistry approaches was envisioned to further increase the potency of C1-74-2. Importantly, through judicious choice of building blocks, the compound potency was increased without increasing its hydrophobicity. Second, this study provides another demonstration that relatively small, structurally constrained cyclic and bicyclic peptides (MW<2000) can recognize flat protein surfaces in an antibody-like manner and therefore serve as effective PPI inhibitors. Third, this work illustrates the importance of having access to both natural and unnatural building blocks (e.g., D-amino acids and β-amino acids) during library design and the power of chemical synthesis in generating such libraries. Pharmaceutical Compositions Also disclosed herein are pharmaceutical compositions comprising the disclosed compounds. That is, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed compound. In some examples, a pharmaceutical composition can be provided comprising a prophylactically effective amount of at least one disclosed compound. Also disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and any of the compounds disclosed herein, wherein the compound is present in an effective amount. Also disclosed herein are neutraceutical compositions comprising a neutraceutically acceptable carrier and any of the compounds disclosed herein, wherein the compound is present in an effective amount. In some examples of the compositions, the compound is present in an amount greater than about an amount selected from 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400, mg, 500 mg, 750 mg, 1000 mg, 1,500 mg, or 2,000 mg. The disclosed pharmaceutical compositions can further comprise one or more anticancer drugs. Example anticancer drugs include 13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex, Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase, Phenylalanine Mustard, Platinol, Platinol-AQ, Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide, Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab, Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid, Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon, Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa, Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulating factor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine, HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisone sodium phosphate, Hydrocortisone sodium succinate, Hydrocortone phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin, Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG conjugate), Interleukin 2, Interleukin-11, Intron A (interferon alfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine, Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, L-PAM, L-Sarcolysin, Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX, Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan, Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. In some examples, the pharmaceutical composition is administered to a subject. In some examples, the subject is a mammal, fish or bird. In some examples, the mammal is a primate. In some examples, the mammal is a human. In some examples, the human is a patient. In some examples, the pharmaceutical composition is administered following identification of the mammal in need of treatment of cancer or inflammation or an autoimmune disease. In some examples, the pharmaceutical composition is administered following identification of the mammal in need of prevention of cancer or inflammation or an autoimmune disease. In some examples, the mammal has been diagnosed with a need for treatment of cancer or inflammation or an autoimmune disease prior to the administering step. In some examples, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. As used herein, the term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically or neutraceutically acceptable non-toxic bases or acids. When the compound is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically or neutraceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. As used herein, the term “pharmaceutically acceptable non-toxic acids”, includes inorganic acids, organic acids, and salts prepared thereof, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids. In practice, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, or neutraceutically acceptable salts thereof, can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier or neutraceutical carrier according to conventional pharmaceutical compounding techniques or conventional neutraceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions or neutraceutical compositions disclosed herein can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds disclosed herein, and/or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and/or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation. Thus, the pharmaceutical compositions disclosed herein can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds disclosed herein. The compounds disclosed herein, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds. The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen. In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules can be used for oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques A tablet containing any of the compositions disclosed herein can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The pharmaceutical compositions disclosed herein can comprise any of the compounds disclosed herein (or pharmaceutically or neutraceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier or neutraceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. Pharmaceutical compositions suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. In some examples, a preservative can be included to prevent the detrimental growth of microorganisms. Pharmaceutical compositions disclosed herein suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In some examples, the final injectable form can be sterile and can be effectively fluid for easy syringability. In some examples, the pharmaceutical compositions can be stable under the conditions of manufacture and storage; thus, they can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof Pharmaceutical compositions disclosed herein can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. In some examples, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing any of the compounds disclosed herein or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment can be prepared by mixing hydrophilic material and water, together with about 5 wt % to about 10 wt % of the compound, to produce a cream or ointment having a desired consistency. Pharmaceutical compositions disclosed herein can be in a form suitable for rectal administration wherein the carrier is a solid. In some examples, the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carriers) followed by chilling and shaping in molds. In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing any of the compounds disclosed herein, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form. In the treatment of cancer, the dosage level of the active ingredient comprising the compound or compositions disclosed herein can be about 0.01 to 500 mg per kg patient body weight per day and can be administered in single or multiple doses. In some examples, he dosage level will be about 0.1 to about 250 mg/kg per day; such as 0.5 to 100 mg/kg per day. A suitable dosage level can be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0, or 5.0 to 50 mg/kg per day. For oral administration, the compositions can be, for example, in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times per day, such as, for example, once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response. It is understood, however, that the specific dose level for any particular patient will depend upon a variety of factors. Such factors include the age, body weight, general health, sex, and diet of the patient. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease or infection undergoing therapy. Also disclosed herein are methods for the manufacture of a medicament for treating cancer in mammals (e.g., humans) comprising combining one or more disclosed compounds, products, or compositions with a pharmaceutically acceptable carrier or diluent. In some examples, the method for manufacturing a medicament comprises combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent. The disclosed pharmaceutical compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological conditions. It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using. Compositions, Formulations and Methods of Administration In vivo application of the disclosed compounds, and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. For example, the disclosed compounds can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, topical, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the disclosed compounds or compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art. The compounds disclosed herein, and compositions comprising them, can also be administered utilizing liposome technology, slow release capsules, implantable pumps, and biodegradable containers. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time. The compounds can also be administered in their salt derivative forms or crystalline forms. The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example,Remington's Pharmaceutical Scienceby E. W. Martin (1995) describes formulations that can be used in connection with the disclosed methods. In general, the compounds disclosed herein can be formulated such that an effective amount of the compound is combined with a suitable carrier in order to facilitate effective administration of the compound. The compositions used can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically-acceptable carriers and diluents which are known to those skilled in the art. Examples of carriers or diluents for use with the compounds include ethanol, dimethyl sulfoxide, glycerol, alumina, starch, saline, and equivalent carriers and diluents. To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 100% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent. Formulations suitable for administration include, for example, aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the compositions disclosed herein can include other agents conventional in the art having regard to the type of formulation in question. Compounds disclosed herein, and compositions comprising them, can be delivered to a cell either through direct contact with the cell or via a carrier means. Carrier means for delivering compounds and compositions to cells are known in the art and include, for example, encapsulating the composition in a liposome moiety. Another means for delivery of compounds and compositions disclosed herein to a cell comprises attaching the compounds to a protein or nucleic acid that is targeted for delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and that allows the composition to be translocated across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transporting biological moieties across cell membranes for intracellular delivery. Compounds can also be incorporated into polymers, examples of which include poly (D-L lactide-co-glycolide) polymer for intracranial tumors; poly[bis(p-carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin; chitin; and chitosan. For the treatment of oncological disorders, the compounds disclosed herein can be administered to a patient in need of treatment in combination with other antitumor or anticancer substances and/or with radiation and/or photodynamic therapy and/or with surgical treatment to remove a tumor. These other substances or treatments can be given at the same as or at different times from the compounds disclosed herein. For example, the compounds disclosed herein can be used in combination with mitotic inhibitors such as taxol or vinblastine, alkylating agents such as cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracil or hydroxyurea, DNA intercalators such as adriamycin or bleomycin, topoisomerase inhibitors such as etoposide or camptothecin, antiangiogenic agents such as angiostatin, antiestrogens such as tamoxifen, and/or other anti-cancer drugs or antibodies, such as, for example, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN (Genentech, Inc.), respectively, or an immunotherapeutic such as ipilimumab and bortezomib. In certain examples, compounds and compositions disclosed herein can be locally administered at one or more anatomical sites, such as sites of unwanted cell growth (such as a tumor site or benign skin growth, e.g., injected or topically applied to the tumor or skin growth), optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent. Compounds and compositions disclosed herein can be systemically administered, such as intravenously or orally, optionally in combination with a pharmaceutically acceptable carrier such as an inert diluent, or an assimilable edible carrier for oral delivery. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, aerosol sprays, and the like. The tablets, troches, pills, capsules, and the like can also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring can be added. When the unit dosage form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials can be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules can be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir can contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound can be incorporated into sustained-release preparations and devices. Compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly, or intraperitoneally by infusion or injection. Solutions of the active agent or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. The ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. Optionally, the prevention of the action of microorganisms can be brought about by various other antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating a compound and/or agent disclosed herein in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, compounds and agents disclosed herein can be applied in as a liquid or solid. However, it will generally be desirable to administer them topically to the skin as compositions, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Compounds and agents and compositions disclosed herein can be applied topically to a subject's skin to reduce the size (and can include complete removal) of malignant or benign growths, or to treat an infection site. Compounds and agents disclosed herein can be applied directly to the growth or infection site. Preferably, the compounds and agents are applied to the growth or infection site in a formulation such as an ointment, cream, lotion, solution, tincture, or the like. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers, for example. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counter indications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Also disclosed are pharmaceutical compositions that comprise a compound disclosed herein in combination with a pharmaceutically acceptable carrier. In some examples, the pharmaceutical compositions are adapted for oral, topical or parenteral administration. The dose administered to a patient, particularly a human, should be sufficient to achieve a therapeutic response in the patient over a reasonable time frame, without lethal toxicity, and preferably causing no more than an acceptable level of side effects or morbidity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition (health) of the subject, the body weight of the subject, kind of concurrent treatment, if any, frequency of treatment, therapeutic ratio, as well as the severity and stage of the pathological condition. Methods of Using the Compounds and Compositions Also provided herein are methods of use of the compounds or compositions described herein. Also provided herein are methods for treating a disease or pathology in a subject in need thereof comprising administering to the subject an effective amount of any of the compounds or compositions described herein. A very important application is for specific delivery of drugs such as anticancer drugs. The bicyclic peptides disclosed herein can be directed to a cancer-specific or overexpressed surface protein. Then an anticancer drug can be covalently or noncovaelently attached to the bicyclic peptide, e.g., at R, R1, R11, or R12. Also provided herein are methods of treating, preventing, or ameliorating cancer in a subject. The methods include administering to a subject an effective amount of one or more of the compounds or compositions described herein, or a pharmaceutically acceptable salt thereof. The compounds and compositions described herein or pharmaceutically acceptable salts thereof are useful for treating cancer in humans, e.g., pediatric and geriatric populations, and in animals, e.g., veterinary applications. The disclosed methods can optionally include identifying a patient who is or can be in need of treatment of a cancer. Examples of cancer types treatable by the compounds and compositions described herein include bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer. Further examples include cancer and/or tumors of the anus, bile duct, bone, bone marrow, bowel (including colon and rectum), eye, gall bladder, kidney, mouth, larynx, esophagus, stomach, testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord, thyroid, vagina, vulva, uterus, liver, muscle, blood cells (including lymphocytes and other immune system cells). Further examples of cancers treatable by the compounds and compositions described herein include carcinomas, Karposi's sarcoma, melanoma, mesothelioma, soft tissue sarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myeloid, and other), and lymphoma (Hodgkin's and non-Hodgkin's), and multiple myeloma. In some examples, the cancer can be associated with TNF-α induced cell death. The methods of treatment or prevention of cancer described herein can further include treatment with one or more additional agents (e.g., an anti-cancer agent or ionizing radiation). The one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be administered in any order, including simultaneous administration, as well as temporally spaced order of up to several days apart. The methods can also include more than a single administration of the one or more additional agents and/or the compounds and compositions or pharmaceutically acceptable salts thereof as described herein. The administration of the one or more additional agents and the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be by the same or different routes. When treating with one or more additional agents, the compounds and compositions or pharmaceutically acceptable salts thereof as described herein can be combined into a pharmaceutical composition that includes the one or more additional agents. The methods and compounds as described herein are useful for both prophylactic and therapeutic treatment. As used herein the term treating or treatment includes prevention; delay in onset; diminution, eradication, or delay in exacerbation of signs or symptoms after onset; and prevention of relapse. For prophylactic use, a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of an infection. Prophylactic administration can be used, for example, in the chemopreventative treatment of subjects presenting precancerous lesions, those diagnosed with early stage malignancies, and for subgroups with susceptibilities (e.g., family, racial, and/or occupational) to particular cancers. Therapeutic treatment involves administering to a subject a therapeutically effective amount of the compounds and compositions or pharmaceutically acceptable salts thereof as described herein after cancer is diagnosed. Also disclosed herein are methods of treating or preventing a disorder in a subject, such as a human, comprising administering to the subject an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt thereof. In some examples, the subject is an animal, such as a human. In some examples, the subject is identified as having a need for treatment of the disorder. In some examples, the method treats a disorder. In some examples, the disorder is associated with TNF-α-induced cell death, such as dysfunctional regulation of TNF-α-induced cell death. In some examples, the disorder is associated with uncontrolled cellular proliferation, such as cancer. In some examples, the disorder is cancer. In some examples, the disorder is an inflammatory disorder. In some examples, the disorder is an autoimmune disorder, such as a disorder selected from rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, hidradenitis suppurativa, and refractory asthma. In some examples, the subject has been diagnosed with the disorder prior to the administration step. In some examples, the compound is administered in an amount between about 0.01 to 500 mg per kg patient body weight per day and can be administered in single or multiple doses. In some examples, the dosage level can be about 0.1 to about 250 mg/kg per day, such as about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. In some examples, the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day. In some examples, the dosage level can be 0.5 to 100 mg/kg per day. For oral administration, the compositions are can be provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, such as 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times per day, such as once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response. In some examples, the subject is a domesticated animal. In some examples, the domesticated animal is a domesticated fish, domesticated crustacean, or domesticated mollusk. In some examples, the domesticated animal is poultry. In some examples, the poultry is selected from chicken, turkey, duck, and goose. In some examples, the domesticated animal is livestock. In some examples, the livestock animal is selected from pig, cow, horse, goat, bison, and sheep. In some examples, the method further comprises the step of identifying the animal in need of treatment or prevention of cancer. In some examples, the mammal has been diagnosed with a need for treatment and prevention of cancer prior to the administering step. Protection Against TNF-α Induced Cell Death Also disclosed herein are methods for protection against TNFα-induced cell death. The method can comprise administering an effective amount of a compound disclosed herein, a compound made by a method disclosed herein, a library disclosed herein, or a compound identified by methods disclosed herein to a subject identified as having a need for protection against TNFα-induced cell death. In some examples, the amount is therapeutically effective. In some examples, the amount is prophylactically effective. In some examples, the subject is a cell. In some examples, the subject is an animal. In some examples, the subject is a human. Manufacture of a Medicament Also disclosed herein are methods for the manufacture of a medicament for treating or preventing cancer comprising combining a therapeutically effective amount of a disclosed compound or product of a disclosed method with a pharmaceutically acceptable carrier or diluent. Also disclosed herein are methods for manufacturing a medicament associated with treating or preventing cancer or the need to treat or prevent cancer with a pharmaceutically acceptable carrier or diluent. In some examples, the medicament comprises a disclosed compound. Kits Also disclosed are kits that comprise a compound disclosed herein in one or more containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and/or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In another embodiment, a kit includes one or more anti-cancer agents, such as those agents described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, a compound and/or agent disclosed herein is provided in the kit as a solid, such as a tablet, pill, or powder form. In another embodiment, a compound and/or agent disclosed herein is provided in the kit as a liquid or solution. In one embodiment, the kit comprises an ampoule or syringe containing a compound and/or agent disclosed herein in liquid or solution form. Also disclosed herein are kits comprising one or more of the disclosed compounds, and one or more of: a) at least one anticancer compound, b) instructions for treating a disorder associated with cancer, or c) instructions for treating cancer. In some examples, the kit further comprises at least one agent, wherein the compound and the agent are co-formulated. In some examples, the compound and the agent are co-packaged. The agent can be any agent as disclosed herein, known to have a side effect of cancer, an agent known to increase the risk of cancer, agent known to treat cancer in a subject. The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient. It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and/or the disclosed compositions. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric. Materials and Reagents. Certain materials, reagents and kits were obtained from specific vendors as indicated herein. Fmoc-protected amino acids were purchased from Advanced ChemTech (Louisville, KY), Peptides International (Louisville, KY), or Aapptec (Louisville, KY). O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole hydrate (HOBt) were from Aapptec. All solvents and other chemical reagents were obtained from Sigma-Aldrich, Fisher Scientific (Pittsburgh, Pa.), or VWR (West Chester, PA) and were used without further purification unless noted otherwise. N-(9-Fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) was from Advanced ChemTech. Phenyl isothiocyanate was purchased in 1-mL sealed ampoules from Sigma-Aldrich, and a freshly opened ampoule was used in each experiment. FITC, Texas Red-CoA, biotin-CoA and actinomycin D (A1410) were purchased from Sigma-Aldrich. Cell culture media, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-EDTA, Dulbecco's phosphate-buffered saline (DPBS; 2.67 mM potassium chloride, 1.47 mM potassium phosphate monobasic, 137 mM sodium chloride, 8.06 mM sodium phosphate dibasic) were purchased from Thermo Scientific (Rockford, IL). Individual Peptide Synthesis and Labeling. Bicyclic peptides were synthesized on 50 mg of Rink amide resin LS (0.2 mmol/g) using standard Fmoc chemistry. For bicyclic peptides C1 to C74, the N-terminal Fmoc group was removed with 20% piperidine in DMF (5+15 mM) and trimesic acid was coupled onto the N-terminal amine using HBTU as the coupling agent (5 equiv trimesic acid, 5 equiv HBTU, and 10 equiv DIPEA in 2 mL of DMF). The Alloc groups on the side chains of cyclization residues (Lys and Dap) were removed by treatment with Pd(PPh3)4and phenylsilane (0.1 and 10 equiv, respectively) in anhydrous DCM (2×2 h). The peptide was cyclized by incubating the resin with a solution of PyBOP/HOBt/DIPEA (5, 5, and 10 equiv, respectively) in 2 mL of DMF for 3 h on a carousel shaker. For bicyclic peptides C1-74-1 to C1-74-6, the Mmt groups on the side chains of cyclization residues (Lys and Dap) were removed by treatment with 2% TFA in DCM (6×5 min). The peptide was cyclized as described above and the Alloc group on the side chain of dap at position-3 was removed by treatment with Pd(PPh3)4as described above. Peptide synthesis was then continued at the side chain of dap using standard Fmoc/HBTU chemistry. FITC-labeled bicyclic peptides were synthesized using Mmt-protected Lys and dap as the cyclization residues and an Alloc-protected Lys at the C-terminus. After the synthesis of the linear peptide was complete, the Mmt groups on the side chains of Lys and dap were removed by 2% TFA and the peptides were cyclized with PyBOP as described above. The Alloc group on the side chain of the C-terminal Lys was removed by treatment with Pd(PPh3)4as described above and the resulting resin (20 mg) was treated with a mixture containing 10 mg FITC and 100 μL of DIPEA in 500 μL of DMF for 2 h at room temperature. Peptide cleavage off the resin and side chain deprotection were carried out by treatment for 2 h with 3 mL of a cocktail containing 82.5:5:5:5:2.5 (v/v) TFA/thioanisole/water/phenol/ethanedithiol. After evaporation of the solvents, the crude peptide was triturated with cold ethyl ether (3×2 mL) and purified by reversed-phase HPLC on a C18 column (which was eluted with a linear gradient of 10-60% acetonitrile in doubly distilled water containing 0.05% TFA over 50 min). All peptides had ≥95% purity as judged by analytical HPLC. The authenticity of each peptide was confirmed by MALDI-TOF mass spectrometry. Protein Expression, Purification and Labeling. Recombinant TNFα containing an N-terminal 13-aa ybbR tag was expressed inEscherichia coli, purified, and specifically labeled with a biotin or Texas red at the ybbR tag as previously described (Lian, W.; et al., supra). Fluorescence Anisotropy. FITC-labeled bicyclic peptide (50 or 100 nM) were incubated with varying concentrations of TNFα (0-5 μM) in 30 mM HEPES, pH 7.4, 150 mM NaCl, and 2 mM magnesium acetate for 2 h. The FA values were measured on a Molecular Devices Spectramax M5 spectrofluorimeter, with excitation and emission wavelengths at 485 and 525 nm, respectively. Equilibrium dissociation constants (KD) were determined by plotting the FA values as a function of TNFα concentration and fitting the data to the following equation Y=(Amin+(Amax×QbQf-Amin)((L+x+Kd)-((L+x+Kd)2-4Lx)2L))(1+(QbQf-1)((L+x+Kd)-((L+x+Kd)2-4Lx)2L)) where Y is the measured anisotropy at a given TNFα concentration x; L is the bicyclic peptide concentration; Qb/Qfis the correction fact for dye-protein interaction; Amaxis the maximum anisotropy when all the peptides are bound to TNFα, while Aminis the minimum anisotropy. Competition experiments were performed by incubating 100 nM Asn-Asn-Asn-Lys(FITC)-labeled anticachexin C1 (Table 1), 2 μM of TNFα, and varying concentrations of unlabeled competitor peptide (0-20 μM) in the above buffer for 2 h. The FA values were plotted against the competitor peptide concentration to determine the IC50values. Inhibition of TNFα-TNRF1. Inhibition of TNFα-TNRF1 interaction by ELISA was carried out as previously described (Lian, W.; et al., supra). Library Synthesis. The bicyclic peptide library was synthesized on 0.3 g of TentaGel S NH2Resin (90 μm, 0.28 mmol/g), with all reactions performed at room temperature unless otherwise noted. The linker sequence (BBFRM) was first added to the resin by standard Fmoc chemistry. To spatially segregate the beads into outer and inner layers, the resin (after removal of the N-terminal Fmoc group) was washed with DMF (2×5 mL) and water (2×5 mL), and soaked in 5 mL of water overnight. The resin was quickly drained and suspended in a solution of Fmoc-OSu (0.5 eq) and DIPEA (0.5 eq) in 5 mL of 1:1 (v/v) DCM/diethyl ether. The mixture was incubated on a carousel shaker for 30 min and the beads were washed with 1:1 DCM/diethyl ether (3×5 mL) and DMF (8×5 mL). Next, allyloxycarbonyl-N-hydroxysuccinimide (Alloc-OSu, 3 equiv) and DIPEA (3 equiv) in 2 mL of 1:1 DCM/DMF was added to the resin. After 1 h, the reaction was repeated once to ensure complete reaction. The Fmoc group was removed from the surface peptides by treating with 5 mL of 20% (v/v) piperidine in DMF (5+15 min). The resin was next incubated in a mixture of Ac-Val-OH (5 equiv), Fmoc-Val-OH (0.1 equiv), HATU (5 equiv) and DIPEA (5 equiv) dissolved in 2 mL of DMF for 1 h. After removal of the N-terminal Fmoc group, properly protected Fmoc-amino acids (Scheme 2) and trimesic acid were sequentially coupled to the bead surface by standard Fmoc/HBTU chemistry, using 5 equiv of Fmoc-amino acid/trimesic acid, 5 equiv of HBTU, and 10 equiv of DIPEA in 4 mL of DMF (2 h). After the entire peptide sequence was synthesized, the Mmt groups on the side chains of cyclization residues (Lys and Dap) were removed by treatment with 5 mL of DCM containing 2% TFA for 5 min and the 2% TFA treatment was repeated five times. For peptide cyclization, the resin was incubated with a solution of PyBOP/HOBt/DIPEA (5, 5, 10 equiv, respectively) in 4 mL of DMF on a carousel shaker for 3 h. Next, the Alloc groups on the side chain of dap (position-3) and the N-terminus of inner linker sequence (BBFRM) were removed by treatment with Pd(PPh3)4and phenylsilane (0.1 and 10 equiv, respectively) in 4 mL of anhydrous DCM for 2 h (twice). The random sequence was then coupled to the exposed amines by the split-and-pool method (Lam, K. S.; et al., “A new type of synthetic peptide library for identifying ligand-binding activity.”Nature1991, 354:82-84; Houghten, R. A.; et al., “Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery.”Nature1991, 354:84-86; Furka, A.; et al., “General method for rapid synthesis of multicomponent peptide mixtures.”Int. J. Pep. Prot. Res.1991, 37:487-493). Briefly, the resin was split into 20 equal aliquots (15 mg each) and placed into 20 different reaction vessels. A different X3 building block as specified inFIG.4Bwas coupled to each aliquot by using 5 equiv of Fmoc-amino acid, 5 equiv of HATU, and 10 equiv of DIPEA in 0.5 mL of DMF (2 h). The coupling reaction was repeated once to ensure complete coupling at each step. The resin from the 20 reactors were pooled into a single vessel and treated with 20% piperidine to remove the N-terminal Fmoc group. After washing and drying, the resin was again split into 20 equal portions by weighing and a different X2 residue was coupled to each portion. This pool-and-split procedure was repeated again to couple the X1 residue. Finally, the resin was pooled and the N-terminal Fmoc group was removed by piperidine. Side-chain deprotection was carried out by incubating the resin in 10 mL of a modified reagent K [78.5:7.5:5:5:2.5:1:1 (v/v) TFA/phenol/water/thioanisole/ethanedithiol/anisole/triisopropylsilane] for 3 h. The resulting peptide library was washed with TFA (5 mL) and DCM (3×5 mL) and dried under vacuum before storage at −20° C. Library Screening. Library resin (20 mg, ˜60,000 beads) was swollen in DCM, washed exhaustively with DMF, doubly distilled H2O, and incubated in 1 mL of blocking buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 0.05% Tween 20 and 0.1% gelatin) containing 500 nM biotinylated TNFα at 4° C. overnight. After quickly washing with the blocking buffer (1 mL), the beads were suspended in 1 mL of the blocking buffer containing streptavidin-alkaline phosphatase (1 μg/mL final concentration) at 4° C. for 10 mM The beads were washed with 1 mL of the blocking buffer (3×) and 1 mL of a staining buffer (30 mM Tris, pH 8.5, 100 mM NaCl, 5 mM MgCl2, 20 μM ZnCl2) (3×). Next, 1 mL of the staining buffer and 100 μL of a BCIP stock solution (5 mg/mL) were added to the beads in a petri dish (exposed to air) and the mixture was incubated with gentle rocking. After 30 min, beads of the most intense turquoise color were manually removed with a micropipette under a dissecting microscope. After washing with DMF (3×1 mL), 8 M guanidine hydrochloride (1 mL with incubation for 30 min), ddH2O (3×1 mL), and PBS (1 mL), the beads were incubated overnight at 4° C. with 60 nM Texas red-labeled TNFα in the blocking buffer. The beads were examined under an Olympus SZX12 microscope equipped with a fluorescence illuminator (Olympus America, Center Valley, PA) and the most intensely fluorescent beads (19 beads) were manually isolated and subjected to sequencing analysis by partial Edman degradation-mass spectrometry (Thakkar, A.; et al., “Traceless capping agent for peptide sequencing by partial Edman degradation and mass spectrometry.”Anal. Chem.2006, 78:5935-5939). MTT Assay. WEHI-13VAR fibroblasts were purchased from American Type Culture Collection (ATCC). For maintenance, cells were grown in full medium as RPMI-1640 supplemented with 10% FBS and 1% Abs. For toxicity assay, cells were seeded in a 96-well plate at a density of 5×103cells/well in 100 μL of RPMI-1640/1% Abs supplemented with 10% or 5% FBS and allowed to grow overnight. Varying concentrations of recombinant human TNFα (0-250 ng/mL) or commercial mouse TNFα were (0-100 μg/mL) were incubated with 10 μM peptide and actinomycin D (1 μg/mL) in 100 μL of corresponding growth medium for 1 h at 37° C. Next, the medium in the 96-well plate was removed and replaced with the above fresh mixture and the cells were incubated overnight. Next day, 10 μL of the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT, final concentration 0.5 mg/ml) was added to each well and incubated at 37° C. for 3 h, following by the addition of 100 μL of the MTT solubilization solution. The plate was kept in the incubator overnight to let the formazan crystals to completely dissolve, and the absorbance at 570 nm was measured on a TECAN plate reader. The LD50was obtained as plotting the viability as a function of the TNFα concentration. Immunoblotting. WEHI-13VAR cells were cultured in full growth media (RPMI, 5% FBS, 1% Pen/Strep) in 12-well plates to reach 80% confluence. TNFα (5 ng/mL final concentration) and varying concentrations of peptide inhibitor (0-6 μM) were pre-incubated in 0.5 mL of the growth medium for 30 mM in the CO2incubator. The mixture was then used to treat the cells at 37° C. and in the presence of 5% CO2. After 1.5 h, the solution was removed and the cells were washed with cold DPBS twice. The cells were lysed in 50 μL of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, and 5% glycerol) containing a protease inhibitor cocktail (Complete mini, Roche, product number 04693159001) and a phosphatase inhibitor cocktail (PhosSTOP, Roche, product number 04906845001). After incubation on ice for 30 mM, the cell lysate was centrifuged at 15,000 rpm for 10 min in a microcentrifuge. An equal volume of 2×SDS-loading buffer was added to the supernatant. The total cellular proteins were separated by SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane, which was subsequently immunoblotted using anti-JNK and anti-p-JNK Thr183/Tyr185 antibodies (both from Santa Cruz Biotechnology). The same samples were also separated on a different SDS-PAGE gel and stained by Coomassie brilliant blue to check the sample loading in all lanes. To examine the effect of C1-74-2 on NF-kB translocation, HT29 colon cancer cells were cultured in DMEM supplemented with 10% FBS and 1% Abs in a 6-well plate to reach 80% confluence. The cells were then starved in serum free DMEM supplemented with 1% Abs for 24 hr. Commercial mouse TNFα (1 ng/mL final concentration), and varying concentrations of peptide (0-50 μM) were pre-incubated in 1.5 mL of serum free DMEM for 1 h at 37° C. DMSO was kept at 0.2% (vol/vol) in all tubes. The mixture was then added to cells and incubated for 30 min. The cells were harvested immediately, and for each well all fractions were collected and combined. The separation of subcellular fractions was achieved by stepwise lysis of the cell pellet. The cells were first lysed in 100 μL of hypotonic buffer (20 mM Tris pH 8.0, 4 mM MgCl2, 6 mM CaCl2, 0.5 mM DTT) and 100 μL of dounce lysis buffer (0.6 M Sucrose, 0.2% NP-40, 0.5 mM DTT) supplemented with 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail. After centrifugation at 1000 g for 5 min, the supernatant (cytoplasmic fraction) was carefully removed. The precipitates were resuspended in 100 μL of RIPA lysis buffer supplemented with 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail, and lysed by sonication for 2 sec at 50% amplitude. The mixture were kept on ice for 30 min and then centrifuged at 15,000 rpm for 10 mM, and the resulting supernatant was the nuclear fraction. The protein concentration was measured by BCA kit, and all samples were adjusted to same concentration and added with equal volume of 2×SDS-loading buffer to boil for 10 mM. Equal amount of proteins (˜30 μg for cytoplasmic fraction and 15 μg for nuclear fraction) were separated by SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane, which was immunoblotted using anti-NF-κB antibody, and Histone H3 and GAPDH were used as loading control for nuclear and cytoplasmic fraction, respectively. Luciferase Assay. NF-κB reporter (Luc)-HEK293 cells30 (BPS Bioscience, San Diego, CA) were seeded at a density of 5,000 cells per well in Greiner white 96-well cell culture microplates in 50 μL of growth medium (DMEM+10% FBS+1% P/S). Cells were incubated overnight at 37° C. in the presence of 5% CO2. TNFα (5 ng/mL) and different concentrations of compound C1-74-2 (0-6 μM) were incubated for 1 h in 5 μL of growth medium (DMEM+5% FBS+1% P/S) at 37° C. in the presence of 5% CO2. The growth medium in the plate was replaced with 50 μL of fresh medium (DMEM+5% FBS+1% P/S). The pre-incubated TNFα and peptide mixture (5 μL) was added into each well, with 5 μL of growth medium only added as the control. The cells were incubated at 37° C. with 5% CO2for 2 h. Fifty μL of ONE-Step™ Luciferase Assay reagent (BPS Bioscience, San Diego, CA) was added into each well. The whole plate was incubated at room temperature for 5 min and the luminescence generated was measured on a Tecan M1000 Pro microplate reader. Serum Stability Test. Diluted human serum (25%) was centrifuged at 15,000 rpm for 10 min, and the supernatant was collected. A peptide stock solution was diluted into the supernatant to a final concentration of 5 μM and incubated at 37° C. At various time points (0-8 h), 200-μL aliquots were withdrawn and mixed with 50 μL of 15% trichloroacetic acid and 200 μL of acetonitrile. After incubation at 4° C. overnight, the mixture was centrifuged at 15,000 rpm for 10 min in a microcentrifuge, and the supernatant was analyzed by reversed-phase HPLC equipped with an analytical C18 column (Waters). The amount of remaining intact peptide (%, relative that of time zero control) was determined by integrating the area underneath the peptide peak (monitored at 214 nm). Results Identification of Significant Binding Residues of Anticachexin C1 by Alanine Scan. To identify residues significant for TNFα binding, each residue of anticachexin C1 (other than D-alanine at position-3, L-lysine at position-4, and L-2,3-diaminopropionic acid (Dap) at position-10) was individually replaced with an alanine (or D-alanine). To facilitate binding analysis by fluorescence anisotropy (FA), a tetrapeptide NNNK (SEQ ID NO: 1) was added to the C-terminus of each bicyclic peptide and the lysine side chain was labeled with fluorescein isothiocyanate (FITC) during solid-phase synthesis (Scheme 1). The three asparagine residues were added to improve the aqueous solubility of the bicyclic peptides. The bicyclic peptide was prepared by first synthesizing the full-length linear sequence on Rink amide resin using standard Fmoc/HBTU chemistry. The side chains of the two internal cyclization residues, L-lysine and Dap, were protected with monomethoxytrityl (Mmt) groups, whereas the C-terminal lysine was protected with an allyloxycarbonyl (Alloc) group. After removal of the N-terminal Fmoc group, a trimesic acid was coupled to the N-terminal amine using HBTU as the coupling agent. Next, the Mmt groups were selectively removed from the Lys and Dap side chains by 2% TFA and the peptide was cyclized by treatment with (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP). The Alloc group on the C-terminal lysine side chain was removed by treatment with Pd(PPh3)4and the bicyclic peptide was selectively labeled at the C-terminal lysine by treating the resin with FITC. The NNNK (SEQ ID NO: 1)-tagged anticachexin C1 bound to TNFα with a KD value of 0.80 μM, similar to the previously reported value (0.45 μM) (Lian, W., et al., supra). The two tyrosine side chains (at positions 2 and 5) are most significant for TNFα binding, as their removal decreased the affinity by 14- and 12-fold, respectively (Table 1). Additionally, D-phenylalanine at position-6, D-lysine at position-8, and histidine at position-9 also contribute significantly to TNFα binding (4- to 7-fold reduction of affinity upon substitution of alanine or D-alanine). On the other hand, phenylglycine (Phg)-1 and glycine-7 play more minor roles in binding. TABLE 1Effect of Alanine Substitution on Anticachexin C1 ActivityModificationKD(μM)Fold of Affinity ReductionC10.80 ± 0.30Phg1 → Ala1.0 ± 0.31.2Tyr2 → Ala13 ± 614Tyr5 → Ala11 ± 412D-Phe6 → D-Ala3.4 ± 1.04.0Gly7 → Ala1.2 ± 0.51.5D-Lys8 → D-Ala5.9 ± 2.07.0His9 → Ala4.4 ± 1.15.0 Optimization of Position-1. The optimization process was begun from the N-terminus (position-1) because of the apparently minor role played by Phg in TNFα binding. It was thought that Phg might be replaced with a variety of other amino acids without negatively impacting the TNFα binding affinity, e.g., hydrophilic residues which would improve the aqueous solubility of the resulting compounds. Surprisingly, removal (i.e., substitution of Gly) or replacement of the phenyl ring with any hydrophilic side chain (e.g., substitution of Ser, Thr, Asn, Asp, or Arg) greatly decreased TNFα binding (by ≥5-fold) (Table 2). On the other hand, replacement of the phenyl group with other hydrophobic side chains resulted in much smaller reduction in affinity, with n-butyl (norleucine) being ˜2-fold better than the phenyl group (compare compounds C1 and C1-5). These results suggest that the side chain of residue-1 likely makes hydrophobic contacts with the TNFα surface. L-norleucine (Nle) was selected as the optimal residue for position-1 because, compared to Phg, Nle is also less susceptible to Cα epimerization during peptide synthesis. Optimization of Positions-3 and 4. Next, D-alanine was replaced at position-3 with amino acids containing small hydrophilic side chains (D-Ser, D-Thr, D-Asn, and L-Ser), to improve the aqueous solubility (Table 2, C1-11 through C1-17). These substitutions resulted in modest decreases in TNFα binding affinity, with D-Ser being least disruptive (˜2-fold reduction). Inversion of the stereochemical configuration of D-alanine (to L-Ala or L-Ser) greatly reduced the binding affinity (C1-18 and C1-19, respectively). Therefore, a D-serine was temporarily selected as the “optimal” residue at position-3 to increase the solubility of the compounds during the subsequent optimization process. Next, the effect of ring size on the TNFα binding affinity was assessed by replacing the L-lysine at position-4 with L-ornithine (Orn) or L-Dap. Reduction of the ring size resulted in progressive decrease in binding affinity (Table 2, C1-20 and C1-21). Therefore, L-lysine was retained as the optimal moiety for peptide bicyclization. TABLE 2Activity of Anticachexin C1 Analogues with Modifications at Residues 1,3, and 4CmpdR1R3n =KD(μM)CmpdR1R3n =KD(μM)C1PhgD-Ala31.4 ± 0.4C1-11NleD-Ser31.5 ± 0.4C1-1GlyD-Ala37.1 ± 4.6C1-12NleD-Asn31.4 ± 0.4C1-2AlaD-Ala31.8 ± 0.6C1-13ValD-Ser32.4 ± 1.1C1-3AbuD-Ala32.0 ± 0.3C1-14ValD-Thr32.9 ± 1.4C1-4ValD-Ala31.7 ± 0.5C1-15ValD-Asn33.2 ± 1.2C1-5NleD-Ala30.85 ± 0.08C1-16LeuD-Asn32.7 ± 0.9C1-6SerD-Ala36.2 ± 2.5C1-17PheD-Asn32.2 ± 1.1C1-7ThrD-Ala37.2 ± 4.4C1-18NleAla3>20C1-8AspD-Ala37.0 ± 3.1C1-19NleSer36.8 ± 4.2C1-9AsnD-Ala37.9 ± 3.0C1-20NleD-Ser23.5 ± 0.7C1-10ArgD-Ala3>10C1-21NleD-Ser05.9 ± 2.7 Optimization of Positions-2, 5, and 6. Because Tyr-2 is significant for TNFα binding, relatively conservative substitutions of phenylalanine, 4-fluorophenylalanine (Fpa), 4-chlorophenylalanine (Cpa), 3,4-difluorophenylalanine (F2pa), and histidine were made (Table 3, compounds C1-22 to C1-26). Phe and Fpa afforded similar binding affinity as Tyr, while the other substitutions caused significant loss of affinity. As expected, more drastic structural changes (e.g., substitution of Leu or Gln) also caused substantial reduction in binding affinity (C1-27 and C1-28). Based on these results, Tyr was kept at position-2 because of its better aqueous solubility than Phe or Fpa. Next, the moderately important D-Phe was replaced at position-6 with D-Tyr, D-His, D-Leu, or D-Gln and found that substitution of a D-His improved the binding affinity by ˜2-fold (Table 3, compounds C1-29 to C1-32). D-His was thus selected as the optimal residue at position-6, because the imidazole ring offers additional benefits of aqueous solubility and metabolic stability. To test whether the Tyr at position-5 can be further improved, it was replaced with Fpa, Cpa, Trp, or 2-naphthylalanine (Nal) (Table 3, compounds C1-33 to C1-36) and discovered that substitution of Trp increased the TNFα binding affinity by 3-fold. Various combinations of substitutions at positions 2, 5, and 6 failed to further improve the potency of the compounds (C1-37 to C1-42). Thus, compound C1-35, which features optimal residues at positions 2, 5, and 6 (Tyr, Trp, and D-His, respectively), emerged as a relatively potent TNFα inhibitor (KD=260 nM). TABLE 3Activity of Anticachexin C1 Analogues with Modifications at Residues 2,5, and 6CmpdR2R5R6KD(μM)CmpdR2R5R6KD(μM)C1-11TyrTyrD-Phe1.5 ± 0.4C1-32TyrTyrD-Gln1.3 ± 0.3C1-22PheTyrD-Phe1.7 ± 0.7C1-33TyrFpaD-His0.76 ± 0.19C1-23FpaTyrD-Phe1.2 ± 0.5C1-34TyrCpaD-His1.6 ± 0.6C1-24CpaTyrD-Phe4.8 ± 1.0C1-35TyrTrpD-His0.26 ± 0.08C1-25F2paTyrD-Phe5.4 ± 2.1C1-36TyrNalD-His1.3 ± 0.6C1-26HisTyrD-Phe2.1 ± 0.4C1-37FpaTyrD-Gln1.3 ± 0.7C1-27LeuTyrD-Phe3.7 ± 0.4C1-38FpaFpaD-His2.1 ± 0.7C1-28GlnTyrD-Phe4.3 ± 0.7C1-39FpaTyrD-His1.7 ± 0.7C1-29TyrTyrD-Tyr2.0 ± 0.4C1-40CpaTyrD-His4.1 ± 1.1C1-30TyrTyrD-His0.76 ± 0.16C1-41TrpTyrD-His0.43 ± 0.15C1-31TyrTyrD-Leu2.5 ± 0.3C1-42NalTrpD-His>10 Optimization of Positions 7-9. Substitution of sarcosine (Sar) or small L-amino acids [e.g., Ala and Ser] for Gly-7 had relatively minor effect (≤2-fold) and could either increase or decrease the TNFα binding affinity depending on whether Tyr or Trp was at position-5 (Table 1 and Table 4). Replacement with larger residues (e.g., Val and Leu), however, resulted in progressive loss of binding (by 3- to 6-fold), suggesting that a small L-amino acid at position-7 may be needed to accommodate a certain conformation of the bicycle. Either enhancing the positive charge at position 8 (by replacing the D-lysine with D-arginine) or removal of the charge (i.e., substitution of D-norleucine) almost completely abolished TNFα binding (Table 4, compounds C1-48 and C1-49). The side chain of D-lysine likely engages in a specific hydrogen bonding interaction(s) with a TNFα residue(s). Replacement of histidine at position-9 with a phenylalanine (compound C1-50) also greatly reduced the TNFα binding affinity (by ˜30-fold). The Gly-D-Lys-His motif was therefore left unchanged at positions 7-9, because it contributes greatly to TNFα binding and aqueous solubility. TABLE 4Activity of Anticachexin C1 Analogues with Modifications at Residues 7-9CmpdR5R7R8R9KD(μM)C1-30TyrGlyD-LysHis0.76 ± 0.16C1-35TrpGlyD-LysHis0.26 ± 0.08C1-43TyrSarD-LysHis0.33 ± 0.10C1-44TyrAlaD-LysHis0.32 ± 0.10C1-45TrpSerD-LysHis0.42 ± 0.13C1-46TrpValD-LysHis0.88 ± 0.22C1-47TrpLeuD-LysHis1.7 ± 0.7C1-48TyrGlyD-ArgHis>10C1-49TyrGlyD-NleHis>10C1-50TrpGlyD-LysPhe7.5 ± 2.2 Extension at C-Terminus (Position 11). To improve the potency of C1-35, the possibility of extending the bicyclic structure at the C-terminus was explored and it was envisioned that a proper exocyclic appendage might engage in additional interactions with TNFα. Initially, a hydrophilic amino acid Asp, Asn, Arg, or 8-amino-3,6-dioxaoctanoic acid (miniPEG) was inserted between the C-terminal Dap and Lys(FITC), hoping to also improve the aqueous solubility of the compounds (Table 5, compounds C1-51 to C1-54). Surprisingly, insertion of any of the amino acids greatly decreased TNFα binding (by 3- to >40-fold), with Asn being least disruptive (3-fold reduction in affinity). Addition of hydrophobic amino acids (Phe, D-Phe, Nle, or Ile) also decreased the binding affinity to various degrees (C1-55 to C1-58). The notable exception was β-alanine (β-Ala) which, when inserted at position-11 (C1-59), substantially increased the TNFα binding affinity (by ˜4-fold). It was hypothesized that the carboxamide group of β-Ala (and less effectively the free α-carboxamide of Lys(FITC) in C1-1 through C1-50) might interact with TNFα through a hydrogen bond(s). Note that in a previous peptide library, the bicyclic peptides were attached to the solid support through a β-Ala at the same position (Lian, W.; et al., supra). An Asn at this position provides a similar carboxamide group through its side chain (see Table 1 for structure). To test this notion and further improve the inhibitor potency, a series of compounds containing different β-amino acids at position-11 were synthesized and tested (Table 5, compounds C1-60 to C1-65). The results showed that, in general, β-amino acids at position-11 afford good TNFα binding activities, with the exception of 4-amino-1-methylpyrrole-3-carboxylic acid (C1-65) whose rigid planar structure might prevent the carboxamide group from hydrogen bonding with the yet unidentified residue(s) in TNFα. Since all side-chain modifications of β-Ala decreased TNFα binding, it was conclude that β-Ala is a beneficial residue at position-11 (Table 5, KD=70 nM for C1-59). TABLE 5Effect of Residue 11 on TNFα Binding AffinityCmpdR11KD(μM)CmpdR11IC50(μM)C1-35Lys(FITC)0.26 ± 0.08C1-66β-Ala0.085 ± 0.050C1-51Asp-Lys(FITC)3.5 ± 1.8C1-67β-Ala-β-Ala1.5 ± 0.9C1-52Asn-Lys(FITC)0.79 ± 0.12C1-68β-Ala-Ala2.6 ± 1.1C1-53Arg-Lys(FITC)>10C1-69β-Ala-Phe1.3 ± 1.1C1-54miniPEG-Lys(FITC)5.1 ± 1.2C1-70β-Ala-D-Phe>10C1-55Phe-miniPEG-Lys(FITC)1.0 ± 0.4C1-56D-Phe-miniPEG-0.63 ± 0.19Lys(FITC)C1-57Ile-miniPEG-Lys(FITC)0.51 ± 0.29C1-58Nle-miniPEG-Lys(FITC)1.4 ± 0.5C1-59β-Ala-miniPEG-0.07 ± 0.01Lys(FITC)C1-600.38 ± 0.14C1-610.42 ± 0.21C1-620.21 ± 0.08C1-712.4 ± 1.3C1-630.16 ± 0.15C1-720.11 ± 0.03C1-640.34 ± 0.23C1-651.9 ± 0.9C1-731.7 ± 0.7 Because of its high-throughput capability, FA analysis had been used to generate all of the SAR data described above. However, FA analysis is less reliable when the KD values become lower than the peptide ligand concentration used in the assay reactions (typically 50 nM), especially for TNFα-cyclic peptide interaction which, for yet unknown reasons, produced relatively small FA increases even when the fluorescent probe is fully bound (˜2-fold). To confirm the SAR data obtained by FA analysis, some of the compounds in Table 5 (as well as new compounds) were resynthesized without the Lys(FITC) label and employed a more sensitive ELISA-based assay to determine the IC50values for inhibition of the TNFα-TNFR1 interaction. Briefly, biotinylated TNFα was immobilized onto a NeutrAvidin-coated surface. TNFR1 conjugated with horseradish peroxidase (HRP) (0.5 nM) was added along with different concentrations of a peptide inhibitor. After washing, the amount of bound TNFR1-HRP was quantitated by ELISA. Compound C1-66, which has the same core structure as C1-59, has an IC50value of 85 nM (Table 5). In agreement with the FA results, either extension at the C-terminus of β-Ala with another amino acid (C1-67 to C1-70) or side-chain modification of β-Ala (C1-71 to C1-73) decreased TNFα binding. As expected, reversion of D-Ser at position-3 back to D-Ala slightly increased the inhibitor potency, resulting in bicyclic peptide C1-74 as a potent TNFα inhibitor (IC50=70 nM). Extension at Position-3. Synthesis and Screening of a 2nd-Generation Library. Finally, it was envisioned that “growing” C1-74 at one of its side chains might generate additional contacts with the TNFα surface and further increase the inhibitor potency. D-Ala at position 3 was selected for this purpose because the SAR data suggested that the D-Ala side chain points toward the solvent and tolerates a variety of substitutions. To identify a proper “appendage” that enhances C1-74 binding to TNFα, a second-generation bicyclic peptide library was constructed in which all library members contained the common core structure of C1-74, but different tripeptide sequences attached to the side chain of residue 3 (Scheme 2). The library was synthesized on 300 mg of 90-μm TentaGel resin (˜900,000 beads) by modifying a previously reported procedure (Lian, W.; et al., supra). Briefly, the resin was spatially segregated into outer and inner layers and the N-terminal amine of a linker sequence (BBFRM (SEQ ID NO: 4) where B is β-Ala) was differentially protected with Fmoc and Alloc groups, respectively (Liu, R.; et al., “A novel peptide-based encoding system for “one-bead one-compound” peptidomimetic and small molecule combinatorial libraries.”J. Am. Chem. Soc.2002, 124:7678-7680; Joo, S. H.; et al., “High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry.”J. Am. Chem. Soc.2006, 128:13000-13009). Following removal of the Fmoc group from the surface layer, the ligand density in the surface layer was reduced by 50-fold (to improve the stringency of library screening) by capping the surface amines with a 49:1 (mol/mol) mixture of Ac-valine and Fmoc-Val (Chen, X.; et al., “On-bead screening of combinatorial libraries: Reduction of nonspecific binding by decreasing surface ligand density.”J. Comb. Chem.2009, 11:604-611). After removal of the Fmoc group again, a linear peptide corresponding to the sequence of C1-74 was synthesized on the surface layer, except that the D-alanine at position-3 was replaced with Alloc-protected (R)-2,3-diaminopropionic acid (dap). Next, the Mmt groups were removed under mild acidic conditions from the L-lysine and C-terminal L-Dap positions and the peptide was bicyclized by using trimesic acid as the scaffold (Lian, W.; et al., supra). Finally, the Alloc groups on dap at position-3 and the N-terminus of the linker sequence in the bead interior were removed by treatment with Pd(PPh3)4and a random tripeptide sequence was coupled to the dap side chain as well as the inner linker sequence by the split-and-pool synthesis method (Lam, K. S.; et al., “Anew type of synthetic peptide library for identifying ligand-binding activity.”Nature1991, 354:82-84; Houghten, R. A.; et al., “Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery.”Nature1991, 354:84-86; Furka, A.; et al., “General method for rapid synthesis of multicomponent peptide mixtures.”Int. J. Pep. Prot. Res.1991, 37:487-493). To maximize the proteolytic stability of the library compounds, D-α-amino acids (4-iodo-D-phenylalanine, 4-cyano-D-phenylalanine, 2-fluoro-D-phenylalanine (fpa), 3,4-difluoro-D-phenylalanine (f2pa), D-Trp, D-Asp, D-Val, D-Thr, D-Pro, D-Ser, D-Leu, D-Phe, D-Ala, D-Tyr, D-Nal, D-Glu, D-Asn, D-Lys, D-Arg, D-His) were used at the two N-terminal positions (X1and X2) (FIG.4A). At the C-terminal position (X3), a structurally diverse set of unnatural amino acids (including 3-amino benzoic acid (Abz), L-β-homoisoleucine, (R)-nipecotic acid (Nip), 4-amino-1-methylpyrrole-3-carboxylic acid, γ-aminobutyric acid, β-Ala, D-homophenylalanine, D-Thr, D-Pro, D-Ser, D-Leu, D-Phe, (S)-3-amino-5-phenylpentanoic acid (apa), D-Tyr, D-Nal, D-Glu, D-Asn, D-Lys, D-Arg, and D-His) were employed (FIG.4B). The resulting library has a theoretical diversity of 8,000 and each library bead carries a unique bicyclic peptide on its surface and the corresponding linear encoding tripeptide sequence in the interior. The library was subjected to two rounds of screening against TNFα. During the first round, ˜20 mg of the library (˜60,000 beads) was incubated with 500 nM biotinylated TNFα, followed by the addition of streptavidin-alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Lam, K. S.; et al., supra). The 38 most intensely turquoise colored beads were manually isolated, washed, and subjected to a second round of screening, during which the 38 beads were incubated with Texas-red labeled TNFα (60 nM). The 19 most intensely fluorescent beads were selected and the encoding peptides in their interior were sequenced by partial Edman degradation-mass spectrometry analysis (Thakkar, A.; et al., “Traceless capping agent for peptide sequencing by partial Edman degradation and mass spectrometry.”Anal. Chem.2006, 78:5935-5939), giving six complete sequences (Table 6, compounds C1-74-1 to C1-74-6). Four representative sequences [val-his-Nip (C1-74-2,FIG.1A), f2pa-ala-phe (C1-74-3), and fpa-asn-apa (C1-74-4), and asp-asp-tyr (C1-74-5)] were individually resynthesized for further evaluation. TABLE 6Structures and Activities of Hits Identified from 2nd-Generation LibraryCmpdX1-X2-X3(R3)IC50(nM)LD50(ng/mL)C1-74None (D-Ala at70 ± 203.2 ± 1.1position-3)C1-74-1NDNDC1-74-212 ± 214 ± 6C1-74-3150 ± 432.6 ± 0.9C1-74-430 ± 152.9 ± 1.0C1-74-51350 ± 4901.9 ± 0.6C1-74-6NDND The four peptides were tested for inhibition of the TNFα-TNFR1 interaction by using the ELISA assay. Compound C1-74-2 was most potent, showing an IC50value of 12±2 nM (FIG.1B), representing a ˜6-fold improvement over C1-74 (Table 5). Compound C1-74-4 (IC50=30 nM) is also more potent than the parent inhibitor, but only by ˜2-fold. On the other hand, compounds C1-74-3 and C1-74-5 were actually less active than C1-74. These results demonstrate that extension of C1-74 at position-3 is effective for further increasing the TNFα binding affinity, but a properly appended structure is helpful for achieving such affinity enhancement. When labeled at the free N-terminal amine with 6-carboxyfluorescein and tested for binding to TNFα by FA, C-74-2 showed a KD value of 40±7 nM (FIG.5). It also competed with FITC-labeled C1 for binding to TNFα in an FA based competition assay (FIG.6A) and inhibited the binding of anti-TNFα antibody infliximab to a surface-immobilized TNFα in a concentration-dependent manner (FIG.6B). Taken together, the above observations strongly suggest that C1-74-2 binds TNFα at a site that overlaps with the TNFR-binding site. Biological Evaluation. One of the biological functions of TNFα is to induce death signaling. Inhibitors against the TNFα-TNFR interaction are expected to protect cells against TNFα induced cell death. Therefore, anticachexin C1, C1-74, and C1-74-2 through C1-74-5 were compared for their ability to protect cultured WEHI-13VAR fibroblasts, which are highly sensitive to TNFα in the presence of actinomycin-D, against TNFα-induced cell death (Khabar, K. S. A.; et al., “WEHI-13VAR: A Stable and Sensitive Variant of WEHI 164 Clone 13 Fibrosarcoma for Tumor Necrosis Factor Bioassay.”Immunol. Lett.1995, 46:107-110). The cells were treated with a fixed concentration of peptides (10 μM) and varying concentrations of TNFα (0-1 μM), and the fraction of live cells was quantitated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the absence of any peptide, TNFα showed an LD50value 1.9 ng/mL (FIG.1C). Addition of 10 μM anticachexin C1-74-2 shifted the LD50value by almost an order of magnitude, to 14 ng/mL. In comparison, C1-74, C1-74-3, C1-74-4, and C1-74-5 were much less effective (LD50values 1.9-3.2 ng/mL, Table 6). The MTT assay was also conducted at a fixed concentration of TNFα (0.4 ng/mL) but varying concentrations of peptides (0-10 μM). All of the peptides protected the cells from TNFα-induced cell death in a concentration-dependent manner C1-74-2 was again most effective, showing an EC50value of ˜50 nM and almost complete protection at ≥100 nM (FIG.1D). In comparison, C1-74 had an EC50value of ˜1 μM, whereas the parent compound (C1) showed significant protection only at ≥10 μM concentration. The effect of anticachexin C1-74-2 on other TNFα induced signaling pathways, namely the JNK and NF-κB pathways, were also assessed. WEHI-13VAR cells were treated with TNFα in the presence of increasing concentrations of C1-74-2 (0-20 μM) and the cell lysates were probed with antibodies specific for phosphorylated JNK (p-JNK) as well as total JNK. The p-JNK level, but not the total JNK protein level, decreased with the C1-74-2 concentration, demonstrating that C1-74-2 is indeed capable of inhibiting the activation of JNK signaling pathway (FIGS.2A and2B). To test the effect of C1-74-2 on the NF-κB pathway, a luciferase reporter assay, in which HEK293 cells were transfected with a luciferase gene under the control of NF-κB, was used (Takada, Y.; et al., “EvodiamineAbolishes Constitutive and Inducible NF-κB Activation by Inhibiting IκBα Kinase Activation, Thereby Suppressing NF-κB-regulated Antiapoptotic and Metastatic Gene Expression, Up-regulating Apoptosis, and Inhibiting Invasion.”J. Biol. Chem.2005, 280:17203-17212). Treatment of the NF-κB reporter (Luc)-HEK293 cells with 5 ng/mL TNFα alone caused a 17-fold increase in luciferase activity (FIG.2C). However, pre-treatment of the cells with C1-74-2 (0-6 μM) followed by 5 ng/mL TNFα significantly decreased the magnitude of the TNFα-induced luciferase activity, with ˜50% reduction of the luciferase activity at 6 μM C1-74-2. Compound C1-74-2 was tested for proteolytic stability and potential cytotoxicity. C1-74-2 is remarkably stable against proteolysis; incubation in human serum for 8 h at 37° C. resulted in only ˜15% degradation (FIG.3A). As a comparison, a linear peptide and clinical candidate, Antp-NBD (Habineza Ndikuyeze, G.; et al., “A Phase I Clinical Trial of Systemically Delivered NEMO Binding Domain Peptide in Dogs with Spontaneous Activated B-Cell like Diffuse Large B-Cell Lymphoma.”PLoS One2014, 9:e95404), was subjected to the same test. Antp-NBD showed a half-life of ˜15 min and was completely degraded within 2 h. The exceptional stability of C1-74-2 is likely due to a combination of structural rigidity of the bicyclic system and the presence of multiple D-amino acids in the sequence. This property should facilitate its potential application as an oral drug for treatment of IBD. C1-74-2 is apparently non-toxic to mammalian cells. Treatment of WEHI-13VAR cells (without TNFα) with up to 25 μM C1-74-2 for up to 72 h did not result on significant reduction in cell viability (FIG.3B). Interestingly, C1-74 and the structurally similar C1-66 (which contains a D-serine at position-3) both showed significant toxicity toward WEHI-13VAR cells at ≥1 μM concentration, likely due to off-target effects. Thus, the tripeptide appendage at position-3 not only improves the binding affinity to TNFα but also appears to block nonspecific binding to the off target(s). TNFα inhibitor C1-74-2 was tested for its inhibitory activity against murine TNFα, a necessary step before testing C1-74-2 in a mouse model of inflammatory bowel disease. As shown inFIG.7A, the addition of 10 μM C1-74-2 into the growth medium protected WEHI-13VAR fibroblasts from TNFα-induced cell death, shifting the LD50value of TNFα from 10 ng/mL (no inhibitor) to 31 ng/mL (with 10 μM C1-74-2). The magnitude of the shift was somewhat smaller than with human TNFα, suggesting that C1-74-2 binds to murine TNFα with lower affinity than human TNFα. Next, C1-74-2 was tested for its ability to block TNFα-induced NF-kB nuclear translocation in HT29 cells. As shown inFIGS.7B and7C, treatment of HT29 cells with 1 ng/mL murine TNFα resulted in a decrease in the cytoplasmic concentration of NF-kB, with a concomitant increase in the nuclear concentration. However, addition of C1-74-2 dose-dependently blocked the nuclear translocation of NF-kB, as expected from its inhibition of TNFα function. It will be appreciated that variants of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. | 165,212 |
11859020 | DETAILED DESCRIPTION Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential. As used herein, the term “peptide” refers to a polymer of amino acid residues joined by amide linkages, which may optionally be chemically modified to achieve desired characteristics. The term “amino acid residue,” includes but is not limited to amino acid residues contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include unnatural amino acids or residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, β-alanine, β-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2′-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term “pharmaceutically acceptable” is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration. As used herein, “subject” refers to an animal, such as a mammal (including a human), that has been or will be the object of treatment, observation or experiment. “Subject” and “patient” may be used interchangeably, unless otherwise indicated. Mammals include, but are not limited to, mice, rodents, rats, simians, humans, farm animals, dogs, cats, sport animals, and pets. The methods described herein may be useful in human therapy and/or veterinary applications. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. The terms “therapeutically effective amount” and “effective amount” are used interchangeably and refer to an amount of a compound that is sufficient to effect treatment as defined below, when administered to a patient (e.g., a human) in need of such treatment in one or more doses. The therapeutically effective amount will vary depending upon the patient, the disease being treated, the weight and/or age of the patient, the severity of the disease, or the manner of administration as determined by a qualified prescriber or caregiver. The term “treatment” or “treating” means administering a compound disclosed herein for the purpose of: (i) delaying the onset of a disease, that is, causing the clinical symptoms of the disease not to develop or delaying the development thereof; (ii) inhibiting the disease, that is, arresting the development of clinical symptoms; and/or (iii) relieving the disease, that is, causing the regression of clinical symptoms or the severity thereof. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described herein. Autism Spectrum Disorders One in 59 children in the U.S. are diagnosed with autism spectrum disorder (ASD). Phelan-McDermid Syndrome (PMS) is an etiologically-defined form of ASD caused by loss of function of the Shank3 gene and is estimated to account for up to 2% of ASD diagnoses. Currently, there are no disease-modifying treatments for ASD or for PMS. Diverse clinical and developmental symptoms of PMS and ASD are managed through a host of expensive interventions with varying degrees of success. Due to the immense etiological heterogeneity, development of therapeutics for ASD is extremely challenging. However, drug development for etiologically-defined subtypes of ASD, such as PMS, could be achievable, and could pave the way for treatment of other forms of autism. With the high prevalence of ASD diagnoses among the general population, and with over 80% co-occurrence with other developmental, neurologic, genetic and psychiatric diagnoses, the economic and social burden of the disease is enormous. The annual costs for children with ASD in the U.S. have been estimated to be $11.5-$60.9B (Lavelle et al., Pediatrics, (2014), 133(3):e520-529; Buescher et al., JAMA Pediatr., (2014), 168(8):721-728) Children and adolescents with ASD have median annual medical expenditures exceeding those of typically developing peers by a factor of 8.0-10.0×. Phelan-McDermid Syndrome is a rare and complex neurodevelopmental disorder characterized by global developmental delay, variable degrees of intellectual disability (ID), absent or delayed speech, ASD, epilepsy, sensory processing, attention and motor deficits, hypotonia, regression, brain abnormalities, mild dysmorphic features, feeding and gastrointestinal problems, and a range of other co-morbid clinical conditions (Drapeau et al., eNeuro, (2018), 5(3): ENEURO.0046-18.2018; Harony-Nicolas et al., J. Child. Neurol., (2015), 30(14):1861-1870; Kolevzon et al., Mol. Autism, (2014), 5(1):54). Indeed, PMS is one of the most frequent and penetrant monogenic causes of autism and ID, representing up to 2% of cases of ASD (Leblond et al., PloS Genet., (2014), 10(9):e1004580). Development of the first effective pharmacological treatment for PMS would thus have an impact for the management of PMS and, potentially, ASD. Insulin-Like Growth Factor Binding Protein (IGFBP) Insulin-like growth factors (IGFs) are key growth-promoting peptides that act as both endocrine hormones and autocrine/paracrine growth factors. In the bloodstream and in local tissues, most IGF molecules are bound by one of the members of the IGF-binding protein (IGFBP) family. IGFBPs 1-7 range in mass from ˜22 to 29 kDA (213-289 amino acid length) and share a similar structure. These binding proteins have highly conserved N- and C-domains, each of which contain internal disulfide links. (See, e.g., www.peprotech.com) Peptides In one aspect, provided herein is an isolated peptide fragment of an insulin-like growth factor binding protein (IGFBP). In another aspect, provided herein is an isolated peptide fragment of IGFBP2. In another aspect, provided herein is an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein is an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein is an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1), or an isolated fragment thereof. In another aspect, provided herein is an isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO:1, and the fragment has a length of 4-16 amino acids. In some embodiments, the fragment is cyclized. In another aspect, provided herein is an isolated peptide comprising an amino acid sequence of SEQ ID NO:1, or an isolated fragment thereof, wherein the peptide or fragment has a length of 4-18 amino acids and is cyclized. In another aspect, provided herein is an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. This includes 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 1. In another aspect, provided herein is an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In another aspect, provided herein is an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another aspect, provided herein is an isolated peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein is an isolated peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2), or an isolated fragment thereof. In another aspect, provided herein is an isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO:2, and the fragment has a length of 3-5 amino acids. In some embodiments, the fragment is cyclized. In another aspect, provided herein is an isolated peptide comprising an amino acid sequence of SEQ ID NO:2, or an isolated fragment thereof, wherein the peptide or fragment has a length of 3-7 amino acids and is cyclized. In another aspect, provided herein is an isolated peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. In another aspect, provided herein is an isolated peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. In some embodiments, the peptide disclosed herein comprises D- and L-amino acids. In some embodiments, the peptide disclosed herein comprises only L-amino acids. In some embodiments, the peptide disclosed herein is cyclized. In some embodiments, the peptide disclosed herein is not cyclized. In some embodiments, the peptide disclosed herein further comprises modifications on the N-terminus, the C-terminus, or both. For example, in one embodiment, the peptide further comprises an acyl group (such as, but not limited to, an acetyl group) on the N-terminus. In another embodiment, the peptide further comprises an amido group on the C-terminus. In some embodiments, the peptide disclosed herein includes any form of a peptide having substantial homology to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a peptide which is “substantially homologous” is about 50% homologous, more preferably about 70% homologous, even more preferably about 80% homologous, more preferably about 90% homologous, even more preferably, about 95% homologous, and even more preferably about 99% homologous to SEQ ID NO: 1 or SEQ ID NO:2. As known in the art the “similarity” between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include peptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment of interest, more preferably different from the original sequence in less than 25% of residues per segment of interest, more preferably different by less than 10% of residues per segment of interest, most preferably different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence and/or the ability to stimulate the differentiation of a stem cell into the osteoblast lineage. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)]. In some embodiments, a peptide disclosed herein is a variant comprising one or more deletions relative to a reference amino acid sequence. A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to a reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids residues or nucleotides. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C-terminal truncation of a reference polypeptide or a 5′-terminal or 3′-terminal truncation of a reference polynucleotide). In some embodiments, a peptide disclosed herein is a variant comprising a fragment of a reference amino acid sequence. A “fragment” is a portion of an amino acid sequence or a polynucleotide which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous amino acid residues of a reference peptide, respectively. Fragments may be preferentially selected from certain regions of a molecule. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide. In some embodiments, a peptide disclosed herein is a variant comprising one or more insertions or additions relative to a reference sequence. The words “insertion” and “addition” refer to changes in an amino acid or sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues. The peptides disclosed herein may be variants comprising one or more unnatural amino acids formed by post-translational modification or by introducing one or more unnatural amino acids during translation or during chemical synthesis. A variety of approaches are available for introducing unnatural amino acids during protein translation. The peptides disclosed herein may be variants comprising one or more selected from halogens, optional substitutions with C1-C3alkyl (further optionally substituted with one or more halogen or amino (NH2) groups, or a combination thereof), optional substitutions with hydroxyl groups, optional substitutions with amino (NH2) groups, and optional deletions of one or more of alkyl, hydroxyl, or amino groups. The variants comprising one or more halogens may include at least one radioactive isotopic halogen, such as 18-Fluorine. A peptide of the present invention may be synthesized by any technique known to those of skill in the art and by methods as disclosed herein. Methods for synthesizing the disclosed peptides may include chemical synthesis of proteins or peptides, the expression of peptides through standard molecular biological techniques, and/or the isolation of proteins or peptides from natural sources. The disclosed peptides thus synthesized may be subject to further chemical and/or enzymatic modification. Various methods for commercial preparations of peptides and polypeptides are known to those of skill in the art. A peptide of the present invention may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing. The peptides of the present invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes orXenopusegg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction. The peptides disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation, lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein), and benzylation (e.g., replacement of a hydrogen atom with a benzyl group). In some embodiments, proline is replaced with 2-amino-thiophene-3-carboxylate (Nadimpally et al., Chemistry Select, (2017), 3594-3596). In some embodiments, a benzene ring on select amino acid residue(s) is modified to include one or more fluorine atoms. In further embodiments, at least one of the fluorine atoms is 18-Fluorine. Compositions In some embodiments, a peptide described herein is formulated as a pharmaceutically acceptable composition when combined with at least one pharmaceutically acceptable carrier and/or excipient. Such pharmaceutically acceptable carrier(s) and/or excipient(s) are non-toxic and do not interfere with the efficacy of active ingredient (e.g., the peptides disclosed herein). The precise nature of the pharmaceutically acceptable carrier(s) and/or excipient(s) depends on the route of administration. The compositions can be formulated for any pharmaceutically acceptable route of administration, such as for example, by oral, parenteral, pulmonary, topical, rectal, nasal, vaginal administration, or via implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneally, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injections. The compositions disclosed herein may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. In pharmaceutical dosage forms, the peptide disclosed herein may be administered in the form of its pharmaceutically acceptable salt (such as, but not limited to, an acetate salt) and/or as a pharmaceutically acceptable solvate of the salt thereof or of the free base form thereof, or the peptide may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting. For oral administration, liquid or solid dose formulations may be used. Some non-limiting examples of oral dosage formulations include tablets, gelatin capsules, pills, troches, elixirs, suspensions, syrups, wafers, chewing gum and the like. The peptide can be mixed with a suitable pharmaceutical carrier (vehicle) or excipient as understood by practitioners in the art. Non-limiting examples of carriers and excipients include starch, milk, sugar, certain types of clay, gelatin, lactic acid, stearic acid or salts thereof, including magnesium or calcium stearate, talc, vegetable fats or oils, gums and glycols. For systemic, intrathecal, topical, intranasal, subcutaneous, or transdermal administration, formulations of the peptides useful in the methods of the present invention may utilize conventional diluents, carriers, or excipients etc., such as are known in the art can be employed to deliver the peptides. For example, the formulations may comprise one or more of the following: a stabilizer, a surfactant (such as a nonionic, ionic, anionic, cationic, or zwitterionic surfactant), and optionally a salt and/or a buffering agent. The peptide may be delivered in the form of a solution or in a reconstituted lyophilized form. In some embodiments, the stabilizer may, for example, be an amino acid, such as for instance, glycine or an oligosaccharide, such as for example, sucrose, tetralose, lactose or a dextran. Alternatively, the stabilizer may be a sugar alcohol, such as for instance, mannitol, sorbitol, xylitol, or a combination thereof. In some embodiments, the stabilizer or combination of stabilizers constitutes from about 0.1% to about 10% by weight of the formulation, or any percentage in between these two values. In some embodiments, the surfactant is a nonionic surfactant, such as a polysorbate. Some examples of suitable surfactants include polysorbates (e.g., Tween20, Tween80); a polyethylene glycol or a polyoxyethylene polyoxypropylene glycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10% (w/v), or any percentage in between these two values. A salt or buffering agent may be any salt or buffering agent, such as for example, sodium chloride, or sodium/potassium phosphate, respectively. In certain embodiments, the buffering agent maintains the pH of the pharmaceutical composition in the range of about 5.5 to about 7.5, or any pH in between these two values. The salt and/or buffering agent is also useful to maintain the osmolality at a level suitable for administration to a human or an animal. In some embodiments, the salt or buffering agent is present at a roughly isotonic concentration of about 150 mM to about 300 mM. The formulations of the peptides useful in the methods of the present invention may additionally comprise one or more conventional additives. Some non-limiting examples of such additives include a solubilizer such as, for example, glycerol; an antioxidant such as for example, benzalkonium chloride (a mixture of quaternary ammonium compounds, known as “quats”), benzyl alcohol, chloretone or chlorobutanol; anaesthetic agent such as for example a morphine derivative; or an isotonic agent etc., such as described above. As a further precaution against oxidation or other spoilage, the pharmaceutical compositions may be stored under nitrogen gas in vials sealed with impermeable stoppers. The amount of any individual excipient in the composition will vary depending on the role of the excipient, the dosage requirements of the active agent components, and particular needs of the composition. Generally, however, the excipient will be present in the composition in an amount of about 1% to about 99% by weight, from about 5% to about 98% by weight, or from about 15 to about 95% by weight of the excipient. In general, the amount of excipient present in a composition of the disclosure is selected from the following: at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 95% by weight. In another aspect, provided herein are formulations comprising, consisting essentially of, or consisting of a peptide disclosed herein and at least one pharmaceutically acceptable excipient for intravenous, intramuscular, subcutaneous, or intranasal administration. In some embodiments, the formulation is for intravenous administration. In some embodiments, the formulation is for intramuscular administration. In some embodiments, the formulation is for subcutaneous administration. In some embodiments, the formulation is for intranasal administration. One or more additional active agents may be administered with a peptide disclosed herein, either sequentially or concomitantly. In some embodiments, the peptide disclosed herein and the one or more additional active agents are administered within a single composition. Non-limiting examples of additional active agents include sodium chloride and carboxymethyl cellulose. In some embodiments, a peptide disclosed herein can be administered to a patient in an effective amount ranging from about 0.1 mg/kg to about 500 mg/kg per day. This includes 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg/kg. Generally, a therapeutically effective amount of a peptide disclosed herein will range from a total daily dosage of about 0.1 mg/day to 500 mg/day, about 1-25 mg/day, about 3-15 mg/day, about 3-20 mg/day, about 30-720 mg/day, about 60-600 mg/day, or about 100-480 mg/day, or more. In some embodiments, a therapeutically effective amount of a peptide disclosed herein will range from about 1-240 mg/day, about 30-240 mg/day, about 30-200 mg/day, about 30-120 mg/day, about 1-120 mg/day, about 50-150 mg/day, about 60-150 mg/day, about 60-120 mg/day, or about 60-100 mg/day, administered as either a single dosage or as multiple dosages. In some embodiments, multiple dosages include two, three, or four doses per day. In some embodiments, the therapeutically effective amount of a peptide disclosed herein is at least 0.1 mg/day, at least 0.5 mg/day, at least 1 mg/day, at least 5 mg/day, at least 10 mg/day, at least 15 mg/day, at least 20 mg/day, at least 30 mg/day, at least 40 mg/day, at least 50 mg/day, at least 60 mg/day, at least 70 mg/day, at least 80 mg/day, at least 90 mg/day, at least 100 mg/day, at least 110 mg/day, at least 120 mg/day, at least 130 mg/day, at least 140 mg/day, at least 150 mg/day, at least 160 mg/day, at least 170 mg/day, at least 180 mg/day, at least 190 mg/day, at least 200 mg/day, at least 225 mg/day, at least 250 mg/day, at least 275 mg/day, at least 300 mg/day, at least 325 mg/day, at least 350 mg/day, at least 375 mg/day, at least 400 mg/day, at least 425 mg/day, at least 450 mg/day, at least 475 mg/day, or at least 500 mg/day. Of course, the dosage may be changed according to the patient's age, weight, susceptibility, symptom, or the efficacy of the compound. The peptides and compositions disclosed herein may be used to prepare formulations and medicaments that treat depression, central nervous system disorders, or neurodevelopmental disorders. In some embodiments, the peptides and compositions disclosed herein are used to prepare formulations and medicaments that treat autism spectrum disorders. In some embodiments, the peptides and compositions disclosed herein are used to prepare formulations and medicaments that treat Phelan-McDermid Syndrome. Methods In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide or a composition disclosed herein. In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating depression in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence has at least 85% sequence identity. In another aspect, provided herein are methods of treating a central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide or a composition disclosed herein. In some embodiments, the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. In another aspect, provided herein are methods of treating a central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide or a composition disclosed herein. In some embodiments, the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, post-traumatic stress disorder (PTSD), encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. In another aspect, provided herein are methods of treating a central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating a central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating a central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another aspect, provided herein are methods of treating central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating central nervous system disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence has at least 85% sequence identity. In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide or a composition disclosed herein. In some embodiments, the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating a neurodevelopmental disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence has at least 85% sequence identity. In some embodiments, the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. In some embodiments, the motor disorders are developmental coordination disorder or stereotypic movement disorder. In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide or a composition disclosed herein. In some embodiments, the autism spectrum disorder is classical autism or Autistic Disorder. In some embodiments, the autism spectrum disorder is Asperger Syndrome. In some embodiments, the autism spectrum disorder is Childhood Disintegrative Disorder. In some embodiments, the autism spectrum disorder is Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS). In some embodiments, the autism spectrum disorder is Fragile X Syndrome. In some embodiments, the autism spectrum disorder is Rett Syndrome. In some embodiments, the autism spectrum disorder is Kanner syndrome. In some embodiments, the autism spectrum disorder is Phelan-McDermid Syndrome. In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating an autism spectrum disorder in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence has at least 85% sequence identity. In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide or a composition disclosed herein. In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of an isolated peptide having a length of 18 amino acids to 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). In another aspect, provided herein are methods of treating Phelan-McDermid Syndrome in a subject in need thereof, the methods comprising, consisting essentially of, or consisting of administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. In some embodiments, the amino acid sequence has at least 85% sequence identity. The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention. EXAMPLES Animals Adult male (2-3 month old) Sprague-Dawley (SD) rats were purchased from Harlan (USA) for the Porsolt Forced Swim Test. Rats were housed in Lucite cages with aspen wood chip bedding, maintained on a 12:12 light:dark cycle (lights on at 5 AM), and given ad libitum access to Purina lab chow (USA) and tap water throughout the study. All experiments were approved by the Sai Life Sciences (India) Animal Care and Use Committees. Example 1 Porsolt Forced Swim Test Porsolt forced swim testing was conducted as described in (Burgdorf et al., Neuropsychopharmacology, (2013), 38(5):729-742). Rats were dosed with IGFBP1-7 (1 microgram/kg i.v.; peprotech, USA) (3 mg/kg, i.v.), ketamine (10 m/kg i.v.; Sigma, USA) or 0.9% sterile saline vehicle (1 ml/kg, i.v.). Injections were made in the lateral tail vein and rats were tested 1 hr post-dosing. Animals were placed in a 46 cm tall×20 cm in diameter clear glass tube filled to 30 cm with tap water (23±1° C.) for 15 min on the first day (habituation) and 5 min on the subsequent test day. Water was changed after every other animal. Animals were videotaped, and tapes were scored offline by a blind experimenter with high inter-rater reliability (Pearson's r>0.9). Floating time (sec) was defined as the minimal amount of effort required to keep the animal's head above and diving (number of incidences) was registered when the whole body of the animal was submersed and the animal's head was facing towards the bottom of the tank. As shown inFIG.1A, IGFBP2, IGFBP3, IGFBP5, IGFBP6, and IGFBP7 produced an equivalent antidepressant-like effect in the Porsolt Test as ketamine compared to saline vehicle as measured by floating time; F(8, 54)=11.01, P<0.05; Fisher's PLSD post hoc test IGFBP2, IGFBP3, IGFBP5, or IGFBP7 vs vehicle, P<0.05; and IGFBP2, IGFBP3, IGFBP5, or IGFBP 7 vs ketamine, P>0.05. As shown inFIG.1B, IGFBP showed a greater percentage of animals that exhibited more than 1 dive per 5 min test session than vehicle, which is potentially indicative of dissociative/sedative side effects. Therefore, IGFB2, IGFBP3, and IGFBP5 show antidepressant-like effects equivalent to vehicle without dissociative/sedative side effects. The raw data is shown in Table 1. TABLE 1Rat#behaviorvehBP1BP2BP3BP4BP5BP6BP7ket1floating193.967.541.642.8175.75510.952.579.52floating140.4179.143.523.3189.58.44594.431.23floating161.2130.725.784.95927.727.29.859.34floating190.696.868.2140.564.725.735.530.878.15floating136.8187.983.6142.3172.927.736.360.139.66floating138.2156.859.829.332.414.425.624.530.57floating220.4143.445.535.8137.2104.636.6123.8551diving0000000002diving1000000103diving0001011114diving0000001105diving0000000006diving0000001007diving000000100 Example 2 Sequence Homology Mapping and Chemistry Based on the results of the Porsolt test in Example 1, IGFBP2, IGFBP3, and IGFBP5 were identified as the best binding proteins for inducing robust antidepressant-like effects without side effects. Given the structural similarities between all 7 IGFBPs, an amino acid sequence homology was performed to identify sequences that were homologues between IGFBP2, IGFBP3, and IGFBP5 and were not consistently shared with IGFBP1, IGFBP6 and IGFBP7. Based on this analysis, three peptides were identified (KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1); PKKLRP (SEQ ID NO: 2); RGD). These peptides were synthesized using standard solid phase peptide chemistry and were assessed in the Porsolt Test as described above. As shown inFIG.2, KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) (low dose), and PKKLRP (SEQ ID NO: 2) (low dose and high dose) produced an equivalent or superior antidepressant-like effect in the Porsolt test as IGFBP2 compared to saline vehicle as measured by floating time; F(7, 56)=95.3, P<0.05; Fisher's PLSD post hoc test vs vehicle, P<0.05; and vs IGFBP2, P>0.05. Diving behavior was not apparent in this experiment. The raw data is shown in Table 2. TABLE 2RatKH . . .KH . . .PK . . .PK . . .RGDRGD#behaviorvehBP254540202009901floating160.235.624.2149.126.29125.8133.62floating172.428.243.3101.518.218.4108.281.63floating15965.833.4105.815.545.6140.6143.44floating16644.353.813022.42.3139.7149.65floating133.479.127.4147.97.67.7127.8123.76floating157.137.229.2108.716.617.3115.41377floating15267.126.594.86.64.3132.91698floating178.22225.2121.410.247.8113.1132.7 Example 3 Comparison of Dendritic Spine Morphologies Between Shank3−/−, Shank3+/− Mice and Wild Type (WT) Controls Primary neuronal cultures from frontal cortices of E21 embryonic mice are generated and cultured in vitro for 28 days on poly-D-lysine (PDL)-laminin coated glass coverslips, fed with Neurobasal medium supplemented with 2% B27 Supplement, penicillin/streptomycin (100 U/mL and 100 mg/mL, respectively), and 2 mM GlutaMAX-I (see, e.g., Russell et al., Biol. Psychiatry, (2018), 83(6):499-508; Smith et al., Neuron, (2014), 84(2):399-415). Neurons are then transfected with a plasmid expressing enhanced Green Fluorescent Protein (eGFP) and after 2 days, treated with a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) suspended in media. Dose-response curves are generated for at least four concentrations of each peptide (1, 10, 100, 1000 nM), the positive control (IGFBP2, 1000 nM) or media. Treated neurons are fixed and stained with antibodies for GFP (Abcam) and anti-PSD95 (Antibodies Incorporated), followed by fluorescent Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies (Invitrogen). Images of healthy GFP-positive pyramidal neurons are captured with a Zeiss LSM5 Pascal confocal microscope using a 63× oil-immersion objective (N.A. 1.4) and are reconstructed using MetaMorph (Molecular Devices). Dendritic spine morphometric analysis (area, length, width and linear density) is performed using MetaMorph. Cultures directly compared are stained simultaneously and are imaged with the same acquisition parameters. For each condition, 3-10 neurons each from 2-5 separate experiments are used. Experiments are performed blind to conditions. Spine parameter data, such as spine size and density, is analyzed with GraphPad Prism by a one-way ANOVA, followed by Bonferroni correction for multiple comparisons. Comparisons between WT, Shank3−/− and Shank3+/− cultures are performed using a two-tailed unpaired t-test. It is expected that Shank3−/− and Shank3+/− mice will have reduced spine sizes and linear densities. It is expected that a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) administration will increase spine sizes and linear density. Example 4 Determination of the Ability of a Peptide Disclosed Herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) to Rescue in Vivo Dendritic Spine Morphology and In Vivo Auditory LTP Deficits in Shank3-Deficient Mice Adult Shank3−/−, Shank3+/− and WT control mice are dosed with either a peptide disclosed herein (1, 10, 100, 1000 pg/kg, i.v.), the positive control (IGFBP2, 1000 pg/kg, i.v.) or sterile saline vehicle via lateral tail vein using the i.v. dosing protocol in mice published in Rajagopal et al. (Behav. Brain Res., (2016), 299:105-110). Vehicle dosed WT mice are used to determine the effect of Shank3 knockout on these phenotypes. Dendritic Spine Morphology. Dendritic spine analyses are conducted as described in Burgdorf et al. (Neuroscience, (2015), 308:202-211). Twenty-four hours after dosing mice are transcardially perfused and the brains are processed for dendritic spine morphology quantification using ballistic dye labeling. Brains are sectioned using a tissue Vibratome (Leica VT1000) to collect 300 μm thick sections from the anterior to posterior extremes of each brain. Ballistic dye labeling (DiI and DiO coated on tungsten particles) are performed using a gene gun (Bio-Rad) to label target neurons. Laser-scanning confocal microscopy (Olympus FV1000) is performed using a 63× objective (1.42 NA). Microscopy is performed blind to experimental conditions. A minimum of 5 cells per animal is sampled. Medial prefrontal cortex (MPFC) samples (50 μm) are analyzed. Blind deconvolution (AutoQuant) is applied to raw 3-dimensional digital images. Individual spines are measured manually for head diameter, spine length, and spine neck diameter from image Z-stacks using software. An optimal dose for a particular peptide disclosed herein may be obtained through these studies and that dose used in the auditory LTP study described below. Secondary endpoints (non-tuff dendrites in pyramidal, as well as proximal and distal dentate in the dentate gyms) may also be used to determine the optimal dose. Auditory LTP. Noninvasive methods for measuring synaptic plasticity via LTP in mice. Mice are anesthetized using isoflurane, and cortical EEG is implanted via skull screws (Pinnacle). Auditory evoked potentials are recorded from a frontal cortex skull screw using a cerebellar skull screw as a ground. EEG signals are captured via a tethered system (Pinnacle); auditory evoked potentials are recorded from frontal cortex skull screws using a cerebellum skull screw as a ground/reference. Data is acquired at 1,000 samples per second using an A&M amplifier with a high (0.1 Hz) and low pass (100 Hz) filters. Data is recorded using Data Wave acquisition software and is analyzed using Brain Products Analyzer 2 software. LTP is induced by an auditory tetanus (6-kHz, 50 ms in duration), presented 10 times per second for 5 min (total of 3,000 tones) using a similar paradigm as Clapp et al. (Eur. J. Neurosci., (2005), 22(5):1135-1140). Mismatch negativity testing occurs immediately before tetanus (pre-tetanus) and 1 hr after tetanus (post-tetanus) following a specific protocol. Post-pre tetanus difference waves are generated to determine the range (in milliseconds) in which LTP occurred. Secondary endpoints include mismatch negativity and quantitative EEG. Data is analyzed using an ANOVA with each experimental group entered as an independent factor with Fisher PLSD post-hoc tests. α=0.05. The optimal dose of each peptide is to be defined as the lowest dose that (1) shows a significant effect compared to vehicle, (2) is not statistically inferior to higher doses, and (3) shows at least 85% of the maximal effect of the most effective dose. A post hoc test comparing the peptide(s) and vehicle is used to determine if the peptide(s) facilitated auditory LTP. It is expected that the peptides disclosed herein will fully restore or at least significantly enhance spine density and auditory LTP in mice. The approach described above can be applied identically in rodent and human studies, as the same noninvasive auditory LTP method can be used in humans in future studies. Treatment regimens that reverse spine deficits and LTP in mice are expected to lead to behavioral improvements, as determined in Example 5 below. Example 5 Determination of the Ability of Peptides Disclosed Herein to Rescue In Vivo Learning, Memory and Vocalization Quality Measures in Shank3-Deficient Mice This study measures novel object recognition (NOR) for memory, as well as home cage ultrasonic vocalizations (USVs) for speech in Shank3-deficient mice. Adult Shank3−/−, Shank3+/− and WT control mice are dosed with a peptide disclosed herein (optimal dose determined in Example 3), the positive control (IGFBP2, 1000 pg/kg, i.v.) or sterile saline vehicle via lateral tail vein using the i.v. dosing protocol in mice described in Rajagopal et al. (Behav. Brain Res., (2016), 299:105-110). Methods to measure NOR are described Rajagopal et al. (Behav. Brain Res., (2016), 299:105-110). The primary endpoint is the D2 discrimination index. Methods for ultrasonic vocalization recording are described in Srivastava et al. (J. Neurosci., (2012), 32(34):11864-11878). Heterospecific rough-and-tumble play is conducted, and testing occurs 3 hours post-dosing or 1 day after the last rough-and-tumble play session. The experimenter is blind to the treatment condition. High-frequency USVs are recorded and analyzed by sonogram in a blind manner as described in Burgdorf et al. (Neuroscience, (2011), 192:515-523). Animals are not habituated to play stimulation before dosing and testing. Rate of USVs, spectrographic properties of USVs, and social contact time are measured and the primary endpoint is rates of ultrasonic calls. An increase in 50-kHz USVs that occurs across trial blocks reflects positive emotional learning. Secondary endpoint measures of social contact time and sonographic features of USVs include loudness, peak frequency, and bandwidth. Data is analyzed using an ANOVA with each experimental group entered as an independent factor with Fisher PLSD post-hoc tests α=0.05. A post hoc test comparing the peptide(s) and vehicle is used to determine if the peptide(s) affected the primary endpoints for the NOR or USV experiments. It is expected that peptides disclosed herein will rescue or enhance at least one of these phenotypes. Alternatively, behavioral learning was tested in trace eyeblink conditioning, Morris water maze, and/or alternating t-maze tasks as described in Burgdorf et al. (Neurobiol. Aging, (2011), 32(4):698-706). It is expected that peptides disclosed herein will enhance learning in all three tasks. Example 6 Assessment of Sleep Slow Wave Activity (SWA) Parameters and Central Brain-Derived Neurotrophic Factor (BDNF) in Human Adult Subjects with Major Depressive Disorder (MDD) Sleep SWA parameters and BDNF may serve as non-invasive indices for testing the efficacy of antidepressant therapy. Following an adaptation night, whole night sleep recordings are obtained for adult subjects (diagnosed with MDD without psychotic features) on the night before compound infusion as well as on the two following nights. The adult subjects are each administered a single intravenous infusion of compound (a peptide disclosed herein, e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)). Electroencephalogram (EEG) recordings are performed approximately 12 hours after compound infusion. Two EEGs (C3/A2 and C4/A1), two electrooculograms and one submental electromyogram are recorded. Slow wave parameters are calculated by applying a procedure adopting fixed parameters derived from sleep EEG standard guidelines (Reidner et al.,Sleep,2007, 1643-1657). BDNF is collected using a vacutainer system before compound infusion as well as 230 minutes after compound infusion. These blood samples are analyzed using an anti-BDNF sandwich ELISA kit. Example 7 Assessment of Experience-Dependent Neuroplasticity in Healthy Human Adult Subjects In a randomized, double-blind study, healthy adult subjects receive a single dose of (a) a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) or (b) placebo. EEG testing begins 3 hours after compound or placebo administration, followed by cognitive testing. To explore potential delayed effects of compound on memory consolidation, adult subjects return to testing site to repeat the cognitive tasks. Adult subjects complete a visual long term potentiation (LTP) task using high-frequency visual stimulation (HFvS) to induce potentiation of visual cortex neurons, followed by a weather prediction task (WPT), an information integration task (IIT), and an n-back task (e.g., a spatial working memory task). Example 8 Assessment in Rat Models of Post-Traumatic Stress Disorder (PTSD) As shown inFIGS.3A-3D, PKKLRP (SEQ ID NO: 2) increased positive emotional learning 1 hr post dosing in rats as measured by rates of hedonic ultrasonic vocalizations that occur during heterospecific play, which captures both pro-social and vocal learning relevant to autism [F(1, 21)=14.9, P<0.05; Fisher's PLSD post hoc test for 0.01, 0.1, 1, 10, and 30 mg/kg vs. vehicle, P<0.05;FIG.3A)]. Center crosses, an index of an anxiolytic drug effect relevant to PTSD, were also increased across these same dose levels in the same assay [F(1, 21)=13.8, P<0.05; Fisher's PLSD post hoc test for 0.01, 0.1, 1, 10, and 30 mg/kg vs. vehicle, P<0.05;FIG.3B)]. PKKLRP (SEQ ID NO: 2) at 1 mg/kg IV one hour before the first extinction session also increased contextual fear extinction (a well validated model of PTSD) across each test day [F(1, 10)=32.3, P<0.05; Fisher's PLSD post hoc test for session 1, 2, 3, 4, 5, and 6 for drug vs. vehicle, P<0.05;FIG.3C)], as well as spontaneous recovery comparing the difference in freezing 14 days post dosing versus extinction session 6 [F(1, 10)=32.3, P<0.05;FIG.3D]. These experiments were conducted as described in Burgdorf et al. (Int. J. Neuropsychopharmacol., 2017, 20:476-484). Example 9 Human Clinical Trial for Phelan-McDermid Syndrome (PMS) In a double-blind, placebo-controlled, crossover design trial, human subjects (confirmed to have SHANK3 deletions or mutations based on chromosomal microarray or high-throughput or targeted sequencing) are intravenously administered a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) over a period of three months and placebo over a period of three months in random order, separated by a 4-week washout period. Efficacy measurements are taken at baseline of each treatment phase, and at weeks 4, 8, and 12 of each treatment phase. Social impairment and restrictive behaviors were measured by the Aberrant Behavior Checklist and the Repetitive Behavior Scale, respectively. Example 10 Human Clinical Trial for Obsessive-Compulsive Disorder (OCD) In a randomized, double-blind, placebo-controlled, crossover design trial, drug-free OCD adult subjects with near-constant obsessions receive two intravenous infusions, one of saline and a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)), spaced at least 1-week apart. The OCD visual analog scale (OCD-VAS) and the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) are used to assess OCD symptoms. Example 11 Human Clinical Trial for Chronic Post-Traumatic Stress Disorder (PTSD) In a randomized, double-blind crossover design trial with an active placebo control, adult subjects (free of concomitant psychotropic medications for 2 weeks prior to randomization for the duration of the study) receive two intravenous infusions of (a) a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) or (b) midazolam, spaced two weeks apart. Eligible adult subjects have a primary diagnosis of PTSD assessed with the Structured Clinical Interview for DSM-IV-TR Axis I Disorders-Patient Version and a score of at least 50 on the Clinician-Administered PTSD Scale (CAPS). The primary outcome is PTSD symptom severity 24 hours after infusion, assessed with the Impact of Event Scale-Revised (IES-R). Secondary outcome measures include the Montgomery-Asberg Depression Rating Scale (MADRS), the Quick Inventory of Depressive Symptomology, Self-Report (QIDS-SR), and the Clinical Global Impression-Severity (CGI-S) and -Improvement (GCI-I) scales administered by a study clinician 24 hours, 48 hours, 72 hours, and 7 days after infusion. The IES-R is also administered 48 hours, 72 hours, and 7 days after infusion. The CAPS is administered at baseline and 7 days after infusion. Patients who score 50 or higher on the CAPS 2 weeks after the first infusion receive an infusion of the second study drug. Patients whose symptoms remain significantly improved 2 weeks after infusion (indicated by a CAPS score of <50 at 2 weeks) are considered to have completed the study after 1 infusion. Example 12 Human Clinical Trial for Treatment-Resistant Major Depression In a randomized, placebo-controlled, double-blind crossover design trial, adult subjects (drug-free for two weeks prior to the study) receive an intravenous infusion of (a) a peptide disclosed herein (e.g., KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1) or PKKLRP (SEQ ID NO: 2)) or (b) placebo on two test days, a week apart. Adult subjects are rated at baseline and at 40, 80, 110, and 230 minutes and 1, 2, 3, and 7 days post-infusion. Rating scales include the 21-item Hamilton Depression Rating Scale (HDRS) as the primary outcome measure, and the secondary outcome measures: The Beck Depression Inventory (BDI), Brief Psychiatric Rating Scale (BPRS) positive symptoms subscale, Young Mania Rating Scale (YMRS), and the visual analog scale. While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made therein in accordance with ordinary skill in the art without departing from the present invention in its broader aspects as defined in the following claims. The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Additionally, the phrase “consisting essentially of” will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed invention. The phrase “consisting of” excludes any element not specified. The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, or compositions, which can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure. Para. A. An isolated peptide having a length of 18 amino acids to 40 amino acids, comprising an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1). Para. B. An isolated peptide consisting of an amino acid sequence of KHGLYNLKQCKMSLNGQ (SEQ ID NO: 1), or an isolated fragment of the peptide. Para. C. The peptide of Para. B, or the fragment thereof, wherein the fragment has a length of 4-16 amino acids. Para. D. An isolated peptide having a length of 18 amino acids to 40 amino acids, comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1. Para. E. The peptide of Para. D comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1. Para. F. The peptide of Para. D comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1. Para. G. The peptide of any one of Paras. A-F, further comprising N-terminal carboxylation, C-terminal amidation, one or more halogens, or a combination thereof. Para. H. The peptide of any one of Paras. A-G, wherein the peptide is cyclized. Para. I. An isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO:1, and the fragment has a length of 4-16 amino acids. Para. J. The fragment of Para. I, further comprising N-terminal carboxylation, C-terminal amidation, one or more halogens, or a combination thereof. Para. K. The fragment of Para. I or Para. J, wherein the fragment is cyclized Para. L. A pharmaceutical composition comprising a peptide of any one of Paras. A-H or a fragment of any one of Paras. I-K and at least one pharmaceutically acceptable excipient. Para. M. A method of treating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. A-H or a fragment of any one of Paras. I-K or a composition of Para. L. Para. N. A method of treating post-traumatic stress disorder (PTSD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. A-H or a fragment of any one of Paras. I-K or a composition of Para. L. Para. O. A method of treating a central nervous system disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. A-H or a fragment of any one of Paras. I-K or a composition of Para. L. Para. P. The method of Para. O, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. Q. The method of Para. O, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, post-traumatic stress disorder (PTSD), encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. R. A method of treating a neurodevelopmental disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. A-H or a fragment of any one of Paras. I-K or a composition of Para. L. Para. S. The method of Para. R, wherein the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. Para. T. The method of Para. S, wherein the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. Para. U. The method of Para. S, wherein the motor disorders are developmental coordination disorder or stereotypic movement disorder. Para. V. A method of treating Phelan-McDermid Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. A-H or a fragment of any one of Paras. I-K or a composition of Para. L. Para. W. A method of treating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. X. A method of treating a central nervous system disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. Y. The method of Para. X, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. Z. A method of treating a neurodevelopmental disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. AA. The method of Para. Z, wherein the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. Para. AB. The method of Para. AA, wherein the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. Para. AC. The method of Para. AA, wherein the motor disorders are developmental coordination disorder or stereotypic movement disorder. Para. AD. A method of treating Phelan-McDermid Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. AE. A method of treating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. AF. A method of treating a central nervous system disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. AG. The method of Para. AF, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. AH. A method of treating a neurodevelopmental disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. AI. The method of Para. AH, wherein the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. Para. AJ. The method of Para. AI, wherein the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. Para. AK. The method of Para. AI, wherein the motor disorders are developmental coordination disorder or stereotypic movement disorder. Para. AL. A method of treating Phelan-McDermid Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. AM. A method of treating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. Para. AN. The method of Para. AM, wherein the peptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. Para. AO. A method of treating a central nervous system disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. Para. AP. The method of Para. AO, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. AQ. A method of treating a neurodevelopmental disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. Para. AR. The method of Para. AQ, wherein the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. Para. AS. The method of Para. AR, wherein the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. Para. AT. The method of Para. AR, wherein the motor disorders are developmental coordination disorder or stereotypic movement disorder. Para. AU. A method of treating Phelan-McDermid Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. Para. AV. The method of any one of Paras. W-AU, wherein the peptide further comprises N-terminal carboxylation, C-terminal amidation, one or more halogens, or a combination thereof. Para. AW. The method of any one of Paras. W-AV, wherein the peptide is cyclized. Para. AX. A method of treating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO: 2, and the fragment has a length of 3-5 amino acids. Para. AY. A method of treating a central nervous system disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount an isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO: 2, and the fragment has a length of 3-5 amino acids. Para. AZ. The method of Para. AY, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. BA. A method of treating a neurodevelopmental disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO: 2, and the fragment has a length of 3-5 amino acids. Para. BB. The method of Para. BA, wherein the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. Para. BC. The method of Para. BB, wherein the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. Para. BD. The method of Para. BB, wherein the motor disorders are developmental coordination disorder or stereotypic movement disorder. Para. BE. A method of treating Phelan-McDermid Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO: 2, and the fragment has a length of 3-5 amino acids. Para. BF. The method of any one of Paras. AX-BE, wherein the fragment further comprises N-terminal carboxylation, C-terminal amidation, one or more halogens, or a combination thereof. Para. BG. The method of any one of Paras. AX-BF, wherein the fragment is cyclized. Para. BH. A isolated peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. BI. An isolated peptide consisting of an amino acid sequence of PKKLRP (SEQ ID NO: 2). Para. BJ. An isolated peptide having a length of 7 amino acids to 20 amino acids and comprising an amino acid sequence having at least 65% sequence identity to SEQ ID NO: 2. Para. BK. The peptide of Para. BJ comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2. Para. BL. The peptide of any one of Paras. BH-BK, further comprising N-terminal carboxylation, C-terminal amidation, one or more halogens, or a combination thereof. Para. BM. The peptide of any one of Paras. BH-BL, wherein the peptide is cyclized. Para. BN. An isolated fragment of a peptide, wherein the peptide consists of an amino acid sequence of SEQ ID NO: 2, and the fragment has a length of 3-5 amino acids. Para. BO. The fragment of Para. BN, further comprising N-terminal carboxylation, C-terminal amidation, one or more halogens, or a combination thereof. Para. BP. The fragment of Para. BN or Para. BO, wherein the peptide is cyclized. Para. BQ. A pharmaceutical composition comprising a peptide of any one of Paras. BH-BM or a fragment of any one of Paras. BN-BP and at least one pharmaceutically acceptable excipient. Para. BR. A method of treating depression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. BH-BM or a fragment of any one of Paras. BN-BP or a composition of Para. BQ. Para. BS. A method of treating post-traumatic stress disorder (PTSD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. BH-BM or a fragment of any one of Paras. BN-BP or a composition of Para. BQ. Para. BT. A method of treating a central nervous system disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. BH-BM or a fragment of any one of Paras. BN-BP or a composition of Para. BQ. Para. BU. The method of Para. BT, wherein the central nervous system disorder is selected from autism spectrum disorders, bipolar disorder, catalepsy, depression, post-traumatic stress disorder (PTSD), encephalitis, epilepsy/seizures, locked-in syndrome, meningitis, migraine, multiple sclerosis, myelopathy, neurodegenerative disorders, schizophrenia, obsessive-compulsive disorder, and tic disorders, or any combination thereof. Para. BV. A method of treating a neurodevelopmental disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. BH-BM or a fragment of any one of Paras. BN-BP or a composition of Para. BQ. Para. BW. The method of Para. BV, wherein the neurodevelopmental disorder is selected from intellectual disability, autism spectrum disorders, motor disorders, tic disorders, traumatic brain injury, Down syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadal syndromes, fetal alcohol spectrum disorder, and Minamata disease caused by mercury, or any combination thereof. Para. BX. The method of Para. BW, wherein the autism spectrum disorders are classical autism or Autistic Disorder; Asperger Syndrome; Childhood Disintegrative Disorder; Pervasive Developmental Disorder—Not Otherwise Specified (PDD-NOS); Fragile X Syndrome; Rett Syndrome; Kanner syndrome; or Phelan-McDermid Syndrome. Para. BY. The method of Para. BW, wherein the motor disorders are developmental coordination disorder or stereotypic movement disorder. Para. BZ. A method of treating Phelan-McDermid Syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of any one of Paras. BH-BM or a fragment of any one of Paras. BN-BP or a composition of Para. BQ. Other embodiments are set forth in the following claims. | 92,598 |
11859021 | DEFINITIONS For convenience, certain terms employed in the specification, examples and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Throughout this disclosure, various patents, patent applications and publications are referenced. The disclosures of these patents, patent applications and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. This disclosure will govern in the instance that there is any inconsistency between the patents, patent applications and publications cited and this disclosure. The term “about” when immediately preceding a numerical value means a range of plus or minus an acceptable degree of variation in the art. In embodiments, the term “about” encompasses 10% of that value, e.g., “about 50” means 45 to 55, “about 25,000” means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as “about 49, about 50, about 55, . . . ”, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. The phrase “pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. “Salts” include those obtained by reacting a compound functioning as a base, with an inorganic or organic acid to form a salt, or those obtained by reacting a compound functioning as an acid, with an inorganic or organic base to form a salt. “Salts” include derivatives of an active agent, wherein the active agent is modified by making acid or base addition salts thereof. Preferably, the salts are pharmaceutically acceptable salts. Such salts include, but are not limited to, pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts. Acid addition salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, sulfuric, nitric acids and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic, citric, fumaric, glycolic, lactic, maleic, malic, malonic, mandelic, oxalic, picric, pyruvic, salicylic, succinic, methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic acid, tartaric, ascorbic, pamoic, bismethylene salicylic, ethanedisulfonic, gluconic, citraconic, aspartic, stearic, palmitic, EDTA, glycolic, p-aminobenzoic, glutamic, benzenesulfonic, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acids, sulphates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, ketoglutarates and the like. Base addition salts include but are not limited to, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris-(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine dicyclohexylamine and the like. Examples of metal salts include lithium, sodium, potassium, magnesium, calcium salts and the like. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts and the like. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline and the like. Standard methods for the preparation of pharmaceutically acceptable salts and their formulations are well known in the art, and are disclosed in various references, including for example, “Remington: The Science and Practice of Pharmacy”, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, PA. The term “carrier” or “vehicle” as used interchangeably herein encompasses carriers, excipients, adjuvants, and diluents or a combination of any of the foregoing, meaning a material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ or portion of the body. In addition to the adjuvants, excipients and diluents known to one skilled in the art, the carrier includes nanoparticles of organic and inorganic nature. For example, in embodiments the present disclosure provides nanoparticle carriers (e.g., HDL-derived nanoparticles) as delivery vehicles for an active agent (e.g., a compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or Table 1). In embodiments, the agent is encapsulated within the nanoparticle carrier. In other embodiments, the agent is bound to the surface of the nanoparticle carrier. The association of the agent and the nanoparticle carrier may be effected by a variety of means, including noncovalent bonding, and trapping the agent in the interior of the delivery vehicle and the like. In embodiments, the association is sufficiently stable so that agent remains associated with the delivery vehicle until it is delivered to the target site in the treated subject. The terms “pharmaceutical combination,” “therapeutic combination” or “combination” as used herein, refers to a single dosage form comprising at least two therapeutically active agents, or separate dosage forms comprising at least two therapeutically active agents together or separately for use in combination therapy. Administration of a combination therapy includes: administration in the same or different composition(s) and/or combinations, either sequentially, simultaneously, or continuously, through the same or different routes. For example, one therapeutically active agent may be formulated into one dosage form and the other therapeutically active agent may be formulated into a single or different dosage forms. For example, one therapeutically active agent may be formulated into a solid oral dosage form whereas the second therapeutically active agent may be formulated into a solution dosage form for parenteral administration. In embodiments, the combination therapy optionally includes one or more pharmaceutically acceptable carriers or excipients, non-pharmaceutically active compounds, and/or inert substances. As used herein, the phrase “a disorder characterized by cell proliferation” or “a condition characterized by cell proliferation” include, but are not limited to, cancer, benign and malignant tumors. Examples of cancer and tumors include, but are not limited to, cancers or tumor growth of the bladder, blood vessels, bone, brain, breast, cervix, chest, colon, endometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovary, pancreas, prostate, rectum, colorectum, skin, stomach, testicles, throat, thyroid, urothelium, and uterus. The terms “treat”, “treating” or “treatment” in reference to a particular disease or disorder includes prevention of the disease or disorder, and/or lessening, improving, ameliorating or abrogating the symptoms and/or pathology of the disease or disorder. Generally the terms as used herein refer to ameliorating, alleviating, lessening, and removing symptoms of a disease or condition. A candidate compound described herein may be in a therapeutically effective amount in a formulation or medicament, which is an amount that can lead to a biological effect, such as apoptosis of certain cells (e.g., cancer cells), reduction of proliferation of certain cells, or lead to ameliorating, alleviating, lessening, or removing symptoms of a disease or condition, for example sepsis. The terms also can refer to reducing or stopping a cell proliferation rate (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor). The term “patient” or “subject” as used herein, includes all mammals and more particularly includes humans. The methods described herein may be useful for both human therapy and veterinary applications. In one embodiment, the subject is a human. As used herein, “prevention” or “preventing” refers to a reduction of the risk of acquiring a given disease or disorder. For example, causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease. As used herein, “therapeutically effective amount” means the amount of a compound or a therapeutically active agent that, when administered to a subject for treating a disease or other undesirable medical condition, is sufficient to have a beneficial effect with respect to that disease or condition. The therapeutically effective amount will vary depending on the type of the selected compound or a therapeutically active agent, the disease or condition and its severity, and the age, weight, etc. of the patient to be treated. By “optional” or “optionally” it is meant that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which is does not. For example, “optionally substituted aryl” encompasses both “aryl” and “substituted aryl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents, that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and/or inherently unstable. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation. When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example, “C1-C6alkyl” is intended to encompass C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6alkyl. The term “acyl” as used herein refers to R—C(O)— groups such as, but not limited to, (alkyl)-C(O)—, (alkenyl)-C(O)—, (alkynyl)-C(O)—, (aryl)-C(O)—, (cycloalkyl)-C(O), (heteroaryl)-C(O)—, and (heterocyclyl)-C(O)—, wherein the group is attached to the parent molecular structure through the carbonyl functionality. In embodiments, it is a C1-10acyl radical which refers to the total number of chain or ring atoms of the, for example, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, or heteroaryl, portion plus the carbonyl carbon of acyl. For example, a C4-acyl has three other ring or chain atoms plus carbonyl. “Alkyl” or “alkyl group” refers to a fully saturated, straight or branched hydrocarbon chain. In embodiments, an alkyl group contains from one to thirty carbon atoms. In embodiments, an alkyl groups has from one to twelve carbon atoms, and which is attached to the rest of the molecule by a single bond. For example, alkyls comprising any number of carbon atoms from 1 to 12 are included. An alkyl comprising up to 12 carbon atoms is a C1-C12alkyl, an alkyl comprising up to 10 carbon atoms is a C1-C10alkyl, an alkyl comprising up to 6 carbon atoms is a C1-C6alkyl and an alkyl comprising up to 5 carbon atoms is a C1-C5alkyl. A C1-C5alkyl includes C5alkyls, C4alkyls, C3alkyls, C2alkyls and C1alkyl (i.e., methyl). A C1-C6alkyl includes all moieties described above for C1-C5alkyls but also includes C6alkyls. A C1-C10alkyl includes all moieties described above for C1-C5alkyls and C1-C6alkyls, but also includes C7, C8, C9and C10alkyls. Similarly, a C1-C12alkyl includes all the foregoing moieties, but also includes C11and C12alkyls. Non-limiting examples of C1-C12alkyl include methyl, ethyl, n-propyl, i-propyl, sec-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, n-pentyl, t-amyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted. In embodiments, “alkyl” is a straight-chain hydrocarbon. In embodiments, “alkyl” is a branched hydrocarbon. “Alkylene” or “alkylene chain” refers to a fully saturated, straight or branched divalent hydrocarbon chain. In embodiments, an alkylene groups has from one to twelve carbon atoms. Non-limiting examples of C1-C12alkylene include methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain can be optionally substituted. “Alkenyl” or “alkenyl group” refers to a straight or branched hydrocarbon chain. In embodiments, an alkenyl group contains from one to thirty carbon atoms. In embodiments, an alkenyl group contains from two to twelve carbon atoms, and having one or more carbon-carbon double bonds, such as a straight or branched group of 2-8 carbon atoms, referred to herein as C2-C8alkenyl. Each alkenyl group is attached to the rest of the molecule by a single bond. Alkenyl group comprising any number of carbon atoms from 2 to 12 are included. An alkenyl group comprising up to 12 carbon atoms is a C2-C12alkenyl, an alkenyl comprising up to 10 carbon atoms is a C2-C10alkenyl, an alkenyl group comprising up to 6 carbon atoms is a C2-C6alkenyl and an alkenyl comprising up to 5 carbon atoms is a C2-C5alkenyl. A C2-C5alkenyl includes C5alkenyls, C4alkenyls, C3alkenyls, and C2alkenyls. A C2-C6alkenyl includes all moieties described above for C2-C5alkenyls but also includes C6alkenyls. A C2-C10alkenyl includes all moieties described above for C2-C5alkenyls and C2-C6alkenyls, but also includes C7, C8, C9and C10alkenyls. Similarly, a C2-C12alkenyl includes all the foregoing moieties, but also includes C11and C12alkenyls. Non-limiting examples of C2-C12alkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, and 11-dodecenyl. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted. “Alkynyl” or “alkynyl group” refers to a straight or branched hydrocarbon chain. In embodiments, an alkynyl group contains from one to thirty carbon atoms. In embodiments, an alkynyl group contains from two to twelve carbon atoms, and having one or more carbon-carbon triple bonds such as a straight or branched group of 2-8 carbon atoms, referred to herein as C2-C8alkynyl. Each alkynyl group is attached to the rest of the molecule by a single bond. Alkynyl group comprising any number of carbon atoms from 2 to 12 are included. An alkynyl group comprising up to 12 carbon atoms is a C2-C12alkynyl, an alkynyl comprising up to 10 carbon atoms is a C2-C10alkynyl, an alkynyl group comprising up to 6 carbon atoms is a C2-C6alkynyl and an alkynyl comprising up to 5 carbon atoms is a C2-C5alkynyl. A C2-C5alkynyl includes C5alkynyls, C4alkynyls, C3alkynyls, and C2alkynyls. A C2-C6alkynyl includes all moieties described above for C2-C5alkynyls but also includes C6alkynyls. A C2-C10alkynyl includes all moieties described above for C2-C5alkynyls and C2-C6alkynyls, but also includes C7, C8, C9and C10alkynyls. Similarly, a C2-C12alkynyl includes all the foregoing moieties, but also includes Cu and C12alkynyls. Non-limiting examples of C2-C12alkenyl include ethynyl, propynyl, butynyl, pentynyl and the like. Unless stated otherwise specifically in the specification, an alkyl group can be optionally substituted. “Aryl” refers to a hydrocarbon ring system comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring, which is attached to the rest molecule by a single bond. For purposes of this invention, the aryl can be a monocyclic, bicyclic, tricyclic, tetracyclic ring system or other multicyclic ring system, which can include fused or bridged ring systems. Aryls include, but are not limited to, aryls derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless stated otherwise specifically in the specification, the aryl can be optionally substituted. “Aralkyl” or “arylalkyl” refers to a group of the formula —Rb—Rcwhere Rbis an alkylene group as defined above and R, is one or more aryls as defined above, for example, benzyl, diphenylmethyl and the like. Unless stated otherwise specifically in the specification, an aralkyl group can be optionally substituted. “Carbocyclyl,” “carbocyclic ring” or “carbocycle” refers to a rings structure, wherein the atoms which form the ring are each carbon, and which is attached to the rest of the molecule by a single bond. Carbocyclic rings can comprise from 3 to 20 carbon atoms in the ring. Carbocyclic rings include aryls and cycloalkyl, cycloalkenyl and cycloalkynyl as defined herein. Unless stated otherwise specifically in the specification, a carbocyclyl group can be optionally substituted. “Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclic fully saturated hydrocarbon consisting solely of carbon and hydrogen atoms, which can include fused, spirocyclic, or bridged ring systems, having from three to twenty carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkyl include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkyl group can be optionally substituted. “Cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon consisting solely of carbon and hydrogen atoms, having one or more carbon-carbon double bonds, which can include fused, spirocyclic, or bridged ring systems, having from three to twenty carbon atoms, preferably having from three to ten carbon atoms, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkenyl include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cycloctenyl, and the like. Polycyclic cycloalkenyls include, for example, bicyclo[2.2.1]hept-2-enyl and the like. Unless otherwise stated specifically in the specification, a cycloalkenyl group can be optionally substituted. “Cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon consisting solely of carbon and hydrogen atoms, having from 3 to 20 carbon atoms and one or more carbon-carbon triple bonds, which can include fused, spirocyclic, or bridged ring systems, and which is attached to the rest of the molecule by a single bond. Monocyclic cycloalkynyls include, for example, cycloheptynyl, cyclooctynyl, and the like. Unless otherwise stated specifically in the specification, a cycloalkynyl group can be optionally substituted. “Heterocyclyl,” “heterocyclic ring” or “heterocycle” refers to a stable 3- to 20-membered aromatic or non-aromatic ring which consists of 2 to 12 carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Heterocycles can be aromatic (heteroaryls) or non-aromatic. Unless stated otherwise specifically in the specification, the heterocyclyl can be a monocyclic, bicyclic, tricyclic, tetracyclic ring system or other multi-cyclic ring system, which can include fused, spirocyclic, or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl can be optionally oxidized; the nitrogen atom can be optionally quaternized; and the heterocyclyl can be partially or fully saturated. Examples of such heterocyclyls include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, biotinyl, dihydrofuranyl, dihydroindolyl, dihydropyranyl, dihydrothienyl, dithiazolyl, homopiperidinyl, pyranyl, pyrazolinyl, thiopyranyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrrolidin-2-only, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, tetrahydroisoquinolyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclyl group can be optionally substituted. “Heteroaryl” refers to a 5- to 20-membered ring system comprising hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of this disclosure, the heteroaryl can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; the heteroaryl may contain one or more non-aromatic rings (e.g., cycloalkyl or heterocyclyl) fused to the aromatic ring. The nitrogen, carbon or sulfur atoms in the heteroaryl can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e. thienyl). Unless stated otherwise specifically in the specification, a heteroaryl group can be optionally substituted. “Heteroarylalkyl” refers to a group of the formula —Rb—Rfwhere Rbis an alkylene chain as defined above and Rfis a heteroaryl as defined above. Unless stated otherwise specifically in the specification, a heteroarylalkyl group can be optionally substituted. The term “substituted” used herein means any of the above groups (i.e., alkyl, alkenyl, alkynyl, aryl, arylalkyl, carbocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, N-heterocyclyl, heteroaryl, etc) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, “substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with NRgRh, NRgC(═O)Rh, NRgC(═O)NRgRh, NRgC(═O)ORh, NRgSO2Rh, OC(═O)NRgRh, ORg, SRg, SORg, SO2Rg, OSO2Rg, SO2ORg, ═NSO2Rg, and SO2NRgRh. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced with C(═O)Rg, C(═)ORg, C(═O)NRgRh, CH2SO2Rg, CH2SO2NRgRh. In the foregoing, Rgand Rhare the same or different and independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. “Substituted” further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl group. In addition, “substituted” means any of the above groups in which two hydrogen atoms are each replaced by a bond to form a fused ring system containing the atoms to which the hydrogens were attached. Moreover, each of the foregoing substituents can also be optionally substituted with one or more of the above substituents. The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” depending on the configuration of substituents around the stereogenic carbon atom. The present disclosure encompasses various stereoisomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures of enantiomers or diastereomers may be designated “(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. In embodiments, an enantiomer or stereoisomer may be provided substantially free of the corresponding enantiomer. In embodiments, the compound is a racemic mixture of (S)- and (R)-isomers. In other embodiments, provided herein is a mixture of compounds wherein individual compounds of the mixture exist predominately in an (S)- or (R)-isomeric configuration. For example, the compound mixture has an (S)-enantiomeric excess of greater than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or more. In other embodiments, the compound mixture has an (S)-enantiomeric excess of greater than about 55% to about 99.5%, greater than about 60% to about 99.5%, greater than about 65% to about 99.5%, greater than about 70% to about 99.5%, greater than about 75% to about 99.5%, greater than about 80% to about 99.5%, greater than about 85% to about 99.5%, greater than about 90% to about 99.5%, greater than about 95% to about 99.5%, greater than about 96% to about 99.5%, greater than about 97% to about 99.5%, greater than about 98% to greater than about 99.5%, greater than about 99% to about 99.5%, or more. In other embodiments, the compound mixture has an (R)-enantiomeric purity of greater than about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or more. In some other embodiments, the compound mixture has an (R)-enantiomeric excess of greater than about 55% to about 99.5%, greater than about 60% to about 99.5%, greater than about 65% to about 99.5%, greater than about 70% to about 99.5%, greater than about 75% to about 99.5%, greater than about 80% to about 99.5%, greater than about 85% to about 99.5%, greater than about 90% to about 99.5%, greater than about 95% to about 99.5%, greater than about 96% to about 99.5%, greater than about 97% to about 99.5%, greater than about 98% to greater than about 99.5%, greater than about 99% to about 99.5% or more. Individual stereoisomers of compounds of the present disclosure can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by: (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary; (2) salt formation employing an optically active resolving agent; or (3) direct separation of the mixture of optical enantiomers on chiral chromatographic columns. Stereoisomeric mixtures can also be resolved into their component stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Stereoisomers can also be obtained from stereomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods. Geometric isomers can also exist in the compounds of the present disclosure. The present disclosure encompasses the various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the “Z” or “E” configuration wherein the terms “Z” and “E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the E and Z isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as “cis” or “trans,” where “cis” represents substituents on the same side of the double bond and “trans” represents substituents on opposite sides of the double bond. The arrangements of substituents around a carbocyclic ring are designated as “cis” or “trans.” The term “cis” represents substituents on the same side of the plane of the ring and the term “trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated “cis/trans.” The compounds disclosed herein may exist as tautomers and both tautomeric forms are intended to be encompassed by the scope of the present disclosure, even though only one tautomeric structure is depicted. As used herein, the term “isotopic variant” is meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. Such compounds may be useful as, for example, analytical tools, probes in biological assays, or therapeutic agents. For example, an “isotopic variant” of a compound can contain one or more nonradioactive isotopes, such as for example, deuterium (2H or D), carbon-13 (13C), nitrogen-15 (15N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be2H/D, any carbon may be13C, or any nitrogen may be15N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.3H, and carbon-14, i.e.14C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such as11C,18F,15O and13N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. The term “triglyceride” as used herein means an ester derived from glycerol and three fatty acids. The fatty acids may be the same or different. The notation used in this specification to describe a triglyceride is the same as that used below to describe a fatty acid. Fatty acids can attach to the glycerol molecule in any order, e.g., any fatty acid can react with any of the hydroxyl groups of the glycerol molecule for forming an ester linkage. For example. In a non-limiting example, a triglyceride can comprise glycerol with any combination of the following fatty acids: C18:1, C14:1, C16:1, polyunsaturated, and saturated. A triglyceride of C18:1 fatty acid simply means that the fatty acid components of the triglyceride are derived from or based upon a C18:1 fatty acid. That is, a C18:1 triglyceride is an ester of glycerol and three fatty acids of 18 carbon atoms each with each fatty acid having one double bond. Similarly, a C14:1 triglyceride is an ester of glycerol and three fatty acids of 14 carbon atoms each with each fatty acid having one double bond. Likewise, a C16:1 triglyceride is an ester of glycerol and three fatty acids of 16 carbon atoms each with each fatty acid having one double bond. Triglycerides of C18:1 fatty acids in combination with C14:1 and/or C16:1 fatty acids means that: (a) a C18:1 triglyceride is mixed with a C14:1 triglyceride or a C16:1 triglyceride or both; or (b) at least one of the fatty acid components of the triglyceride is derived from or based upon a C18:1 fatty acid, while the other two are derived from or based upon C14:1 fatty acid and/or C16:1 fatty acid. The term “fatty acid” and like terms mean a carboxylic acid with a long aliphatic tail that is either saturated or unsaturated. The term “long aliphatic tail” and “fatty acid chain” are used interchangeably herein. Fatty acids and fatty acid chains may be esterified to phospholipids and triglycerides. As used herein, the fatty acid chain length includes from C4 to C30 (e.g., C6 to C30), saturated or unsaturated, cis or trans, unsubstituted or substituted, branched or unbranched hydrocarbon chain (e.g., the fatty acid chain length includes from C4 to C30 (e.g., C6 to C30), saturated or unsaturated, cis or trans, unsubstituted or substituted with 1-6 side chains). For example, in embodiments, examples of a fatty acid chain include, but are not limited to, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, C26, C27, C28, C29, or C30 saturated or unsaturated, cis or trans, unsubstituted or substituted hydrocarbon chain. Unsaturated fatty acids and fatty acid chains have one or more double bonds between carbon atoms. Saturated fatty acids and fatty acid chains do not contain any double bonds. In embodiments, a fatty acid may be described herein by the capital letter “C” for carbon atom, followed by a number describing the number of carbon atoms in the fatty acid, followed by a colon and another number for the number of double bonds in the fatty acid. For example, C16:1 denotes a fatty acid of 16 carbon atoms with one double bond, e.g., palmitoleic acid. The number after the colon in this notation neither designates the placement of the double bond(s) in the fatty acid nor whether the hydrogen atoms bonded to the carbon atoms of the double bond are cis to one another. Other examples of this notation include C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:3 (a-linolenic acid) and C20:4 (arachidonic acid). The term “sterols” such as, but not limited to cholesterol, can also be utilized in the methods and compounds described herein. Sterols are animal or vegetable steroids which only contain a hydroxyl group but no other functional groups at C-3. In general, sterols contain 27 to 30 carbon atoms and one double bond in the 5/6 position and occasionally in the 7/8, 8/9 or other positions. Besides these unsaturated species, other sterols are the saturated compounds obtainable by hydrogenation. One example of a suitable animal sterol is cholesterol. Typical examples of suitable phytosterols, which are preferred from the applicational point of view, are ergosterols, campesterols, stigmasterols, brassicasterols and, preferably, sitosterols or sitostanols and, more particularly, β-sitosterols or β-sitostanols. Besides the phytosterols mentioned, their esters are preferably used. The acid component of the ester may go back to carboxylic acids corresponding to formula (CA-I): RI CO—OH (CA-I); in which RI CO is an aliphatic, linear or branched acyl group containing 2 to 30 carbon atoms and O and/or 1, 2 or 3 double bonds. Typical examples are acetic acid, propionic acid, hexanoic acid, butyric acid, valeric acid, caproic acid, caprylic acid, 2-ethyl hexanoic acid, capric acid, cyclopentanepropionic acid, lauric acid, isotridecanoic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselic acid, linoleic acid, conjugated linoleic acid (CLA), linolenic acid, elaeosteric add, arachic acid, gadoleic acid, behenic acid and erucic acid. The term “phospholipid” refers to an amphiphilic compound that consists of two hydrophobic fatty acid “tails” and a hydrophilic “head” consisting of a phosphate group. The two components are joined together by a glycerol molecule. The phosphate groups can be modified with simple organic molecules such as choline, ethanolamine or serine. Choline refers to an essential, bioactive nutrient having the chemical formula R—(CH2)2—N(CH2)4. When a phospho-moiety is R— it is called phosphocholine. “Lysolipids”, as used herein, include (acyl-, single chain) such as in non-limiting embodiments 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (MHPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC) and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (SHPC). The term “apolipoprotein A-I” or “apoA-I”, and also “apoliprotein Al” or “apoAl”, refers to a protein that is encoded by the APOAI gene in humans. DETAILED DESCRIPTION Conventionally, immune systems in vertebrate animals are subdivided into two parts. The first part, innate immunity, provides an initial response to an infection within minutes to hours. Its cellular component comprises natural killer (NK) cells, innate lymphoid cells (ILCs) and phagocytes such as monocytes, macrophages and neutrophils. The innate immune system acts as a rapid first line of defense, triggered through recognition of either pathogens or endogenous danger signals by pattern recognition receptors (PRRs). Upon detecting pathogen-associated molecular patterns (PAMPs), PRRs initiate an innate immune response, which involves activating the subsequent adaptive immune system by antigen presentation, co-stimulation, and cytokine excretion. In addition, PRRs also recognize damage-associated molecular patterns (DAMPs), leading to non-infectious inflammatory responses. The second stage of the response to infection involves the immune system's second part the adaptive response in which T and B lymphocytes specifically recognize a pathogen, proliferate and become activated against that pathogen. These cells also build immunological memory of that specific infection. The specificity of the adaptive immune system response is mediated by recombination of the immunoglobulin genes at the lymphocyte level. Immunological memory results in a quicker and quantitatively better immune response (as compared with the primary response alone) against a previously encountered antigen. Though the innate immune system was long believed to lack memory, recent studies show that innate immune cells undergo metabolic and epigenetic rewiring, adjusting their functional programs in a process termed ‘trained immunity’ that is considered de facto innate immune memory. Trained Immunity is defined by a secondary long-term hyper-responsiveness, as manifested by increased cytokine excretion caused by metabolic and epigenetic rewiring, to re-stimulation after a primary insult of myeloid cells and their progenitors and stem cells in the bone marrow, spleen and blood. Trained Immunity (also called innate immune memory) is also defined by a long-term increased responsiveness (e.g. high cytokine production) after restimulation with a secondary stimulus of myeloid innate immune cells, being induced by a primary insult stimulating these cells or their progenitors and stem cells in the bone marrow and spleen, and mediated by epigenetic, metabolic and transcriptional rewiring. Trained immunity is regulated and maintained through induction of training properties to progenitor cells in the bone marrow, resulting in durable reprogramming that exceeds the myeloid cell lifespan in the bloodstream. Although trained immunity can be induced with a range of ‘training agents’ in cultured myeloid cells, its systemic induction requires bone marrow progenitor cell engagement. In one aspect, the present disclosure provides compounds (e.g., of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or Table 1) that activate nucleotide-binding oligomerization domain-containing protein 2 (NOD2). The present disclosure also provides nanobiologic compositions comprising a nanoparticle carrier (e.g., HDL-derived nanoparticle) comprising a compound of the present disclosure (e.g., of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or Table 1). Nanobiologic compositions of the present disclosure comprising compounds of the present disclosure (e.g., of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or Table 1) that activate nucleotide-binding oligomerization domain-containing protein 2 (NOD2) are designed to exhibit bone marrow proclivity. These nanomaterials can be administered (e.g., intravenously) to promote trained immunity. Therapeutically inducing trained immunity may find use, for example, in overcoming immunoparalysis in sepsis and infections, in treating cell proliferation disorders (such as cancer), and augmenting immune responses. Compounds In embodiments, the present disclosure provides a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein:R1is —H or —C(O)—RX;R2and R3are each independently selected from the group consisting of —H, alkyl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5are each alkyl;R6and R11are each independently —H, or alkyl;R7is a fatty acid chain, —Y—N(R6)—C(O)—O-alkylene-C(H)(OR8)-alkylene-OR9, —Y—N(R6)—C(O)—RX, —Y—O—P(O)(OH)—O-alkylene-C(H)(OR8)-alkylene-OR9, or —Y— triazolyl-L;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;R22, R33, R33′, R44, R44′, R55, and R55′are each independently H or RA;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, aryl, and triazolyl is optionally substituted with one or more RA, wherein RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro. In embodiments, the present disclosure provides a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein:R1is —H or —C(O)—RX;R2and R3are each independently selected from the group consisting of —H, alkyl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5are each alkyl;R6and R11are each independently —H, or alkyl;R7is a C9-30fatty acid chain, —Y—N(R11)—C(O)—O-alkylene-C(H)(OR8)-alkylene-OR9, —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C16-30fatty acid chain, —(CR10R10)2—O—P(O)(OH)—O-alkylene-C(R10)(ORZ)-alkylene-ORZ′, or —Y-triazolyl-L;RZand RZ′are each independently C8-30fatty acid chain or —C(O)—C16-30fatty acid chain;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;R10, R22, R33, R33′, R44, R44′, R55, and R55′are each independently H or RA;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, aryl, and triazolyl is optionally substituted with one or more RA, wherein RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro;wherein, when R7is C9-30fatty acid chain, R2is —H. In embodiments of the compound of Formula (I), the compound is of formula (IA): or a pharmaceutically acceptable salt thereof, wherein:R1is —H or —C(O)—RX;R2and R3are each independently selected from the group consisting of —H, alkyl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5are each alkyl;R6and R11are each independently —H, or alkyl;R7is a C9-30fatty acid chain, —Y—N(R11)—C(O)—O-alkylene-C(H)(OR8)-alkylene-OR9, —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C16-30fatty acid chain, —(CR10R10)2—O—P(O)(OH)—O-alkylene-C(R10)(ORZ)-alkylene-ORZ′, or —Y-triazolyl-L;RZand RZ′are each independently C8-30fatty acid chain or —C(O)—C16-30fatty acid chain;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;R10, R22, R33, R33′, R44, R44′, R55, and R55′are each independently H or RA;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, aryl, and triazolyl is optionally substituted with one or more RA, wherein RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro;wherein, when R7is C9-30fatty acid chain, R2is —H. In embodiments, the compound of formula (I) is a compound of formula (IB): or a pharmaceutically acceptable salt thereof. In embodiments of the compounds of formula (I), (IA), or (IB), Y is alkylene optionally substituted with —C(O)N(RC)(RD). In embodiments, Y is —C1-6alkylene. In embodiments, Y is —CH2—. In embodiments, Y is In embodiments, Y is or In embodiments, Y is In embodiments of the compounds of formula (I), (IA), or (IB), R7is —Y-triazolyl-L. In embodiments of the compound of formula (I), (IA), or (IB), R7comprises a cholesteryl moiety or at least one fatty acid chain comprising at least 17 carbons. In embodiments, R7comprises a cholesteryl moiety. In embodiments, R7comprises at least one fatty acid chain comprising at least 17 carbons. In embodiments, R7comprises at least two fatty acid chain comprising at least 17 carbons. In embodiments, R7comprises at least two C17fatty acid chains. In embodiments, the C17fatty acid chain is derived from stearic acid or oleic acid. In embodiments, the C17fatty acid chain is derived from stearic acid. In embodiments, the C17fatty acid chain is derived from oleic fatty acid. In embodiments, R7comprises two C17fatty acid chains derived from stearic acid. In embodiments of the compound of formula (I), (IA), or (IB), R7is an alkyl group having at least 16 carbons. In embodiments of the compound of formula (I), (IA), or (IB), R7is an alkenyl group having at least 16. In embodiments of the compound of formula (I), (IA), or (IB), R7is an alkyl group having at least 18 carbons. In embodiments of the compound of formula (I), R7is an alkenyl group having at least 18 carbons. In embodiments of the compound of formula (I), (IA), or (IB), R7is C9-30fatty acid chain. In embodiments of the compound of formula (I), R7is a —C9-30alkyl or a C9-30alkenyl. In embodiments, R7is a —C9-30alkyl or a C9-30alkenyl provided that when R7is a —C9-30alkyl then R2is —H. In embodiments of the compound of formula (I), R7is a —C9-30alkyl. In embodiments, R7is a —C9-30alkyl and R2is —H. In embodiments of the compound of formula (I), R7is a —C9-30alkenyl. In embodiments, R7is a —C15-30alkyl group. In embodiments, R7is a —C15-30alkyl group and R2is —H. In embodiments, R7is a —C15-30alkenyl group. In embodiments, R7is a —C17-19alkyl. In embodiments, R7is a —C17-19alkyl and R2is —H. In embodiments, R7is a —C17-19alkenyl. In embodiments, R7is a —C18alkyl group. In embodiments, R7is a —C18alkyl group and R2is —H. In embodiments, R7is a —C18alkenyl group. In embodiments of the compound of formula (I), (IA), or (IB), R7is: In embodiments of the compound of formula (I), (IA), or (IB), R7is: In embodiments of the compound of formula (I), (IA), or (IB), R7is: and R2is —H. In embodiments of the compound of formula (I), (IA), or (IB), R7is —Y—N(R11)—C(O)—O— alkylene-C(H)(OR8)-alkylene-OR9. In embodiments of the compound of formula (I), (IA), or (IB), R7is —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C16-30fatty acid chain. In embodiments, R7is —C(H)(C(O)NH2)—C5alkylene-N(R11)—C(O)—C17-30fatty acid. In embodiments, R7is —C(H)(C(O)NH2)-alkylene-N(R11)—C(O)—C17-30fatty acid. In embodiments of the compound of formula (I), (IA), or (IB), R7is —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C16-30alkyl. In embodiments, R7is —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C17-30alkyl. In embodiments, R7is —C(H)(C(O)NH2)-alkylene-N(R11)—C(O)—C17-30alkyl. In embodiments, R7is —C(H)(C(O)NH2)—C4alkylene-N(R11)—C(O)—C17-30alkyl. In embodiments of the compound of formula (I), R7is —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C16-30alkyl and R2is alkylene-aryl (e.g., benzyl). In embodiments, R7is —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C17-30alkyl and R2is alkylene-aryl (e.g., benzyl). In embodiments, R7is —C(H)(C(O)NH2)-alkylene-N(R11)—C(O)—C17-30alkyl and R2is alkylene-aryl (e.g., benzyl). In embodiments, R7is —C(H)(C(O)NH2)—C4alkylene-N(R11)—C(O)—C17-30alkyl and R2is alkylene-aryl (e.g., benzyl). In embodiments of the compound of formula (I), (IA), or (IB), R7is —(CR10R10)2—O—P(O)(OH)—O-alkylene-C(R10)(ORZ)-alkylene-ORZ′. In embodiments of the compound of formula (I), R7is —CH2CH2—O—P(O)(OH)—O—CH2—C(H)(ORZ)—CH2—ORZ′. In embodiments, RZand RZ′are each independently C12-20alkyl or —C(O)—C16-30fatty acid chain. In embodiments, RZand RZ′are each independently C18alkyl or —C(O)—C17alkyl. In embodiments, RZand RZ′are each independently a —C(O)—C16-30alkyl. In embodiments, RZand RZ′are both a —C(O)—C17alkyl. In embodiments of the compound of formula (I), (IA), or (IB), R7is —Y—N(R6)—C(O)—O— alkylene-C(H)(OR8)-alkylene-OR9. In embodiments, R8and R9are each independently C8-30alkyl or —C(O)—C8-30alkyl. In embodiments, R8and R9are each independently C12-20alkyl or —C(O)—C11-20alkyl. In embodiments, R8and R9are each independently C18alkyl or —C(O)—C17alkyl. In embodiments, R8and R9are both —C(O)—C17alkyl. In embodiments of the compounds of formula (I), (IA), or (IB), RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl. In embodiments of the compounds of formula (I), (IA), or (IB), RAis independently selected for each occurrence from the group consisting of halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl. In embodiments the compound of formula (I) is a compound of formula (II): or a pharmaceutically acceptable salt thereof wherein:X1is —N— and X2is —C—; or X1is —C— and X2is —N—;R2and R3are each independently selected from the group consisting of —H, alkyl, aryl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5′are each alkyl;R6and R11are each independently —H, or alkyl;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, and aryl is optionally substituted with one or more RA;RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro. In embodiments, the compound of formula (II) is: In embodiments, the compound of formula (II) is a compound of formula (II-1), or a pharmaceutically acceptable salt thereof In embodiments, the compound of formula (II-1), is or a pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (II) is a compound of formula (II-2): In embodiments, the compound of formula (II-2) is: or a pharmaceutically acceptable salt thereof. In embodiments the compound of formula (I) is a compound of formula (IIA): or a pharmaceutically acceptable salt thereof wherein:X1is —N— and X2is —C—; or X1is —C— and X2is —N—R2and R3are each independently selected from the group consisting of —H, alkyl, aryl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5′are each alkyl;R6and R11are each independently —H, or alkyl;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, and aryl is optionally substituted with one or more RA;RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro. In embodiments, the compound of formula (IIA) is: In embodiments the compound of formula (IIA) is a compound of formula (IIA-1), or a pharmaceutically acceptable salt thereof: In embodiments, the compound of formula (IIA-1) is: or a pharmaceutically acceptable salt thereof. In embodiments the compound of formula (IIA) is a compound of formula (IIA-2), or a pharmaceutically acceptable salt thereof: In embodiments, the compound of formula (IIA-2) is: or a pharmaceutically acceptable salt thereof. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), Y is alkylene. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), Y is C1-6alkylene. In embodiments, Y is C1-5alkylene. In embodiments, Y is C1-3alkylene. In embodiments, Y is alkylene optionally substituted with —C(O)N(RC)(RD), wherein RCand RDare defined herein. In embodiments, Y is —CH2—. In embodiments, Y is In embodiments, Y is In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), RAis independently selected for each occurrence from the group consisting of hydrogen, halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), RAis independently selected for each occurrence from the group consisting of halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), alkyl, alkylene, alkylene-aryl, and aryl is optionally substituted with one or more RA; In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), RBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), RAis —H. In some embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R2is alkyl, aryl, or alkylene-aryl. In embodiments, aryl is optionally substituted with alkyl. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R2and R3are each independently selected from the group consisting of —H, alkyl, aryl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R2is —H or benzyl. In embodiments of the compounds of formula (I), R2is —H. In embodiments, R2is benzyl. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R4, R5, and R5′are each alkyl. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R4is alkyl. In embodiments, R4is methyl. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R3is —H. In embodiments R6is —H. In embodiments, R3and R6are both —H. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R10, R22, R33, R33′, R44, R44′, R55, and R55′are each —H. In embodiments of the compound of formula (I), (II), (IA), (IB), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L comprises a cholesteryl moiety or at least one fatty acid chain comprising at least 13 carbons. In embodiments, L comprises a cholesteryl moiety. In embodiments, L comprises at least one fatty acid chain comprising at least 13 carbons. In embodiments, L comprises at least two fatty acid chains comprising at least 15 carbons. In embodiments, L comprises at least one C17fatty acid chain. In embodiments, L comprises at least two C17fatty acid chains. In embodiments, L comprises at least one fatty acid chains independently selected from a C17alkyl or a C17alkenyl. In embodiments, L comprises at least two fatty acid chains independently selected from a C17alkyl or a C17alkenyl. In embodiments, the C17fatty acid chain is derived from stearic acid or oleic acid. In embodiments, the C17fatty acid chain is derived from stearic acid. In embodiments, the C17fatty acid chain is derived from oleic fatty acid. In embodiments, L comprises two C17fatty acid chains derived from stearic acid. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is selected from the group consisting of C8-30fatty acid chain, —CH2—C(O)—W, —CH2—O—C(O)—W, —CH2CH2—N— CH2CH2—C(O)—NR11—CH2CH2—NR11—C(O)—W)2, and —CH2CH2—N—(CH2CH2—C(O)—W)2. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is a C8-30fatty acid chain. In embodiments, L is a C8-30alkyl or a C8-30alkenyl. In embodiments, L is a C15-20alkyl or a C15-20alkenyl. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, or -alkylene-N-(alkylene-C(O)—W)2. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is -alkylene-C(O)—W. In embodiments, L is —C1-6alkylene-C(O)—W. In embodiments, L is —CH2—C(O)—W. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is -alkylene-O—C(O)—W. In embodiments, L is —C1-6alkylene-O—C(O)—W. In embodiments, L is —CH2—O—C(O)—W. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2. In embodiments, L is —C2-6-alkylene-N—(—C2-alkylene-C(O)—NR11—C2-alkylene-NR11—C(O)—W)2. In embodiments, L is —CH2—CH2—N—(CH2—CH2—C(O)—NR11—CH2—CH2—NR11—C(O)—W)2. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is -alkylene-N-(alkylene-C(O)—W)2. In embodiments, L is —C1-6alkylene-N—(C1-6alkylene-C(O)—W)2. In embodiments, L is —CH2—CH2—N—(CH2—CH2—C(O)—W)2. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), L is a C18fatty acid chain. In embodiments, L is a C18alkyl or a C18alkenyl. In embodiments, L is —CH2(CH2CH2)8—CH3. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is a C8-30fatty acid chain. In embodiments, W is a C8-30alkyl or a C8-30alkenyl. In embodiments, W is a C8-30alkyl. In embodiments, W is a C8-30alkenyl. In embodiments, W is a C12-18fatty acid chain. In embodiments, W is a C12-18alkyl or a C12-18alkenyl. In embodiments, W is a C12-18alkyl. In embodiments, W is a C12-18alkenyl. In embodiments, W is a Cis fatty acid chain. In embodiments, W is a C17fatty acid chain. In embodiments, W is a C17alkyl or a C17alkenyl. In embodiments, W is a C17alkyl. In embodiments, W is a C17alkenyl. In embodiments, W is —(CH2CH2)8—CH3. In embodiments, W is a fatty acid chain comprising at least 15 carbons. In embodiments, W is a fatty acid chain comprising at least 18 carbons. In embodiments, W is a fatty acid chain comprising at least 17 carbons. In embodiments, W is a fatty acid chain comprising at least 18 carbons. In embodiments of the compounds of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is: In embodiments, RXand RX′is each independently a fatty acid chain. In embodiments, RXand RX′is each independently a fatty acid chain comprising at least 15 carbons. In embodiments, RXand RX′is each independently a fatty acid chain comprising at least 17 carbons. In some embodiment, RXand RX′is each independently a —C8-30fatty acid chain. In embodiments, RXand RX′is each independently a —C8-30alkyl or a —C8-30alkenyl. In embodiments, RXand RX′are both a —C8-30alkyl. In embodiments, RXand RX′are both a —C8-30alkenyl. In embodiments, RXand RX′is each independently a C12-18fatty acid chain. In embodiments, RXand RX′is each independently a —C12-18alkyl or a —C12-18alkenyl. In embodiments, RXand RX′are a —C12-18alkyl. In embodiments, RXand RX′are a —C12-18alkenyl. In embodiments, RXand RX′is each independently a C17fatty acid chain. In embodiments, RXand RX′are each independently a C17alkyl or a C17alkenyl. In embodiments, RXand RX′are a C17alkyl. In embodiments, RXand RX′are a C17alkenyl. In embodiments, the C17chains are independently derived from stearic acid or oleic acid. In embodiments, the C17chains are derived from stearic acid. In embodiments, the C17chains are derived from oleic acid. In embodiments, RXand RX′are both —(CH2CH2)8—CH3. In embodiments of the compounds of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is a sterol. In embodiments of the compounds of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is cholesterol: In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is a phospholipid is selected from the group consisting of: a phosphatidylcholine (PC), a phosphatidylglycerol (PG), a phosphatidylserine (PS), a phosphatidylethanolamine (PE), a phosphatidic acid (PA), and a lysophosphatidylcholine. In embodiments, W is a phosphatidylcholine (PC). In embodiments, W is a phosphatidylglycerol (PG). In embodiments, W is a phosphatidylserine (PS). In embodiments, W is a phosphatidylethanolamine (PE). In embodiments, W is a phosphatidic acid (PA). In embodiments, W is a lysophosphatidylcholine. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), W is: whereinYQ1, YQ2, and YQ3are each independently alkylene. In embodiments, YQ1is C2-6alkylene, and YQ2, and YQ3are each independently —C1-3alkylene. In embodiments, RXand RX′are each independently a fatty acid chain having at least 15 carbons, or in particular embodiments RXand RX′are each independently a fatty acid chain having at least 17 carbons. In embodiments, RXand RX′are each independently a C8-30fatty acid chain. In embodiments, RXand RX′are each independently a C8-30alkyl or a C8-30alkenyl. In embodiments, RXand RX′are each independently a C15-30alkyl or a C15-30alkenyl. In embodiments, RXand RX′are each independently a C15-20alkyl or a C15-20alkenyl. In embodiments, RXand RX′are each independently a C17alkyl or a C17alkenyl. In embodiments, RXand RX′are both —(CH2CH2)8—CH3. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), the W is: In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), the W is: or a pharmaceutically acceptable salt thereof; wherein RXand RX′are each independently a C8-30fatty acid chain. In embodiments, the fatty acid is saturated. In embodiments, RXand RX′are each independently a C8-30alkyl or a C8-30alkenyl. In embodiments, RXand RX′are each independently a C15-20alkyl or a C15-20alkenyl. In embodiments, RXand RX′are each independently a C17alkyl or a C17alkenyl. In embodiments, RXand RX′are both —(CH2CH2)8—CH3. In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R8and R9are each independently RXor —C(O)—RX; In embodiments of the compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2), R6and R11are each independently —H, or alkyl. In embodiments, the present disclosure provides a compound selected from the group consisting of: or stereoisomer thereof (e.g., an alpha or beta anomer thereof, or a tautomer thereof). In embodiments, the present disclosure provides a compound selected from the group consisting of: or a stereoisomer thereof (e.g., an anomer thereof, or mixture of anomers thereof). In embodiments, the present disclosure provides a compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or a stereoisomer thereof. In embodiments, the present disclosure provides a compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or a diastereomer, or tautomer thereof. In embodiments, provided herein is one or more compounds selected from Table 1. In embodiments, provided herein is one or more pharmaceutically acceptable salts of a compound selected from Table 1. In embodiments, provided herein is one or more compounds of selected from Table 1, or a stereoisomer, or a pharmaceutically acceptable salt thereof. TABLE 1CompoundsNo.Compound1MDP- C18 [click]2MDP- DSPE [click]3MDP- chol [click]4MDP- DSPE2[click]5MDP- Chol2[click]6MDP- DSG [click]7MDP (Bn)- DSPE [click]8MDP (Bn)- chol [click]9MTP- b-C18 [invclick]10MTP- b-C1811MDP- C1812MDP- DSPE13MTP- b-DSG14MTP (Bn)-a- DPPE15MTP- a-chol16MDP (Bn)- chol17MTP- a- DSPE18MTP (Bn)-a- DSPE19MDP (Bn)- DSPE Muramyl tripeptide phosphatidylethanolamine; N—(N-Acetylmuramoyl)-L-alanyl-D-alpha-glutaminyl-N-[(7R)-4-hydroxy-4-oxido-10-oxo-7-[(1-oxohexadecyl)oxy]-3,5,9-trioxa-4-phosphapentacos-1-yl]-L-alaninamide (MTP-a-DPPE or Mifamurtide): Molecular weight: 1238 Dalton. C Log P=10.59 (uncharged) and 4.80 (negatively charged). Mifamurtide (CAS-number [83461-56-7]) was prepared as according to literature procedures (e.g. Brundish, D. E.; Wade, R. (1985) J Label Compd Radiopharm. 22 (1): 29-35. doi:10.1002/jlcr.2580220105). The lipophilicity of this molecule is relatively low at C Log P 4.80 in physiological circumstances. N-Acetylmuramyl-L-Alanyl-D-Isoglutamine-6-O-Stearoyl (MDP-C18[mur]) Molecular weight: 759 Dalton. C Log P=5.39 (uncharged) and 1.39 (negatively charged). MDP-C18[mur] (CAS-number [60398-08-5]) was prepared as according to literature procedures (e.g. Matsumoto K. et al. (1981) Infect Immun. 32(2):748-58). The lipophilicity of this molecule is low at C Log P 1.39 in physiological circumstances is unlikely to be sufficient to ensure its robust incorporation into HDL-derived nanoparticles. Romurtide (CAS-number [78113-36-7]) Molecular weight: 887 Dalton. C Log P=3.90 (uncharged) and 0.61 (negatively charged) has a lipophilicity (C Log P 0.61 in physiological circumstances). The lipophilicity of this compound is low with a C Log P of the charged molecule that is close to 0. Murabutide (CAS-number [74817-61-1]) Molecular weight: 549 Dalton. C Log P=−1.53 (uncharged) has a C Log P value that is negative. This molecule is hydrophilic given its C Log P value that is below 0. In embodiments, compounds of the present disclosure (such as one or more compounds of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), (IIA-2) or Table 1) activate nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Anomers and Open/Closed Ring Structures In embodiments, the molecules of the present disclosure bear an —OH substituent at the anomeric hemi-acetal carbon of the muramyl sugar group, i.e. when R2═H, it is understood that both anomeric isomers alpha and beta are included in the compounds of the present disclosure. Furthermore, in these cases where R2═H, it is known in the art that such molecules (in aqueous environments) actually exist in both the closed ring isomer as well as the open ring structure. Again, it is understood that both the open and closed ring isomers are included in the compounds of the present disclosure. Below, in non-limiting examples, the top structures show the alpha and beta anomers, and the bottom structures show the general anomeric ring-closed structure (left) and the open ring structure (right). Molecular Weight The compounds of the invention preferably have a molecular weight higher than 500 Dalton, higher than 700 Dalton, higher than 950 Dalton, or higher than 1,200 Dalton. The compounds of the invention preferably have a molecular weight lower than 10,000 Dalton, lower than 5,000 Dalton, lower than 2,500 Dalton, or lower than 1,750 Dalton. Hydrophobicity The compounds of the disclosure are, in particular embodiments, hydrophobic in nature. Hydrophobicity can be estimated by calculation of the C Log P value. This can be done in software programs such as for example Perkin Elmer's ChemDraw or ChemDraw Professional (v 18). The higher the C Log P value of a compound, the more hydrophobic a compound is. In embodiments, the compounds of the present disclosure have a C Log P value higher than about 1, higher than about 3, higher than about 5, higher than about 7, higher than about 9, or higher than about 11. The C Log P value represents the n-octanol/water partition coefficient (Log Po/w) of a molecule, and is a calculated value as opposed to Log P values, i.e. values assessed by experimentation. Accordingly, C Log P values may deviate from Log P values. Importantly, however, C Log P values give a good comparison between the lipophilicities of molecules. C Log P values can be assessed for molecules in either their uncharged or their charged state. This is the case for molecules that have ionogenic groups, such as molecules with carboxylic acid (—COOH) or phosphate (—OP(O)OH—O—) groups. At physiological pH (about 7.4) these particular groups are deprotonated and thus become charged. Also alkyl(ated) amine groups become charged at physiological pH, in this case by protonation. At physiological pH, the molecules of the invention have C Log P values that are lower than 20, or lower than 15, or lower than 10. Moreover, at physiological pH, the molecules of the invention have C Log P values that are higher than 3, or higher than 4, or higher than 5, or higher than 5.5. Nanobiologic Compositions Provided herein are nanobiologic compositions comprising a nanoparticle carrier and one or more compounds of the present disclosure (such as a compound of formula (I), (IA), (IB), (II), (II-1), (II-2), (IIA), (IIA-1), or (IIA-2) as disclosed herein or Table 1). In embodiments, the compounds of the present disclosure can be formulated in a nanoparticle carrier, which can include, but is not limited to polyplexes, colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes, lipoplexes, lipid nanoparticles, lipid nanocapsules, lipidoids, rapidly eliminated lipid nanoparticles (reLNPs), micro- and nano-emulsions, and the like, HDL-derived nanoparticles, polymeric nanoparticles, including poly (lactic-co-glycolic acid) (PLGA) nanoparticles such as PLGA microspheres, poly(lactide) (PLA) nanoparticles, poly(F-caprolactone) (PCL) nanoparticles, poly(butyl cyanoacrylate) (PBCA) nanoparticles, demdrimers, hyperbranched polyglycerol (HPG) nanoparticles, PEG-polyaspartate micellar nanoparticles, cationic polymers including for example poly(L-lysine), polyethylenimine (PEI), DEAE-dextran, poly(amino esters) (PBAE) and chitosan, cyclodextrin nanoparticles, metallic nanopartides, surfactant based emulsions, virus like particles (e.g., particles that are primarily made up of viral structural proteins but that are not infectious or have low infectivity), peptide or protein-based particles such as albumin nanopartides, nanowires, gold nanoparticles, magnetic nanoparticles, core-shell nanoparticles, carbon nanotubes, nanocrystals, hyaluronidase, and combinations thereof. In embodiments, the compounds of the present disclosure may be formulated in a nanoparticle carrier, such as those described in U.S. Pat. Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, 5,795,587, 10,485,884, US2018/0263907, US2016/0317647 US2019/0290593, US2020/0253884, US2020/0376146, and WO2018/071549 the contents of each of which are incorporated herein by reference. In embodiments, the nanoparticle carrier is a high-density lipoprotein (HDL)-derived nanoparticle. The high-density lipoprotein (HDL)-derived nanoparticles are envisioned as delivery vehicles that may, for example, improve the therapeutic index of small-molecule immunomodulatory compounds and/or confer innate immune cell-specific delivery. By conferring targeting specificity for innate immune cells (such as myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood and/or spleen), the therapeutic agents encapsulated or incorporated in the HDL-derived nanoparticles may be deposited in a concentrated and localized fashion. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises apoA-I or a peptide mimetic of apoA-I. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises apoA-I. Human apoA-I can be isolated or prepared by any method known in the art. In embodiments, human apoA-I is isolated from human HDL. Another known method comprises the synthesis of apoA-I by recombinant protein expression, for example inE. coliorganisms. When expressed in bacteria the apoA-I may include an N-terminal methionine or a formyl-methionine. The presence of the methionine group can be assessed by mass spectroscopic (MS) methods that are known in the art. The position of the methionine in the protein sequence can be assessed after digestion of apoA-I with subsequent analysis of the peptide mixture with MS, as is also known in the art. In embodiments, purifications of apoA-I, including any of its variations, may comprise any method known in the art (e.g. use of hydrophobic interaction chromatography, ion exchange columns, precipitations, etc.). Production methods may or may not comprise the use of affinity tags that enable the purification of the proteins; such tags require removal after purification to restore the human apoA-I identity. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises ApoA-1 Milano. Suitable apoA-I mimetic polypeptides may have the sequence shown in Table 2 (SEQ ID NOS: 256 to 263, and 342 to 346) or in SEQ ID NOS: 1 to 341. TABLE 2ApoA-I mimeticsSEQ ID NOAmino Acid Sequence256DWLKAFYDKVAEKLKEAF (18A)257Ac-DWLKAFYDKVAEKLKEAF-NH2(2F)260AC-DWFKAFYDKVAEKFKEAF-NH2(4F)258Ac-DWFKAFYDKVAEKLKEAF-NH2(3F3)259Ac-DWLKAFYDKVAEKFKEAF-NH2(3F14)261Ac-DWLKAFYDKVFEKFKEFF-NH2(5F)262Ac-DWLKAFYDKFFEKFKEFF-NH2(6F)263Ac-DWFKAFYDKFFEKFKEFF-NH2(7F)342Ac-FWLKAFYDKVAEKLKEAF-NH2(3F-1)343AC-DFLKAFYDKVAEKLKEAF-NH2(3F-2)344Ac-DWFRAFYDKVAEKFREAF-NH2(4F-R)q345Ac-DWFKAFYDRVAERFKEAF-NH2(4F-R′)q346Ac-DWLXAFYDXVAEXLXEAF-NH2(2F′) In embodiments, the apoA-I mimetic is DWLKAFYDKVAEKLKEAF (SEQ ID NO. 256). In embodiments, the apoA-I mimetic is Ac-DWLKAFYDKVAEKLKEAF-NH2(SEQ ID NO. 257). In embodiments, the apoA-I mimetic is Ac-DWFKAFYDKVAEKFKEAF-NH2(SEQ ID NO. 260). In embodiments, apoA-I mimetics are optionally acetylated on the N-terminus, or optionally amidated on the C-terminus. In embodiments, the apoA-I mimetics are acetylated on the N-terminus. In embodiments, the apoA-I mimetics are amidated on the C-terminus. In embodiments, the apoA-I mimetics are acetylated on the N-terminus and amidated on the C-terminus. In embodiments, the HDL-derived nanoparticles of the present disclosure comprise one or more phospholipids. All phospholipids ranging in chain length from C4 to C30, saturated or unsaturated, cis or trans, unsubstituted or substituted with 1-6 side chains, and with or without the addition of lysolipids are contemplated for use in the nanoparticles described herein. Additionally, other synthetic variants and variants with other phospholipid headgroups are also contemplated. In embodiments, the HDL-derived nanoparticle comprises a phospholipid. In embodiments, the HDL-derived nanoparticle comprises a phospholipid and a lysolipid. Non-limiting examples of the phospholipids that may be used in the present composition include phosphatidylcholines (PC), phosphatidylglycerols (PG), phosphatidylserines (PS), phosphatidylethanolamines (PE). In embodiments phosphatidic acid/esters (PA (may be used. In embodiments, the phospholipid or lysolipid is one or more of the following: DDPC CAS-3436-44-0 1,2-Didecanoyl-sn-glycero-3-phosphocholine, DEPA-NA CAS-80724-31-8 1,2-Dierucoy 1-sn-glycero-3-phosphate (Sodium Salt), DEPC CAS-56649-39-9 1,2-Dierucoyl-sn-glycero-3-phosphocholine, DEPE CAS-988-07-2 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine, DEPG-NA 1,2-Dierucoyl-sn-glycero-3-phospho-rac-(l-glycerol) (Sodium Salt), DLOPC CAS-998-06-11,2-Dilinoleoyl-sn-glycero-3-phosphocholine, DLPA-NA 1,2-Dilauroyl-sn-glycero-3-phosphate (Sodium Salt), DLPC CAS-18194-25-7 1,2-Dilauroyl-sn-glycero-3-phosphocholine, DLPE 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine, DLPG-NA 1,2-Dilauroyl-sn-glycero-3-phospho-rac-(l-glycerol) (Sodium Salt), DLPG-NH4 1,2-Dilauroyl-sn-glycero-3-phospho-rac-(l-glycerol) (Ammonium Salt), DLPS-NA 1,2-Dilauroyl-sn-glycero-3-phosphoserine (Sodium Salt), DMPA-NA CAS-80724-3 1,2-Dimyristoyl-sn-glycero-3-phosphate (Sodium Salt), DMPC CAS-18194-24-6 1,2-Dimyristoyl-sn-glycero-3-phosphocholine, DMPE CAS-988-07-2 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPG-NA CAS-67232-80-8 1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(l-glycerol) (Sodium Salt), DMPG-NH4 1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(l-glycerol) (Ammonium Salt), DMPG-NH4/NA 1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(l-glycerol) (Sodium/Ammonium Salt), DMPS-NA 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (Sodium Salt), DOPA-NA 1,2-Dioleoyl-sn-glycero-3-phosphate (Sodium Salt), DOPC CAS-4235-95-4 1,2-Dioleoyl-sn-glycero-3-phosphocholine, DOPE CAS-4004-5-1 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, DOPG-NA CAS-62700-69-0 1,2-Dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol)(Sodium Salt), DOPS-NA CAS-70614-14-1 1,2-Dioleoyl-sn-glycero-3-phosphoserine (Sodium Salt), DPPA-NA CAS-71065-87-7 1,2-Dipalmitoyl-sn-glycero-3-phosphate (Sodium Salt), DPPC CAS-63-89-8 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, DPPE CAS-923-61-5 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPG-NA CAS-67232-81-9 1,2-Dipalmitoyl-sn-glycero-3-phospho-rac-(l-glycerol) (Sodium Salt), DPPG-NH4 CAS-73548-70-6 1,2-Dipalmitoylsn-glycero-3-phospho-rac-(l-glycerol) (Ammonium Salt), DPPS-NA 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (Sodium Salt), DSPA-NA CAS-108321-18-2 1,2-Distearoyl-snglycero-3-phosphate (Sodium Salt), DSPC CAS-816-94-4 1,2-Distearoyl-sn-glycero-3-phosphocholine, DSPE CAS-1069-79-0 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, DSPG-NA CAS-67232-82-0 1,2-Distearoyl-sn-glycero-3-phospho-rac-(l-glycerol) (Sodium Salt), DSPG-NH4 CAS-108347-80-4 1,2-Distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) (Ammonium Salt), DSPS-NA 1,2-Distearoyl-sn-glycero-3-phosphoserine (Sodium Salt), EPC Egg-PC, HEPC Hydrogenated Egg PC, HSPC Hydrogenated Soy PC, LYSOPC MYRISTIC CAS-18194-24-6 1-Myristoyl-sn-glycero-3-phosphocholine, LYSOPC PALMITIC CAS-17364-16-8 1-Palmitoyl-sn-glycero-3-phosphocholine, LYSOPC STEARIC CAS-19420-57-6 1-Stearoyl-sn-glycero-3-phosphocholine, Milk Sphingomyelin, MPPC 1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine, MSPC 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine, PMPC 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, POPC CAS-26853-31-6 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPE 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPG-NA CAS-81490-05-31-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(l-glycerol)] (Sodium Salt), PSPC 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, SMPC 1-Stearoyl-2-myristoyl-snglycero-3-phosphocholine, SOPC 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, SPPC1-S tearoy 1-2-palmitoy 1-sn-glycero-3-phosphocholine. In some preferred embodiments, specific non-limiting examples of phospholipids include: dimyristoylphosphatidylcholine (DMPC), soy lecithin, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dilaurylolyphosphatidylcholine (DLPC), dioleoylphosphatidylcholine (DOPC), dilaurylolylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), dipalmitoyl sphingomyelin (DPSP), distearoyl sphingomyelin (DSSP), and mixtures thereof. In embodiments, the phospholipid is 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), and the lysolipid is 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC). In embodiments, the phospholipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and the lysolipid is 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC). In embodiments, when the present composition comprises (or consists essentially of, or consists of) two or more types of lipids (such as a phospholipid, or a lysolipid), the weight ratio of two types of phospholipids ranges from about 1:10 to about 10:1, including about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, to about 10:1, including all values and ranges therebetween. In embodiments, the HDL-derived nanoparticles comprise DMPC, and MHPC, and the weight ratio of DMPC to MHPC may range from about 1:10 to about 10:1, from about 2:1 to about 4:1, from about 1:1 to about 5:1, from about 2:1 to about 5:1, from about 6:1 to about 10:1, from about 7:1 to about 10:1, from about 8:1 to about 10:1, from about 7:1 to about 9:1, or from about 8:1 to about 9:1. The weight ratio of DMPC to MHPC may be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1, including all values and ranges therebetween. In embodiments, the HDL-derived nanoparticles comprise POPC and PHPC, and the weight ratio of POPC to PHPC may range from about 1:10 to about 10:1, from about 2:1 to about 4:1, from about 1:1 to about 5:1, from about 2:1 to about 5:1, from about 6:1 to about 10:1, from about 7:1 to about 10:1, from about 8:1 to about 10:1, from about 7:1 to about 9:1, or from about 8:1 to about 9:1. The weight ratio of POPC to PHPC may be about 1:10, about 1:9, about 1:8, about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In embodiments, the phospholipids in nanoparticles of the present disclosure comprise (or consists essentially of, or consists of) a mixture of a two-chain diacyl-phospholipid and a single chain acyl-phospholipid/lysolipid. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises apoA-I or a peptide mimetic of apoA-I, and a phospholipid. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises apoA-I or a peptide mimetic of apoA-I, a phospholipid, and a compound of Formula (I), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2). In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; iii) a lysolipid, and iv) cholesterol. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; iii) a lysolipid, iv) cholesterol and a compound of Formula (I), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2). In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; and iii) cholesterol. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; iii) cholesterol, and a compound of Formula (I), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2). In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; iii) a lysolipid, iv) a hydrophobic matrix core and v) cholesterol. In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; iii) a lysolipid, iv) a hydrophobic matrix core v) cholesterol and a compound of Formula (I), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2). In embodiments, the high-density lipoprotein (HDL)-derived nanoparticle comprises i) apoA-I or a peptide mimetic of apoA-I; ii) a phospholipid; iii) a lysolipid, iv) a triglyceride v) cholesterol, and a compound of Formula (I), (II), (II-1), (II-2), (II-A), (IIA-1) or (IIA-2). In embodiments, the structure and properties of the HDL-derived nanoparticles (e.g., particle size, rigidity, viscosity, loading, etc.) can be modified by incorporating a hydrophobic matrix. As used herein, hydrophobic matrix refers to a core or filler or structural modifier of the nanobiologic. Non-limiting examples of suitable hydrophobic matrix molecules include, triglycerides, fatty acid esters, hydrophobic polymers, sterol esters, or combinations thereof. For example, the inclusion of one or more triglycerides and/or one or more polymers in the nanoparticles disclosed herein, may facilitate modulation of nanoparticle size (e.g., from about 10 nm to over 100 nm) and shape (from discoisal to spherical). In turn, the size, rigidity, and viscosity of the HDL-derived nanoparticle may also affect loading and biodistribution. In a non-limiting example, a HDL-derived nanoparticle comprising phospholipids and apoA-I may have a diameter of about 10 nm to about 50 nm, and adding a hydrophobic matrix molecule (such as triglycerides), swells the HDL-derived nanoparticle from a minimum of about 10 nm to at least about 30 nm. Adding more triglycerides can further increase the diameter of the HDL-derived nanoparticle to at least 50 nm, at least 75 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 300 nm, and up to 400 nm, including all values and ranges therebetween. Any suitable synthetic or natural fatty acid or fatty acid ester, known in the art are contemplated for use in the HDL-derived nanoparticles of the present disclosure. Non-limiting examples of fatty acids of use include: arachidonic acid, oleic acid, arachidic acid, lauric acid, sad, capric acid, myristic acid, Palmic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, three decanoins, glycerin mono-fatty acid ester, Dilaurin, 1-Sunsoft 767, laurocapram (1-dodecyl-aza-cycloheptane-2-ketone), acylcarnitines, acyl group choline or C1-C10arrcostab (such as isopropyl myristate IPM), monoglyceride, diglyceride or its pharmaceutically acceptable salt. Any suitable synthetic or natural triglycerides, known in the art are contemplated for use in the HDL-derived nanoparticles of the present disclosure. Non-limiting examples of triglycerides of use include: tricaprylin, tristearin, triolein, tripalmitin, 1,2-dipalmitoolein, 1,3-dipalmitoolein, 1-palmito-3-stearo-2-olein, 1-palmito-2-stearo-3-olein, 2-palmito-1-stearo-3-olein, trilinolein, 1,2-dipalmitolinolein, 1-palmito-dilinolein, 1-stearo-dilinolein, 1,2-diacetopalmitin, 1,2-distearo-olein, 1,3-distearo-olein, trimyristin, trilaurin and combinations thereof. Suitable triglycerides may be added to the present compositions in neat form. Additionally, or alternatively, oils and/or processed oils containing suitable triglycerides may be added to the compositions. Non-limiting examples of oils include coconut oil, corn germ oil, olive oil, palm seed oil, cottonseed oil, palm oil, rapeseed oil, sunflower, whale oil, soybean oi, peanut oil, linseed oil, tall oil, and combinations thereof. The hydrophobic polymer or polymers may be selected from the group of polymers approved for human use (i.e. biocompatible and FDA-approved). Such polymers include, for example, but are not limited to the following polymers, derivatives of such polymers, co-polymers, block co-polymers, branched polymers, and polymer blends: polyalkenedicarboxlates, polyanhydrides, poly(aspartic acid), polyamides, polybutylenesuccinates (PBS), polybutylenesuccinates-co-adipate (PBSA), poly(8-caprolactone) (PCL), polycarbonates including poly-alkylene carbonates (PC), polyesters including aliphatic polyesters and polyester-amides, polyethylenesuccinates (PES), polyglycolides (PGA), polyimines and polyalkyleneimines (Pl, PAI), polylactides (PLA (polylactic acid), PLLA, PDLLA), polylactic-co-glycolic acid (PLGA), poly(l-lysine), polymethacrylates, polypeptides, polyorthoesters, poly-p-dioxanones (PPDO), (hydrophobic) modified polysaccharides, polysiloxanes and poly-alkyl-siloxanes, polyureas, polyurethanes, and polyvinyl alcohols, and biodegradable polyalkyl-cyanoacrylate. In embodiments of the HDL-derived nanoparticle of the present disclosure, the addition of cholesterol to the nanoparticle carrier stabilizes the composition and improves entrapment efficiency. Typically, the HDL-derived nanoparticle comprises from about 1 mol % to about 100 mol % of cholesterol relative to phospholipid (e.g., relative to DMPC), including about 1% mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol %, about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55 mol %, about 60 mol %, about 65 mol %, about 70 mol %, about 75 mol %, about 80 mol %, about 85 mol %, about 90 mol %, about 95 mol %, to about 100 mol % (i.e. 1:1 mol/mol mixture of cholesterol and phospholipd (e.g. DMPC) including all ranges and values therebetween. In embodiments, the HDL-derived nanoparticle comprises from about 1 mol % to about 30 mol % cholesterol. In embodiments, the HDL-derived nanoparticle comprises from about 15 mol % to about 25 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 20 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 10 mol % to about 35 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 15 mol % to about 30 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 15 mol % to about 25 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 28 mol % to about 23 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 20 mol % to about 27 mol % cholesterol, relative to phospholipid. In embodiments, the HDL-derived nanoparticle is cholesterol free. In embodiments, the molar ratio of cholesterol:phospholipid, in the HDL-derived nanoparticle is about 0:1, about 0.025:1, about 0.05:1, about 0.075:1, about 0.1:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1, about 0.25:1, about 0.275:1, about 0.3:1, about 0.325:1, about 0.35:1, about 0.375:1, about 0.4:1, about 0.425:1, about 0.45:1, about 0.475:1 or about 0.5:1, including all values therebetween. In embodiments, the molar ratio of cholesterol:phospholipids ranges from about 0:1 to about 0.5:1, including about 0:1, about 0.025:1, about 0.05:1, about 0.075:1, about 0.1:1, about 0.125:1, about 0.15:1, about 0.175:1, about 0.2:1, about 0.225:1, about 0.25:1, about 0.275:1, about 0.3:1, about 0.325:1, about 0.35:1, about 0.375:1, about 0.4:1, about 0.425:1, about 0.45:1, about 0.475:1 to about 0.5:1, including all ranges therebetween. In embodiments, the molar ratio of cholesterol:phospholipids ranges from about 0.05:1 to about 0.25:1. In embodiments, the molar ratio of cholesterol is about 0.2:1. In embodiments, the HDL-derived nanoparticle comprises one or more phospholipids and cholesterol in a molar ratio in the range of about 1:0.05 to about 1:0.25. In embodiments, the HDL-derived nanoparticle comprises one or more phospholipids and cholesterol in a molar ratio of about 1:0.2. In embodiments, the weight percentage of cholesterol ranges from about 0% (w/w) to about 15% (w/w) of the nanoparticle, lipid, or composition, including from about 1% (w/w), about 1.5% (w/w), about 2% (w/w), about 2.5% (w/w), about 3% (w/w), about 3.5% (w/w), about 4% (w/w), about 4.5% (w/w), about 5% (w/w), about 5.5% (w/w), about 6% (w/w), about 6.5% (w/w), about 7% (w/w), about 7.5% (w/w), about 8% (w/w), about 8.5% (w/w), about 9% (w/w), about 9.5% (w/w), about 10% (w/w), about 10.50% (w/w), about 110% (w/w/), about 11.50% (w/w), about 12% (w/w), about 12.5% (w/w), about 13% (w/w), about 13.5% (w/w), about 14% (w/w), about 14.5% (w/w), to about 15% (w/w). In embodiments, the weight percentage of cholesterol ranges from about 0% (w/w) to about 15%, (w/w) of the nanoparticle, lipid, or composition, including from about 1% (w/w), about 1.5% (w/w), about 2% (w/w), about 2.5% (w/w), about 3% (w/w), about 3.5% (w/w), about 4% (w/w), about 4.5% (w/w), about 5% (w/w), about 5.5% (w/w), about 6% (w/w), about 6.5% (w/w), about 7% (w/w), about 7.5% (w/w), about 8% (w/w), about 8.5% (w/w), about 9% (w/w), about 9.5% (w/w), about 10% (w/w), about 10.5% (w/w), about 11% (w/w/), about 11.5% (w/w), about 12% (w/w), about 12.5% (w/w), about 13% (w/w), about 13.5% (w/w), about 14% (w/w), about 14.5% (w/w), to about 15% (w/w). In embodiments, the weight percentage is the weight percentage of cholesterol relative to phospholipids. In embodiments, the weight percentage of cholesterol ranges from about 1 to 10% cholesterol (w/w %) of the composition. the weight percentage of cholesterol ranges from about 2 to 8% cholesterol (w/w %) of the composition. In embodiments, the weight percentage of cholesterol ranges from about 3.5 to 7.5% cholesterol (w/w %) of the composition. In embodiments, the weight percentage of cholesterol ranges from about 5 to 10% cholesterol (w/w %) of the composition. In embodiments, the weight percentage of cholesterol is about 3.6 (w/w %) of the composition. In embodiments, the weight percentage of cholesterol is about 7.2 (w/w %) of the composition. In embodiments, the weight percentage of cholesterol is about 5.9 (w/w %) of the composition. In embodiments, the size and circulating time of the nanoparticles can be modulated, for example, by controlling the ratio of lipids-to-APOAl and the ratio of lipids to polymer or lipids to triglyceride. In embodiments, the HDL-derived nanoparticle comprises from about a 5:1 to 1000:1 ratio (e.g., on a molar basis) of phospholipids:apoA-I or a mimetic of apoA-I, including about 5:1, about 10:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, about 100:1, about 110:1, about 120:1, about 130:1, about 140:1, about 150:1, about 160:1, about 170:1, about 180:1, about 190:1, about 200:1, about 210:1, about 220:1 about 230:1, about 240:1, about 250:1, about 260:1, about 270:1, about 280:1, about 290:1, about 300:1, about 310:1, about 320:1, about 330:1, about 340:1, about 350:1, about 360:1, about 370:1, about 380:1, about 390:1, about 400:1, about 410:1, about 420:1, about 430:1, about 440:1, about 450:1, about 460:1, about 470:1, about 480:1, about 490:1, about 500:1, about 510:1, about 520:1, about 530:1, about 540:1, about 550:1, about 560:1, about 570:1, about 580:1, about 590:1, about 600:1, about 610:1, about 620:1, about 630:1, about 640:1, about 650:1, about 660:1, about 670:1, about 680:1, about 690:1, about 700:1, about 710:1, about 720:1 about 730:1, about 740:1, about 750:1, about 760:1, about 770:1, about 780:1, about 790:1, about 800:1, about 810:1, about 820:1, about 830:1, about 840:1, about 850:1, about 860:1, about 870:1, about 880:1, about 890:1, about 900:1, about 910:1, about 920:1, about 930:1, about 940:1, about 950:1, about 960:1, about 970:1, about 980:1, about 990:1, to about 1000:1, including all subranges and values therebetween. In embodiments, the HDL-derived nanoparticle comprises from about a 10:1 to 1000:1 ratio (e.g., on a molar basis) of phospholipids:apoA-I or a mimetic of apoA-I. In embodiments, the HDL-derived the nanoparticle comprises from about a 70:1 to 125:1 ratio (e.g., on a molar basis) of phospholipids:apoA-I. In embodiments, the HDL-derived the nanoparticle comprises from about a 5:1 to 10:1 ratio (e.g., on a molar basis) of mimetic of apoA-I. In embodiments, the HDL-derived nanoparticle comprises from about a 2:1 to 3:1 ratio by weight of phospholipids:apoA-I or a mimetic of apoA-I. In embodiments, the HDL-derived nanoparticle comprises from about, or at least about 0.1 mol % to about 100 mol % of a compound of Formula I relative to phospholipid (e.g., DMPC), including about, or at least about 0.1 mol %, about or at least about 0.5 mol %, about or at least about 0.75 mol %, about, or at least about 1% mol %, about, or at least about 2 mol %, about, or at least about 3 mol %, about, or at least about 4 mol %, about, or at least about 5 mol %, about, or at least about 6 mol %, about, or at least about 7 mol %, about, or at least about 8 mol %, about, or at least about 9 mol %, about, or at least about 10 mol %, about, or at least about 11 mol %, about, or at least about 12 mol %, about, or at least about 13 mol %, about, or at least about 14 mol %, about, or at least about 15 mol %, about, or at least about 16 mol %, about, or at least about 17 mol %, about, or at least about 18 mol %, about, or at least about 19 mol %, about, or at least about 20 mol %, about, or at least about 21 mol %, about, or at least about 22 mol %, about, or at least about 23 mol %, about, or at least about 24 mol %, about, or at least about 25 mol %, about, or at least about 26 mol %, about, or at least about 27 mol %, about, or at least about 28 mol %, about, or at least about 29 mol %, to about, at least about 30 mol %, about or at least about or at least about 35 mol %, about or at least about 40 mol %, about or at least about 45 mol %, about or at least about 50 mol %, about or at least about 55 mol %, about or at least about 60 mol %, about or at least about 65 mol %, about or at least about 70 mol %, about or at least about 75 mol %, about or at least about 80 mol %, about or at least about 85 mol %, about or at least about 90 mol %, about or at least about 95 mol %, to about or at least about 100 mol % (1:1 mol/mol mixture of compound and phospholipd (e.g. DMPC)), including all ranges and values therebetween. In embodiments, the HDL-derived nanoparticle comprises from about 10 mol % to about 30 mol % of a compound of Formula I relative to phospholipid. In embodiments, the HDL-derived nanoparticle comprises from about 12 mol % to about 25 mol % compound, relative to phospholipid. In embodiments, the nanoparticle size ranges from about 5 nm to about 500 nm in diameter, including about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, to about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 320 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, about 400 nm, about 410 nm, about 420 nm, about 430 nm, about 440 nm, about 450 nm, about 460 nm, about 470 nm, about 480 nm, about 490 nm, to about 500 nm, including all ranges and values therebetween. In embodiments, the nanoparticle size is less than about 50 nm. In embodiments, the nanoparticle size is about 50 nm to about 100 nm, or about 5 nm to about 30 nm. In embodiments, the nanoparticle sizes are measured by dynamic light scattering (DLS). In embodiments, to target immune cells in tissue with limited access to circulation, nanoparticles having long blood half-lives and small size (<50 nm) may be used. In embodiments, to target immune cells in well-perfused tissues, nanoparticles having short blood half-lives and large size (about 100 nm) may be used. These tissues include spleen, liver, kidney, lungs, and bone marrow. In embodiments, the HDL-derived nanoparticle is discoidal in shape. In embodiments, the HDL-derived nanoparticle is spherical in shape. In embodiments the HDL-derived nanoparticle morphology is visualized by transmission electron microscopy (TEM). In embodiments, the length of the HDL-derived nanoparticle is about 5 to about 100 nm in length, including about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, to about 100 nm in length, including all ranges and values therebetween. In embodiments, the HDL-derived nanoparticle is about 10 nm to 80 nm in length. In embodiments, the HDL-derived nanoparticle is about 15 nm to 50 nm in length. In embodiments, the HDL-derived nanoparticle is longer than about 10 nm, or longer than about 15 nm. In embodiments, the HDL-derived nanoparticle has a thicknesses of about 1 nm to 10 nm, including about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, to about 10 nm, including all ranges and values therebetween. In embodiments, the thicknesses of the HDL-particles are about 1 to 10 nm, or 2 to 7 nm, or 3 to 6 nm. In embodiments, the dimensions (e.g., length and thickness) are recorded by cryo-TEM. In embodiments, the HDL-particles have a worm-like morphology by cryo-TEM. In embodiments, the HDL-derived nanoparticle is discoidal in shape with a diameter between about 5 nm to about 50 nm (e.g., as measured by dynamic light scattering (DLS)), including about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 40 nm, to about 50 nm, including all subranges and values therebetween. In embodiments, the nanodisc is about 5 nm to about 30 nm in diameter. In embodiments, the HDL-derived nanoparticle is spherical in shape with a diameter between about 10 nm to about 400 nm in diameter (e.g., as measured by dynamic light scattering (DLS)), including about 10 nm, about 15 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, about 310 nm, about 330 nm, about 340 nm, about 350 nm, about 360 nm, about 370 nm, about 380 nm, about 390 nm, to about 400 nm in diameter, including all values and ranges therebetween. In embodiments, the nanosphere is between about 15 nm to about 250 nm in diameter. In embodiments, the nanosphere is between about 30 nm, about 100 nm in diameter. Stability of the HDL-derived nanoparticle can be assessed by performing DLS measurements. In embodiments, the HDL-derived nanoparticle is stable for at least about 1 week, or at least about 2 weeks, or at least about 5 weeks e.g., by DLS. In embodiments, the nanobiologic composition promotes a hyper-responsive innate immune response in the patient in need thereof. In embodiments, the hyper-responsive innate immune response is promoted for at least about 7 to about 30 days. In embodiments, the hyper-responsive innate immune response is promoted for at least 30 to 100 days. In embodiments, the hyper-responsive innate immune response is promoted for more than 100 days and up to 3 years. In embodiments, the nanobiologic composition is administered once and wherein the hyper-responsive innate immune response is promoted for at least 30 days. In embodiments, the nanobiologic composition is administered at least once per day in each day of a multiple-dosing regimen, and wherein the hyper-responsive innate immune response is promoted for at least 30 days. Production methods can prepare uniform size HDL-derived nanoparticles, or a non-uniform sized mixture of HDL-derived nanoparticles, either by not filtering, or by preparing a range of different sized HDL-derived nanoparticles and re-combining them in a post-production step. The larger the size of the HDL-derived nanoparticles, the more drug can be incorporated. However, larger sizes e.g. >120 nm, can limit, prevent or slow diffusion of the HDL-derived nanoparticles into the tissues of the patient being treated. Smaller HDL-derived nanoparticles do not hold as much drug per particle, but are able to access the bone marrow, blood, or spleen, or other localized tissue affected by trained immunity, e.g. myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood and/or spleen, and so forth (biodistribution). Using a non-uniform mixture of nanoparticles sizes in a single administration or regimen can produce an immediate reduction in innate immune hyper-responsiveness, and simultaneously produce a durable, long-term reduction in innate immune hyper-responsiveness that can last days, weeks, months, and years, wherein the nanobiologic has reversed, modified, or reregulated the metabolic, epigenetic, and inflammasome pathways of the hematopoietic stem cells (HSC), the common myeloid progenitors (CMP), and the myeloid cells such as monocytes, macrophages and other short-lived circulating cells. In embodiments, the maximum loading capacity of the HDL-derived nanoparticle can be determined dividing the volume of the interior of the HDL-derived nanoparticle by the volume of a drug-load spheroid. Particle: assume a 100 nm spherical particle having 2.2 nm-3.0 nm phospholipid wall, yielding a 94 nm diameter interior with volume (L) @4/3n(r)3. Drug: assume STIMULATOR at 12×12×35 Angstrom or as a cylinder 1.2×1.2×3.5 nm, where multiple drug molecule cylinders, e.g. seven or nine, etc. could assume a 3.5 nm diameter spheroid having a radius of 1.75 nm Vol (small) @ 4/3n(r)3. Maximum Loading Capacity (calc): −487 k 3.5 nm spheroids within a 100 nm particle. Formulations When employed as pharmaceuticals, the compounds and HDL-derived nanoparticles of the present disclosure are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. In embodiments, the pharmaceutical composition comprises a nanobiologic composition of the present disclosure, and a pharmaceutically acceptable carrier. Generally, the compounds of this invention are administered in a pharmaceutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound-administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. The pharmaceutical compositions of this invention can be administered by a variety of routes including oral, rectal, intraocular, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intradermal, directly into cerebrospinal fluid, intratracheal, and intranasal. Depending on the intended route of delivery, the compounds of this invention are preferably formulated as either injectable or oral compositions or as salves, as lotions or as patches all for transdermal administration. In embodiments, the composition is administered intraveneously or intraarterially. The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, a compound as described herein is usually a minor component (from about 0.1 to about 50% by weight or preferably from about I to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form. Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like. Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as an ointment, the active ingredients will typically be combined with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base. Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention. Nanoparticles described herein can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety. The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17thedition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference. For injection, nanoparticles described herein can be provided in an injection grade saline solution, in the form of an injectable liposome solution, slow-release polymer system or the like. Nanoparticles described herein can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences. Methods Provided herein are methods of treating a subject susceptible to or afflicated with immune-related diseases and conditions, including, for example, immunoparalysis in sepsis and infections, cell proliferation disorders (such as cancer), and other diseases and conditions caused by defective trained immunity. In embodiments, the present disclosure provides methods for treating a cell-proliferation disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a nanobiologic composition comprising a high-density lipoprotein (HDL)-derived nanoparticle comprising a compound of the present disclosure (such as a compound of Formula I). In embodiments, the compounds, compositions provided herein are useful for treating cancer by inducing trained immunity. In embodiments, the cell proliferation disorder is cancer. In embodiments, the cancer is one or more of the following cancers: advanced malignancy, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastase, glioblastoma multiforms, glioblastoma, brain stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant giolma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B-Cell lymphoma, diffuse large B-Cell lymphoma, low grade follicular lymphoma, malignant melanoma, malignant mesothelioma, malignant pleural effusion mesothelioma syndrome, peritoneal carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scelroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressive, hormone refractory prostate cancer, resected high-risk soft tissue sarcoma, unrescectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube cancer, androgen independent prostate cancer, androgen dependent stage IV non-metastatic prostate cancer, hormone-insensitive prostate cancer, chemotherapyinsensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma. In embodiments, the cancer is selected from the group consisting of bladder cancer, cancer of the blood vessels, bone cancer, brain cancer, breast cancer, cervical cancer, chest cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer, kidney cancer, liver cancer, cancer of the lymph nodes, lung cancer, mouth cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, colorectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, urothelial cancers, and uterine cancer. In embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, pancreatic cancer, and glioblastoma. In embodiments, the cancer is metastatic. In embodiments, the cancer is refractory or resistance to chemotherapy or radiation; in particular, refractory to thalidomide. In embodiments, the cancer is selected from the group consisting of bladder cancer, cancer of the blood vessels, bone cancer, brain cancer, breast cancer, cervical cancer, chest cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer, kidney cancer, liver cancer, cancer of the lymph nodes, lung cancer, mouth cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, colorectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, urothelial cancers, and uterine cancer. In embodiments, the cancer is selected from the group consisting of breast cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, pancreatic cancer, and glioblastoma. In embodiments, the present disclosure provides methods for treating sepsis comprising administering to a subject in need thereof a therapeutically effective amount of a nanobiologic composition of the present disclosure. In embodiments, the patient has sepsis associated with a bacterial, viral or fungal infection of the lungs, abdomen, kidney, or bloodstream. The compounds of the present disclosure and their carriers disclosed herein may be used to augment immune responses. Accordingly, disclosed herein are methods of inducing immune responses comprising administering an immunogenic composition to a subject, wherein the composition comprises (i) at least one antigen and (ii) a compound disclosed herein, optionally in a nanoparticle carrier, such as a HDL-derived nanoparticle or liposome. The antigens are typically from pathogens, though neoantigens from subjects having cancer may also be used. Illustrative pathogen antigens may be from a virus, a bacteria, a parasite or a yeast. In aspects, the antigen may be a secreted from a pathogen; for example, an exotoxin or an endotoxin. Exemplary viruses include Adenovirus, Adeno-associated virus (AAV), Chikungunya, Dengue, Influenza, Ebola, Epstein-Barr, Hanta, Hepatitis (e.g., Hepatitis A, B, C, D, E), CMV, HPV (e.g., one or more of HPV1-18), Coronavirus, (e.g., SARS, MERS, COVID-19), Polio, Rabies, Zika. Exemplary bacteria includeVibrio cholerae, E. coli, Salmonellaspp.,N. gonorrheae, N. meningitidis, Streptococcus pyogenes, Mycobacterium tuberculosis, Legionella pneumophila, Brucella bortus, andListeria monocytogenes. The antigen may be, for example, a polypeptide, including a glycosylated peptide, or a carbohydrate. In aspects, the immunogenic composition may contain a nucleic acid that encodes an antigen, typically polypeptide that is transcribed and/or translated from the nucleic acid. The nucleic acid may be a DNA or an RNA, or a derivative of DNA or of RNA. Common derivatives of RNA include covalent modification to the molecule to enhance stability and/or expression. In aspects, the nucleic acid encoding the polypeptide may be within a plasmid or a viral vector, such as adenoviral vectors, adeno-associated virus vectors, baculoviral vectors, lentiviral vectors, and the like. In aspects, the administration may be preventative; for example, to vaccinate the subject prior to exposure to the pathogen. In other aspects, the administration may be a treatment; for example, inducing an immune response against a tumor carrying neo-antigens in a subject suffering from cancer. In embodiments, the nanobiologic composition is administered in a treatment regimen comprising two or more doses to the patient to generate an accumulation of drug in myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood and/or spleen. In embodiments, the nanobiologic composition is administered intravenously or intra-arterially. Injection dose levels range from about 0.1 mg/kg/hour to at least 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 24 to 96 hours. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient. Oral dose levels range from about 0.01 to about 20 mg/kg of the compound of the invention, including all ranges and values there between. For example, dose levels range from about 0.1 to about 10 mg/kg or from about 1 to about 5 mg/kg. Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses. Modes of administration suitable for mucosal sites are also envisioned herein and include without limitation: intra-anal swabs, enemas, intranasal sprays, and aerosolized or vaporized compounds and/or compositions for delivery to the lung mucosa. One of skill in the art would choose an appropriate delivery models based on a variety of parameters, including the organ or tissue site in a patient with a disease or condition that is most severely affected by the disease or condition. The compounds of this invention can be administered as the sole active agent or they can be administered in combination with one or more additional pharmaceutical agents, including other compounds that demonstrate the same or a similar therapeutic activity and are determined to safe and efficacious for such combined administration. In embodiments, the additional pharmaceutical agent is an inhibitor of a checkpoint protein. In embodiments, the methods provided herein further comprise co-administering a cancer drug as a combination therapy with the nanobiologic composition. A compound or composition described herein can be provided in a kit. In some embodiment the kit includes (a) a compound described herein, or a composition that includes a compound described herein (wherein, e.g., the compound can be an NOD2 modulator described herein), and, optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein and/or the use of a compound or composition described herein for the methods described herein. In embodiments, the informational material can include information about production of the compound. In embodiments, the informational material relates to methods for administering the compound. In embodiments, the informational material can include instructions to administer a compound or composition described herein in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In embodiments, the informational material can include instructions to administer a compound described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein. The kit can include one or more containers for the composition containing a compound or composition described herein. In embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of a compound or composition described herein. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of a compound described herein. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. Also provided herein is a process for manufacturing a nanobiologic composition of the present disclosure, the process comprising:a) forming a lipid film comprising: i) a compound of the present disclosure; ii) one or more phospholipids; optionally iii) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or a combination thereof; and optionally iv) cholesterol; under conditions effective to form the lipid film; andb) dissolving the lipid film in a solvent to form a lipid solution; and contacting the lipid solution with apoA-I or a peptide mimetic of apoA-I under conditions effective to form a HDL-derived nanoparticle comprising a compound of the present disclosure. In embodiments, provided herein is a nanobiologic composition prepared according to the methods disclosed herein. EXAMPLES The therapeutic agents described herein and nanoparticles comprising same may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis. Materials and Methods All chemicals were purchased from commercial sources and used without further purification unless specified. N-methylmorpholine was redistilled, collecting the fraction from 110° C. to 112° C. Cholesterol azidoacetate was synthesized according known procedures (RSC Adv. 2015, 5, 12094), as was 1-azidooctadecane. Dry solvents were obtained with an MBRAUN Solvent Purification System (MB-SPS). Toluene was dried over 4 Å molecular sieves before use. Glassware used for reactions carried out under argon atmosphere was dried with a heat gun prior to use. Thin-layer chromatography (TLC) was performed using 60-F254 silica gel plates from Merck and visualized by UV light at 254 nm, permanganate staining and/or cerium molybdate (CeMo) staining. Normal and reversed-phase automated column chromatography was conducted on a Biotage Isolera One or Grace Reveleris X2 Flash Chromatography System using Biotage Sfar Silica, Buchi FlashPure ID Silica or Buchi FlashPure ID C18 columns. Elution gradients are specified in column volumes (CVs). Non-stabilized THF was used for the water/THF gradients. NMR spectra were recorded on Bruker 400 MHz Ultrashield spectrometer (400 MHz for 1H NMR). Deuterated solvents used are indicated in each case. Chemical shifts (6) are expressed in ppm and are referred to the residual peak of the solvent. Peak multiplicity is abbreviated as s: singlet; d: doublet; t: triplet; dt: doublet of triplets; ddt: doublet of doublets of triplets; td: triplet of doublets; tt: triplet of triplets; q: quartet; ABq: AB quartet; dq: doublet of quartets; qd: quartet of doublets; sept: septet; m: multiplet; bs: broad singlet. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were obtained on a PerSeptive Biosystems Voyager DE-PRO spectrometer using α-cyano-4-hydroxycinnamic acid (CHCA) or trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]-malononitrile (DCTB) as matrix. Gas chromatography-mass spectrometry (GC-MS) measurements were conducted on a Shimadzu GC-17A gas chromatograph with a Shimadzu AOC-20i auto injector, Shimadzu GCMS-QP5000 gas chromatograph mass spectrometer and Phenomenex Zebron ZB-35 column (1=30 meters, ID=0.25 mm, film thickness=0.25 μm). High-pressure liquid chromatography mass spectrometry (HPLC-ESI-MS) experiments using a water/acetonitrile gradient were performed a Shimadzu setup with 2×LC-20 AD pumps, DGU-20A3 degasser, SIL-20AC autosampler, SPD-M20A PDA and ThermoScientific LCQ fleet MS. Column: Phenomenex Kinetex 5 um EVO C18 100 Å LC (50×2.1 mm). Gradient: water/MeCN (+0.1% formic acid) from 5 to 100% MeCN, 0.300 mL/min. Electrospray ionization (ESI) was used to create the charges for MS-detection. HPLC-MS and HPLC-ELSD experiments with a water/THF or water/MeOH gradient were performed on a Shimadzu Nexera-i LC-2040C 3D Plus with Shimadzu LCMS-8045. Column: Alltech Alltima C18 (150×3.2 mm; 5 um; no. 88383). Gradient: water/THF (+0.1% TFA) or water/MeOH (+0.1% TFA), 0.400 mL/min. This HPLC setup was also used in combination with ELSD (evaporative light scattering detection). Alternatively, HPLC-MS(SIM) and HPLC-ELSD were performed on a Phenomenex Kinetex 5 micrometer EVO C18 100A LC-column (50×2.1 mm) employing a gradient from A to B eluent, where A=20 mM NH4HCO2in H2O with 0.1 v/v % formic acid, and B=2-propanol/MeCN/H2O 85:15:5, also with 20 mM NH4HCO2and 0.1 v/v % formic acid. Abbreviations HPLC=high performance liquid chromatography; ELSD=evaporative light scattering detection; ESI-MS=electrospray ionization mass spectrometry; SIM=selected ion mode; NMR=nuclear magnetic resonance. MDP=Muramyl dipeptide muramyl (or N-Acetylmuramyl-L-alanyl-D-isoglutamine) CAS [53678-77-6]. Was either prepared according to standard peptide synthesis or bought from commercial sources. NHS=N-hydroxy succinimide; DiC or DIC=N,N′-diisopropylcarbodiimide; EDC=N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (used hydrochloride); PyBOP=(benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate); SPPS=solid phase peptide synthesis. TEA=triethyl-amine; THF=tetrahydrofuran; MeOH=methanol; DMF=dimethylformamide; FA=formic acid; TFA=trifluoro-acetic acid. Building Blocks DSPE-azidoacetate (DSPE-CO—CH2—N3) (2R)-3-(((2-Aminoethoxy)(hydroxy)phosphoryl)oxy)propane-1,2-diyl distearate (260 mg, 0.35 mmol), 2,3,5,6-tetrafluorophenyl 2-azidoacetate (prepared according to D. J. Vugts et al., Bioconjugate Chem. 2011, 22, 2072-2081; 87 mg, 0.35 mmol, 1 eq) and N,N-diisopropylethylamine (184 μL, 1.1 mmol, 3 eq) were combined in chloroform (2 mL). The mixture was stirred at 50° C. for 1 h during which the white suspension cleared. Chloroform (200 mL) was added and the organic layer was gently washed twice with 1 M HCl (100 mL). After drying with MgSO4, filtration and removal of the solvent in vacuo, the compound was purified with column chromatography (flash SiO2) using an elution gradient of 5% to 40% MeOH in chloroform. This yielded pure DSPE-azidoacetate (244 mg, 0.29 mmol, 84%) as a white solid.1H-NMR (400 MHz, CDCl3/CD3OD 9:1): δ=5.23 (dt, J=9.0, 4.6 Hz, 1H), 4.35 (dd, J=12.0, 3.7 Hz, 1H), 4.22-3.99 (m, 5H), 3.95 (s, 2H), 3.53 (t, J=5.1 Hz, 2H), 2.33 (q, J=7.6 Hz, 4H), 1.61 (td, J=7.4, 4.2 Hz, 4H), 1.48-1.16 (m, 56H), 0.88 (t, J=6.7 Hz, 6H).13C-NMR (101 MHz, CDCl3): δ=173.7, 173.4, 168.3, 69.7, 69.6, 66.1, 66.0, 65.2, 62.1, 52.5, 40.01, 39.95, 34.3, 34.2, 34.1, 32.1, 29.9, 29.80, 29.7, 29.62, 29.59, 29.50, 29.47, 29.46, 29.4, 29.30, 29.26, 25.00, 24.97, 24.9, 22.8, 14.2.31P-NMR (162 MHz, CDCl3): δ=−0.48. MALDI-TOF MS: m/z Calc. for C43H83N4O9P 830.59; Obs. [M+Na]+853.62, [M−H+2Na]+875.58. MDP-propargyl A 50 mL round-bottomed flask was filled with MDP (0.113 g, 0.23 mmol, 1.00 eq). The material was dissolved in dry DMF (˜1.5 mL, 0.15 M) and the flask purged with argon. EDC·HCl (0.066 g, 0.34 mmol, 1.50 eq) and N,N-diisopropylethylamine (0.050 g, 0.068 mL, 0.39 mmol, 1.70 eq) and 4-(N,N-dimethylamino)pyridine (0.0028 g, 0.023 mmol, 0.10 eq) were added, and the resulting clear solution stirred at RT for 5 min. Next, prop-2-yn-1-amine (0.018 g, 0.021 mL, 0.32 mmol, 1.40 eq) was added syringe. Stirring was continued at RT. After 21 h reaction time, LC-MS (water/MeOH) confirmed full conversion of the MDP starting material. The reaction mixture was concentrated in vacuo, giving crude product as a yellow glass. The material was purified twice by automated column chromatography (reversed-phase (C18); product:C18-silica 1:100; detection: 200-400 nm), eluting with water/MeOH 90/10-82/18. Pure fractions were lyophilized, giving pure product as a white solid (0.050 g, 41%). 1H NMR (400 MHz, MeOD) δ 5.16 (d, J=3.4 Hz, 1H), 4.42-4.23 (m, 3H), 3.95 (t, J=2.3 Hz, 2H), 3.94-3.57 (m, 5H), 3.52-3.39 (m Hz, 1H), 2.58 (t, J=2.6 Hz, 1H), 2.33-2.26 (m, 2H), 2.25-2.13 (m, 1H), 2.00-1.85 (m, 4H), 1.45-1.33 ppm (m, 6H).13C NMR (100 MHz, MeOD) δ 175.26, 174.83, 173.89, 173.06, 172.08, 91.01, 79.15, 78.92, 76.68, 71.86, 70.83, 70.21, 63.35, 61.22, 54.13, 52.66, 49.48, 31.54, 28.11, 27.10, 23.85, 21.46, 18.31, 16.21 ppm. HPLC-MS (water/MeCN): t (product)=0.76 and 1.02 min. Found: m/z=512.08 [M−H2O+H]+; 552.33 [M+Na]+(pos. mode); 325.17 [M-muramyl]− (neg. mode). MDP(Bn) MDP(Bn) was synthesized using standard SPPS methods in an 100 mL glass reactor vessel with glass frit filter bottom. Sufficient agitation of the reaction mixture was ensured by applying a constant argon flow through the glass frit filter, whilst excess reagent and washing solutions were removed by vacuum filtration. The crude MDP(Bn) was purified twice by automated column chromatography (reversed-phase (C18); product:C18-silica 1:200; detection: 200-400 nm), eluting with water/MeCN+0.1% formic acid 90/10-82/18. Pure fractions were lyophilized, giving pure product as a fluffy, white material (0.309 g, 67%). 1H NMR (400 MHz, DMF-d7) δ 8.18 (d, J=8.6 Hz, 1H), 8.15 (d, J=8.6 Hz, 1H), 7.75 (d, J=6.5 Hz, 1H), 7.53-7.28 (m, 6H), 7.11-7.03 (m, 1H), 4.86 (d, J=3.5 Hz, 1H), 4.76 (d, J=12.3 Hz, 1H), 4.72-4.56 (m, 1H), 4.51 (d, J=12.3 Hz, 1H), 4.47-4.32 (m, 3H), 4.01 (ddd, J=10.7, 8.4, 3.5 Hz, 1H), 3.83 (dd, J=11.6, 2.2 Hz, 1H), 3.78-3.61 (m, 3H), 3.60-3.40 (m, 1H), 2.39 (t, J=7.8 Hz, 2H), 2.24-2.12 (m, 1H), 1.97-1.83 (m, 4H), 1.40 (d, J=7.0 Hz, 3H), 1.34 ppm (d, J=6.7 Hz, 3H).13C NMR (100 MHz, DMF-d7) δ 174.30, 173.74, 173.60, 172.82, 170.19, 138.47, 128.58, 127.93, 127.79, 97.05, 80.16, 77.36, 73.94, 70.66, 68.61, 61.76, 53.64, 52.59, 49.41, 35.63, 30.57, 30.47, 27.78, 22.65, 19.05, 18.04 ppm. HPLC-MS (water/MeCN): t(product)=3.11 min. Found: m/z=583.08 [M+H]+. MDP(Bn)-propargyl A 5 mL round-bottomed flask was charged with MDP(Bn) (0.110 g, 0.19 mmol, 1.00 eq) under argon atmosphere. The material was dissolved in dry DMF (0.5 mL). PyBOP (0.127 g, 0.25 mmol, 1.30 eq) and N,N-diisopropylethylamine (0.049 g, 0.066 mL, 0.38 mmol, 2.00 eq) were added, resulting in a clear, colorless solution. The mixture was stirred for 5 minutes at RT. Next, prop-2-yn-1-amine (0.021 g, 0.024 mL, 0.38 mmol, 2.00 eq) was added and the resulting light yellow mixture stirred at RT. After 2 h reaction time, the reaction mixture was concentrated in vacuo, giving crude product as a beige, sticky solid. The material was purified by automated column chromatography (reversed-phase (C18); product:C18-silica 1:100; detection: 200-400 nm), eluting with water/MeCN 90/10-80/20. Pure fractions were lyophilized, giving pure product as a white solid (0.096 g, 82%). 1H NMR (400 MHz, DMF-d7) δ 8.25 (t, J=5.5 Hz, 1H), 8.20-8.15 (m, 2H), 7.75 (d, J=6.6 Hz, 1H), 7.51-7.28 (m, 6H), 7.08-7.03 (m, 1H), 5.47 (d, J=6.3 Hz, 1H), 4.86 (d, J=3.5 Hz, 1H), 4.76 (d, J=12.3 Hz, 1H), 4.63 (t, J=6.0 Hz, 1H), 4.51 (d, J=12.4 Hz, 1H), 4.47-4.27 (m, 3H), 4.05-3.95 (m, 3H), 3.83 (ddd, J=11.5, 5.7, 2.2 Hz, 1H), 3.75-3.61 (m, 3H), 3.54-3.46 (m, 1H), 3.04 (t, J=2.5 Hz, 1H), 2.32-2.26 (m, 2H), 2.21-2.11 (m, 1H), 1.95-1.83 (m, 4H), 1.39 (d, J=7.0 Hz, 3H), 1.34 ppm (d, J=6.8 Hz, 3H).13C NMR (100 MHz, DMF-d7) δ 173.79, 173.59, 172.74, 171.93, 170.19, 138.47, 128.58, 127.93, 127.80, 97.05, 81.35, 80.16, 77.36, 73.94, 72.14, 70.63, 68.62, 61.76, 53.65, 52.94, 49.38, 35.63, 32.21, 30.47, 28.31, 28.25, 22.66, 19.01, 18.06 ppm. HPLC-MS (water/MeCN): t (product)=3.35 min. Found: m/z=620.17 [M+H]+. MTP-b on Resin MTP-b on-resin was synthesized using standard SPPS methods in an 100 mL glass reactor vessel with glass frit filter bottom. Sufficient agitation of the reaction mixture was ensured by applying a constant argon flow through the glass frit filter, whilst excess reagent and washing solutions were removed by vacuum filtration. After performing the final post-coupling wash, the resin was washed again with DCM (2×20 mL) and dried in an argon flow. The material was stored at −20° C. A sample was cleaved from the resin using TFA/TIPS/water 95/2.5/2.5 (0.1 mL, 10 min) and checked with HPLC-MS (water/MeCN). HPLC-MS (water/MeCN): t (product)=3.65 min. Found: m/z=874.42 [M+H]+(pos. mode); m/z=918.08 [M+HCOO]−(neg. mode). MTP-b-N3 A 20 mL PE syringe with PE frit was charged with on-resin MTP-b (429 mg, approx. 0.15 mmol MTP-b) and the resin was swollen in DMF (12 mL) for 30 min. The resin was treated twice with 2% hydrazine hydrate solution in DMF (12 mL) for 15 min. After filtration the resin was washed with DMF (4×12 mL) for 1 min. A solution of CuSO4·5H2O (0.6 mg, 2.4 μmol, 1.5 mol %), imidazole-1-sulfonyl azide HCl-salt (170 mg, 0.77 mmol, 5 eq) and N,N-diisopropylethylamine (0.34 mL, 1.9 mmol, 12 eq) in DMF (12 mL) was added to the resin and the beads were agitated at room temperature for 24 h (slight overpressure was relieved every now and then). After filtration the resin was washed with DMF (5×12 mL) for 1 min and dichloromethane (4×10 mL) for 1 min. The resin was then subjected to cleavage in TFA/TIPS/H2O 95:2.5:2.5 (4 mL) for 2 h. After filtration the resin was washed with TFA (4 mL) for 5 min. The combined TFA filtrates were concentrated in vacuo (keeping the temperature as low as possible to avoid TFA-ester formation). Automated column chromatography (reversed-phase (C18); detection: λ=200 nm), using an elution gradient of 5% to 60% MeCN in H2O (both containing 0.1% TFA) yielded impure compound. This was further purified with RP-HPLC using an elution gradient of 26% to 35% MeCN in H2O (both containing 0.1% TFA) yielding pure product (37.5 mg, 51 μmol, 34%) as a white fluffy solid after lyophilization.1H-NMR (400 MHz, DMF-d7/D2O 9:1): δ=8.45 (t, J=9.1 Hz, 2H), 8.26 (d, J=7.9 Hz, 1H), 7.99 (d, J=6.5 Hz, 1H), 7.81 (d, J=2.8 Hz, 2H), 7.64-7.45 (m, 5H), 7.31 (d, J=17.2 Hz, 2H), 5.82 (d, J=6.3 Hz, 1H), 5.03 (d, J=3.5 Hz, 1H), 4.93 (d, J=12.4 Hz, 1H), 4.68 (d, J=12.4 Hz, 1H), 4.63-4.46 (m, 4H), 4.19 (dd, J=10.7, 3.6 Hz, 1H), 4.00 (dd, J=6.8, 6.3 Hz, 1H), 3.92-3.79 (m, 3H), 3.66 (t, J=9.0 Hz, 1H), 3.52 (t, J=6.8 Hz, 2H), 2.53 (t, J=7.5 Hz, 2H), 2.37 (dtd, J=16.5, 7.9, 4.3 Hz, 1H), 2.10 (s, 3H), 2.09-1.93 (m, 2H), 1.88-1.55 (m, 5H), 1.58 (d, J=7.1 Hz, 3H), 1.52 (d, J=6.7 Hz, 3H).13C-NMR (100 MHz, DMF-d7/D2O 9:1): δ=174.9, 174.24, 174.17, 174.00, 173.93, 173.14, 173.06, 172.9, 172.8, 170.90, 170.8, 138.2, 128.6, 127.9, 127.8, 96.8, 80.0, 77.3, 73.7, 70.3, 68.6, 61.5, 53.6, 53.5, 53.2, 53.13, 53.10, 52.7, 52.6, 51.2, 49.4, 49.3, 32.1, 31.7, 28.5, 28.2, 23.2, 22.53, 22.48, 18.9, 17.80, 17.76. ESI-MS: m/z Calc. for C32H49N9O11735.36; Obs. [M+H]+736.25, [M+Na]+758.42. DSG 4-nitrophenylcarbonate A 25 mL round-bottom flask was filled with a solution of commercially available [(2S)-3-hydroxy-2-octadecanoyloxypropyl] octadecanoate (0.601 g, 0.96 mmol, 1.00 eq) in chloroform (6.5 mL, ˜0.15 M). Pyridine (0.122 g, 0.125 mL, 1.54 mmol, 1.60 eq) was added and the resulting clear solution cooled in icewater. Next, solid 4-nitrophenyl chloroformate (0.252 g, 1.25 mmol, 1.30 eq) was added in small portions. The light yellow reaction mixture was stirred at RT overnight. Full conversion of the alcohol was confirmed by1H NMR (CDCl3). Subsequently, the reaction mixture was precipitated in MeOH (100 mL) and collected by filtration through glass filter. The material was washed with MeOH (30 mL total) and Et2O (10 mL total) and dried in a vacuum oven 30° C. Product (0.713 g, 94%) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J=9.1 Hz, 2H), 7.39 (d, J=9.1 Hz, 2H), 5.38 (p, J=5.2 Hz, 1H), 4.50 (dd, J=11.7, 3.9 Hz, 1H), 4.41-4.32 (m, 2H), 4.22 (dd, J=12.0, 5.6 Hz, 1H), 2.35 (dt, J=9.8, 7.5 Hz, 4H), 1.68-1.58 (m, 4H), 1.37-1.17 (m, 56H), 0.88 ppm (t, J=6.7 Hz, 6H).13C NMR (101 MHz, CDCl3) δ 173.24, 172.92, 155.35, 152.29, 145.56, 125.36, 121.76, 68.33, 66.97, 61.63, 34.16, 34.03, 31.93, 29.71, 29.68, 29.64, 29.49, 29.37, 29.29, 29.13, 29.07, 24.87, 22.70, 14.12 ppm. 2,3,5,6-Tetrafluorophenyl stearate Trietylamine (2.8 mL, 5 eq.) was added slowly to a solution of stearoyl chloride (1.26 g, 4.2 mmol) and tetrafluorophenol (0.72 g, 1.04 eq.) in DCM (10 mL), causing the immediate formation of a white precipitate. The heterogeneous reaction mixture was stirred for another 2 hours, diluted with 25 mL DCM and extracted with water (50 mL), 0.1 M HCl (2×50 mL), dried with MgSO4 and evaporated to dryness. The resulting solid was redissolved in 50 mL diethyl ether and extracted again with 1M NaHCO3(50 mL), 0.1 M HCl (50 mL), water (50 mL) and brine (2×50 mL). The organic phase was dried with MgSO4 (augmented with a small amount of activated carbon) and evaporated to dryness. The resulting crude material was redissolved in chloroform and flushed over a silica plug and again evaporated to dryness to afford 1.2 g (67%) of the desired compound as a white solid.1H NMR (400 MHz, CDCl3) δ 6.98 (tt, J=9.9, 7.0 Hz, 1H), 2.66 (t, J=7.4 Hz, 2H), 1.78 (p, J=7.4 Hz, 2H), 1.26 (s, 28H), 0.88 (t, J=6.7 Hz, 3H) ppm.19F NMR (376 MHz, CDCl3) δ−139.21 (ddd, J=24.0, 11.8, 7.8 Hz), −153.03-−153.20 (m).13C NMR (101 MHz, CDCl3) δ 169.56, 147.23 (m), 144.74 (m), 141.94, 141.86 (m), 139.41 (m), 129.78 (m), 103.01 (t), 33.42, 31.92, 29.69, 29.65, 29.61, 29.54, 29.39, 29.36, 29.14, 28.85, 24.78, 22.68, 14.09 ppm. Synthetic Approaches First approach. Compounds of the invention can be prepared by using the starting reactants MDP or MDP(Bn)—vide supra. These molecules have a functional carboxylic acid group that originates from the glutamic acid (Glu) building block. The COOH-group enables conjugation to amine-functional reactants that comprise lipophilic groups. Such lipophilic groups can for example be C18-moieties, for examples those derived from stearic acid, oleic acid, stearyl alcohol, oleyl alcohol, stearyl amine or oleyl amine; or sterol moieties, for example those derived from cholesterol. Saturated linear lipophilic moieties are preferred, as well as moieties derived from cholesterol. Particularly useful building blocks are PE-phospholipids such as DSPE ([1069-79-0]) or DOPE ([4004-05-1]); these molecules are already amine functional. Mixed acyl PE-phospholipids can also be useful (e.g. 16:0-18:1 PE or 18:0-18:1 PE or 18:0-16:0 PE). Another useful building block is cholesterol. Yet other useful building blocks are diglycerides, such as 1,2-dioctadecanoyl-sn-glycerol (18:0 DG [51063-97-9]) or 1-2-dioleoyl-sn-glycerol (18:1 DG [24529-88-2]). Mixed acyl diglycerides can also be used (e.g. 16:0-18:1 DG or 18:0-18:1 DG or 18:0-16:0 DG). PE-phospholipids as well as the diglycerides have two lipophilic chains, and such building blocks are preferably used in this approach. This first approach is illustrated in Examples 10 to 19. Second approach. In a particularly suitable alternative modular approach, copper-catalyzed azide-alkyne cycloadditions (“click reactions”) are employed to connect the MDP (or MTP) reactant to the lipophilic reactant. Here, MDP, MDP(Bn), MTP or MTP(Bn) building blocks are used that have azide (—N3) or alkyne (—C≡C—H) functionalities. Non-limiting examples of such molecules are MDP-propargyl, MDP(Bn)-propargyl or MTP-b-N3— vide supra. In the copper-catalyzed click reactions, these molecules can be coupled to alkyne- or azide-functional molecules that comprise lipophilic groups. Using the click-reaction, azide- or alkyne functional intermediates are targeted that can be prepared easily and modularly and that are stable. This allows simple isolation and storage of intermediates. Furthermore, the copper-catalyzed click cycloadditions can be performed—and are best performed—in an aqueous environment (such as e.g. THF/water or tBuOH/water) or in an aqueous two-phase liquid/liquid solvent combination (such as e.g. dichloromethane/water). In these reaction media, both the hydrophilic MDP (or MTP) reactant (with or without Bn-group) and the lipophilic reactant are conveniently soluble, highly improving ease of conjugation and reaction yields. In this click-approach, the lipophilic groups comprised in the azide or alkyne reactants can be C14, C16 or C18-moieties, such as those derived from stearic acid, palmitic acid, myristic acid, oleic acid, palmitoleic acid, myristoleic acid, stearyl alcohol or amine, palmityl alcohol or amine, myristyl alcohol or amine, oleyl alcohol or amine, palmitoleyl alcohol or amine, myristoleyl alcohol or amine; or sterol moieties, for example those derived from cholesterol. Saturated linear lipophilic moieties are preferred, as well as moieties derived from cholesterol. Particularly useful building blocks are PE-phospholipids, mixed acyl PE-phospholipids, diglycerides (DG) or mixed-acyl diglycerides, with C14, C16 and/or C18 moieties in them, as well as cholesterol. Lipophilic azide or alkyne reactants that comprise two lipophilic chains or that comprise a cholesteryl group are preferred. This second approach is illustrated in Examples 1 to 9. Note that both approaches allow the introduction of an extra amino-acid unit attached to the glutamic acid unit of MDP or MDP(Bn). Suitable amino acid units are those derived from L-Lysine or L-Alanine. With an extra amino acid unit connected, the MDP (muramyl dipeptide) moiety is converted to an MTP (muramyl tripeptide) moiety, either with or without Bn-group. Illustrations are found in Examples 9-10, 13-15 and 17-18. Example 1. Synthesis of MDP-C18 [Click] (1) Molecular weight: 825 Dalton. C Log P=4.15. This Synthesis Illustrates General Conditions for Cu-Click Type Reactions. A 5 mL vial was charged with MDP-propargyl (0.011 g, 0.02 mmol, 1.00 eq). To this was added L-ascorbic acid (0.4 M aq. solution, 104 μL, 41.5 μmol ascorbic acid, 2.00 eq). To the resulting slightly opaque solution, a solution of 1-azidooctadecane (0.012 g, 0.04 mmol, 2.00 eq) in DCM (0.8 mL) was added, followed by aqueous copper(II) sulfate pentahydrate solution (0.2 M, 104 μL, 20.8 μmol Cu, 1.00 eq). The bi-layered reaction mixture was then stirred at 1400 rpm at RT, resulting in a light yellow/green emulsion. After 16 h, the reaction mixture was concentrated in a stream of N2, giving crude product as a light brown brown sludge. The material was taken up in chloroform/MeOH 4:1 and impregnated on celite (90 mg, ˜1:5 loading ratio). Purification was done by automated column chromatography (product:silica 1:500; detection: 200-400 nm), eluting with chloroform/MeOH/water 90/9/1-70/27/3, giving product (0.005 g, 31%) as a white solid. 1H NMR (400 MHz, MeOD) δ 7.64-7.61 (m, 1H), 5.33 (d, J=3.4 Hz, 1H), 4.58-4.21 (m, 7H), 3.85-3.46 (m, 6H), 2.35-2.26 (m, 2H), 2.23-2.08 (m, 1H), 2.06-1.84 (m, 6H), 1.42-1.36 (m, 6H), 1.35-1.22 (m, 30H), 0.88 ppm (t, J=6.8 Hz, 3H).13C NMR (100 MHz, MeOD) δ 175.75, 174.66, 174.04, 173.58, 171.91, 144.63, 122.53, 91.00, 76.01, 71.84, 71.18, 67.16, 61.98, 54.15, 52.98, 50.67, 34.78, 32.20, 32.03, 30.34, 29.80, 29.76, 29.72, 29.65, 29.52, 29.46, 29.12, 27.49, 26.60, 22.83, 22.79, 22.69, 19.34, 16.95, 16.57, 14.15 ppm. HPLC-MS (water/MeCN): t (product)=5.64 min. Found: m/z=825.33 [M+H]+. Example 2. Synthesis of MDP-DSPE [Click] (2) Molecular weight: 1361 Dalton. C Log P=11.56 (uncharged) and 5.78 (negatively charged). MDP-propargyl (24.4 mg, 46 μmol) was dissolved in 0.4 M ascorbic acid (0.24 mL, 2 eq) and a solution of DSPE-azidoacetate (38.5 mg, 46 μmol, 1 eq) in dichloromethane (0.5 mL) was added. Under vigorous stirring 0.2 M CuSO4·5H2O (0.24 mL, 1 eq) was added and the two-phase system was vigorously stirred at room temperature for 19 h. The solvents were removed in vacuo and the greenish solid was subjected to column chromatography (flash SiO2) using an elution gradient of 20% to 50% MeOH in chloroform, concluded by (45% MeOH+5% H2O) in chloroform (a significant amount of compound only elutes after the addition of H2O). This yielded impure product which was further purified with automated column chromatography (reversed-phase (C18); product:C18-silica 1:200; detection: λ=210 nm), using an elution gradient of 25% to 70% THF in H2O. This yielded pure product (19.5 mg, 14 μmol, 31%) as a white fluffy solid after lyophilization. 1H NMR (400 MHz, CDCl3+MeOD) δ 7.93 (s, 1H), 5.29-5.22 (m, 2H), 5.16 (s, 2H), 4.51-4.41 (m, 4H), 4.38-4.25 (m, 2H), 4.20 (dd, J=12.1, 6.8 Hz, 1H), 4.05-3.89 (m, 4H), 3.88-3.77 (m, 2H), 3.77-3.63 (m, 6H), 3.55-3.42 (m, 3H), 2.34 (q, J=7.3 Hz, 6H), 2.22 (dddd, J=18.4, 13.5, 8.4, 5.7 Hz, 1H), 1.98 (d, J=5.7 Hz, 4H), 1.93 (s, 0H), 1.62 (q, J=6.4 Hz, 5H), 1.46-1.36 (m, 6H), 1.28 (s, 61H), 0.89 (t, J=6.8 Hz, 6H). Peaks between 4.9 and 4.6 ppm are not visible due to overlap with the H2O peak. MALDI-TOF MS: m/z Calc. for C65H118N9O19P 1359.83; Obs. [M+Na]+1382.83, [M−H+2Na]+1404.84. HPLC-MS (H2O/THF, gradient: 65-95% THF): t (product)=2.33 min; m/z=1360.80 [M+H]+(SIM mode). Example 3. Synthesis of MDP-Chol [Click] (3) Molecular weight: 999 Dalton. C Log P=5.04. MDP-propargyl (25 mg, 47 μmol) was dissolved in 0.4 M ascorbic acid (0.24 mL, 2 eq) and a solution of cholesterol azidoacetate (26.6 mg, 57 μmol, 1.2 eq) in dichloromethane (0.5 mL) was added. Under vigorous stirring 0.2 M CuSO4·5H2O (0.24 mL, 1 eq) was added and the two-phase system was vigorously stirred at room temperature for 17 h. H2O/brine 1:1 (50 mL) was added and the bluish aqueous layer was extracted with chloroform/MeOH 2:1 (5×20 mL). The combined organic layers were dried using Na2SO4, filtrated and the solvent was removed in vacuo. The resulting colorless solid was purified with repeated column chromatography (flash SiO2) using an elution gradient of 6% to 20% MeOH in chloroform. This yielded pure product (24.4 mg, 24 μmol, 52%) as a white fluffy solid after lyophilization from THF/H2O.1H-NMR (400 MHz, THF-d8/D2O 95:5): δ=7.81 (s, 1H), 5.28 (d, J=4.9 Hz, 1H), 5.16 (s, 2H), 5.10 (d, J=3.4 Hz, 1H), 4.59-4.46 (m, 1H), 4.46-4.16 (m, 5H), 3.75-3.17 (m, 6H), 2.26 (d, J=8.2 Hz, 2H), 2.20 (t, J=7.5 Hz, 2H), 2.12-1.99 (m, 1H), 1.98-1.69 (m, 8H), 1.58-1.33 (m, 5H), 1.32-1.23 (m, 7H), 1.18 (s, 5H), 1.13-0.97 (m, 4H), 0.94 (s, 3H), 0.84 (d, J=6.5 Hz, 3H), 0.77 (dd, J=6.6, 1.4 Hz, 8H), 0.61 (s, 3H).13C-NMR (100 MHz, THF-d8/D2O 95:5): δ=175.2, 174.9, 173.7, 173.4, 171.9, 166.6, 165.1, 139.6, 122.4, 91.0, 78.7, 76.7, 75.4, 72.0, 70.2, 61.1, 56.8, 56.2, 54.0, 52.6, 50.5, 50.2, 49.5, 42.2, 39.8, 39.4, 37.7, 36.8, 36.4, 36.1, 35.8, 34.4, 31.9, 31.8, 29.6, 28.1, 27.9, 27.5, 27.4, 22.5, 22.2, 22.1, 21.9, 20.9, 18.8, 18.7, 18.2, 16.8, 13.5, 11.3. MALDI-TOF MS: m/z Calc. for C51H82N8O12998.60; Obs. [M+Na]+1021.58. HPLC-MS (H2O/THF, gradient: 65-95% THF): t (prod)=2.20 min; m/z=999.60 [M+H]+(SIM mode). Note: THF-d8/D2O 95:5 was found to be an optimal solvent combination for NMR characterization. Nevertheless, the spectrum suffers from overlap and is very complicated. Therefore, integration is tentative. Example 4. Synthesis of MDP-DSPE2[Click] (4) Molecular weight: 2192 Dalton. Example 5. MDP-Chol2[Click] Molecular weight: 1669 Dalton. C Log P=15.31. Example 6. MDP-DSG[Click] (6) Molecular weight: 1238 Dalton. C Log P=12.45. Example 7. MDP(Bn)-DSPE [Click] (7) Molecular weight: 1451 Dalton. C Log P=13.85 (uncharged) and 8.06 (negatively charged). Following the general conditions for Cu-click reactions, MDP(Bn)-propargyl (0.028 g, 0.045 mmol, 1.00 eq) and DSPE-azidoacetate (0.039 g, 0.047 mmol, 1.05 eq) were reacted overnight. During the reaction, some material precipitated out, resulting in a white suspension/emulsion. Afterwards, the reaction mixture was diluted with chloroform/MeOH 1:1. The resulting clear solution was impregnated on celite (˜200 mg, 1:3 loading ratio). The impregnated crude product was first purified by automated column chromatography (reversed-phase (C18); product:C18-silica 1:200; detection: ELSD and UV 200-400 nm), eluting with water/THF 60/40-20/80. The combined product fractions were lyophilized and then purified again by automated column chromatography (normal phase (silica); product:silica 1:300; detection: ELSD), eluting with chloroform/MeOH/water 90/9/1-75/22.5/2.5. Pure fractions were concentrated in vacuo, dissolved in water/THF 70/30 and lyophilized. Thus, pure product was obtained as a white fluffy solid (0.027 g, 41%). 1H NMR (400 MHz, CDCl3+MeOD 1:1) δ 7.99 (d, J=8.2 Hz, 1H), 7.90 (s, 1H), 7.43-7.23 (m, 5H), 5.24 (m, 1H), 5.13 (s, 2H), 4.93 (d, J=3.5 Hz, 1H), 4.73 (d, J=12.0 Hz, 1H), 4.51 (d, J=12.0 Hz, 2H), 4.48-4.40 (m, 2H), 4.37-4.15 (m, 3H), 4.05-3.89 (m, 5H), 3.86-3.73 (m, 2H), 3.71-3.53 (m, 3H), 3.49-3.42 (m, 2H), 2.37-2.28 (m, 6H), 2.25-2.11 (m, 1H), 2.05-1.89 (m, 4H), 1.67-1.56 (m, 4H), 1.52-1.19 (m, 72H), 0.89 ppm (t, J=6.8 Hz, 6H). 13C NMR (100 MHz, MeOD) δ 174.97, 174.73, 173.93, 173.66, 173.57, 173.46, 171.92, 166.34, 137.16, 128.28, 128.09, 127.84, 124.43, 96.38, 79.09, 76.52, 72.55, 70.40, 69.75, 69.28, 63.66, 63.44, 62.56, 61.14, 53.18, 52.60, 52.13, 49.45, 40.52, 34.62, 34.12, 33.96, 31.81, 31.73, 29.57, 29.53, 29.43, 29.41, 29.23, 29.21, 29.03, 29.00, 27.12, 24.82, 24.77, 22.52, 22.20, 18.70, 16.75, 13.69 ppm. MALDI-TOF: m/z calcd for C72H124N9O19P+2Na+−H+: 1494.85 [M+2Na−H]+; found: 1494.94. HPLC-MS (water/THF, gradient: 55-95% THF): t (product)=5.09 min; m/z=1450.9 [M+H]+and 1472.9 [M+Na]+(SIM mode). Example 8. Synthesis of MDP(Bn)-Chol[Click] (8) Molecular weight: 1089 Dalton. C Log P=6.83. Following the general conditions for Cu-click reactions, MDP(Bn)-propargyl (0.030 g, 0.048 mmol, 1.00 eq) and cholesterol azidoacetate (0.025 g, 0.053 mmol, 1.10 eq) were reacted overnight, resulting in a white emulsion. The reaction mixture was then concentrated in vacuo and impregnated on celite (150 mg). The impregnated crude product was purified by automated column chromatography (reversed-phase (C18); product:C18-silica 1:200; detection: 200-400 nm), eluting with water/THF 70/30-15/85. The combined product fractions were lyophilized and then purified again by automated column chromatography (normal phase (silica); product:silica 1:350; detection: ELSD), eluting with chloroform/MeOH 96/4-86/14. Pure fractions were concentrated in vacuo, giving pure product as a white solid (0.028 g, 53%). 1H NMR (400 MHz, CDCl3+MeOD 1:1) δ 7.78 (s, 1H), 7.39-7.28 (m, 5H), 5.38 (d, J=5.1 Hz, 1H), 5.16 (d, J=1.6 Hz, 2H), 4.93 (d, J=3.6 Hz, 1H), 4.70 (d, J=11.8 Hz, 2H), 4.56-4.37 (m, 3H), 4.30 (dt, J=8.9, 4.4 Hz, 1H), 4.27-4.17 (m, 2H), 4.02 (dd, J=10.1, 3.6 Hz, 1H), 3.93-3.67 (m, 24H), 3.67-3.53 (m, 3H), 3.40 (d, J=3.2 Hz, 0H), 2.44-2.32 (m, 2H), 2.27 (td, J=7.1, 3.7 Hz, 2H), 2.12 (dtd, J=14.9, 7.5, 4.3 Hz, 1H), 2.07-1.74 (m, 8H), 1.72-1.42 (m, 5H), 1.38 (dd, J=13.0, 7.0 Hz, 7H), 1.34-0.94 (m, 13H), 0.92 (d, J=6.4 Hz, 3H), 0.87 (dd, J=6.6, 1.8 Hz, 6H), 0.69 ppm (s, 3H).13C NMR (100 MHz, CDCl3+MeOD 1:1) δ 175.17, 174.60, 173.71, 173.62, 171.76, 166.21, 145.18, 139.13, 137.21, 128.65, 128.37, 128.25, 124.35, 123.47, 96.91, 79.14, 76.83, 76.49, 72.44, 69.79, 61.63, 56.82, 56.28, 53.17, 52.57, 51.14, 50.14, 49.78, 42.45, 39.84, 39.65, 38.01, 36.97, 36.68, 36.32, 35.93, 34.85, 32.02, 31.97, 29.81, 28.34, 28.14, 27.74, 27.58, 24.39, 23.95, 22.87, 22.80, 22.77, 22.61, 21.16, 19.34, 18.98, 18.80, 16.90, 11.94 ppm. MALDI-TOF: m/z calcd for C58H88N8O12+Na+: 1111.64 [M+Na]+; found: 1111.65. HPLC-MS (water/THF, gradient: 65-95% THF): t (product)=2.79 min; m/z=1089.70 [M+H]+(SIM mode). Example 9. Synthesis of MTP-b-C18[Invclick] (9) Molecular weight: 1058 Dalton. C Log P=5.68. MTP-b-N3 (26 mg, 35 μmol) and prop-2-yn-1-yl stearate (11.3 mg, 35 μmol, 1 eq) were suspended in THF (0.36 mL) and 0.4 M ascorbic acid (0.18 mL, 2 eq) was added yielding a clear solution. Prop-2-yn-1-yl stearate was prepared using known procedures. Under vigorous stirring 0.2 M CuSO4·5H2O (0.18 mL, 1 eq) was added and the mixture was vigorously stirred at room temperature (initially gelation occurred but gentle heating resulted in a yellow solution). After 1 h, HPLC-MS (THF/H2O) indicated the absence of starting compounds and the opaque solution was lyophilized. The crude product was adsorbed onto Celite from chloroform/MeOH 2:1 and subjected to column chromatography (flash SiO2) using an elution gradient of 10% to 25% MeOH in chloroform. Column chromatography was repeated using a similar gradient yielding pure product (31.5 mg, 30 μmol, 84%) as a white fluffy solid after lyophilization from THF/H2O.1H-NMR (400 MHz, THF-d8/D2O 4:1): δ=7.92 (s, 1H), 7.31 (d, J=7.5 Hz, 2H), 7.23 (t, J=7.5 Hz, 2H), 7.15 (t, J=7.3 Hz, 1H), 5.04 (s, 2H), 4.74 (d, J=3.5 Hz, 1H), 4.63 (d, J=12.2 Hz, 1H), 4.40 (d, J=12.2 Hz, 1H), 4.31-4.15 (m, 6H), 3.96 (dd, J=10.5, 3.6 Hz, 1H), 3.66 (d, J=3.2 Hz, 2H), 3.59-3.45 (m, 3H), 2.22 (dt, J=15.3, 7.7 Hz, 4H), 2.09 (tt, J=12.9, 6.0 Hz, 1H), 1.87-1.70 (m, 6H), 1.64-1.54 (m, 1H), 1.47 (q, J=7.3 Hz, 2H), 1.32 (d, J=7.2 Hz, 3H), 1.28 (d, J=6.7 Hz, 3H), 1.18 (s, 30H), 0.78 (t, J=6.6 Hz, 3H).13C-NMR (100 MHz, THF-d8/D2O 4:1): δ=175.5, 174.9, 174.8, 173.73, 173.66, 173.2, 171.7, 142.3, 137.8, 128.1, 128.0, 127.4, 124.3, 96.6, 80.0, 77.3, 72.8, 69.1, 68.8, 60.9, 57.2, 53.2, 53.1, 52.2, 49.7, 49.5, 33.6, 31.8, 31.5, 30.9, 29.7, 29.54, 29.50, 29.4, 29.24, 29.20, 29.0, 27.7, 22.54, 22.49, 21.9, 18.6, 16.9, 13.5. MALDI-TOF MS: m/z Calc. for C53H87N9O131057.64; Obs. [M+Na]+1080.63, [M+K]+1096.65. HPLC-MS (H2O/THF, gradient: 65-95% THF): t (prod)=2.15 min; m/z=1058.60 [M+H]+(SIM mode). Example 10. Synthesis of MTP-b-C18 (10) Molecular weight: 976 Dalton. C Log P=5.31 A 10 mL PE syringe with PE frit was charged with MTP-b on-resin (137 mg, approx. 0.0493 mmol MTP-b, 1.00 eq). The resin was swollen in DMF (5 mL) for 30 min. Next, the resin was treated twice with 2% hydrazine hydrate solution in DMF (10 mL) for 15 min. The hydrazine solution was removed and the resin washed with DMF (4×5 mL). Next, a solution of 2,3,5,6-tetrafluorophenyl stearate (0.064 g, 0.15 mmol, 3.00 eq) and 4-methylmorpholine (0.030 g, 0.033 mL, 0.30 mmol, 6.00 eq) in DMF/DCM (1+1 mL; ˜0.075 M) was added. The beads were agitated overnight at RT. Afterwards, the supernatant was removed and the resin washed with DMF/DCM 50/50 (4×5 mL) and DCM (2×5 mL). The resin was then treated with TFA/TIPS/water 95/2.5/2.5 (200 uL) for 1 h. The filtrate was collected and the resin washed with additional cleavage cocktail. The combined filtrates were concentrated in vacuo, giving the crude product as a white solid. The material was impregnated on celite (200 mg, 1:4 loading ratio) from THF/water solution (95/5). The impregnated crude product was purified by automated column chromatography (reversed-phase (C18); product:C18-silica 1:250; detection: ELSD), eluting with water/THF 50/50-10/90. The combined product fractions were lyophilized and then purified again by automated column chromatography (normal phase (silica); product:silica 1:500; detection: ELSD), eluting with dichloromethane/MeOH 90/10-70/30. Pure fractions were concentrated in vacuo, giving the product as a white solid (0.016 g, 33%).1H NMR (400 MHz, CDCl3+TFA-d3) δ 7.40-7.24 (m, 5H), 4.94-4.89 (m, 1H), 4.71-4.65 (m, 1H), 4.57-4.17 (m, 6H), 4.03-3.74 (m, 4H), 3.38 (bs, 2H), 2.60-2.22 (m, 5H), 2.30 (s, 1H), 2.11-1.55 (s, 11H), 1.50-1.17 (m, 36H), 0.87 ppm (t, J=6.6 Hz, 3H). HPLC-MS (water/MeCN): t (product)=5.64 min. Found: m/z=976.33 [M+H]+(pos. mode); 1020.25 [M+HCOO]−(neg. mode). Example 11. Synthesis of MDP-C18 (11) Molecular weight: 744 Dalton. C Log P=4.79. MDP (10 mg, 20 μmol), octadecyl amine (5.2 mg, 0.95 eq.), NHS (2.4 mg, 1 eq.) and EDC-HCl (7.9 mg, 2 eq.) were stirred in DMF (0.7 mL) at 50° C. for 3 hours. The reaction mixture was subsequently allowed to cool to room temperature and stirred for another 16 hours. The resulting dispersion was heated to 40° C. to redissolve all the precipitated solids and subsequently precipitated with 5 mL ether. The collected precipitate was washed 2 more times with ether, dried and then suspended in demineralized water, collected by centrifugation, resuspended in demineralized water, and again collected by centrifugation. The resulting solid was lyophilized to remove all water to afford 13.8 mg (96%) of the desired compound as a white powder.1H NMR (400 MHz, DMF-d7) δ 8.31 (d, J=7.7 Hz, minor isomer), 8.23 (d, J=8.0 Hz, major isomer), 8.12 (d, J=7.9 Hz, minor isomer), 8.09 (d, J=7.6 Hz, major isomer), 7.96 (d, J=6.6 Hz, minor isomer), 7.90 (d, J=6.5 Hz, major isomer), 7.77 (m, 1H), 7.47 (m, 1H), 7.10 (m, minor isomer), 7.01 (m, major isomer), 6.86 (d, J=6.0 Hz, minor isomer), 6.74 (dd, J=4.1, 1.2 Hz, major isomer), 5.44-5.29 (m, 1H), 5.16 (t, J=3.7 Hz, major isomer), 4.79 (t, J=6.1 Hz, minor isomer), 4.60 (dd, J=8.2, 6.0 Hz, minor isomer), 4.57-4.22 (m, 4H), 3.93-3.57 (m, 5H), 3.45 (m, 1H), 3.13 (m, 2H), 2.42-2.07 (m, 3H), 2.00-1.78 (m, 4H), 1.56-1.07 (m, 38H), 0.88 (m, 3H) ppm.13C NMR (101 MHz, DMF-d7) δ 174.25, 174.20, 173.93, 173.89, 172.75, 172.56, 172.07, 172.02, 171.76, 170.11, 96.92, 91.66, 82.46, 79.55, 77.39, 77.00, 72.97, 71.30, 70.95, 62.05, 57.49, 54.49, 53.16, 53.03, 49.54, 39.26, 32.55, 32.05, 29.83, 29.49, 28.62, 28.41, 27.18, 22.86, 22.76, 22.73, 19.19, 17.80, 17.62, 13.93 ppm. ESI-MS: m/z 743.50 (calc.), found 744.42 (M+H+), 788.33 (M−FA−). Example 12. Synthesis of MDP-DSPE (12) Molecular weight: 1223 Dalton. C Log P=12.96 (uncharged) and 7.18 (negatively charged). MDP (14 mg, 29 μmol), NHS (5.6 mg, 1.7 eq.), and DIC (7.3 mg, 2 eq) were stirred in 0.9 mL DMF for 2 hours to activate the MDP. The resulting mixture was added to a dispersion of DSPE (17 mg, 0.8 eq.) in 2.7 mL tert-butanol with TEA (9 mg, 3.1 eq.) at 50° C. and stirred for 3½ hours at that temperature. The resulting mixture was evaporated to dryness and the resulting material was purified by repeated column chromatography (SiO2, CHCl3/MeOH/H2O, 70/30/5, 5:4:1 and gradient 95/5/0 to 60/40/0) to afford 6 mg (21%) of the desired compound as a white fluffy material after lyophilization from water/THF.1H-NMR (400 MHz, CDCl3/CD3OD 5:) δ 5.29 (d, major isomer), 5.24 (m, 1H), 4.54 (d, minor isomer), 4.47 (m, obscured by HDO), 4.41 (dd, obscured by HDO), 4.31 (m, obscured by HDO), 4.19 (dd, 1H), 4.00-3.90 (m, mixture of isomers), 3.87-3.75 (m, mixture of isomers), 3.72 (m, 1H), 3.63 (m, 1H), 3.53-3.30 (m, obscured by CD3OD), 2.37-2.26 (m, 5H), 2.25-2.10 (m, 2H), 2.10-1.92 (m, 4H), 1.61 (m, 4H), 1.50-1.15 (br. m, 62H), 0.88 (t, 6H) ppm.31P-NMR (162 MHz, CDCl3/CD3OD 5:1) δ 0.14 (br. m) ppm. 13C-NMR (101 MHz, CDCl3/CD3OD 5:1) δ 174.63, 174.00, 173.27, 173.08, 172.92, 172.89, 171.34, 90.19, 75.19, 71.09, 70.18, 69.73, 69.65, 63.24, 62.76, 61.86, 60.84, 53.13, 52.04, 48.77, 48.19, 39.71, 33.48, 33.32, 31.29, 31.14, 28.91, 28.87, 28.76, 28.74, 28.57, 28.54, 28.37, 28.34, 26.44, 24.14, 24.10, 21.88, 21.78, 18.35, 15.98, 13.14 ppm. MALDI-MS: m/z 1221.77 (calc.), found 1220.82 (M−H−), negative mode. HPLC-ELSD (C18, 65-95% THF/H2O): single peak plus shoulder for the alpha and beta isomers. Example 13. Synthesis of MTP-b-DSG (13) Molecular weight: 1361 Dalton. C Log P=15.05. A 20 mL PE syringe with PE frit was charged with MTP-b on-resin (0.291 g, approx. 0.11 mmol MTP-b, 1.00 eq). The resin was swollen in DMF (5 mL) for 30 min. Next, the resin was treated twice with 2% hydrazine hydrate solution in DMF (5 mL) for 15 min. The hydrazine solution was removed and the resin washed with DMF (4×5 mL). Next, a solution of DSG 4-nitrophenylcarbonate (0.249 g, 0.32 mmol, 3.00 eq) and N,N-diisopropylethylamine (0.081 g, 0.110 mL, 0.63 mmol, 6.00 eq) in chloroform (4 mL) was added. The beads were agitated overnight at RT. Afterwards, the bright yellow supernatant was removed and the resin washed with chloroform (3×5 mL), MeOH (3×5 mL) and again chloroform (2×5 mL). The resin was treated with TFA/TIPS/water 95/2.5/2.5 (5 mL) for 45 min. The supernatant was injected into ice-cold diethyl ether (100 mL) under stirring, resulting in the slow formation of a white flocculate. This cleavage and precipitation procedure was repeated twice. The solids were collected by filtration through a disposable PE filter, giving crude product as a white solid. The material was impregnated on celite (250 mg, 1:2.5 loading ratio) from chloroform/MeOH (1:2) solution. The impregnated crude product was purified by automated column chromatography (reversed-phase (C18); product:C18-silica 1:150; detection: 200-400 nm), eluting with water/THF 60/40-0/100. The combined product fractions were lyophilized and then purified again by automated column chromatography (normal phase (silica); product:silica 1:250; detection: 200-400 nm), eluting with chloroform/MeOH 95/5-60/40. Pure fractions were concentrated in vacuo, giving the product as a white solid (0.030 g, 21%). 1H NMR (400 MHz, CDCl3+MeOD) δ 7.39-7.27 (m, 5H), 5.25 (p, J=5.3 Hz, 1H), 4.89 (d, J=3.5 Hz, 1H), 4.73 (d, J=11.9 Hz, 1H), 4.50 (d, J=11.9 Hz, 1H), 4.42-4.31 (m, 2H), 4.31-4.19 (m, 4H), 4.15 (dd, J=11.9, 6.2 Hz, 2H), 4.10-3.97 (m, 1H), 3.87-3.75 (m, 2H), 3.72-3.53 (m, 3H), 3.12 (t, J=6.9 Hz, 2H), 2.37-2.27 (m, 6H), 2.26-2.13 (m, 1H), 1.93 (s, 3H), 1.92-1.74 (m, 2H), 1.71-1.56 (m, 6H), 1.56-1.47 (m, 2H), 1.43 (d, 7.0 Hz, 3H), 1.40 (d, 7.0 Hz, 3H), 1.36-1.19 (s, 56H), 0.89 ppm (t, J=6.7 Hz, 6H).13C NMR (100 MHz, CDCl3+MeOD) δ 175.80, 174.79, 174.30, 173.86, 173.76, 173.44, 173.25, 171.70, 156.58, 137.07, 128.36, 128.14, 127.95, 96.52, 79.36, 76.83, 72.38, 69.48, 69.43, 69.37, 62.50, 62.29, 61.30, 53.52, 53.24, 51.71, 49.46, 40.33, 34.13, 33.97, 31.82, 31.29, 31.08, 29.58, 29.54, 29.52, 29.41, 29.39, 29.25, 29.20, 29.18, 29.09, 29.01, 28.98, 28.16, 24.80, 24.77, 22.88, 22.55, 22.35, 18.61, 16.89, 13.77 ppm. HPLC-MS (water/THF, gradient: 55-95% THF): t (product)=6.20 min. Found: m/z=1360.9 [M+H]+and 1382.9 [M+Na]+(SIM mode). Example 14. Synthesis of MTP(Bn)-a-DPPE (14) Molecular weight: 1328 Dalton. C Log P=12.88 (uncharged) and 7.09 (negatively charged) Building block (CBz)-Ala-DPPE N-CBz-protected L-alanine (390 mg, 1.7 mmol) and N-hydroxysuccinimide (222 mg, 1.89 mmol, 1.1 eq) were dissolved in chloroform (6 mL), yielding an almost clear solution. N,N′-Diisopropylcarbodiimide (DIC; 0.32 mL, 2.0 mmol, 1.2 eq) was added and the mixture was stirred at r.t. for 40 min (after 1 min the solution turns hazy and after 25 min 1H-NMR shows full conversion). This solution was then added to a 60° C. solution containing 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE; 1.06 g, 1.5 mmol, 0.9 eq) and triethylamine (600 μL, 0.71 mmol, 2.5 eq) in chloroform (12 mL; DPPE dissolved at reflux and triethylamine was added at a lowered temperature). The resulting clear solution was stirred at 60° C. for 1 h (the solution remains clear and after 1 h1H-NMR shows full conversion). Chloroform (360 mL) was added and the organic layer was gently washed with 0.1 M HCl (100 mL). The organic layer was dried using Na2SO4, filtrated and the solvent was removed in vacuo. Column chromatography (flash SiO2) using an elution gradient of 2% to 30% methanol in chloroform yielded the title compound that was partly contaminated with triethylamine. The impure fractions were dissolved in chloroform and the organic layer was gently washed with 0.1 M HCl. The organic layer was dried using Na2SO4, filtrated and the solvent was removed in vacuo. This effectively removed triethylamine and the pure fractions were combined yielding pure product (1.22 g, 1.4 mmol, 91%) as a colorless waxy solid. 1H-NMR (400 MHz, DMSO-d6): δ=8.06 (t, J=5.7 Hz, 1H), 7.47-7.22 (m, 6H), 5.15 (dq, J=8.3, 4.6 Hz, 1H), 5.01 (q, J=12.6 Hz, 2H), 4.28 (dd, J=12.0, 3.2 Hz, 1H), 4.11 (dd, J=12.1, 7.0 Hz, 1H), 4.06-3.92 (m, 3H), 3.82 (q, J=6.4 Hz, 2H), 3.33-3.18 (m, 2H), 2.27 (dt, J=12.8, 5.0 Hz, 4H), 1.50 (q, J=6.9 Hz, 4H), 1.32-1.16 (m, 51H), 0.85 (t, J=6.7 Hz, 6H).31P-NMR (162 MHz, DMSO-d6): δ=−1.4. Building Block Ala-DPPE In a 2-neck round-bottom flask (CBz)-Ala-DPPE (308 mg, 0.34 mmol) and Pd/C (374 mg, 10% Pd, pre-wetted Degussa/Evonik type) were combined in chloroform/ethanol 1:2 (36 mL). The flask was evacuated and back-filled with Ar three times. A H2-balloon was attached, the flask was evacuated and back-filled with H2three times and the mixture was stirred under a positive H2pressure for 3 h at room temperature. The solution was filtrated over Celite which was copiously washed with ethanol, chloroform/ethanol 1:1 and chloroform. The combined filtrates were evaporated to dryness, the resulting compound was dissolved in chloroform/ethanol 2:1 (90 mL) and dried using Na2SO4. The solution was filtrated over Celite which was copiously washed with chloroform/ethanol 2:1. The filtrate was evaporated to dryness yielding the product (224 mg, 0.29 mmol, 86%) as a slightly yellowish waxy solid, which contained trace amounts of Pd. 1H-NMR (400 MHz, DMSO-d6): δ=8.60 (br, 1H), 8.08 (br, 2H), 5.15 (br, 1H), 4.28 (d, J=13.2 Hz, 1H), 4.12 (dd, J=6.9 Hz, 1H), 4.00 (m, 2H), 3.88 (m, 2H), 3.80 (br, 1H), 3.09 (br, 1H), 2.35-2.23 (m, 4H), 1.50 (br, 4H), 1.41-1.14 (m, 51H), 0.85 (t, J=6.6 Hz, 6H).31P-NMR (DMSO-d6): δ=−1.4. MTP(Bn)-a-DPPE (14) MDP(Bn) (20.0 mg, 34 μmol) and Ala-DPPE (26.2 mg, 34 μmol, 1.0 eq) were combined in DMAc (0.3 mL) and N,N-diisopropylethylamine (24 μL, 0.14 mmol, 4 eq) and PyBOP (22 mg, 41 μmol, 1.2 eq) were added consecutively. The resulting suspension was stirred at 50° C. for 1 h, after which the mixture had almost cleared. The volatiles were removed in vacuo (oil pump, 45° C.) and the mixture was flushed once with chloroform. Column chromatography (flash SiO2) using an elution gradient of 15% to 40% methanol in chloroform was followed by automated column chromatography (reversed-phase C18; product:C18-silica 1:200; detection: λ=200-220 nm), using an elution gradient of 30% to 80% THF in H2O. This yielded product 14 (8.0 mg, 6 μmol, 18%) as a white fluffy solid after lyophilization. HPLC-MS: t[product]=3.92 min.; m/z=1327.80 [M+H]+(SIM mode). HPLC-ELSD: t[prod]=3.48 min; 99.2% relative peak area. Example 15. MTP-a-chol Molecular weight: 1018 Dalton. C Log P=5.64. N-(2-aminoethyl)-cholesterol carbamate Building Block A solution of cholesterol chloroformate (0.95 g, 2.1 mmol) in 20 mL DCM was slowly added to a solution of ethylenediamine (2 mL, 14 eq.) in 30 mL DCM in about 2 hours. The reaction was allowed to proceed for another 30 minutes, after which the reaction mixture was evaporated to dryness. The resulting white material was purified via column chromatography (SiO2, CHCl3/MeOH/formic acid 78:20:2), yielding 720 mg (72%) of the desired compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 5.46-5.27 (m, 1H), 4.99 (br. s, 1H), 4.50 (br. m, 1H), 3.22 (q, J=5.6 Hz, 2H), 2.82 (t, J=5.9 Hz, 2H), 2.43-2.19 (m, 2H), 2.06-1.75 (m, 5H), 1.64-0.80 (m, 35H), 0.68 (s, 3H) ppm.13C NMR (101 MHz, CDCl3) δ 156.41, 139.83, 122.47, 74.31, 56.68, 56.13, 50.00, 43.63, 42.30, 41.79, 39.73, 39.51, 38.57, 36.99, 36.56, 36.18, 35.79, 31.90, 31.87, 28.22, 28.17, 28.00, 24.28, 23.82, 22.81, 22.55, 21.03, 19.33, 18.71, 11.85. MALDI: m/z=472.40 (calc.), found: 495.39 (M+Na+). A prominent peak is observed at m/z=369.37, which is attributed to a 3,4-eliminated product formed in MALDI (not observed in NMR). This building block can be coupled to N-Boc-L-Alanine (CAS [15761-38-3]) via amidation; next the Boc-group can be deprotected; finally, the formed amine functional molecule can be coupled to MDP to arrive at MTP-a-chol. Example 16. MDP(Bn)-chol Molecular weight: 1037 Dalton. C Log P=7.69. The N-(2-aminoethyl)-cholesterol carbamate building block (see Example 15) can be coupled to MDP(Bn) via amidation, arriving at molecule MDP(Bn)-chol. Example 17 (MTP-a-DSPE), Example 18 (MTP(Bn)-a-DSPE) and Example 19 (MDP(Bn)-DSPE) MTP-a-DSPE: MW is 1294 Dalton. C Log P=12.70 and 6.92 (uncharged and charged). MTP(Bn)-a-DSPE: MW is 1384 Dalton. C Log P=14.99 and 9.21 (uncharged and charged). MDP(Bn)-DSPE: MW is 1313 Dalton. C Log P=15.25 and 9.46 (uncharged and charged). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE; CAS [1069-79-0]) can be connected to N-Boc-L-Alanine (CAS [15761-38-3]) via amidation; next the Boc-group can be deprotected; finally, the formed amine functional molecule can be either coupled to MDP, to arrive at MTP-a-DSPE, or to MDP(Bn), to arrive at MTP(Bn)-a-DSPE. Alternatively, DSPE can be coupled to MDP(Bn) via amidation, arriving at molecule MDP(Bn)-DSPE. Example 20: Lipophilicity Studies The C log P values of the exemplary compounds of the disclosure were assessed, using Perkin Elmer ChemDraw Professional, version 18.0.0231 (4029) software. The results show values from about 4.15 to 18.28. At the physiological pH of about 7.4 (i.e. COOH and PO3H groups become charged), 2 molecules of the invention have C Log P values between 4 and 5, three have values between 10 and 20, and the rest of the molecules have values between 5 and 10. Experimentally, one can also compare lipophilicities of molecules by performing HPLC using the same elution gradient. Molecules that have higher affinities with the hydrophobic C18-material of the column are more lipophilic and as a result have a higher retention time. The below Table shows that the Example molecules of the invention (Entries 1-4) have higher retention times as compared to the Comparative Example molecules (Entries 5 and 6), and are thus more lipophilic. Methods: HPLC-MS(SIM) and HPLC-ELSD were performed on a Phenomenex Kinetex 5 micrometer EVO C18 100A LC-column (50×2.1 mm) employing the same gradient from A to B eluent, where A=20 mM NH4HCO2in H2O with 0.1 v/v % formic acid, and B=2-propanol/MeCN/H2O 85:15:5, also with 20 mM NH4HCO2and 0.1 v/v % formic acid. TABLE 3HPLC-MS or HPLC-ELSD retention times of molecules with C16,C18 and/or benzyl lipophilic units.CLogPCLogP (atHPLC-Lipophilic(uncharged)pH = 7.4)HPLC-MSELSDEntryExampleunit(s)(—)(—)t (min)t (min)12C18 (2x)11.565.784.464.5827C18 (2x)/Bn13.858.065.165.16313C18 (2x)/Bn15.0515.05n.d.5.49414C16 (2x)/Bn12.887.093.613.55Comp-1C16 (2x)10.594.82.853.046Comp-2C185.391.390.95n.d.n.d. = not determined;cmpd-1 = mifamurtide;cmpd-2 = MDP-C18[mur] Example 21. Aqueous Solubility Studies Compounds of the disclosure were tested for their solubility in PBS-buffer and in water, applying low concentrations. First, compounds were weighed in a vial and PBS buffer (137, 2.7, 10 and 1.8 mM in NaCl, KCl, Na2HPO4, KH2PO4, respectively; pH=7.4) was added, such that the concentration would become 0.2 mg/mL in case full dissolution would take place. The sample was shaken, left to stand for an hour, shaken again, and then the appearance of the solution at room temperature (RT) was checked. Next, the sample was warmed for 1 minute in a water bath of 37° C., and the appearance of the solution was checked again. In the below table the results are compiled. None of the tested compounds spontaneously dissolve in PBS, not at room temperature and not at 37° C. In contrast, the tested Comparative Example compounds dissolve spontaneously under these conditions. Further treatment of the sample solutions with a heat gun did not give dissolution for entries 4, 5 and 6, while entries 2 and 3 gave hazy solutions after cooling down to RT. TABLE 4Solubility tests on solutions in PBS, at RT and at 37° C.CLogPAppearanceAppearanceEntryExampleat pH = 7.4at RTat 37° C.1Ex-25.78hazyhazy2Ex-35.04suspensionhazy3Ex-78.06suspensionhazy4Ex-86.83suspensionsuspension5Ex-114.79suspensionsuspension6Ex-1315.05suspensionsuspension7Comp-14.80hazyclear8Comp-21.39clearclearcmpd-1 = mifamurtide;cmpd-2 = MDP-C18[mur] Next, compounds were weighed and dissolved in chloroform/methanol. The solutions were left to dry in a vial forming a film of the materials. The vials were put in vacuo to remove traces of organic solvent. Demineralized water was added, such that the concentration of the compounds would become 0.3 mM in case full dissolution would take place (0.3 mM corresponds to 0.3 mg/mL for a MW=1000 Dalton compound). The vial was briefly sonicated in a water bath, left to stand overnight, and once again sonicated (sonication at RT). The appearances of the solutions at RT were checked to assess solubilities. In the below table the results are compiled. None of the tested compounds of the disclosure spontaneously dissolve in water at room temperature. In contrast, the tested Comparative Example compounds dissolve spontaneously under these conditions. TABLE 5Solubility tests on solutions in water at RT.CLogPAppearanceEntryExampleat pH = 7.4at RT1Ex-25.78Gelly/hazy/precipitate2Ex-35.04Gelly/hazy/precipitate3Ex-78.06Hazy4Ex-86.83Precipitate5Ex-95.68Precipitate6Ex-105.31Precipitate7Ex-114.79Precipitate8Ex-1315.05Gel/precipitate9Comp-Ex-14.80Clear10Comp-Ex-21.39Clearcmpd-1 = mifamurtide;cmpd-2 = MDP-C18[mur] Finally, comparative Examples 1 and 2 were also tested with respect to their solubilities in PBS (0.01 M, pH=7.4) and demineralized water at a level of 1 mg/mL. The same results as indicated in the above two tables were found at this concentration. Taken together, these results show that a series of compounds of the invention do not spontaneously dissolve in PBS or water at concentrations as low as 0.2 mg/mL (and higher). In contrast, comparative Example materials are soluble in PBS or water to give clear and transparent solutions at concentrations as high as at least 0.2 mg/mL or even 1 mg/mL. Because the disclosed compounds have a low solubility in aqueous solution, their physico-chemical properties find particular use in producing stable HDL-derived NPs. Without being bound by theory, it is thought that the disclosed compounds provide improved anchoring into the NPs, reducing leakage, and providing products with greater stability and shelf-life. Example 22. Degradation by Augmented Oxidation Test The reference compounds MDP and MDP(Bn) as well as the Bn-substituted compounds from Example 7 (i.e. MDP(Bn)-DSPE[click] and Example 14 (i.e. MTP(Bn)-a-DPPE) were mixed with 12% hydrogen peroxide in water and were heated to 80° C. for 4 hours, in order to get a fast degradation of the molecules by oxidation, mimicking slower in-vivo oxidation events. The resulting reaction mixtures were diluted with acetonitrile and water (1:1) for the MDP and MDP(Bn) test solutions, or with iPrOH, acetonitrile and water (40:7.5:52.5) with 0.1% formic acid and 20 mM ammonium formate for the MDP(Bn)-DSPE[click] and the MTP(Bn)-a-DPPE solutions. The 4 diluted samples were analyzed by HPLC-MS. For reference, the 4 starting materials were also analyzed by HPLC-MS, as well as MDP-DSPE[click] and MTP-a-DPPE, i.e. the de-benzylated reference compounds to the Bn-substituted test molecules. For all 4 test solutions, the un-affected starting compounds were traced. In addition, multiple derivatives with masses of +14, +16, +28, +30 and +32 were found, indicating oxidations from CH2to CO moieties (+14) and from C—H to C—OH moieties (+16), and combinations of these oxidation events. The tested MDP(Bn)-DSPE[click] and the MTP(Bn)-a-DPPE compounds mainly degraded via oxidation of the Bn-group to a benzoate group (+14) followed by hydrolysis of the benzoate (−104). This was testified by the dominant presence of the de-benzylated MDP-DSPE[click] and MTP-a-DPPE compounds as degradation products: corroborating retention times in HPLC were found as well as corroborating masses in MS (−90 relative to the starting compounds). The results indicate that the Bn-groups in the compounds of the invention have the highest propensity for in-vivo oxidative degradation. After Bn-oxidation and cleavage, the regular MDP- or MTP-group is formed, and these groups will degrade in-vivo in a similar fashion as other MDP/MTPs—that are known in the art—do. Example 23. Nanobiologic Synthesis Method 1—Film The phospholipids, (pro-)drug and optional triglycerides or polymer are dissolved (typically in chloroform, ethanol or acetonitrile). This solution is then evaporated under vacuum to form a film of the components. Subsequently, a buffer solution is added to hydrate the film and generate a vesicle suspension. The phospholipids, (pro-)drug and optional triglycerides or polymer are dissolved (typically in chloroform, ethanol or acetonitrile). This solution is infused—or added drop-wise—to a mildly heated buffer solution under stirring, until complete evaporation of the organic solvents, generating a vesicle suspension. To the vesicle suspension, generated using A or B, apolipoprotein A-I (apoA-I) (note that apoA-I can also already be in B)—use dropwise to avoid denature, is added and the resulting mixture is sonicated for 30 minutes using a tip sonicator while being thoroughly cooled using an external ice-water bath. The obtained solution containing the nanobiologics and other by products is transferred to a Sartorius Vivaspin tube with a molecular weight cut-off depending on the estimated size of the nanobiologics (typically Vivaspin tubes with cut-offs of 10.000-100.000 kDa are used). The tubes are centrifuged until ˜90% of the solvent volume has passed through the filter. Subsequently, a volume of buffer, roughly equal to the volume of the remaining solution, is added and the tubes are spun again until roughly half the volume has passed through the filter. This is repeated twice after which the remaining solution is passed through a polyethersulfone 0.22 μm syringe filter, resulting in the final nanobiologic solution. Method 2—Microfluidics In an alternative approach, the phospholipids, (pro-)drug and optional triglycerides, cholesterol, steryl esters, or polymer are dissolved (typically in ethanol or acetonitrile) and loaded into a syringe. Additionally, a solution of apolipoprotein A-I (apoA-I) in phosphate buffered saline is loaded into a second syringe. Using microfluidics pumps, the content of both syringes is mixed using a microvortex platform. The obtained solution containing the nanobiologics and other by products is transferred to a Sartorius Vivaspin tube with a molecular weight cut-off depending on the estimate size of the particles (typically Vivaspin tubes with cut-offs of 10.000-100.000 kDa are used). The tubes are centrifuged until ˜90% of the solvent volume has passed through the filter. Subsequently, a volume of phosphate buffered saline roughly equal to the volume of the remaining solution is added and the tubes are spun again until roughly half the volume has passed through the filter. This is repeated twice after which the remaining solution is passed through a polyethersulfone 0.22 μm syringe filter, resulting in the final nanobiologic solution. Method 3—Microfluidizer In another method according to the invention, microfluidizer technology is used to prepare the nanoscale assembly and the final nanobiologic composition. Microfluidizers are devices for preparing small particle size materials operating on the submerged jet principle. In operating a microfluidizer to obtain nanoparticulates, a premix flow is forced by a high pressure pump through a so-called interaction chamber consisting of a system of channels in a ceramic block which split the premix into two streams. Precisely controlled shear, turbulent and cavitational forces are generated within the interaction chamber during microfluidization. The two streams are recombined at high velocity to produce shear. The so-obtained product can be recycled into the microfluidizer to obtain smaller and smaller particles. Advantages of microfluidization over conventional milling processes include substantial reduction of contamination of the final product, and the ease of production scaleup. Formulation 1 The below Table provides details on the preparation of HDL-derived nanoparticle formulations. First, DMPC, cholesterol and the compound of the invention were dissolved in the given molar ratios in ethanol (entries A, B and D) or in ethanol/DMSO 4/1 (entries C, E and F), while protein apoA-1 was separately dissolved in PBS buffer (pH=7.5). In these formulations, the amount of applied apoA-I was related to the amount of DMPC, by weight. The organic solution was mixed with the PBS buffer solution by bringing them together by T-junction mixing. Purification of the resulting solutions was performed by TFF (tangential flow fractionation), thereby getting rid of the organic solvents and dissolving the nanoparticles in PBS. Concentration of the NP solutions was performed by spin-filter centrifugation. Finally, the HDL-derived nanoparticle solutions were filtered over 0.2 micrometer Acrodisk PES filters. The final HDL-derived nanoparticle solutions had typical recoveries of the used compounds (Examples 2, 3, 7, 8 and 13), of DMPC and of cholesterol that exceeded 80%. Recoveries were determined by HPLC (for the compounds), and using assays that are known in the art (for DMPC and cholesterol). Concentrations of the final HDL-derived nanoparticle solutions were about 2 to 4 mg/mL in compound. TABLE 6Formulation compositions of HDL-derived nanoparticlesAPO-A1 *DMPCCompoundCholesterolmg-to-EntryCompoundmol %mol %mol %mgANone900101-to-2(Blank)BExample 29010101-to-2CExample 39010101-to-2DExample 79010101-to-2EExample 89010101-to-2FExample 139010101-to-2* APO-A1 is employed in about half the amount in weight (mg) as DMPC The stability of the nano-biologics as assessed by dynamic light scattering (DLS). The formulations of entries A to F were characterized by DLS over a time period of 8 weeks. The nanoparticles in the Example 2, 3, 7 and 13 formulations had Z-averaged (intensity weighted mean hydrodynamic size) diameters of about 20, 30, 20 and 45 nm, respectively. The dimensions of these nanoparticles stayed constant in time, with also the dispersity in particle size (PDI) remaining constant. The unloaded particles (Entry A) were also stable in time (at about 30 nm diameter). The nanoparticles in the Example 8 formulation showed diameters that grew as of the 2-week time point to the 5-week time-point, from about 50 to about 225 nm. Dimensions stabilized as of the 5-week time point. Using other processing conditions, also this Example 8 material can most likely be formulated to stable 10 to 50 nm sized particles. DLS-determined Z-averaged diameters and PDI-values for nanoparticles in formulations A to F are shown inFIG.2. Tip sonication formulation A: DSPC ([816-94-4]; 2.7 mg), cholesterol (0.26 mg) and the compound (0.46 mg) were dissolved in a glass vial with chloroform/methanol (9:1). The solvents were removed by an argon gas flow and the resulting film was dried in vacuo for >1 h. A solution of apoA-I PBS (6 mL) was added to the vial, which was subsequently bath sonicated for 5 minutes, incubated at 37° C. for 20 minutes, and then tip-sonicated for 10 minutes. The resulting dispersion was centrifuged to remove larger aggregates. The supernatant was transferred to a Vivapin 20 ultrafiltration unit (cutoff 10 kDa) and spun down to a volume of approximately 1 mL. The resulting dispersion was diluted with PBS and spun down to 1 mL, and this procedure was repeated twice. Finally, the volume was diluted to 2 mL using PBS to afford the desired nanoparticle solution. Tip sonication formulation B: DMPC (2.7 mg), cholesterol (0.30 mg) and the compound (0.57 mg) were dissolved in a glass vial with chloroform/methanol (9:1). The solvents were removed by an argon gas flow and the resulting film was dried in vacuo for >1 h. A solution of peptide-2F (an apoA-I mimetic 18-mer; sequence 257 in Table 2) in PBS (6 mL) was added to the vial, which was subsequently bath sonicated for 5 minutes, incubated at 37° C. for 20 minutes, and then tip-sonicated for 5 minutes. The resulting dispersion was centrifuged to remove larger aggregates. The supernatant was transferred to a Vivapin 20 ultrafiltration unit (cutoff 10 kDa) and spun down to approximately 1 mL. The resulting dispersion was diluted with PBS and spun down to 1 mL, and this procedure was repeated twice. Finally, the volume was diluted to 2 mL using PBS to afford the desired nanoparticle solution. T-junction formulation C: DMPC, cholesterol and the compound were dissolved in ethanol, while apoA-1 was dissolved in PBS buffer (pH=7.5). The organic solution was mixed with the buffer solution applying T-junction mixing. Purification of the resulting solutions was performed by TFF (tangential flow filtration), thereby getting rid of the organic solvents. Samples were concentrated by spin-filtration. The final HDL-derived nanoparticle solutions had typical recoveries for compound, DMPC and cholesterol that exceeded 75%. Concentrations of the final HDL-derived nanoparticle solutions were about 2 to 4 mg/mL in the compound. TABLE 7Formulations of HDL-derived nanoparticlePC-apoA-IDLS#typeCompoundCholesterolor 2F *nmEntrymol %mol %mol %mg-to-mg(error)ADSPCExample 220apoA-I51 (19)100102-to-5 *BDMPCExample 7202F12.9 (0.7)100102-to-5 *CDMPCExample 220apoA-I16.4 (4.2)80201-to-1 *** apoA-I or the 2F-peptide are employed in mg-to-mg phosphocholine PC);** apoA-I is employed in mg-to-mg compound;#number averaged diameter. The above examples highlight, that DSPC can be used instead of DMPC (POPC can also be employed e.g.), that peptidomimetics instead of apoA-I can be used, and that high levels of compounds of the disclosure can be incorporated. Furthermore, tip-sonication can be used as processing technique, instead of e.g. T-junction mixing or micro-fluidic mixing. APOLIPOPROTEIN A-I (apoA-I) ISOLATION Human apoA-I was isolated from human HDL concentrates (Bioresource Technology) following a previously described procedure (Zamanian-Daryoush et al., 2013). Briefly, a potassium bromide solution (density: 1.20 g/mL) was layered on top of the concentrate and purified HDL was obtained by ultracentrifugation. The purified fraction was added to a chloroform/methanol solution for delipidation. The resulting milky solution was filtered and the apoA-I precipitate was allowed to dry overnight. The protein was renatured in 6 M guanidine hydrochloride, and the resulting solution dialyzed against PBS. Finally, the apoA-I PBS solution was filtered through a 0.22 μm filter and the protein's identity and purity were established by gel electrophoresis and size exclusion chromatography. Example 24. Cryo-TEM Measurements on HDL-Derived Nanoparticle Formulations The molecule compounds of Example 2 and Example 7, respectively, i.e. MDP-DSPE[click] and MDP(Bn)-DSPE[click] were formulated together with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC; (CAS [18194-24-6]) and APO-A1, and with varying amounts of cholesterol, to create HDL-derived nanoparticle formulations. The following table shows the employed relative molar amounts of the DMPC, compound and cholesterol components. APO-Al was used in twice the amount (in mg) as the compound of the invention (in mg). The dynamic scattering data (DLS) of the processed formulation are also given: the recorded diameter and its error in brackets are based on the number-averaged DLS data. The DLS-recorded polydispersity in the dimensions of the particles is also given. TABLE 8Formulations for Cryo-TEM measurementsAPO-A1DMPCCompoundCholesterol*DLSDLSEntryCompoundmol %mol %mol %mg-to-mgnm (error)PDIAExample 2901002-to-110.1 (2.1)0.42BExample 7901002-to-19.9 (1.8)0.57CExample 29010102-to-111.9 (3.0)0.32DExample 79010102-to-111.4 (2.1)0.42EExample 29010202-to-122.2 (6.1)0.18FExample 79010202-to-131.0 (6.0)0.48* APO-A1 is employed in twice the amount in weight (mg) as the compound Formulation: DMPC, cholesterol and the compound were dissolved in ethanol (entries A, C and E) or in ethanol/DMSO (entries B, D, F), while apoA-1 was dissolved in PBS buffer (pH=7.5). The organic solution was mixed with the buffer solution applying T-junction mixing. Purification of the resulting solutions was performed by TFF (tangential flow filtration), thereby getting rid of the organic solvents. Samples were concentrated by spin-filtration. The final HDL-derived nanoparticle solutions had typical recoveries of the used compounds (Example 2 and 7), DMPC and cholesterol that exceeded 75%. Recoveries were determined by HPLC (for the compounds), and using assays that are known in the art (for DMPC and cholesterol). Concentrations of the final HDL-derived nanoparticle solutions were about 2 to 4 mg/mL in the compound (Example 2 or 7). FIG.1shows the recorded Cryo-TEM pictures for the HDL-derived nanoparticle of entries A to F. The 50 nm bar applies to all 6 pictures. When 0% cholesterol is used, spherical disc-like particles are dominantly observed (A and B; sizes about 5 to 10 nm). Applying 10% of cholesterol mostly shows slightly extended discs (C and D; lengths about 5 to 25 nm and thicknesses about 5 nm). Using 20% cholesterol shows clearly extended worm-like particles. The lengths of these worm-like particles is about 20 to about 50 nm and the thicknesses are about 5 nm (picture C). In picture F, the lengths of the worms are about 50 to 100 nm; again, the thicknesses are about 5 nm. In F one also observes that the worm-like particles aggregate to larger stacks. The results highlight that the HDL-derived nanoparticle particle dimensions can be steered using the cholesterol content of the formulation (compare C with E, and D with F), but also with the lipophilicity of the compound and/or the substitution of the R2position in Formula (I) of the compounds of the invention (clear: compare E with F; less clear: in D the particles seem to be a bit more extended than in C). Method: Just before processing the samples, 200-mesh lacey carbon supported copper grids (Electron Microscopy Sciences) were surface plasma treated for 40 seconds using a Cressington 208 carbon coater. Next, 3 μL of the HDL-derived nanoparticle sample solutions was transferred to the grids. A thin film of sample solution was then vitrified on the grid by plunge vitrification in liquid ethane, using an automated vitrification robot (FEI Vitrobot Mark IV). Processed films were stored until measurement took place. Cryo-TEM imaging of the prepared films was carried out on a CryoTITAN microscope (Thermo Fisher) equipped with a field emission gun (FEG), a post-column Gatan imaging filter (model 2002), and a post-GIF 2 k×2 k Gatan CCD camera (model 794). Example 25: In Vitro NOD2 Activation Assay The stimulation of human NOD2 (hNOD2) by compounds disclosed herein was studied by monitoring activation of NF-κB in HEK-Blue™ hNOD2 cells (Invitrogen). 50,000 HEK-Blue™ hNOD2 cells were seeded in HEK-Blue™ Detection Medium in flat-bottom tissue culture plates. Concentration ranges of the test article (compounds diluted from DMSO-solutions (17.8 mmoL/L), first with demineralized water and then with PBS to the desired concentrations) were added to the cells in the tissue culture plates. Cells were incubated overnight at 37° C. and 5% CO2. The following day, supernatants were collected in an ELISA plate and the OD was measured at 620 nm using a spectrophotometer. The signal in this assay is based on NOD2 stimulation with a ligand which subsequently activates NF-κB and AP-1, resulting in the production of SEAP. Levels of SEAP were then determined with HEK-Blue™ Detection medium (Invitrogen). The hydrolysis of the substrate in the medium by SEAP produces a purple/blue color that was then measured with an Absorbance microplate reader. OD values are mapped based on test article concentration and are depicted inFIG.3. Assay performance was validated with muramyl dipeptide (CAS number [53678-77-6]). All tested compounds of the invention (APIs) are capable of activating NOD2. Potency for Example 2, 3 and 7 compounds is comparable. Example 8 and 13 compounds are also able to activate NOD2, but seemingly to a lesser extent than the Example 2, 3 and 7 compounds. Example 26: Combination Activity of (i) HDL-Derived Nanoparticles and (ii) Immune Checkpoint Inhibition in the B16F10 Mouse Model Formulations In-Vivo Study 1. The formulations for these in-vivo studies were prepared by T-junctions mixing, followed by tangential flow filtration (TFF). Processing and purification procedures were applied as those highlighted in the formulations for the Cryo-TEM measurements (Table 8). Compounds used were those of Example 2, 3, 7, 8 and 13 (i.e. these Example materials are collectively named APIs in these descriptions of the in-vivo studies). For the 5 administered formulations, the following relative ratio of components have been used. DMPCCompoundCholesterolapoA-1*EntryCompoundMol %Mol %Mol %mg-to-mg1Example 29010102-to-12Example 39010102-to-13Example 79010102-to-14Example 89010101-to-15Example 139010101-to-1*relative to the compound Protocol and Results The panel of Example APIs was formulated to HDL-derived nanoparticles (or nanobiologics, NBs) that were screened for their anti-tumor activity in combination with immune checkpoint inhibitors in the B16F10 syngeneic mouse tumor model. To this end, B16F10 murine melanoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM)(Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycine (P/S). At the day of injection, cells were harvested and resuspended at 1×106viable cells/mL in PBS with 0.5% FBS. During counting the cells were checked for viability using Trypan Blue Solution, 0.4% (Gibco). At the start of the experiment 1×105B16F10 tumor cells in 100 μL PBS supplemented with 0.5% fetal bovine serum (FBS) were injected subcutaneously in the flank of 7-week-old female C57BL/6 mice (The Jackson Laboratory). Seven days after tumor inoculation, mice were randomized in groups with similar average group size (n=10). Average tumor size of groups was 3.26 mm3. After randomization mice were ear notched and weighed. Subsequently doses were calculated and aliquoted. Aliquoted doses were stored until use at 4° C. The study consisted of the following: a PBS control group, an immune checkpoint inhibitors group (CI) and 6 treatment groups. The immune checkpoint inhibitors treated mice received an intraperitoneal injection on day 2, 4 and 8, using doses of 200 μg anti-CTLA-4 (clone, 9H10, BioXcell) and/or 200 μg anti-PD-1 (clone, RMP1-14, BioXcell). Treatment groups consisted of NBs prepared from the Example 2 compound, NBs from Example 3, NBs from Example 7, NBs from Example 8, NBs from Example 13 combined with the immune checkpoint inhibitors therapy (CI) as described above. Dosing for treatment groups was about 9 mg MDP/kg (or about 27 mg/kg of the respective APIs contained in the NBs) in question on day 0, 2 and 4. Tumor growth curves are depicted inFIG.4A-Eand in each graph the same PBS and CI groups have been mapped to allow for comparison between graphs. PBS treated animals or animals that were treated with immune checkpoint inhibitors alone did not show tumor growth inhibition. The groups of animals treated with combination therapy all showed tumor growth inhibition and it was most pronounced for those groups in which NBs of Example 2 (FIG.4A), NBs of Example 7 (FIG.4C) or NBs of Example 8 (FIG.4D) was part of the combination therapy. Note that the above results on the combination therapy were obtained with an applied formulation that contained 10 mol % cholesterol relative to the applied 90 mol % DMPC (see the above Table), and thus these particles had dimensions of approximately 5 to maximally 10 nm (see the Cryo-TEM panels C and D inFIG.1). Example 27: Single Agent Activity of Nanobiologics in B16F10 Mouse Model The formulations for these in-vivo studies were prepared by T-junctions mixing, followed by tangential flow filtration (TFF). Processing and purification procedures were applied as those highlighted in the formulations for the Cryo-TEM measurements (Table 8). The below table shows the employed relative ratios of components to prepare the HDL-derived nanoparticles. DMPCCompoundCholesterolapoA-1*DLS #DLS #EntryCompoundMol %Mol %Mol %mm-to-mg(nm)PDI (—)1Example 29010102-to-111.9 (3.0)0.322Example 79010102-to-111.4 (2.1)0.423Example 29010202-to-122.2 (6.1)0.184Example 28020201-to-116.4 (4.2)0.215Example 78020201-to-124.4 (4.4)0.47*relative to the compound;# number averaged diameters. Protocol and Results Two APIs (the Example 2 and Example 7 compounds) were used to generate a set of different HDL-derived nanoparticle formulations (or nanobiologic formulations; nanobiologics; NBs) to determine their potencies. The resulting nanobiologics were screened for their single agent anti-tumor activity in the B16F10 syngeneic mouse tumor model. To this end B16F10 murine melanoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM)(Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycine (P/S). The day of injection cells were harvested and resuspended at 1×106viable cells/mL in PBS with 0.5% FBS. During counting cells were checked for viability using a Cell Counter and Analyzer (Casy). At the start of the experiment 1×105B16F10 tumor cells in 100 μL PBS supplemented with 0.5% fetal bovine serum (FBS) were injected subcutaneously in the flank of 7-week-old female C57BL/6J mice (Charles River). Seven days after tumor inoculation, mice were randomized in groups with similar average group size. Groups consisted of 8-10 mice. Average tumor size of groups was 6.33 mm3. After randomization mice were tattooed with a number on the tail. Subsequently doses were calculated and aliquoted. Aliquoted doses were stored until use at 4° C. The study consisted of the following: a PBS control group and 5 treatment groups: NBs of Example 2 (formulation Entries 1, 3 and 4) and NBs of Example 7 (formulation Entries 2 and 5). Dosing for treatment groups was about 3 mg MDP/kg (or about 9 mg/kg of the respective APIs contained in the NBs) in question on day 0, 2 and 4. Tumor size was measured at set times during the course of the study. Tumor growth curves are depicted inFIG.5A-Cand in each graph the same PBS group is mapped to allow for comparison between graphs. Apart from the NBs of Example 2 (formulation Entry 1), all nanobiologics clearly showed a reduction in the tumor growth in comparison with the PBS control group. For both Example 2 (FIG.5A) and Example 7 (FIG.5B) the nanobiologics formulations with 20 mol % cholesterol performed best.FIG.5Cshows that NBs of Example 2 slightly outperform NBs of Example 7 (using the formulations of Entries 4 and 5, respectively). Note that in FigureFIG.5A, the Example-2 formulation containing 10 mol % cholesterol (Entry 1 in the above Table) showed a minor single agent activity (compare to PBS). Surprisingly, and in comparison, the Example-2 formulations with 20 mol % cholesterol (Entries 3 and 4 in the above Table) show extraordinarily enhanced tumor suppressions. For the Example-7 formulations, this can also be seen (FIG.5B). Apparently, the dimensions of the formed particles play a decisive role in the activity of the prepared HDL-derived nanoparticles: the 10% cholesterol formulations give spherical or only slightly stretched disk-like particles with 5 to 10 nm dimensions, while the 20% cholesterol formulations give elongated worm-like particles about 15 to 50 nm in length, and about 5 nm in thickness (compare panels C and E inFIG.1; cryo-TEM data). The disclosed HDL-derived nanoparticles have certain dimensions, and these features find particular use in producing stable and potent HDL-derived NPs. Without being bound by theory, it is thought that the disclosed HDL-derived nanoparticles provide improved (multivalent) presentation of the MDP (or MDP(Bn) or MTP or MTP(Bn)) moieties to cells, dramatically improving their potency. Embodiments 1. A compound of formula (I): or a pharmaceutically acceptable salt thereof,wherein:R1is —H or —C(O)—RX;R2and R3are each independently selected from the group consisting of —H, alkyl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5are each alkyl;R6and R11are each independently —H, or alkyl;R7is a C9-30fatty acid chain, —Y—N(R11)—C(O)—O-alkylene-C(H)(OR8)-alkylene-OR9, —C(R10)(C(O)NH2)-alkylene-N(R11)—C(O)—C16-30fatty acid chain, —(CR10R10)2—O—P(O)(OH)—O-alkylene-C(R10)(ORZ)-alkylene-ORZ, or —Y-triazolyl-L;RZis a C8-30fatty acid or —C(O)—C16-30fatty acid chain;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11)—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;R10, R22, R33, R33′, R44, R44′, R55, and R55′are each independently H or RA;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, aryl, and triazolyl is optionally substituted with one or more RA, wherein RAis independently selected for each occurrence from the group consisting of halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro;wherein, when R7is C9-30fatty acid chain, R2is —H.2. The compound of embodiment 1, wherein the compound of formula (I) is a compound of formula (IA): or a pharmaceutically acceptable salt thereof.3. The compound of embodiment 1 or 2, wherein R2is —H or benzyl.4. The compound of any one of embodiments 1-3, wherein R2is —H.5. The compound of any one of embodiments 1-4, wherein R10, R22, R33, R33′, R44, R44′, R55, and R55′are each —H.6. The compound of any one of embodiments 1-5, wherein R4is alkyl.7. The compound of any one of embodiments 1-6, wherein R4is methyl.8. The compound of any one of embodiments 1-7, wherein R3and R6are both —H.9. The compound of any one of embodiments 1-8, wherein Y is alkylene optionally substituted with —C(O)N(RC)(RD).10. The compound of embodiment 9, wherein Y is —CH2— or 11. The compound of embodiment 10, wherein Y is —CH2—.12. The compound of any one of embodiments 1-11, wherein R7is —Y-triazolyl-L;13. The compound of any one of embodiments 1-12, wherein the compound of formula (I) is a compound of formula (II): or a pharmaceutically acceptable salt thereof,wherein:X1is —N— and X2is —C—; or X1is —C— and X2is —N—.14. The compound of embodiment 13, wherein the compound of formula (I) is a compound of formula (IIA): or a pharmaceutically acceptable salt thereof.15. The compound of embodiment 13 or 14, wherein Y is C1-6alkylene.16. The compound of any one of embodiments 13-15, wherein RAis —H.17. The compound of any one of embodiments 1-16, wherein L is selected from the group consisting of C8-30fatty acid chain, —CH2—C(O)—W, —CH2—O—C(O)—W, —CH2CH2—N—CH2CH2—C(O)—NR11—CH2CH2—NR11—C(O)—W)2, and —CH2CH2—N—(CH2CH2—C(O)—W)2.18. The compound of embodiment 17, wherein L is a C12-18fatty acid chain.19. The compound of embodiment 17, wherein L is —CH2(CH2CH2)8—CH3.20. The compound of any one of embodiments 1-19, wherein W is a C8-30fatty acid chain.21. The compound of any one of embodiments 1-19, wherein W is a C12-18fatty acid chain.22. The compound of any one of embodiments 1-19, wherein W is: 23. The compound of embodiment 22, wherein RXand RX′is each independently a —C8-30fatty acid chain24. The compound of embodiment 22 or 23, wherein RXand RX′is each independently a C12-18fatty acid chain.25. The compound of embodiment 24, wherein RXand RX′are both —(CH2CH2)8—CH3.26. The compound of any one of embodiments 1-19, wherein W is cholesterol: 27. The compound of any one of embodiments 1-19, wherein W is a phospholipid selected from the group consisting of: a phosphatidylcholine (PC), a phosphatidylglycerol (PG), a phosphatidylserine (PS), a phosphatidylethanolamine (PE), a phosphatidic acid (PA), and a lysophosphatidylcholine.28. The compound of embodiment 27, wherein W is a phospholipid having the structure: or a pharmaceutically acceptable salt thereof;wherein RXand RX′are each independently a C8-30fatty acid chain.29. The compound of embodiment 28, wherein RXand RX′are each independently a C12-18fatty acid.30. The compound of embodiment 28 or 29, wherein the fatty acid is saturated.31. The compound of embodiment 28, wherein RXand RX′are both —(CH2CH2)8—CH3.32. The compound of embodiment any one of embodiments 1-11, wherein R7is —C(H)(C(O)NH2)—C5alkylene-N(R11)—C(O)—C17-30fatty acid.33. The compound of any one of embodiments 1-11, wherein R7is —CH2CH2—O—P(O)(OH)—O—CH2—C(H)(ORZ)—CH2—ORZ.34. The compound of any one of embodiments 1-33 selected from the group consisting of: 35. A nanobiologic composition comprising a high-density lipoprotein (HDL)-derived nanoparticle, wherein the nanoparticle comprises a compound of formula (I): or a pharmaceutically acceptable salt thereof,wherein:R1is —H or —C(O)—RX;R2and R3are each independently selected from the group consisting of —H, alkyl, alkylene-aryl, —C(O)-alkyl, and —C(O)-aryl;R4, R5, and R5are each alkyl;R6and R11are each independently —H, or alkyl;R7is a fatty acid chain, —Y—N(R6)—C(O)—O-alkylene-C(H)(OR8)-alkylene-OR9, —Y—N(R6)—C(O)—RX, —Y—O—P(O)(OH)—O-alkylene-C(H)(OR8)-alkylene-OR9, or —Y— triazolyl-L;Y is alkylene;L is selected from the group consisting of a fatty acid chain, -alkylene-C(O)—W, -alkylene-O—C(O)—W, -alkylene-N-(alkylene-C(O)—NR11-alkylene-NR11—C(O)—W)2, and -alkylene-N-(alkylene-C(O)—W)2;W is a fatty acid chain, —O-alkylene-C(H)(OR8)-alkylene-OR9, a phospholipid, or a sterol;R8and R9are each independently RXor —C(O)—RX;R10, R22, R33, R33′, R44, R44′, R55, and R55′are each independently H or RA;RXis a fatty acid chain;wherein each aforementioned alkyl, alkylene, alkylene-aryl, aryl, and triazolyl is optionally substituted with one or more RA, wherein RAis independently selected for each occurrence from the group consisting of halo, alkoxy, haloalkoxy, cyano, hydroxyl, —N(RC)(RD), —C(O)N(RC)(RD), —N(RC)C(O)RB, —OC(O)NRCRD, —NRCC(O)ORB, —OC(O)RB, —C(O)ORB, —C(O)RB, —CO2H, —NO2, —SH, S(O)XRB(wherein X is 0, 1, or 2), aryl, heteroaryl, heteroarylalkyl, cycloalkyl, heterocycloalkyl, and RB;RCand RDare independently selected for each occurrence from the group consisting of hydrogen, alkyl, haloalkyl —C(O)RB, and —C(O)ORB; or RCand RDare taken together with the nitrogen to which they are attached to form a heterocyclic ring optionally substituted with RA; andRBis alkyl, alkenyl, or alkynyl optionally substituted with one or more fluoro.36. A nanobiologic composition, comprising a high-density lipoprotein (HDL)-derived nanoparticle, wherein the nanoparticle comprises a compound of any one of embodiments 1-34.37. The nanobiologic composition of embodiment 35 or 36, wherein the HDL-derived nanoparticle comprises one or more phospholipids.38. The nanobiologic composition of embodiment 37, wherein the phospholipid is independently selected from the group consisting of a phosphatidylcholine, a phosphatidylethanolamine, a phosphatidylinositol, a phosphatidylserine, a sphingomyelin or other ceramide, a phospholipid-containing oil, a phosphatidylglycerol, a phosphatidic acid, a lysophosphatidylcholine, and combinations thereof.39. The nanobiologic composition of embodiment 35 or 36, comprising a phospholipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and a lysolipid selected from the group consisting of 1-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHPC).40. The nanobiologic composition of any one of embodiments 35-39, wherein the HDL-derived nanoparticle comprises apoA-I or a peptide mimetic of apoA-I.41. The nanobiologic composition of any one of embodiments 35-40, wherein the HDL-derived nanoparticle further comprises one or more triglycerides, fatty acid esters, hydrophobic polymers, sterol esters, or combinations thereof.42. The nanobiologic composition of any one of embodiments 35-41, wherein the HDL-derived nanoparticle further comprises cholesterol.43. The nanobiologic composition of embodiment 42, wherein the HDL-derived nanoparticle comprises one or more phospholipids and cholesterol in a molar ratio in the range of about 1:0.05 to about 1:0.25.44. The nanobiologic composition of embodiment 43, wherein the HDL-derived nanoparticle comprises one or more phospholipids and cholesterol in a molar ratio of about 1:0.2.45. The nanobiologic composition of any one of embodiments 1-44, wherein the HDL-derived nanoparticle is a nanodisc or nanosphere.46. The nanobiologic composition of embodiment 45, wherein the nanodisc or nanosphere is about 8 nm to about 400 nm in diameter.47. A pharmaceutical composition comprising a compound of any one of embodiments 1-34 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.48. A method for treating a cell-proliferation disorder in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the nanobiologic composition of any one of embodiments 35-46.49. The method of embodiment 48, wherein the cell-proliferation disorder is cancer.50. The method of embodiment 49, wherein the cancer is selected from the group consisting of bladder cancer, cancer of the blood vessels, bone cancer, brain cancer, breast cancer, cervical cancer, chest cancer, colon cancer, endometrial cancer, esophageal cancer, eye cancer, head cancer, kidney cancer, liver cancer, cancer of the lymph nodes, lung cancer, mouth cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, colorectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, urothelial cancers, and uterine cancer.51. The method of embodiment 49, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, pancreatic cancer, and glioblastoma.52. The method of any one of embodiments 48-51, wherein the method further comprises co-administering a cancer drug as a combination therapy with the nanobiologic composition.53. A method for treating sepsis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the nanobiologic composition of any one of embodiments 35-46.54. The method of embodiment 53, wherein the patient has sepsis associated with a bacterial, viral or fungal infection of the lungs, abdomen, kidney, or bloodstream.55. The method of any one of embodiments 48-54, wherein the nanobiologic composition promotes a hyper-responsive innate immune response in the patient in need thereof.56. The method of embodiment 55, wherein the hyper-responsive innate immune response is promoted for at least about 7 to about 30 days.57. The method of embodiment 55, wherein the hyper-responsive innate immune response is promoted for at least 30 to 100 days.58. The method of embodiment 55, wherein the hyper-responsive innate immune response is promoted for more than 100 days and up to 3 years.59. The method of embodiment 55, wherein the nanobiologic composition is administered once and wherein the hyper-responsive innate immune response is promoted for at least 30 days.60. The method of embodiment 55, wherein the nanobiologic composition is administered at least once per day in each day of a multiple-dosing regimen, and wherein the hyper-responsive innate immune response is promoted for at least 30 days.61. The method of any one of embodiments 48-58, wherein the nanobiologic composition is administered in a treatment regimen comprising two or more doses to the patient to generate an accumulation of drug in myeloid cells, myeloid progenitor cells, and hematopoietic stem cells in the bone marrow, blood and/or spleen.62. The method of any one of embodiments 48-61, wherein the nanobiologic composition is administered intravenously or intra-arterially.63. A method for activating a NOD2 receptor in a subject in need thereof, comprising administering to the subject an effective amount of the nanobiologic composition of any one of embodiments 35-46.64. A process for manufacturing a nanobiologic composition of any one of embodiments 35-46, the process comprising:a) forming a lipid film comprising: i) a compound of any one of embodiments 1-34; ii) one or more phospholipids; optionally iii) a hydrophobic matrix comprising one or more triglycerides, fatty acid esters, hydrophobic polymers, or sterol esters, or a combination thereof, and optionally iv) cholesterol; under conditions effective to form the lipid film; andb) dissolving the lipid film in a solvent to form a lipid solution; and contacting the lipid solution with apoA-I or a peptide mimetic of apoA-I under conditions effective to form a HDL-derived nanoparticle comprising a compound of any one of embodiments 1-34.65. A nanobiologic composition prepared according to embodiment 64.66. A kit comprising a nanobiologic composition of any one of embodiments 35-46.67. The method of claim52wherein the cancer drug is a checkpoint inhibitor.68. The method of claim67wherein the checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-CTLA-4 antibody, and combinations thereof. | 226,379 |
11859022 | DETAILED DESCRIPTION The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety. Unless otherwise disclosed, the terms “a” and “an” as used herein are intended to encompass one or more (i.e., at least one) of a referenced feature. Where present, all ranges are inclusive and combinable, except as otherwise noted. For example, when a range of “1 to 5” (i.e., 1-5) is recited, the recited range should be construed as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”, “1-3 & 5”, and the like. The terms “graft copolymer”, “branched copolymer” and the like herein generally refer to a copolymer comprising a “backbone” (or “main chain”) and side chains branching from the backbone. Examples of graft copolymers herein have a backbone comprising an alpha-1,3-glucan ether or ester compound, and at least one side chain of alpha-1,3-glucan comprising at least about 50% alpha-1,3 glycosidic linkages. In some aspects, a backbone can have an alpha-1,3-glucan extension, since the non-reducing end of the ether/ester glucan backbone is contemplated to be able to prime alpha-1,3-glucan synthesis by a glucosyltransferase enzyme. A backbone can be a [non-derivatized alpha-1,3-glucan]-[alpha-1,3-glucan ether/ester derivative] linear copolymer in some instances. An alpha-1,3-glucan ether or ester compound of a backbone herein can optionally be referred to in shorthand as an “alpha-glucan derivative”. In an alternative aspect of the present disclosure, any feature herein characterizing a graft copolymer can likewise characterize (if appropriate and applicable) a [non-derivatized alpha-1,3-glucan]-[alpha-1,3-glucan ether/ester derivative] linear copolymer that does not have any alpha-1,3-glucan side chains (such a copolymer is not a graft copolymer). The terms “alpha-1,3-glucan side chain” and “alpha-1,3-glucan arm” and the like can be used interchangeably herein. An alpha-1,3-glucan side chain(s) is contemplated to be (i) joined directly to a glucose unit of the backbone via alpha-glycosidic linkage (e.g., alpha-1,6, alpha-1,4, or alpha-1,2) (in some cases, such linkage might result from the promiscuous activity of an alpha-1,3-glucan-synthesizing glucosyltransferase enzyme); (ii) extensions of pre-existing branches (e.g., alpha-1,2, -1,4, and/or -1,6), as such branches present non-reducing ends that can possibly prime alpha-1,3-glucan synthesis by a glucosyltransferase enzyme; and/or (iii) possibly joined in an uncharacterized manner. The terms “alpha-glucan”, “alpha-glucan polymer” and the like are used interchangeably herein. An alpha-glucan is a polymer comprising glucose monomeric units linked together by alpha-glycosidic linkages. In typical embodiments, an alpha-glucan herein comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages. Examples of alpha-glucan polymers herein include alpha-1,3-glucan used to prepare an ether or ester derivative, which is consequently used as a backbone in a graft copolymer, and alpha-1,3-glucan side arms of a graft copolymer. The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and the like are used interchangeably herein. Alpha-1,3-glucan is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 30% of the glycosidic linkages are alpha-1,3. Alpha-1,3-glucan in certain embodiments comprises at least about 90% or 95% alpha-1,3 glycosidic linkages. Most or all of the other linkages in alpha-1,3-glucan herein typically are alpha-1,6, though some linkages may also be alpha-1,2 and/or alpha-1,4. Alpha-1,3-glucan as presently defined can characterize (i) an alpha-1,3-glucan side chain herein and (ii) an alpha-1,3-glucan used to prepare an ether or ester derivative, which is consequently used as a backbone in a graft copolymer. In some aspects, alpha-1,3-glucan can characterize an alpha-1,3-glucan “homopolymer”, which is alpha-1,3-glucan that is not part of (i) a graft copolymer or (ii) part of a [non-derivatized alpha-1,3-glucan]-[alpha-1,3-glucan ether/ester derivative) linear copolymer. An alpha-1,3-glucan used to prepare an ether or ester derivative herein typically is linear (no branches) or substantially linear. “Completely linear” alpha-1,3-glucan has no branches, before being ether- or ester-derivatized and consequently used to produce a graft copolymer. A “substantially linear” alpha-1,3-glucan herein has 5% or less branches (e.g., alpha-1,2, -1,4, and/or -1,6), before being ether- or ester-derivatized and consequently used to produce a graft copolymer. Branches of a substantially linear alpha-1,3-glucan herein typically are short, being one (pendant) to three glucose monomers in length, and comprise less than about 5% of all the glucose monomers of the entire glucan molecule. An alpha-1,3-glucan ether or ester derivative used in a glucosyltransferase reaction herein for alpha-1,3-glucan synthesis (thereby producing a copolymer) can optionally be characterized as a “primer”, “acceptor”, or other like term. An “alpha-1,2 branch” (and like terms) herein comprises a glucose that is alpha-1,2-linked to an alpha-1,3-glucan; an alpha-1,2 branch herein can also be referred to as an alpha-1,2,3 linkage. An “alpha-1,6 branch” (and like terms) as referred to herein comprises a glucose that is alpha-1,6-linked to an alpha-1,3-glucan; an alpha-1,6 branch herein can also be referred to as an alpha-1,6,3 linkage. An “alpha-1,4 branch” (and like terms) as referred to herein comprises a glucose that is alpha-1,4-linked to an alpha-1,3-glucan; an alpha-1,4 branch herein can also be referred to as an alpha-1,4,3 linkage. The percent branching in an alpha-glucan or graft copolymer herein refers to that percentage of all the glycosidic linkages therein that represent branch points. The terms “glycosidic linkage”, “glycosidic bond” and the like refer to the covalent bonds connecting the sugar monomers within a saccharide compound (oligosaccharides and/or polysaccharides). Examples of glycosidic linkages include alpha-linked glucose oligomers with 1,6-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,6” linkages); 1,3-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,3” linkages); 1,4-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,4” linkages); and 1,2-alpha-D-glycosidic linkages (herein also referred to as “alpha-1,2” linkages). The glycosidic linkages of a glucan polymer herein can also be referred to as “glucosidic linkages”. Herein, “alpha-D-glucose” is referred to as “glucose”. Alpha-1,2 linkages typically only occur at branch points, and do not occur in tandem (i.e., two or more consecutive glucose monomers are not joined by consecutive alpha-1,2 linkages). The linkage profile of an alpha-glucan or graft copolymer herein can be determined using any method known in the art. For example, a linkage profile can be determined using methods using nuclear magnetic resonance (NMR) spectroscopy (e.g.,13C NMR or1H NMR). These and other methods that can be used are disclosed in, for example,Food Carbohydrates: Chemistry, Physical Properties, and Applications(S. W. Cui, Ed., Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, FL, 2005), which is incorporated herein by reference. The “molecular weight” of large alpha-glucan and graft copolymers herein can be represented as weight-average molecular weight (Mw) or number-average molecular weight (Mn), the units of which are in Daltons or grams/mole. Alternatively, such molecular weight can be represented as DPw (weight average degree of polymerization) or DPn (number average degree of polymerization). The molecular weight of smaller polymers such as oligosaccharides typically can be provided as “DP” (degree of polymerization), which simply refers to the number of glucoses comprised within the alpha-glucan; “DP” can also characterize the molecular weight of a polymer on an individual molecule basis. Various means for calculating these various molecular weight measurements can be employed such as high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC), and/or by following the procedure disclosed in the below Examples. The term “sucrose” herein refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar. Sucrose can alternatively be referred to as “alpha-D-glucopyranosyl-(1→2)-beta-D-fructofuranoside”. “Alpha-D-glucopyranosyl” and “glucosyl” are used interchangeably herein. The terms “glucosyltransferase”, “glucosyltransferase enzyme”, “GTF”, “glucansucrase” and the like are used interchangeably herein. The activity of a glucosyltransferase herein catalyzes the reaction of the substrate sucrose to make the products alpha-glucan and fructose. Other products (by-products) of a GTF reaction can include glucose, various soluble gluco-oligosaccharides, and leucrose. Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide (which is typically removed by cleavage processes), a variable domain, a catalytic domain, and a glucan-binding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al.,Nucleic Acids Res.37:D233-238, 2009). The term “glucosyltransferase catalytic domain” herein refers to the domain of a glucosyltransferase enzyme that provides alpha-glucan-synthesizing activity to a glucosyltransferase enzyme. A glucosyltransferase catalytic domain typically does not require the presence of any other domains to have this activity. The terms “enzymatic reaction”, “glucosyltransferase reaction”, “glucan synthesis reaction”, “reaction composition”, “reaction formulation” and the like are used interchangeably herein and generally refer to a reaction that initially comprises water, sucrose, at least one active glucosyltransferase enzyme, and optionally other components such as an alpha-1,3-glucan ether- or ester-derivative (as a primer). Components that can be further present in a glucosyltransferase reaction typically after it has commenced include fructose, glucose, leucrose, soluble gluco-oligosaccharides (e.g., DP2-DP7) (such may be considered as products or by-products, depending on the glucosyltransferase used), and/or insoluble alpha-glucan product(s) of DP8 or higher. It would be understood that certain glucan products, such as alpha-1,3-glucan with a degree of polymerization (DP) of at least 8 or 9, are water-insoluble and thus not dissolved in a glucan synthesis reaction, but rather may be present out of solution (e.g., by virtue of having precipitated from the reaction). It is in a glucan synthesis reaction where the step of contacting water, sucrose and a glucosyltransferase enzyme is performed. The term “under suitable reaction conditions” as used herein refers to reaction conditions that support conversion of sucrose to alpha-glucan product(s) via glucosyltransferase enzyme activity. It is during such a reaction that glucosyl groups originally derived from the input sucrose are enzymatically transferred and used in alpha-glucan polymer synthesis; glucosyl groups as involved in this process can thus optionally be referred to as the glucosyl component or moiety (or like terms) of a glucosyltransferase reaction. The “yield” of insoluble alpha-glucan product in a glucosyltransferase reaction in some aspects herein represents the molar yield based on the converted sucrose. The molar yield of an alpha-glucan product can be calculated based on the moles of insoluble alpha-glucan product divided by the moles of the sucrose converted. Moles of converted sucrose can be calculated as follows: (mass of initial sucrose−mass of final sucrose)/molecular weight of sucrose [342 g/mol]. This molar yield calculation can be considered as a measure of selectivity of the reaction toward the alpha-glucan. In some aspects, the “yield” of insoluble alpha-glucan product in a glucosyltransferase reaction can be based on the glucosyl component of the reaction. Such a yield (yield based on glucosyl) can be measured using the following formula: Insoluble Alpha-Glucan Yield=((IS/2−(FS/2+LE/2+GL+SO))/(IS/2−FS/2))×100%. The fructose balance of a glucosyltransferase reaction can be measured to ensure that HPLC data, if applicable, are not out of range (90-110% is considered acceptable). Fructose balance can be measured using the following formula: Fructose Balance=((180/342×(FS+LE)+FR)/(180/342×IS))×100%. In the above two formulae, IS is [Initial Sucrose], FS is [Final Sucrose], LE is [Leucrose], GL is [Glucose], SO is [Soluble Oligomers] (gluco-oligosaccharides), and FR is [Fructose]; the concentrations of each foregoing substrate/product provided in double brackets are in units of grams/L and as measured by HPLC, for example. Terms used herein regarding “ethers” (e.g., alpha-1,3-glucan ether-derivative and like terms) are defined as in U.S. Patent Appl. Publ. Nos. 2014/179913, 2016/0304629, 2016/0311935, 2015/0239995, 2018/0230241, and/or 2018/0237816, which are incorporated herein by reference. An alpha-1,3-glucan ether compound has a DoS of about 0.001 to about 3.0 with one or more different types of organic group(s). An alpha-1,3-glucan ether compound is termed an “ether” herein by virtue of comprising the substructure —CG—O—C—, where “—CG—” represents carbon 2, 4, or 6 of a glucose monomeric unit of the alpha-1,3-glucan ether compound, and where “—C—” is comprised in the organic group. An “organic group” group (e.g., an ether-linked organic group) in some aspects refers to a chain of one or more carbons that (i) has the formula —CnH2n+1(i.e., an alkyl group, which is completely saturated) or (ii) is mostly saturated but has one or more hydrogens substituted with another atom or functional group (i.e., a “substituted alkyl group”). Such substitution can be with one or more hydroxyl groups, oxygen atoms (thereby forming an aldehyde or ketone group), carboxyl groups, or other alkyl groups. A “hydroxy alkyl” group herein refers to a substituted alkyl group in which one or more hydrogen atoms of the alkyl group are substituted with a hydroxyl group. A “carboxy alkyl” group herein refers to a substituted alkyl group in which one or more hydrogen atoms of the alkyl group are substituted with a carboxyl group. Terms used herein regarding “esters” (e.g., alpha-1,3-glucan ester-derivative and like terms) are defined as in U.S. Pat. Appl. Publ. Nos. 2014/0187767 and/or 2018/0155455, and/or Int. Patent Appl. Publ. No. WO2018/098065, which are incorporated herein by reference. An alpha-1,3-glucan ester compound herein has a DoS of about 0.001 to about 3.0 with one or more different types of acyl group(s). An alpha-1,3-glucan ester compound is termed an “ester” herein by virtue of comprising the substructure —CG—O—CO—C—, where “—CG—” represents carbon 2, 4, or 6 of a glucose monomeric unit of the alpha-1,3-glucan ester compound, and where “—CO—C—” is comprised in the acyl group. The term “degree of substitution” (DoS) as used herein refers to the average number of hydroxyl groups that are substituted (with organic groups via ether linkage, or with acyl groups via ester linkage) in each monomeric unit (glucose) of an alpha-1,3-glucan ether- or ester-derivative herein. DoS herein specifically refers to substitution with organic groups or acyl groups, and does not refer to any substituting saccharide groups (alpha-1,3-glucan side chain) (the DoS of an alpha-1,3-glucan ether or ester backbone of a graft copolymer herein is strictly based on its substitution with organic or acyl groups). In this sense, it can be said that the DoS of an alpha-1,3-glucan ether- or ester-derivative is the same both before and after its use as a primer for alpha-1,3-glucan side arm synthesis. The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)]×100%. The terms “percent by weight”, “weight percentage (wt %)”, “weight-weight percentage (% w/w)” and the like are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution. The term “weight/volume percent”, “w/v %” and the like are used interchangeably herein. Weight/volume percent can be calculated as: ((mass [g] of material)/(total volume [mL] of the material plus the liquid in which the material is placed))×100%. The material can be insoluble in the liquid (i.e., be a solid phase in a liquid phase, such as with a dispersion), or soluble in the liquid (i.e., be a solute dissolved in the liquid). The terms “aqueous liquid”, “aqueous fluid” and the like as used herein can refer to water or an aqueous solution. An “aqueous solution” herein can comprise one or more dissolved salts, where the maximal total salt concentration can be about 3.5 wt % in some embodiments. Although aqueous liquids herein typically comprise water as the only solvent in the liquid, an aqueous liquid can optionally comprise one or more other solvents (e.g., polar organic solvent) that are miscible in water. Thus, an aqueous solution can comprise a solvent having at least about 10 wt % water. An “aqueous composition” herein has a liquid component that comprises at least about 10 wt % water, for example. Examples of aqueous compositions include mixtures, solutions, dispersions (e.g., colloidal dispersions), suspensions and emulsions, for example. As used herein, the term “colloidal dispersion” refers to a heterogeneous system having a dispersed phase and a dispersion medium, i.e., microscopically dispersed insoluble particles are suspended throughout another substance (e.g., an aqueous composition such as water or aqueous solution). An example of a colloidal dispersion herein is a hydrocolloid. All, or a portion of, the particles of a colloidal dispersion such as a hydrocolloid can comprise a graft copolymer herein. The terms “dispersant” and “dispersion agent” are used interchangeably herein to refer to a material that promotes the formation and/or stabilization of a dispersion. “Dispersing” herein refers to the act of preparing a dispersion of a material in an aqueous liquid. An alpha-glucan or graft copolymer that is “insoluble”, “aqueous-insoluble”, “water-insoluble” (and like terms) (e.g., alpha-1,3-glucan with a DP of 8 or higher) does not dissolve (or does not appreciably dissolve) in water or other aqueous conditions, optionally where the aqueous conditions are further characterized to have a pH of 4-9 (e.g., pH 6-8) (i.e., non-caustic) and/or a temperature of about 1 to 85° C. (e.g., 20-25° C.). In contrast, alpha-glucans such as certain oligosaccharides herein that are “soluble”, “aqueous-soluble”, “water-soluble” and the like (e.g., alpha-1,3-glucan with a DP less than 8) appreciably dissolve under these conditions. The term “viscosity” as used herein refers to the measure of the extent to which a fluid (aqueous or non-aqueous) resists a force tending to cause it to flow. Various units of viscosity that can be used herein include centipoise (cP, cps) and Pascal-second (Pa·s), for example. A centipoise is one one-hundredth of a poise; one poise is equal to 0.100 kg·m−1·s−1. The term “zeta potential” as used herein refers to the electrical potential difference between a dispersion medium and the stationary layer of fluid attached to a graft copolymer particle dispersed in the dispersion medium. In general, a dispersed graft copolymer herein with a high zeta potential (negative or positive) is more electrically stabilized compared to a dispersed material with low zeta potentials (closer to zero). Since the repulsive forces of a high zeta potential material in a dispersion tend to exceed its attractive forces, such a dispersion is relatively more stable than a dispersion of low zeta potential material, which tends to more easily flocculate/coagulate. The terms “particle”, “particulate” and other like terms are interchangeably used herein. “Particle size” (and like terms) in some aspects can refer to particle diameter and/or the length of the longest particle dimension. Average particle size can be based on the average of diameters and/or longest particle dimensions of at least about 50, 100, 500, 1000, 2500, 5000, or 10000 or more particles, for example. The term “household care product” and like terms typically refer to products, goods and services relating to the treatment, cleaning, caring and/or conditioning of a home and its contents. The foregoing include, for example, chemicals, compositions, products, or combinations thereof having application in such care. The terms “fabric”, “textile”, “cloth” and the like are used interchangeably herein to refer to a woven material having a network of natural and/or artificial fibers. Such fibers can be in the form of thread or yarn, for example. A “fabric care composition” and like terms refer to any composition suitable for treating fabric in some manner. Examples of such a composition include laundry detergents and fabric softeners, which are examples of laundry care compositions. The terms “heavy duty detergent”, “all-purpose detergent” and the like are used interchangeably herein to refer to a detergent useful for regular washing of white and/or colored textiles at any temperature. The terms “low duty detergent”, “fine fabric detergent” and the like are used interchangeably herein to refer to a detergent useful for the care of delicate fabrics such as viscose, wool, silk, microfiber or other fabric requiring special care. “Special care” can include conditions of using excess water, low agitation, and/or no bleach, for example. A “detergent composition” herein typically comprises at least a surfactant (detergent compound) and/or a builder. A “surfactant” herein refers to a substance that tends to reduce the surface tension of a liquid in which the substance is dissolved. A surfactant may act as a detergent, wetting agent, emulsifier, foaming agent, and/or dispersant, for example. The term “personal care product” and like terms typically refer to products, goods and services relating to the treatment, cleaning, cleansing, caring or conditioning of a person. The foregoing include, for example, chemicals, compositions, products, or combinations thereof having application in such care. An “oral care composition” herein is any composition suitable for treating a soft or hard surface in the oral cavity such as dental (teeth) and/or gum surfaces. The terms “sequence identity”, “identity” and the like as used herein with respect to a polypeptide amino acid sequence are as defined and determined in U.S. Patent Appl. Publ. No. 2017/0002336, which is incorporated herein by reference. Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced. Alternatively, a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function/activity of the disclosed sequence, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence. Any polypeptide amino acid sequence disclosed herein not beginning with a methionine can typically further comprise at least a start-methionine at the N-terminus of the amino acid sequence. In contrast, any polypeptide amino acid sequence disclosed herein beginning with a methionine can optionally lack such a methionine residue. The terms “aligns with”, “corresponds with”, and the like can be used interchangeably herein. Some embodiments herein relate to a glucosyltransferase comprising at least one amino acid substitution at a position corresponding with at least one particular amino acid residue of SEQ ID NO:62. An amino acid position of a glucosyltransferase or subsequence thereof (e.g., catalytic domain or catalytic domain plus glucan-binding domains) (can refer to such an amino acid position or sequence as a “query” position or sequence) can be characterized to correspond with a particular amino acid residue of SEQ ID NO:62 (can refer to such an amino acid position or sequence as a “subject” position or sequence) if (1) the query sequence can be aligned with the subject sequence (e.g., where an alignment indicates that the query sequence and the subject sequence [or a subsequence of the subject sequence] are at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical), and (2) if the query amino acid position directly aligns with (directly lines up against) the subject amino acid position in the alignment of (1). In general, one can align a query amino acid sequence with a subject sequence (SEQ ID NO:62 or a subsequence of SEQ ID NO:62) using any alignment algorithm, tool and/or software described disclosed herein (e.g., BLASTP, ClustalW, ClustaIV, Clustal-Omega, EMBOSS) to determine percent identity. Just for further example, one can align a query sequence with a subject sequence herein using the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970) as implemented in the Needle program of the European Molecular Biology Open Software Suite (EMBOSS [e.g., version 5.0.0 or later], Rice et al., Trends Genet. 16:276-277, 2000). The parameters of such an EMBOSS alignment can comprise, for example: gap open penalty of 10, gap extension penalty of 0.5, EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The numbering of particular amino acid residues of SEQ ID NO:62 herein is with respect to the full-length amino acid sequence of SEQ ID NO:62. The first amino acid (i.e., position 1, Met-1) of SEQ ID NO:62 is at the start of the signal peptide. Unless otherwise disclosed, substitutions herein are with respect to the full-length amino acid sequence of SEQ ID NO:62. A “non-native glucosyltransferase” herein (“mutant”, “variant”, “modified” and like terms can likewise be used to describe such a glucosyltransferase) has at least one amino acid substitution at a position corresponding with a particular amino acid residue of SEQ ID NO:62. Such at least one amino acid substitution typically is in place of the amino acid residue(s) that normally (natively) occurs at the same position in the native counterpart (parent) of the non-native glucosyltransferase (i.e., although SEQ ID NO:62 is used as a reference for position, an amino acid substitution herein is with respect to the native counterpart of a non-native glucosyltransferase) (considered another way, when aligning the sequence of a non-native glucosyltransferase with SEQ ID NO:62, determining whether a substitution exists at a particular position does not depend in-and-of-itself on the respective amino acid residue in SEQ ID NO:62, but rather depends on what amino acid exists at the subject position within the native counterpart of the non-native glucosyltransferase). The amino acid normally occurring at the relevant site in the native counterpart glucosyltransferase often (but not always) is the same as (or conserved with) the particular amino acid residue of SEQ ID NO:62 for which the alignment is made. A non-native glucosyltransferase optionally can have other amino acid changes (mutations, deletions, and/or insertions) relative to its native counterpart sequence. The term “isolated” means a substance (or process) in a form or environment that does not occur in nature. A non-limiting example of an isolated substance includes any non-naturally occurring substance such as a graft copolymer herein (as well as the enzymatic reactions and other processes used in preparation thereof). It is believed that the embodiments disclosed herein are synthetic/man-made (could not have been made except for human intervention/involvement), and/or have properties that are not naturally occurring. The term “increased” as used herein can refer to a quantity or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms “increased”, “elevated”, “enhanced”, “greater than”, “improved” and the like are used interchangeably herein. New forms of alpha-1,3-glucan are desired to enhance the economic value and performance characteristics of this material in various applications. Compositions comprising alpha-1,3-glucan in the form of a graft copolymer are presently disclosed to address this need. Certain embodiments of the present disclosure concern a composition comprising a graft copolymer that comprises: (i) a backbone comprising an alpha-1,3-glucan ether or ester compound that has a degree of substitution (DoS) of about 0.001 to about 3.0, and (ii) one or more alpha-1,3-glucan side chains comprising at least about 50% alpha-1,3 glycosidic linkages. Significantly, graft copolymers of the present disclosure have several advantageous properties, including enhanced dispersion functions (e.g., viscosity and stability). A graft copolymer herein has a backbone comprising an alpha-1,3-glucan ether compound or ester compound that has a DoS of about 0.001 to about 3.0. An alpha-1,3-glucan ether or ester compound can comprise about, or at least about, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% alpha-1,3 glycosidic linkages, for example. In some aspects, accordingly, there can be less than about 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0% glycosidic linkages that are not alpha-1,3. Typically, the glycosidic linkages that are not alpha-1,3 are mostly or entirely alpha-1,6. In certain embodiments, an alpha-1,3-glucan ether- or ester-derivative has no glycosidic branch points or less than about 5%, 4%, 3%, 2%, or 1% glycosidic branch points as a percent of the glycosidic linkages in the derivative. In aspects in which a backbone comprises 50% alpha-1,3 glycosidic linkages, such a backbone typically does not comprise alternan (alternating alpha-1,3 and -1,6 linkages). An alpha-1,3-glucan ether compound or ester compound of a graft copolymer backbone herein can have a weight-average degree of polymerization (DPw) of about, at least about, or less than about, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1250, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000, for example. DPw can optionally be expressed as a range between any two of these values. Merely as examples, the DPw can be about 11-4000, 15-4000, 11-1300, 15-1300, 400-1300, 500-1300, 600-1300, 700-1300, 400-1200, 500-1200, 600-1200, 700-1200, 400-1000, 500-1000, 600-1000, 700-1000, 400-900, 500-900, 600-900, 700-900, 11-25, 12-25, 11-22, 12-22, 11-20, 12-20, 20-300, 20-200, 20-150, 20-100, 20-75, 30-300, 30-200, 30-150, 30-100, 30-75, 50-300, 50-200, 50-150, 50-100, 50-75, 75-300, 75-200, 75-150, 75-100, 100-300, 100-200, 100-150, 150-300, 150-200, or 200-300. Any of the preceding linkage and/or molecular weight features of an alpha-1,3-glucan ether- or ester-derivative can likewise characterize an alpha-1,3-glucan polymer used to prepare the alpha-1,3-glucan ether- or ester-derivative. Any glucosyltransferase disclosed herein for synthesizing alpha-1,3-glucan side chains comprising at least about 50% alpha-1,3 linkages can be used to synthesize alpha-1,3-glucan for ether- or ester-derivatization. A backbone of a graft copolymer in some aspects can be comprised entirely of an alpha-1,3-glucan ether- or ester-derivative as presently disclosed. However, in some aspects, a backbone can comprise other elements. For example, a graft copolymer backbone can comprise non-derivatized alpha-1,3-glucan originating from a/the non-reducing end of an alpha-1,3-glucan ether- or ester-derivative, by virtue of the glucan derivative (at a non-reducing end) serving to prime alpha-1,3-glucan synthesis during synthesis of the graft copolymer (during the step of adding alpha-1,3-glucan side chains). In such aspects, the DoS of the backbone can be considered to be the DoS of the alpha-1,3-glucan derivative as it existed before the addition of non-derivatized alpha-1,3-glucan during side chain synthesis. In some alternative aspects, an alpha-1,3-glucan ether compound or ester compound of a graft copolymer backbone herein can comprise at least about 30% alpha-1,3 linkages and a percentage of alpha-1,6 linkages that brings the total of both the alpha-1,3 and -1,6 linkages in the side chain to 100%. For example, the percentage of alpha-1,3 and -1,6 linkages can be about 30-40% and 60-70%, respectively. Glucosyltransferases contemplated to be useful for producing alpha-1,3-glucan for preparing such derivatives are disclosed in U.S. Patent Appl. Publ. No. 2015/0232819, which is incorporated herein by reference. In some alternative aspects, a graft copolymer herein can have, as a backbone, an ether- or ester-derivative of another type of alpha-glucan, such as dextran (alpha-glucan with at least 90%, 95%, 99% or 100% alpha-1,6 linkages), alpha-1,4-glucan (e.g., with at least 90%, 95%, 99% or 100% alpha-1,4 linkages), alternan, or reuteran. Any of the above DPw values/ranges can apply to any of these types of backbones. The backbone of a graft copolymer in some aspects can comprise an alpha-1,3-glucan ether compound. The DoS of an alpha-1,3-glucan ether with one or more etherified organic groups can be about 0.001 to about 3.0, for example. The DoS in some aspects can be about, or at least about, or up to about, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 (DoS can optionally be expressed as a range between any two of these values). Examples of DoS ranges herein include 0.05-2.6, 0.05-2.4, 0.05-2.2, 0.05-2.0, 0.05-1.8, 0.05-1.6, 0.05-1.4, 0.05-1.3, 0.05-1.2, 0.05-1.1, 0.05-1.0, 0.05-0.8, 0.05-0.6, 0.05-0.4, 0.1-2.6, 0.1-2.4, 0.1-2.2, 0.1-2.0, 0.1-1.8, 0.1-1.6, 0.1-1.4, 0.1-1.3, 0.1-1.2, 0.1-1.1, 0.1-1.0, 0.1-0.8, 0.1-0.6, 0.1-0.4, 0.2-2.6, 0.2-2.4, 0.2-2.2, 0.2-2.0, 0.2-1.8, 0.2-1.6, 0.2-1.4, 0.2-1.3, 0.2-1.2, 0.2-1.1, 0.2-1.0, 0.2-0.8, 0.2-0.6, 0.2-0.4, 0.3-2.6, 0.3-2.4, 0.3-2.2, 0.3-2.0, 0.3-1.8, 0.3-1.6, 0.3-1.4, 0.3-1.3, 0.3-1.2, 0.3-1.1, 0.3-1.0, 0.3-0.8, 0.3-0.6, and 0.3-0.4. An ether group can be anionic, uncharged (nonionic), or cationic; the charge of an ether group can be as it exists when the graft copolymer ether derivative is in an aqueous composition herein, for example, further taking into account the pH of the aqueous composition (in some aspects, the pH can be 4-10 or 5-9). An organic group etherified to an alpha-1,3-glucan backbone of a graft copolymer herein can be, for example, any of those as disclosed in U.S. Patent Appl. Publ. Nos. 2014/179913, 2016/0304629, 2016/0311935, 2015/0232785, 2015/0239995, 2018/0237816, and 2019/0202942, and Int. Patent Appl. Publ. Nos. WO2017/218389 and WO2017/218391, which are incorporated herein by reference. An organic group etherified to an alpha-1,3-glucan backbone of a graft copolymer herein can comprise an alkyl group such as a methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, or decyl group, for example (these are examples of uncharged groups). In some aspects, an organic group can be a substituted alkyl group in which there is a substitution on one or more carbons of the alkyl group. The substitution(s) may be one or more hydroxyl, aldehyde, ketone, and/or carboxyl groups. For example, a substituted alkyl group can be a hydroxy alkyl group, dihydroxy alkyl group, or carboxy alkyl group. Examples of suitable hydroxy alkyl groups include hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl and hydroxypentyl groups (these are examples of uncharged groups). Other examples include dihydroxy alkyl groups (diols) such as dihydroxymethyl, dihydroxyethyl, dihydroxypropyl, dihydroxybutyl and dihydroxypentyl groups (these are examples of uncharged groups). Examples of suitable carboxy alkyl groups include carboxymethyl (—CH2COOH), carboxyethyl, carboxypropyl, carboxybutyl and carboxypentyl groups (these are examples of anionic groups). An organic group in some aspects can comprise an aryl group such as a benzyl group. An organic group etherified to an alpha-1,3-glucan backbone of a graft copolymer herein can be a positively charged (cationic) organic group in some aspects. A positively charged group can be, for example, any of those as disclosed in U.S. Patent Appl. Publ. No. 2016/0311935, which is incorporated herein by reference. A positively charged group can comprise a substituted ammonium group, for example. Examples of substituted ammonium groups are primary, secondary, tertiary and quaternary ammonium groups. An ammonium group can be substituted with one, two, or three alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), for example. One of the groups of a substituted ammonium group comprises one carbon, or a chain of carbons, in ether linkage to alpha-1,3-glucan, such a carbon or carbon chain can be —CH2—, —CH2CH—, —CH2CH2CH2—, —CH2CH2CH2CH2—, or —CH2CH2CH2CH2CH2—, for example. A carbon or carbon chain in this context can optionally have at least one substitution with an oxygen atom (e.g., alcohol group) and/or alkyl group (e.g., methyl, ethyl, propyl, butyl). One or more positively charged organic groups in some aspects can be trimethylammonium hydroxypropyl groups (structure I, when each of R2, R3and R4is a methyl group). An alpha-1,3-glucan ether backbone of a graft copolymer in certain aspects can contain one type of etherified organic group. Non-limiting examples of such a backbone is carboxymethyl alpha-1,3-glucan or benzyl alpha-1,3-glucan. Alternatively, an alpha-1,3-glucan ether backbone can contain two or more different types of etherified organic groups (e.g., combinations of [i] carboxymethyl and benzyl groups, [ii] hydroxypropyl and hydroxyethyl groups, [iii] hydroxypropyl and ethyl groups, [iv] propyl and ethyl groups, or [v] propyl and hydroxyethyl groups). In some aspects, an alpha-1,3-glucan ether backbone can comprise at least one nonionic organic group and at least one anionic group as ether groups. In some aspects, an alpha-1,3-glucan ether backbone can comprise at least one nonionic organic group and at least one positively charged organic group as ether groups. Thus, an alpha-1,3-glucan ether backbone herein can optionally be amphiphilic. Any suitable process for ether-derivatizing polysaccharides can be employed to prepare an alpha-1,3-glucan ether backbone for graft copolymer production, such as disclosed in U.S. Pat. Nos. 2,961,439, 2,344,179, 2,203,703, 2,203,704, 2,380,879 and 2,974,134, U.S. Patent Appl. Publ. Nos. 2014/179913, 2016/0304629, 2016/0311935, 2015/0232785, 2015/0239995, 2018/0237816, and 2019/0202942, and Int. Patent Appl. Publ. Nos. WO2017/218389 and WO2017/218391, all of which are incorporated herein by reference. The backbone of a graft copolymer in some aspects can comprise an alpha-1,3-glucan ester compound. The DoS of an alpha-1,3-glucan ester with one or more acyl groups can be about 0.001 to about 3.0, for example. The DoS with an acyl group in some aspects can have the same value or range as listed above for ether derivatives. An acyl group esterified to an alpha-1,3-glucan backbone of a graft copolymer herein can be, for example, any of those as disclosed in U.S. Patent Appl. Publ. Nos. 2014/0187767 and 2018/0155455, and Int. Patent Appl. Publ. No. WO2018/098065, which are incorporated herein by reference. Examples of acyl groups herein include methanoyl (formyl), ethanoyl (acetyl, —CO—CH3), propanoyl (propionyl), butanoyl (butyryl), pentanoyl (valeryl), hexanoyl (caproyl), heptanoyl (enanthyl), octanoyl (caprylyl), nonanoyl (pelargonyl), decanoyl (capryl), undecanoyl, dodecanoyl (lauroyl), tridecanoyl, tetradecanoyl (myristyl), pentadecanoyl, hexadecanoyl (palmityl), heptadecanoyl, octadecanoyl (stearyl), nonadecanoyl, eicosanoyl (arachidyl), uneicosanoyl, docosanoyl (behenyl), tricosanoyl, tetracosanoyl (lignoceryl), pentacosanoyl and hexacosanoyl (cerotyl) groups, for example. Additional examples of acyl groups herein include branched acyl groups (e.g., 2-methylpropanoyl, 2-methylbutanoyl, 2,2-dimethylpropanoyl, 3-methylbutanoyl, 2-methylpentanoyl, 3-methylpentanoyl group, 4-methylpentanoyl, 2,2-dimethylbutanoyl, 2,3-dimethylbutanoyl, 3,3-dimethylbutanoyl group, 2-ethylbutanoyl group, 2-ethylhexanoyl), cyclic acyl groups (e.g., cyclopropanoyl, cyclobutanoyl, cyclopentanoyl, cyclohexanoyl, cycloheptanoyl), and aryl acyl groups (e.g., benzoyl). Additional examples of acyl groups herein include —CO—CH2—CH2—COOH, —CO—CH2—CH2—CH2—COOH, —CO—CH2—CH2—CH2—CH2—COOH, —CO—CH2—CH2—CH2—CH2—CH2—COOH, —CO—CH2—CH2—CH2—CH2—CH2—CH2—COOH, —CO—CH═CH—COOH, —CO—CH═CH—CH2—COOH, —CO—CH═CH—CH2—CH2—COOH, —CO—CH═CH—CH2—CH2—CH2—COOH, —CO—CH═CH—CH2—CH2—CH2—CH2—COOH, —CO—CH2—CH═CH—COOH, —CO—CH2—CH═CH—CH2—COOH, —CO—CH2—CH═CH—CH2—CH2—COOH, —CO—CH2—CH═CH—CH2—CH2—CH2—COOH, —CO—CH2—CH2—CH═CH—COOH, —CO—CH2—CH2—CH═CH—CH2—COOH, —CO—CH2—CH2—CH═CH—CH2—CH2—COOH, —CO—CH2—CH2—CH2—CH═CH—COOH, —CO—CH2—CH2—CH2—CH═CH—CH2—COOH, —CO—CH2—CH2—CH2—CH2—CH═CH—COOH, and any other acyl group that can be formed using a cyclic organic anhydride as an ester-derivatization agent. An alpha-1,3-glucan ester backbone of a graft copolymer in certain aspects can contain one type of acyl group (e.g., acetyl group or benzoyl group). Alternatively, a graft copolymer ester compound can contain two or more different types of acyl groups (e.g., combinations of two of three of acetyl, propionyl, and/or butyryl groups). Any suitable process for ester-derivatizing polysaccharides can be employed to prepare an alpha-1,3-glucan ester backbone for graft copolymer production, such as disclosed in U.S. Patent Appl. Publ. Nos. 2014/0187767 and 2018/0155455, and Int. Patent Appl. Publ. No. WO2018/098065, which are incorporated herein by reference. A graft copolymer as presently disclosed comprises one or more alpha-1,3-glucan side chains comprising at least about 50% alpha-1,3 glycosidic linkages. An alpha-1,3-glucan side chain in certain aspects can comprise about, or at least about, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% alpha-1,3 glycosidic linkages. In some aspects, accordingly, an alpha-1,3-glucan side chain has less than about 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0% glycosidic linkages that are not alpha-1,3. Typically, the glycosidic linkages that are not alpha-1,3 are mostly or entirely alpha-1,6. In certain embodiments, an alpha-1,3-glucan side chain has no branch points or less than about 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the side chain. Glucosyltransferases contemplated to be useful for producing alpha-1,3-glucan side chains comprising at least about 50% alpha-1,3 linkages as above are disclosed herein and in U.S. Pat. Nos. 7,000,000 and 8,871,474, and Int. Patent Appl. Publ. No. WO2017/079595, all of which are incorporated herein by reference. The DP of one or more alpha-1,3-glucan side chains in certain aspects can individually be about, or at least about, or less than about, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, or 1650. DP can optionally be expressed as a range between any two of these values. Merely as examples, the DP of one or more alpha-1,3-glucan side chains can individually be about 400-1650, 500-1650, 600-1650, 700-1650, 400-1250, 500-1250, 600-1250, 700-1250, 400-1200, 500-1200, 600-1200, 700-1200, 400-1000, 500-1000, 600-1000, 700-1000, 400-900, 500-900, 600-900, 700-900, 11-25, 12-25, 11-22, 12-22, 11-20, 12-20, 20-300, 20-200, 20-150, 20-100, 20-75, 30-300, 30-200, 30-150, 30-100, 30-75, 50-300, 50-200, 50-150, 50-100, 50-75, 75-300, 75-200, 75-150, 75-100, 100-300, 100-200, 100-150, 150-300, 150-200, or 200-300. The DPw of a plurality of alpha-1,3-glucan side chains of a graft copolymer can be referred to, if desired; any of the foregoing DP values, which characterize side chains on an individual basis, can optionally be considered a DPw of all the side chains of a copolymer. In some aspects in which a graft copolymer has a plurality of alpha-1,3-glucan side chains, the individual DP values of the side chains are similar to each other (e.g., the DP values vary by less than 2.5%, 5%, 10%, 15%, or 20%). In some aspects, an alpha-1,3-glucan side chain can comprise at least about 30% alpha-1,3 linkages and a percentage of alpha-1,6 linkages that brings the total of both the alpha-1,3 and -1,6 linkages in the side chain to 100%. For example, the percentage of alpha-1,3 and -1,6 linkages can be about 30-40% and 60-70%, respectively. Glucosyltransferases contemplated to be useful for producing alpha-1,3-glucan side chains comprising at least about 30% alpha-1,3 linkages are disclosed in U.S. Patent Appl. Publ. No. 2015/0232819, which is incorporated herein by reference. One or more alpha-1,3-glucan side chains in some aspects are contemplated to be (A) joined directly to a glucose unit of the backbone via alpha-glycosidic linkage (e.g., alpha-1,6, alpha-1,4, or alpha-1,2) (in some cases, such linkage might result from the promiscuous activity of an alpha-1,3-glucan-synthesizing glucosyltransferase enzyme), (B) extensions of pre-existing branches (e.g., alpha-1,2, -1,4, and/or -1,6), as such branches present non-reducing ends that can possibly prime alpha-1,3-glucan synthesis by a glucosyltransferase enzyme; and/or (C) possibly joined in another manner. In some aspects, alpha-1,3-glucan side chains are all linked to the backbone via the linkage type of (A) or (B), or via a combination of both (A) and (B) linkage types. Regarding the latter, a combination of both linkage types can comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of linkage type (A), with the balance of the other linkages being of type (B). The number of alpha-1,3-glucan side chains of a graft copolymer herein can be about, at least about, or up to about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the number of monomeric units of the alpha-1,3-glucan derivative component of the graft copolymer. In some aspects, a graft copolymer is contemplated to comprise about, at least about, or less than about, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% branch points; such branchpoints can be those of alpha-1,3-glucan side arms (directly linked to backbone and/or linked via a pre-existing branch on the backbone) and/or branches not having alpha-1,3-glucan extended therefrom (e.g., alpha-1,2, -1,4, or -1,6 branch). A graft copolymer as presently disclosed can be aqueous insoluble or aqueous soluble, but typically is aqueous insoluble. Graft copolymer insolubility can be under non-caustic aqueous conditions, such as the conditions of a glucosyltransferase reaction herein (e.g., pH 4-8, see below). In some aspects, a graft copolymer is insoluble in aqueous conditions at a temperature up to about 50, 60, 70, 80, 90, 100, 110, or 120° C. An aqueous composition herein such as an aqueous solution can comprise a solvent having about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 wt % water, for example. The DPw of a graft copolymer herein can be the sum of the DPw of any alpha-1,3-glucan ether- or ester-derivative backbone herein (such DPw can optionally be that of the alpha-1,3-glucan prior to its ether- or ester-derivatization) plus the DP/DPw of any alpha-1,3-glucan side chain(s) herein, for example. Merely as examples, the DPw of a graft copolymer herein can be about 1000-3000, 1500-3000, 2000-3000, 2500-3000, 1000-2500, 1500-2500, 2000-2500, 1000-2000, 1500-2000, or 1000-1500. Other examples include any of the DPw values disclosed in the below Examples for a graft copolymer product. A graft copolymer herein can comprise about, or at least about, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% (e.g., by weight, or by mole percent [mol %]) (or a range between any two of these values) of alpha-1,3-glucan ether- or ester-derivative backbone, for example. In some aspects, a graft copolymer can comprise about 1-60%, 1-50%, 1-40%, 1-30%, 10-60%, 10-50%, 10-40%, 10-30%, 20-60%, 20-50%, 20-40%, or 20-30% (by wt % or mol %) of alpha-1,3-glucan ether- or ester-derivative backbone. A graft copolymer as presently disclosed can be a product of any of the enzymatic reaction processes disclosed below, for example. Certain embodiments of the present disclosure concern a method of producing (preparing) a graft copolymer as described herein. Such a graft polymer production method can comprise: (a) contacting (in the context of a reaction composition) at least (i) water, (ii) sucrose, (iii) an alpha-1,3-glucan ether or ester compound that has a DoS of about 0.001 to about 3.0 (serves as a primer), and (iv) a glucosyltransferase enzyme that synthesizes alpha-1,3-glucan comprising at least about 50% alpha-1,3 glycosidic linkages, thereby producing a graft copolymer as presently disclosed, optionally wherein the viscosity of the reaction composition increases by at least 10% at least 1 hour following the contacting step; and (b) optionally, isolating the graft copolymer produced in step (a). Step (a) can optionally be characterized as performing a reaction (or preparing/providing a reaction composition) comprising at least water, sucrose, an alpha-1,3-glucan ether or ester compound that has a DoS of about 0.001 to about 3.0, and a glucosyltransferase enzyme that synthesizes alpha-1,3-glucan with at least about 50% alpha-1,3 glycosidic linkages. A graft polymer production method herein can optionally further comprise, prior to step (a), ether- or ester-derivatizing alpha-1,3-glucan to provide an alpha-1,3-glucan ether or ester compound (for use in step [a]). Any feature of a graft polymer production method herein (e.g., features of a backbone, alpha-1,3-glucan side chains, graft copolymer) can be as described elsewhere herein. For example, the alpha-1,3-glucan derivative can be an ether (e.g., an anionic ether such as carboxymethyl ether) and/or comprise over 99% alpha-1,3 glycosidic linkages, and the side arms can comprise over 99% alpha-1,3 glycosidic linkages. A method herein of producing a graft copolymer can also be characterized as a method of producing alpha-1,3-glucan, if desired. A glucosyltransferase enzyme for producing alpha-1,3-glucan side chains of a graft copolymer herein can be derived from any microbial source, such as bacteria. Examples of bacterial glucosyltransferase enzymes are those derived from aStreptococcusspecies,Leuconostocspecies orLactobacillusspecies. Examples ofStreptococcusspecies includeS. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticusandS. sanguinis. Examples ofLeuconostocspecies includeL. mesenteroides, L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicumandL. fructosum. Examples ofLactobacillusspecies includeL. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentumandL. reuteri. A glucosyltransferase enzyme for producing alpha-1,3-glucan side chains of a graft copolymer herein can in some aspects comprise an amino acid sequence as disclosed in any of U.S. Patent Appl. Publ. Nos. 2014/0087431, 2017/0166938, 2017/0002335 2018/0072998 and 2019/0078062 (corresponds to U.S. patent application Ser. No. 16/127,288), all of which are incorporated herein by reference. In some aspects, a glucosyltransferase enzyme herein can comprise an amino acid sequence that is 100% identical to, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% identical to, SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 26, 28, 30, 34, or 59 (Table 1), and have glucosyltransferase activity. It is noted that a glucosyltransferase enzyme with SEQ ID NO:2, 4, 8, 10, 14, 20, 26, 28, 30, or 34 can synthesize alpha-1,3-glucan side chains comprising at least about 90% (˜100%) alpha-1,3 linkages. The amino acid sequence of a glucosyltransferase enzyme in certain aspects has been modified such that the enzyme produces more products (alpha-1,3-glucan and fructose), and less by-products (e.g., glucose, oligosaccharides such as leucrose), from a given amount of sucrose substrate. For example, one, two, three, four, or more amino acid residues of the catalytic domain of a glucosyltransferase herein can be modified/substituted to obtain an enzyme that produces more products (insoluble alpha-glucan and fructose). Examples of a suitable modified glucosyltransferase enzyme are disclosed in the below Examples (Tables A and B). A modified glucosyltransferase enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables A and/or B (or in Table 3 of U.S. Patent Appl. Publ. No. 2018/0072998 [incorporated herein by reference, corresponding to application Ser. No. 15/702,893]) that is/are associated with an alpha-1,3-glucan yield of at least 40% (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62). A set of amino acid modifications as listed in Tables A or B can be used, for example. The amino acid sequence of a glucosyltransferase enzyme in certain aspects has been modified such that the enzyme produces alpha-1,3-glucan with a molecular weight (DPw) that is lower than the molecular weight of alpha-1,3-glucan produced by its corresponding parent glucosyltransferase. Examples of a suitable modified glucosyltransferase enzyme are disclosed in the below Examples (Tables C and D). A modified glucosyltransferase enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables C and/or D that is/are associated with an alpha-1,3-glucan product molecular weight that is at least 5% less than the molecular weight of alpha-1,3-glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62). A set of amino acid modifications as listed in Table D can be used, for example. The amino acid sequence of a glucosyltransferase enzyme in certain aspects has been modified such that the enzyme produces alpha-1,3-glucan with a molecular weight (DPw) that is higher than the molecular weight of alpha-1,3-glucan produced by its corresponding parent glucosyltransferase. Examples of a suitable modified glucosyltransferase enzyme are disclosed in the below Examples (Tables E and F). A modified glucosyltransferase enzyme, for example, can comprise one or more amino acid substitutions corresponding with those in Tables E and/or F that is/are associated with an alpha-1,3-glucan product molecular weight that is at least 5% higher than the molecular weight of alpha-1,3-glucan produced by parent enzyme (the position numbering of such at least one substitution corresponds with the position numbering of SEQ ID NO:62). A set of amino acid modifications as listed in Table 5 of U.S. Patent Appl. Publ. No. 2019/0078062 (incorporated herein by reference, corresponds to application Ser. No. 16/127,288) can be used, for example. In some aspects, a modified glucosyltransferase (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above regarding yield or molecular weight), and (ii) comprises or consists of a glucosyltransferase catalytic domain that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to amino acid residues 55-960 of SEQ ID NO:4, residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20. Each of these subsequences are the approximate catalytic domains of each respective reference sequence, and are believed to be able to produce alpha-1,3-glucan comprising at least about 50% (e.g., ≥90% or ≥95%) alpha-1,3 linkages, and optionally further have a DPw of at least 100. In some aspects, a modified glucosyltransferase (i) comprises at least one amino acid substitution or a set of amino acid substitutions (as described above), and (ii) comprises or consists of an amino acid sequence that is at least about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO:62 or a subsequence thereof such as SEQ ID NO:4 (without start methionine thereof) or positions 55-960 of SEQ ID NO:4 (approximate catalytic domain). The temperature of a reaction composition herein can be controlled, if desired, and can be about 5-50° C., 20-40° C., 30-40° C., 20-30° C., 20-25° C., 20° C., 25° C., 30° C., 35° C., or 40° C., for example. The initial concentration of sucrose in a reaction composition herein can be about, at least about, or less than about, 10, 15, 20, 25, 30, 40, 45, 50, 55, 60, 80, 90, 95, 100, 105, 110, 125, 150, 200, 300, 400, 500, 600, 10-50, 10-40, 10-30, 10-25, 15-50, 15-40, 15-30, or 15-25 g/L, or a range between any two of these values. Merely as examples, the initial sucrose concentration can be about 10-150, 40-60, 45-55, 90-110, or 95-105 g/L, for example. “Initial concentration of sucrose” refers to the sucrose concentration in a reaction composition just after all the reaction components have been added/combined (e.g., at least water, sucrose, an alpha-1,3-glucan ether or ester compound, glucosyltransferase enzyme). The initial concentration of an alpha-1,3-glucan ether or ester compound as presently disclosed in a reaction composition can be about, or at least about, 0.1, 0.5, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 g/L, or a range between any two of these values. Merely as examples, the initial concentration of an alpha-1,3-glucan ether or ester compound can be about 0.1-15, 0.5-15, 1-15, 2-15, 4-15, 0.1-10, 0.5-10, 1-10, 2-10, 4-10, 0.1-5, 0.5-5, 1-5, 2-5, 4-5, 0.1-2.5, 0.5-2.5, 1-2.5, or 2-2.5 g/L. Any of the foregoing values (in g/L) can optionally rather be expressed in terms of w/v %. An alpha-1,3-glucan ether or ester compound typically is soluble in aqueous conditions as presently disclosed (e.g., reaction composition); such a compound is dissolved (along with sucrose, buffer and enzyme components) in setting up a reaction composition. The pH of a reaction composition in certain embodiments can be about 4.0-9.0, 4.0-8.5, 4.0-8.0, 5.0-8.0, 5.5-7.5, or 5.5-6.5. In some aspects, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. The pH can be adjusted or controlled by the addition or incorporation of a suitable buffer, including but not limited to: phosphate, tris, citrate, or a combination thereof. The buffer concentration in a reaction composition herein can be about 0.1-300 mM, 0.1-100 mM, 10-100 mM, 5 mM, 10 mM, 20 mM, or 50 mM, for example. A reaction composition can be contained within any vessel (e.g., an inert vessel/container) suitable for applying one or more of the reaction conditions disclosed herein. An inert vessel in some aspects can be of stainless steel, plastic, or glass (or comprise two or more of these components) and be of a size suitable to contain a particular reaction. For example, the volume/capacity of an inert vessel (and/or the volume of a reaction composition herein), can be about, or at least about, 1, 10, 50, 100, 500, 1000, 2500, 5000, 10000, 12500, 15000, or 20000 liters. An inert vessel can optionally be equipped with a stirring device. Any of the foregoing features, for example, can be used to characterize an isolated reaction herein. A reaction composition herein can contain one, two, or more different glucosyltransferase enzymes that produce alpha-1,3-glucan side chains, for example. In some aspects, only one or two glucosyltransferase enzymes is/are comprised in a reaction composition. A reaction composition herein can be, and typically is, cell-free (e.g., no whole cells present). Completion of a reaction in certain aspects can be determined visually (e.g., no more accumulation of insoluble graft copolymer product), and/or by measuring the amount of sucrose left in the solution (residual sucrose), where a percent sucrose consumption of at least about 90%, 95%, or 99% can indicate reaction completion. In some aspects, a reaction can be considered complete when its sucrose content is at or below about 2-5 g/L. A reaction of the disclosed process can be conducted for about, at least about, or up to about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 36, 48, 60, 72, 96, 120, 144, 168, 1-4, 1-3.5, 1-3, 1.5-4, 1.5-3.5, 1.5-3, 2-4, 2-3.5, or 2-3 hours, for example. A reaction can optionally be terminated and/or otherwise treated to stop glucosyltransferase activity by heating it to at least about 65° C. for at least about 30-60 minutes. Examples of other conditions and/or components suitable for synthesizing alpha-1,3-glucan side chains in graft copolymer production herein are disclosed in U.S. Patent Appl. Publ. Nos. 2014/0087431, 2017/0166938 and 2017/0002335, which are incorporated herein by reference. Graft copolymer produced in a reaction composition herein can optionally be isolated. In certain embodiments, isolating graft copolymer can include at least conducting a step of centrifugation, filtration, fractionation, chromatographic separation, dialysis, evaporation, or dilution. Isolation of insoluble graft copolymer can include at least conducting a step of preparing a cake of graft copolymer. Cake preparation can include at least conducting a step of centrifugation (cake is pelleted graft copolymer) and/or filtration (cake is filtered graft copolymer). Isolation can optionally further comprise washing the centrifuged and/or filtered graft copolymer one, two, or more times with water or other aqueous liquid. A wash volume can optionally be at least about 10-100% of the volume of the reaction composition used to produce the graft copolymer. Washing can be done by various modes, as desired, such as by displacement or re-slurry washing. In some aspects, the aqueous portion of the resulting cake has no (detectable) dissolved sugars, or about 0.1-1.5, 0.1-1.25, 0.1-1.0, 0.1-0.75, 0.1-0.5, 0.2-0.6, 0.3-0.5, 0.2, 0.3, 0.4, 0.5, or 0.6 wt % dissolved sugars. Such dissolved sugars can include sucrose, fructose, leucrose, and/or soluble gluco-oligosaccharides, for example. Isolation herein can optionally further comprise drying graft copolymer, and/or preparing a dispersion of graft copolymer. An isolated graft copolymer herein as provided in a dry form, can comprise no more than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01 wt % water, for example. In some aspects, a graft copolymer is provided in an amount of at least 1 gram (e.g., at least about 2.5, 5, 10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000, 7500, 10000, 25000, 50000, or 100000 g); such an amount can be a dry amount, for example. In some aspects, a graft copolymer that has been isolated (optionally characterized as “purified”) can be present in a composition at a wt % (dry weight basis) of at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.8%, or 99.9%. Such isolated graft copolymer can be used as an ingredient/component in a product/application, for example. Certain embodiments of the present disclosure concern a method of providing an aqueous composition that comprises a graft copolymer. Such a method comprises (a) providing a graft copolymer as presently disclosed, and (b) dispersing or dissolving the graft copolymer into an aqueous liquid, thereby producing an aqueous composition that comprises a graft copolymer. This method can optionally be characterized as a dispersion method, in aspects in which the graft copolymer provided in step (a) is insoluble in aqueous conditions as presently disclosed. Alternatively, the method can optionally be characterized as a dissolution method, in aspects in which the graft copolymer provided in step (a) is soluble in aqueous conditions as presently disclosed. A graft copolymer provided for use in a dispersion or dissolution method herein can be dry or wet. A dry form of graft copolymer can comprise no more than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01 wt % water, for example. A wet form of a graft copolymer can be a “cake” in some aspects. A cake herein refers to a preparation of graft copolymer in condensed, compacted, packed, squeezed, and/or compressed form that comprises at least (i) about 50%-90% by weight aqueous fluid (e.g., water or water-based solution), and (ii) about 10%-50% by weight graft copolymer. A cake can optionally be referred to as a “filter cake” or “wet cake”. Any suitable method can be employed to perform step (b) of dispersing a graft copolymer. In some aspects, such dispersal can be performed by applying high shear and/or other forms of mixing/agitation. High shear can be of about, or at least about, 8, 9, 10, 11, or 12 kJ/kg in specific energy, and/or can comprise mixing at about, or up to about, 3000, 4000, 6000, 8000, 10000, 12000, 14000, or 15000 rpm, for example. High shear and/or mixing/agitation can be applied for about 1, 2, 3, 4, 5, 6, 8, or 10 minutes, or 2-4 minutes, for example. Suitable means for shearing/mixing/agitating include, for example, a disperser, sonicator (e.g., ultrasonicator) (e.g., 40-60 W, ˜50 μl) homomixer, homogenizer (e.g., rotary or piston, rotar-stator), microfluidizer, planetary mixer, colloid mill, jet mill, vortex, and/or any methodology as described in the below Examples and/or in International Patent Appl. Publ. No. WO2016/030234, U.S. Pat. Nos. 5,767,176, 6,139,875, and/or 8722092, and/or U.S. Patent Appl. Publ. Nos. 2017/0055540 and/or 2018/0021238, which are all incorporated herein by reference. In some aspects, high shear mixing (such as applied by any of the foregoing means) is not used to disperse a graft copolymer to achieve elevated viscosity; gentle mixing/agitation such at a low rpm/frequency (e.g., less than about 100, 50, or 30 rpm) is used to disperse the graft copolymer in such aspects. A dispersion produced herein can optionally be a colloidal dispersion. An aqueous composition produced by a dispersion or dissolution method herein can comprise about, at least about, or less than about, 0.1, 0.25, 0.4, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 w/v % graft copolymer, for example, or a range between any two of these values. Merely as examples, an aqueous composition can comprise about 0.1-2.5, 0.1-2.25, 0.1-2.0, 0.1-1.75, 0.1-1.5, 0.1-1.25, 0.1-1.0, 0.1-0.75, 0.1-0.5, 0.25-2.5, 0.25-2.25, 0.25-2.0, 0.25-1.75, 0.25-1.5, 0.25-1.25, 0.25-1.0, 0.25-0.75, 0.25-0.5, 0.5-2.5, 0.5-2.25, 0.5-2.0, 0.5-1.75, 0.5-1.5, 0.5-1.25, 0.5-1.0, or 0.5-0.75 w/v % graft copolymer. In some aspects, the viscosity of the aqueous composition produced in step (b) of a dispersion or dissolution method is at least about 10%, 50%, 75%, 100%, 500%, 1000%, 10000%, or 100000%, or 1000000% (or any integer between 10% and 100000%) higher than the viscosity of the aqueous liquid as it existed before step (b) of dispersing or dissolving. Very large percent increases in viscosity can be obtained with the disclosed method when the aqueous liquid has little to no viscosity before step (b). The viscosity of an aqueous composition comprising a graft copolymer can be about, or at least about, 100, 200, 300, 400, 500, 600, 700, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, or 100000 centipoise (cP), for example. Viscosity can be as measured with an aqueous composition at any temperature between about 3° C. to about 80° C., for example (e.g., 4-30° C., 15-30° C., 15-25° C.). Viscosity typically is as measured at atmospheric pressure (about 760 torr) or a pressure that is within ±10% thereof. Viscosity can be measured using a viscometer or rheometer, for example, and can optionally be as measured ata shear rate (rotational shear rate) of about 0.1, 0.5, 1.0, 5, 10, 50, 100, 500, 1000, 0.1-500, 0.1-100, 1.0-500, or 1.0-100 s−1(1/s), for example. Viscosity can optionally be measured following the procedure outlined in the below Examples. It is notable that a graft copolymer herein typically has enhanced viscosity (at any given shear rate) compared to alpha-1,3-glucan (each polymer provided in the same amount and having been produced under the same or similar glucosyltransferase reaction conditions, except for the presence of an alpha-1,3-glucan ether or ester primer in the graft copolymer-producing reaction). Such viscosity enhancement can be about, or at least about, a 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, or 12-fold increase in viscosity (at any given shear rate) for a graft copolymer as compared to alpha-1,3-glucan. It is notable that dry forms (e.g., dried at least once following synthesis) and “never-dried” wet forms (the polymer has never been dried following its synthesis) of graft copolymer herein are typically both able to increase viscosity to the same or similar (e.g., within ±10%) extent, whereas alpha-1,3-glucan produced in a reaction without a glucan ether or ester primer typically does not exhibit this beneficial feature; alpha-1,3-glucan that has been dried at least once typically is not capable of increasing viscosity to the same or similar (e.g., within ±10%) extent as never-dried wet forms of alpha-1,3-glucan. A graft copolymer herein can be present in a composition, such as an aqueous composition (e.g., dispersion such as colloidal dispersion) or dry composition, at about, at least about, or less than about, 0.01, 0.05, 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 1.2, 1.25, 1.4, 1.5, 1.6, 1.75, 1.8, 2.0, 2.25, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt % or w/v %, for example, or a range between any two of these values. Merely as examples, an aqueous composition can comprise about 0.1-2.5, 0.1-2.25, 0.1-2.0, 0.1-1.75, 0.1-1.5, 0.1-1.25, 0.1-1.0, 0.1-0.75, 0.1-0.5, 0.25-2.5, 0.25-2.25, 0.25-2.0, 0.25-1.75, 0.25-1.5, 0.25-1.25, 0.25-1.0, 0.25-0.75, 0.25-0.5, 0.5-2.5, 0.5-2.25, 0.5-2.0, 0.5-1.75, 0.5-1.5, 0.5-1.25, 0.5-1.0, or 0.5-0.75 w/v % or wt % graft copolymer. The liquid component of an aqueous composition can be an aqueous fluid such as water or aqueous solution, for instance. The solvent of an aqueous solution typically is water, or can comprise about, or at least about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 98, or 99 wt % water, for example. A graft copolymer as comprised in a dispersion herein typically is aqueous insoluble. A composition herein can optionally further comprise alpha-1,3-glucan homopolymer. An aqueous solution in some aspects has no (detectable) dissolved sugars, or about 0.1-1.5, 0.1-1.25, 0.1-1.0, 0.1-0.75, 0.1-0.5, 0.2-0.6, 0.3-0.5, 0.2, 0.3, 0.4, 0.5, or 0.6 wt % dissolved sugars. Such dissolved sugars can include sucrose, fructose, leucrose, and/or soluble gluco-oligosaccharides, for example. An aqueous solution in some aspects can have one or more salts/buffers (e.g., Na+, Cl−, NaCl, phosphate, tris, citrate) (e.g., 0.1, 0.5, 1.0, 2.0, or 3.0 wt %) and/or a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 4.0-9.0, 4.0-8.5, 4.0-8.0, 5.0-9.0, 5.0-8.5, 5.0-8.0, 6.0-9.0, 6.0-8.5, or 6.0-8.0, for example. A composition comprising a graft copolymer (e.g., a dispersion if the polymer is insoluble, or a solution if the polymer is soluble) can have a viscosity of about, or at least about, 100, 200, 300, 400, 500, 600, 700, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, or 100000 centipoise (cP), for example. Viscosity can be as measured with an aqueous composition at any temperature between about 3° C. to about 80° C., for example (e.g., 4-30° C., 15-30° C., 15-25° C.). Viscosity typically is as measured at atmospheric pressure (about 760 torr) or a pressure that is within ±10% thereof. Viscosity can be measured using a viscometer or rheometer, for example, and can optionally be as measured at a shear rate (rotational shear rate) of about 0.1, 0.5, 1.0, 5, 10, 50, 100, 500, 1000, 0.1-500, 0.1-100, 1.0-500, or 1.0-100 s−1(1/s), for example. Viscosity can optionally be measured following the procedure outlined in the below Examples. The zeta potential of a graft copolymer in an aqueous composition (e.g., dispersion) in some aspects can be over ±15 mV, ±17.5 mV, ±20 mV, ±22.5 mV, ±25 mV, ±27.5 mV, ±30 mV, ±32.5 mV, ±35 mV, ±37.5 mV, or ±40 mV. Simply for illustration purposes, it should be understood that a zeta potential over ±15 mV, for example, excludes zeta potentials ranging from −15 mV to +15 mV. In some aspects, the zeta potential is ±20 to ±40 mV, ±20 to ±30 mV, ±25 to ±40 mV, or ±25 to ±30 mV. In general, it is contemplated that the zeta potential of a graft copolymer comprising an anionic alpha-1,3-glucan derivative backbone has a greater negative value than −15 mV, and that the zeta potential of a graft copolymer comprising a cationic alpha-1,3-glucan derivative backbone has a greater positive value than +15 mV. The foregoing zeta potential values can in some aspects can be associated with aqueous compositions having a pH of about 6-8 or 5-9. It is notable that a graft copolymer herein typically has a greater positive or negative zeta potential value compared to alpha-1,3-glucan (each polymer provided in the same manner and having been produced under the same or similar glucosyltransferase reaction conditions, except for the presence of an alpha-1,3-glucan ether or ester primer in the graft copolymer-producing reaction). Such enhancement can be by an absolute difference in zeta potential values of at least 10, 15, 20, 25, 30, or 40, for example. Zeta potential herein can be measured as described in the below Examples, and/or as disclosed, for example, in U.S. Pat. Nos. 6,109,098 and/or 4,602,989, and/or Int. Patent Appl. Publ. Nos. WO2014/097402 and/or EP0869357, which are incorporated herein by reference. In some aspects, the particle size of a graft copolymer in an aqueous composition (e.g., dispersion) is less than 2.0, 1.8, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 micron. Particle size can be 0.15-0.5, 0.15-0.4, 0.15-0.3, 0.2-0.5, 0.2-0.4, or 0.2-0.3 in some aspects. Particle size herein can optionally be in terms of a “D50” (diameter-50) value of a sample of graft copolymer particles. A D50 measurement for a sample of graph copolymer particles herein is the particle diameter at which (i) 50% of the sample's mass is comprised of particles with a diameter less than this measurement, and (ii) 50% of the sample's mass is comprised of particles with a diameter greater than this measurement. It is notable that graft copolymer particles herein typically are of a reduced size compared to the particle size of alpha-1,3-glucan (each polymer provided in the same manner and having been produced under the same or similar glucosyltransferase reaction conditions, except for the presence of an alpha-1,3-glucan ether or ester primer in the graft copolymer-producing reaction). Such size reduction can be by about at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9%, for example. Without intending to be held to any particular theory, it is contemplated that retention of small particle size by graft copolymer particles herein (as opposed to agglomerating/flocculating to form larger particles) is due at least in part to the enhanced zeta potential of the particles. Graft copolymer particles can be prepared using a mixing/agitation means as disclosed herein such as sonication, for example, and particle size can be measured as described in the below Examples, and/or as disclosed, for example, in U.S. Pat. Nos. 6,109,098, 9,869,625, and/or 5831150, which are incorporated herein by reference. A composition comprising a graft copolymer herein can, in some aspects, be non-aqueous (e.g., a dry composition). Examples of such embodiments include powders, granules, microcapsules, flakes, or any other form of particulate matter. Other examples include larger compositions such as pellets, bars, kernels, beads, tablets, sticks, or other agglomerates. A non-aqueous or dry composition typically has less than 2.0, 1.5, 1.0, 0.5, 0.25, 0.10, 0.05, or 0.01 wt % water comprised therein. A composition comprising a graft copolymer herein can, in some aspects, comprise one or more salts such as a sodium salt (e.g., NaCl, Na2SO4). Other non-limiting examples of salts include those having (i) an aluminum, ammonium, barium, calcium, chromium (II or III), copper (I or II), iron (II or III), hydrogen, lead (II), lithium, magnesium, manganese (II or III), mercury (I or II), potassium, silver, sodium strontium, tin (II or IV), or zinc cation, and (ii) an acetate, borate, bromate, bromide, carbonate, chlorate, chloride, chlorite, chromate, cyanamide, cyanide, dichromate, dihydrogen phosphate, ferricyanide, ferrocyanide, fluoride, hydrogen carbonate, hydrogen phosphate, hydrogen sulfate, hydrogen sulfide, hydrogen sulfite, hydride, hydroxide, hypochlorite, iodate, iodide, nitrate, nitride, nitrite, oxalate, oxide, perchlorate, permanganate, peroxide, phosphate, phosphide, phosphite, silicate, stannate, stannite, sulfate, sulfide, sulfite, tartrate, or thiocyanate anion. Thus, any salt having a cation from (i) above and an anion from (ii) above can be in a composition, for example. A salt can be present in an aqueous composition herein at a wt % of about, or at least about, 0.01, 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, 3.0, 3.5, 0.01-3.5, 0.5-3.5, 0.5-2.5, or 0.5-1.5 wt % (such wt % values typically refer to the total concentration of one or more salts), for example. A composition herein comprising a graft copolymer can optionally contain one or more active enzymes. Examples of suitable enzymes include proteases, cellulases, hemicellulases, peroxidases, lipolytic enzymes (e.g., metallolipolytic enzymes), xylanases, lipases, phospholipases, esterases (e.g., arylesterase, polyesterase), perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases (e.g., choline oxidase), phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, metalloproteinases, amadoriases, glucoamylases, arabinofuranosidases, phytases, isomerases, transferases and amylases. If an enzyme(s) is included, it may be comprised in a composition herein at about 0.0001-0.1 wt % (e.g., 0.01-0.03 wt %) active enzyme (e.g., calculated as pure enzyme protein), for example. In fabric care applications, cellulase can be present in an aqueous composition in which a fabric is treated (e.g., wash liquor) at a concentration that is minimally about 0.01-0.1 ppm total cellulase protein, or about 0.1-10 ppb total cellulase protein (e.g., less than 1 ppm), to maximally about 100, 200, 500, 1000, 2000, 3000, 4000, or 5000 ppm total cellulase protein, for example. A composition comprising a graft copolymer herein, such as an aqueous composition or a non-aqueous composition (above), can be in the form of a household care product, personal care product, industrial product, pharmaceutical product, or food product, for example, such as described in any of U.S. Patent Appl. Publ. Nos. 2018/0022834, 2018/0237816, 20180079832, 2016/0311935, 2016/0304629, 2015/0232785, 2015/0368594, 2015/0368595, 2019/0202942, and/or 2016/0122445, and/or Int. Patent Appl. Publ. Nos. WO2016/160737, WO2016/160738, WO2016/133734, and/or WO2016/160740, which are all incorporated herein by reference. In some aspects, a composition comprising a graft copolymer can comprise at least one component/ingredient of a household care product, personal care product, industrial product, pharmaceutical product, or food product as disclosed in any of the foregoing publications and/or as presently disclosed. Graft copolymers disclosed herein are believed to be useful for providing one or more of the following physical properties to a personal care product, pharmaceutical product, household product, industrial product, or food product: thickening, freeze/thaw stability, lubricity, moisture retention and release, texture, consistency, shape retention, emulsification, binding, suspension, dispersion, gelation, reduced mineral hardness, for example. Examples of a concentration or amount of a graft copolymer in a product can be any of the weight percentages provided herein, for example. Personal care products herein are not particularly limited and include, for example, skin care compositions, cosmetic compositions, antifungal compositions, and antibacterial compositions. Personal care products herein may be in the form of, for example, lotions, creams, pastes, balms, ointments, pomades, gels, liquids, combinations of these and the like. The personal care products disclosed herein can include at least one active ingredient, if desired. An active ingredient is generally recognized as an ingredient that causes an intended pharmacological effect. In certain embodiments, a skin care product can be applied to skin for addressing skin damage related to a lack of moisture. A skin care product may also be used to address the visual appearance of skin (e.g., reduce the appearance of flaky, cracked, and/or red skin) and/or the tactile feel of the skin (e.g., reduce roughness and/or dryness of the skin while improved the softness and subtleness of the skin). A skin care product typically may include at least one active ingredient for the treatment or prevention of skin ailments, providing a cosmetic effect, or for providing a moisturizing benefit to skin, such as zinc oxide, petrolatum, white petrolatum, mineral oil, cod liver oil, lanolin, dimethicone, hard fat, vitamin A, allantoin, calamine, kaolin, glycerin, or colloidal oatmeal, and combinations of these. A skin care product may include one or more natural moisturizing factors such as ceramides, hyaluronic acid, glycerin, squalane, amino acids, cholesterol, fatty acids, triglycerides, phospholipids, glycosphingolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharide, sodium lactate, or sodium pyrrolidone carboxylate, for example. Other ingredients that may be included in a skin care product include, without limitation, glycerides, apricot kernel oil, canola oil, squalane, squalene, coconut oil, corn oil, jojoba oil, jojoba wax, lecithin, olive oil, safflower oil, sesame oil, shea butter, soybean oil, sweet almond oil, sunflower oil, tea tree oil, shea butter, palm oil, cholesterol, cholesterol esters, wax esters, fatty acids, and orange oil. A personal care product herein can also be in the form of makeup, lipstick, mascara, rouge, foundation, blush, eyeliner, lip liner, lip gloss, other cosmetics, sunscreen, sun block, nail polish, nail conditioner, bath gel, shower gel, body wash, face wash, lip balm, skin conditioner, cold cream, moisturizer, body spray, soap, body scrub, exfoliant, astringent, scruffing lotion, depilatory, permanent waving solution, antidandruff formulation, antiperspirant composition, deodorant, shaving product, pre-shaving product, after-shaving product, cleanser, skin gel, rinse, dentifrice composition, toothpaste, or mouthwash, for example. An example of a personal care product (e.g., a cleanser, soap, scrub, cosmetic) comprises a carrier or exfoliation agent (e.g., jojoba beads [jojoba ester beads]) (e.g., about 1-10, 3-7, 4-6, or 5 wt %); such an agent may optionally be dispersed within the product. A personal care product in some aspects can be a hair care product. Examples of hair care products herein include shampoo, hair conditioner (leave-in or rinse-out), cream rinse, hair dye, hair coloring product, hair shine product, hair serum, hair anti-frizz product, hair split-end repair product, mousse, hair spray, and styling gel. A hair care product can be in the form of a liquid, paste, gel, solid, or powder in some embodiments. A hair care product as presently disclosed typically comprises one or more of the following ingredients, which are generally used to formulate hair care products: anionic surfactants such as polyoxyethylenelauryl ether sodium sulfate; cationic surfactants such as stearyltrimethylammonium chloride and/or distearyltrimethylammonium chloride; nonionic surfactants such as glyceryl monostearate, sorbitan monopalmitate and/or polyoxyethylenecetyl ether; wetting agents such as propylene glycol, 1,3-butylene glycol, glycerin, sorbitol, pyroglutamic acid salts, amino acids and/or trimethylglycine; hydrocarbons such as liquid paraffins, petrolatum, solid paraffins, squalane and/or olefin oligomers; higher alcohols such as stearyl alcohol and/or cetyl alcohol; superfatting agents; antidandruff agents; disinfectants; anti-inflammatory agents; crude drugs; water-soluble polymers such as methyl cellulose, hydroxycellulose and/or partially deacetylated chitin; antiseptics such as paraben; ultra-violet light absorbers; pearling agents; pH adjustors; perfumes; and pigments. A pharmaceutical product herein can be in the form of an emulsion, liquid, elixir, gel, suspension, solution, cream, or ointment, for example. Also, a pharmaceutical product herein can be in the form of any of the personal care products disclosed herein, such as an antibacterial or antifungal composition. A pharmaceutical product can further comprise one or more pharmaceutically acceptable carriers, diluents, and/or pharmaceutically acceptable salts. A graft copolymer disclosed herein can also be used in capsules, encapsulants, tablet coatings, and as an excipients for medicaments and drugs. A household and/or industrial product herein can be in the form of drywall tape-joint compounds; mortars; grouts; cement plasters; spray plasters; cement stucco; adhesives; pastes; wall/ceiling texturizers; binders and processing aids for tape casting, extrusion forming, injection molding and ceramics; spray adherents and suspending/dispersing aids for pesticides, herbicides, and fertilizers; fabric care products such as fabric softeners and laundry detergents; hard surface cleaners; air fresheners; polymer emulsions; gels such as water-based gels; surfactant solutions; paints such as water-based paints; protective coatings; adhesives; sealants and caulks; inks such as water-based ink; metal-working fluids; films or coatings; or emulsion-based metal cleaning fluids used in electroplating, phosphatizing, galvanizing and/or general metal cleaning operations, for example. A graft copolymer disclosed herein can be comprised in a personal care product, pharmaceutical product, household product, or industrial product in an amount that provides a desired degree of thickening and/or dispersion, for example. Examples of a concentration or amount of a graft copolymer in a product can be any of the weight percentages provided above, for example. Compositions disclosed herein can be in the form of a fabric care composition. A fabric care composition herein can be used for hand wash, machine wash and/or other purposes such as soaking and/or pretreatment of fabrics, for example. A fabric care composition may take the form of, for example, a laundry detergent; fabric conditioner; any wash-, rinse-, or dryer-added product; unit dose or spray. Fabric care compositions in a liquid form may be in the form of an aqueous composition as disclosed herein. In other aspects, a fabric care composition can be in a dry form such as a granular detergent or dryer-added fabric softener sheet. Other non-limiting examples of fabric care compositions herein include: granular or powder-form all-purpose or heavy-duty washing agents; liquid, gel or paste-form all-purpose or heavy-duty washing agents; liquid or dry fine-fabric (e.g. delicates) detergents; cleaning auxiliaries such as bleach additives, “stain-stick”, or pre-treatments; substrate-laden products such as dry and wetted wipes, pads, or sponges; sprays and mists. A detergent composition herein may be in any useful form, e.g., as powders, granules, pastes, bars, unit dose, or liquid. A liquid detergent may be aqueous, typically containing up to about 70 wt % of water and 0 wt % to about 30 wt % of organic solvent. It may also be in the form of a compact gel type containing only about 30 wt % water. A detergent composition herein typically comprises one or more surfactants, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60%, while in alternative embodiments the level is from about 1% to about 50%, while in still further embodiments the level is from about 5% to about 40%, by weight of the detergent composition. A detergent will usually contain 0 wt % to about 50 wt % of an anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenylsuccinic acid, or soap. In addition, a detergent composition may optionally contain 0 wt % to about 40 wt % of a nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (as described for example in WO92/06154, which is incorporated herein by reference). A detergent composition herein typically comprises one or more detergent builders or builder systems. In some aspects, oxidized poly alpha-1,3-glucan can be included as a co-builder, in which it is used together with one or more additional builders such as any disclosed herein. Oxidized poly alpha-1,3-glucan compounds for use herein are disclosed in U.S. Patent Appl. Publ. No. 2015/0259439. In some embodiments incorporating at least one builder, the cleaning compositions comprise at least about 1%, from about 3% to about 60%, or even from about 5% to about 40%, builder by weight of the composition. Builders (in addition to oxidized poly alpha-1,3-glucan) include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicates, polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4, 6-trisulphonic acid, and carboxymethyloxysuccinic acid, various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof. Indeed, it is contemplated that any suitable builder will find use in various embodiments of the present disclosure. Additional examples of a detergent builder or complexing agent include zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst). In some embodiments, builders form water-soluble hardness ion complexes (e.g., sequestering builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphospate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphate, etc.). It is contemplated that any suitable builder will find use in the present disclosure, including those known in the art (See, e.g., EP2100949). In some embodiments, suitable builders can include phosphate builders and non-phosphate builders. In some embodiments, a builder is a phosphate builder. In some embodiments, a builder is a non-phosphate builder. A builder can be used in a level of from 0.1% to 80%, or from 5% to 60%, or from 10% to 50%, by weight of the composition. In some embodiments, the product comprises a mixture of phosphate and non-phosphate builders. Suitable phosphate builders include mono-phosphates, di-phosphates, tri-polyphosphates or oligomeric-polyphosphates, including the alkali metal salts of these compounds, including the sodium salts. In some embodiments, a builder can be sodium tripolyphosphate (STPP). Additionally, the composition can comprise carbonate and/or citrate, preferably citrate that helps to achieve a neutral pH composition. Other suitable non-phosphate builders include homopolymers and copolymers of polycarboxylic acids and their partially or completely neutralized salts, monomeric polycarboxylic acids and hydroxycarboxylic acids and their salts. In some embodiments, salts of the above mentioned compounds include ammonium and/or alkali metal salts, i.e., lithium, sodium, and potassium salts, including sodium salts. Suitable polycarboxylic acids include acyclic, alicyclic, hetero-cyclic and aromatic carboxylic acids, wherein in some embodiments, they can contain at least two carboxyl groups which are in each case separated from one another by, in some instances, no more than two carbon atoms. A detergent composition herein can comprise at least one chelating agent. Suitable chelating agents include, but are not limited to copper, iron and/or manganese chelating agents and mixtures thereof. In embodiments in which at least one chelating agent is used, the composition comprises from about 0.1% to about 15%, or even from about 3.0% to about 10%, chelating agent by weight of the composition. A detergent composition herein can comprise at least one deposition aid. Suitable deposition aids include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof. A detergent composition herein can comprise one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. Additional dye transfer inhibiting agents include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof; chelating agents examples of which include ethylene-diamine-tetraacetic acid (EDTA); diethylene triamine penta methylene phosphonic acid (DTPMP), hydroxy-ethane diphosphonic acid (HEDP); ethylenediamine N,N′-disuccinic acid (EDDS); methyl glycine diacetic acid (MGDA); diethylene triamine penta acetic acid (DTPA); propylene diamine tetraacetic acid (PDT A); 2-hydroxypyridine-N-oxide (HPNO); or methyl glycine diacetic acid (MGDA); glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamic acid tetrasodium salt (GLDA); nitrilotriacetic acid (NTA); 4,5-dihydroxy-m-benzenedisulfonic acid; citric acid and any salts thereof; N-hydroxyethyl ethylenediaminetri-acetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEI DA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP) and derivatives thereof, which can be used alone or in combination with any of the above. In embodiments in which at least one dye transfer inhibiting agent is used, a composition herein may comprise from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3%, by weight of the composition. A detergent composition herein can comprise silicates. In some of these embodiments, sodium silicates (e.g., sodium disilicate, sodium metasilicate, and/or crystalline phyllosilicates) find use. In some embodiments, silicates are present at a level of from about 1% to about 20% by weight of the composition. In some embodiments, silicates are present at a level of from about 5% to about 15% by weight of the composition. A detergent composition herein can comprise dispersants. Suitable water-soluble organic materials include, but are not limited to the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. A detergent composition herein may additionally comprise one or more enzymes. Examples of enzymes include proteases, cellulases, hemicellulases, peroxidases, lipolytic enzymes (e.g., metallolipolytic enzymes), xylanases, lipases, phospholipases, esterases (e.g., arylesterase, polyesterase), perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases (e.g., choline oxidase, phenoloxidase), phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, beta-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases, metalloproteinases, amadoriases, glucoamylases, alpha-amylases, beta-amylases, galactosidases, galactanases, catalases, carageenases, hyaluronidases, keratinases, lactases, ligninases, peroxidases, phosphatases, polygalacturonases, pullulanases, rhamnogalactouronases, tannases, transglutaminases, xyloglucanases, xylosidases, metalloproteases, arabinofuranosidases, phytases, isomerases, transferases and/or amylases in any combination. In some embodiments, a detergent composition can comprise one or more enzymes (e.g., any disclosed herein), each at a level from about 0.00001% to about 10% by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In some other embodiments, a detergent composition can also comprise each enzyme at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5%, by weight of the composition. Enzymes that may be comprised in a detergent composition herein may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol; a sugar or sugar alcohol; lactic acid; boric acid or a boric acid derivative (e.g., an aromatic borate ester). A detergent composition in certain embodiments may comprise one or more other types of polymers in addition to a graft copolymer as disclosed herein. Examples of other types of polymers useful herein include carboxymethyl cellulose (CMC), dextran, poly(vinylpyrrolidone) (PVP), polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers. A detergent composition herein may contain a bleaching system. For example, a bleaching system can comprise an H2O2source such as perborate or percarbonate, which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, a bleaching system may comprise peroxyacids (e.g., amide, imide, or sulfone type peroxyacids). Alternatively still, a bleaching system can be an enzymatic bleaching system comprising perhydrolase, for example, such as the system described in WO2005/056783. A detergent composition herein may also contain conventional detergent ingredients such as fabric conditioners, clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibiters, optical brighteners, or perfumes. The pH of a detergent composition herein (measured in aqueous solution at use concentration) is usually neutral or alkaline (e.g., pH of about 7.0 to about 11.0). It is believed that a graft copolymer herein can be included as an anti-redeposition agent and/or clay soil removal agent in a detergent composition such as a fabric care composition, if desired (such agents can optionally be characterized as whiteness maintenance agents in certain aspects). Examples of other suitable anti-redeposition and/or clay soil removal agents herein include polyethoxy zwitterionic surfactants, water-soluble copolymers of acrylic or methacrylic acid with acrylic or methacrylic acid-ethylene oxide condensates (e.g., U.S. Pat. No. 3,719,647), cellulose derivatives such as carboxymethylcellulose and hydroxypropylcellulose (e.g., U.S. Pat. Nos. 3,597,416 and 3,523,088), and mixtures comprising nonionic alkyl polyethoxy surfactant, polyethoxy alkyl quaternary cationic surfactant and fatty amide surfactant (e.g., U.S. Pat. No. 4,228,044). Non-limiting examples of other suitable anti-redeposition and clay soil removal agents are disclosed in U.S. Pat. Nos. 4,597,898 and 4,891,160, and Int. Patent Appl. Publ. No. WO95/32272, all of which are incorporated herein by reference. Particular forms of detergent compositions that can be adapted for purposes disclosed herein are disclosed in, for example, US20090209445A1, US20100081598A1, U5700187862, EPI 504994B1, WO2001085888A2, WO2003089562A1, WO2009098659A1, WO2009098660A1, WO2009112992A1, WO2009124160A1, WO2009152031A1, WO2010059483A1, WO2010088112A1, WO2010090915A1, WO2010135238A1, WO2011094687A1, WO2011094690A1, WO2011127102A1, WO2011163428A1, WO2008000567A1, WO2006045391A1, WO2006007911A1, WO2012027404A1, EP174069061, WO2012059336A1, U5673064661, WO2008087426A1, WO2010116139A1, and WO2012104613A1, all of which are incorporated herein by reference. Laundry detergent compositions herein can optionally be heavy duty (all purpose) laundry detergent compositions. Exemplary heavy duty laundry detergent compositions comprise a detersive surfactant (10%-40% wt/wt), including an anionic detersive surfactant (selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof), and optionally non-ionic surfactant (selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohol, e.g., C8-C18 alkyl ethoxylated alcohols and/or C6-C12 alkyl phenol alkoxylates), where the weight ratio of anionic detersive surfactant (with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersive surfactant is greater than 1:1. Suitable detersive surfactants also include cationic detersive surfactants (selected from a group of alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary sulphonium compounds, and/or mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (selected from a group of alkanolamine sulpho-betaines); ampholytic surfactants; semi-polar non-ionic surfactants and mixtures thereof. A detergent herein such as a heavy duty laundry detergent composition may optionally include, a surfactancy boosting polymer consisting of amphiphilic alkoxylated grease cleaning polymers (selected from a group of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylenimines in the range of 0.05 wt %-10 wt %) and/or random graft polymers (typically comprising of hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated C1-C6 carboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols such as glycerol, and mixtures thereof; and hydrophobic side chain(s) selected from the group consisting of: C4-C25 alkyl group, polypropylene, polybutylene, vinyl ester of a saturated C1-C6 mono-carboxylic acid, C1-C6 alkyl ester of acrylic or methacrylic acid, and mixtures thereof. A detergent herein such as a heavy duty laundry detergent composition may optionally include additional polymers such as soil release polymers (include anionically end-capped polyesters, for example SRP1, polymers comprising at least one monomer unit selected from saccharide, dicarboxylic acid, polyol and combinations thereof, in random or block configuration, ethylene terephthalate-based polymers and co-polymers thereof in random or block configuration, for example REPEL-O-TEX SF, SF-2 AND SRP6, TEXCARE SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 AND SRN325, MARLOQUEST SL), anti-redeposition agent(s) herein (0.1 wt % to 10 wt %), include carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof, vinylpyrrolidone homopolymer, and/or polyethylene glycol, molecular weight in the range of from 500 to 100,000 Da); and polymeric carboxylate (such as maleate/acrylate random copolymer or polyacrylate homopolymer). A detergent herein such as a heavy duty laundry detergent composition may optionally further include saturated or unsaturated fatty acids, preferably saturated or unsaturated C12-C24 fatty acids (0 wt % to 10 wt %); deposition aids (examples for which include polysaccharides, cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DAD MAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, in random or block configuration, cationic guar gum, cationic starch, cationic polyacrylamides, and mixtures thereof. A detergent herein such as a heavy duty laundry detergent composition may optionally further include dye transfer inhibiting agents, examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof; chelating agents, examples of which include ethylene-diamine-tetraacetic acid (EDTA), diethylene triamine penta methylene phosphonic acid (DTPMP), hydroxy-ethane diphosphonic acid (HEDP), ethylenediamine N,N′-disuccinic acid (EDDS), methyl glycine diacetic acid (MGDA), diethylene triamine penta acetic acid (DTPA), propylene diamine tetraacetic acid (PDTA), 2-hydroxypyridine-N-oxide (HPNO), or methyl glycine diacetic acid (MGDA), glutamic acid N,N-diacetic acid (N,N-dicarboxymethyl glutamic acid tetrasodium salt (GLDA), nitrilotriacetic acid (NTA), 4,5-dihydroxy-m-benzenedisulfonic acid, citric acid and any salts thereof, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), and derivatives thereof. A detergent herein such as a heavy duty laundry detergent composition may optionally include silicone or fatty-acid based suds suppressors; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti-foam (0.001 wt % to about 4.0 wt %), and/or a structurant/thickener (0.01 wt % to 5 wt %) selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof). Such structurant/thickener would be, in certain embodiments, in addition to the one or more graft copolymers comprised in the detergent. A structurant can also be referred to as a structural agent. A detergent herein can be in the form of a heavy duty dry/solid laundry detergent composition, for example. Such a detergent may include: (i) a detersive surfactant, such as any anionic detersive surfactant disclosed herein, any non-ionic detersive surfactant disclosed herein, any cationic detersive surfactant disclosed herein, any zwitterionic and/or amphoteric detersive surfactant disclosed herein, any ampholytic surfactant, any semi-polar non-ionic surfactant, and mixtures thereof; (ii) a builder, such as any phosphate-free builder (e.g., zeolite builders in the range of 0 wt % to less than 10 wt %), any phosphate builder (e.g., sodium tri-polyphosphate in the range of 0 wt % to less than 10 wt %), citric acid, citrate salts and nitrilotriacetic acid, any silicate salt (e.g., sodium or potassium silicate or sodium meta-silicate in the range of 0 wt % to less than 10 wt %); any carbonate salt (e.g., sodium carbonate and/or sodium bicarbonate in the range of 0 wt % to less than 80 wt %), and mixtures thereof; (iii) a bleaching agent, such as any photobleach (e.g., sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines, xanthenes dyes, and mixtures thereof), any hydrophobic or hydrophilic bleach activator (e.g., dodecanoyl oxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethy hexanoyl oxybenzene sulfonate, tetraacetyl ethylene diamine-TAED, nonanoyloxybenzene sulfonate-NOBS, nitrile quats, and mixtures thereof), any source of hydrogen peroxide (e.g., inorganic perhydrate salts, examples of which include mono or tetra hydrate sodium salt of perborate, percarbonate, persulfate, perphosphate, or persilicate), any preformed hydrophilic and/or hydrophobic peracids (e.g., percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, and mixtures thereof); and/or (iv) any other components such as a bleach catalyst (e.g., imine bleach boosters examples of which include iminium cations and polyions, iminium zwitterions, modified amines, modified amine oxides, N-sulphonyl imines, N-phosphonyl imines, N-acyl imines, thiadiazole dioxides, perfluoroimines, cyclic sugar ketones, and mixtures thereof), and a metal-containing bleach catalyst (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations along with an auxiliary metal cations such as zinc or aluminum and a sequestrate such as EDTA, ethylenediaminetetra(methylenephosphonic acid). Compositions disclosed herein can be in the form of a dishwashing detergent composition, for example. Examples of dishwashing detergents include automatic dishwashing detergents (typically used in dishwasher machines) and hand-washing dish detergents. A dishwashing detergent composition can be in any dry or liquid/aqueous form as disclosed herein, for example. Components that may be included in certain embodiments of a dishwashing detergent composition include, for example, one or more of a phosphate; oxygen- or chlorine-based bleaching agent; non-ionic surfactant; alkaline salt (e.g., metasilicates, alkali metal hydroxides, sodium carbonate); any active enzyme disclosed herein; anti-corrosion agent (e.g., sodium silicate); anti-foaming agent; additives to slow down the removal of glaze and patterns from ceramics; perfume; anti-caking agent (in granular detergent); starch (in tablet-based detergents); gelling agent (in liquid/gel based detergents); and/or sand (powdered detergents). Dishwashing detergents such as an automatic dishwasher detergent or liquid dishwashing detergent can comprise (i) a non-ionic surfactant, including any ethoxylated non-ionic surfactant, alcohol alkoxylated surfactant, epoxy-capped poly(oxyalkylated) alcohol, or amine oxide surfactant present in an amount from 0 to 10 wt %; (ii) a builder, in the range of about 5-60 wt %, including any phosphate builder (e.g., mono-phosphates, di-phosphates, tri-polyphosphates, other oligomeric-polyphosphates, sodium tripolyphosphate-STPP), any phosphate-free builder (e.g., amino acid-based compounds including methyl-glycine-diacetic acid [MGDA] and salts or derivatives thereof, glutamic-N,N-diacetic acid [GLDA] and salts or derivatives thereof, iminodisuccinic acid (IDS) and salts or derivatives thereof, carboxy methyl inulin and salts or derivatives thereof, nitrilotriacetic acid [NTA], diethylene triamine penta acetic acid [DTPA], B-alaninediacetic acid [B-ADA] and salts thereof), homopolymers and copolymers of poly-carboxylic acids and partially or completely neutralized salts thereof, monomeric polycarboxylic acids and hydroxycarboxylic acids and salts thereof in the range of 0.5 wt % to 50 wt %, or sulfonated/carboxylated polymers in the range of about 0.1 wt % to about 50 wt %; (iii) a drying aid in the range of about 0.1 wt % to about 10 wt % (e.g., polyesters, especially anionic polyesters, optionally together with further monomers with 3 to 6 functionalities—typically acid, alcohol or ester functionalities which are conducive to polycondensation, polycarbonate-, polyurethane- and/or polyurea-polyorganosiloxane compounds or precursor compounds thereof, particularly of the reactive cyclic carbonate and urea type); (iv) a silicate in the range from about 1 wt % to about 20 wt % (e.g., sodium or potassium silicates such as sodium disilicate, sodium meta-silicate and crystalline phyllosilicates); (v) an inorganic bleach (e.g., perhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate salts) and/or an organic bleach (e.g., organic peroxyacids such as diacyl- and tetraacylperoxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid); (vi) a bleach activator (e.g., organic peracid precursors in the range from about 0.1 wt % to about 10 wt %) and/or bleach catalyst (e.g., manganese triazacyclononane and related complexes; Co, Cu, Mn, and Fe bispyridylamine and related complexes; and pentamine acetate cobalt(III) and related complexes); (vii) a metal care agent in the range from about 0.1 wt % to 5 wt % (e.g., benzatriazoles, metal salts and complexes, and/or silicates); and/or (viii) any active enzyme disclosed herein in the range from about 0.01 to 5.0 mg of active enzyme per gram of automatic dishwashing detergent composition, and an enzyme stabilizer component (e.g., oligosaccharides, polysaccharides, and inorganic divalent metal salts). Compositions disclosed herein can be in the form of an oral care composition, for example. Examples of oral care compositions include dentifrices, toothpaste, mouth wash, mouth rinse, chewing gum, and edible strips that provide some form of oral care (e.g., treatment or prevention of cavities [dental caries], gingivitis, plaque, tartar, and/or periodontal disease). An oral care composition can also be for treating an “oral surface”, which encompasses any soft or hard surface within the oral cavity including surfaces of the tongue, hard and soft palate, buccal mucosa, gums and dental surfaces. A “dental surface” herein is a surface of a natural tooth or a hard surface of artificial dentition including a crown, cap, filling, bridge, denture, or dental implant, for example. An oral care composition herein can comprise about 0.01-15.0 wt % (e.g., ˜0.1-10 wt % or ˜0.1-5.0 wt %, ˜0.1-2.0 wt %) of one or more graft copolymers as disclosed herein, for example. One or more graft copolymers comprised in an oral care composition can sometimes be provided therein as a thickening agent and/or dispersion agent, which may be useful to impart a desired consistency and/or mouth feel to the composition. One or more other thickening or dispersion agents can also be provided in an oral care composition herein, such as a carboxyvinyl polymer, carrageenan (e.g., L-carrageenan), natural gum (e.g., karaya, xanthan, gum arabic, tragacanth), colloidal magnesium aluminum silicate, or colloidal silica, for example. An oral care composition herein may be a toothpaste or other dentifrice, for example. Such compositions, as well as any other oral care composition herein, can additionally comprise, without limitation, one or more of an anticaries agent, antimicrobial or antibacterial agent, anticalculus or tartar control agent, surfactant, abrasive, pH-modifying agent, foam modulator, humectant, flavorant, sweetener, pigment/colorant, whitening agent, and/or other suitable components. Examples of oral care compositions to which one or more graft copolymers can be added are disclosed in U.S. Patent Appl. Publ. Nos. 2006/0134025, 2002/0022006 and 2008/0057007, which are incorporated herein by reference. An anticaries agent herein can be an orally acceptable source of fluoride ions. Suitable sources of fluoride ions include fluoride, monofluorophosphate and fluorosilicate salts as well as amine fluorides, including olaflur (N′-octadecyltrimethylendiamine-N,N,N′-tris(2-ethanol)-dihydrofluoride), for example. An anticaries agent can be present in an amount providing a total of about 100-20000 ppm, about 200-5000 ppm, or about 500-2500 ppm, fluoride ions to the composition, for example. In oral care compositions in which sodium fluoride is the sole source of fluoride ions, an amount of about 0.01-5.0 wt %, about 0.05-1.0 wt %, or about 0.1-0.5 wt %, sodium fluoride can be present in the composition, for example. An antimicrobial or antibacterial agent suitable for use in an oral care composition herein includes, for example, phenolic compounds (e.g., 4-allylcatechol; p-hydroxybenzoic acid esters such as benzylparaben, butylparaben, ethylparaben, methylparaben and propylparaben; 2-benzylphenol; butylated hydroxyanisole; butylated hydroxytoluene; capsaicin; carvacrol; creosol; eugenol; guaiacol; halogenated bisphenolics such as hexachlorophene and bromochlorophene; 4-hexylresorcinol; 8-hydroxyquinoline and salts thereof; salicylic acid esters such as menthyl salicylate, methyl salicylate and phenyl salicylate; phenol; pyrocatechol; salicylanilide; thymol; halogenated diphenylether compounds such as triclosan and triclosan monophosphate), copper (II) compounds (e.g., copper (II) chloride, fluoride, sulfate and hydroxide), zinc ion sources (e.g., zinc acetate, citrate, gluconate, glycinate, oxide, and sulfate), phthalic acid and salts thereof (e.g., magnesium monopotassium phthalate), hexetidine, octenidine, sanguinarine, benzalkonium chloride, domiphen bromide, alkylpyridinium chlorides (e.g. cetylpyridinium chloride, tetradecylpyridinium chloride, N-tetradecyl-4-ethylpyridinium chloride), iodine, sulfonamides, bisbiguanides (e.g., alexidine, chlorhexidine, chlorhexidine digluconate), piperidino derivatives (e.g., delmopinol, octapinol), magnolia extract, grapeseed extract, rosemary extract, menthol, geraniol, citral, eucalyptol, antibiotics (e.g., augmentin, amoxicillin, tetracycline, doxycycline, minocycline, metronidazole, neomycin, kanamycin, clindamycin), and/or any antibacterial agents disclosed in U.S. Pat. No. 5,776,435, which is incorporated herein by reference. One or more antimicrobial agents can optionally be present at about 0.01-10 wt % (e.g., 0.1-3 wt %), for example, in the disclosed oral care composition. An anticalculus or tartar control agent suitable for use in an oral care composition herein includes, for example, phosphates and polyphosphates (e.g., pyrophosphates), polyaminopropanesulfonic acid (AMPS), zinc citrate trihydrate, polypeptides (e.g., polyaspartic and polyglutamic acids), polyolefin sulfonates, polyolefin phosphates, diphosphonates (e.g., azacycloalkane-2,2-diphosphonates such as azacycloheptane-2,2-diphosphonic acid), N-methyl azacyclopentane-2,3-diphosphonic acid, ethane-1-hydroxy-1,1-diphosphonic acid (EHDP), ethane-1-amino-1,1-diphosphonate, and/or phosphonoalkane carboxylic acids and salts thereof (e.g., their alkali metal and ammonium salts). Useful inorganic phosphate and polyphosphate salts include, for example, monobasic, dibasic and tribasic sodium phosphates, sodium tripolyphosphate, tetrapolyphosphate, mono-, di-, tri- and tetra-sodium pyrophosphates, disodium dihydrogen pyrophosphate, sodium trimetaphosphate, sodium hexametaphosphate, or any of these in which sodium is replaced by potassium or ammonium. Other useful anticalculus agents in certain embodiments include anionic polycarboxylate polymers (e.g., polymers or copolymers of acrylic acid, methacrylic, and maleic anhydride such as polyvinyl methyl ether/maleic anhydride copolymers). Still other useful anticalculus agents include sequestering agents such as hydroxycarboxylic acids (e.g., citric, fumaric, malic, glutaric and oxalic acids and salts thereof) and aminopolycarboxylic acids (e.g., EDTA). One or more anticalculus or tartar control agents can optionally be present at about 0.01-50 wt % (e.g., about 0.05-25 wt % or about 0.1-15 wt %), for example, in the disclosed oral care composition. A surfactant suitable for use in an oral care composition herein may be anionic, non-ionic, or amphoteric, for example. Suitable anionic surfactants include, without limitation, water-soluble salts of C8-20alkyl sulfates, sulfonated monoglycerides of C8-20fatty acids, sarcosinates, and taurates. Examples of anionic surfactants include sodium lauryl sulfate, sodium coconut monoglyceride sulfonate, sodium lauryl sarcosinate, sodium lauryl isoethionate, sodium laureth carboxylate and sodium dodecyl benzenesulfonate. Suitable non-ionic surfactants include, without limitation, poloxamers, polyoxyethylene sorbitan esters, fatty alcohol ethoxylates, alkylphenol ethoxylates, tertiary amine oxides, tertiary phosphine oxides, and dialkyl sulfoxides. Suitable amphoteric surfactants include, without limitation, derivatives of C8-20aliphatic secondary and tertiary amines having an anionic group such as a carboxylate, sulfate, sulfonate, phosphate or phosphonate. An example of a suitable amphoteric surfactant is cocoamidopropyl betaine. One or more surfactants are optionally present in a total amount of about 0.01-10 wt % (e.g., about 0.05-5.0 wt % or about 0.1-2.0 wt %), for example, in the disclosed oral care composition. An abrasive suitable for use in an oral care composition herein may include, for example, silica (e.g., silica gel, hydrated silica, precipitated silica), alumina, insoluble phosphates, calcium carbonate, and resinous abrasives (e.g., a urea-formaldehyde condensation product). Examples of insoluble phosphates useful as abrasives herein are orthophosphates, polymetaphosphates and pyrophosphates, and include dicalcium orthophosphate dihydrate, calcium pyrophosphate, beta-calcium pyrophosphate, tricalcium phosphate, calcium polymetaphosphate and insoluble sodium polymetaphosphate. One or more abrasives are optionally present in a total amount of about 5-70 wt % (e.g., about 10-56 wt % or about 15-30 wt %), for example, in the disclosed oral care composition. The average particle size of an abrasive in certain embodiments is about 0.1-30 microns (e.g., about 1-20 microns or about 5-15 microns). An oral care composition in certain embodiments may comprise at least one pH-modifying agent. Such agents may be selected to acidify, make more basic, or buffer the pH of a composition to a pH range of about 2-10 (e.g., pH ranging from about 2-8, 3-9, 4-8, 5-7, 6-10, or 7-9). Examples of pH-modifying agents useful herein include, without limitation, carboxylic, phosphoric and sulfonic acids; acid salts (e.g., monosodium citrate, disodium citrate, monosodium malate); alkali metal hydroxides (e.g. sodium hydroxide, carbonates such as sodium carbonate, bicarbonates, sesquicarbonates); borates; silicates; phosphates (e.g., monosodium phosphate, trisodium phosphate, pyrophosphate salts); and imidazole. A foam modulator suitable for use in an oral care composition herein may be a polyethylene glycol (PEG), for example. High molecular weight PEGs are suitable, including those having an average molecular weight of about 200000-7000000 (e.g., about 500000-5000000 or about 1000000-2500000), for example. One or more PEGs are optionally present in a total amount of about 0.1-10 wt % (e.g. about 0.2-5.0 wt % or about 0.25-2.0 wt %), for example, in the disclosed oral care composition. An oral care composition in certain embodiments may comprise at least one humectant. A humectant in certain embodiments may be a polyhydric alcohol such as glycerin, sorbitol, xylitol, or a low molecular weight PEG. Most suitable humectants also may function as a sweetener herein. One or more humectants are optionally present in a total amount of about 1.0-70 wt % (e.g., about 1.0-50 wt %, about 2-25 wt %, or about 5-15 wt %), for example, in the disclosed oral care composition. A natural or artificial sweetener may optionally be comprised in an oral care composition herein. Examples of suitable sweeteners include dextrose, sucrose, maltose, dextrin, invert sugar, mannose, xylose, ribose, fructose, levulose, galactose, corn syrup (e.g., high fructose corn syrup or corn syrup solids), partially hydrolyzed starch, hydrogenated starch hydrolysate, sorbitol, mannitol, xylitol, maltitol, isomalt, aspartame, neotame, saccharin and salts thereof, dipeptide-based intense sweeteners, and cyclamates. One or more sweeteners are optionally present in a total amount of about 0.005-5.0 wt %, for example, in the disclosed oral care composition. A natural or artificial flavorant may optionally be comprised in an oral care composition herein. Examples of suitable flavorants include vanillin; sage; marjoram; parsley oil; spearmint oil; cinnamon oil; oil of wintergreen (methylsalicylate); peppermint oil; clove oil; bay oil; anise oil; eucalyptus oil; citrus oils; fruit oils; essences such as those derived from lemon, orange, lime, grapefruit, apricot, banana, grape, apple, strawberry, cherry, or pineapple; bean- and nut-derived flavors such as coffee, cocoa, cola, peanut, or almond; and adsorbed and encapsulated flavorants. Also encompassed within flavorants herein are ingredients that provide fragrance and/or other sensory effect in the mouth, including cooling or warming effects. Such ingredients include, without limitation, menthol, menthyl acetate, menthyl lactate, camphor, eucalyptus oil, eucalyptol, anethole, eugenol, cassia, oxanone, Irisone®, propenyl guaiethol, thymol, linalool, benzaldehyde, cinnamaldehyde, N-ethyl-p-menthan-3-carboxamine, N,2,3-trimethyl-2-isopropylbutanamide, 3-(1-menthoxy)-propane-1,2-diol, cinnamaldehyde glycerol acetal (CGA), and menthone glycerol acetal (MGA). One or more flavorants are optionally present in a total amount of about 0.01-5.0 wt % (e.g., about 0.1-2.5 wt %), for example, in the disclosed oral care composition. An oral care composition in certain embodiments may comprise at least one bicarbonate salt. Any orally acceptable bicarbonate can be used, including alkali metal bicarbonates such as sodium or potassium bicarbonate, and ammonium bicarbonate, for example. One or more bicarbonate salts are optionally present in a total amount of about 0.1-50 wt % (e.g., about 1-20 wt %), for example, in the disclosed oral care composition. An oral care composition in certain embodiments may comprise at least one whitening agent and/or colorant. A suitable whitening agent is a peroxide compound such as any of those disclosed in U.S. Pat. No. 8,540,971, which is incorporated herein by reference. Suitable colorants herein include pigments, dyes, lakes and agents imparting a particular luster or reflectivity such as pearling agents, for example. Specific examples of colorants useful herein include talc; mica; magnesium carbonate; calcium carbonate; magnesium silicate; magnesium aluminum silicate; silica; titanium dioxide; zinc oxide; red, yellow, brown and black iron oxides; ferric ammonium ferrocyanide; manganese violet; ultramarine; titaniated mica; and bismuth oxychloride. One or more colorants are optionally present in a total amount of about 0.001-20 wt % (e.g., about 0.01-10 wt % or about 0.1-5.0 wt %), for example, in the disclosed oral care composition. Additional components that can optionally be included in an oral composition herein include one or more enzymes (above), vitamins, and anti-adhesion agents, for example. Examples of vitamins useful herein include vitamin C, vitamin E, vitamin B5, and folic acid. Examples of suitable anti-adhesion agents include solbrol, ficin, and quorum-sensing inhibitors. The present disclosure also concerns a method of treating a material. This method comprises contacting a material with an aqueous composition comprising at least one graft copolymer as disclosed herein. A material contacted with an aqueous composition in a contacting method herein can comprise a fabric in certain embodiments. A fabric herein can comprise natural fibers, synthetic fibers, semi-synthetic fibers, or any combination thereof. A semi-synthetic fiber herein is produced using naturally occurring material that has been chemically derivatized, an example of which is rayon. Non-limiting examples of fabric types herein include fabrics made of (i) cellulosic fibers such as cotton (e.g., broadcloth, canvas, chambray, chenille, chintz, corduroy, cretonne, damask, denim, flannel, gingham, jacquard, knit, matelassé, oxford, percale, poplin, plissé, sateen, seersucker, sheers, terry cloth, twill, velvet), rayon (e.g., viscose, modal, lyocell), linen, and Tencel®; (ii) proteinaceous fibers such as silk, wool and related mammalian fibers; (iii) synthetic fibers such as polyester, acrylic, nylon, and the like; (iv) long vegetable fibers from jute, flax, ramie, coir, kapok, sisal, henequen, abaca, hemp and sunn; and (v) any combination of a fabric of (i)-(iv). Fabric comprising a combination of fiber types (e.g., natural and synthetic) include those with both a cotton fiber and polyester, for example. Materials/articles containing one or more fabrics herein include, for example, clothing, curtains, drapes, upholstery, carpeting, bed linens, bath linens, tablecloths, sleeping bags, tents, car interiors, etc. Other materials comprising natural and/or synthetic fibers include, for example, non-woven fabrics, paddings, paper, and foams. An aqueous composition that is contacted with a fabric can be, for example, a fabric care composition (e.g., laundry detergent, fabric softener). Thus, a treatment method in certain embodiments can be considered a fabric care method or laundry method if employing a fabric care composition therein. A fabric care composition herein is contemplated to effect one or more of the following fabric care benefits (i.e., surface substantive effects): wrinkle removal, wrinkle reduction, wrinkle resistance, fabric wear reduction, fabric wear resistance, fabric pilling reduction, extended fabric life, fabric color maintenance, fabric color fading reduction, reduced dye transfer, fabric color restoration, fabric soiling reduction, fabric soil release, fabric shape retention, fabric smoothness enhancement, anti-redeposition of soil on fabric, anti-greying of laundry, improved fabric hand/handle, and/or fabric shrinkage reduction. Examples of conditions (e.g., time, temperature, wash/rinse volumes) for conducting a fabric care method or laundry method herein are disclosed in WO1997/003161 and U.S. Pat. Nos. 4,794,661, 4,580,421 and 5,945,394, which are incorporated herein by reference. In other examples, a material comprising fabric can be contacted with an aqueous composition herein: (i) for at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes; (ii) at a temperature of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95° C. (e.g., for laundry wash or rinse: a “cold” temperature of about 15-30° C., a “warm” temperature of about 30-50° C., a “hot” temperature of about 50-95° C.), (iii) at a pH of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (e.g., pH range of about 2-12, or about 3-11); (iv) at a salt (e.g., NaCl) concentration of at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0 wt %; or any combination of (i)-(iv). The contacting step in a fabric care method or laundry method can comprise any of washing, soaking, and/or rinsing steps, for example. Contacting a material or fabric in still further embodiments can be performed by any means known in the art, such as dissolving, mixing, shaking, spraying, treating, immersing, flushing, pouring on or in, combining, painting, coating, applying, affixing to, and/or communicating an effective amount of a graft copolymer herein with the fabric or material. In still further embodiments, contacting may be used to treat a fabric to provide a surface substantive effect. As used herein, the term “fabric hand” or “handle” refers to a person's tactile sensory response towards fabric which may be physical, physiological, psychological, social or any combination thereof. In one embodiment, the fabric hand may be measured using a PhabrOmeter® System for measuring relative hand value (available from Nu Cybertek, Inc. Davis, CA) (American Association of Textile Chemists and Colorists [AATCC test method “202-2012, Relative Hand Value of Textiles: Instrumental Method”]). In certain embodiments of treating a material comprising fabric, a graft copolymer component(s) of the aqueous composition adsorbs to the fabric. This feature is believed to render graft copolymers herein useful as anti-redeposition agents and/or anti-greying agents in fabric care compositions disclosed (in addition to their viscosity-modifying effect). An anti-redeposition agent or anti-greying agent herein helps keep soil from redepositing onto clothing in wash water after the soil has been removed. It is further contemplated that adsorption of one or more graft copolymers herein to a fabric enhances mechanical properties of the fabric. Adsorption of a graft copolymer to a fabric herein can be measured using a colorimetric technique (e.g., Dubois et al., 1956, Anal. Chem.28:350-356; Zemljič et al., 2006, Lenzinger Berichte85:68-76; both incorporated herein by reference), for example, or any other method known in the art. Other materials that can be contacted in the above treatment method include surfaces that can be treated with a dish detergent (e.g., automatic dishwashing detergent or hand dish detergent). Examples of such materials include surfaces of dishes, glasses, pots, pans, baking dishes, utensils and flatware made from ceramic material, china, metal, glass, plastic (e.g., polyethylene, polypropylene, polystyrene, etc.) and wood (collectively referred to herein as “tableware”). Thus, the treatment method in certain embodiments can be considered a dishwashing method or tableware washing method, for example. Examples of conditions (e.g., time, temperature, wash volume) for conducting a dishwashing or tableware washing method herein are disclosed in U.S. Pat. No. 8,575,083, which is incorporated herein by reference. In other examples, a tableware article can be contacted with an aqueous composition herein under a suitable set of conditions such as any of those disclosed above with regard to contacting a fabric-comprising material. Other materials that can be contacted in the above treatment method include oral surfaces such as any soft or hard surface within the oral cavity including surfaces of the tongue, hard and soft palate, buccal mucosa, gums and dental surfaces (e.g., natural tooth or a hard surface of artificial dentition such as a crown, cap, filling, bridge, denture, or dental implant). Thus, a treatment method in certain embodiments can be considered an oral care method or dental care method, for example. Conditions (e.g., time, temperature) for contacting an oral surface with an aqueous composition herein should be suitable for the intended purpose of making such contact. Other surfaces that can be contacted in a treatment method also include a surface of the integumentary system such as skin, hair or nails. Thus, certain embodiments of the present disclosure concern material (e.g., fabric) that comprises a graft copolymer herein. Such material can be produced following a material treatment method as disclosed herein, for example. A material can comprise a graft copolymer in certain aspects if the copolymer is adsorbed to, or otherwise in contact with, the surface of the material. Certain embodiments of a method of treating a material herein further comprise a drying step, in which a material is dried after being contacted with the aqueous composition. A drying step can be performed directly after the contacting step, or following one or more additional steps that might follow the contacting step (e.g., drying of a fabric after being rinsed, in water for example, following a wash in an aqueous composition herein). Drying can be performed by any of several means known in the art, such as air drying (e.g., ˜20-25° C.), or at a temperature of at least about 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 170, 175, 180, or 200° C., for example. A material that has been dried herein typically has less than 3, 2, 1, 0.5, or 0.1 wt % water comprised therein. Fabric is a preferred material for conducting an optional drying step. An aqueous composition used in a treatment method herein can be any aqueous composition disclosed herein, such as in the above embodiments. Thus, the graft copolymer component(s) of an aqueous composition can be any as disclosed herein. Examples of aqueous compositions include detergents (e.g., laundry detergent or dish detergent) and water-containing dentifrices such as toothpaste. Non-limiting examples of compositions and methods disclosed herein include:1. A composition comprising a graft copolymer that comprises: (i) a backbone comprising an alpha-1,3-glucan ether or ester compound that has a degree of substitution (DoS) of about 0.001 to about 3.0, and (ii) one or more alpha-1,3-glucan side chains comprising at least about 50% alpha-1,3 glycosidic linkages.2. The composition of embodiment 1, wherein the alpha-1,3-glucan ether or ester compound comprises at least about 50% alpha-1,3 glycosidic linkages.3. The composition of embodiment 1 or 2, wherein the weight-average degree of polymerization (DPw) of the alpha-1,3-glucan ether or ester compound is at least about 15.4. The composition of embodiment 1, 2, or 3, wherein the backbone comprises an alpha-1,3-glucan ether compound.5. The composition of embodiment 1, 2, 3, or 4, wherein the alpha-1,3-glucan ether compound comprises an ether-linked group that is anionic or cationic when the compound is comprised in an aqueous composition.6. The composition of embodiment 1, 2, 3, 4, or 5, wherein the alpha-1,3-glucan ether compound comprises an ether-linked carboxymethyl group.7. The composition of embodiment 1, 2, 3, 4, 5, or 6, wherein: (a) the alpha-1,3-glucan side chains comprise at least about 90% alpha-1,3 glycosidic linkages, and/or (b) the DP or DPw of the one or more alpha-1,3-glucan side chains is at least about 100.8. The composition of embodiment 1, 2, 3, 4, 5, 6, or 7, wherein the composition is an aqueous composition, optionally wherein the graft copolymer is insoluble in the aqueous composition.9. The composition of embodiment 8, wherein aqueous composition is a dispersion of the graft copolymer, optionally wherein the dispersion comprises less than 1.5 w/v % of the graft copolymer.10. The composition of embodiment 8 or 9, wherein: (a) the zeta potential of the graft copolymer in the aqueous composition is over ±15 mV, and/or (b) the particle size of the graft copolymer in the aqueous composition is less than 1 micron.11. The composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, wherein the composition is a household care product, personal care product, industrial product, pharmaceutical product, or food product.12. The composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, wherein the graft copolymer is produced in a reaction composition comprising at least water, sucrose, the alpha-1,3-glucan ether or ester compound, and a glucosyltransferase enzyme that synthesizes alpha-1,3-glucan with at least about 50% alpha-1,3 glycosidic linkages, optionally wherein the composition is said reaction composition.13. A method of producing a graft copolymer, the method comprising: (a) contacting, in a reaction composition, at least (i) water, (ii) sucrose, (iii) an alpha-1,3-glucan ether or ester compound that has a degree of substitution (DoS) of about 0.001 to about 3.0, and (iv) a glucosyltransferase enzyme that synthesizes alpha-1,3-glucan comprising at least about 50% alpha-1,3 glycosidic linkages, whereby a graft copolymer according to the composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 is produced, optionally wherein the viscosity of the reaction composition increases by at least 10% at least 1 hour following the contacting step; and (b) optionally, isolating the graft copolymer produced in step (a).14. A method of providing an aqueous composition, the method comprising: (a) providing a graft copolymer according to the composition of embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and (b) dispersing or dissolving the graft copolymer into an aqueous liquid, thereby producing the aqueous composition.15. The method of embodiment 14, wherein the graft copolymer provided in step (a) is dry.16. The method of embodiment 14 or 15, wherein the graft copolymer provided in step (a) is insoluble in aqueous conditions, and wherein the graft copolymer is dispersed into the aqueous liquid in step (b).17. The method of embodiment 14, 15, or 16, wherein the aqueous composition produced in step (b) comprises less than 1.5 w/v % of the graft copolymer.18. The method of embodiment 14, 15, 16, or 17, wherein the viscosity of the aqueous composition produced in step (b) is at least 10% higher than the viscosity of the aqueous liquid. EXAMPLES The present disclosure is further exemplified in the below Examples. It should be understood that these Examples, while indicating certain aspects herein, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the disclosed embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosed embodiments to various uses and conditions. General Methods (Aside Those Listed in the Below Examples) Analysis of Glucan Molecular Weight by Size-Exclusion Chromatography (SEC) Insoluble glucan polymer wet cake isolated from glucosyltransferase reactions was dissolved at 2 mg/mL with overnight shaking in neat DMSO with 2% lithium chloride (LiCl) from Aldrich (Milwaukee, Wis.) to form a glucan polymer solution. This solution (100 μL, 40° C.) was then injected into an Alliance™ 2695 HPLC (Waters Corporation, Milford, MA) coupled with three on-line detectors: a differential refractometer (DR) 2414™ from Waters Corp., a multiangle light-scattering photometer Heleos II™ 18 angles from Wyatt Technologies (Santa Barbara, CA), and a differential capillary viscometer ViscoStar™ from Wyatt Tech, all operating at 50° C. The mobile phase (DMAc containing 0.11 wt % LiCl) passed at a flow rate of 0.5 mL/min through four styrene-divinyl benzene columns in series; specifically, one KD-802, one KD-801, and two linear KD-806M columns (Shodex, Japan). The molecular weight distribution of the glucan polymer sample, as well as its average molar masses (Mn, Mw and Mz) were determined using Astra version 7.1 software package from Wyatt (triple detection method without column calibration). Values of average degree of polymerization, e.g., DPw, were calculated by dividing a corresponding average molar mass by 162. This SEC protocol yielded the following glucan polymer measurements: average molecular weights, molecular weight distribution, intrinsic viscosity, Mark-Houwink and conformation plots, radius of gyration, branching frequency. The DPw and PDI values of all graft copolymer products disclosed herein include the DPw/PDI values of both the glucan ether primer and the alpha-1,3-glucan synthesized off the primer. Determination of Glycosidic Linkages Glycosidic linkages in glucan products synthesized by a glucosyltransferase were determined by1H NMR (nuclear magnetic resonance) spectroscopy. Dry glucan polymer (6 to 8 mg) was dissolved in 0.75 mL of 3 wt % lithium chloride (LiCl) in deuterated dimethyl sulfoxide (DMSO-d6) by stirring overnight at ambient temperature. Deuterated water (D2O) was then added (0.05 mL), and the sample was heated at 80° C. for about one hour to exchange protonated hydroxyls on the glucan polymer and ensure complete dissolution. 600 μL of the resulting clear homogeneous solution was transferred to a 5-mm NMR tube.1H NMR spectra was used to quantify glycosidic linkage and a 2D1H,13C homo/hetero-nuclear suite of experiments was used to identify glucan linkages. The data were collected at 80° C. and processed on a Bruker Avance III NMR spectrometer, operating at either 500 MHz or 600 MHz. The systems are equipped with a proton-optimized cryoprobe. Percent Primer Incorporation The percent incorporation of primer herein was determined using1H NMR (nuclear magnetic resonance) spectroscopy measurements. Dry glucan product was first fully depolymerized to monomeric anhydroglucose (AGU) units by sulfuric acid hydrolysis. Approximately 20 mg of polymer was dissolved by stirring in 0.175 mL of a 60% deuterated sulfuric acid (D2SO4) solution in 99.99% deuterated water (D2O) at ambient temperatures until clear (typically about 1 hour). The homogenous solution was diluted to 12% D2SO4by adding 0.6 mL of D2O and 0.1 mL of D2O containing 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (DSS) used as an internal NMR chemical shift standard. The sample was then moved to a stirring heat block set at 90° C. for 2 hours. The resulting pale-yellow solution was transferred to a 5-mm NMR tube. Quantitative 1-D1H NMR data were then collected on a Bruker DRX at 500 MHz or Bruker Avance III at 600 MHz. In general, the degree of substitution (DoS) of the CMG (carboxymethyl glucan) primer was determined to measure the number of moles AGU for CMG. DoS of CMG is defined as the moles of CM (carboxymethyl) divided by the moles of AGU. Moles of CMG in CMGPG (CMG-primed glucan) was determined as ratio of moles CM divided by the CMG primer DoS. The moles of grafted glucan (alpha-1,3-glucan side-chains/arms) was found from the difference of total moles AGU measured in the NMR of the CMGPG and moles AGU of CMG primer. Finally, the mol % CMG was determined from moles CMG divided by the total moles of AGU multiplied by 100. Specifically, the mol % CMG incorporation in CMGPG was determined from the NMR spectrum as follows: Mol % CMG=((Moles CMG)/(total Moles AGU))*100. The CMG CH2group exhibits a complex series of multiplets due to regioselectivity of the CM moiety on positions 2, 4, and/or 6 and anomeric configuration of the AGU. Moles CM=∫CM/2=(∫(δ 4.53 ppm to δ 4.32 ppm)+∫(∫ 4.27 ppm to δ 4.23 ppm))/2. Moles CMG=Moles CM/DoS. The mole ratio of AGU was measured as the sum of all observed α/β anomeric protons. Moles AGU=∫(δ 5.44 ppm to δ 4.57 ppm). Carboxymethyl Alpha-1,3-Glucan Ether (CMG) Water-soluble CMG was prepared by entering unmodified alpha-1,3-glucan (˜100% alpha-1,3 glycosidic linkages) with a DPw of about 760 or 1220 into etherification reactions similar to, or the same as, the reactions disclosed in U.S. Patent Appl. Publ. Nos. 2016/0304629 and 2014/0179913, which are incorporated herein by reference. The etherification agent was sodium chloroacetate. CMG with a degree of substitution (DoS) with carboxymethyl groups of about 0.25, 0.31, 0.70, or 1.1 was prepared (respectively termed herein as CMG25, CMG31, CMG70 and CMG110). High Yielding Alpha-1,3-Glucan-Producing Glucosyltransferase Enzymes The amino acid sequence of the glucosyltransferase used to prepare amino acid substitutions was SEQ ID NO:4 (GTF 6855), which essentially is an N-terminally truncated (signal peptide and variable region removed) version of the full-length wild type glucosyltransferase (represented by SEQ ID NO:62) fromStreptococcus salivariusSK126 (see Table 1). Substitutions made in SEQ ID NO:4 can be characterized as substituting for native amino acid residues, as each amino acid residue/position of SEQ ID NO:4 (apart from the Met-1 residue of SEQ ID NO:4) corresponds accordingly with an amino acid residue/position within SEQ ID NO:62. In reactions comprising at least sucrose and water, the glucosyltransferase of SEQ ID NO:4 typically produces alpha-glucan having about 100% alpha-1,3 linkages and a DPwof 400 or greater (e.g., refer to U.S. Patent No. U.S. Patent Appl. Publ. No. 2017/0002335, which is incorporated herein by reference). This alpha-glucan product, which is insoluble, can be isolated following enzymatic synthesis via filtration, for example. Briefly, certain combinations of amino acid substitutions were made to SEQ ID NO:4 (GTF 6855). These substitutions are listed in Tables A and B below. Each variant enzyme listed in Table A was entered into a glucan synthesis reaction with parameters that were the same as, or similar to, the following: vessel, 250-mL indented shake flask agitated at 120 rpm; initial pH, 5.7; reaction volume, 50 mL; sucrose, 75 g/L; GTF, 1.5 mL lysate ofE. colicells heterologously expressing enzyme; KH2PO4, 20 mM; temperature, 30° C., time, about 20-24 hours. The alpha-1,3-glucan yields of these reactions (measured by HPLC analysis) are provided in Table A. TABLE AAlpha-1,3-Glucan Yields of GTF 6855 (SEQ ID NO: 4)Variants with Multiple Amino Acid SubstitutionsAlpha-1,3-GTFaGlucanbYieldcA510D/F607Y/R741S72.6%A510D/F607Y/N743S79.2%A510D/F607Y/D948G88.2%A510D/R741S/D948G74.5%A510D/F607Y/R741S/D948G82.8%A510E/F607Y/R741S/R1172C78.2%A510D/F607Y/D820G/D948G87.8%A510D/F607Y/D948G/R1172C88.6%A510D/F607Y/N743S/D948G/R1172C89.4%A510D/F607Y/R741S/L784Q/F929L/R1172C79.3%aEach listed GTF is a version of GTF 6855 (SEQ ID NO: 4) comprising substitutions at respective positions, where each position number is in correspondence with the residue numbering of SEQ ID NO: 62. The wild type residue is listed first (before residue position number) and the substituting residue is listed second (after the residue position number).bInsoluble alpha-1,3-glucan product with 100% alpha-1,3 linkages.cAlpha-1,3-glucan yield based on glucosyl. The average yield of unmodified GTF 6855 (SEQ ID NO: 4, no substitutions) was about 29%. Each variant enzyme listed in Table B was entered into a glucan synthesis reaction with parameters that were the same as, or similar to, the following: vessel, 500-mL jacketed reactor with Teflon®-pitched blade turbine (45-degree angle) on a glass stir rod and agitated at 50-200 rpm; initial pH, 5.5; reaction volume, 500 mL; sucrose, 108 g/L, KH2PO4, temperature, 39° C., time, about 18-24 hours; filtrate from a previous alpha-1,3-glucan synthesis reaction, 50 vol %. The alpha-1,3-glucan yields of these reactions (measured by HPLC analysis) are provided in Table B. TABLE BAlpha-1,3 Glucan Yields of GTF 6855 (SEQ ID NO: 4) Variants with Multiple Amino Acid-SubstitutionsAlpha-1,3-GlucanbGTFaYieldcA510DQ588LF607YR741SD948GR722HT877KM1253IK1277N88%A510DQ588LF607YR741SD948GR722HT877KV1188EM1253IQ957P92%A510DQ588LF607YR741SD948GT877KV1188EM1253IQ957P91%A510DQ588LF607YR741SD948GM1253I89%A510DQ588LF607WR741SD948G91%Q588LF607YR741SD948G91%A510DQ588LF607YR741SD948GN628DT635AT877KM1253IF929LR1172C92%A510DQ588LF607WR741SD948GS631TS710GR722HT877KV1188EM1253I94%A510DQ588LF607WR741SD948GS631TS710GR722HT877KV1188E93%A510DQ588LF607WR741SD948GS631TS710GT877KV1188EM1253I96%A510DQ588LF607YR741SD948G89%A510DQ588LF607YR741SD948GV1188E88%A510DQ588LF607WR741SD948GS631TS710GV1188E96%A510DQ588LF607WR741SD948GS710GR722HT877KM1253I96%A510DQ588LF607YR741SD948GS631TR722HT877KV1188EM1253I96%A510DQ588LF607WR741SD948GS631TT877KV1188EM1253I94%A510DQ588LF607WR741SD948GS631TV1188E98%A510DQ588LF607YR741SD948GS631TR722HT877KV1188EM1253I95%A510DQ588LF607WR741SD948GV1188EM1253I93%aEach listed GTF is a version of GTF 6855 (SEQ ID NO: 4) comprising substitutions at respective positions, where each position number is in correspondence with the residue numbering of SEQ ID NO: 62.bInsoluble alpha-1,3 glucan product.cAlpha-1,3-glucan yield based on glucosyl. Glucosyltransferase Enzymes that Produce Lower Molecular Weight Alpha-1,3-Glucan As discussed above, SEQ ID NO:4 (GTF 6855) is an amino acid sequence of a glucosyltransferase that can be used to prepare amino acid substitutions. Briefly, certain combinations of amino acid substitutions can be made to SEQ ID NO:4 to provide a glucosyltransferase that produces alpha-1,3-glucan of lower molecular weight (as compared to alpha-1,3-glucan product of non-modified GTF 6855). These substitutions are listed in Tables C and D below. For collecting the data in these tables, each variant enzyme was entered into a glucan synthesis reaction with parameters that were the same as, or similar to, the following: vessel, 50-mL indented shake flask agitated at 75 rpm; initial pH, 5.7; reaction volume, 10 mL; sucrose, 400 g/L; GTF, 0.3 mL of culture supernatant (prepared from lysate ofE. colicells heterologously expressing enzyme); KH2PO4, 5 mM; temperature, 35° C.; time, about two days; de-activation, heated at 80° C. for 30 minutes. Insoluble glucan polymers produced in the reactions were individually harvested, water-washed, and analyzed for molecular size (DPw) via a standard SEC approach (see Tables C and D for DPw data). A glucosyltransferase with any of the amino acid substitution(s) listed in Tables C and D is contemplated to be useful in practicing the presently disclosed subject matter. TABLE CDPw of Insoluble Alpha-1,3-Glucan Produced by GTF 6855 (SEQID NO: 4) and Single Amino Acid-Substituted Variants thereofGTFDPwGTFDPwGTFDPw6855a350S553A127N573A123L513Ab194S553C125N573A125L513C119S553C126N573D108L513C159S553E105N573D134L513D147S553E122N573G126L513D640S553FcN573G120L513E129S553FcN573H148L513E130S553HcN573IcL513F171S553HcN573K145L513G138S553I79N573K148L513H153S553I97N573LcL513H175S553M129N573McL513I186S553M140N573N222L513K143S553N77N573P100L513K160S553N69N573T102L513M183S553R63N573T109L513M210S553T226N573V91L513NcS553T124N573W249L513N372S553V86N573W237L513P173S553Y110L513Q138S553Y52L513Q152L513R134L513R141L513S138L513S152L513T146L513V175L513VcL513W146L513W171L513Y1566855a350K578A110Q616A175D575Ab74K578A113Q616A440D575A199K578C132Q616C81D575C97K578D156Q616D115D575CcK578E95Q616E50D575C94K578EcQ616G66D575E90K578F103Q616GcD575E88K578G113Q616H61D575F74K578G103Q616I82D575G90K578H212Q616K58D575G89K578H187Q616K59D575H70K578I179Q616L61D575H134K578L177Q616L62D575I76K578M135Q616M164D575I98K578M141Q616N269D575K52K578N185Q616N211D575K95K578P126Q616P75D575L74K578P128Q616P78D575LcK578Q111Q616R103D575M66K578R214Q616R167D575M72K578R294Q616S72D575N90K578S105Q616T79D575N191K578S105Q616V88D575NcK578T131Q616V97D575P50K578T157Q616W60D575R65K578V146Q616W101D575R71K578V145Q616Y65D575S104K578W106D575S96K578W122D575V54K578Y145D575W69D575W167D575Y124D575Y69aGTF 6855, SEQ ID NO: 4. The DPw of insoluble alpha-1,3-glucan produced by GTF 6855 averaged to be about 350.bEach listed GTF with a substitution is a version of GTF 6855 comprising a substitution at a respective position, where the position number is in correspondence with the residue numbering of SEQ ID NO: 62.cInsoluble alpha-1,3-glucan not produced or detected. TABLE DDPw of Insoluble Alpha-1,3-Glucan Produced by Multiple AminoAcid-Substituted Variants of GTF 6855 (SEQ ID NO: 4)GTFaDPwP550LN557IN581P12L535PS553CN558DD575VT585PK697R12P550VS553RN581PT585P12P550LS553FN581P12P550VN557ET585P12P550LN557ED575VT585P13L538PP550LS553Y13P544LP550VS553CN573IT585PS589G13P550VG576DT585P13P550LN558DT585PT679I13P550LN557ET585PS589G14P550LN557ET569LN581P14P550LN557ET569LT585P14P550VS553TN558DT585PG730D14E577GP550LN557IT569LN573I14P550LS553CD575AT585PS589G14S553RN573VK578NS631GT660A14P550VS553RW571VG576D14P550VN557EK578DT585P14P550VN558DN573PT585P14P550LN558DW571VN581PK593E14P550VS553EN581P15P550LN573IT585PW725R15P550LN557IN573P15N557EN573VN581P15P550LN557IG576DQ643L15P550VS553NT585PV586GS710G15P550VS553CD575AT585P15S553RN573VK578NS631GT660A15P550LS553KD575AY580H16P550VD575AT585PS589GK713E16P550VS553NN573IY693C16P544LP550LN557EN573IT585P16P550LN558DW571VN581PT585P16S504GP550VN557QN581P16P550LS553RD575A16P550VN558DW571DD575AT585P16P544LP550VN557QN581P17P550VS553KT585P17P550VS553NT585P17P550LT569LN573I17P550LN558DD575V17L537PP550LN558DN573I17P550LS553CW571CG576DT585P17P550LN557QW571CG576DT585P17P550LN558DW571VN581P17A542VP550VN558DW571VT585P17P550VN558DW571DG576D18P550VS553NN573I18P550VN557QD575VA669T18P550VN581PI636T18P550VN557EN581P18P550VN573IT585P18P550LS553KN558DK578RY700N19P550VS553TN558DW571V19P514LP550VN557QT585PD602N20P550LN557IT569AG576D20P550VN557TN558DW571D21P550LN557ED575AT585P21I545VP550VN557QT585PD638N21P544LP550VN557IT585P22Y518CP550VN581PT585P22P550LN557ED575A22aEach listed GTF is a version of GTF 6855 (SEQ ID NO: 4) comprising substitutions at respective positions, where each position number is in correspondence with the residue numbering of SEQ ID NO: 62. Glucosyltransferase Enzymes that Produce Higher Molecular Weight Alpha-1,3-Glucan As discussed above, SEQ ID NO:4 (GTF 6855) is an amino acid sequence of a glucosyltransferase that can be used to prepare amino acid substitutions. Briefly, certain amino acid substitutions can be made to SEQ ID NO:4 to provide a glucosyltransferase that produces alpha-1,3-glucan of higher molecular weight (as compared to alpha-1,3-glucan product of non-modified GTF 6855). These substitutions are listed in Tables E and F below. For collecting the data in these tables, each variant enzyme was entered into a glucan synthesis reaction with parameters as disclosed in U.S. Patent Appl. Publ. No. 2019/0078062 (corresponds to U.S. patent application Ser. No. 16/127,288), which is incorporated herein by reference. A glucosyltransferase with any of the amino acid substitution(s) listed in Tables E and F is contemplated to be useful in practicing the presently disclosed subject matter. TABLE EDPw of Insoluble Alpha-1,3-Glucan Produced by GTF 6855 (SEQID NO: 4) and Single Amino Acid-Substituted Variants thereofGTFDPwGTFDPwGTFDPwGTFDPw6855a626T635H539P1499Y587A510E625V186Ab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aGTF 6855, SEQ ID NO: 4. The DPw of insoluble alpha-1,3-glucan produced by GTF 6855 averaged to be about 626.bEach listed GTF with a substitution is a version of GTF 6855 comprising a substitution at a respective position, where the position number is in correspondence with the residue numbering of SEQ ID NO: 62. TABLE FDPw of Insoluble Alpha-1,3-Glucan Produced by GTF 6855 (SEQID NO: 4) and Single Amino Acid-Substituted Variants thereofGTFDPw6855a558E567Qb1001I591V859L661P842N743D700N743S937N743T874R741A831R741P871R741Q886R741S887R741T693T563A910V586T874aGTF 6855, SEQ ID NO: 4.bEach listed GTF with a substitution is a version of GTF 6855 comprising a substitution at a respective position, where the position number is in correspondence with the residue numbering of SEQ ID NO: 62. Example 1 Glucosyltransferase-Catalyzed Synthesis of Alpha-Glucan Using Alpha-Glucan Ether as Primer This Example describes using a glucosyltransferase to synthesize alpha-glucan using alpha-glucan ether as a primer. In particular, a glucosyltransferase enzyme that produces alpha-1,3-glucan was employed in a reaction comprising at least water, sucrose and carboxymethyl alpha-1,3-glucan (CMG) primer. The alpha-1,3-glucan material produced in this Example is contemplated to include a copolymer comprising a carboxymethylated alpha-1,3-glucan backbone and linear alpha-1,3-glucan side chain(s)/arm(s) (graft copolymer). It is also contemplated that copolymer was produced in which linear alpha-1,3-glucan was synthesized from the non-reducing end of the CMG primer; such a product is believed to include graft copolymer species, and/or species in which alpha-1,3-glucan arms were not synthesized off of the CMG primer. The copolymer products herein are collectively referred to as carboxymethyl glucan-primed glucan (CMGPG). A series of eight reactions was performed; each reaction contained 50 or 100 g/L sucrose, 5 mM sodium phosphate buffer (pH 5.5), CMG primer (0, 0.5, 2.0, or 5.0 g/L), and 100 U/L of a glucosyltransferase (GTF). The CMG primer (termed herein as “CMG25”), which comprised carboxymethylated linear alpha-1,3-glucan (DPw of about 760), was aqueous-soluble and had degree of substitution of about 0.25. The GTF was anS. salivariusGTF modified in its catalytic domain such that the enzyme could produce more products (fructose and alpha-1,3-glucan with about 100% alpha-1,3 linkages), and less by-products (e.g., glucose, oligosaccharides such as leucrose and DP2-7 gluco-oligosaccharides), from sucrose substrate, as compared to the enzyme's unmodified counterpart. The General Methods section describes preparation of this non-native glucosyltransferase (Table A). Each reaction was prepared by charging a 250-mL disposable shake flask with 10 or 20 mL of a sterile-filtered 500-g/L sucrose stock solution; 5 mL of a 100-mM sodium phosphate buffer stock solution (pH 5.5); 0, 50, 200, or 500 mg of CMG25 dry powder; and a sufficient amount of water for a final volume of 100 mL. Solids were dissolved by vigorous shaking, after which GTF (0.226 mL, 100 U/L final concentration) was added to initiate each polymerization reaction; a 0.5-mL aliquot was taken from each solution just prior to GTF addition. All reactions were carried out in an incubator at 30° C. with shaking at 100 rpm. A 0.5-mL aliquot was withdrawn from each reaction 1 hour after start; a 0.1-mL aliquot was withdrawn at 2, 3 and 24 hours after reaction start. These aliquots were analyzed by HPLC. At 2 hours after reaction start, a 2.25-mL aliquot was withdrawn from both reactions containing 5 g/L CMG25 primer for rheological analysis. All aliquot samples upon their acquisition were placed in a heat block and heated to 80° C. for 10 minutes to stop the reactions. After 24 hours, all the reactions were stopped by placing them in an 80° C. water bath for 30 minutes. The reactions containing 5 g/L CMG25 primer appeared as a hard semi-opaque gel. All the deactivated reactions were loaded onto 10-micron CHEMRUS disposable fritted filters and fully vacuum-filtered. The solids were then washed and filtered three times with 100 mL pure water to remove any water-soluble moieties, resulting in polymer wetcakes. Products of reactions having 5 g/L CMG25 primer were not homogenous and difficult to wash. Approximately 300-500 mg of each wetcake was set aside for SEC analysis, while the remaining material was dried in a 40° C., −45 cmHg vacuum oven for ˜3 days. Approximately 300 mg of each dry product was set aside for NMR analysis. Wetcake samples were dissolved in 1% LiCl/DMSO and analyzed by SEC (General Methods) to determine apparent molecular weight and DPw of each polymer product. Dry polymer samples were dissolved in 3% LiCl/DMSO-d6. A small amount of D2O was added. Each solution was then analyzed by1H NMR (General Methods) to determine the linkage profile of each of the polymer products and to calculate the mole percent (mol %) of CMG25 incorporation. HPLC samples were prepared by diluting the 1-, 2- and 3-hour samples 10-fold with deionized, submicron-filtered water. The 0- and 24-hour samples were not diluted prior to preparation. All the samples were vortexed to ensure homogeneity, loaded onto a 0.45-micron Corning SPIN-X centrifuge filter, and centrifuged for 30 minutes at 12,000 rpm in a micro-centrifuge. The filtrate from each sample was loaded into a glass HPLC vial fitted with a low volume insert. The samples were analyzed using an Agilent 1200 series instrument equipped with an RI detector. Analysis was performed on two columns in parallel: BioRad AMINEX 87C (column 1) for separation of <DP3 sugars and BioRad AMINEX 42A (column 2) for separation of DP2-DP8+ oligosaccharides/polysaccharides. Linear calibration curves were constructed for sucrose (0.1-50 g/L), fructose (0.1-100 g/L), leucrose (0.1-100 g/L), and glucose (0.1-100 g/L) for quantitation on column 1. Linear calibration curves were constructed for DP2 (0.04-10 g/L), DP3 (0.04-10 g/L), DP4 (0.04-10 g/L), DP5 (0.04-10 g/L), DP6 (0.04-10 g/L), DP7 (0.04-10 g/L) and DP8+(0.25-10 g/L) for quantitation on column 2. Moieties with a DP greater than or equal to 8 (DP8+) were not well resolved on column 2. For quantification, a calibration curve was established using a lab-prepared dextran material (linear alpha-1,6 dextran with ˜20% alpha-1,2 branches, MW ˜17 kDa). Table 2 (below) provides some results of the above analyses. CMG25 primer incorporation in the copolymer products was evidenced by NMR. TABLE 2Profiles of Glucosyltransferase Reactions (24-hour) Containing CMG25 PrimerStart-of-ReactionAlpha-1,3-SucroseCMG25GlucanConsumptionAlpha-1,3-Glucan ProductSucrosePrimerProduct YieldRateMol % CMG25(g/L)(g/L)(Glucosyl)(g/L/h)DPwPDIIncorporation500.081%5.712421.90.0%0.582%6.813542.22.8%2.085%6.815392.38.0%5.082%7.312652.316.0%1000.082%9.211591.80.0%0.583%10.721152.81.6%2.082%10.512852.64.0%5.083%7.115062.612.0% Shear viscosity of the 2-hour samples of reactions with 5 g/L CMG25 primer (50 and 100 g/L sucrose) were obtained using a Kinexus PRO+ rheometer fitted with cone and plate geometry. Results of this analysis are shown inFIGS.1A and1B. A dramatic increase in viscosity was observed for copolymer products of reactions with 5 g/L CMG25 primer at an early time point (2 hours), as confirmed by observing >10000 centipoise (cP) of shear viscosity at a 0.1/s shear rate. All shear viscosity measurements in this and the following Examples were made using the above equipment on homogenized samples of insoluble polymer products or samples of whole reactions. Viscosity results were obtained by sweeping from low to high (0.1/s→100/s) for one set of measurements, followed by sweeping from high to low (100/s→0.1/s), this procedure thus provided acquisition of replicate data. Since the values for each sweep in all cases herein were generally similar, only the high-to-low sweep viscosity measurements are shown in the figures (FIGS.1-5) for clarity. In this Example, a series of water-insoluble copolymers was produced in reactions comprising CMG25 primer and a glucosyltransferase that synthesizes alpha-1,3-glucan. Based on observations made in the SEC analyses, these copolymers (CMGPG) are contemplated to comprise a carboxymethylated alpha-1,3-glucan backbone and linear alpha-1,3-glucan side chain(s)/arm(s) (graft copolymer). CMGPG may further comprise an alpha-1,3-glucan extension from the non-reducing end of the CMG25 primer; while most of these species are contemplated to have alpha-1,3-glucan arms, some species might not have these arms. Example 2 Glucosyltransferase-Catalyzed Synthesis of Alpha-Glucan Using Alpha-Glucan Ether as Primer (2-L Scale) This Example describes a scale-up of select reactions described in Example 1, which were done in shake flasks (100-mL scale), to a 2-L scale in reactors. A similar set of conditions to that described in Example 1 was used to synthesize CMGPG. Two reactions were prepared containing 22.5 or 100 g/L sucrose, 5 mM sodium phosphate buffer (pH 5.5), 10 g/L CMG25 primer and 100 U/L a GTF enzyme. The CMG primer and GTF enzyme were the same as those used in Example 1. Each reaction was prepared by dissolving 45 or 200 grams of sucrose and 20 grams of CMG25 in about 1 L of pure water using a magnetic stirrer. 10 mL of a 1 M sodium phosphate buffer stock solution (pH 5.5) was then added. The final volume was adjusted to 2 L using a volumetric flask. These solutions were each charged into a 2-L glass-jacketed reactor fitted with an overhead stirrer. A 0.5-mL aliquot was withdrawn from each solution at t=0 time point for HPLC analysis. An extra 5 mL was withdrawn at t=0 time point from the low sucrose (22.5 g/L) solution for rheological testing. Each reaction was then initiated by charging it with 4.535 mL of the GTF enzyme (100 U/L final concentration). The reactions were carried out at 30° C. Samples (1 mL) were withdrawn from each reaction to micro-centrifuge tubes at 1, 2, 3 and 24 hours following the start of the reactions. Each sample was heated to 80° C. for 10 minutes to deactivate the GTF enzyme. For HPLC, the samples were loaded onto 0.45-micron Corning SPIN-X centrifuge filters and centrifuged for 30 minutes at 12000 rpm on a table microcentrifuge. The filtrate from each sample was loaded into an HPLC glass vial fitted with a low volume insert. Significant viscosity increases were observed for both reactions at early time points. In the 100 g/L sucrose reaction, viscosity broke between 2 and 3 hours after reaction initiation, with observable polymer precipitation followed by formation of a hard semi-opaque gel. This phenomenon caused the overhead stirring paddle to seize. Stirring was stopped for the remainder of the reaction. In the 22.5 g/L sucrose reaction, no break in viscosity was observed during this time period and through the end of 24 hours; the reaction mixture remained as an apparently homogenously-dispersed, high viscosity gel. Some increase in opacity was observed. At 24 hours reaction time, both reaction vessels were heated to 80° C. for 30 minutes to deactivate the GTF enzyme to stop the reactions. Approximately 40 grams of reaction mixture were withdrawn from the completed, highly viscous, 22.5 g/L sucrose reaction for rheological characterization. Acetonitrile (2 L) was added to the remaining reaction mixture to precipitate the CMGPG product (to facilitate its isolation for analysis, given the high viscosity), stirred for 24 hours, and allowed to settle for 48 hour before separating the solids by centrifugation at 5000 rpm at −20° C. in 1-L centrifuge bottles. After decanting the supernatant, which contained soluble sugars and oligosaccharides, CMGPG (appeared as a gel layer) was collected from the bottom of the centrifuge tubes. This gel was placed in a vacuum oven at 40° C., -45 cmHg for about 3 days, until dry. HPLC, NMR and SEC analyses were performed as described in Example 1. Table 3 (below) provides some results of these analyses. TABLE 3Profiles of Glucosyltransferase Reactions(24-hour) Containing 10 g/L CMG25 PrimerStart-of-Alpha-1,3-SucroseReactionGlucanConsumptionAlpha-1,3-Glucan ProductSucroseProduct YieldRateMol % CMG25(g/L)(Glucosyl)(g/L/h)DPwPDIIncorporation22.563%4.115453.052.0%10084%8.216283.116.0% Rheology tests were performed on the 0- and 24-hour samples of the low sucrose (22.5 g/L) reactions (these samples are whole samples of each reaction; i.e., insoluble glucan product was not purified and homogenized before viscosity testing). Shear viscosity from 0.1 to 100 s−1was determined using the method described in Example 1; results are shown inFIG.2. The 24-hour sample had shear viscosity about 100 times higher than that of the 0-hour sample at shear rate of 0.1/s. The final concentration of grafted polymer was approximated to be 1.6% in the 2-L reaction (22.5 g/L sucrose) based on a recovery of about 32 grams of dry, pure material from the reaction at 24 hours. Example 3 Dispersion and Viscosity Analysis of Alpha-Glucan Ether-Primed Alpha-Glucan This Example describes, among other things, the recovery of high viscosity conditions upon dispersion/re-suspension of the CMGPG that was isolated and dried from the 22.5 g/L sucrose reaction described in Example 2 (i.e., the high viscosity observed in the terminated reaction, which contained “never-dried” CMGPG product, could be achieved by dispersing the CMGPG following its isolation from the reaction mixture and being dried). This represents an advantage over alpha-1,3-glucan that is synthesized without an ether primer; such non-ether-primed alpha-1,3-glucan not only has lower viscosity compared to CMGPG (in their respective never-dried states such as in reaction mixtures), but also is more difficult to disperse after drying (thereby not achieving the viscosity level of its never-dried forerunner). Also, this Example describes a lack of viscosity increase when mixing non-primed alpha-1,3-glucan reaction mixtures with CMG primer, which indicates that the enhanced viscosity observed with CMGPG is likely due to CMGPG itself. Two GTF reactions were performed to generate mixtures containing non-primed alpha-1,3-glucan (i.e., glucan was synthesized in reactions that did not contain primer). Similar conditions were used as described in Example 2, except that CMG primer was omitted. Briefly, two 250-mL disposable shake flasks were charged with 1 or 2.25 g of sucrose (10 or 22.5 g/L final concentration), 5 mL of 100 mM sodium phosphate buffer stock (pH 5.5), and a sufficient volume of deionized, submicron-filtered water to provide a final volume of 100 mL. A 0.5-mL aliquot was taken from each solution before 0.226 mL of GTF enzyme (100 U/L final concentration, same enzyme as used in Example 2) was added to initiate each polymerization reaction. Each reaction was carried out in an incubator shaker at 30° C. with shaking at 100 rpm. After 24 hours, a 1-mL sample was withdrawn from each reaction and heated to 80° C. for 10 minutes to stop the reaction. Also at 24 hours, the entire reactions were terminated by placing the reaction vessels into an 80° C. water bath. The terminated reactions were considered to be alpha-1,3-glucan reaction mixtures, given the presence of the aqueous-insoluble glucan product. The 0.5-mL and 1-mL samples were centrifuged at 12000 rpm for 10 minutes on a microcentrifuge to remove insoluble material. Supernatants were filtered through 0.45-micron PTFE Whatman® syringeless filters and then analyzed by HPLC as described in Example 1. Several preparations were made, as described in Tables 4 and 5 below, to test the effects of mixing the alpha-1,3-glucan reaction mixtures prepared above with CMG primer (same as used in Example 1), as well as to test the ability of isolated CMGPG (as produced in the 22.5 g/L sucrose reaction of Example 2) to regain its reaction mixture viscosity after resuspension/dispersion in water. TABLE 4Preparation ComponentsBatchAlpha-1,3-GlucanStarting MaterialID(w/v %)Alpha-1,3-glucan reaction mixture−010.38%(non-primed, 10 g/L sucrose reaction,terminated, Example 3)Alpha-1,3-glucan reaction mixture−020.75%(non-primed, 22.5 g/L sucrose reaction,terminated, Example 3)CMG dry powder−303dry powder(DoS = 0.25, DPw 760; CMG25)CMGPG dry powderdry powder(from 22.5 g/L sucrose reaction as described inExample 2) TABLE 5Aqueous Preparations of Alpha-1,3-Glucan (Non-Primed), CMG, and/or CMGPGMediumFinalTotal of Alpha-1,3-Glucan,SampleCMG or CMGPG(Glucan ReactionVolumeCMG, and/or CMGPGID(w/v %)Mixture or H2O)(mL)(w/v %)10.0%−01b500.38%20.0%−02b500.75%3CMG, 1% batch −303a−01b501.39%4CMG, 1% batch −303a−02b501.75%5CMG, 1% batch −303aH2O501.00%6CMG, 1.7% batch −303aH2O501.72%7CMGPGa, 1%H2O501.02%8CMGPGa, 0.5%H2O500.50%aAs listed in Table 4.bAs listed in Table 4 under Batch ID. All the preparations of Table 5 (Sample 1-8) were homogenized at about 13500 rpm for at least 2 minutes, or until appearance was homogenous. Upon the initial addition of water to the dry CMGPG samples, a low viscosity, non-homogenous slurry was formed; the dry particles appeared to swell, but did not disperse. Upon homogenization (13500 rpm) of these CMGPG slurries, viscosity rapidly built up, forming an apparently homogenous dispersion resembling the reaction mixture from which the CMGPG was originally isolated. Shear viscosity measurements of all the homogenized samples (above) were obtained as described in Example 1, and are shown inFIGS.3A-D. Samples 2-8 were analyzed using plate/plate geometry, while Sample 1 was analyzed using cup/vane geometry.FIG.3Acompares the viscosity profile of the end-of-reaction mixture from non-primed 10 g/L sucrose reaction (Sample 1) with the viscosity profile of the same end-of-reaction mixture as further mixed with 1 w/v % CMG25 primer (Sample 3). Sample 3 represents a hypothetical end-of-reaction mixture in which a GTF reaction with 10 g/L sucrose and 1 w/v % CMG25 primer was conducted, but no priming occurred.FIG.3Bcompares the viscosity profile of the end-of-reaction mixture from non-primed 22.5 g/L sucrose reaction (Sample 2) with the viscosity profile of the same end-of-reaction mixture as further mixed with 1 w/v % CMG25 primer (Sample 4). Sample 4 represents a hypothetical end-of-reaction mixture in which a GTF reaction with 22.5 g/L sucrose and 1 w/v % CMG25 primer was conducted, but no priming occurred. The data inFIGS.3A-Bindicate that addition of 1 w/v % CMG25 to terminated, non-primed alpha-1,3-glucan reaction mixtures only mildly changes shear viscosity profile. If, for sake of argument, priming did not occur in Example 2, the end-of-reaction mixture of the 22.5 g/L sucrose+CMG reaction in Example 2 would have exhibited a viscosity profile similar to the profile of Sample 3 inFIG.3B, but that was not the case. Indeed, the actual viscosity profile of that reaction (FIG.2, 24-hr) was significantly higher than the profile of Sample 4 (FIG.3B). For instance, as a simple but direct comparison, the viscosity at a shear rate of 0.1 s−1was 235 cP for Sample 4 (FIG.3B), but was 40720 cP for the primed end-of-reaction mixture in Example 2 (FIG.2).FIG.3Cshows the viscosity profiles of CMGPG preparations at 0.5 w/v % (Sample 8) and 1 w/v % (Sample 7), respectively. The CMGPG in these samples was the graft copolymer product of the 22.5 g/L sucrose+CMG priming reaction of Example 2. The data indicate that CMGPG was able to dramatically increase viscosity even at 0.5 w/v % and 1 w/v % concentrations. Therefore, CMGPG was confirmed to be the highly viscous material in the end-of-reaction mixture of Example 2. A 1 w/v % CMGPG preparation had a similar or comparable viscosity profile (Sample 7,FIG.3D) as the profile of the end-of-reaction mixture in Example 2 (FIG.2) in which the CMGPG content was estimated to be -1.6 w/v %. This observation suggests that CMGPG powder could be dispersed to regain the viscosity that it exhibited when prepared in its original reaction-mixture state (“never-dried” state).FIG.3Dcompares the viscosity profile of 1 w/v % CMGPG (Sample 7) with CMG25 primer at 1 w/v % (Sample 5) and 1.7 w/v % (Sample 6). The 1 w/v % CMGPG preparation (Sample 7) was much more viscous than CMG25 at the same 1 w/v % concentration (Sample 5). The 1.7 w/v % CMG25 preparation of Sample 6 represents a reaction in which no priming occurred; for Sample 6, the non-primed alpha-1,3-glucan portion of the end-of-reaction mixture of Example 2 was replaced by CMG25 primer. In this test, the addition of about 0.7 w/v % of CMG25 to the starting concentration of 1 w/v % was not able to boost the viscosity level to that of the 1 w/v % CMGPG dispersion. Since the final CMGPG concentration of the actual end-of-reaction mixture of Example 2 was estimated to be ˜1.6 w/v %, which was very similar to the 1.7 w/v % CMGPG concentration in Sample 6, these two tests can be used for a direct comparison on viscosity. The viscosity at 0.1 s−1shear rate was 40720 cP for the end-of-reaction mixture of Example 2 (FIG.2), but only 591 cP for the 1.7 w/v % CMG25 (Sample 6,FIG.3D). This observation again highlights the high viscosity nature of CMGPG as compared with that of CMG25 at a higher concentration of ˜1.6 w/v %. To summarize this Example, the priming reactions of Example 2 were further confirmed by the viscosity changes observed among samples representing different reaction conditions, either real or hypothetical. Isolated and dried CMGPG powder was demonstrated to be highly viscous when dispersed into an aqueous solution, even at the low concentrations of 0.5 w/v % and 1 w/v %. CMGPG preparations were significantly more viscous than solutions of CMG25 primer at the same/similar concentrations. CMGPG was confirmed to be the main cause of the high viscosity of the end-of-reaction mixtures of Example 2. Example 4 Effects of Increased Alpha-Glucan Ether DoS (˜0.31) on Priming of Alpha-Glucan Synthesis in Glucosyltransferase Reactions This Example, coupled with Examples 1 and 2, describes how alpha-1,3-glucan synthesis reactions that contain alpha-glucan primer are affected by the DoS of the primer. Herein, a soluble carboxymethylated linear alpha-1,3-glucan was used as a primer (˜760 DPw, ˜0.31 DoS, termed “CMG31” herein) for the enzymatic synthesis of insoluble alpha-1,3-glucan. The CMG31 primer only differs from the primer used in the above Examples (CMG25) by its higher DoS of ˜0.31. Alpha-1,3-glucan products of this Example likely represent additional forms of CMGPG as described in the above Examples (i.e., graft copolymer with a carboxymethylated alpha-1,3-glucan backbone and alpha-1,3-glucan chain(s)/arm(s)). Similar to the observations in the above Examples, the graft copolymer products of this Example were observed to dramatically increase reaction viscosity. A series of three reactions was performed with about 10, 20, or 100 g/L sucrose, 5 mM sodium phosphate buffer (pH 5.5), 10 g/L of CMG31 primer (see above) and 100 U/L of a GTF enzyme. The GTF enzyme was the same as that used in Example 1. Each reaction mixture was prepared by charging a 250-mL disposable shake flask with 2, 4, or 20 mL of a sterile-filtered 500 g/L sucrose stock solution, 5 mL of a 100 mM sodium phosphate buffer stock solution (pH 5.5), 1 g of CMG31 dry powder and a sufficient volume of deionized, submicron-filtered water to provide a final volume of 100 mL. The solids were dissolved with vigorous shaking, after which a 0.5-mL aliquot was taken from each solution (t=0 time point) and 0.226 mL of the GTF enzyme (100 U/L final concentration) was added to initiate polymerization. All reactions were carried out in an incubator at 30° C. with shaking at 100 rpm. A 1-mL aliquot was withdrawn from each reaction at 1, 2, 3, and 24 hours after reaction initiation for HPLC analysis. All aliquot samples were placed in a heat block at 80° C. for 10 minutes to deactivate the GTF. After 24 hours, all reaction vessels were placed in an 80° C. water bath for 30 minutes to stop the reactions. The reaction with 100 g/L sucrose appeared as a hard semi-opaque gel, while the other two reactions appeared to be viscous dispersions (thus, all three reactions could be characterized as reaction mixtures). The 100 g/L sucrose reaction was processed as described in Example 1 to generate dry polymer. Each of the deactivated 10 g/L and 20 g/L sucrose reactions had about 5 mL withdrawn for rheology analysis and about 1 mL withdrawn for SEC analysis, before being dried in a 40° C., -45 cmHg vacuum oven for about 3 days. HPLC, SEC, NMR and rheological analyses were performed largely as described in Example 1, except rheology was measured with cone/plate geometry. Table 6 (below) andFIG.4provide some results of these analyses. TABLE 6Profiles of Glucosyltransferase Reactions(24-hour) Containing 10 g/L CMG31 PrimerStart-of-Alpha-1,3-SucroseReactionGlucanConsumptionAlpha-1,3-Glucan ProductSucroseProduct YieldRateMol % CMG31(g/L)(Glucosyl)(g/L/h)DPwPDIIncorporation10.245%1.2019421.55670.97%21.664%1.8913271.86654.84%107.377%3.7911471.69422.58% The reactions appeared, for the most part, to proceed in a manner similar to that of the reactions described in Examples 1 and 2. For example, high viscosities were observed for samples of low sucrose reactions (10 g/L and 20 g/L sucrose) at 24 hours; this observation was confirmed by rheology data (FIG.4). This observation, along with NMR data showing CMG31 primer incorporation into the insoluble products, for example, support the conclusion that the glucosyltransferase reactions in this Example produced the graft copolymer, CMGPG. However, compared to the glucosyltransferase reactions of Examples 1 and 2, which employed a CMG primer with a DoS of about 0.25, use of the higher DoS primer, CMG31 (DoS ˜0.31), seemed to reduce the sucrose consumption rate of the glucosyltransferase reactions in this Example. Example 5 Effects of Increased Alpha-Glucan Ether DoS (˜0.70) on Priming of Alpha-Glucan Synthesis in Glucosyltransferase Reactions This Example, coupled with Examples 1, 2 and 4, describes how alpha-1,3-glucan synthesis reactions that contain alpha-glucan primer are affected by the DoS of the primer. Herein, soluble carboxymethylated linear alpha-1,3-glucan was used as a primer (1220 DPw, ˜0.70 DoS, termed “CMG70” herein) for the enzymatic synthesis of insoluble alpha-1,3-glucan. The CMG70 primer differs from the primers used in the above Examples (CMG25 and CMG31) by its higher DoS of ˜0.70 and DPw of 1220. Alpha-1,3-glucan products of this Example likely represent additional forms of CMGPG as described in the above Examples (i.e., graft copolymer with a carboxymethylated alpha-1,3-glucan backbone and alpha-1,3-glucan chain(s)/arm(s)). Similar to the observations in the above Examples, the graft copolymer products of this Example were observed to dramatically increase reaction viscosity. A series of three reactions was performed with about 10, 20, or 100 g/L sucrose, 5 mM sodium phosphate buffer (pH 5.5), 10 g/L of CMG70 primer (see above) and 100 U/L of a GTF enzyme. The GTF enzyme was the same as that used in Example 1. Each reaction mixture was prepared by charging a 250-mL disposable shake flask with 2, 4, or 20 mL of a sterile-filtered 500 g/L sucrose stock solution, 5 mL of a 100 mM sodium phosphate buffer stock solution (pH 5.5), 1 g of CMG70 dry powder and a sufficient volume of deionized, submicron-filtered water to provide a final volume of 100 mL. The solids were dissolved with vigorous shaking, after which a 0.5-mL aliquot was taken from each solution (t=0 time point) and 0.226 mL of the GTF enzyme (100 U/L final concentration) was added to initiate polymerization. All reactions were carried out in an incubator at 30° C. with shaking at 100 rpm. A 1-mL aliquot was withdrawn from each reaction at 1, 2, 3, and 24 hours after reaction initiation for HPLC analysis. All aliquot samples were placed in a heat block at 80° C. for 10 minutes to deactivate the GTF. After 24 hours, all reaction vessels were placed in an 80° C. water bath for 30 minutes to stop the reactions. The reaction with 100 g/L sucrose appeared as a hard semi-opaque gel, while the reaction with 20 g/L sucrose appeared as a viscous opaque dispersion. The reaction with 10 g/L sucrose appeared as an opaque dispersion with relatively unchanged viscosity relative to the starting point. The deactivated 100 g/L sucrose reaction was processed as described in Example 1 to generate dry polymer. The deactivated 10 g/L and 20 g/L sucrose reactions had about 5 mL withdrawn for rheology analysis and about 1 mL withdrawn for SEC analysis, before being dried in their entirety in a 40° C., −45 cmHg vacuum oven for about 3 days. HPLC, SEC, NMR and rheological analyses were performed largely as described in Example 1, except rheology was measured with cone/plate geometry. Table 7 (below) andFIG.5provide some results of these analyses. TABLE 7Profiles of Glucosyltransferase Reactions(24-hour) Containing 10 q/L CMG70 PrimerStart-of-End-of-Alpha-1,3-SucroseReactionReactionGlucanConsumptionAlpha-1,3-Glucan ProductSucroseSucroseProduct YieldRateMol % CMG70(g/L))(g/L)(Glucosyl)(g/L/h)DPwPDIIncorporation11518%0.611950.41.70161.43%22941%0.751290.31.65041.43%1132069%3.491109.61.53215.71% All the reactions were found not to go to completion (Table 7, note end-of-reaction sucrose levels), and proceeded at a significantly reduced sucrose consumption rate relative to what was described in Examples 1, 2 and 4. These results might indicate some level of inhibition of glucosyltransferase enzymatic activity as primer DoS is increased. However, all the reactions still were able to produce at least some amount of CMGPG (also note the results in Example 7 below regarding use of primer CMG110). That the −10 g/L sucrose reaction did not produce a high viscosity reaction mixture (FIG.5) probably was due to the low yield of product in this reaction (Table 7). Example 6 Glucosyltransferase-Catalyzed Synthesis of Alpha-Glucan Using Alpha-Glucan Ether as Primer and Zeta Potential Analysis of the Alpha-Glucan Product This Example describes glucosyltransferase-catalyzed synthesis of alpha-1,3-glucan graft copolymer using CMG31 (above) as a primer (i.e., CMGPG was produced). The graft copolymer products of these reactions, CMGPG, were then subject to zeta potential and particle size analyses, showing that CMGPG has significantly altered structural features as compared to non-primed alpha-1,3-glucan. A series of three reactions was performed with 108 g/L sucrose, 2 mM sodium phosphate buffer (pH 5.8), 250 U/L of a GTF, and CMG31 primer (i.e., CMG with -760 DPw and -0.31 DoS). The GTF was anS. salivariusGTF modified in its catalytic domain such that the enzyme could produce more products (fructose and alpha-1,3-glucan with about 100% alpha-1,3 linkages), and less by-products (e.g., glucose, oligosaccharides such as leucrose and DP2-7 gluco-oligosaccharides), from sucrose substrate, as compared to the enzyme's unmodified counterpart. The General Methods section describes preparation of this non-native glucosyltransferase (Table B). CMG31 primer was added in 0%, 1% and 5% concentrations in relation to the 45 g/L alpha-1,3-glucan that was expected to be produced by each reaction. Each reaction was prepared with a master stock solution that was made by dissolving 324 g sucrose and 0.82 g anhydrous sodium phosphate in deionized water to 3 L using an overhead mixer. The pH of the master stock solution was adjusted to 5.8 by addition of 0.1 N sodium hydroxide solution. 1-L aliquots were sterile-filtered from the master stock solution and charged into three 1000-mL jacketed glass reactors equipped with a PBT impeller and overhead mixer. Dry CMG31 powder was added to each reactor in the amounts of 0, 45 and 225 mg and dissolved in the media. All the reactors were connected to a recirculating water bath, which maintained the reaction temperature at 38.5° C. with agitation. A 5.0-mL aliquot was taken from each solution just before addition of 1.80 mL GTF enzyme (250 U/L final concentration) to initiate the polymerization reactions. A 5.0-mL aliquot was withdrawn at 1, 2, 3, 3.5, 4, 5 and 24 hours after starting each reaction. All these samples were placed in a heat block and heated to 80° C. for 10 minutes to deactivate the enzyme, and then analyzed by HPLC for sugars and soluble oligomers. After 24 hours, all reaction vessels were heated to 75° C. for 30 minutes to stop the reactions, by adjusting the temperature of the recirculating water baths. All the deactivated reactions were then vacuum-filtered with 2-L Büchner funnels and Whatman® Grade 541 filter paper. The filtered insoluble products were then washed and filtered with 4 L deionized water to remove any water-soluble moieties, thereby providing polymer wetcakes. Approximately 5-10 g of each wetcake was set aside for SEC analysis, about 1-2 g of each wetcake was set aside for particle size distribution (PSD) analysis, and about 15-28 g of the wetcake material was dried in an 85° C., -45 cmHg vacuum oven for about 24 hours. Wet cake was also set aside for zeta potential analysis. Wetcakes were analyzed by SEC to determine apparent molecular weight and DPw of the final products. HPLC samples were vortexed to ensure homogeneity, loaded onto a Corning SPIN-X UF6 centrifuge filter, and centrifuged for 10 minutes at 4400 rpm. The filtrate from each sample was loaded into a glass HPLC vial and analyzed using an Agilent 1200 series instrument equipped with an RI detector. Analysis was performed on two columns in parallel: BioRad AMINEX 87C (column 1) for separation of <DP3 sugars and BioRad AMINEX 42A (column 2) for separation of DP2-DP8+ oligo/polysaccharides. Linear calibration curves were constructed for sucrose (0.1-50 g/L), fructose (0.1-100 g/L), leucrose (0.1-100 g/L), and glucose (0.1-100 g/L) for quantitation on column 1. Linear calibration curves were constructed for DP2 (0.04-10 g/L), DP3 (0.04-10 g/L), DP4 (0.04-10 g/L), DP5 (0.04-10 g/L), DP6 (0.04-10 g/L), DP7 (0.04-10 g/L) and DP8+(0.25-10 g/L) for quantitation on column 2. Moieties with a DP greater than or equal to 8 (DP8+) were not well resolved on column 2. For quantification, a calibration curve was established using a lab-prepared dextran material (linear alpha-1,6 dextran with -20% alpha-1,2 branches, MW ˜17 kDa). Particle size distribution (PSD) was measured on a Malvern MASTERSIZER 2000 laser diffraction device (Malvern Instruments, Westborough, Mass.), accordingly (e.g., see ISO 13320-1:1999, Particle size analysis—Laser diffraction methods—Part 1: General principles; incorporated herein by reference). Particulars of the PSD measurement procedure were as follows: Instrument SettingsParticle Refractive Index: 0 (Fraunhofer optical model).Dispersant: clean deionized water (typically pH 5.5, in some cases adjusted to pH 3.7).Dispersant Refractive Index: 1.33.Signal averaging: 15000 snaps per measurement (15 sec).Dispersion Unit: Malvern HYDRO S general-purpose automated sample dispersion unit.Pump/Stirrer Speed: 2000 rpm. Procedure1. Fill sample reservoir with clean water.2. Start measurement: measure the MASTERSIZER's background signals.3. Gently shake vial containing sonicated or non-sonicated sample of resuspended wet cake by hand for 5 seconds to mix well and re-suspend samples.4. Add sample dropwise to HYDRO S sample reservoir until 5-10% obscuration is obtained.5. Prompt unit to continue with diffraction measurement. PSD is automatically calculated by MASTERSIZER and data is stored for analysis. PSD analysis was performed with or without sonication (50 W horn) for three minutes prior to analysis with the laser diffraction device. Zeta potential was measured with a ZETASIZER NANO ZS instrument (Malvern, Westborough, Mass.). For each product, a wet cake sample was used to prepare a dilute, see-through dispersion, which was then adjusted to pH 8 with KOH. This preparation was then sonicated for one minute using a Cole Parmer GEX 750 Ultrasonic Processor (750 Watts, 20 kHz, Amp 49%). Each sample was then placed in a cuvette having a pair of immersed platinum electrodes. An alternating electric potential was applied across the electrodes, and the motion of the particles was observed using a back-scattered laser light and an auto-correlation technique similar to dynamic light-scattering. The electrophoretic mobility of particles was measured by phase-sensitive detection, and this mobility was used to determine the zeta potential. Three consecutive 10 to 15-minute cycles of measurement were taken for each sample; these measurements were averaged together. Tables 8 and 9 (below) provide some results of the above analyses. TABLE 8Profiles of Glucosyltransferase Reactions(24-hour) Containing CMG31 PrimerAlpha-1,3-Start-of-ReactionGlucanSucroseAlpha-1,3-Glucan ProductCMG31ProductConsumptionZetaSucrosePrimerYieldRatePotential(g/L)Conc.a(Glucosyl)(g/L/h)DPwPDI(mV)1080%92.6%12.29056.51−3.251%92.5%10.38965.07−17.395%94.3%8.59152.54−40.10aPercent based on ratio of CMG31 primer mass to mass of expected alpha-1,3-glucan product (based on expected 45 g/L production by reaction). TABLE 9Particle Size Distribution (PSP) of Insoluble Alpha-1,3-GlucanProducts of Glucosyltransferase Reactions (24-hour)Alpha-1,3-Glucan ProductCMG31UnsonicatedSonicated (3 min w/horn)PrimerD10D50D90D10D50D90Conc.a(μm)(μm)(μm)(μm)(μm)(μm)0%22.26964.368170.3382.4744.6388.8071%46.97690.793150.6330.0820.2702.3725%13.72345.214218.5670.0740.1794.553aPercent based on ratio of CMG31 primer mass to mass of expected alpha-1,3-glucan product (based on expected 45 g/L production by reaction). As shown in Table 8, there was a dramatic difference in the zeta potential of non-primed alpha-1,3-glucan compared to the zeta potentials of alpha-1,3-glucan synthesized off CMG primer (i.e., CMGPG). The CMGPG product zeta potential was generally proportional to the concentration of CMG primer used in the reaction. Since the zeta potential of the dispersed CMGPG was greater (more than ±15 mV) compared to the zeta potential of dispersed non-CMG-primed product, dispersions of CMGPG should be more stable (e.g., less prone to aggregation over time). In addition, as shown in Table 9, the PSD of insoluble products was affected by the incorporation of CMG into the alpha-1,3-glucan polymer. This incorporation allowed for a reduction in CMGPG particle size to less than one micron (compared to non-primed polymer) as observed when imparting mild energy to the system in the form of sonication. These PSD data likely reflect, at least in part, the greater zeta potential of CMGPG. Example 7 Glucosyltransferase-Catalyzed Synthesis of Alpha-Glucan Using Alpha-Glucan Ether as Primer and Structural Analysis of the Alpha-Glucan Product This Example describes glucosyltransferase-catalyzed synthesis of alpha-1,3-glucan graft copolymer using CMG31 (above) or CMG of ˜1.1 DoS and ˜1220 DPw (“CMG110” herein) as a primer (i.e., CMGPG was produced). The graft copolymer products of these reactions were then subject to zeta potential analysis, showing that CMGPG has significantly altered structural features as compared to non-primed alpha-1,3-glucan. A series of ten reactions was performed with 108 g/L sucrose, 2 mM sodium phosphate buffer (pH 5.8), 250 U/L of a GTF, and a CMG primer (CMG31 or CMG110). The GTF enzyme was the same as that used in Example 6. CMG primer was added in 0%, 1% and 5% concentrations in relation to the 45 g/L alpha-1,3-glucan that was expected to be produced by each reaction at 38.5° C. and 28.0° C. Each reaction was prepared with a master stock solution that was made by dissolving 1040 g sucrose and 2.72 g anhydrous sodium phosphate in deionized water to 10 L using an overhead mixer. The pH of the master stock solution was adjusted to 5.8 by addition of 0.1 N sodium hydroxide solution. 1-L aliquots were sterile-filtered from the master stock solution and charged into ten 1000-mL jacketed glass reactors equipped with a PBT impeller and overhead mixer. Dry CMG powder was added to each reactor in the amounts of 0, 45 and 225 mg and dissolved in the media. All the reactors were connected to a recirculating water bath, which maintained the reaction temperature at 38.5° C. or 28.0° C. with agitation. A 5.0-mL aliquot was taken from each solution just before addition of 1.80 mL GTF enzyme (250 U/L final concentration) to initiate the polymerization reactions. A 5.0-mL aliquot was withdrawn at 1, 2, 3, 3.5, 4, 5 and 24 hours after starting each reaction. All these samples were placed in a heat block and heated to 80° C. for 10 minutes to deactivate the enzyme, and then analyzed by HPLC for sugars and soluble oligomers. After 24 hours, all reaction vessels were heated to 75° C. for 30 minutes to stop the reactions, by adjusting the temperature of the recirculating water baths. Preparation for, and performance of, HPLC, SEC, PSD and zeta potential analyses were as described in Example 6. Table 10 (below) provides some results of the above analyses. TABLE 10Profiles of Glucosyltransferase Reactions(24-hour) Containing CMG31 or CMG110 PrimerSucroseAlpha-1,3-Glucan ProductReaction ConditionsConsumptionZetaPrimerTemp.RatePotentialPrimerConc.a(° C.)(g/L/h)DPwPDI(mV)—0%28.06.417194.82−0.66CMG311%28.016.616232.76−1.42CMG315%38.515.39270.37−35.33CMG1101%28.017.920432.23−27.50CMG315%28.019.313372.34−33.77—0%38.532.09051.681.40CMG1105%28.018.117882.77−28.53CMG1101%38.510.512940.36−29.30CMG1105%38.53.312420.32−36.57CMG311%38.516.79631.35−16.40aPercent based on ratio of CMG primer mass to mass of expected alpha-1,3-glucan product (based on expected 45 g/L production by reaction). As shown in Table 10, there was a dramatic difference in the zeta potential of non-CMG-primed alpha-1,3-glucan compared to the zeta potentials of alpha-1,3-glucan synthesized off CMG primer (i.e., CMGPG). The zeta potentials of CMGPG products in dispersion were more greatly negative compared to the zeta potentials of dispersed non-CMG-primed alpha-1,3-glucan products. Example 8 (Comparative) Carboxymethyl Cellulose Ether Does Not Successfully Prime Glucosyltransferase-Catalyzed Synthesis of Alpha-Glucan This Example describes an attempt to use a glucosyltransferase to synthesize alpha-1,3-glucan graft copolymer using carboxymethyl cellulose (CMC) as a primer. This approach produced little, if any, CMC-primed alpha-1,3-glucan product, thereby indicating that cellulose ethers likely are not suitable for priming synthesis of alpha-1,3-glucan. CMC was tested as a primer in glucosyltransferase priming reactions having largely the same conditions as described for the CMG70-primed reactions described in Example 5. Briefly, a series of three reactions was performed with about 10, 20, or 100 g/L sucrose, 5 mM sodium phosphate buffer (pH 5.5), 10 g/L of a CMC90 primer (CMC, DP ˜1540, DoS 0.9; Sigma-Aldrich, catalog no. 419303), and 100 U/L of a GTF enzyme. The GTF enzyme was the same as that used in Example 1. Another series of reactions was performed in the same manner, but using CMC70 (CMC, DP ˜560, DoS 0.7; Sigma-Aldrich, catalog no. 419273) as primer instead of CMC90. Since the CMC90 and CMC70 primers had DoS values similar to the DoS of primer CMG70, and the reaction conditions were almost the same as those tested with CMG70, the comparison in priming reactions was quite direct. In both reaction series using either CMC90 or CMC70 primer, increasing the sucrose concentration increased both alpha-1,3-glucan yield (glucosyl basis) and initial reaction rates. The highest glucan yields (˜80%) were observed in reactions with ˜100 g/L of sucrose, and more than 90% of sucrose was consumed after 24 hours in all the reactions. However, NMR analyses (data not shown) showed no obvious signs of CMC incorporation in the final insoluble glucan products of any of the reactions. The only exception to this observation was the glucan product of the reaction comprising CMC90 and ˜100 g/L sucrose; this product seemed to possibly have a trace amount of CMC incorporation. Instead of incorporation, though, this very low level CMC90 could be due to insufficient washing of the insoluble product (to remove residual CMC90 primer) prior to NMR testing. Regardless of the reason, the detected CMC90 level was insignificant as compared to the levels of CMG70 primer detected in insoluble products in Example 5. Additionally, the six reactions with CMC primers visually appeared to be the same as reactions that produce non-primed alpha-1,3-glucan (i.e., homopolymer); there was no dramatic viscosity increase or appearance changes as observed above with successful CMG priming reactions. All this evidence suggests that CMC does not prime, or does not appreciably prime, glucosyltransferase-catalyzed alpha-1,3-glucan synthesis. | 206,872 |
11859023 | BRIEF SUMMARY Disclosed herein is method for conjugating a metal chelating agent to a functionalized dextran by reacting a chelator with an aminated dextran backbone, where the chelator comprises a one, and only one, derivatized carboxylic acid group to form a chelator-dextran complex. In certain aspects, the dextran-chelator complex is substantially free of intra- or intermolecular crosslinking. In certain aspects, the functionalized dextran is an amine dextran, an alkynyl dextran, or a thiol dextran. In exemplary implementations, the functionalized dextran is an amine dextran. In further embodiments, one and only one carboxylic acid group on the chelating agent is derivatized as a N-hydroxysuccinimide (NHS) ester. According to certain embodiments, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In certain implementations, the chelator further comprises a plurality of carboxylic acid groups, each conjugated to a protecting group. In certain embodiments of these implementations, the method further includes removing the protecting groups from the chelator-dextran complex. In certain embodiments the protecting groups are chosen from a list consisting of t-butyl esters, benzyl esters, phenyl esters, allyl esters, silyl esters, methyl esters, trifluoromethyl esters, ortho esters, oxazolines, and thioesters. In further embodiments, the protecting groups are t-butyl groups. According to certain further embodiments, the chelator is diethylenetriaminepentaacetic acid (DTPA). In certain implementations, prior to reacting the chelator with the aminated dextran backbone, the chelator is synthesized by activating a chelator with a plurality of carboxylic acid groups conjugated to protecting groups and a single active carboxylic acid groups to form a mono-NHS-chelator. In certain embodiments, the protecting groups may be t-butyl esters, benzyl esters, phenyl esters, allyl esters, silyl esters, methyl esters, trifluoromethyl esters and ortho esters, oxazolines, and/or thioesters. In exemplary embodiments of these implementations, each of the plurality of protecting groups is removed, prior to reaction with the dextran backbone. In certain embodiments, the chelator is DTPA. Further disclosed herein is a method for synthesizing a monomeric a mannosylated dextran (e.g., tilmanocept) by reacting a chelator with an aminated dextran backbone, wherein the chelator comprises a plurality of carboxylic acid groups, each conjugated to a protecting group and a single activated mono-N-hydroxysuccinimide (NHS) ester, to form a chelator-dextran complex; removing the protecting groups from each of the plurality of carboxylic acid groups; and adding a plurality of mannose moieties to the chelator dextran complex through amidate linkage to the dextran backbone. In certain embodiments, the chelator is DTPA. In certain embodiments, the protecting groups are chosen from a list consisting of: t-butyl esters, benzyl esters, phenyl esters, allyl esters, silyl esters, methyl esters, trifluoromethyl esters ortho esters, oxazolines, and thioesters. In certain embodiments, the protecting groups are t-butyl groups. Further disclosed herein is a substantially pure monomeric compound comprising a dextran backbone having one or more CD206 targeting moieties and one or more diagnostic moieties attached thereto. In certain implementations, the compound is a compound of Formula (II): whereineach X is independently H, L1-A, or L2-R; each L1 and L2 are independently linkers;each A independently comprises a detection moiety or H; each R independently comprises a CD206 targeting moiety or H; andn is an integer greater than zero; andwherein at least one R is a CD206 targeting moiety and at least one A is a diagnostic moiety or a therapeutic moiety. In certain embodiments, at least about 60% of the compound is between about 10 and about 30 kDa. In further embodiments, the dextran backbone of the compound is about 3.5 kDa. DETAILED DESCRIPTION Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more —OCH2CH2O— units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more —CO(CH2)8CO— moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted). In defining various terms, “A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents. “R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1 is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group. The term “alkyl” as used herein denotes an unbranched or branched chain, saturated, monovalent hydrocarbon residue containing 1 to 10 carbon atoms. The term “lower alkyl” denotes a straight or branched chain hydrocarbon residue containing 1 to 6 carbon atoms. “C1-10alkyl” as used herein refers to an alkyl composed of 1 to 10 carbons. Examples of alkyl groups include, but are not limited to, lower alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, t-butyl or pentyl, isopentyl, neopentyl, hexyl, heptyl, and octyl. The term “arylalkyl” or “aralkyl” as used herein denotes the radical R′R″—, wherein R′ is an aryl radical as defined herein, and R″ is an alkylene radical as defined herein with the understanding that the attachment point of the arylalkyl moiety will be on the alkylene radical. Examples of arylalkyl radicals include, but are not limited to, benzyl, phenylethyl and 3-phenylpropyl. The term “alkoxy group” as used herein means an —O-alkyl group, wherein alkyl is as defined above such as methoxy, ethoxy, n-propyloxy, i-propyloxy, n-butyloxy, i-butyloxy, t-butyloxy, pentyloxy, hexyloxy, including their isomers. “C1-10alkoxy” as used herein refers to an-O-alkyl wherein alkyl is C1-10. The term “alkylene” as used herein denotes a divalent linear or branched saturated hydrocarbon radical, having from four to six carbons inclusive, unless otherwise indicated. Examples of alkylene radicals include propylene, butylene, pentylene or hexylene. The term “cycloalkyl” as used herein denotes a saturated carbocyclic ring containing 3 to 7 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. “C3-7cycloalkyl” as used herein refers to an cycloalkyl composed of 3 to 7 carbons in the carbocyclic ring. The term “alkanol” as used herein means an HO-alkyl group, wherein alkyl is as defined above such as methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol, t-butanol, including their isomers. The term “urethane” as used herein refers to a group ROC(═O)NH— where the nitrogen atom is an alpha-amino group of an amino acid. R in the urethane is alkyl as used herein preferably tert-butyl (boc) or R is benzyl (cbz). An equivalent definition for “urethane” as used herein is an alkoxycarbonyl or benzyloxycarbonyl linked to an amino group. The term “orthoester” as used herein refers to a group RC(OR′)3 wherein R is alkyl or hydrogen and R′ is alkyl. The term “aprotic (or nonpolar) solvent” means organic solvents such as diethyl ether, ligroin, pentane, hexane, cyclohexane, heptane, octane, benzene, toluene, dioxane, tetrahydrofuran, carbon tetrachloride. The term “derivative” of a compound as used herein means a compound obtainable from the original compound by a simple chemical process. The term “acylating agent” as used herein refers to either an anhydride, acid halide or an activated derivative of an N-protected alpha amino acid. The term “anhydride” as used herein refers to compounds of the general structure RC(O)—O—C(O)R wherein R is an N-protected alpha amino. The term “acid halide” as used herein refers to compounds of the general structure RC(O)X wherein X is a halogen As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted). Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention. As used herein, “protecting group” refers to a moiety attached to a functional group to prevent an otherwise unwanted reaction of that functional group. As used herein, the term “pharmaceutically acceptable carrier” or “carrier” refers to sterile aqueous or nonaqueous solutions, colloids, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers. As used herein, the term “subject” refers to the target of administration, e.g., an animal. Thus the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the subject has been diagnosed with a need for treatment of one or more cancer disorders prior to the administering step. As used herein, the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result. For example, an object that is “substantially” enclosed would mean that the object is either completely enclosed or nearly completely enclosed. The exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained. The use of “substantially” is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a composition that is “substantially free of crosslinking” would either completely lack crosslinking, or so nearly completely lack crosslinking that the effect would be the same as if it completely lacked crosslinking. In other words, a composition that is “substantially free” of an ingredient or element may still actually contain such item as long as there is no measurable effect thereof. “Tilmanocept” refers to a non-radiolabeled active pharmaceutical ingredient (API) of the LYMPHOSEEK® diagnostic agent. Tilmanocept is a mannosylaminodextran, a subset of mannosylated dextrans. It has a dextran backbone to which a plurality of amino-terminated leashes (—O(CH2)3S(CH2)2NH2) are attached to the core glucose hydroxyl elements. In addition, mannose moieties are conjugated to amino groups of a number of the leashes, and the chelator diethylenetriamine pentaacetic acid (DTPA) may be conjugated to the amino group of other leashes not containing the mannose. Tilmanocept generally, has a dextran backbone, in which a plurality of the glucose residues comprise an amino-terminated leash: the mannose moieties are conjugated to the amino groups of the leash via an amidine linker: the chelator diethylenetriamine pentaacetic acid (DTPA) is conjugated to the amino groups of the leash via an amide linker: Tilmanocept has the chemical name dextran 3-[(2-aminoethyl)thio]propyl 17-carboxy-10,13,16-tris(carboxymethyl)-8-oxo-4-thia-7,10,13,16-tetraazaheptadec-1-yl 3-[[2-[[1-imino-2-(D-mannopyranosylthio)ethyl]amino]ethyl]thio]propyl ether complexes, and tilmanocept Tc99m has the following molecular formula: [C6H10O5]n.(C19H28N4O9S99mTc)b.(C13H24N2O5S2)c.(C5H11NS)aand contains 3-8 conjugated DTPA molecules (b); 12-20 conjugated mannose molecules (c); and 0-17 amine side chains (a) remaining free. Tilmanocept has the following general structure: Certain of the glucose moieties may have no attached amino-terminated leash. When the molecular weight of tilmanocept increases, it penetrates less efficiently into target tissues and lesions causing less of the injected dose to be available to localize to CD206 expressing macrophages. This is expected to reduce the radiographic signal that can be detected by imaging studies. Also, because high molecular weight material is excreted less efficiently into the urine, more of the injected dose is retained in circulation causing the blood pool background signal to increase. The expected result is a decreased signal to noise ratio that would limit imaging sensitivity and accuracy. In addition, because there are limited barriers between the Kupffer and mesangial cells to circulating blood coupled with reduced rates of excretion, more of the injected dose would localize to the liver and kidneys. This off-target exposure to radiopharmaceutical may further reduce the sensitivity of imaging the desired tissues due to shine through effects from the liver and kidney. The same issues would arise if non-radioactive metal ions were being targeted to CD206 expressing lesional macrophages. To remedy all of these deficiencies, what is desired is a synthesis procedure for tilmanocept and related constructs that prevents crosslinking and oligomerization. Such a procedure would create uniformly (or near uniformly) monomeric products without the high molecular weight oligomerized forms. This disclosure describes new chemical reagents and a modified synthesis protocol that enable the conjugation of chelators (i.e. DTPA and DOTA) to the amine dextran precursor of tilmanocept and related mannosylated dextran molecular constructs that nearly or completely eliminates crosslinking. There are no molecular differences between tilmanocept created by the instantly disclosed processes and the method described in the '990 Patent except that the products prepared by the new method are not crosslinked. The novelty of the products described in this disclosure is that the products so formed are not crosslinked and thus more uniform in molecular weight profile. The utility of this method is that the tilmanocept and other constructs synthesized by the disclosed methods will have improved biodistributions and pharmacokinetic attributes, thus enhanced performance characteristics. Disclosed herein is a method for conjugating a metal chelating agent to a functionalized dextran by reacting a chelator with a functionalized dextran backbone, wherein the chelator comprises one and only one derivatized carboxylic acid group, to form a chelator-dextran complex that is substantially free of intra- or intermolecular crosslinking. In certain aspects, the functionalized dextran is an amine dextran, an alkynyl dextran, or a thiol dextran. In exemplary implementations, the functionalized dextran is an amine dextran. According to certain implementations of this embodiment the one and only one derivatized carboxylic acid group is a mono-N-hydroxysuccinimide (NHS) ester. In certain embodiments, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). In certain implementations of these embodiments, the chelator further comprises a plurality of carboxylic acid groups, each conjugated to a protecting group. According to certain embodiments, the protecting groups are t-butyl esters. Other commonly employed carboxylic acid protecting groups (e.g.,benzyl esters, phenyl esters, allyl esters, silyl esters, methyl esters, trifluoromethyl esters and ortho esters, oxazolines, thioesters) may be utilized. In certain aspects, the method further comprises removing the protecting groups from the chelator-dextran complex. According to certain further embodiments, the chelator is diethylenetriaminepentaacetic acid (DTPA). In certain implementations, various combinations of functionalized dextrans, mono-derivativitized chelators, and protecting groups are possible. These combinations include but are not limited to those shown in the following schemes: In certain implementations, prior to reacting the chelator with a functionalized dextran backbone, the chelator with a plurality of carboxylic acid groups conjugated to protecting groups and a single free carboxylic acid group is functionalized to form a mono-activated chelator (e.g. one and only one of the plurality of carboxylic acid groups is activated for attachment to the dextran backbone). In exemplary aspects, the mono-activated chelator is a mono-NHS-chelator. In certain exemplary implementations, each of the plurality of protecting groups is removed, prior to reaction with the dextran backbone. Further disclosed herein is a method for synthesizing a monomeric tilmanocept by reacting a chelator with an aminated dextran backbone, wherein the chelator comprises a plurality of carboxylic acid groups, each conjugated to a protecting group and a single activated mono-N-hydroxysuccinimide (NHS) ester, to form a chelator-dextran complex; the protecting groups from each of the plurality of carboxylic acid groups; and adding a plurality of mannose moieties to the chelator dextran complex through amidate linkage to the dextran backbone. In exemplary implementations, the chelator is DTPA. In further implementations, the protecting groups are t-butyl groups. Further disclosed herein is a substantially pure monomeric compound comprising a compound of Formula (I): whereineach X is independently H, L1-A, or L2-R;each L1 and L2 are independently linkers;each A independently comprises a therapeutic agent or a detection moiety or H;each R independently comprises a mannose-binding C-type lectin receptor targeting moiety or H;and n is an integer greater than zero; andwherein at least one R comprises a mannose-binding C-type lectin receptor targeting moiety selected from the group consisting of mannose, fucose, and N-acetylglucosamine and at least one A comprises a therapeutic agent. Further disclosed herein is a substantially pure monomeric compound comprising a dextran backbone having one or more CD206 targeting moieties and one or more diagnostic moieties or therapeutic moiety attached thereto. In certain aspects, the compound is a compound of Formula (II): whereineach X is independently H, L1-A, or L2-R; each L1 and L2 are independently linkers;each A independently comprises a detection moiety or H; each R independently comprises a CD206 targeting moiety or H; and n is an integer greater than zero; and wherein at least one R is a CD206 targeting moiety and at least one A is a diagnostic moiety or therapeutic moiety. In certain aspects, the at least one A is a gamma-emitting agent. In further aspects, at least one A is a PET agent. In yet further aspects, at least one A is an isotope. In exemplary implementations, the at least one A is selected from the group consisting99mTc,210Bi,212Bi,213Bi,214Bi,131Ba,140Ba,11C,14C,51Cr,67Ga,68Ga,153Gd,88Y,90Y,91Y,123I,124I,125I,131I,111In,115mIn,18F,13N,105Rh,153Sm,67Cu,64Cu,166Ho,177Lu,223Ra,62Rb,186Re and188Re,32P,33P,46Sc,47Sc,72Se,75Se,35S,89Sr,182Ta,123mTe,127Te,129Te,132Te,65Zn and89Zr,95Zr. In certain embodiments, at least about 60% of the disclosed compound is between about 10 and about 30 kDa. In certain implementations, constructs will have an Mw of about 7 kDa and will have the majority of their mases between 5-10 kDa. In certain implementations, the dextran backbone is about 5 kDa. There are many potential medical indications besides SLN identification for which mannosylated dextrans conjugated with chelators may provide clinical utility. These alternative indications encompass any disease state in which CD206 expressing cells aggregate. Examples of such indications include, but are not limited to, cancer, atherosclerosis, rheumatoid arthritis and many others. Depending on the specific indication, the metal ions chelated to the mannosylated dextran constructs may be either radioactive or not. Examples of potential radioactive metal ions that could be chelated to treat these various metal medical indications include, but are not limited to,99mTc,210Bi,212Bi,213Bi,214Bi,131Ba,140Ba,11C,14C,51Cr,67Ga,68Ga,153Gd,88Y,90Y,91Y,123I,124I,125I,131I,111In,115mIn,18F,13N,105Rh,153Sm,67Cu,64Cu,166Ho,177Lu,223Ra,62Rb,186Re and188Re,32P,33P,46Sc,47Sc,72Se,75Se,35S,89Sr,182Ta,123mTe,127Te,129Te,132Te,65Zn and89Zr,95Zr. Examples of nonradioactive metal ions that may be enable various medical indications include, but are not limited to stable isotopes of Fe, Cu, Ag, Cr, Zn, Cd, Ni, Mo, Mn, As, Sb, Bi, Ga, In, Pd, Ru and OS. Also, depending on the specific medical indication being addressed, the preferred size of the dextran backbone on which the mannosylated dextran construct is synthesized may vary from 1.0 kDa to 500 kDa. Finally, for some medical indications, a chelator other than DTPA may be preferred. A common example of an alternative chelator that may be used is 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The current disclosure describes improved synthesis pathways that avoid crosslinking for conjugating DTPA and DOTA to mannosylated dextrans. The same synthesis strategy disclosed herein may have utility for conjugating a wide variety of other chelators to mannosylated dextrans while avoiding crosslinking. Our studies have shown that the activation and addition of unprotected metal-chelators such as DTPA to amine dextran is largely responsible for the generation of these polymeric species and the molecular weight variability observed in tilmanocept batches (Step 3, Scheme 1). While strategies are available to limit the crosslinking of the amine chains on dextran, the current tilmanocept synthesis protocol for activating one of the five free carboxylic acid moieties on a given chelator is problematic, resulting in multiply activated sites. Multiply activated chelators (e.g. DTPA) can react with multiple amine groups on the amine dextran, tilmanocept precursor (Step 3 in Scheme 1) resulting in intra- and intermolecular crosslinking as shown in Scheme 2. This reaction between multiply activated chelators and the amine dextran is the most significant if not the sole source of oligomerization of tilmanocept observed in the products of the current synthesis protocol. In contrast, appending a metal-binding agent bearing a single, activated acid would ameliorate the oligomerization of amine dextran, producing a polymer product of desired Mw profile and with polydispersity comparable to the starting dextran (i.e. PDI 1.3-1.4). DTPA is not the only chelator that can be conjugated to amine dextrans to create imaging and therapeutic agents capable of delivering radioactive and non-radioactive metal ions to specific targets such as CD206 expressing cells. For some indications, other chelators, such as DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, 4b in Scheme 1) may be preferred. Furthermore, the molecular weight of the starting dextran backbone can be modified. For example, starting dextran backbones with Mw of about 3.5 kDa, 5 kDa, 10 kDa, 20 kDa, 40 kDa or larger may be preferred for various indications. In addition, the final constructs can be targeted to various receptors by replacing some or all of the mannose moieties on tilmanocept with other targeting moieties. Examples of other targeting moieties could include but are not limited to other sugars (e.g. galactose), peptides, nucleic acids, and ligands for somatostatin receptors (SSR). The current invention describes a synthesis strategy and compositions of matter for conjugating chelators to amine dextrans without undesirable crosslinking. Other attributes of the final molecular construct are possible. EXAMPLES The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of certain examples of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. What follows are the first examples describing a novel synthesis strategy for conjugating chelators to amine dextrans without undesired crosslinking. The Examples describe the conjugation of DTPA and DOTA to amine dextrans and have immediate utility for improving synthesis of tilmanocept and related products by enabling the creation of final synthesis products that are predominately monomeric and not oligomerized or crosslinked. Example 1: Uniformly Defined Molecular Weight DTPA and Mannose Derivatized 10 kDa Dextran, (Tilmanocept) During the manufacture of tilmanocept, DTPA is activated as a mixed anhydride using isobutylchloroformate (IBCF) at low temperature for condensation with amino groups on amine dextran (Scheme 1). There are 5 carboxylic acid groups on a DTPA molecule, which can react independently with IBCF to become activated. Thus, any number or all (0-5) of these carboxylic acid groups may become activated during this activation process. When two or more carboxylic acid groups become activated, crosslinking as shown in Scheme 2 is possible, resulting in a range of oligomeric species of increased molecular weight. Intramolecular cros slinking can also occur, which may negatively impact the ability of the molecule to bind avidly to CD206. While varying the molar ratio of IBCF to DTPA in the reaction can alter the portion of DTPA molecules with two or more activated carboxylic acid groups, it is important to note that there is no molar ratio of IBCF to DTPA that does not result in a portion of the DTPA molecules being activated at two or more sites. This is the deficiency of the previous approaches, such as that described in the '990 Patent, that is remedied by the current invention. The starting amine-dextran 3 in this reaction is generated in two steps from commercially available 10 kDa dextran and has been previously described (Scheme 1), involving the allylation of dextran with PDI≤1.4 in aqueous sodium hydroxide (Step 1, Scheme 1) followed by radical addition of 2-aminoethanethiol (Step 2, Scheme 1). The chemical composition and molar ratios of the reagents utilized to produce amine-dextran 3 are the same regardless of the size of the starting dextran polymer. On average, 40-60% of the glucose units in this material bears an amine-terminated chain (25-37 per 10 kDa dextran) and the overall PDI is approximately equal to the starting dextran. In certain embodiments, a single carboxylic acid group on DTPA is activated as the mono-N-hydroxysuccinimide (NHS) ester prepared from commercially available diethylenetriamine-N,N,N,N″-tetra-tert-butyl acetate-N′-acetic acid (DTPA-Tetra, CAS [174267-71-1], Scheme 3) and adapted from a protocol for synthesis of the mono-NHS ester of DOTA. In this way, four of the five acid groups are protected as t-butyl esters, leaving one free carboxylic acid available for activation and addition to amine dextran. The transformation to NHS ester is readily achieved using numerous common protocols, solvents and reagents. However, activation in dry dichloromethane with a slight excess of NHS, triethylamine (TEA) and N,N′-dicyclohexylcarbodiimide (DCC) at room temperature provides the desired product with minimal effort, only requiring filtration of the urea by-product and concentration in vacuo. The fully-protected mono-NHS DTPA 6 may be directly added to the dextran backbone forming 4′ followed by removal of the t-butyl protecting groups (Route A, Scheme 3), or deprotected and added to amine dextran 3 as the tetra free acid 7 (Route B, Scheme 3). In route A, the protected DTPA mono-NHS ester 6 (0.5 M in DMF) is added portion wise to a 3:2 0.1 M sodium bicarbonate-carbonate pH 8.6 buffer/DMF solution of amine dextran 3. Reaction progress is determined by monitoring changes in the amine concentration. At the desired loading of DTPA to the backbone (ave. 3-8 per 10 kDa dextran chain), the material is isolated by UF concentration using a 3 kD molecular weight cut-off (MWCO) membrane and lyophilized. The t-butyl protecting groups of the dextran-bound DTPAs are subsequently removed in DMSO-TFA or 85% phosphoric acid solution followed by neutralization, UF concentration and lyophilization. Although this is a feasible route to DTPA dextran 4a, it is advisable to avoid exposure of the dextran to the highly acidic conditions utilized for removal of the t-butyl groups on 4′. In route B, addition of the mono-NHS tetra acid 7 to amine dextran requires t-butyl deprotection of 6 as a first step in anhydrous DCM/TFA, concentration and precipitation from methanol/diethyl ether. The resulting off-white product is added as a solid to a 0.1 M sodium bicarbonate-carbonate pH 8.6 buffer solution of amine dextran 3 and isolated as described above. In route B, addition of the mono-NHS tetra acid 7 to amine dextran requires t-butyl deprotection of 6 as a first step in anhydrous DCM/TFA, concentration and precipitation from methanol/diethyl ether. The resulting off-white product is added as a solid to a 0.1 M sodium bicarbonate-carbonate pH 8.6 buffer solution of amine dextran 3 and isolated as described above. Preparation of mono-NHS DTPA 7: 150 mg (0.24 mmol) of diethylenetriamine-N,N,N,N″-tetra-tert-butyl acetate-N′-acetic acid (DTPA-Tetra, CAS [174267-71-1]) is charged as a powder into a dry flask and dissolved in 6.9 ml of anhydrous dichloromethane under inert atmosphere. 31 mg (0.27 mmol, 1.1 equivalent) of N-hydroxysuccinimide is added to the flask followed by 55 mg (0.27 mmol, 1.1 equivalent) of N,N′-dicyclohexylcarbodiimide and 74 ul (0.53 mmol, 2.2 equivalents) of triethylamine. The solution is stirred at room temperature under inert atmosphere for 15-20 hours. The insoluble dicyclohexylurea by-product is filtered from the mixture and the clear filtrate solution cooled on an ice-bath. 2 volumes of dry TFA is slowly added to the chilled NHS-activated DTPA-Tetra 6 solution and stirring continued under inert gas for 4 hours. The solvents are removed via vacuum concentration at room temperature followed by drying on hi-vacuum for 15 hours resulting in crude 7 as a glassine solid. After dissolving briefly in methanol, 7 is isolated as an off-white powder after filtration by precipitation with diethyl ether. Preparation of DTPA Dextran 4a: 150 mg of amine dextran 3 is solubilized in 0.1M sodium carbonate-bicarbonate pH 8.6 buffer at 25 mg/ml using brief sonication and stirring at ambient temperature for 30 minutes. A sample of the starting dextran solution is removed as a reference of reaction progress. Solid mono-NHS DTPA 7 is charged to the amine dextran solution in portions while frequently monitoring the change in amine content versus the starting solution by fluorescamine assay (CAS [38183-12-9]). Using an ethanolamine standard curve, the approximate DTPA loading is determined by the percentage of remaining amine and the known average number of amine-terminated chains on the starting dextran. When the desired DTPA loading is achieved (typically 3-8), the reaction is concentrated with 0.1M aqueous sodium carbonate via 3 kDa MWCO ultrafiltration to remove free DTPA followed by concentration with purified water until the filtrate runs a neutral pH. The retentate product solution is frozen and lyophilized providing an off-white foam. The total DTPA in 4a is determined by spectrophotometric iron-chelation assay at 380 nm and free (unbound) DTPA by HPLC. 151 mg, 4.9 DTPA per dextran chain. Example 2: Uniformly Defined Molecular Weight DOTA and Mannose Derivatized 3.5 and 10 kDa Dextrans The synthesis of the DOTA mimetic of dextrans, functionalized with DOTA rather than DTPA diverges from tilmanocept at the point of appending the metal-chelating agent via an amide bond to an amine-dextran backbone (Scheme 4). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester, DOTA-NHS, CAS [170908-81-3], the commercially available fully deprotected mono-NHS ester of DOTA is added as a solid to a 0.1 M sodium bicarbonate-carbonate pH 8.6 buffer solution of amine dextran. Reaction progress is determined by changes in amine concentration. At the desired loading of DOTA to the backbone the material is isolated by UF concentration using a 3 kD MWCO membrane and lyophilized. Preparation of DOTA Dextran 4b on 10 kDa dextran backbone: 150 mg of amine dextran 3 is solubilized in 0.1M sodium carbonate-bicarbonate pH 8.6 buffer at 25 mg/ml using brief sonication and stirring at ambient temperature for 30 minutes. A sample of the starting dextran solution is removed as a reference of reaction progress. Solid DOTA-NHS is charged to the amine dextran solution in portions while frequently monitoring the change in amine content versus the starting solution by fluorescamine assay (CAS [38183-12-9]). Using an ethanolamine standard curve, the approximate DOTA loading is determined by the percentage of remaining amine and the known average number of amine-terminated chains on the starting dextran. When the target DOTA loading is achieved (typically 3-8), the reaction is concentrated with 0.1M aqueous sodium carbonate via 3 kDa MWCO ultrafiltration to remove free DOTA followed by concentration with purified water until the filtrate runs a neutral pH. The retentate product solution is frozen and lyophilized providing an off-white foam. The total DOTA in 4b is determined by spectrophotometric iron-chelation assay at 380 nm and free (unbound) DOTA by HPLC. 153 mg, 7.0 DOTA per dextran chain. Preparation of DOTA Dextran 4b on 3.5 kDa dextran backbone: 150 mg of amine dextran 3 is solubilized in 0.1M sodium carbonate-bicarbonate pH 8.6 buffer at 25 mg/ml using brief sonication and stirring at ambient temperature for 30 minutes. Solid DOTA-NHS (48 mg, 0.063 mmol) is charged to the amine dextran solution and allowed to stir at room temperature for 12 hours. The reaction is washed 3 times by diluting in 0.1M aqueous sodium carbonate and concentrating via 3 kDa MWCO ultrafiltration to remove free DOTA followed by concentration with purified water until the filtrate runs a neutral pH. The retentate product solution is frozen and lyophilized providing an off-white foam. The total DOTA in 4b is determined by spectrophotometric iron-chelation assay at 380 nm and free (unbound) DOTA by HPLC. 93 mg, 1.9 DOTA per dextran chain. The final step for the synthetic preparation of DTPA-mannose dextran (tilmanocept 5a) and its DOTA counterpart 5b in examples 1 and 2 involve the addition of mannose to the dextran chain via amidate linkages which does not increase polydispersity and the procedure has been previously described (Scheme 1). Analysis of Uniformly Defined Molecular Weight DTPA and DOTA Mannose Dextrans 5a/5b: Dextran constructs 5a and 5b prepared in examples 1 and 2 were fractionated on a series of MWCO Amicon® Ultra-15 centrifugal filters to obtain a semi-quantitative Mw distribution profile. In each study, approximately 100 mg of lyophilized tilmanocept product was solubilized in 12 ml of purified water and loaded onto the top of a pre-washed 100 kDa MWCO spin-filter. The material was centrifuged at 3250×g in an Eppendorf A-4-62 rotor for 15-40 minutes as minimally required for a final retentate volume of 0.5 ml above the membrane. The fall-through (filtrate) was transferred to a vial and 12 ml of purified water added to the retentate on the top of the spin-filter. The centrifugation was repeated a second and third time, retaining the filtrate in 3 separate vials. The retentate that had not passed through the membrane was transferred to a 4th vial quantitatively using purified water rinses. The vials were then frozen and lyophilized to determine the amount of material in the fall-through vials and retentate. The lyophilized fall-through material was subsequently solubilized in 12 of purified water and transferred to the top of a pre-washed 50 kDa MWCO filter and the centrifugation procedure repeated as above. In this way, a known amount of construct is fractionated into retentate pools of decreasing MW by passing through a 100, 50, 30, 10 and 3 kDa MWCO centrifugal filters. FIGS.1a-cshow the series of collected retentates and final fall-through for tilmanocept 5a prepared using the DTPA penta-acid reagent for addition to amine dextran (FIG.1a) and DTPA-tilmanocept and the DOTA derivative 5b prepared using the mono-activated NHS ester of the chelating agent as described in this disclosure (FIG.1bandFIG.1c). The material inFIG.1adepicts a close to ideal case and was selected as an example of very low polydispersity after activation of unprotected DTPA with IBCF, where higher levels of oligomeric material above 50 kDa is typically observed. Although this 5a batch was successfully prepared with low PDI, the Mw is skewed higher than calculated. In comparison, the constructs synthesized in Examples 1 and 2 of this disclosure utilizing mono-activated DTPA/DOTA (FIGS.1aand1b) are much less disperse, with over 60% of the retentate collected bracketing the calculated/targeted Mw range. This study shows that the new method for appending metal-chelators to amine dextran is vastly superior to the current tilmanocept processing step 3 (Scheme 1), delivering a uniform Mw distribution that is only dependent on the inherent polydispersity of the starting dextran, and not the efficiency of chemical addition of the chelating agent. Example 3 A mannosylated dextran construct carrying a DOTA chelator was synthesized according to the presently disclosed methods starting with a 3.5 kDa dextran backbone. The final construct had a calculated Mw of 8.7 kDa. This construct was labeled with [68]Ga. Tilmanocept (10 kDa dextran backbone carrying a DTPA chelator) was prepared by the method described in the 990 patent. This construct had an observed Mw of 37.5 kDa and a calculated Mw of ≈20 kDa indicating that a portion of the molecules were crosslinked species. Tilmanocept was labeled with [99m]Tc. These constructs were injected intravenously (IV) into Babl/c mice that had been implanted with CT27 syngeneic tumors. Syngeneic tumors contain numerous CD206+ tumor associated macrophages (TAMs). In addition, CD206+ macrophages occur normally in most tissues of the body including the large intestine. The liver contains large numbers of macrophage-like CD206+ Kupffer cells which are exposed directly to the blood flow providing the injected mannosylated dextrans with unobstructed access to the CD206 receptors expressed by these cells. In most tissues and in tumors, mannosylated dextrans must exit the blood flow and penetrate into the tissues or tumors to encounter CD206 expressing cells (mostly macrophages), Four animals were injected with each labeled construct. Animals were imaged by PET/CT ([68]Ga) or SPECT/CT ([99m]Tc] approximately 1 hour after injection. Standard image analyses determined the amount of the injected dose that localized to each organ or tumor that was then expressed as the percent of the injected dose per gram of tissue (% ID/g) that had localized to the respective organ or tumor as shown inFIG.2. InFIG.2, the % ID/g that had localized to the liver was nearly the same for both constructs. This was expected because there are no barriers separating the mannosylated dextrans from CD206 on Kupffer cells. However, for the large intestine and the CT26 tumors, the mannosylated dextrans had to exit the blood flow and penetrate the respective tissues in order to encounter a CD206+ macrophage and localize. As shown inFIG.2, the non-crosslinked DOTA construct (Mw 8.7 kDa) had significantly greater localization (≈3×) than the crosslinked construct with a Mw of 37.5 kDa.FIG.2shows the percent of injected dose per gram (% ID/g) localization of mannosylated dextrans in Balb/c mice with CT26 syngeneic tumors. These tumors contain CD206+ tumor associated macrophages (TAMs). A: a non-crosslinked mannosylated dextran construct with a DOTA chelator built on 3.5 kDa dextran backbone and labeled with [68]Ga (calculated Mw=8.7 kDa). B: Tilmanocept (10 kDa dextran backbone) with a DTPA chelator labeled with [99m]Tc (measured Mw=37.5 kDa). | 50,008 |
11859024 | DEFINITIONS To define more clearly the terms used herein, the following definitions are provided. Unless otherwise indicated, the following definitions are applicable to this disclosure. If a term is used in this disclosure but is not specifically defined herein, the definition from the IUPAC Compendium of Chemical Terminology, 2nd Ed (1997), can be applied, as long as that definition does not conflict with any other disclosure or definition applied herein, or render indefinite or non-enabled any claim to which that definition is applied. To the extent that any definition or usage provided by any document incorporated herein by reference conflicts with the definition or usage provided herein, the definition or usage provided herein controls. Herein, features of the subject matter are described such that, within particular aspects, a combination of different features can be envisioned. For each and every aspect and/or feature disclosed herein, all combinations that do not detrimentally affect the designs, compositions, processes, and/or methods described herein are contemplated with or without explicit description of the particular combination. Additionally, unless explicitly recited otherwise, any aspect and/or feature disclosed herein can be combined to describe inventive features consistent with the present disclosure. While compositions and methods are described herein in terms of “comprising” various components or steps, the compositions and methods also can “consist essentially of” or “consist of” the various components or steps, unless stated otherwise. For example, a catalyst composition consistent with aspects of the present invention can comprise; alternatively, can consist essentially of; or alternatively, can consist of; catalyst component I, catalyst component II, an activator, and a co-catalyst. The terms “a,” “an,” “the,” etc., are intended to include plural alternatives, e.g., at least one, unless otherwise specified. For instance, the disclosure of “an activator-support” or “a metallocene compound” is meant to encompass one, or mixtures or combinations of more than one, activator-support or metallocene compound, respectively, unless otherwise specified. Generally, groups of elements are indicated using the numbering scheme indicated in the version of the periodic table of elements published inChemical and Engineering News,63(5), 27, 1985. In some instances, a group of elements can be indicated using a common name assigned to the group; for example, alkali metals for Group 1 elements, alkaline earth metals for Group 2 elements, transition metals for Group 3-12 elements, and halogens or halides for Group 17 elements. For any particular compound disclosed herein, the general structure or name presented is also intended to encompass all structural isomers, conformational isomers, and stereoisomers that can arise from a particular set of substituents, unless indicated otherwise. Thus, a general reference to a compound includes all structural isomers unless explicitly indicated otherwise; e.g., a general reference to pentane includes n-pentane, 2-methyl-butane, and 2,2-dimethylpropane, while a general reference to a butyl group includes an n-butyl group, a sec-butyl group, an iso-butyl group, and a tert-butyl group. Additionally, the reference to a general structure or name encompasses all enantiomers, diastereomers, and other optical isomers whether in enantiomeric or racemic forms, as well as mixtures of stereoisomers, as the context permits or requires. For any particular formula or name that is presented, any general formula or name presented also encompasses all conformational isomers, regioisomers, and stereoisomers that can arise from a particular set of substituents. The term “substituted” when used to describe a group, for example, when referring to a substituted analog of a particular group, is intended to describe any non-hydrogen moiety that formally replaces a hydrogen in that group, and is intended to be non-limiting. A group or groups can also be referred to herein as “unsubstituted” or by equivalent terms such as “non-substituted,” which refers to the original group in which a non-hydrogen moiety does not replace a hydrogen within that group. Unless otherwise specified, “substituted” is intended to be non-limiting and include inorganic substituents or organic substituents as understood by one of ordinary skill in the art. The term “hydrocarbon” whenever used in this specification and claims refers to a compound containing only carbon and hydrogen. Other identifiers can be utilized to indicate the presence of particular groups in the hydrocarbon (e.g., halogenated hydrocarbon indicates the presence of one or more halogen atoms replacing an equivalent number of hydrogen atoms in the hydrocarbon). The term “hydrocarbyl group” is used herein in accordance with the definition specified by IUPAC: a univalent group formed by removing a hydrogen atom from a hydrocarbon (that is, a group containing only carbon and hydrogen). Non-limiting examples of hydrocarbyl groups include alkyl, alkenyl, aryl, and aralkyl groups, amongst other groups. The term “polymer” is used herein generically to include olefin homopolymers, copolymers, terpolymers, and the like, as well as alloys and blends thereof. The term “polymer” also includes impact, block, graft, random, and alternating copolymers. A copolymer is derived from an olefin monomer and one olefin comonomer, while a terpolymer is derived from an olefin monomer and two olefin comonomers. Accordingly, “polymer” encompasses copolymers and terpolymers derived from any olefin monomer and comonomer(s) disclosed herein. Similarly, the scope of the term “polymerization” includes homopolymerization, copolymerization, and terpolymerization. Therefore, an ethylene polymer includes ethylene homopolymers, ethylene copolymers (e.g., ethylene/α-olefin copolymers), ethylene terpolymers, and the like, as well as blends or mixtures thereof. Thus, an ethylene polymer encompasses polymers often referred to in the art as LLDPE (linear low density polyethylene) and HDPE (high density polyethylene). As an example, an olefin copolymer, such as an ethylene copolymer, can be derived from ethylene and a comonomer, such as 1-butene, 1-hexene, or 1-octene. If the monomer and comonomer were ethylene and 1-hexene, respectively, the resulting polymer can be categorized as an ethylene/1-hexene copolymer. The term “polymer” also includes all possible geometrical configurations, unless stated otherwise, and such configurations can include isotactic, syndiotactic, and random symmetries. Moreover, unless stated otherwise, the term “polymer” also is meant to include all molecular weight polymers, and is inclusive of lower molecular weight polymers. The term “co-catalyst” is used generally herein to refer to compounds such as aluminoxane compounds, organoboron or organoborate compounds, ionizing ionic compounds, organoaluminum compounds, organozinc compounds, organomagnesium compounds, organolithium compounds, and the like, that can constitute one component of a catalyst composition, when used, for example, in addition to an activator-support. The term “co-catalyst” is used regardless of the actual function of the compound or any chemical mechanism by which the compound may operate. The terms “chemically-treated solid oxide,” “treated solid oxide compound,” and the like, are used herein to indicate a solid, inorganic oxide of relatively high porosity, which can exhibit Lewis acidic or Brønsted acidic behavior, and which has been treated with an electron-withdrawing component, typically an anion, and which is calcined. The electron-withdrawing component is typically an electron-withdrawing anion source compound. Thus, the chemically-treated solid oxide can comprise a calcined contact product of at least one solid oxide with at least one electron-withdrawing anion source compound. Typically, the chemically-treated solid oxide comprises at least one acidic solid oxide compound. The “activator-support” of the present invention can be a chemically-treated solid oxide. The terms “support” and “activator-support” are not used to imply these components are inert, and such components should not be construed as an inert component of the catalyst composition. The term “activator,” as used herein, refers generally to a substance that is capable of converting a metallocene component into a catalyst that can polymerize olefins, or converting a contact product of a metallocene component and a component that provides an activatable ligand (e.g., an alkyl, a hydride) to the metallocene, when the metallocene compound does not already comprise such a ligand, into a catalyst that can polymerize olefins. This term is used regardless of the actual activating mechanism. Illustrative activators include activator-supports, aluminoxanes, organoboron or organoborate compounds, ionizing ionic compounds, and the like. Aluminoxanes, organoboron or organoborate compounds, and ionizing ionic compounds generally are referred to as activators if used in a catalyst composition in which an activator-support is not present. If the catalyst composition contains an activator-support, then the aluminoxane, organoboron or organoborate, and ionizing ionic materials are typically referred to as co-catalysts. The term “metallocene” as used herein describes compounds comprising at least one η1to η5-cycloalkadienyl-type moiety, wherein η1to η5-cycloalkadienyl moieties include cyclopentadienyl ligands, indenyl ligands, fluorenyl ligands, and the like, including partially saturated or substituted derivatives or analogs of any of these. Possible substituents on these ligands can include H, therefore this invention comprises ligands such as tetrahydroindenyl, tetrahydrofluorenyl, octahydrofluorenyl, partially saturated indenyl, partially saturated fluorenyl, substituted partially saturated indenyl, substituted partially saturated fluorenyl, and the like. In some contexts, the metallocene is referred to simply as the “catalyst,” in much the same way the term “co-catalyst” is used herein to refer to, for example, an organoaluminum compound. The terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, do not depend upon the actual product or composition resulting from the contact or reaction of the initial components of the disclosed or claimed catalyst composition/mixture/system, the nature of the active catalytic site, or the fate of the co-catalyst, catalyst component I, catalyst component II, or the activator (e.g., activator-support), after combining these components. Therefore, the terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, encompass the initial starting components of the composition, as well as whatever product(s) may result from contacting these initial starting components, and this is inclusive of both heterogeneous and homogenous catalyst systems or compositions. The terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, can be used interchangeably throughout this disclosure. The term “contact product” is used herein to describe compositions wherein the components are contacted together in any order, in any manner, and for any length of time, unless otherwise specified. For example, the components can be contacted by blending or mixing. Further, contacting of any component can occur in the presence or absence of any other component of the compositions described herein. Combining additional materials or components can be done by any suitable method. Further, the term “contact product” includes mixtures, blends, solutions, slurries, reaction products, and the like, or combinations thereof. Although “contact product” can include reaction products, it is not required for the respective components to react with one another. Similarly, the term “contacting” is used herein to refer to materials which can be blended, mixed, slurried, dissolved, reacted, treated, or otherwise combined in some other manner. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the typical methods, devices, and materials are herein described. All publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described invention. Several types of ranges are disclosed in the present invention. When a range of any type is disclosed or claimed, the intent is to disclose or claim individually each possible number that such a range could reasonably encompass, including end points of the range as well as any sub-ranges and combinations of sub-ranges encompassed therein. For example, when a chemical moiety having a certain number of carbon atoms is disclosed or claimed, the intent is to disclose or claim individually every possible number that such a range could encompass, consistent with the disclosure herein. For example, the disclosure that a moiety is a C1to C18hydrocarbyl group, or in alternative language, a hydrocarbyl group having from 1 to 18 carbon atoms, as used herein, refers to a moiety that can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbon atoms, as well as any range between these two numbers (for example, a C1to C8hydrocarbyl group), and also including any combination of ranges between these two numbers (for example, a C2to C4and a C12to C16hydrocarbyl group). Similarly, another representative example follows for the ratio of Mw/Mn of an ethylene polymer consistent with aspects of this invention. By a disclosure that the ratio of Mw/Mn can be in a range from about 5 to about 15, the intent is to recite that the ratio of Mw/Mn can be any ratio in the range and, for example, can be equal to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. Additionally, the ratio of Mw/Mn can be within any range from about 5 to about 15 (for example, from about 6 to about 10), and this also includes any combination of ranges between about 5 and about 15 (for example, the Mw/Mn ratio can be in a range from about 6 to about 9, or from about 11 to about 14). Further, in all instances, where “about” a particular value is disclosed, then that value itself is disclosed. Thus, the disclosure that the ratio of Mw/Mn can be from about 5 to about 15 also discloses a ratio of Mw/Mn from 5 to 15 (for example, from 6 to 10), and this also includes any combination of ranges between 5 and 15 (for example, the Mw/Mn ratio can be in a range from 6 to 9, or from 11 to 14). Likewise, all other ranges disclosed herein should be interpreted in a manner similar to these examples. The term “about” means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement errors, and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about,” the claims include equivalents to the quantities. The term “about” can mean within 10% of the reported numerical value, preferably within 5% of the reported numerical value. DETAILED DESCRIPTION OF THE INVENTION The present invention is directed generally to dual metallocene ethylene-based polymers having similar processability to chromium-based polymers, but with improved ESCR and toughness properties. Articles produced from these ethylene-based polymers can include blow molded products, such as blow molded bottles. Generally, metallocene-derived ethylene-based polymers with long chain branches have those long chain branches concentrated in the high molecular weight fraction of the polymer. Advantageously, the ethylene polymers disclosed herein have substantially no long chain branching in the high molecular weight fraction of the polymer; instead, significant amounts of long chain branching are present in lower molecular weight fractions of the polymer. These ethylene polymers can be produced, for example, with a dual metallocene catalyst system in a single reactor. It was found that using a first metallocene catalyst that preferentially produces lower molecular weight polyethylene with relatively high LCB content in combination with a second metallocene catalyst that preferentially produces higher molecular weight, essentially linear polyethylene can result in the unique combination of polymer properties described herein. Ethylene Polymers Generally, the polymers disclosed herein are ethylene-based polymers, or ethylene polymers, encompassing homopolymers of ethylene as well as copolymers, terpolymers, etc., of ethylene and at least one olefin comonomer. Comonomers that can be copolymerized with ethylene often can have from 3 to 20 carbon atoms in their molecular chain. For example, typical comonomers can include, but are not limited to, propylene, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, and the like, or combinations thereof. In an aspect, the olefin comonomer can comprise a C3-C18olefin; alternatively, the olefin comonomer can comprise a C3-C10olefin; alternatively, the olefin comonomer can comprise a C4-C10olefin; alternatively, the olefin comonomer can comprise a C3-C10α-olefin; alternatively, the olefin comonomer can comprise a C4-C10α-olefin; alternatively, the olefin comonomer can comprise 1-butene, 1-hexene, 1-octene, or any combination thereof, or alternatively, the comonomer can comprise 1-hexene. Typically, the amount of the comonomer, based on the total weight of monomer (ethylene) and comonomer, can be in a range from about 0.01 to about 20 wt. %, from about 0.1 to about 10 wt. %, from about 0.5 to about 15 wt. %, from about 0.5 to about 8 wt. %, or from about 1 to about 15 wt. %. In one aspect, the ethylene polymer of this invention can comprise an ethylene/α-olefin copolymer, while in another aspect, the ethylene polymer can comprise an ethylene homopolymer, and in yet another aspect, the ethylene polymer of this invention can comprise an ethylene/α-olefin copolymer and an ethylene homopolymer. For example, the ethylene polymer can comprise an ethylene/1-butene copolymer, an ethylene/1-hexene copolymer, an ethylene/1-octene copolymer, an ethylene homopolymer, or any combination thereof, alternatively, an ethylene/1-butene copolymer, an ethylene/1-hexene copolymer, an ethylene/1-octene copolymer, or any combination thereof, or alternatively, an ethylene/1-hexene copolymer. An illustrative and non-limiting example of an ethylene polymer (e.g., comprising an ethylene copolymer) of the present invention can have a melt index of less than or equal to about 1 g/10 min, a density in a range from about 0.94 to about 0.965 g/cm3, a Mw in a range from about 100,000 to about 250,000 g/mol, a relaxation time from about 0.5 to about 3 sec, and an average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 300,000 to 900,000 g/mol that is greater (for instance, at least 50% greater, or at least 75% greater, or at least 100% greater, or at least 150% greater, or at least 200% greater) than that in a molecular weight range of 1,000,000 to 2,000,000 g/mol. Another illustrative and non-limiting example of an ethylene polymer (e.g., comprising an ethylene copolymer) of the present invention can have a melt index of less than or equal to about 1 g/10 min, a density in a range from about 0.94 to about 0.965 g/cm3, a Mw in a range from about 100,000 to about 250,000 g/mol, a relaxation time from about 0.5 to about 3 sec, an average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 1,000,000 to 2,000,000 g/mol of less than or equal to about 5, and a maximum ratio of ηE/3η at an extensional rate of 0.1 sec in a range from about 1.2 to about 10. These ethylene polymers also can have any of the polymer properties listed below and in any combination, unless indicated otherwise. The densities of ethylene-based polymers disclosed herein often are greater than or equal to about 0.94 g/cm3, and less than or equal to about 0.965 g/cm3. Yet, in particular aspects, the density can be in a range from about 0.942 to about 0.965 g/cm3, from about 0.94 to about 0.96 g/cm3, from about 0.95 to about 0.965 g/cm3, from about 0.955 to about 0.962 g/cm3, or from about 0.955 to about 0.96 g/cm3. Ethylene polymers described herein often can have a melt index (MI) of less than or equal to about 1 g/10 min, less than or equal to about 0.7 g/10 min, or less than or equal to about 0.5 g/10 min. In further aspects, ethylene polymers described herein can have a melt index (MI) in a range from about 0.1 to about 0.7 g/10 min, from about 0.1 to about 0.5 g/10 min, from about 0.2 to about 0.7 g/10 min, or from about 0.2 to about 0.4 g/10 min. While not being limited thereto, the ethylene polymer also can have a high load melt index (HLMI) in a range from about 10 to about 65 g/10 min; alternatively, from about 35 to about 55 g/10 min; alternatively, from about 20 to about 60 g/10 min; or alternatively, from about 40 to about 55 g/10 min. The ratio of high load melt index (HLMI) to melt index (MI), referred to as the ratio of HLMI/MI, is not particularly limited, but typically ranges from about 80 to about 220, from about 100 to about 200, from about 120 to about 170, or from about 130 to about 160. In this HLMI/MI ratio, the melt index is not equal to zero. In an aspect, ethylene polymers described herein can have a ratio of Mw/Mn, or the polydispersity index, in a range from about 5 to about 15, from about 6 to about 12, from about 6 to about 10, from about 7 to about 13, or from about 7 to about 10. Additionally or alternatively, the ethylene polymer can have a ratio of Mz/Mw in a range from about 3.5 to about 10, from about 4 to about 8, from about 4 to about 6, or from about 4.5 to about 5.5. In an aspect, ethylene polymers described herein can have a weight-average molecular weight (Mw) in a range from about 125,000 to about 250,000 g/mol, from about 100,000 to about 200,000 g/mol, from about 110,000 to about 190,000 g/mol, or from about 125,000 to about 175,000 g/mol. Additionally or alternatively, the ethylene polymer can have a number-average molecular weight (Mn) in a range from about 10,000 to about 30,000 g/mol, from about 10,000 to about 25,000 g/mol, from about 15,000 to about 25,000 g/mol, or from about 15,000 to about 20,000 g/mol. Additionally or alternatively, the ethylene polymer can have a z-average molecular weight (Mz) in a range from about 500,000 to about 2,000,000 g/mol, from about 600,000 to about 1,200,000 g/mol, from about 650,000 to about 1,000,000 g/mol, or from about 700,000 to about 900,000 g/mol. Ethylene polymers consistent with certain aspects of the invention often can have a bimodal molecular weight distribution (as determined using gel permeation chromatography (GPC) or other related analytical technique). Often, in a bimodal molecular weight distribution, there is a valley between the peaks, and the peaks can be separated or deconvoluted. Typically, a bimodal molecular weight distribution can be characterized as having an identifiable high molecular weight component (or distribution) and an identifiable low molecular weight component (or distribution). Illustrative unimodal MWD curves and bimodal MWD curves are shown in U.S. Pat. No. 8,383,754, incorporated herein by reference in its entirety. While not limited thereto, ethylene polymers described herein can have a zero-shear viscosity at 190° C. in a range from about 1×103to about 1×108Pa-sec, from about 1×104to about 1×107Pa-sec, or from about 1×104to about 1×106Pa-sec. Moreover, these ethylene polymers can have a CY-a parameter from about 0.15 to about 0.45, from about 0.2 to about 0.4, from about 0.22 to about 0.35, or from about 0.22 to about 0.32. Additionally or alternatively, these ethylene polymers can have a relatively short relaxation time given their relatively high molecular weight, with the relaxation time typically in a range from about 0.5 to about 3 sec, such as from about 0.75 to about 2.5 sec, from about 1 to about 2 sec, or from about 1 to about 1.5 sec. Additionally or alternatively, these ethylene polymers can be characterized by a viscosity at HLMI (eta @ HLMI or η@ HLMI) at 190° C. in a range from about 250 to about 800 Pa-sec, and more often, in a range from about 300 to about 750, from about 300 to about 500, from about 300 to about 450, or from about 350 to about 450 Pa-sec. Additionally or alternatively, these ethylene polymers can have a viscosity at 100 sec−1(eta @ 100 or η @ 100) at 190° C. in a range from about 750 to about 1750, from about 850 to about 1300, from about 1000 to about 1500, or from about 1000 to about 1300 Pa-sec. Additionally or alternatively, these ethylene polymers can have a ratio of η@ 0.1/η@ 100 (the viscosity at 0.1 sec−1divided by the viscosity at 100 sec−1) in a range from about 20 to about 45, from about 20 to about 35, from about 22 to about 32, or from about 25 to about 30. These rheological parameters are determined from viscosity data measured at 190° C. and using the Carreau-Yasuda (CY) empirical model as described herein. The average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the ethylene polymer in a molecular weight range of 1,000,000 to 2,000,000 g/mol can be less than or equal to about 5 (there is effectively no LCB in the high molecular weight fraction of the polymer). All average numbers of LCBs disclosed herein are number-average numbers. In some aspects, the average number of LCBs per 1,000,000 total carbon atoms of the polymer in the molecular weight range of 1,000,000 to 2,000,000 g/mol can be less than or equal to about 4, less than or equal to about 3.5, less than or equal to about 3, or less than or equal to about 1. In further aspects, the average number of LCBs in this molecular weight range can be below the detection limit. The average number of LCBs per 1,000,000 total carbon atoms of the ethylene polymer in a molecular weight range of 300,000 to 900,000 g/mol can be greater (by any amount disclosed herein, e.g., at least 50%, at least 75%, at least 100%, at least 150%, or at least 200%, and often up to 400-800%, or more) than the average number of LCBs per 1,000,000 total carbon atoms in a molecular weight range of 1,000,000 to 2,000,000 g/mol. In some aspects, the average number of LCBs per 1,000,000 total carbon atoms of the ethylene polymer in a molecular weight range of 300,000 to 900,000 g/mol can be at least 50% greater (or at least 75% greater, or at least 100% greater, or at least 150% greater, or at least 200% greater, and often up to 400-800% greater) than that in a molecular weight range of 1,000,000 to 2,000,000 g/mol. As disclosed herein, all average numbers of LCBs are number-average numbers. The average number of LCBs per 1,000,000 total carbon atoms of the ethylene polymer in the molecular weight range of 300,000 to 900,000 g/mol is not particularly limited, but often falls within a range from about 3 to about 15; alternatively, from about 4 to about 13; alternatively, from about 4 to about 10; alternatively, from about 5 to about 9; or alternatively, from about 6 to about 8. Likewise, the average number of LCBs per 1,000,000 total carbon atoms of the ethylene polymer in the molecular weight range of 400,000 to 600,000 g/mol is not particularly limited, but often falls within a range from about 4 to about 15; alternatively, from about 5 to about 14; alternatively, from about 5 to about 12; alternatively, from about 7 to about 10; or alternatively, from about 8 to about 9. In the overall polymer (using the Janzen-Colby model), the ethylene polymers typically have levels of long chain branches (LCBs) in a range from about 4 to about 20 LCBs, from about 5 to about 15 LCBs, from about 6 to about 14 LCBs, or from about 8 to about 12 LCBs, per 1,000,000 total carbon atoms. Unexpectedly, the ethylene polymers described herein can have a maximum ratio of ηE/3η at an extensional rate of 0.1 sec−1in a range from about 1.2 to about 10. For Newtonian fluids, the ratio of extensional viscosity to 3 times the shear viscosity is equal to 1, while strain hardening due to long chain branching can lead to ratios of greater than 1. In one aspect, the maximum ratio of ηE/3η at the extensional rate of 0.1 sec−1can range from about 1.2 to about 10, or from about 1.5 to about 8, while in another aspect, the maximum ratio can range from about 1.5 to about 5, or from about 1.2 to about 4, and in yet another aspect, the maximum ratio can range from about 1.2 to about 3, or from about 1.4 to about 3.5, and in still another aspect, the maximum ratio can range from about 1.4 to about 3, or from about 1.5 to about 2.5. These ratios of extensional viscosity to three times the shear viscosity are determined using a Sentmanat Extensional Rheometer (SER) at 150° C. Additionally, while not being limited thereto, the ethylene polymer can be characterized further by a maximum ratio of ηE/3η at an extensional rate of 0.03 sec−1in a range from about 1.2 to about 10; alternatively, from about 1.5 to about 8; alternatively, from about 2 to about 7; alternatively, from about 2 to about 5; alternatively, from about 2.5 to about 4.5; or alternatively, from about 3 to about 4. Moreover, the ethylene polymers (e.g., ethylene copolymers) typically can have a flat short chain branching distribution (flat SCBD; uniform comonomer distribution). A flat SCBD can be characterized by a slope of a plot of the number of short chain branches (SCBs) per 1000 total carbon atoms versus the logarithm of molecular weight of the ethylene polymer (determined via linear regression over the range from D15 to D85) that is in a range from about −0.6 to about 0.6, and/or a percentage of data points deviating from the average short chain branch content by greater than 0.5 SCBs per 1000 total carbon atoms (determined over the range from D15 to D85) that is less than or equal to about 20%, and/or a percentage of data points deviating from the average short chain branch content by greater than 1 SCB per 1000 total carbon atoms (determined over the range from D15 to D85) that is less than or equal to about 10%. Polymers having a flat or uniform SCBD are disclosed, for example, in U.S. Pat. Nos. 9,217,049 and 9,574,031, which are incorporated herein by reference in their entirety. Aspects of this invention also are directed to the performance of the ethylene polymer (e.g., an ethylene/1-hexene copolymer) on representative blow molding equipment, as described herein below. The ethylene polymers can have a cycle time from about 13 to about 20, from about 14 to about 19, from about 15 to about 18, or from about 16 to about 17 seconds; unexpectedly, these polymers can have cycle times that are substantially the same as that of comparable chromium-based resins. Additionally or alternatively, ethylene polymers described herein can have a part weight in a range from about 95 to about 115, from about 100 to about 115, from about 95 to about 110, or from about 100 to about 110 grams. Additionally or alternatively, ethylene polymers described herein can have a layflat (top) in a range from about 5.2 to about 6, from about 5 to about 5.7, or from about 5.2 to about 5.7 inches. Consistent with aspects of this disclosure, the ethylene polymers can have a “bottle” environmental stress crack resistance (ESCR) of at least 200 hours. Moreover, in some aspects, the ethylene polymers can have an ESCR of at least 250 hours, at least 300 hours, at least 400 hours, or at least 500 hours, and often can range as high as 600 to 1000 hours. The “bottle” ESCR test is typically stopped after a certain number of hours is reached, and given the long duration of the test, the upper limit of ESCR (in hours) is generally not determined. The “bottle” ESCR test is conducted in 10% Igepal at 140° F. (ASTM D2561), which is a much more stringent test than ESCR testing conducted using a 100% igepal solution. Additionally or alternatively, the ethylene polymers can have a “bent strip” (ESCR) of at least 50 hours, such as at least 60 hours, at least 75 hours, at least 85 hours, or at least 100 hours, and often can range as high as 150 to 300 hours. As above, the “bent strip” ESCR test is typically stopped after a certain number of hours is reached, and given the long duration of the test, the upper limit of ESCR (in hours) is generally not determined. The “bent strip” ESCR test is conducted in 10% Igepal at 50° C. for a 75 mil thickness (ASTM D1693). In an aspect, the ethylene polymer can be a reactor product (e.g., a single reactor product), for example, not a post-reactor blend of two polymers, for instance, having different molecular weight characteristics. As one of skill in the art would readily recognize, physical blends of two different polymer resins can be made, but this necessitates additional processing and complexity not required for a reactor product. Additionally, the ethylene polymer can further contain any suitable additive, non-limiting examples of which include an antioxidant, an acid scavenger, an antiblock additive, a slip additive, a colorant, a filler, a polymer processing aid, a UV additive, and the like, as well as any combination thereof. Moreover, the ethylene polymers can be produced with a metallocene catalyst system containing zirconium and hafnium, discussed further below. Ziegler-Natta, chromium, and titanium metallocene based catalysts systems are not required. Therefore, the ethylene polymer can contain no measurable amount of chromium or titanium (catalyst residue), i.e., less than 0.1 ppm by weight. In some aspects, the ethylene polymer can contain, independently, less than 0.08 ppm, less than 0.05 ppm, or less than 0.03 ppm, of chromium and titanium. Articles and Products Articles of manufacture can be formed from, and/or can comprise, the olefin polymers (e.g., ethylene polymers) of this invention and, accordingly, are encompassed herein. For example, articles which can comprise the polymers of this invention can include, but are not limited to, an agricultural film, an automobile part, a bottle, a container for chemicals, a drum, a fiber or fabric, a food packaging film or container, a food service article, a fuel tank, a geomembrane, a household container, a liner, a molded product, a medical device or material, an outdoor storage product (e.g., panels for walls of an outdoor shed), outdoor play equipment (e.g., kayaks, bases for basketball goals), a pipe, a sheet or tape, a toy, or a traffic barrier, and the like. Various processes can be employed to form these articles. Non-limiting examples of these processes include injection molding, blow molding, rotational molding, film extrusion, sheet extrusion, profile extrusion, thermoforming, and the like. Additionally, additives and modifiers often are added to these polymers in order to provide beneficial polymer processing or end-use product attributes. Such processes and materials are described inModern Plastics Encyclopedia, Mid-November 1995 Issue, Vol. 72, No. 12; andFilm Extrusion Manual—Process, Materials, Properties, TAPPI Press, 1992; the disclosures of which are incorporated herein by reference in their entirety. In some aspects of this invention, an article of manufacture can comprise any of olefin polymers (or ethylene polymers) described herein, and the article of manufacture can be or can comprise a blow molded product. Also contemplated herein is a method for forming or preparing an article of manufacture comprising any polymer disclosed herein. For instance, a method can comprise (i) contacting a catalyst composition with an olefin monomer (e.g., ethylene) and an optional olefin comonomer under polymerization conditions in a polymerization reactor system to produce an olefin polymer (e.g., an ethylene polymer), wherein the catalyst composition can comprise catalyst component I, catalyst component II, an activator (e.g., an activator-support comprising a solid oxide treated with an electron-withdrawing anion), and an optional co-catalyst (e.g., an organoaluminum compound); and (ii) forming an article of manufacture comprising the olefin polymer (or ethylene polymer). The forming step can comprise blending, melt processing, extruding, molding (e.g., blow molding), or thermoforming, and the like, including combinations thereof. Any suitable additive can be combined with the polymer in the melt processing step (extrusion step), such as antioxidants, acid scavengers, antiblock additives, slip additives, colorants, fillers, processing aids, UV inhibitors, and the like, as well as combinations thereof. Catalyst Systems and Polymerization Processes In accordance with aspects of the present invention, the olefin polymer (e.g., the ethylene polymer) can be produced using a dual catalyst system. In these aspects, catalyst component I can comprise any suitable single atom bridged or two atom bridged metallocene compound with two indenyl groups, or any single atom bridged or two atom bridged metallocene compound disclosed herein with two indenyl groups. Catalyst component II can comprise any suitable single atom bridged metallocene compound with a fluorenyl group and a cyclopentadienyl group, and with an alkenyl substituent on the single atom bridge and/or on the cyclopentadienyl group, or any single atom bridged metallocene compound disclosed herein with a fluorenyl group and a cyclopentadienyl group, and with an alkenyl substituent on the single atom bridge and/or on the cyclopentadienyl group. The catalyst system also can comprise any suitable activator or any activator disclosed herein, and optionally, any suitable co-catalyst or any co-catalyst disclosed herein. Referring first to catalyst component II, which can comprise a single atom bridged metallocene compound with a fluorenyl group and a cyclopentadienyl group; an alkenyl substituent can be present on the single atom bridge, or on the cyclopentadienyl group, or both. In one aspect, the fluorenyl group can be substituted, while in another aspect, the fluorenyl group can be unsubstituted. Additionally, the bridged metallocene compound of catalyst component II can contain zirconium, hafnium, or titanium, or alternatively, zirconium or hafnium. Further, the single atom bridge can be a single carbon atom or a single silicon atom, although not limited thereto. In some aspects, this bridging atom can have two substituents independently selected from H or any C1to C18hydrocarbyl group disclosed herein (e.g., one substituent, or both substituents, can be a phenyl group). The alkenyl substituent on the cyclopentadienyl group (or on the bridging atom) can be any suitable alkenyl group, such as a C3to C18alkenyl group, or a C3to C8terminal alkenyl group. Catalyst component II can comprise, in particular aspects of this invention, a bridged metallocene compound having formula (II): Within formula (II), M, Cp, RX, RY, E, and each X are independent elements of the bridged metallocene compound. Accordingly, the bridged metallocene compound having formula (II) can be described using any combination of M, Cp, RX, RY, E, and X disclosed herein. In accordance with aspects of this invention, the metal in formula (II), M, can be Ti, Zr, or Hf. In one aspect, for instance, M can be Zr or Hf, while in another aspect, M can be Ti; alternatively, M can be Zr; or alternatively, M can be Hf. Each X in formula (II) independently can be a monoanionic ligand. In some aspects, suitable monoanionic ligands can include, but are not limited to, H (hydride), BH4, a halide, a C1to C36hydrocarbyl group, a C1to C36hydrocarboxy group, a C1to C36hydrocarbylaminyl group, a C1to C36hydrocarbylsilyl group, a C1to C36hydrocarbylaminylsilyl group, —OBR12, or —OSO2R1, wherein R1is a C1to C36hydrocarbyl group. It is contemplated that each X can be either the same or a different monoanionic ligand. In addition to representative selections for each X that are disclosed herein, additional suitable hydrocarbyl groups, hydrocarboxy groups, hydrocarbylaminyl groups, hydrocarbylsilyl groups, and hydrocarbylaminylsilyl groups are disclosed, for example, in U.S. Pat. No. 9,758,600, incorporated herein by reference in its entirety. In one aspect, each X independently can be H, BH4, a halide (e.g., F, Cl, Br, etc.), a C1to C18hydrocarbyl group, a C1to C18hydrocarboxy group, a C1to C18hydrocarbylaminyl group, a C1to C18hydrocarbylsilyl group, or a C1to C18hydrocarbylaminylsilyl group. Alternatively, each X independently can be H, BH4, a halide, OBR12, or OSO2R1, wherein R1is a C1to C18hydrocarbyl group. In another aspect, each X independently can be H, BH4, a halide, a C1to C12hydrocarbyl group, a C1to C12hydrocarboxy group, a C1to C12hydrocarbylaminyl group, a C1to C12hydrocarbylsilyl group, a C1to C12hydrocarbylaminylsilyl group, OBR12, or OSO2R1, wherein R1is a C1to C12hydrocarbyl group. In another aspect, each X independently can be H, BH4, a halide, a C1to C10hydrocarbyl group, a C1to C10hydrocarboxy group, a C1to C10hydrocarbylaminyl group, a C1to C10hydrocarbylsilyl group, a C1to C10hydrocarbylaminylsilyl group, OBR12, or OSO2R1, wherein R1is a C1to C10hydrocarbyl group. In yet another aspect, each X independently can be H, BH4, a halide, a C1to C8hydrocarbyl group, a C1to C8hydrocarboxy group, a C1to C8hydrocarbylaminyl group, a C1to C8hydrocarbylsilyl group, a C1to C8hydrocarbylaminylsilyl group, OBR12, or OSO2R1, wherein R1is a C1to C8hydrocarbyl group. In still another aspect, each X independently can be a halide or a C1to C18hydrocarbyl group. For example, each X can be C1. In one aspect, each X independently can be H, BH4, a halide, or a C1to C36hydrocarbyl group, hydrocarboxy group, hydrocarbylaminyl group, hydrocarbylsilyl group, or hydrocarbylaminylsilyl group, while in another aspect, each X independently can be H, BH4, or a C1to C18hydrocarboxy group, hydrocarbylaminyl group, hydrocarbylsilyl group, or hydrocarbylaminylsilyl group. In yet another aspect, each X independently can be a halide; alternatively, a C1to C18hydrocarbyl group; alternatively, a C1to C18hydrocarboxy group; alternatively, a C1to C18hydrocarbylaminyl group; alternatively, a C1to C18hydrocarbylsilyl group; or alternatively, a C1to C18hydrocarbylaminylsilyl group. In still another aspect, each X can be H; alternatively, F; alternatively, C1; alternatively, Br; alternatively, I; alternatively, BH4; alternatively, a C1to C18hydrocarbyl group; alternatively, a C1to C18hydrocarboxy group; alternatively, a C1to C18hydrocarbylaminyl group; alternatively, a C1to C18hydrocarbylsilyl group; or alternatively, a C1to C18hydrocarbylaminylsilyl group. Each X independently can be, in some aspects, H, a halide, methyl, phenyl, benzyl, an alkoxy, an aryloxy, acetylacetonate, formate, acetate, stearate, oleate, benzoate, an alkylaminyl, a dialkylaminyl, a trihydrocarbylsilyl, or a hydrocarbylaminylsilyl; alternatively, H, a halide, methyl, phenyl, or benzyl; alternatively, an alkoxy, an aryloxy, or acetylacetonate; alternatively, an alkylaminyl or a dialkylaminyl; alternatively, a trihydrocarbylsilyl or hydrocarbylaminylsilyl; alternatively, H or a halide; alternatively, methyl, phenyl, benzyl, an alkoxy, an aryloxy, acetylacetonate, an alkylaminyl, or a dialkylaminyl; alternatively, H; alternatively, a halide; alternatively, methyl; alternatively, phenyl; alternatively, benzyl; alternatively, an alkoxy; alternatively, an aryloxy; alternatively, acetylacetonate; alternatively, an alkylaminyl; alternatively, a dialkylaminyl; alternatively, a trihydrocarbylsilyl; or alternatively, a hydrocarbylaminylsilyl. In these and other aspects, the alkoxy, aryloxy, alkylaminyl, dialkylaminyl, trihydrocarbylsilyl, and hydrocarbylaminylsilyl can be a C1to C36, a C1to C18, a C1to C12, or a C1to C8alkoxy, aryloxy, alkylaminyl, dialkylaminyl, trihydrocarbylsilyl, and hydrocarbylaminylsilyl. Moreover, each X independently can be, in certain aspects, a halide or a C1to C18hydrocarbyl group; alternatively, a halide or a C1to C8hydrocarbyl group; alternatively, F, Cl, Br, I, methyl, benzyl, or phenyl; alternatively, C1, methyl, benzyl, or phenyl; alternatively, a C1to C18alkoxy, aryloxy, alkylaminyl, dialkylaminyl, trihydrocarbylsilyl, or hydrocarbylaminylsilyl group; alternatively, a C1to C8alkoxy, aryloxy, alkylaminyl, dialkylaminyl, trihydrocarbylsilyl, or hydrocarbylaminylsilyl group; or alternatively, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, phenyl, tolyl, benzyl, naphthyl, trimethylsilyl, triisopropylsilyl, triphenylsilyl, or allyldimethylsilyl. In formula (II), Cp can be a cyclopentadienyl group, optionally with an alkenyl substituent. In some aspects, Cp can contain no additional substituents, other than the alkenyl substituent. In other aspects, Cp can be further substituted with one substituent, two substituents, and so forth. If present, each substituent on Cp independently can be H, a halide, a C1to C36hydrocarbyl group, a C1to C36halogenated hydrocarbyl group, a C1to C36hydrocarboxy group, or a C1to C36hydrocarbylsilyl group. Importantly, each substituent on Cp can be either the same or a different substituent group. Moreover, each substituent can be at any position on the cyclopentadienyl ring structure that conforms with the rules of chemical valence. In general, any substituent on Cp, independently, can be H or any halide, C1to C36hydrocarbyl group, C1to C36halogenated hydrocarbyl group, C1to C36hydrocarboxy group, or C1to C36hydrocarbylsilyl group described herein. In addition to representative substituents that are disclosed herein, additional suitable hydrocarbyl groups, halogenated hydrocarbyl groups, hydrocarboxy groups, and hydrocarbylsilyl groups are disclosed, for example, in U.S. Pat. No. 9,758,600, incorporated herein by reference in its entirety. In one aspect, for example, each substituent on Cp independently can be a C1to C12hydrocarbyl group or a C1to C12hydrocarbylsilyl group. In another aspect, each substituent on Cp independently can be a C1to C8alkyl group or a C3to C8alkenyl group. In yet another aspect, each substituent on Cp independently can be H, Cl, CF3, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an ethenyl group, a propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, a phenyl group, a tolyl group, a benzyl group, a naphthyl group, a trimethylsilyl group, a triisopropylsilyl group, a triphenylsilyl group, or an allyldimethylsilyl group. Similarly, RXand RYin formula (II) independently can be H or any halide, C1to C36hydrocarbyl group, C1to C36halogenated hydrocarbyl group, C1to C36hydrocarboxy group, or C1to C36hydrocarbylsilyl group disclosed herein. In one aspect, for example, RXand RYindependently can be H or a C1to C12hydrocarbyl group. In another aspect, RXand RYindependently can be a C1to C10hydrocarbyl group or, alternatively, a C1to C6alkyl group. In yet another aspect, RXand RYindependently can be H, Cl, CF3, a methyl group, an ethyl group, a propyl group, a butyl group (e.g., t-Bu), a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an ethenyl group, a propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, a phenyl group, a tolyl group, a benzyl group, a naphthyl group, a trimethylsilyl group, a triisopropylsilyl group, a triphenylsilyl group, or an allyldimethylsilyl group, and the like. In still another aspect, RXand RYindependently can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an ethenyl group, a propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, a phenyl group, a tolyl group, or a benzyl group. Bridging group E in formula (II) can be a bridging group having the formula >EARARB, wherein EAcan be C, Si, or Ge, and RAand RBindependently can be H or a C1to C18hydrocarbyl group. In some aspects of this invention, RAand RBindependently can be a C1to C12hydrocarbyl group; alternatively, RAand RBindependently can be a C1to C8hydrocarbyl group; alternatively, RAand RBindependently can be a phenyl group, a C1to C8alkyl group, or a C3to C8alkenyl group; alternatively, RAand RBindependently can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an ethenyl group, a propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, a phenyl group, a cyclohexylphenyl group, a naphthyl group, a tolyl group, or a benzyl group; or alternatively, RAand RBindependently can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a phenyl group, or a benzyl group. In these and other aspects, RAand RBcan be either the same or different. Illustrative and non-limiting examples of bridged metallocene compounds having formula (II) and/or suitable for use as catalyst component II can include the following compounds (Me=methyl, Ph=phenyl; t-Bu=tert-butyl): and the like, as well as combinations thereof. Catalyst component II is not limited solely to the bridged metallocene compounds such as described above. Other suitable bridged metallocene compounds are disclosed in U.S. Pat. Nos. 7,026,494, 7,041,617, 7,226,886, 7,312,283, 7,517,939, and 7,619,047, which are incorporated herein by reference in their entirety. Referring now to catalyst component I, which can comprise, in particular aspects of this invention, a single atom bridged or two atom bridged (two atom chain) metallocene compound with two indenyl groups. In some aspects, the metallocene compound contains two unsubstituted indenyl groups. The bridge can be a single carbon atom; alternatively, a single silicon atom; alternatively, a two carbon atom bridge; or alternatively, a two silicon atom bridge. Independently, any bridging atom (or atoms) can have two substituents independently selected from H or a C1to C18hydrocarbyl group, or from H or a C1to C8hydrocarbyl group; alternatively, two substituents independently selected from H or a C1to C6alkyl group; or alternatively, two substituents independently selected from a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an ethenyl group, a propenyl group, a butenyl group, a pentenyl group, a hexenyl group, a heptenyl group, an octenyl group, a nonenyl group, a decenyl group, a phenyl group, a cyclohexylphenyl group, a naphthyl group, a tolyl group, or a benzyl group. The two substituents can be either the same or different. If the metallocene compound is a two carbon atom bridged metallocene compound, the bridging group can have the formula —CRCRD—CRERF—, wherein RC, RD, RE, and RFindependently can be H or any C1to C18hydrocarbyl group disclosed herein (and similarly for a two silicon atom bridge). For instance, RC, RD, RE, and RFindependently can be H or a C1to C6alkyl group, or alternatively, H or a methyl group. In other aspects, at least one indenyl group is substituted (thus, one or both indenyl groups can be substituted). As above, the bridge can be a single carbon atom, a single silicon atom, a two carbon atom bridge, or a two silicon atom bridge, and further, each bridging atom (or atoms) can have two substituents independently selected from H or a C1to C18hydrocarbyl group (e.g., a C1to C6alkyl group). Any substituent on either indenyl group also can be independently selected from H or a C1to C18hydrocarbyl group (e.g., a C1to C6alkyl group). While not limited thereto, catalyst component I typically contains zirconium. Illustrative and non-limiting examples of metallocene compounds suitable for use as catalyst component I can include the following compounds: and the like, as well a combination thereof. Catalyst component I is not limited solely to the bridged metallocene compounds such as described above. Other suitable metallocene compounds are disclosed in U.S. Pat. Nos. 8,288,487 and 8,426,538, which are incorporated herein by reference in their entirety. According to an aspect of this invention, the weight ratio of catalyst component I to catalyst component II in the catalyst composition can be in a range from about 25:1 to about 1:25, from about 10:1 to about 1:10, from about 8:1 to about 1:8, from about 5:1 to about 1:5, from about 3:1 to about 1:3; from about 2:1 to about 1:2, from about 1.5:1 to about 1:1.5, from about 1.25:1 to about 1:1.25, or from about 1.1:1 to about 1:1.1. In another aspect, catalyst component II is the minor component of the catalyst composition, and in such aspects, the weight ratio of catalyst component I to catalyst component II in the catalyst composition can be in a range from about 1:1 to about 10:1, from about 1.2:1 to about 5:1, from about 1.5:1 to about 4:1, or from about 1.5:1 to about 2.5:1. Additionally, the dual catalyst system contains an activator. For example, the catalyst system can contain an activator-support, an aluminoxane compound, an organoboron or organoborate compound, an ionizing ionic compound, and the like, or any combination thereof. The catalyst system can contain one or more than one activator. In one aspect, the catalyst system can comprise an aluminoxane compound, an organoboron or organoborate compound, an ionizing ionic compound, and the like, or a combination thereof. Examples of such activators are disclosed in, for instance, U.S. Pat. Nos. 3,242,099, 4,794,096, 4,808,561, 5,576,259, 5,807,938, 5,919,983, and 8,114,946, the disclosures of which are incorporated herein by reference in their entirety. In another aspect, the catalyst system can comprise an aluminoxane compound. In yet another aspect, the catalyst system can comprise an organoboron or organoborate compound. In still another aspect, the catalyst system can comprise an ionizing ionic compound. In other aspects, the catalyst system can comprise an activator-support, for example, an activator-support comprising a solid oxide treated with an electron-withdrawing anion. Examples of such materials are disclosed in, for instance, U.S. Pat. Nos. 7,294,599, 7,601,665, 7,884,163, 8,309,485, 8,623,973, and 9,023,959, which are incorporated herein by reference in their entirety. For instance, the activator-support can comprise fluorided alumina, chlorided alumina, bromided alumina, sulfated alumina, fluorided silica-alumina, chlorided silica-alumina, bromided silica-alumina, sulfated silica-alumina, fluorided silica-zirconia, chlorided silica-zirconia, bromided silica-zirconia, sulfated silica-zirconia, fluorided silica-titania, fluorided-chlorided silica-coated alumina, fluorided silica-coated alumina, sulfated silica-coated alumina, or phosphated silica-coated alumina, and the like, as well as any combination thereof. In some aspects, the activator-support can comprise a fluorided solid oxide and/or a sulfated solid oxide. Various processes can be used to form activator-supports useful in the present invention. Methods of contacting the solid oxide with the electron-withdrawing component, suitable electron withdrawing components and addition amounts, impregnation with metals or metal ions (e.g., zinc, nickel, vanadium, titanium, silver, copper, gallium, tin, tungsten, molybdenum, zirconium, and the like, or combinations thereof), and various calcining procedures and conditions are disclosed in, for example, U.S. Pat. Nos. 6,107,230, 6,165,929, 6,294,494, 6,300,271, 6,316,553, 6,355,594, 6,376,415, 6,388,017, 6,391,816, 6,395,666, 6,524,987, 6,548,441, 6,548,442, 6,576,583, 6,613,712, 6,632,894, 6,667,274, 6,750,302, 7,294,599, 7,601,665, 7,884,163, and 8,309,485, which are incorporated herein by reference in their entirety. Other suitable processes and procedures for preparing activator-supports (e.g., fluorided solid oxides, sulfated solid oxides, etc.) are well known to those of skill in the art. The present invention can employ catalyst compositions containing catalyst component I, catalyst component II, an activator (one or more than one), and optionally, a co-catalyst. When present, the co-catalyst can include, but is not limited to, metal alkyl, or organometal, co-catalysts, with the metal encompassing boron, aluminum, zinc, and the like. Optionally, the catalyst systems provided herein can comprise a co-catalyst, or a combination of co-catalysts. For instance, alkyl boron, alkyl aluminum, and alkyl zinc compounds often can be used as co-catalysts in such catalyst systems. Representative boron compounds can include, but are not limited to, tri-n-butyl borane, tripropylborane, triethylborane, and the like, and this include combinations of two or more of these materials. While not being limited thereto, representative aluminum compounds (e.g., organoaluminum compounds) can include trimethylaluminum, triethylaluminum, tri-n-propylaluminum, tri-n-butylaluminum, triisobutylaluminum, tri-n-hexylaluminum, tri-n-octylaluminum, diisobutylaluminum hydride, diethylaluminum ethoxide, diethylaluminum chloride, and the like, as well as any combination thereof. Exemplary zinc compounds (e.g., organozinc compounds) that can be used as co-catalysts can include, but are not limited to, dimethylzinc, diethylzinc, dipropylzinc, dibutylzinc, dineopentylzinc, di(trimethylsilyl)zinc, di(triethylsilyl)zinc, di(triisoproplysilyl)zinc, di(triphenylsilyl)zinc, di(allyldimethylsilyl)zinc, di(trimethylsilylmethyl)zinc, and the like, or combinations thereof. Accordingly, in an aspect of this invention, the dual catalyst composition can comprise catalyst component I, catalyst component II, an activator-support, and an organoaluminum compound (and/or an organozinc compound). In another aspect of the present invention, a catalyst composition is provided which comprises catalyst component I, catalyst component II, an activator-support, and an organoaluminum compound, wherein this catalyst composition is substantially free of aluminoxanes, organoboron or organoborate compounds, ionizing ionic compounds, and/or other similar materials; alternatively, substantially free of aluminoxanes; alternatively, substantially free or organoboron or organoborate compounds; or alternatively, substantially free of ionizing ionic compounds. In these aspects, the catalyst composition has catalyst activity, discussed herein, in the absence of these additional materials. For example, a catalyst composition of the present invention can consist essentially of catalyst component I, catalyst component II, an activator-support, and an organoaluminum compound, wherein no other materials are present in the catalyst composition which would increase/decrease the activity of the catalyst composition by more than about 10% from the catalyst activity of the catalyst composition in the absence of said materials. Catalyst compositions of the present invention generally have a catalyst activity greater than about 250 grams of ethylene polymer (homopolymer and/or copolymer, as the context requires) per gram of activator-support per hour (abbreviated g/g/hr). In another aspect, the catalyst activity can be greater than about 350, greater than about 450, or greater than about 550 g/g/hr. Yet, in another aspect, the catalyst activity can be greater than about 700 g/g/hr, greater than about 1000 g/g/hr, or greater than about 2000 g/g/hr, and often as high as 3500-6000 g/g/hr. Illustrative and non-limiting ranges for the catalyst activity include from about 500 to about 5000, from about 750 to about 4000, or from about 1000 to about 3500 g/g/hr, and the like. These activities are measured under slurry polymerization conditions, with a triisobutylaluminum co-catalyst, using isobutane as the diluent, at a polymerization temperature of about 95° C. and a reactor pressure of about 590 psig. Moreover, in some aspects, the activator-support can comprise sulfated alumina, fluorided silica-alumina, or fluorided silica-coated alumina, although not limited thereto. This invention further encompasses methods of making these catalyst compositions, such as, for example, contacting the respective catalyst components in any order or sequence. In one aspect, for example, the catalyst composition can be produced by a process comprising contacting, in any order, catalyst component I, catalyst component II, and the activator, while in another aspect, the catalyst composition can be produced by a process comprising contacting, in any order, catalyst component I, catalyst component II, the activator, and the co-catalyst. Olefin polymers (e.g., ethylene polymers) can be produced from the disclosed catalyst systems using any suitable olefin polymerization process using various types of polymerization reactors, polymerization reactor systems, and polymerization reaction conditions. One such olefin polymerization process for polymerizing olefins in the presence of a catalyst composition of the present invention can comprise contacting the catalyst composition with an olefin monomer and optionally an olefin comonomer (one or more) in a polymerization reactor system under polymerization conditions to produce an olefin polymer, wherein the catalyst composition can comprise, as disclosed herein, catalyst component I, catalyst component II, an activator, and an optional co-catalyst. This invention also encompasses any olefin polymers (e.g., ethylene polymers) produced by any of the polymerization processes disclosed herein. As used herein, a “polymerization reactor” includes any polymerization reactor capable of polymerizing (inclusive of oligomerizing) olefin monomers and comonomers (one or more than one comonomer) to produce homopolymers, copolymers, terpolymers, and the like. The various types of polymerization reactors include those that can be referred to as a batch reactor, slurry reactor, gas-phase reactor, solution reactor, high pressure reactor, tubular reactor, autoclave reactor, and the like, or combinations thereof; or alternatively, the polymerization reactor system can comprise a slurry reactor, a gas-phase reactor, a solution reactor, or a combination thereof. The polymerization conditions for the various reactor types are well known to those of skill in the art. Gas phase reactors can comprise fluidized bed reactors or staged horizontal reactors. Slurry reactors can comprise vertical or horizontal loops. High pressure reactors can comprise autoclave or tubular reactors. Reactor types can include batch or continuous processes. Continuous processes can use intermittent or continuous product discharge. Polymerization reactor systems and processes also can include partial or full direct recycle of unreacted monomer, unreacted comonomer, and/or diluent. A polymerization reactor system can comprise a single reactor or multiple reactors (2 reactors, more than 2 reactors, etc.) of the same or different type. For instance, the polymerization reactor system can comprise a slurry reactor, a gas-phase reactor, a solution reactor, or a combination of two or more of these reactors. Production of polymers in multiple reactors can include several stages in at least two separate polymerization reactors interconnected by a transfer device making it possible to transfer the polymers resulting from the first polymerization reactor into the second reactor. The desired polymerization conditions in one of the reactors can be different from the operating conditions of the other reactor(s). Alternatively, polymerization in multiple reactors can include the manual transfer of polymer from one reactor to subsequent reactors for continued polymerization. Multiple reactor systems can include any combination including, but not limited to, multiple loop reactors, multiple gas phase reactors, a combination of loop and gas phase reactors, multiple high pressure reactors, or a combination of high pressure with loop and/or gas phase reactors. The multiple reactors can be operated in series, in parallel, or both. Accordingly, the present invention encompasses polymerization reactor systems comprising a single reactor, comprising two reactors, and comprising more than two reactors. The polymerization reactor system can comprise a slurry reactor, a gas-phase reactor, a solution reactor, in certain aspects of this invention, as well as multi-reactor combinations thereof. According to one aspect, the polymerization reactor system can comprise at least one loop slurry reactor comprising vertical or horizontal loops. Monomer, diluent, catalyst, and comonomer can be continuously fed to a loop reactor where polymerization occurs. Generally, continuous processes can comprise the continuous introduction of monomer/comonomer, a catalyst, and a diluent into a polymerization reactor and the continuous removal from this reactor of a suspension comprising polymer particles and the diluent. Reactor effluent can be flashed to remove the solid polymer from the liquids that comprise the diluent, monomer and/or comonomer. Various technologies can be used for this separation step including, but not limited to, flashing that can include any combination of heat addition and pressure reduction, separation by cyclonic action in either a cyclone or hydrocyclone, or separation by centrifugation. A typical slurry polymerization process (also known as the particle form process) is disclosed, for example, in U.S. Pat. Nos. 3,248,179, 4,501,885, 5,565,175, 5,575,979, 6,239,235, 6,262,191, 6,833,415, and 8,822,608, each of which is incorporated herein by reference in its entirety. Suitable diluents used in slurry polymerization include, but are not limited to, the monomer being polymerized and hydrocarbons that are liquids under reaction conditions. Examples of suitable diluents include, but are not limited to, hydrocarbons such as propane, cyclohexane, isobutane, n-butane, n-pentane, isopentane, neopentane, and n-hexane. Some loop polymerization reactions can occur under bulk conditions where no diluent is used. According to yet another aspect, the polymerization reactor system can comprise at least one gas phase reactor (e.g., a fluidized bed reactor). Such reactor systems can employ a continuous recycle stream containing one or more monomers continuously cycled through a fluidized bed in the presence of the catalyst under polymerization conditions. A recycle stream can be withdrawn from the fluidized bed and recycled back into the reactor. Simultaneously, polymer product can be withdrawn from the reactor and new or fresh monomer can be added to replace the polymerized monomer. Such gas phase reactors can comprise a process for multi-step gas-phase polymerization of olefins, in which olefins are polymerized in the gaseous phase in at least two independent gas-phase polymerization zones while feeding a catalyst-containing polymer formed in a first polymerization zone to a second polymerization zone. Representative gas phase reactors are disclosed in U.S. Pat. Nos. 5,352,749, 4,588,790, 5,436,304, 7,531,606, and 7,598,327, each of which is incorporated by reference in its entirety herein. According to still another aspect, the polymerization reactor system can comprise a high pressure polymerization reactor, e.g., can comprise a tubular reactor or an autoclave reactor. Tubular reactors can have several zones where fresh monomer, initiators, or catalysts are added. Monomer can be entrained in an inert gaseous stream and introduced at one zone of the reactor. Initiators, catalysts, and/or catalyst components can be entrained in a gaseous stream and introduced at another zone of the reactor. The gas streams can be intermixed for polymerization. Heat and pressure can be employed appropriately to obtain optimal polymerization reaction conditions. According to yet another aspect, the polymerization reactor system can comprise a solution polymerization reactor wherein the monomer/comonomer are contacted with the catalyst composition by suitable stirring or other means. A carrier comprising an inert organic diluent or excess monomer can be employed. If desired, the monomer/comonomer can be brought in the vapor phase into contact with the catalytic reaction product, in the presence or absence of liquid material. The polymerization zone can be maintained at temperatures and pressures that will result in the formation of a solution of the polymer in a reaction medium. Agitation can be employed to obtain better temperature control and to maintain uniform polymerization mixtures throughout the polymerization zone. Adequate means are utilized for dissipating the exothermic heat of polymerization. The polymerization reactor system can further comprise any combination of at least one raw material feed system, at least one feed system for catalyst or catalyst components, and/or at least one polymer recovery system. Suitable reactor systems can further comprise systems for feedstock purification, catalyst storage and preparation, extrusion, reactor cooling, polymer recovery, fractionation, recycle, storage, loadout, laboratory analysis, and process control. Depending upon the desired properties of the olefin polymer, hydrogen can be added to the polymerization reactor as needed (e.g., continuously, pulsed, etc.). Polymerization conditions that can be controlled for efficiency and to provide desired polymer properties can include temperature, pressure, and the concentrations of various reactants. Polymerization temperature can affect catalyst productivity, polymer molecular weight, and molecular weight distribution. Various polymerization conditions can be held substantially constant, for example, for the production of a particular grade of the olefin polymer (or ethylene polymer). A suitable polymerization temperature can be any temperature below the de-polymerization temperature according to the Gibbs Free energy equation. Typically, this includes from about 60° C. to about 280° C., for example, or from about 60° C. to about 120° C., depending upon the type of polymerization reactor(s). In some reactor systems, the polymerization temperature generally can be within a range from about 70° C. to about 100° C., or from about 75° C. to about 95° C. Suitable pressures will also vary according to the reactor and polymerization type. The pressure for liquid phase polymerizations in a loop reactor is typically less than 1000 psig (6.9 MPa). Pressure for gas phase polymerization is usually at about 200 to 500 psig (1.4 MPa to 3.4 MPa). High pressure polymerization in tubular or autoclave reactors is generally run at about 20,000 to 75,000 psig (138 to 517 MPa). Polymerization reactors can also be operated in a supercritical region occurring at generally higher temperatures and pressures. Operation above the critical point of a pressure/temperature diagram (supercritical phase) can offer advantages to the polymerization reaction process. Olefin monomers that can be employed with catalyst compositions and polymerization processes of this invention typically can include olefin compounds having from 2 to 30 carbon atoms per molecule and having at least one olefinic double bond, such as ethylene or propylene. In an aspect, the olefin monomer can comprise a C2-C20olefin; alternatively, a C2-C20alpha-olefin; alternatively, a C2-C10olefin; alternatively, a C2-C10alpha-olefin; alternatively, the olefin monomer can comprise ethylene; or alternatively, the olefin monomer can comprise propylene (e.g., to produce a polypropylene homopolymer or a propylene-based copolymer). When a copolymer (or alternatively, a terpolymer) is desired, the olefin monomer and the olefin comonomer independently can comprise, for example, a C2-C20alpha-olefin. In some aspects, the olefin monomer can comprise ethylene or propylene, which is copolymerized with at least one comonomer (e.g., a C2-C20alpha-olefin, a C3-C20alpha-olefin, etc.). According to one aspect of this invention, the olefin monomer used in the polymerization process can comprise ethylene. In this aspect, the comonomer can comprise a C3-C10alpha-olefin; alternatively, the comonomer can comprise 1-butene, 1-pentene, 1-hexene, 1-octene, 1-decene, styrene, or any combination thereof, alternatively, the comonomer can comprise 1-butene, 1-hexene, 1-octene, or any combination thereof, alternatively, the comonomer can comprise 1-butene; alternatively, the comonomer can comprise 1-hexene; or alternatively, the comonomer can comprise 1-octene. EXAMPLES The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations to the scope of this invention. Various other aspects, embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present invention or the scope of the appended claims. Melt index (MI, g/10 min) was determined in accordance with ASTM D1238 at 190° C. with a 2,160 gram weight, and high load melt index (HLMI, g/10 min) was determined in accordance with ASTM D1238 at 190° C. with a 21,600 gram weight. Density was determined in grams per cubic centimeter (g/cm3) on a compression molded sample, cooled at 15° C. per hour, and conditioned for 40 hours at room temperature in accordance with ASTM D1505 and ASTM D4703. Molecular weights and molecular weight distributions were obtained using a PL-GPC 220 (Polymer Labs, an Agilent Company) system equipped with a IR4 detector (Polymer Char, Spain) and three Styragel HMW-6E GPC columns (Waters, MA) running at 145° C. The flow rate of the mobile phase 1,2,4-trichlorobenzene (TCB) containing 0.5 g/L 2,6-di-t-butyl-4-methylphenol (BHT) was set at 1 mL/min, and polymer solution concentrations were in the range of 1.0-1.5 mg/mL, depending on the molecular weight. Sample preparation was conducted at 150° C. for nominally 4 hr with occasional and gentle agitation, before the solutions were transferred to sample vials for injection. An injection volume of about 400 μL was used. The integral calibration method was used to deduce molecular weights and molecular weight distributions using a Chevron Phillips Chemical Company's HDPE polyethylene resin, MARLEX® BHB5003, as the standard. The integral table of the standard was pre-determined in a separate experiment with SEC-MALS. Mn is the number-average molecular weight, Mw is the weight-average molecular weight, Mz is the z-average molecular weight, and Mp is the peak molecular weight (location, in molecular weight, of the highest point of the molecular weight distribution curve). Melt rheological characterizations were performed as follows. Small-strain (less than 10%) oscillatory shear measurements were performed on an Anton Paar MCR rheometer using parallel-plate geometry. All rheological tests were performed at 190° C. The complex viscosity |η*| versus frequency (ω) data were then curve fitted using the modified three parameter Carreau-Yasuda (CY) empirical model to obtain the zero shear viscosity—η0, characteristic viscous relaxation time—τη, and the breadth parameter—a (CY-a parameter). The simplified Carreau-Yasuda (CY) empirical model is as follows. η*(ω)=η0[1+(τηω)a](1-n)/a, wherein: |η*(ω)|=magnitude of complex shear viscosity;η0=zero shear viscosity;τη=viscous relaxation time (Tau(η));a=“breadth” parameter (CY-a parameter);n=fixes the final power law slope, fixed at 2/11; andω=angular frequency of oscillatory shearing deformation. Details of the significance and interpretation of the CY model and derived parameters can be found in: C. A. Hieber and H. H. Chiang,Rheol. Acta,28, 321 (1989); C. A. Hieber and H. H. Chiang,Polym. Eng. Sci.,32, 931 (1992); and R. B. Bird, R. C. Armstrong and O. Hasseger,Dynamics of Polymeric Liquids, Volume1, Fluid Mechanics,2nd Edition, John Wiley & Sons (1987); each of which is incorporated herein by reference in its entirety. A creep adjustment was used to extend the low frequency range of rheological characterization to 10−4sec−1, as described in U.S. Pat. No. 9,169,337, incorporated herein by references in its entirety. Polymer viscosities at 0.1 sec−1, at 100 sec−1, and at HLMI were determined from the Carreau-Yasuda model at 190° C. with creep adjustment, described hereinabove. The long chain branches (LCBs) per 1,000,000 total carbon atoms of the overall polymer were calculated using the method of Janzen and Colby (J. Mol. Struct., 485/486, 569-584 (1999), incorporated herein by reference in its entirety), from values of zero shear viscosity, ηo(determined from the Carreau-Yasuda model with creep adjustment, described hereinabove) and measured values of Mw obtained using a Dawn EOS multiangle light scattering detector (Wyatt). LCB content and LCB distribution were determined using the method established by Yu, et al (Yu, DesLauriers, Rohlfing, Polymer, 2015, 46, 5165-5192, incorporated herein by reference in its entirety). Briefly, in the SEC-MALS system, a DAWN EOS photometer (Wyatt Technology, Santa Barbara, CA) was attached to a Waters 150-CV plus GPC system (Milford, MA) or a PL-210 GPC system (Polymer Labs, an Agilent company) through ahot-transfer line controlled at 145° C. Degassed mobile phase 1,2,4-trichlorobenzene (TCB) containing 0.5 wt % of BHT (butylated hydroxytoluene) was pumped through an inline filter before passing through a SEC column bank. Polymer solutions injected to the system were brought downstream to the columns by the mobile phase for fractionation. The fractionated polymers first eluted through the MALS photometer where light scattering signals were recorded before passing through the differential refractive index detector (DRI) or an IR4 detector (Polymer Characterization SA, Spain) where their concentrations were quantified. The DAWN EOS system was calibrated with neat toluene at room temperature to convert the measured voltage to intensity of scattered light. During the calibration, toluene was filtered with a 0.02 um filter (Whatman) and directly passed through the flowcell of the EOS system. At room temperature, the Rayleigh ratio is given by 1.406×10−5cm−1. A narrow polystyrene (PS) standard (American Polymer Standards) with MW of 30,000 g/mol at a concentration about 5˜10 mg/mL in TCB was employed to normalize the system at 145° C. At the given chromatographic conditions, the radius of gyration (Rg) of the polystyrene (PS) was estimated to be 5.6 nm. The differential refractive index detector (DRI) was calibrated with a known quantity of PE standard. By averaging the total chromatographic areas of recorded chromatograms for at least five injections, the DRI constant (αRI) was obtained using the equation below (equation 1): αRI=(dndc)c/IRIEquation1 where IRIis the DRI detector intensity, c is the polymer concentration, and dn/dc is the refractive index increment of PE in TCB at the measuring temperature. At a flow rate set at 0.7 mL/min, the mobile phase was eluted through three (3) 7.5 mm×300 mm 20 μm mixed A columns (Polymer Labs, an Agilent company). PE solutions with nominal concentrations of 1.5 mg/mL were prepared at 150° C. for 4 h. At each chromatographic slice, both the absolute molecular weight (M) and the root mean square (RMS) radius, aka, radius of gyration, Rg, were obtained from the Debye plots. The linear PE control employed was CPChem Marlex™ HiD9640, a high-density PE with broad MWD. The refractive index increment dn/dc used in this study was 0.097 mL/g for PE dissolved in TCB at 135° C. The Zimm-Stockmayer approach (Zimm, Stockmayer,J. Chem. Phys.1949, 17, 1301, incorporated herein by reference in its entirety) was employed to determine the amount of LCB in the polyethylene resins. In SEC-MALS, both M and Rgwere measured simultaneously at each slice of a chromatogram. At the same molecular weight, Rgof a branched polymer is smaller than that of a linear polymer. The branching index (gM) factor is defined as the ratio of the mean square radius of gyration of the branched polymer to that of the linear one at the same molecular weight using equation 2, gM≡(〈Rg2〉b〈Rg2〉l)MEquation2 where the subscripts b and l represent the branched and linear polymer, respectively. The weight-average LCB per molecule (B3w) was calculated using Equation 3 using an in-house software, gM=6B3w{12(2+B3wB3w)1/2ln[(2+B3w)1/2+(B3w)1/2(2+B3w)1/2-(B3w)1/2]-1}(3) LCB frequency (λMi, number of LCB per 1,000 total carbons) was calculated using equation 4 using the B3wvalue obtained from equation 3, λMi=1,000×M0×B3w/Mi(4) where M0is the unit molecular weight of polyethylene, Miis the molecular weight of the ithslice. Since the presence of SCB in a polymer can affect its Rg-MW relationship, the SCB effect was corrected before using equation 3 and 4 for LCB and LCB distribution calculation for PE copolymers. To correct the SCB effect on the branching index across the MWD, two relationships are needed: one is the relationship between the branching-index correction factor (ΔgM) and the SCB content (xSCB), and the other is the relationship between SCB content and molecular weight, both of which were determined experimentally. Mathematically, the product of these two relationships gives the branching index correction factor (ΔgM) as a function of MW, as shown in equation 5, d(ΔgM)d(M)=d(xSCB)d(M)×d(ΔgM)d(xSCB)(5) where xSCBis the SCB content (i.e., number of SCB per 1,000 total carbons) of the copolymer in question. To establish the relationship between ΔgMand xSCB, PE standards that met the following criteria were used: the standards contain essentially no LCB and have flat SCB distribution and known SCB contents. At least five SCB standards were used for the SCB effect correction. The SCB content for these SCB standards ranged from 0 to 34 SCB/1,000 total carbon atoms. Short chain branch content and short chain branching distribution (SCBD) across the molecular weight distribution were determined via an IR5-detected GPC system (IR5-GPC) using the method established by Yu (Y. Yu, Macromolecular Symposium, 2020, 390, 1900014), wherein the GPC system was a PL220 GPC/SEC system (Polymer Labs, an Agilent company) equipped with three Styragel HMW-6E columns (Waters, MA) for polymer separation. A thermoelectric-cooled IR5 MCT detector (IR5) (Polymer Characterisation SA, Spain) was connected to the GPC columns via a hot-transfer line. Chromatographic data was obtained from two output ports of the IR5 detector. First, the analog signal goes from the analog output port to a digitizer before connecting to Computer “A” for molecular weight determinations via the Cirrus software (Polymer Labs, now an Agilent Company) and the integral calibration method using a HDPE Marlex™ BHB5003 resin (Chevron Phillips Chemical) as the molecular weight standard. The digital signals, on the other hand, go via a USB cable directly to Computer “B” where they are collected by a LabView data collection software provided by Polymer Char. Chromatographic conditions were set as follows: column oven temperature of 145° C.; flowrate of 1 mL/min; injection volume of 0.4 mL; and polymer concentration of about 2 mg/mL, depending on sample molecular weight. The temperatures for both the hot-transfer line and IR5 detector sample cell were set at 150° C., while the temperature of the electronics of the IR5 detector was set at 60° C. Short chain branching content was determined via an in-house method using the intensity ratio of CH3(ICH3) to CH2(ICH2) coupled with a calibration curve. The calibration curve was a plot of SCB content (xSCB) as a function of the intensity ratio of ICH3/ICH2. To obtain a calibration curve, a group of polyethylene resins (no less than 5) of SCB level ranging from zero to ca. 32 SCB/1,000 total carbons (SCB Standards) were used. All these SCB Standards have known SCB levels and flat SCBD profiles pre-determined separately by NMR and the solvent-gradient fractionation coupled with NMR (SGF-NMR) methods. Using SCB calibration curves thus established, profiles of short chain branching distribution across the molecular weight distribution were obtained for resins fractionated by the IR5-GPC system under exactly the same chromatographic conditions as for these SCB standards. A relationship between the intensity ratio and the elution volume was converted into SCB distribution as a function of MWD using a predetermined SCB calibration curve (i.e., intensity ratio of ICH3/ICH2vs. SCB content) and MW calibration curve (i.e., molecular weight vs. elution time) to convert the intensity ratio of ICH3/ICH2and the elution time into SCB content and the molecular weight, respectively. Extensional viscosity was measured on a rotational rheometer (Physica MCR-500, Anton Paar) using the extensional viscosity fixture, a Sentimanat Extensional Rheometer (model SER-3 universal testing platform, Xpansion Instruments). The SER attachment makes it possible to easily measure the transient extensional viscosity as a function of time. Test samples were prepared via compression molding at 182° C. The pellets samples were allowed to melt at a relatively low pressure for 1 min and then subjected to a high molding pressure for additional 2 min. Then, the hot press was turned off for slow cooling. The cooled plaque was retrieved from the press on the following day. Rectangular strips with dimensions of 12.77×18 mm were cut out of the molded plaque, and the thickness of the sample was measured. The SER testing platform has two drums that rotate in the opposing direction (M. L. Sentmanat, “Miniature universal testing platform: from extensional melt rheology to solid-state deformation behavior,”Rheol. Acta43, 657 (2004); M. L. Sentmanat, B. N. Wang, G. H. McKinley, “Measuring the transient extensional rheology of polyethylene melts using the SER universal testing platform,”J. Rheol.49, 585 (2005); both incorporated herein by reference in their entirety). The rectangular samples were tested by clipping onto the two posts of the fixture, then closing the oven to heat to 150° C., where it was annealed at 150° C. for 30 sec to allow the temperature to reach equilibrium. The sample was then stretched at constant Hencky strain rates {dot over (ε)}Hbetween 0.03 and 25 s−1at 150° C. The torque M resulting from the force of tangential stretching of the sample between the rotating drums F was recorded by the rotational rheometer: M(t)=2RF(t) (A) where the radius of drums R=5.155 mm. The Hencky strain rate {dot over (ε)}Hat constant drum rotating speed Ω is ɛ.H=2ΩRL(B) where the length of the stretching zone between the rotating drums L=12.72 mm. The transient extensional viscosity ηE*(t) was obtained for given Hencky strain rate as ηE+(t)=σE(t)ɛ.E=F(t)A(t,T)ɛ.E(C) where A(t,T) is the cross-sectional area of the sample which thermally expands upon melting and exponentially decreases with stretching: A(t,T)=Aoexp(-ɛ.Et)(ρsρ(T))2/3(D) where A0and ρsare the initial cross-sectional area and the density of the sample measured at room temperature in solid state. The melt density ρ(T) is given by ρ(T)=ρ0−Δρ(T−273.15)T. Therefore, the transient extensional viscosity ηE*(t) as a function of time was calculated at each extension rate as ηE+(t)=M-Moffset2Rɛ.EA0exp(-ɛ.Et)(ρ(T)ρs)2/3(E) where Moffsetis a pre-set torque which can be applied prior to the actual test. To compare the extensional response to the linear viscoelastic (LVE) limit, the LVE envelop 3η+(t) was obtained from the relaxation spectrum of the dynamic frequency sweep data measured at 150° C. as η+(t)=∑i=1NGiλi[1-exp(-t/λi)](F) where the set of Giand λidefine the relaxation spectrum of the material. In general, it has been observed that when long chain branching exists in the polymer, the transient extensional viscosity deviates from the LVE drastically by increasing slope just before breakage. This behavior is called the strain hardening. In contrast, for linear resins the transient extensional viscosity growth curves show no strain hardening by continuing to follow the LVE envelop (3η+(t)) according to the Trouton's rule. Metals content, such as the amount of catalyst residue in the ethylene polymer or the article of manufacture (on a ppm basis), can be determined by ICP analysis on a PerkinElmer Optima 8300 instrument. Polymer samples can be ashed in a Thermolyne furnace with sulfuric acid overnight, followed by acid digestion in a HotBlock with HCl and HNO3(3:1 v:v). Fluorided silica-coated alumina activator-supports (FSCA) used in Examples 1-2 were prepared as follows. Bohemite was obtained from W.R. Grace & Company under the designation “Alumina A” and having a surface area of 300 m2/g, a pore volume of 1.3 mL/g, and an average particle size of 100 microns. The alumina was first calcined in dry air at about 600° C. for approximately 6 hours, cooled to ambient temperature, and then contacted with tetraethylorthosilicate in isopropanol to equal 25 wt. % SiO2. After drying, the silica-coated alumina was calcined at 600° C. for 3 hours. Fluorided silica-coated alumina (7 wt. % F) was prepared by impregnating the calcined silica-coated alumina with an ammonium bifluoride solution in methanol, drying, and then calcining for 3 hours at 600° C. in dry air. Afterward, the fluorided silica-coated alumina (FSCA) was collected and stored under dry nitrogen, and was used without exposure to the atmosphere. Pilot plant polymerizations were conducted in a 30-gallon slurry loop reactor at a production rate of approximately 30 pounds of polymer per hour. Polymerization runs were carried out under continuous particle form process conditions in a loop reactor (also referred to as a slurry process) by contacting separate metallocene solutions, an organoaluminum solution (triisobutylaluminum, TIBA), and an activator-support (fluorided silica-coated alumina, FSCA) in a 1-L stirred autoclave (30 min residence time) with output to the loop reactor. Ethylene used was polymerization grade ethylene which was purified through a column of AZ 300 (activated at 300-500° F. in nitrogen). 1-Hexene was polymerization grade 1-hexene (obtained from Chevron Phillips Chemical Company) which was purified by nitrogen purging and storage over AZ 300 activated at 300-500° F. in nitrogen. Liquid isobutane was used as the diluent. Certain polymerization conditions for Examples 1-2 are provided in Table I below (mole % ethylene and ppm by weight of triisobutylaluminum (TIBA) are based on isobutane diluent). The polymerization conditions also included a reactor pressure of 590 psig, a polymerization temperature of 97° C., a feed rate of 30 lb/hr ethylene, and 2.5-3.5 ppm total of MET 1 and MET 2 (based on the weight of isobutane diluent). The structures for MET 1 and MET 2, used in Examples 1-2, are shown below: TABLE I1-HexeneH2Weight ratioC2H4TIBAExample(lb/hr)(lb/hr)MET 1/MET 2mole %ppm10.160.00211.8811.7314320.150.00202.0112.22144 Blow molded 1-gallon containers were produced under suitable conditions on a Uniloy reciprocating blow molding machine. The parison was extruded using a 2.5″ diverging die and then blown into a mold to produce the 1-gallon containers weighing approximately 105 g at the following set of process controls: 360° F. extruder temperature; 2.1-2.2 shot size; 160 g total parison weight; 45 rpm screw speed; 200 (±15) psig back pressure. Drop impact testing was performed on the 1-gallon containers that were blow molded from the polymers of Examples 1-3, generally in accordance with ASTM D2463. Examples 1-4 Comparative Example 3 was a commercially-available chromium-catalyzed ethylene/1-hexene copolymer resin from Chevron-Phillips Chemical Company LP, and Comparative Example 4 was a linear dual-metallocene blow molding resin (with no long chain branching). For the polymers of Examples 1-3, Table II summarizes various molecular weight, LCB (Janzen-Colby), rheology, melt index, density, ESCR, and blow molded bottle properties, while Table III summarizes an extrusion and blow molding processing comparison, andFIG.1illustrates the molecular weight distribution curves (amount of polymer versus the logarithm of molecular weight) for the polymers of Examples 1-3. As compared to chromium-based Example 3, the ethylene/1-hexene copolymers of Examples 1-2 had less LCBs per million total carbon atoms (via Janzen-Colby), lower relaxations times, higher CY-a parameters, and significantly better ESCR properties. Using the chromium polymer of Example 3 as a benchmark, Table III shows that the polymers of Examples 1-2 had unexpectedly lower extrusion pressure (psi) and equivalent part weights, cycle times, layflats, and output rates (measured at 100 rpm with a 0.022″ die gap). The processing similarities are also shown by the relatively small rheology differences between Examples 2-3 and Comparative Example 1 inFIG.10. FIG.2illustrates the short chain branch distributions for the polymers of Examples 1-2. Surprisingly, these polymers have a substantially flat SCBD, in which the SCB content is generally constant with increasing molecular weight. FIG.3illustrates a plot of the molecular weight distribution and long chain branch distribution of the polymer of Example 1, whileFIG.4illustrates a plot of the molecular weight distribution and long chain branch distribution of the polymer of Example 2. The concentration of long chain branch content in the ˜300,000-900,000 g/mol molecular weight range (but not in the very high molecular weight fraction) of the inventive polymers of Examples 1-2 is illustrated in these figures. Further,FIG.5illustrates a plot of the radius of gyration versus the molecular weight for a linear standard and the polymers of Examples 1-2, and demonstrates the deviation of the polymers of Examples 1-2 from the linear standard, due to the presence of LCB in the ˜300,000-900,000 g/mol range. FromFIGS.3-4, Table IV summarizes the LCB content of the respective ethylene polymers in certain molecular weight ranges. As an example, the number-average number of LCBs per 1,000,000 total carbon atoms of the respective polymers inFIGS.3-4in the molecular weight range of 300,000 to 900,000 g/mol and in the molecular weight range of 1,000,000 to 2,000,000 g/mol can be calculated based on Equations I and II, respectively, and are summarized in Table IV. λ_=∑MW=300kg/molMW=900kg/molλi(dwd(LogM))i(d(LogM))i∑MW=300kg/molMW=900kg/mol(dwd(LogM))i(d(LogM))iEquationIλ_=∑MW=1000kg/molMW=2000kg/molλi(dwd(LogM))i(d(LogM))i∑MW=1000kg/molMW=2000kg/mol(dwd(LogM))i(d(LogM))iEquationII where {tilde over (λ)} is the number-average LCB number in the respective molecular weight range and λiis LCB at slice i. As shown in Table IV, the number-average number of LCBs per 1,000,000 total carbon atoms of the polymers inFIGS.3-4in the molecular weight range of 300,000 to 900,000 g/mol (or 400,000 to 600,000 g/mol) is significantly—and unexpectedly—greater than that in the molecular weight range of 1,000,000 to 2,000,000 g/mol. FIGS.6-8illustrates extensional viscosity plots, respectively, for the polymers of Example 1, Example 2, and Comparative Example 4. Extensional rheology was used as a means to quantify the amount of LCBs, since for a Newtonian fluid, the ratio of extensional viscosity will be equal to 3 times the shear viscosity; the ratio of ηE/3η will be equal to 1 for a Newtonian fluid (see the linear polymer of Comparative Example 4 inFIG.8, with no long chain branching). For molten polymers with strain hardening due to the presence of LCBs, the ratio of ηE/3η will be greater than 1.FIGS.6-7are extensional viscosity plots at for the polymers of Examples 1-2, determined using SER. The minor scatter in the baseline was due to the limited amount of samples for the SER experiments. FromFIGS.6-7,FIG.9was prepared to summarize the maximum ratio of ηE/3η at extensional rates in the 0.03 to 10 sec−1range for the polymers of Examples 1-2. A higher ratio equates to more strain hardening, and therefore, higher levels of LCBs. For these inventive polymers, unexpectedly, the maximum ratio of ηE/3η at the extensional rate of 0.03 sec−1ranged from 3 to 4, and ranged from 1.5 to 2.5 at an extensional rate of 0.1 sec−1. While not wishing to be bound by theory, higher levels of LCB—and thus higher extensional viscosity—can be obtained herein by decreasing the concentration of ethylene in the reactor. Additionally or alternatively, increasing comonomer levels (e.g., more 1-hexene) for a given ethylene concentration (the ratio of [1-hexene]/[ethylene]) can be used to increase levels of LCB in the polymer. Levels of LCB also can be adjusted by varying the ratio of the metallocene compounds in the catalyst system. Thus, the ethylene copolymers disclosed herein offer a beneficial combination of density, melt flow, molecular weight, relaxation time, long chain branching, and extensional rheology properties, resulting in processability comparable to chromium-based polymers, but with improved ESCR and toughness properties. TABLE IIMn/1000Mw/1000Mz/1000Mp/1000LCB/millionExample(g/mol)(g/mol)(g/mol)(g/mol)Mw/MnMz/Mwcarbon atoms118.315678340.28.525.028.2218.714974044.58.034.9710.9320.713463552.56.474.7437η0τηη @ 0.1 secη @ 100 secη @ 0.1/η @ HLMIExample(Pa-sec)(sec)CY-a(Pa-sec)(Pa-sec)η @ 100(Pa-sec)11.02E+051.20.3133710118128.536721.40E+051.40.2632780116528.140131.43E+063.70.1435480131826.9757Bent StripYieldBottleBottleBottle DropHLMIMIDensityESCRStrengthESCRToploadImpactExample(g/10 min)(g/10 min)(g/cc)(hr)(psi)(hr)(lb)(ft)1450.300.95831154260499175>122500.360.9581794310———3320.330.9556<50413016517511.9 TABLE IIIMeltWeightDieParisonPartScrewCycleHeadTopBottomMeltTemp.SettingGapWeightWeightChargeTimePressureLayflatLayflatOutputStrengthExample(° F.)(%)(in)(g)(g)(sec)(sec)(psi)(in)(in)(g/min)(sec)14111.20.015160.8105.515.317.044505.525.79129514.3240500.013161.5109.615.216.946005.465.70129714.734161.20.015160.2105.114.616.350405.225.55130231.5 TABLE IVAverage LCBs per 1,000,000ExampleExampletotal carbon atoms12(a) 300,000-900,000 g/mol range7.836.66(b) 1,000,000-2,000,00 g/mol range1.892.45Percentage (a)/(b)414%272%(a) 400,000-600,000 g/mol range8.988.32(b) 1,000,000-2,000,000 g/mol range1.892.45Percentage (a)/(b)475%340% The invention is described above with reference to numerous aspects and specific examples. Many variations will suggest themselves to those skilled in the art in light of the above detailed description. All such obvious variations are within the full intended scope of the appended claims. Other aspects of the invention can include, but are not limited to, the following (aspects are described as “comprising” but, alternatively, can “consist essentially of” or “consist of”): Aspect 1. An ethylene polymer having:a melt index of less than or equal to about 1 g/10 min;a density in a range from about 0.94 to about 0.965 g/cm3;a Mw in a range from about 100,000 to about 250,000 g/mol;a relaxation time from about 0.5 to about 3 sec; andan average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 300,000 to 900,000 g/mol that is greater (by any amount disclosed herein, e.g., at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, etc.) than that in a molecular weight range of 1,000,000 to 2,000,000 g/mol. Aspect 2. An ethylene polymer having:a melt index of less than or equal to about 1 g/10 min;a density in a range from about 0.94 to about 0.965 g/cm3;a Mw in a range from about 100,000 to about 250,000 g/mol;a relaxation time from about 0.5 to about 3 sec;an average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 1,000,000 to 2,000,000 g/mol of less than or equal to about 5; anda maximum ratio of >E/3η at an extensional rate of 0.1 sec−1in a range from about 1.2 to about 10. Aspect 3. The polymer defined in aspect 1 or 2, wherein the ethylene polymer has a melt index (MI) in any range disclosed herein, e.g., less than or equal to about 0.7 g/10 nm, less than or equal to about 0.5 g/10 min, from about 0.1 to about 0.5 g/10 min, from about 0.2 to about 0.4 g/10 min, etc. Aspect 4. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a high load melt index (HLMI) in any range disclosed herein, e.g., from about 10 to about 65 g/10 min, from about 35 to about 55 g/10 min, from about 20 to about 60 g/10 min, from about 40 to about 55 g/10 min, etc. Aspect 5. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a ratio of HLMI/MI in any range disclosed herein, e.g., from about 100 to about 200, from about 120 to about 170, from about 130 to about 160, etc. Aspect 6. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a density in any range disclosed herein, e.g., from about 0.942 to about 0.965 g/cm3, from about 0.94 to about 0.96 g/cm3, from about 0.95 to about 0.965 g/cm3, from about 0.955 to about 0.962 g/cm3, from about 0.955 to about 0.96 g/cm3, etc. Aspect 7. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a ratio of Mw/Mn in any range disclosed herein, e.g., from about 5 to about 15, from about 6 to about 12, from about 6 to about 10, from about 7 to about 10, etc. Aspect 8. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a ratio of Mz/Mw in any range disclosed herein, e.g., from about 3.5 to about 10, from about 4 to about 8, from about 4 to about 6, from about 4.5 to about 5.5, etc. Aspect 9. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a Mz in any range disclosed herein, e.g., from about 500,000 to about 2,000,000 g/mol, from about 600,000 to about 1,200,000 g/mol, from about 650,000 to about 1,000,000 g/mol, from about 700,000 to about 900,000 g/mol, etc. Aspect 10. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a Mw in any range disclosed herein, e.g., from about 125,000 to about 250,000 g/mol, from about 100,000 to about 200,000 g/mol, from about 110,000 to about 190,000 g/mol, from about 125,000 to about 175,000 g/mol, etc. Aspect 11. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a Mn in any range disclosed herein, e.g., from about 10,000 to about 30,000 g/mol, from about 10,000 to about 25,000 g/mol, from about 15,000 to about 25,000 g/mol, from about 15,000 to about 20,000 g/mol, etc. Aspect 12. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has an average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 300,000 to 900,000 g/mol in any range disclosed herein, e.g., from 3 to about to 15, from about 4 to about 10, from about 5 to about 9, from about 6 to about 8, etc. Aspect 13. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has an average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 400,000 to 600,000 g/mol in any range disclosed herein, e.g., from 4 to about 15, from about 5 to about 12, from about 7 to about 10, from about 8 to about 9, etc. Aspect 14. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has an average number of long chain branches (LCBs) per 1,000,000 total carbon atoms of the polymer in a molecular weight range of 1,000,000 to 2,000,000 g/mol in any range disclosed herein, e.g., less than or equal to about 5, less than or equal to about 4, less than or equal to about 3.5, less than or equal to about 3, etc. Aspect 15. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer contains from about 5 to about 15 LCBs, from about 6 to about 14 LCBs, from about 8 to about 12 LCBs, etc., per 1,000,000 total carbon atoms. Aspect 16. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a maximum ratio of ηE/3η at an extensional rate of 0.1 sec−1in any range disclosed herein, e.g., from about 1.2 to about 10, from about 1.5 to about 8, from about 1.5 to about 5, from about 1.2 to about 4, from about 1.2 to about 3, from about 1.4 to about 3.5, from about 1.4 to about 3, from about 1.5 to about 2.5, etc. Aspect 17. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a maximum ratio of ηE/3η at an extensional rate of 0.03 sec−1in any range disclosed herein, e.g., from about 1.2 to about 10, from about 1.5 to about 8, from about 2 to about 7, from about 2 to about 5, from about 2.5 to about 4.5, from about 3 to about 4, etc. Aspect 18. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a CY-a parameter in any range disclosed herein, e.g., from about 0.15 to about 0.45, from about 0.2 to about 0.4, from about 0.22 to about 0.35, from about 0.22 to about 0.32, etc. Aspect 19. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a zero-shear viscosity in any range disclosed herein, e.g., from about 1×103to about 1×108Pa-sec, from about 1×104to about 1×107Pa-sec, from about 1×104to about 1×106Pa-sec, etc. Aspect 20. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a relaxation time in any range disclosed herein, e.g., from about 0.75 to about 2.5 sec, from about 1 to about 2 sec, from about 1 to about 1.5 sec, etc. Aspect 21. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a viscosity at 100 sec−1(eta @ 100 or η@ 100) in any range disclosed herein, e.g., from about 750 to about 1750, from about 850 to about 1300, from about 1000 to about 1500, from about 1000 to about 1300 Pa-sec, etc. Aspect 22. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a viscosity at HLMI (eta @ HLMI or η@ HLMI) in any range disclosed herein, e.g., from about 300 to about 750, from about 300 to about 500, from about 300 to about 450, from about 350 to about 450 Pa-sec, etc. Aspect 23. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a ratio of η@ 0.1/η@ 100 in any range disclosed herein, e.g., from about 20 to about 45, from about 20 to about 35, from about 22 to about 32, from about 25 to about 30, etc. Aspect 24. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a substantially constant number of short chain branches (SCBs) per 1000 total carbon atoms, or a substantially flat SCBD (short chain branching distribution). Aspect 25. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a part weight in any range disclosed herein, e.g., from about 95 to about 115, from about 100 to about 115, from about 95 to about 110, from about 100 to about 110 g, etc. Aspect 26. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a layflat (top) in any range disclosed herein, e.g., from about 5.2 to about 6, from about 5 to about 5.7, from about 5.2 to about 5.7 inches, etc. Aspect 27. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a cycle time in any range disclosed herein, e.g., from about 13 to about 20, from about 14 to about 19, from about 15 to about 18, from about 16 to about 17 seconds, etc. Aspect 28. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has an environmental stress crack resistance (ESCR—bottle in 10% Igepal, 140° F., ASTM D2561) in any range disclosed herein, e.g., at least 200 hours, at least 250 hours, at least 300 hours, at least 400 hours, at least 500 hours, etc. Aspect 29. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has an environmental stress crack resistance (ESCR—bent strip in 10% Igepal, 50° C., 75 mils, ASTM D1693) in any range disclosed herein, e.g., at least 50 hours, at least 60 hours, at least 75 hours, at least 85 hours, at least 100 hours, etc. Aspect 30. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer contains, independently, less than 0.1 ppm (by weight), less than 0.08 ppm, less than 0.05 ppm, less than 0.03 ppm, etc., of chromium and titanium. Aspect 31. The polymer defined in any one of the preceding aspects, wherein the polymer further comprises any additive disclosed herein, e.g., an antioxidant, an acid scavenger, an antiblock additive, a slip additive, a colorant, a filler, a polymer processing aid, a UV additive, etc., or combinations thereof. Aspect 32. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer has a bimodal molecular weight distribution. Aspect 33. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer is a single reactor product, e.g., not a post-reactor blend of two polymers, for instance, having different molecular weight characteristics. Aspect 34. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer comprises an ethylene/α-olefin copolymer and/or an ethylene homopolymer. Aspect 35. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer comprises an ethylene/1-butene copolymer, an ethylene/1-hexene copolymer, an ethylene/1-octene copolymer, an ethylene homopolymer, or any combination thereof. Aspect 36. The polymer defined in any one of the preceding aspects, wherein the ethylene polymer comprises an ethylene/1-hexene copolymer. Aspect 37. An article (e.g., a blow molded product) comprising the ethylene polymer defined in any one of aspects 1-36. Aspect 38. An article comprising the ethylene polymer defined in any one of aspects 1-36, wherein the article is an agricultural film, an automobile part, a bottle, a container for chemicals, a drum, a fiber or fabric, a food packaging film or container, a food service article, a fuel tank, a geomembrane, a household container, a liner, a molded product, a medical device or material, an outdoor storage product, outdoor play equipment, a pipe, a sheet or tape, a toy, or a traffic barrier. Aspect 39. A catalyst composition comprising: catalyst component I comprising any single atom bridged or two atom bridged metallocene compound disclosed herein with two indenyl groups; catalyst component II comprising any single atom bridged metallocene compound disclosed herein with a fluorenyl group and a cyclopentadienyl group, and with an alkenyl substituent on the single atom bridge and/or on the cyclopentadienyl group; any activator disclosed herein; and optionally, any co-catalyst disclosed herein. Aspect 40. The composition defined in aspect 39, wherein the activator comprises an activator-support, an aluminoxane compound, an organoboron or organoborate compound, an ionizing ionic compound, or any combination thereof. Aspect 41. The composition defined in aspect 39, wherein the activator comprises an aluminoxane compound. Aspect 42. The composition defined in aspect 39, wherein the activator comprises an organoboron or organoborate compound. Aspect 43. The composition defined in aspect 39, wherein the activator comprises an ionizing ionic compound. Aspect 44. The composition defined in aspect 39, wherein the activator comprises an activator-support, the activator-support comprising any solid oxide treated with any electron-withdrawing anion disclosed herein. Aspect 45. The composition defined in aspect 39, wherein the activator comprises fluorided alumina, chlorided alumina, bromided alumina, sulfated alumina, fluorided silica-alumina, chlorided silica-alumina, bromided silica-alumina, sulfated silica-alumina, fluorided silica-zirconia, chlorided silica-zirconia, bromided silica-zirconia, sulfated silica-zirconia, fluorided silica-titania, fluorided silica-coated alumina, fluorided-chlorided silica-coated alumina, sulfated silica-coated alumina, phosphated silica-coated alumina, or any combination thereof. Aspect 46. The composition defined in aspect 39, wherein the activator comprises fluorided alumina, sulfated alumina, fluorided silica-alumina, sulfated silica-alumina, fluorided silica-coated alumina, fluorided-chlorided silica-coated alumina, sulfated silica-coated alumina, or any combination thereof. Aspect 47. The composition defined in aspect 39, wherein the activator comprises a fluorided solid oxide and/or a sulfated solid oxide. Aspect 48. The composition defined in any one of aspects 44-47, wherein the activator further comprises any metal or metal ion disclosed herein, e.g., zinc, nickel, vanadium, titanium, silver, copper, gallium, tin, tungsten, molybdenum, zirconium, or any combination thereof. Aspect 49. The composition defined in any one of aspects 39-48, wherein the catalyst composition comprises a co-catalyst, e.g., any suitable co-catalyst. Aspect 50. The composition defined in any one of aspects 39-49, wherein the co-catalyst comprises any organoaluminum compound disclosed herein. Aspect 51. The composition defined in aspect 50, wherein the organoaluminum compound comprises trimethylaluminum, triethylaluminum, triisobutylaluminum, or a combination thereof. Aspect 52. The composition defined in any one of aspects 44-51, wherein the catalyst composition comprises catalyst component I, catalyst component II, a solid oxide treated with an electron-withdrawing anion, and an organoaluminum compound. Aspect 53. The composition defined in any one of aspects 39-52, wherein catalyst component I has two unsubstituted indenyl groups. Aspect 54. The composition defined in any one of aspects 39-53, wherein catalyst component I has a single carbon or silicon bridging atom. Aspect 55. The composition defined in aspect 54, wherein the carbon or silicon bridging atom has two substituents independently selected from H or a C1to C18hydrocarbyl group, e.g., a C1to C6alkyl group. Aspect 56. The composition defined in any one of aspects 39-53, wherein catalyst component I has a two carbon atom bridge. Aspect 57. The composition defined in any one of aspects 39-52, wherein at least one indenyl group is substituted. Aspect 58. The composition defined in aspect 57, wherein catalyst component I has a single carbon or silicon bridging atom. Aspect 59. The composition defined in aspect 58, wherein the carbon or silicon bridging atom has two substituents independently selected from H or a C1to C18hydrocarbyl group, e.g., a C1to C6alkyl group. Aspect 60. The composition defined in any one of aspects 57-59, wherein any substituent on an indenyl group is independently selected from H or a C1to C18hydrocarbyl group, e.g., a C1to C6alkyl group. Aspect 61. The composition defined in any one of aspects 39-60, wherein catalyst component I contains zirconium. Aspect 62. The composition defined in any one of aspects 39-61, wherein catalyst component II has a single carbon or silicon bridging atom. Aspect 63. The composition defined in aspect 62, wherein the carbon or silicon bridging atom has two substituents independently selected from H or a C1to C18hydrocarbyl group, e.g., a phenyl group. Aspect 64. The composition defined in any one of aspects 39-63, wherein the fluorenyl group is substituted. Aspect 65. The composition defined in any one of aspects 39-64, wherein the alkenyl substituent is a C3to C18alkenyl group, e.g., a C3to C8terminal alkenyl group. Aspect 66. The composition defined in any one of aspects 39-65, wherein catalyst component II contains zirconium or hafnium. Aspect 67. The composition defined in any one of aspects 44-66, wherein the catalyst composition is substantially free of aluminoxane compounds, organoboron or organoborate compounds, ionizing ionic compounds, or combinations thereof. Aspect 68. The composition defined in any one of aspects 39-67, wherein a weight ratio of catalyst component I to catalyst component II in the catalyst composition is in any range disclosed herein, e.g., from about 10:1 to about 1:10, from about 5:1 to about 1:5, from about 1.2:1 to about 5:1, from about 1.5:1 to about 4:1, from about 1.5:1 to about 2.5:1, etc. Aspect 69. The composition defined in any one of aspects 39-68, wherein the catalyst composition is produced by a process comprising contacting, in any order, catalyst component I, catalyst component II, and the activator. Aspect 70. The composition defined in any one of aspects 39-68, wherein the catalyst composition is produced by a process comprising contacting, in any order, catalyst component I, catalyst component II, the activator, and the co-catalyst. Aspect 71. The composition defined in any one of aspects 39-70, wherein a catalyst activity of the catalyst composition is in any range disclosed herein, e.g., from about 500 to about 5000, from about 750 to about 4000, from about 1000 to about 3500 grams, etc., of ethylene polymer per gram of activator-support per hour, under slurry polymerization conditions, with a triisobutylaluminum co-catalyst, using isobutane as a diluent, and with a polymerization temperature of 95° C. and a reactor pressure of 590 psig. Aspect 72. An olefin polymerization process, the process comprising contacting the catalyst composition defined in any one of aspects 39-71 with an olefin monomer and an optional olefin comonomer in a polymerization reactor system under polymerization conditions to produce an olefin polymer. Aspect 73. The process defined in aspect 72, wherein the olefin monomer comprises any olefin monomer disclosed herein, e.g., any C2-C20olefin. Aspect 74. The process defined in aspect 72 or 73, wherein the olefin monomer and the olefin comonomer independently comprise a C2-C20alpha-olefin. Aspect 75. The process defined in any one of aspects 72-74, wherein the olefin monomer comprises ethylene. Aspect 76. The process defined in any one of aspects 72-75, wherein the catalyst composition is contacted with ethylene and an olefin comonomer comprising a C3-C10alpha-olefin. Aspect 77. The process defined in any one of aspects 72-76, wherein the catalyst composition is contacted with ethylene and an olefin comonomer comprising 1-butene, 1-hexene, 1-octene, or a mixture thereof. Aspect 78. The process defined in any one of aspects 72-74, wherein the olefin monomer comprises propylene. Aspect 79. The process defined in any one of aspects 72-78, wherein the polymerization reactor system comprises a batch reactor, a slurry reactor, a gas-phase reactor, a solution reactor, a high pressure reactor, a tubular reactor, an autoclave reactor, or a combination thereof. Aspect 80. The process defined in any one of aspects 72-79, wherein the polymerization reactor system comprises a slurry reactor, a gas-phase reactor, a solution reactor, or a combination thereof. Aspect 81. The process defined in any one of aspects 72-80, wherein the polymerization reactor system comprises a loop slurry reactor. Aspect 82. The process defined in any one of aspects 72-81, wherein the polymerization reactor system comprises a single reactor. Aspect 83. The process defined in any one of aspects 72-81, wherein the polymerization reactor system comprises 2 reactors. Aspect 84. The process defined in any one of aspects 72-81, wherein the polymerization reactor system comprises more than 2 reactors. Aspect 85. The process defined in any one of aspects 72-84, wherein the olefin polymer comprises any olefin polymer disclosed herein. Aspect 86. The process defined in any one of aspects 72-77 and 79-85, wherein the olefin polymer comprises an ethylene homopolymer, an ethylene/1-butene copolymer, an ethylene/1-hexene copolymer, and/or an ethylene/1-octene copolymer. Aspect 87. The process defined in any one of aspects 72-77 and 79-85, wherein the olefin polymer comprises an ethylene/1-hexene copolymer. Aspect 88. The process defined in any one of aspects 72-74 and 78-85, wherein the olefin polymer comprises a polypropylene homopolymer or a propylene-based copolymer. Aspect 89. The process defined in any one of aspects 72-88, wherein the polymerization conditions comprise a polymerization reaction temperature in a range from about 60° C. to about 120° C. and a reaction pressure in a range from about 200 to about 1000 psig (about 1.4 to about 6.9 MPa). Aspect 90. The process defined in any one of aspects 72-89, wherein the polymerization conditions are substantially constant, e.g., for a particular polymer grade. Aspect 91. The process defined in any one of aspects 72-90, wherein no hydrogen is added to the polymerization reactor system. Aspect 92. The process defined in any one of aspects 72-90, wherein hydrogen is added to the polymerization reactor system. Aspect 93. The process defined in any one of aspects 72-92, wherein the olefin polymer produced is defined in any one of aspects 1-36. Aspect 94. An olefin polymer produced by the olefin polymerization process defined in any one of aspects 72-92. Aspect 95. An ethylene polymer defined in any one of aspects 1-36 produced by the process defined in any one of aspects 72-92. Aspect 96. An article comprising the polymer defined in any one of aspects 94-95. Aspect 97. A method or forming or preparing an article of manufacture comprising an olefin polymer, the method comprising (i) performing the olefin polymerization process defined in any one of aspects 72-92 to produce an olefin polymer (e.g., the ethylene polymer of any one of aspects 1-36), and (ii) forming the article of manufacture comprising the olefin polymer, e.g., via any technique disclosed herein. | 123,576 |
11859025 | DEFINITIONS To define more clearly the terms used herein, the following definitions are provided. Unless otherwise indicated, the following definitions are applicable to this disclosure. If a term is used in this disclosure but is not specifically defined herein, the definition from the IUPAC Compendium of Chemical Terminology, 2nd Ed (1997), can be applied, as long as that definition does not conflict with any other disclosure or definition applied herein, or render indefinite or non-enabled any claim to which that definition is applied. To the extent that any definition or usage provided by any document incorporated herein by reference conflicts with the definition or usage provided herein, the definition or usage provided herein controls. Herein, features of the subject matter can be described such that, within particular aspects and/or statements, a combination of different features can be envisioned. For each and every aspect, and/or statement, and/or feature disclosed herein, all combinations that do not detrimentally affect the systems, compositions, processes, and/or methods described herein are contemplated with or without explicit description of the particular combination. Additionally, unless explicitly recited otherwise, any aspect, and/or statement, and/or feature disclosed herein can be combined to describe conceived processes and systems consistent with the present disclosure. The terms “a,” “an,” and “the” are intended to include plural alternatives, e.g., at least one, unless otherwise specified. For instance, the disclosure of “an organoaluminum compound” is meant to encompass one, or combinations of more than one, organoaluminum compound, unless otherwise specified. Generally, groups of elements are indicated using the numbering scheme indicated in the version of the periodic table of elements published inChemical and Engineering News,63(5), 27, 1985. In some instances, a group of elements can be indicated using a common name assigned to the group; for example, alkali metals for Group 1 elements, alkaline earth metals for Group 2 elements, transition metals for Group 3-12 elements, and halogens or halides for Group 17 elements. For any particular compound or group disclosed herein, any name or structure presented is intended to encompass all conformational isomers, regioisomers, stereoisomers, and mixtures thereof that can arise from a particular set of substituents, unless otherwise specified. The name or structure also encompasses all enantiomers, diastereomers, and other optical isomers (if there are any), whether in enantiomeric or racemic forms, as well as mixtures of stereoisomers, as would be recognized by a skilled artisan, unless otherwise specified. For example, a general reference to hexene (or hexenes) includes all linear or branched, acyclic or cyclic, hydrocarbon compounds having six carbon atoms and 1 carbon-carbon double bond; a general reference to pentane includes n-pentane, 2-methyl-butane, and 2,2-dimethylpropane; and a general reference to a butyl group includes an n-butyl group, a sec-butyl group, an iso-butyl group, and a t-butyl group. A chemical “group” is described according to how that group is formally derived from a reference or “parent” compound, for example, by the number of hydrogen atoms formally removed from the parent compound to generate the group, even if that group is not literally synthesized in this manner By way of example, an “alkyl group” formally can be derived by removing one hydrogen atom from an alkane, while an “alkylene group” formally can be derived by removing two hydrogen atoms from an alkane. Moreover, a more general term can be used to encompass a variety of groups that formally are derived by removing any number (“one or more”) hydrogen atoms from a parent compound, which in this example can be described as an “alkane group,” and which encompasses an “alkyl group,” an “alkylene group,” and materials have three or more hydrogens atoms, as necessary for the situation, removed from the alkane. Throughout, the disclosure of a substituent, ligand, or other chemical moiety can constitute a particular “group” implies that the well-known rules of chemical structure and bonding are followed when that group is employed as described. When describing a group as being “derived by,” “derived from,” “formed by,” or “formed from,” such terms are used in a formal sense and are not intended to reflect any specific synthetic methods or procedure, unless specified otherwise or the context requires otherwise. The term “organyl group” is used herein in accordance with the definition specified by IUPAC: an organic substituent group, regardless of functional type, having one free valence at a carbon atom. Similarly, an “organylene group” refers to an organic group, regardless of functional type, derived by removing two hydrogen atoms from an organic compound, either two hydrogen atoms from one carbon atom or one hydrogen atom from each of two different carbon atoms. An “organic group” refers to a generalized group formed by removing one or more hydrogen atoms from carbon atoms of an organic compound. Thus, an “organyl group,” an “organylene group,” and an “organic group” can contain organic functional group(s) and/or atom(s) other than carbon and hydrogen, that is, an organic group can comprise functional groups and/or atoms in addition to carbon and hydrogen. An “organyl group,” “organylene group,” or “organic group” can be aliphatic, (inclusive of being cyclic or acyclic, or linear or branched), or can be aromatic. For the purposes of this application, the term or variations of the term “organyl group consisting of inert functional groups” refers to an organyl group wherein the organic functional group(s) and/or atom(s) other than carbon and hydrogen present in the functional group are restricted to those functional group(s) and/or atom(s) other than carbon and hydrogen which do not complex with a metal compound and/or are inert under the process conditions defined herein. Thus, the term or variation of the term “organyl group consisting of inert functional groups” further defines the particular organyl groups that can be present within the organyl group consisting of inert functional groups. Additionally, the term “organyl group consisting of inert functional groups” can refer to the presence of one or more inert functional groups within the organyl group. Similarly, an “organylene group consisting of inert functional groups” refers to an organic group formed by removing two hydrogen atoms from one or two carbon atoms of an organic compound consisting of inert functional groups and an “organic group consisting of inert functional groups” refers to a generalized organic group consisting of inert functional groups formed by removing one or more hydrogen atoms from one or more carbon atoms of an organic compound consisting of inert functional groups. For purposes of this application, an “inert functional group” is a group which does not substantially interfere with the processes described herein in which the material having an inert functional group takes part and/or does not complex with the metal compound of the metal complex. The term “does not complex with the metal compound” can include groups that could complex with a metal compound but in particular molecules described herein may not complex with a metal compound due to its positional relationship within a ligand. For example, while an ether group can complex with a metal compound, an ether group located at a para position of a substituted phenyl phosphinyl group can be an inert functional group because a single metal compound cannot complex with both the para ether group and the N2-phosphinyl formamidine group of the same metal complex molecule. Thus, the inertness of a particular functional group is not only related to the functional group's inherent inability to complex the metal compound but can also be related to the functional group's position within the metal complex. Non-limiting examples of inert functional groups which do not substantially interfere with processes described herein can include halo (fluoro, chloro, bromo, and iodo), nitro, hydrocarboxy groups (e.g., alkoxy, and/or aroxy, among others), and/or sulfidyl groups, among others. The term “hydrocarbon” whenever used in this specification and claims refers to a compound containing only carbon and hydrogen. Other identifiers can be utilized to indicate the presence of particular groups in the hydrocarbon (e.g. halogenated hydrocarbon indicates that the presence of one or more halogen atoms replacing an equivalent number of hydrogen atoms in the hydrocarbon). The term “hydrocarbyl group” is used herein in accordance with the definition specified by IUPAC: a univalent group formed by removing a hydrogen atom from a hydrocarbon. Similarly, a “hydrocarbylene group” refers to a group formed by removing two hydrogen atoms from a hydrocarbon, either two hydrogen atoms from one carbon atom or one hydrogen atom from each of two different carbon atoms. Therefore, in accordance with the terminology used herein, a “hydrocarbon group” refers to a generalized group formed by removing one or more hydrogen atoms (as necessary for the particular group) from a hydrocarbon. A “hydrocarbyl group,” “hydrocarbylene group,” and “hydrocarbon group” can be acyclic or cyclic groups, and/or can be linear or branched. A “hydrocarbyl group,” “hydrocarbylene group,” and “hydrocarbon group” can include rings, ring systems, aromatic rings, and aromatic ring systems, which contain only carbon and hydrogen. “Hydrocarbyl groups,” “hydrocarbylene groups,” and “hydrocarbon groups” include, by way of example, aryl, arylene, arene, alkyl, alkylene, alkane, cycloalkyl, cycloalkylene, cycloalkane, aralkyl, aralkylene, and aralkane groups, among other groups, as members. The term “alkane” whenever used in this specification and claims refers to a saturated hydrocarbon compound. Other identifiers can be utilized to indicate the presence of particular groups in the alkane (e.g. halogenated alkane indicates that the presence of one or more halogen atoms replacing an equivalent number of hydrogen atoms in the alkane). The term “alkyl group” is used herein in accordance with the definition specified by IUPAC: a univalent group formed by removing a hydrogen atom from an alkane. Similarly, an “alkylene group” refers to a group formed by removing two hydrogen atoms from an alkane (either two hydrogen atoms from one carbon atom or one hydrogen atom from two different carbon atoms). An “alkane group” is a general term that refers to a group formed by removing one or more hydrogen atoms (as necessary for the particular group) from an alkane. An “alkyl group,” “alkylene group,” and “alkane group” can be acyclic or cyclic groups, and/or can be linear or branched unless otherwise specified. Primary, secondary, and tertiary alkyl groups are derived by removal of a hydrogen atom from a primary, secondary, or tertiary carbon atom, respectively, of an alkane. The n-alkyl group can be derived by removal of a hydrogen atom from a terminal carbon atom of a linear alkane. The term “substituted” when used to describe a compound or group, for example, when referring to a substituted analog of a particular compound or group, is intended to describe any non-hydrogen moiety that formally replaces a hydrogen in that group, and is intended to be non-limiting. A group or groups can also be referred to herein as “unsubstituted” or by equivalent terms such as “non-substituted,” which refers to the original group in which a non-hydrogen moiety does not replace a hydrogen within that group. “Substituted” is intended to be non-limiting and include inorganic substituents or organic substituents. The term “olefin” whenever used in this specification and claims refers to hydrocarbons that have at least one carbon-carbon double bond that is not part of an aromatic ring or an aromatic ring system. The term “olefin” includes aliphatic and aromatic, cyclic and acyclic, and/or linear and branched hydrocarbons having at least one carbon-carbon double bond that is not part of an aromatic ring or ring system unless specifically stated otherwise. Olefins having only one, only two, only three, etc., carbon-carbon double bonds can be identified by use of the term “mono,” “di,” “tri,” etc., within the name of the olefin. The olefins can be further identified by the position of the carbon-carbon double bond(s). The term “alkene” whenever used in this specification and claims refers to a linear or branched aliphatic hydrocarbon olefin that has one or more carbon-carbon double bonds. Alkenes having only one, only two, only three, etc., such multiple bonds can be identified by use of the term “mono,” “di,” “tri,” etc., within the name. Alkenes can be further identified by the position of the carbon-carbon double bond(s). Other identifiers can be utilized to indicate the presence or absence of particular groups within an alkene. For example, a haloalkene refers to an alkene having one or more hydrogen atoms replaced with a halogen atom. The term “alpha olefin” as used in this specification and claims refers to an olefin that has a carbon-carbon double bond between the first and second carbon atoms of the longest contiguous chain of carbon atoms. The term “alpha olefin” includes linear and branched alpha olefins unless expressly stated otherwise. In the case of branched alpha olefins, a branch can be at the 2-position (a vinylidene) and/or the 3-position or higher with respect to the olefin double bond. The term “vinylidene” whenever used in this specification and claims refers to an alpha olefin having a branch at the 2-position with respect to the olefin double bond. By itself, the term “alpha olefin” does not indicate the presence or absence of other carbon-carbon double bonds unless explicitly indicated. The term “normal alpha olefin” whenever used in this specification and claims refers to a linear aliphatic mono-olefin having a carbon-carbon double bond between the first and second carbon atoms. It is noted that “normal alpha olefin” is not synonymous with “linear alpha olefin” as the term “linear alpha olefin” can include linear olefinic compounds having a double bond between the first and second carbon atoms and additional double bonds. The term “reaction zone effluent,” and it derivatives (e.g., oligomerization reaction zone effluent, trimerization reaction zone effluent, tetramerization reaction zone effluent, or trimerization and tetramerization reaction zone effluent) generally refers to all the material which exits the reaction zone through a reaction zone outlet/discharge which discharges a reaction mixture and can include reaction zone feed(s) (e.g., olefin, catalyst system or catalyst system components, and/or solvent), and/or reaction product (e.g., oligomer product including oligomers and non-oligomers, trimerization product including trimer and non-trimer, tetramerization product including tetramer and non-tetramer, or trimerization and tetramerization product including trimer and tetramer and non-trimer and tetramer). The term “reaction zone effluent” and its derivatives can be qualified to refer to certain portions by use of additional qualifying terms. For example, while reaction zone effluent refers to all material which exits the reaction zone through the reaction zone outlet/discharge, a reaction zone oligomer product effluent refers to only the oligomer product within the reaction zone effluent. The terms “room temperature” or “ambient temperature” are used herein to describe any temperature from 15° C. to 35° C. wherein no external heat or cooling source is directly applied to the reaction vessel. Accordingly, the terms “room temperature” and “ambient temperature” encompass the individual temperatures and any and all ranges, subranges, and combinations of subranges of temperatures from 15° C. to 35° C. wherein no external heating or cooling source is directly applied to the reaction vessel. The term “atmospheric pressure” is used herein to describe an earth air pressure wherein no external pressure modifying means is utilized. Generally, unless practiced at extreme earth altitudes, “atmospheric pressure” is about 1 atmosphere (alternatively, about 14.7 psi or about 101 kPa). Features within this disclosure that are provided as minimum values can be alternatively stated as “at least” or “greater than or equal to” any recited minimum value for the feature disclosed herein. Features within this disclosure that are provided as maximum values can be alternatively stated as “less than or equal to” or “below” any recited maximum value for the feature disclosed herein. Within this disclosure, the normal rules of organic nomenclature prevail. For instance, when referencing substituted compounds or groups, references to substitution patterns are taken to indicate that the indicated group(s) is (are) located at the indicated position and that all other non-indicated positions are hydrogen. For example, reference to a 4-substituted phenyl group indicates that there is a non-hydrogen substituent located at the 4-position and hydrogens located at the 2, 3, 5, and 6 positions. References to compounds or groups having substitutions at positions in addition to the indicated position can be referenced using comprising or some other alternative language. For example, a reference to a phenyl group comprising a substituent at the 4-position refers to a phenyl group having a non-hydrogen substituent at the 4-position and hydrogen or any non-hydrogen substituent at the 2, 3, 5, and 6 positions. The terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, do not depend upon the actual product or composition resulting from the contact or reaction of the initial components of the disclosed or claimed catalyst composition/mixture/system, the nature of the active catalytic site, or the fate of the organoaluminum compound and the heteroatomic ligand chromium compound complex after combining these components. Therefore, the terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, encompass the initial starting components of the composition, as well as whatever product(s) may result from contacting these initial starting components. The terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, may be used interchangeably throughout this disclosure. The term oligomer refers to a product that contains from 2 to 20 monomer units. The terms “oligomerization product” and “oligomer product” include all products made by the “oligomerization” process, including the “oligomers” and products which are not “oligomers” (e.g., products which contain more than 20 monomer units, or solid polymer). The term “oligomerization,” and its derivatives, refers to processes which produce an oligomer product comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % products comprising from 2 to 20 monomer units. In an example, an “oligomerization” process using ethylene as the monomer produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % oligomers having from 4 to 40 carbon atoms. The term “trimerization,” and its derivatives, refers to a process which produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % products comprising three and only three monomer units. A “trimer” is a product which comprises three and only three monomer units. A “trimerization product” includes all products made by the trimerization process including trimer and products which are not trimer (e.g., dimers or tetramers, solid polymer). In an example, a “trimerization” process using ethylene as the monomer produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % hexenes. The term “tetramerization,” and its derivatives, refers to a process which produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % products comprising four and only four monomer units. A “tetramer” is a product which comprises four and only four monomer units. A “tetramerization product” includes all products made by the tetramerization process including tetramer and products which are not tetramer (e.g., dimers or trimers, solid polymer). In an example, a “tetramerization” process using ethylene as the monomer produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % octenes. The term “trimerization and tetramerization,” and its derivatives, refers to a process which produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % products comprising three and/or four and only three and/or four monomer units. A “trimerization and tetramerization product” includes all products made by the “trimerization and tetramerization” process including trimer, tetramer, and products which are not trimer and tetramer (e.g., dimers, solid polymer). In an example, a “trimerization and tetramerization” process using ethylene as the monomer produces a mixture of products comprising at least 20 wt. %, 35 wt. %, 50 wt. %, or 60 wt. % hexenes and octenes. Within this specification, the word “reactor” refers to a single piece of equipment, such as, for example, a vessel, in which a reaction takes place, but excludes any associated equipment such as piping, pumps, and the like which is external to the vessel. Examples of reactors include stirred tank reactors (e.g., a continuous stirred tank reactor), plug flow reactors, or any other type of reactor. Within this specification, term “reaction zone” refers to the portion of a reaction system where all the necessary reaction components and reaction conditions are present such that the reaction can occur at a desired rate. That is to say that the reaction zone begins where the necessary reaction components and reaction conditions are present to maintain the reaction within 25 percent of the average reaction rate and the reaction system ends where the conditions do not maintain a reaction rate within 25 percent of the average reaction rate (based upon a volume average of the reaction rate of the reaction zone). For example, in terms of an ethylene oligomerization process, the reaction zone begins at the point where sufficient ethylene and active catalyst system is present under the sufficient reaction conditions (e.g., temperature and/or pressure, among others) to maintain oligomer product production at the desired rate and the reaction zone ends at a point where either the catalyst system is deactivated, sufficient ethylene is not present to sustain oligomer product production, or other reaction conditions (e.g., temperature and/or pressure, among others) are not sufficient to maintain the oligomer product production or the desired oligomer product production rate. Within this specification the “reaction zone” can comprise one or more reactors. The term “reaction zone” can be qualified to refer to more specific “reaction zones” by use of additional qualifying terms. For example, the use of the term “oligomerization reaction zone” indicates that the desired reaction within the “reaction zone” is an oligomerization. The term “reaction system” refers to all of the equipment to produce a product. The term “reaction system” includes reactors, reaction zones, and all the associated equipment, associated process lines, and control equipment which can bring the necessary component(s) into and out of the reaction system and control the reaction. Within this specification the “reaction system” can comprise one or more reactor zones, one or more reactors, and associated equipment to produce a product. The term “reaction system” can be qualified to refer to more specific “reaction systems” by use of additional qualifying terms. For example, the use of the term “oligomerization reaction system” indicates that the “reaction system” relates to an oligomerization. Catalyst system productivity is defined herein in units of kilograms of a normal alpha olefin product produced per gram of chromium of the heteroatomic ligand chromium compound complex (or chromium compound) utilized in the catalyst system per hour—kg NAO/g Cr/hr. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the processes and reaction systems, the typical methods and materials are herein described. All publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the present disclosure. DETAILED DESCRIPTION OF THE INVENTION Disclosed herein are ethylene oligomerization reaction systems and ethylene oligomerization processes that utilize a homogeneous chromium-based catalyst system to produce various oligomer products, such as 1-hexene and/or 1-octene. In these oligomerization reaction systems and oligomerization processes, an activation vessel is employed to activate the catalyst system and to improve catalyst productivity and efficiency in the oligomerization reaction zone. Incomplete catalyst activation can often lead to a variety of negative effects in the reaction zone, including the co-generation of by-products such as cyclopentane/cyclopentanes, mixed C10s, C12s, and C14s, and solid polyethylene/polymer. In particular, the formation of polymer leads to reactor wall fouling that can cause reduced run time, loss of heat transfer for cooling the exothermic reaction, and poor overall reactor performance. Therefore, reducing or eliminating the formation of polymer is important for any ethylene oligomerization process and oligomerization reaction system. This is even more important if reactor temperatures are below the melting temperature of the polymers and/or wax products that are being generated. Catalyst system activation of many chromium-based complexes with an organoaluminum compound, such as MAO, is not instantaneous. While not being bound by theory, it is believed that there can be an induction period for the catalyst system, after contact with ethylene, and before the catalyst system reaches it full catalytic activity. This induction period can be very brief (e.g., less than 5 sec), or it can range from 1-5 min to 20-30 min, to 1-2 hr, to 4 hr, or more. It is further believed that activating the catalyst system directly in the oligomerization reactor in the presence of ethylene reduces overall catalyst system performance and leads to inefficient use of the catalyst system components. As disclosed herein, activating the catalyst system in an activation vessel under a controlled set of conditions for a specified residence time that can eliminate some or all of the induction period in the oligomerization reaction zone can improve overall catalyst system performance and/or oligomer product discharge rate. Operating at a residence time that is substantially the same as the induction time of the particular catalyst system (and such that minimal to no ethylene oligomerization occurs in the activation vessel) can ensure that the catalyst system is at peak activity and efficiency (such as observed by oligomer product discharge rate) as it enters the oligomerization reaction zone. Thus, the total active catalytic species present in the oligomerization reaction zone is maximized, resulting in improved overall catalyst system productivity and improved catalyst system component usage (and reduced waste). Catalyst Systems The processes and reaction systems disclosed herein can utilize a catalyst system (or catalyst system mixture) comprising i) a heteroatomic ligand chromium compound complex and an organoaluminum compound or ii) a heteroatomic ligand, a chromium compound, and an organoaluminum compound; alternatively, a heteroatomic ligand chromium compound complex and an organoaluminum compound; or alternatively, a heteroatomic ligand, a chromium compound, and an organoaluminum compound. In an aspect, the catalyst system (or catalyst system mixture) can further comprise (optionally) a catalyst system organic medium. In some aspects, the catalyst system (or catalyst system mixture) comprising i) a heteroatomic ligand chromium compound complex and an organoaluminum compound or ii) a heteroatomic ligand, a chromium compound, and an organoaluminum compound) can be introduced into the reaction mixture within the reaction zone. In other aspects, at least one of i) a heteroatomic ligand chromium compound complex and an organoaluminum compound or ii) a heteroatomic ligand, a chromium compound, and an organoaluminum compound can be separately introduced into a mixture in an activation vessel from other components of the catalyst system (or catalyst system mixture). The heteroatomic ligand, the chromium compound, the heteroatomic ligand chromium compound complex, the heteroatomic ligand of the heteroatomic ligand chromium compound complex, the chromium compound of the heteroatomic ligand chromium compound, the organoaluminum compound, and the optional catalyst system organic medium are independent elements of the processes and reaction systems described herein and are independently described herein. These independently described catalyst system (or catalyst system mixture) elements can be utilized in any combination, and without limitation, to further describe the processes and reaction systems provided herein. Non-limiting examples of suitable catalyst system organic mediums include hydrocarbons, such as aromatic hydrocarbons. Suitable aromatic hydrocarbons can include isolated refinery aromatic streams containing mixtures comprising aromatic hydrocarbons (e.g., aromatic streams comprising C8and C9aromatic hydrocarbons like Total Atosol 100, ExxonMobil A100, and Shell Solv100, or other streams containing xylenes, cumene, or ethylbenzene, among others). Alternatively, suitable aromatic hydrocarbons can include predominately single carbon number aromatic hydrocarbon compound streams (e.g., benzene, toluene, xylenes, cumene, or ethylbenzene). Aromatic hydrocarbons that can be utilized singly or in any combination include benzene, toluene, xylene (including ortho-xylene, meta-xylene, para-xylene, or mixtures thereof), cumene, and ethylbenzene, or combinations thereof; alternatively, benzene; alternatively, toluene; alternatively, xylene (including ortho-xylene, meta-xylene, para-xylene or mixtures thereof); alternatively, cumene; or alternatively, ethylbenzene. In a particular aspect of this disclosure, the catalyst system organic medium can comprise, or consist essentially of, or consist of, ethylbenzene. Generally, the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex can be any heteroatomic ligand, which when utilized in the catalyst systems (or catalyst system mixtures) described herein for the processes and/or reaction systems described herein, can form an oligomer product in the reaction zone. In an aspect, the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex can be a neutral heteroatomic ligand or an anionic heteroatomic ligand; alternatively, a neutral heteroatomic ligand; or alternatively, an anionic heteroatomic ligand. In an aspect, the neutral heteroatomic ligand can comprise one or more heteroatomic complexing moieties; alternatively, two heteroatomic complexing; or alternatively, three heteroatomic complexing moieties. In an aspect, the anionic heteroatomic ligand can also comprise one or more neutral heteroatomic complexing moieties; alternatively, two heteroatomic complexing; or alternatively, three heteroatomic complexing moieties. In an aspect, the each neutral heteroatomic complexing moiety of the neutral ligand or the anionic ligand comprising a neutral heteroatomic complexing moiety independently can be an ether group, a sulfide group, an amine group, an imine group, a phosphine group, a phosphinite group, a phosphonite group, or a phosphite group; alternatively, an ether group, a sulfide group, an amine group, an imine group, or a phosphine group; alternatively, an ether group; alternatively, a sulfide group; alternatively, an amine group; alternatively, an imine group; or alternatively, a phosphine group. In an aspect, the anion atom of the anionic heteroatomic ligand (which forms a covalent or ionic bond with the chromium of the chromium compound) can be an anionic carbon atom, an anionic oxygen atom, or an anion nitrogen atom; alternatively, an anionic carbon atom; alternatively, an anionic oxygen atom; or alternatively, an anion nitrogen atom. In any aspect, the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex can comprise, can consist essentially of, or can be, an N2-phosphinyl formamidine, an N2-phosphinyl amidine, an N2-phosphinyl guanidine, a heterocyclic 2-[(phosphinyl)aminyl]imine, or any combination thereof; alternatively, an N2-phosphinyl formamidine; alternatively, an N2-phosphinyl amidine; alternatively, an N2-phosphinyl guanidine; or alternatively, a heterocyclic 2-[(phosphinyl)aminyl]imine Generally, the an N2-phosphinyl formamidine can have Structure NPF1, the N2-phosphinyl amidine can have Structure NPA1, the N2-phosphinyl guanidine can have Structure Gu1, Structure Gu2, Structure Gu3, Structure Gu4, or Structure Gu5, and the heterocyclic 2-[(phosphinyl)aminyl]imine can have structure HCPA1. In some aspects, the N2-phosphinyl guanidine have Structure Gu2, Structure Gu3, or Structure Gu4; alternatively, Structure Gu1; alternatively, Structure Gu2; alternatively, Structure Gu3; alternatively, Structure Gu4; or alternatively Structure Gu5. In any aspect, the heteroatomic ligand chromium compound complex can comprise, can consist essentially of, or can be, an N2-phosphinyl formamidine chromium compound complex, an N2-phosphinyl amidine chromium compound complex, an N2-phosphinyl guanidine chromium compound complex, a heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex, or any combination thereof; alternatively, an N2-phosphinyl formamidine chromium compound complex; alternatively, an N2-phosphinyl amidine chromium compound complex; alternatively, an N2-phosphinyl guanidine chromium compound complex; alternatively, an N2-phosphinyl guanidine chromium compound complex; or alternatively, a heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex. Generally, the an N2-phosphinyl formamidine chromium compound complex can have Structure NPFCr1, the N2-phosphinyl amidine chromium compound complex can have Structure NPACr1, the N2-phosphinyl guanidine chromium compound complex can have Structure GuCr1, Structure GuCr2, Structure GuCr3, Structure GuCr4, or Structure GuCr5, and the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex can have Structure HCPACr1. In some aspects, the N2-phosphinyl guanidine chromium compound complex have Structure GuCr2, Structure GuCr3, or Structure GuCr4; alternatively, Structure GuCr1; alternatively, Structure GuCr2; alternatively, Structure GuCr3; alternatively, Structure GuCr4; or alternatively Structure GuCr5. Within the N2-phosphinyl formamidines, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, and the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes the nitrogen participating in a double bond with the central carbon atom is referred to as the N1nitrogen and the nitrogen atom participating in a single bond with the central carbon atom is referred to as the N2nitrogen. Similarly, within the N2-phosphinyl guanidines and the N2-phosphinyl guanidine chromium compound complexes, the nitrogen participating in a double bond with the central carbon atom of the guanidine core is referred to as the N1nitrogen, the nitrogen atom participating in a single bond with the central carbon atom of the guanidine core and a bond with the phosphorus atom of the phosphinyl group is referred to as the N2nitrogen, and the remaining nitrogen atom participating in a single bond with the central carbon atom of the guanidine core is referred to as the N3nitrogen. It should be noted that the guanidine group of the guanidine in the N2-phosphinyl guanidines and the N2-phosphinyl guanidine chromium complexes can be a portion of a larger group which does not contain guanidine in it name. For example, while the compound 7-dimethylphosphinylimidazo[1,2-a]imidazole could be classified as a compound having an imidazo[1,2-a]imidazole core (or a compound having a phosphinylimidazo[1,2-a]imidazole group), 7-dimethylphosphinylimidazo[1,2-a]imidazole would still be classified as a compound having a guanidine core (or as a compound having an guanidine group) since it contains the defined general structure of the guanidine compound. R1, R3, R4, and R5within the N2-phosphinyl formamidine structures and the N2-phosphinyl formamidine chromium compound complex structures, R1, R2, R3, R4, and R5within the N2-phosphinyl amidine structures and the N2-phosphinyl amidine chromium compound complex structures, R1, R2a, R2b, R3, R4, R5, L12, L22, and L23within the N2-phosphinyl guanidine structures and the N2-phosphinyl guanidine chromium compound complex structures, and L12, T, R3, R4, and R5within the heterocyclic 2-[(phosphinyl)aminyl]imine structures and heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex structures are independently described herein and can be utilized in any combination and without limitation to further describe the N2-phosphinyl formamidine structures, the N2-phosphinyl formamidine chromium compound complex structures, the N2-phosphinyl amidine structures, the N2-phosphinyl amidine chromium compound complex structures, the N2-phosphinyl guanidine structures, the N2-phosphinyl guanidine chromium compound complex structures, the heterocyclic 2-[(phosphinyl)aminyl]imine structures, and the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex structures disclosed herein. Xp, Q, and q of the N2-phosphinyl formamidine chromium compound complex structures, the N2-phosphinyl amidine chromium compound complex structures, the N2-phosphinyl guanidine chromium compound complex structures, and the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex structures are independently described herein and can be utilized in any combination, and without limitation, to further describe the N2-phosphinyl formamidine chromium compound complex structures, the N2-phosphinyl amidine chromium compound complex structures, the N2-phosphinyl guanidine chromium compound complex structures, and the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex structures disclosed herein. Additionally, the independent descriptions of Xp, Q, and q can be combined, without limitation, with the independently described R1, R2, R2a, R2b, R3, R4, R5, L12, L22, and L23to further describe the appropriate N2-phosphinyl formamidine chromium compound complex structures, the N2-phosphinyl amidine chromium compound complex structures, the N2-phosphinyl guanidine chromium compound complex structures, and the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex structures contemplated herein. Generally, R1of the N2-phosphinyl formamidines, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, and/or the N2-phosphinyl guanidine chromium compound complexes which have an R1group can be an organyl group; alternatively, an organyl group consisting of inert functional groups; or alternatively, a hydrocarbyl group. In an aspect, the R1organyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In an aspect, the R1organyl group consisting of inert functional groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In an aspect, the R1hydrocarbyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In an aspect, R1of the N2-phosphinyl formamidines, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, and/or the N2-phosphinyl guanidine chromium compound complexes which have an R1group can be an alkyl group, a substituted alkyl group, a cycloalkyl group, a substituted cycloalkyl group, an aryl group, a substituted aryl group, an aralkyl group, or a substituted aralkyl group; alternatively an alkyl group or a substituted alkyl group; alternatively, a cycloalkyl group or a substituted cycloalkyl group; alternatively, an aryl group or a substituted aryl group; alternatively, an aralkyl group or a substituted aralkyl group; alternatively, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group; alternatively, an alkyl group; alternatively, a substituted alkyl group, alternatively, a cycloalkyl group; alternatively, a substituted cycloalkyl group; alternatively, an aryl group; alternatively, a substituted aryl group; alternatively, an aralkyl group; or alternatively, a substituted aralkyl group. In any aspect disclosed herein, the R1alkyl group can be a C1to C20, a C1to C10, or a C1to C5alkyl group. In any aspect disclosed herein, the R1substituted alkyl group can be a C1to C20, a C1to C10, or a C1to C5substituted alkyl group. In any aspect disclosed herein, the R1cycloalkyl group can be a C4to C20, a C4to C15, or a C4to C10cycloalkyl group. In any aspect disclosed herein, the R1substituted cycloalkyl group can be a C4to C20, a C4to C15, or a C4to C10substituted cycloalkyl group. In any aspect disclosed herein, the R1aryl group can be a C6to C20, a C6to C15, or a C6to C10aryl group. In any aspect disclosed herein, the R1substituted aryl group can be a C6to C20, a C6to C15, or a C6to C10substituted aryl group. In any aspect disclosed herein, the R1aralkyl group can be a C7to C20, a C7to C15, or a C7to C10aralkyl group. In any aspect disclosed herein, the R1substituted aralkyl group can be a C7to C20, a C7to C15, or a C7to C10substituted aralkyl group. Each substituent of a substituted alkyl group (general or specific), a substituted cycloalkyl group (general or specific), a substituted aryl group (general or specific), and/or substituted aralkyl group (general or specific) can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted R1group. In an aspect, R1can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group; or alternatively, a methyl group, an ethyl group, a n-propyl (1-propyl) group, an iso-propyl (2-propyl) group, a tert-butyl (2-methyl-2-propyl) group, or a neopentyl (2,2-dimethyl-1-propyl) group. In some aspects, the alkyl groups which can be utilized as R1can be substituted. Each substituent of a substituted alkyl group (general or specific) independently can be a halogen or a hydrocarboxy group; alternatively, a halogen; or alternatively, a hydrocarboxy group. Substituent halogens and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted alkyl group which can be utilized as R1. In an aspect, R1can be a cyclopentyl group, a substituted cyclopentyl group, a cyclohexyl group, or a substituted cyclohexyl group; alternatively, a cyclopentyl group or a substituted cyclopentyl group; or alternatively, a cyclohexyl group or a substituted cyclohexyl group. In an aspect, the substituted cycloalkyl group, which can be utilized as R1, can be a 2-substituted cyclohexyl group, a 2,6-disubstituted cyclohexyl group, a 2-substituted cyclopentyl group, or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2,6-disubstituted cyclohexyl group; alternatively, a 2-substituted cyclopentyl group or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2-substituted cyclopentyl group; or alternatively, a 2,6-disubstituted cyclohexyl group or a 2,5-disubstituted cyclopentyl group. In an aspect, one or more substituents of a multi-substituted cycloalkyl group utilized as R1can be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group can be different. Each substituent of a substituted cycloalkyl group having a specified number of ring carbon atoms independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy (general and specific) groups are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted cycloalkyl group (general or specific) which can be utilized as R1. In a non-limiting aspect, R1can be a cyclohexyl group, a 2-alkylcyclohexyl group, or a 2,6-dialkylcyclohexyl group; or alternatively, a cyclopentyl group, a 2-alkylcyclopentyl group, or a 2,5-dialkylcyclopentyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe alkylcyclohexyl groups (general and specific), dialkylcyclohexyl groups (general and specific), alkylcyclopentyl groups (general or specific), and/or dialkylcyclopentyl groups (general and specific) which can be utilized as R1. Generally, the alkyl substituents of a dialkylcyclohexyl group or a dialkylcyclopentyl group can be the same; or alternatively, the alkyl substituents of a dialkylcyclohexyl group or a dialkylcyclopentyl group can be different. In some non-limiting aspects, R1can be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, a 2-tert-butylcyclohexyl group, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In other non-limiting aspects, R1can be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, or a 2-tert-butylcyclohexyl group; or alternatively, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In an aspect, R1can be a phenyl group, a substituted phenyl group; alternatively, a phenyl group; or alternatively, a substituted phenyl group. In an aspect, the substituted phenyl group, which can be utilized as R1, can be a 2-substituted phenyl group, a 3-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, a 2,6-disubstituted phenyl group, a 3,5-disubstituted phenyl group, or a 2,4,6-trisubstituted phenyl group; alternatively, a 2-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, or a 2,6-disubstituted phenyl group; alternatively, a 3-substituted phenyl group or a 3,5-disubstituted phenyl group; alternatively, a 2-substituted phenyl group or a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group or a 2,6-disubstituted phenyl group; or alternatively, a 2,4,6-trisubstituted phenyl group. In an aspect, one or more substituents of a multi-substituted phenyl group utilized as R1can be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group different. Each substituent of a substituted phenyl group (general or specific) independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted phenyl group (general or specific) which can be utilized as R1. In a non-limiting aspect, R1can be a phenyl group, a 2-alkylphenyl group, a 3-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group a 2,6-dialkylphenyl group, a 3,5-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group or a 4-alkylphenyl group; alternatively, a 2,4-dialkylphenyl group or a 2,6-dialkylphenyl group; alternatively, a 3-alkylphenyl group or a 3,5-dialkylphenyl group; alternatively, a 2-alkylphenyl group or a 2,6-dialkylphenyl group; or alternatively, a 2,4,6-trialkylphenyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe any alkyl substituted phenyl group which can be utilized as R. Generally, the alkyl substituents of a dialkylphenyl group (general or specific) or a trialkylphenyl group (general or specific) can be the same; or alternatively, the alkyl substituents of a dialkylphenyl group or trialkylphenyl group can be different. In some non-limiting aspects, R1independently can be a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, a 2-tert-butylphenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group; alternatively, a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, or a 2-tert-butylphenyl group; alternatively, a phenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group. In an aspect, R1can be a benzyl group or a substituted benzyl group; alternatively, a benzyl group; or alternatively, a substituted benzyl group. Each substituent of a substituted benzyl group independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted benzyl group (general or specific) which can be utilized as R1. Generally, R2of the N2-phosphinyl amidines and/or the N2-phosphinyl amidine chromium compound complexes can be an organyl group; alternatively, an organyl group consisting of inert functional groups; or alternatively, a hydrocarbyl group. In an aspect, the R2organyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In an aspect, R2organyl group consisting of inert functional groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In an aspect, R2hydrocarbyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In an aspect, R2of the N2-phosphinyl amidines and/or the N2-phosphinyl amidine chromium compound complexes can be an acyl group or a substituted acyl group; an acyl group; or alternatively, a substituted acyl group. In an aspect, the acyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5acyl group. In an aspect, the substituted acyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5a substituted acyl group. In some aspects, R2of the N2-phosphinyl amidines and/or the N2-phosphinyl amidine chromium compound complexes can be an alkanoyl group, a substituted alkanoyl group, a benzoyl group, or a substituted benzoyl group; alternatively, an alkanoyl group or a substituted alkanoyl group; alternatively, a benzoyl group, or a substituted benzoyl group; alternatively, an alkanoyl group; alternatively, a substituted alkanoyl group; alternatively, a benzoyl group; or alternatively, a substituted benzoyl group. In any aspect disclosed herein, the R2alkanoyl group can be a C1to C20, a C1to C10, or a C1to C5alkanoyl group. In any aspect disclosed herein, the R2substituted alkanoyl group can be a C1to C20, a C1to C10, or a C1to C5substituted R2alkanoyl group. In any aspect disclosed herein, the R2benzoyl group can be a C7to C20, a C7to C15, or a C7to C10benzoyl group. In any aspect disclosed herein, the R2substituted benzoyl group can be a C7to C20, a C1to C15, or a C1to C10substituted R2benzoyl group. Each substituent of a substituted alkanoyl group (general or specific), and/or substituted benzoyl group (general or specific) can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe substituted alkanoyl groups and/or substituted benzoyl group which can be utilized as R2. In an aspect, R2of the N2-phosphinyl amidines and/or the N2-phosphinyl amidine chromium compound complexes can be an alkyl group, a substituted alkyl group, a cycloalkyl group, a substituted cycloalkyl group, an aryl group, a substituted aryl group, an aralkyl group, or a substituted aralkyl group; alternatively, an alkyl group or a substituted alkyl group; alternatively, a cycloalkyl group or a substituted cycloalkyl group; alternatively, an aryl group or a substituted aryl group; alternatively, an aralkyl group or a substituted aralkyl group; or alternatively, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. In other aspects, R2of the N2-phosphinyl amidine and/or the N2-phosphinyl amidine chromium compound complexes can be an alkyl group; alternatively, a substituted alkyl group, alternatively, a cycloalkyl group; alternatively, a substituted cycloalkyl group; alternatively, an aryl group; alternatively, a substituted aryl group; alternatively, an aralkyl group; or alternatively, a substituted aralkyl group. In any aspect disclosed herein, the R2alkyl group can be a C1to C20, a C1to C10, or a C1to C5alkyl group. In any aspect disclosed herein, the R2substituted alkyl group can be a C1to C20, a C1to C10, or a C1to C5substituted alkyl group. In any aspect disclosed herein, the R2cycloalkyl group can be a C4to C20, a C4to C15, or a C4to C10cycloalkyl group. In any aspect disclosed herein, the R2substituted cycloalkyl group can be a C4to C20, a C4to C15, or a C4to C10substituted cycloalkyl group. In any aspect disclosed herein, the R2aryl group can be a C6to C20, a C6to C15, or a C6to C10aryl group. In any aspect disclosed herein, the R2substituted aryl group can be a C6to C20, a C6to C15, or a C6to C10substituted aryl group. In any aspect disclosed herein, the R2aralkyl group can be a C7to C20, a C7to C15, or a C7to C10aralkyl group. In any aspect disclosed herein, the R2substituted aryl group can be a C7to C20, a C7to C15, or a C7to C10substituted aralkyl group. Each substituent of a substituted alkyl group (general or specific), a substituted cycloalkyl group (general or specific), a substituted aryl group (general or specific), and/or substituted aralkyl group (general or specific) can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe R2. In an aspect, R2can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group; or alternatively, a methyl group, an ethyl group, an n-propyl (1-propyl) group, an iso-propyl (2-propyl) group, a tert-butyl (2-methyl-2-propyl) group, or a neopentyl (2,2-dimethyl-1-propyl) group. In some aspects, the alkyl groups which can be utilized as R2can be substituted. Each substituent of a substituted alkyl group independently can be a halogen or a hydrocarboxy group; alternatively, a halogen; or alternatively, a hydrocarboxy group. Substituent halogens and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted alkyl group (general or specific) which can be utilized as R2. In an aspect, R2can be a cyclopentyl group, a substituted cyclopentyl group, a cyclohexyl group, or a substituted cyclohexyl group; alternatively, a cyclopentyl group or a substituted cyclopentyl group; or alternatively, a cyclohexyl group or a substituted cyclohexyl group. In an aspect, the substituted cycloalkyl group, which can be utilized as R2, can be a 2-substituted cyclohexyl group, a 2,6-disubstituted cyclohexyl group, a 2-substituted cyclopentyl group, or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2,6-disubstituted cyclohexyl group; alternatively, a 2-substituted cyclopentyl group or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2-substituted cyclopentyl group; or alternatively, a 2,6-disubstituted cyclohexyl group or a 2,5-disubstituted cyclopentyl group. In an aspect, one or more substituents of a multi-substituted cycloalkyl group utilized as R2can be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group can be different. Each substituent of a cycloalkyl group having a specified number of ring carbon atoms independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted cycloalkyl group (general or specific) which can be utilized as R2. In a non-limiting aspect, R2can be a cyclohexyl group, a 2-alkylcyclohexyl group, or a 2,6-dialkylcyclohexyl group; or alternatively, a cyclopentyl group, a 2-alkylcyclopentyl group, or a 2,5-dialkylcyclopentyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe alkylcyclohexyl groups (general or specific), dialkylcyclohexyl groups (general or specific), alkylcyclopentyl groups (general or specific), and/or dialkylcyclopentyl groups (general or specific) which can be utilized as R2. Generally, the alkyl substituents of a disubstituted cyclohexyl or cyclopentyl group can be the same; or alternatively, the alkyl substituents of a dialkyl cyclohexyl or cyclopentyl group can be different. In some non-limiting aspects, R2can be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, a 2-tert-butylcyclohexyl group, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In other non-limiting aspects, R2can be, a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, or a 2-tert-butylcyclohexyl group; or alternatively, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In an aspect, R2can be a phenyl group, a substituted phenyl group; alternatively, a phenyl group; or alternatively, a substituted phenyl group. In an aspect, the substituted phenyl group, which can be utilized as R2can be a 2-substituted phenyl group, a 3-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, a 2,6-disubstituted phenyl group, a 3,5-disubstituted phenyl group, or a 2,4,6-trisubstituted phenyl group; alternatively, a 2-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, or a 2,6-disubstituted phenyl group; alternatively, a 3-substituted phenyl group or a 3,5-disubstituted phenyl group; alternatively, a 2-substituted phenyl group or a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group or a 2,6-disubstituted phenyl group; or alternatively, a 2,4,6-trisubstituted phenyl group. In an aspect, one or more substituents of a multi-substituted phenyl group utilized as R2can be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group can be different. Each substituent of a substituted phenyl group (general or specific) independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted phenyl group (general or specific) which can be utilized as R2. In a non-limiting aspect, R2can be a phenyl group, a 2-alkylphenyl group, a 3-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group a 2,6-dialkylphenyl group, a 3,5-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group or a 4-alkylphenyl group; alternatively, a 2,4-dialkylphenyl group or a 2,6-dialkylphenyl group; alternatively, a 3-alkylphenyl group or a 3,5-dialkylphenyl group; alternatively, a 2-alkylphenyl group or a 2,6-dialkylphenyl group; or alternatively, a 2,4,6-trialkylphenyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe any alkyl substituted phenyl group which can be utilized as R2. Generally, the alkyl substituents of a dialkylphenyl group (general or specific) or trialkylphenyl group (general or specific) can be the same; or alternatively, the alkyl substituents of a dialkylphenyl group or trialkylphenyl group can be different. In some non-limiting aspects, R2independently can be a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, a 2-tert-butylphenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group; alternatively, phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, or a 2-tert-butylphenyl group; alternatively, a phenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group. In a non-limiting aspect, R2can be a phenyl group, a 2-alkoxyphenyl group, or a 4-alkoxyphenyl group. In some non-limiting aspects, R2can be a phenyl group, a 2-methoxyphenyl group, a 2-ethoxyphenyl group, a 2-isopropoxyphenyl group, a 2-tert-butoxyphenyl group, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-isopropoxyphenyl group, or a 4-tert-butoxyphenyl group; alternatively, a 2-methoxyphenyl group, a 2-ethoxyphenyl group, a 2-isopropoxyphenyl group, or a 2-tert-butoxyphenyl group; or alternatively, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-isopropoxyphenyl group, or a 4-tert-butoxyphenyl group. In another non-limiting aspect, R2can be a phenyl group, a 2-halophenyl group, a 4-halophenyl group, or a 2,6-dihalophenylgroup. Generally, the halides of a dihalophenyl group can be the same; or alternatively, the halides of a dihalophenyl group can be different. In some aspects, R2can be a phenyl group, a 2-fluorophenyl group, a 4-fluorophenyl group, or a 2,6-difluorophenyl group. In an aspect, R2can be a benzyl group or a substituted benzyl group; alternatively, a benzyl group; or alternatively, a substituted benzyl group. Each substituent of a substituted benzyl group independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted benzyl group which can be utilized as R2. In further aspects, R1and R2can be joined to form a ring or a ring system containing the carbon-nitrogen double bond of the N2-phosphinyl amidines and/or the N2-phosphinyl amidine chromium compound complexes. The joining of R1and R2can be designated as L12rand can be an organylene group; alternatively, an organylene group consisting of inert functional groups; alternatively, a hydrocarbylene group; or alternatively, an alkylene group. In an aspect, the L12rorganylene group, when present, can be a C3to C30, a C3to C20, a C3to C15, or a C3to C10organylene group. In some aspects, the L12rorganylene group consisting of inert functional groups, when present, can be a C3to C30, a C3to C20, a C3to C15, or a C3to C10organylene group consisting of inert functional groups. In other aspects, the L12rhydrocarbyl group, when present, independently can be a C3to C30, a C3to C20, a C3to C15, or a C3to C10hydrocarbylene group. In a further aspect, the L12ralkylene group, when present, independently ca n be a C3to C30, a C3to C20, a C3to C15, or a C3to C10alkylene group. In an aspect, L12, can be prop-1,3-ylene group, a propen-1,3-ylene group (—CH2CH═CH—), a but-1,3-ylene group, a but-1-en-1,3-ylene group (—CH═CHCH(CH3)—), a but-1-en-1,3-ylene group (—CH2CH═C(CH3)—), a 3-methylbut-1,3-ylene group (—CH2CH2C(CH3)2—), a 2-methylbut-1,3-ylene group (—CH2CH(CH3)CH(CH3)—), a 3-methylbut-1-en-1,3-ylene group (—CH═CHC(CH3)2—), a 2-methylbut-1,3-ylene group (—CH2C(CH3)═C(CH3)—) a but-1,4-ylene group a but-1-en-1,4-ylene group (—CH═CHCH2CH2)—), a but-2-en-1,4-ylene group (—CH2CH═CHCH2—), or a 1,4-pent-1,4-ylene group. Generally, T of the heterocyclic 2-[(phosphinyl)aminyl]imines and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes can be oxygen or sulfur. In and aspect, T of the heterocyclic 2-[(phosphinyl)aminyl]imines and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes can be oxygen; or alternatively, sulfur. Generally, R2aand/or R2b, of the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes which have an R2aand/or R2bgroup, independently can be hydrogen or an organyl group; alternatively, hydrogen or an organyl group consisting of inert functional groups; alternatively, hydrogen or a hydrocarbyl group; alternatively, hydrogen; alternatively, an organyl group; alternatively, an organyl group consisting of inert functional groups; or alternatively, a hydrocarbyl group. In an aspect, the R2aand/or R2borganyl groups independently can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In some aspects, the R2aand/or R2borganyl groups consisting of inert functional groups independently can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In other aspects, the R2aand/or R2bhydrocarbyl groups independently can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In an aspect, R2aand R2b, of the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes which have an R2aand/or R2borganyl group, independently can be an alkyl group, a substituted alkyl group, a cycloalkyl group, a substituted cycloalkyl group, an aryl group, a substituted aryl group, an aralkyl group, or a substituted aralkyl group; alternatively, an alkyl group or a substituted alkyl group; alternatively, a cycloalkyl group or a substituted cycloalkyl group; alternatively, an aryl group or a substituted aryl group; alternatively, an aralkyl group or a substituted aralkyl group; alternatively, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group; alternatively, an alkyl group; alternatively, a substituted alkyl group, alternatively, a cycloalkyl group; alternatively, a substituted cycloalkyl group; alternatively, an aryl group; alternatively, a substituted aryl group; alternatively, an aralkyl group; or alternatively, a substituted aralkyl group. In any aspect disclosed herein, the R2aand/or R2balkyl group independently can be C1to C20, a C1to C10, or a C1to C5alkyl group. In any aspect disclosed herein, the R2aand/or R2bcycloalkyl group independently can be a C4to C20, a C4to C15, or a C4to C10cycloalkyl group. In any aspect disclosed herein, the R2aand/or R2bsubstituted cycloalkyl group independently can be a C4to C20, a C4to C15, or a C4to C10substituted cycloalkyl group. In any aspect disclosed herein, the R2aand/or R2baryl group independently can be a C6to C20, a C6to C15, or a C6to C10aryl group. In any aspect disclosed herein, the R2aand/or R2bsubstituted aryl group independently can be a C6to C20, a C6to C15, or a C6to C10substituted aryl group. Each substituent of a substituted cycloalkyl group (general or specific) and/or a substituted aryl group (general or specific) can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe R2aand/or R2b. In an aspect, R1and R2aof the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes can be joined to form a group, L12, wherein L12, the N1nitrogen atom, and the N3nitrogen atom form a ring or a ring system. In another aspect, R3and R2bof the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes can be joined to form a group, L23, wherein L23, the N2nitrogen atom, and the N3nitrogen atom form a ring or a ring system. In an aspect, L12and/or L23, of the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes which have an L2group and/or an L23group, independently can be an organylene group; alternatively, an organylene group consisting of inert functional groups; or alternatively, a hydrocarbylene group. The L12and/or L23organylene groups independently can be a C2to C20, a C2to C15, a C2to C10, or a C2to C5organylene group. The L12and/or L23organylene groups consisting of inert functional groups independently can be a C2to C20, a C2to C15, a C2to C10, or a C2to C5organylene group consisting of inert functional groups. The L12and/or L23hydrocarbylene groups independently can be a C2to C20, a C2to C15, a C2to C10, or a C2to C5hydrocarbylene group. In an aspect, L12of the N2-phosphinyl guanidines, the N2-phosphinyl guanidine chromium compound complexes, the heterocyclic 2-[(phosphinyl)aminyl]imines and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes which have an L12, and L23of the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes which have an L23, can have any structure provided in Table 1. In some aspects, L12and/or L23can have Structure 1L, Structure 2L, Structure 3L, Structure 4L or Structure 5L. In some aspects, L12and/or L23can have Structure 2L or Structure 3L; alternatively, Structure 4L or Structure 5L. In other aspects, L12and/or L23can have Structure 1L; alternatively, Structure 2L; alternatively, Structure 3L; alternatively, Structure 4L; or alternatively, Structure 5L. In some N2-phosphinyl guanidine and N2-phosphinyl guanidine chromium compound complex aspects, L12and/or L23can have Structure 6L. It should be noted that when L12or L23has Structure 6L the corresponding R2bor R2ais null because of the double bond link with the N3nitrogen atom of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex. TABLE 1Structures for Linking Groups L12and/or L23.—(CRL1R12)m—Structure 1L—CRL3RL4—CRL5RL6—Structure 2L—CRL3RL4—CRL7RL8—CRL5RL6—Structure 3L—CRL11═CRL12—Structure 4LStructure 5L═CRL27—CRL28═CRL29—Structure 6L Within the structures of Table 1, the undesignated valences of L12and/or L23represent the points at which L12and/or L23, when present, attach to the respective nitrogen atoms of the N2-phosphinyl guanidine and the N2-phosphinyl guanidine chromium compound complex. Additionally, with the structures of Table 1, the undesignated valences of L12represent the points at which L12attach to T and the respective nitrogen atom of the heterocyclic 2-[(phosphinyl)aminyl]imine and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complex. Generally, m can be an integer ranging from 2 to 5. In further aspects, m can be 2 or 3; alternatively, m can be 2; or alternatively, m can be 3. RL1and RL2of the linking group having Structure 1L, RL3, RL4, RL5, and RL6of the linking group having Structure 2L, RL3, RL4, RL5, RL6, RL7, and RL8, of the linking group having Structure 3L, RL11and RL12of the linking group having Structure 4L, RL23, RL24, RL25, and RL26of the linking group having Structure 5L, RL27, RL28, and RL29of the linking group having Structure 6L independently can be a hydrogen or a non-hydrogen substituent group; or alternatively, hydrogen. Non-hydrogen substituent groups (general and specific) are independently disclosed herein and can be utilized without limitation to further describe the linking group having Structure 1L, Structure 2L, Structure 3L, Structure 4L, Structure 5L, and/or Structure 6L. In an aspect, L12and/or L23independently can be an eth-1,2-ylene group (—CH2CH2—), an ethen-1,2-ylene group (—CH═CH—), a prop-1,3-ylene group (—CH2CH2CH2—), a propen-1,2-ylene group (—C(CH3)═CH—), a propen-1,3-ylene group (—CH═CHCH2—), a but-1,3-ylene group (—CH2CH2CH(CH3)—), a 3-methylbut-1,3-ylene group (—CH2CH2C(CH3)2—), or a phen-1,2-ylene group. In some non-limiting aspects, L12and/or L23be an eth-1,2-ylene group (—CH2CH2—), a prop-1,3-ylene group (—CH2CH2CH2—), a propen-1,2-ylene group (—C(CH3)═CH—), a propen-1,3-ylene group (—CH═CHCH2—), a but-1,3-ylene group (—CH2CH2CH(CH3)—), or a 3-methylbut-1,3-ylene group (—CH2CH2C(CH3)2—); alternatively, an eth-1,2-ylene group (—CH2CH2—), an ethen-1,2-ylene group (—CH═CH—), a prop-1,3-ylene group (—CH2CH2CH2—), or a phen-1,2-ylene group; alternatively, an eth-1,2-ylene group (—CH2CH2—) or a prop-1,3-ylene group (—CH2CH2CH2—); alternatively, an ethen-1,2-ylene group (—CH═CH—) or a phen-1,2-ylene group; alternatively, an eth-1,2-ylene group (—CH2CH2—); alternatively, a prop-1,3-ylene group (—CH2CH2CH2—); alternatively, a propen-1,2-ylene group (—C(CH3)═CH—); alternatively, a propen-1,3-ylene group (—CH═CHCH2—); or alternatively, a phen-1,2-ylene group. In an aspect, L12can have a structure that can comprise at least one substituent located on the carbon atom attached to the N1nitrogen atom of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex; alternatively, can comprise only one substituent located on the carbon atom attached to the N1nitrogen atom of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex; or alternatively, can comprise two substituents located on the carbon atom attached to the N1nitrogen atom of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex. In another aspect, L12can have a structure that can consist of one substituent located on the carbon atom attached to the N1nitrogen atom the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex; or alternatively, can consist of two substituents located on the carbon atom attached to the N1nitrogen atom of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex. In an aspect, R2aand R2bof the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes can be joined to form a group, L22, wherein R2a, R2b, and the N3nitrogen (or L22and the N3nitrogen) form a ring or ring system. In an aspect, L22of the N2-phosphinyl guanidines and/or the N2-phosphinyl guanidine chromium compound complexes having an L2group can be an organylene group; alternatively, an organylene group consisting of inert functional groups; or alternatively, a hydrocarbylene group. The L22organylene group can be a C3to C20, a C3to C15, or a C3to C10organylene group. The L22organylene group consisting of inert functional groups can be a C3to C20, a C3to C15, or a C3to C10organylene group consisting of inert functional groups. The L22hydrocarbylene group can be a C4to C20, a C4to C15, or a C4to C10hydrocarbylene group. In an aspect, L22can have any structure provided in Table 2. In some aspects, L22can have Structure 11L, Structure 12L, Structure 13L, Structure 14L, Structure 15L, or Structure 16L. In other aspects, L22can have Structure 11L; alternatively, Structure 12L; alternatively, Structure 13L; alternatively, Structure 14L; or alternatively, Structure 15L. TABLE 2Structures for Linking Groups L22.—(CRL31RL32)n——CRL41RL42—CRL45RL46CRL47RL48CRL43RL44—Structure 11LStructure 12L—CRL41RL42—CRL45RL46—CRL49RL50—CRL47RL48—CRL43RL44—Structure 13L—CRL41RL42—CRL45RL46—O—CRL47RL48—CRL43RL44——CRL51═CRL53—CRL54═CRL52—Structure 14LStructure 15L Within the structures of Table 2, the undesignated valences represent the points at which L22of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex, when present, attach to the N3nitrogen atom of the N2-phosphinyl guanidine and/or the N2-phosphinyl guanidine chromium compound complex. Generally, n can be an integer ranging from 4 to 7. In further aspects, n can be 4 or 5; alternatively, n can be 4; or alternatively, n can be 5. RL31and RL32of the linking group having Structure 11L, RL41, RL42, RL43, RL44, RL45, RL46, RL47, and RL48of the linking group having Structure 12L, RL41, RL42, RL43, RL44, RL45, RL46, RL47, RL48, RL49, and RL50of the linking group having Structure 13L, RL41, RL42, RL43, RL44, RL45, RL46, RL47, and RL48of the linking group having Structure 14L, and RL41, RL42, RL43, RL44, RL45, RL46, RL47, and RL48of the linking group having Structure 15L independently can be a hydrogen or a non-hydrogen substituent group; alternatively, hydrogen. Non-hydrogen substituent groups are independently disclosed herein and can be utilized without limitation to further describe the linking group having Structure 11L, Structure 12L, Structure 13L, Structure 14L, and/or Structure 15L. In an aspect, L22can be a but-1,4-ylene group, a pent-1,4-ylene group, a pent-1,5-ylene group, a hex-2,5-ylene group, a hex-1,5-ylene group, a hept-2,5-ylene group, a buta-1,3-dien-1,4-ylene group, or a bis(eth-2-yl)ether group; or alternatively, a but-1,4-ylene group, a pent-1,5-ylene group, or a bis(eth-2-yl)ether group. Generally, R3of the N2-phosphinyl formamidines, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, the N2-phosphinyl guanidine chromium compound complexes, the heterocyclic 2-[(phosphinyl)aminyl]imines, and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes which have an R3group can be hydrogen or an organyl group; hydrogen or an organyl group consisting of inert functional group; alternatively, hydrogen or a hydrocarbyl group; alternatively, hydrogen; alternatively, an organyl group; alternatively, an organyl group consisting of inert functional group; or alternatively, a hydrocarbyl group. In an aspect, the R3organyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In an aspect, the R3organyl group consisting of inert functional groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In an aspect, the R3hydrocarbyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In other aspects, R3of the N2-phosphinyl formamidine, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, the N2-phosphinyl guanidine chromium compound complexes, the heterocyclic 2-[(phosphinyl)aminyl]imines, and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes which have an R3group can be a C1to C30, a C1to C20, a C1to C15, a C1to C10, or a C1to C5alkyl group. In yet other aspects, R3of the N2-phosphinyl formamidine, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, the N2-phosphinyl guanidine chromium compound complexes, the heterocyclic 2-[(phosphinyl)aminyl]imines, and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes can be a phenyl group or a C6to C20substituted phenyl group; alternatively, a phenyl group or a C6to C15substituted phenyl group; or alternatively, a phenyl group or a C6to C10substituted phenyl group. Substituent groups (general and specific) are provided herein and these substituent groups can be utilized to further describe the substituted phenyl groups which can be utilized as R3the N2-phosphinyl formamidine, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, and/or the N2-phosphinyl guanidine chromium compound complexes having a non-hydrogen R3group. Generally, R4and/or R5of the N2-phosphinyl formamidines, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, the N2-phosphinyl guanidine chromium compound complexes, the heterocyclic 2-[(phosphinyl)aminyl]imines, and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes independently can be an organyl group; alternatively, an organyl group consisting of inert functional groups; or alternatively, a hydrocarbyl group. In an aspect, the R4and/or R5organyl groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In an aspect, the R4and/or R5organyl groups consisting of inert functional groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In an aspect, the R4and/or R5hydrocarbyl groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In an aspect, R4and/or R5of the N2-phosphinyl formamidine, the N2-phosphinyl formamidine chromium compound complexes, the N2-phosphinyl amidines, the N2-phosphinyl amidine chromium compound complexes, the N2-phosphinyl guanidines, the N2-phosphinyl guanidine chromium compound complexes, the heterocyclic 2-[(phosphinyl)aminyl]imines, and/or the heterocyclic 2-[(phosphinyl)aminyl]imine chromium compound complexes independently can be an alkyl group, a substituted alkyl group, a cycloalkyl group, a substituted cycloalkyl group, an aryl group, a substituted aryl group, an aralkyl group, or a substituted aralkyl group; alternatively, an alkyl group or a substituted alkyl group; alternatively, a cycloalkyl group or a substituted cycloalkyl group; alternatively, an aryl group or a substituted aryl group; alternatively, an aralkyl group or a substituted aralkyl group; alternatively, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group; alternatively, an alkyl group; alternatively, a substituted alkyl group, alternatively, a cycloalkyl group; alternatively, a substituted cycloalkyl group; alternatively, an aryl group; alternatively, a substituted aryl group; alternatively, an aralkyl group; or alternatively, a substituted aralkyl group. In any aspect disclosed herein, the R4and/or R5alkyl groups independently can be a C1to C20, a C1to C10, or a C1to C5alkyl group. In any aspect disclosed herein, the R4and/or R5substituted alkyl groups independently can be a C1to C20, a C1to C10, or C1to C5substituted alkyl group. In any aspect disclosed herein, the R4and/or R5cycloalkyl groups independently can be a C4to C20, a C4to C15, or a C4to C10cycloalkyl group. In any aspect disclosed herein, the R4and/or R5substituted cycloalkyl groups independently can be a C4to C20, a C4to C15, or a C4to C10substituted cycloalkyl group. In any aspect disclosed herein, the R4and/or R5aryl groups independently can be a C6to C20, a C6to C15, or a C6to C10aryl group. In any aspect disclosed herein, the R4and/or R5substituted aryl group independently can be a C6to C20, a C6to C15, or a C6to C10substituted aryl group. In any aspect disclosed herein, the R4and/or R5aralkyl groups independently can be a C7to C20, a C7to C15, or a C7to C10aralkyl group. In any aspect disclosed herein, the R4and/or R5substituted aryl groups independently can be a C7to C20, a C7to C15, or a C7to C10substituted aralkyl group. Each substituent of a substituted alkyl group (general or specific), a substituted cycloalkyl group (general or specific), a substituted aryl group (general or specific), and/or substituted aralkyl group (general or specific) can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe R4and/or R5. In an aspect, R4and R5independently can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group; or alternatively, a methyl group, an ethyl group, an n-propyl (1-propyl) group, an iso-propyl (2-propyl) group, a 2-methyl-1-propyl group, a tert-butyl (2-methyl-2-propyl) group, or a neopentyl (2,2-dimethyl-1-propyl) group. In some aspects, the alkyl groups which can be utilized as R4and/or R5can be substituted. Each substituent of a substituted alkyl group independently can be a halogen or a hydrocarboxy group; alternatively, a halogen; or alternatively, a hydrocarboxy group. Substituent halogens and substituent hydrocarboxy (general and specific) groups are independently disclosed herein. These substituent halogens and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted alkyl group which can be utilized as R4and/or R5. In an aspect, R4and R5independently can be a cyclopentyl group, a substituted cyclopentyl group, a cyclohexyl group, or a substituted cyclohexyl group; alternatively, a cyclopentyl group or a substituted cyclopentyl group; or alternatively, a cyclohexyl group or a substituted cyclohexyl group. In an aspect, the substituted cycloalkyl group, which can be utilized for R4and/or R5, can be a 2-substituted cyclohexyl group, a 2,6-disubstituted cyclohexyl group, a 2-substituted cyclopentyl group, or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2,6-disubstituted cyclohexyl group; alternatively, a 2-substituted cyclopentyl group or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2-substituted cyclopentyl group; or alternatively, a 2,6-disubstituted cyclohexyl group or a 2,5-disubstituted cyclopentyl group. In an aspect where the substituted cycloalkyl group (general or specific) has more the one substituent, the substituents can be the same or different; alternatively, the same; or alternatively, different. Each substituent of a cycloalkyl group (general or specific) having a specified number of ring carbon atoms independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted cycloalkyl group (general or specific) which can be utilized as R4and/or R5. In a non-limiting aspect, R4and R5independently can be a cyclohexyl group, a 2-alkylcyclohexyl group, or a 2,6-dialkylcyclohexyl group; or alternatively, a cyclopentyl group, a 2-alkylcyclopentyl group, or a 2,5-dialkylcyclopentyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further described alkylcyclohexyl groups (general or specific), dialkylcyclohexyl groups (general or specific), alkylcyclopentyl groups (general or specific), and/or dialkylcyclopentyl groups (general or specific) which can be utilized as R4and/or R5. Generally, the alkyl substituents of a disubstituted cyclohexyl or cyclopentyl group can be the same; or alternatively, the alkyl substituents of a dialkyl cyclohexyl or cyclopentyl group can be different. In some non-limiting aspects, R4and R5independently can be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, a 2-tert-butylcyclohexyl group, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In other non-limiting aspects, R4and R5independently can be, a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, or a 2-tert-butylcyclohexyl group; or alternatively, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In an aspect, R4and R5independently can be a phenyl group, a substituted phenyl group; alternatively, a phenyl group; or alternatively, a substituted phenyl group. In an aspect, the substituted phenyl group, which can be utilized for R4and/or R5, can be a 2-substituted phenyl group, a 3-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, a 2,6-disubstituted phenyl group, a 3,5-disubstituted phenyl group, or a 2,4,6-trisubstituted phenyl group; alternatively, a 2-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, or a 2,6-disubstituted phenyl group; alternatively, a 3-substituted phenyl group or a 3,5-disubstituted phenyl group; alternatively, a 2-substituted phenyl group or a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group or a 2,6-disubstituted phenyl group; or alternatively, a 2,4,6-trisubstituted phenyl group. In an aspect, one or more substituents of a multi-substituted phenyl group utilized as R4and/or R5can be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group different. Each substituent of a substituted phenyl group (general or specific) independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted phenyl group (general or specific) which can be utilized as R4and/or R5. In a non-limiting aspect, R4and R5independently can be a phenyl group, a 2-alkylphenyl group, a 3-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group a 2,6-dialkylphenyl group, a 3,5-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group or a 4-alkylphenyl group; alternatively, a 2,4-dialkylphenyl group or a 2,6-dialkylphenyl group; alternatively, a 3-alkylphenyl group or a 3,5-dialkylphenyl group; alternatively, a 2-alkylphenyl group or a 2,6-dialkylphenyl group; or alternatively, a 2,4,6-trialkylphenyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe any alkyl substituted phenyl group which can be utilized as R4and/or R5. Generally, the alkyl substituents of a dialkylphenyl group (general or specific) or a trialkylphenyl group (general or specific) can be the same; or alternatively, the alkyl substituents of a dialkylphenyl group (general or specific) or a trialkyl phenyl group (general or specific) can be different. In some non-limiting aspects, R4and R5independently can be a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, a 2-tert-butylphenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group; alternatively, phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, or a 2-tert-butylphenyl group; alternatively, a phenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group. In a non-limiting aspect, R4and R5can be a phenyl group, a 2-alkoxyphenyl group, or a 4-alkoxyphenyl group. In some non-limiting aspects, R4and/or R5can be a phenyl group, a 2-methoxyphenyl group, a 2-ethoxyphenyl group, a 2-isopropoxyphenyl group, a 2-tert-butoxyphenyl group, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-isopropoxyphenyl group, or a 4-tert-butoxyphenyl group; alternatively, a 2-methoxyphenyl group, a 2-ethoxyphenyl group, a 2-isopropoxyphenyl group, or a 2-tert-butoxyphenyl group; or alternatively, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-isopropoxyphenyl group, or a 4-tert-butoxyphenyl group. In a non-limiting aspect, R4and R5independently can be a phenyl group, a 2-halophenyl group, a 4-halophenyl group, or a 2,6-dihalophenylgroup. Generally, the halides of a dihalophenyl group can be the same; or alternatively, the halides of a dihalophenyl group can be different. In some aspects, R4and R5independently can be a phenyl group, a 2-fluorophenyl group, a 4-fluorophenyl group, or a 2,6-difluorophenyl group. In an aspect, R4and R5independently can be a benzyl group or a substituted benzyl group; alternatively, a benzyl group; or alternatively, a substituted benzyl group. Each substituent of a substituted benzyl group independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted benzyl which can be utilized as R4and/or R5. In further aspects, R4and R5can be joined to form a ring or a ring system containing the phosphorus atom. The joining of R4and R5can be designated as L45and can be an organylene group; alternatively, an organylene group consisting of inert functional groups; alternatively, a hydrocarbylene group; or alternatively, an alkylene group. In an aspect, the L45organylene group, when present, can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10organylene group. In an aspect, the L45organylene group consisting of inert functional groups, when present, can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10organylene group consisting of inert functional groups. In an aspect, the L45hydrocarbyl group, when present, independently can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10hydrocarbylene group. In a further aspect, the L45alkylene group, when present, independently can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10alkylene group. In an aspect, L45can be a but-1,4-ylene group, a 1,4-diphenylbut-1,4-ylene group, a 1,4-di(2-methylphenyl)but-1,4-ylene group, 1,4-di(4-methylphenyl)but-1,4-ylene group, 1,4-di(4-t-butylphenyl)but-1,4-ylene group, a 1,4-di(3,5-dimethylphenyl)but-1,4-ylene group, a pent-1,4-ylene group, a 1-phenylpenta-1,4-ylene group, a 4-phenylpenta-1,4-ylene group, a hex-2,5-ylene group, a 2,2′-biphenylene group, a 2,2′-(methandiyl)dipheylene group, or a 2,2′-(1,2-ethandiyl)diphenylene group. In an aspect, the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex can have the formula (R1s)mX1s(L1s)X2s(R2s)nwhile the heteroatomic ligand chromium compound complex can have the formula: In some aspects, the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex can have two groups capable of being described by the formula (R1s)mX1s(L1s)X2s(R2s)n. In instances wherein the heteroatomic ligand can have two groups capable of being described by the formula (R1s)mX1s(L1s)X2s(R2s)n, the two L1sgroups are linked and the heteroatomic ligand and the heteroatomic ligand chromium compound complex can have the formulas: respectively. In the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex having formula (R1s)mX1s(L1s)X2s(R2s)nor having two linked (R1s)mX1s(L1s)X2s(R2s)ngroups, each X1sand each X2sindependently can be selected from the group consisting of N, P, O, and S; each L1scan be an independent linking group between the respective X1ss and X2ss; each m and each n independently can be 1 or 2; and each R1sand each R2sindependently can be a hydrogen, an organyl group (or alternatively, an organyl group consisting of inert functional group; or alternatively, a hydrocarbyl group), or a heterohydrocarbyl group, where when there are two or more R1ss and/or two R2ss, each R1scan be the same or different (alternatively, the same; or alternatively, different) and/or each R2scan be the same or different (alternatively, the same; or alternatively, different). L1s, X1s, X2s, R1s, R2s, m, and n are independent elements of any heteroatomic ligand or any heteroatomic ligand of the heteroatomic ligand chromium compound complex which have an L1s, X1s, X2s, R1s, R2s, m, and/or n and are independently described herein. These independent descriptions of Lis, X1s, X2s, R1s, R2s, m, and n can be utilized without limitation, and in any combination, to further describe any heteroatomic ligand or any heteroatomic ligand of the heteroatomic ligand chromium compound complex which have an L1s, X1s, X2s, R1s, R2s, m, and/or n. Additionally, CrXpis an independent element of the heteroatomic ligand chromium compound complex, and is independently described herein, and can be utilized without limitation, and in any combination with L1s, X1s, X2s, R1s, R2s, m, and n of the heteroatomic ligand to further describe the heteroatomic ligand chromium compound complexes contemplated herein. In an aspect, each X1sand each X2sof any heteroatomic ligand or any heteroatomic ligand of any heteroatomic ligand chromium compound complex described herein having an X1sand/or X2scan be independently selected from N, P, O, and S; alternatively, independently selected from N and P; or alternatively, independently selected from O and S. In some aspects, each X1sand each X2scan be N; alternatively, P; alternatively, O; or alternatively, S. Each m and each n of any heteroatomic ligand or any heteroatomic ligand of any heteroatomic ligand chromium compound complex described herein having an m and/or n can be independently selected from 1 or 2; alternatively, 1; or alternatively, 2. Is some particular aspects, each m and/or each n can be 1 when X1sand/or X2s, respectively, is O or S; alternatively, O; or alternatively, S. In some other particular aspects, each m and/or each n can be 2 when X1sand/or X2s, respectively, is N or P; alternatively, N; or alternatively, P. In a non-limiting aspect, the heteroatomic ligand can have the formula R1sS(L1s)SR2s, (R1s)2P(L1s)P(R2s)2or (R1s)2N(L1s)N(R2s)2; alternatively, R1sS(L1s)SR2s; alternatively, (R1s)2P(L1s)P(R2s)2; or alternatively, (R1s)2N(L1s)N(R2s)2while the heteroatomic ligand chromium compound complex can have any one of the formulas In non-limiting aspects where the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand chromium compound complex has two linked heteroatomic groups, the heteroatomic ligand can have the formula selected from one or more of while the heteroatomic ligand chromium compound complex can have any one of the formulas In an aspect, each L1sof any heteroatomic ligand or any heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein independently can be any group capable of linking group X1sand X2s(and other L1sgroup when the heteroatomic ligand or heteroatomic ligand of the heteroatomic ligand chromium compound complex when there are more than one L1sgroup). In some aspects, each L1sindependently can be an organylene group, an amin-di-yl group, or a phosphin-di-yl group; alternatively, an organylene group consisting of inert functional groups, an amin-di-yl group, or a phosphin-di-yl group; alternatively, a hydrocarbylene group, an amin-di-yl group, or a phosphin-di-yl group; alternatively an amin-di-yl group or a phosphin-di-yl group; alternatively, an organylene group; alternatively, an organylene group consisting of inert functional groups; alternatively, a hydrocarbylene group; alternatively, an amin-di-yl group; or alternatively, a phosphin-di-yl group. When there is more than one L1sgroup in the heteroatomic ligand or heteroatomic ligand of the heteroatomic ligand chromium compound complex each L1sindependently can be an organic, an amine group, or a phosphine group; alternatively, an organic group consisting of inert functional groups, an amine group, or a phosphine group; alternatively, a hydrocarbon group, an amine group, or a phosphine group; alternatively an amine group or a phosphine group; alternatively, an organic group; alternatively, an organic group consisting of inert functional groups; alternatively, a hydrocarbon group; alternatively, an amine group; or alternatively, a phosphine group. In an aspect, the L1sorganylene group or organic group can be a C1to C20, a C1to C15, a C1to C10, or, a C1to C5organylene or organic group. In an aspect, the L1sorganylene group consisting of inert functional groups can be a C1to C20, a C1to C15, a C1to C10, or, a C1to C5organylene or organic group consisting of inert functional groups. In an aspect, the L1shydrocarbylene group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbylene or hydrocarbon group. In an aspect, the amin-di-yl or amine group can be a C1to C30, a C1to C20, a C1to C15, or a C1to C10amin-di-yl or amine group. In an aspect, the phosphin-di-yl or phosphine group can be a C1to C30, a C1to C20, a C1to C15, or a C1to C10phosphin-di-yl or phosphine group. In an aspect, each L1sorganylene or organic group can have the formula -(L3s)NR5s(L4s)- or -(L3s)PR5s(L4s)-; alternatively, -(L3s)NR5s(L4s)-; or alternatively, -(L3s)PR5s(L4s)-. In an aspect, the each amin-di-yl group can have the formula —N(R5s)—. In an aspect, each phosphin-di-yl group can have the formula —P(R5s)—. In these L1sgroup formulas, the dashes represent the undesignated valance to which the X1sand X2sof the heteroatomic ligand or the heteroatomic ligand of the heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein attach. When there is more than one L1sgroup in the heteroatomic ligand or heteroatomic ligand of the heteroatomic ligand chromium compound complex, the R5sof each L1sgroup can be combined into a linking group designated as L2s. In some non-limiting aspects, the heteroatomic ligand can have Structure PNP1, Structure PNP2, Structure NRNRN, Structure PRPRP, Structure SRNRS, Structure PRNRP, and Structure NRPRN; alternatively, Structure PNP1 or Structure PNP2; alternatively, Structure PRPRP, Structure SRNRS, or Structure PRNRP; alternatively, Structure PNP1; alternatively, Structure PNP2; alternatively, Structure NRNRN; alternatively, Structure PRPRP; alternatively, Structure SRNRS; alternatively, Structure PRNRP; or alternatively, Structure NRPRN. In some non-limiting aspects, the heteroatomic ligand chromium compound complex having a heteroatomic ligand (R1s)mX1s(L1s)X2s(R2s)nwhich can be utilized in catalyst systems described herein can have Structure PNCr1, Structure PNPCr2, Structure NRNRNCr, Structure PRPRPCr, Structure SRNRSCr, Structure PRNRPCr, and Structure NRPRNCr; alternatively, Structure PNPCr1 or Structure PNPCr2; alternatively, Structure PRPRPCr, Structure SRNRSCr, or Structure PRNRPCr; alternatively, Structure PNPCr1; alternatively, Structure PNPCr2; alternatively, Structure NRNRNCr; alternatively, Structure PRPRPCr; alternatively, Structure SRNRSCr; alternatively, Structure PRNRPCr; or alternatively, Structure NRPRNCr. The R5s, L2s, L3s, L4s, R11s, R12s, R13s, and R14sare each independent elements of the heteroatomic ligands having Structure PNP1, Structure PNP2, Structure NRNRN, Structure PRPRP, Structure SRNRS, Structure PRNRP, or Structure NRPRN, and/or the heteroatomic ligand of the heteroatomic ligand chromium compound complexes having Structure PNPCr1, Structure PNPCr2, Structure NRNRNCr, Structure PRPRPCr, Structure SRNRSCr, Structure PRNRPCr, and Structure NRPRNCr in which they occur and are independently described herein. The independent descriptions of R5s, L2s, L3s, L4s, R11s, R12s, R13s, and R14scan be utilized without limitation, and in any combination, to further describe the heteroatomic ligand structures and/or the heteroatomic ligand chromium compound complex structure in which they occur. Similarly, X and p are independent elements of the heteroatomic ligand chromium compound complexes having Structure PNCr1, Structure PNPCr2, Structure NRNCRNr, Structure PRPRPCr, Structure SRNRSCr, Structure PRNRPCr, and Structure NRPRNCr and are independently described herein. The independent description of X and p can be utilized without limitation, and in any combination, with the independently described R5s, L2s, L3s, L4s, R11s, R12s, R13s, and R14sprovided herein to further describe any heteroatomic ligand chromium compound complex having Structure PNPCr1, Structure PNPCr2, Structure NRNRNCr, Structure PRPRPCr, Structure SRNRSCr, Structure PRNRPCr, and/or Structure NRPRNCr. Generally, R1s, R2s, R11s, R12s, R13s, and/or R14s, of any heteroatomic ligand structure depicted herein and/or any heteroatomic ligand chromium compound complex depicted herein having an R1s, R2s, R11s, R12s, R13s, and/or R14sgroup, independently can be an organyl group; alternatively, an organyl group consisting of inert functional groups; or alternatively, a hydrocarbyl group. In an aspect, the organyl group which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In an aspect, the organyl group consisting of inert functional groups which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In an aspect, the hydrocarbyl group which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In an aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sof any heteroatomic ligand structure depicted herein and/or any heteroatomic ligand chromium compound complex depicted herein having an R1s, R2s, R11s, R12s, R13s, and/or R14sgroup independently can an alkyl group, a substituted alkyl group, a cycloalkyl group, a substituted cycloalkyl group, an aryl group, a substituted aryl group, an aralkyl group, or a substituted aralkyl group; alternatively, an alkyl group or a substituted alkyl group; alternatively, a cycloalkyl group or a substituted cycloalkyl group; alternatively, an aryl group or a substituted aryl group; alternatively, an aralkyl group or a substituted aralkyl group; or alternatively, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. In other aspects, each R1s, R2s, R11s, R12s, R13s, and/or R14sof any heteroatomic ligand structure depicted herein and/or any heteroatomic ligand chromium compound complex depicted herein having an R1s, R2s, R11s, R12s, R13s, and/or R14sgroup independently can be an alkyl group; alternatively, a substituted alkyl group, alternatively, a cycloalkyl group; alternatively, a substituted cycloalkyl group; alternatively, an aryl group; alternatively, a substituted aryl group; alternatively, an aralkyl group; or alternatively, a substituted aralkyl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14salkyl group independently can be a C1to C20, a C1to C10, or a C1to C5alkyl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14ssubstituted alkyl group independently can be a C1to C20, a C1to C10, or a C1to C5substituted alkyl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14scycloalkyl group independently can be a C4to C20, a C4to C15, or a C4to C10cycloalkyl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14ssubstituted cycloalkyl group independently can be a C4to C20, a C4to C15, or a C4to C10substituted cycloalkyl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14saryl group independently can be a C6to C20, a C6to C15, or a C6to C10aryl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14ssubstituted aryl group independently can be a C6to C20, a C6to C15, or a C6to C10substituted aryl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14saralkyl group independently can be a C7to C20, a C7to C15, or a C7to C10aralkyl group. In any aspect disclosed herein, each R1s, R2s, R11s, R12s, R13s, and/or R14ssubstituted aralkyl group independently can be a C7to C20, a C7to C15, or a C7to C10substituted aralkyl group. Each substituent of a substituted alkyl group (general or specific), a substituted cycloalkyl group (general or specific), a substituted aryl group (general or specific), and/or substituted aralkyl group (general or specific) can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarboxy groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted group (general or specific) which can be utilized R1s, R2s, R11s, R12s, R13s, and/or R14s. In an aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group; or alternatively, a methyl group, an ethyl group, an iso-propyl (2-propyl) group, a tert-butyl (2-methyl-2-propyl) group, or a neopentyl (2,2-dimethyl-1-propyl) group. In some aspects, the alkyl groups which can be utilized as each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be substituted. Each substituent of a substituted alkyl group independently can be a halogen or a hydrocarboxy group; alternatively, a halogen; or alternatively, a hydrocarboxy group. Substituent halogens and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted alkyl group (general or specific) which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14s. In an aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a cyclopentyl group, a substituted cyclopentyl group, a cyclohexyl group, or a substituted cyclohexyl group; alternatively, a cyclopentyl group or a substituted cyclopentyl group; or alternatively, a cyclohexyl group or a substituted cyclohexyl group; alternatively, a cyclopentyl group; alternatively, a substituted cyclopentyl group; alternatively, a cyclohexyl group; or alternatively, a substituted cyclohexyl group. In an aspect, the substituted cycloalkyl group, which can be utilized for any of R1s, R2s, R11s, R12s, R13s, and/or R14s, when present in any heteroatomic ligand described herein, any heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein, any heteroatomic ligand formula or structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein, independently can be a 2-substituted cyclohexyl group, a 2,6-disubstituted cyclohexyl group, a 2-subsituted cyclopentyl group, or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2,6-disubstituted cyclohexyl group; alternatively, a 2-subsituted cyclopentyl group or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2-subsituted cyclopentyl group; or alternatively, a 2,6-disubstituted cyclohexyl group or a 2,5-disubstituted cyclopentyl group. In an aspect, one or more substituents of a multi-substituted cycloalkyl group utilized as R1s, R2s, R11s, R12s, R13s, and/or R14scan be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group can be different. Each substituent of a substituted cycloalkyl group (general or specific) having a specified number of ring carbon atoms independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted cycloalkyl group (general or specific) which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14s. In a non-limiting aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a cyclohexyl group, a 2-alkylcyclohexyl group, or a 2,6-dialkylcyclohexyl group; or alternatively, a cyclopentyl group, a 2-alkylcyclopentyl group, or a 2,5-dialkyleyclopentyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe alkylcyclohexyl groups (general or specific), dialkylcyclohexyl groups (general or specific), alkylcyclopentyl groups (general or specific), and/or dialkylcyclopentyl groups (general or specific) which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14. Generally, the alkyl substituents of a disubstituted cyclohexyl or cyclopentyl group can be the same, or alternatively, the alkyl substituents can be different. In some non-limiting aspects, each R1s, R2s, R11s, R12s, R13s, and R14s, when present in any heteroatomic ligand described herein, any heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein, any heteroatomic ligand formula or structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein, independently can be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, a 2-tert-butylcyclohexyl group, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group; alternatively, a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, or a 2-tert-butylcyclohexyl group; or alternatively, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In an aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a phenyl group or a substituted phenyl group; alternatively, a phenyl group; or alternatively, a substituted phenyl group. In an aspect, the substituted phenyl group which can be utilized for each R1s, R2s, R11s, R12s, R13s, and/or R14s, when present in any heteroatomic ligand described herein, any heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein, any heteroatomic ligand formula or structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein, independently can be a 2-substituted phenyl group, a 3-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, a 2,6-disubstituted phenyl group, a 3,5-disubstituted phenyl group, or a 2,4,6-trisubstituted phenyl group; alternatively, a 2-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, or a 2,6-disubstituted phenyl group; alternatively, a 3-subsituted phenyl group or a 3,5-disubstituted phenyl group; alternatively, a 2-substituted phenyl group or a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group or a 2,6-disubstituted phenyl group; alternatively, a 2-substituted phenyl group; alternatively, a 3-substituted phenyl group; alternatively, a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group; alternatively, a 2,6-disubstituted phenyl group; alternatively, a 3,5-disubstituted phenyl group; or alternatively, a 2,4,6-trisubstituted phenyl group. In an aspect, one or more substituents of a multi-substituted phenyl group utilized as R1s, R2s, R11s, R12s, R13s, and/or R14scan be the same or different; alternatively, all the substituents can be the same; or alternatively, all the substituents can be different. Each substituent of a substituted phenyl group (general or specific) independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy group can be utilized without limitation to further describe a substituted phenyl group (general or specific) which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14s. In a non-limiting aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a phenyl group, a 2-alkylphenyl group, a 3-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, a 3,5-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group or a 4-alkylphenyl group; alternatively, a 2,4-dialkylphenyl group or a 2,6-dialkylphenyl group; alternatively, a 3-alkylphenyl group or a 3,5-dialkylphenyl group; alternatively, a 2-alkylphenyl group or a 2,6-dialkylphenyl group; or alternatively, a 2,4,6-trialkylphenyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe any alkyl substituted phenyl group which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14s. Generally, the alkyl substituents of dialkylphenyl groups (general or specific) or trialkylphenyl groups (general or specific) can be the same, or alternatively, the alkyl substituents can be different. In some non-limiting aspects, each R1s, R2s, R11s, R12s, R13s, and/or R14s, when present in any heteroatomic ligand described herein, any heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein, any heteroatomic ligand formula or structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein, independently can be a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, a 2-tert-butylphenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group; alternatively, a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, or a 2-tert-butylphenyl group; or alternatively, a phenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group. In a non-limiting aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a phenyl group, a 2-alkoxyphenyl group, or a 4-alkoxyphenyl group. In some non-limiting aspects, each R1s, R2s, R11s, R12s, R13s, and/or R14s, when present in any heteroatomic ligand described herein, any heteroatomic ligand of the heteroatomic ligand chromium compound complex described herein, any heteroatomic ligand formula or structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein, independently can be a phenyl group, a 2-methoxyphenyl group, a 2-ethoxyphenyl group, a 2-isopropoxyphenyl group, a 2-tert-butoxyphenyl group, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-isopropoxyphenyl group, or a 4-tert-butoxyphenyl group; alternatively, a 2-methoxyphenyl group, a 2-ethoxyphenyl group, a 2-isopropoxyphenyl group, or a 2-tert-butoxyphenyl group; or alternatively, a 4-methoxyphenyl group, a 4-ethoxyphenyl group, a 4-isopropoxyphenyl group, or a 4-tert-butoxyphenyl group. In a non-limiting aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a phenyl group, a 2-halophenyl group, a 4-halophenyl group, or a 2,6-dihalophenylgroup. Generally, the halides of a dihalophenyl group can be the same, or alternatively, the halides can be different. In some aspects, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a phenyl group, a 2-fluorophenyl group, a 4-fluorophenyl group, or a 2,6-difluorophenyl group. In an aspect, each R1s, R2s, R11s, R12s, R13s, and/or R14sindependently can be a benzyl group or a substituted benzyl group; alternatively, a benzyl group; or alternatively, a substituted benzyl group. Each substituent of a substituted benzyl group (general or specific) independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen, alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted benzyl group (general or specific) which can be utilized as R1s, R2s, R11s, R12s, R13s, and/or R14s. In further aspects, two geminal R1ss, two geminal R2ss, geminal R11sand R12s, and/or geminal R13Sand R14sindependently can be joined to form a ring or a ring system containing the heteroatom to which they are attached. The joining of two geminal R1ss can be designated L11s. The joining of two geminal R2ss can be designated L22s. The joining of geminal R11sand R12scan be designated L12s. The joining of geminal R13sand R14scan be designated L34s. In an aspect, L11s, L22s, L12s, and/or L34sindependently can be an organylene group; alternatively, an organylene group consisting of inert functional groups; alternatively, a hydrocarbylene group; or alternatively, an alkylene group. In an aspect, the L11s, L22s, L12s, and/or L34sorganylene group, when present, independently can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10organylene group. In some aspects, the L11s, L22s, L12s, and/or L34sorganylene group consisting of inert functional groups, when present, independently can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10organylene group consisting of inert functional groups. In other aspects, the L11s, L22s, L12s, and/or L34shydrocarbyl group, when present, independently can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10hydrocarbylene group. In a further aspect, the L11s, L22s, L12s, and/or L34salkylene group, when present, independently can be a C4to C30, a C4to C20, a C4to C15, or a C4to C10alkylene group. In an aspect, L11s, L22s, L12s, and/or L34s, when present, independently can be a can be but-1,4-ylene group, a 1,4-diphenylbut-1,4-ylene group, a 1,4-di(2-methylphenyl)but-1,4-ylene group, 1,4-di(4-methylphenyl)but-1,4-ylene group, 1,4-di(4-t-butylphenyl)but-1,4-ylene group, a 1,4-di(3,5-dimethylphenyl)but-1,4-ylene group, a pent-1,4-ylene group, a 1-phenylpenta-1,4-ylene group, a 4-phenylpenta-1,4-ylene group, a hex-2,5-ylene group, a 2,2′-biphenylene group, a 2,2′-(methandiyl)dipheylene group, or a 2,2′-(1,2-ethandiyl)diphenylene group. Generally, R5s, of any heteroatomic ligand structure depicted herein and any heteroatomic ligand chromium compound complex depicted herein having an R5sgroup, can be an organyl group; alternatively, an organyl group consisting of inert functional groups; or alternatively, a hydrocarbyl group. In an aspect, the R5sorganyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group. In an aspect, the R5sorganyl group consisting of inert functional groups can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5organyl group consisting of inert functional groups. In an aspect, the R5shydrocarbyl group can be a C1to C20, a C1to C15, a C1to C10, or a C1to C5hydrocarbyl group. In an aspect, R5s, of any heteroatomic ligand structure depicted herein and any heteroatomic ligand chromium compound complex depicted herein having an R5sgroup, can be an alkyl group, a substituted alkyl group, a cycloalkyl group, a substituted cycloalkyl group, an aryl group, a substituted aryl group, an aralkyl group, or a substituted aralkyl group; can be an alkyl group or a substituted alkyl group; alternatively, a cycloalkyl group or a substituted cycloalkyl group; alternatively, an aryl group or a substituted aryl group; alternatively, an aralkyl group or a substituted aralkyl group; or alternatively, an alkyl group, a cycloalkyl group, an aryl group, or an aralkyl group. In other aspects, R5sof any heteroatomic ligand structure depicted herein and any heteroatomic ligand chromium compound complex depicted herein having an R5sgroup, can be an alkyl group; alternatively, a substituted alkyl group, alternatively, a cycloalkyl group; alternatively, a substituted cycloalkyl group; alternatively, an aryl group; alternatively, a substituted aryl group; alternatively, an aralkyl group; or alternatively, a substituted aralkyl group. In any aspect disclosed herein, the R5salkyl group can be a C1to C20, a C1to C15, or a C1to C10alkyl group. In any aspect disclosed herein, the R5ssubstituted alkyl group can be a C1to C20, a C1to C15, or a C1to C10substituted alkyl group. In any aspect disclosed herein, the R5scycloalkyl group can be a C4to C20, a C4to C15, or a C4to C10cycloalkyl group. In any aspect disclosed herein, the R5ssubstituted cycloalkyl group can be a C4to C20, a C4to, or a C4to C10substituted cycloalkyl group. In any aspect disclosed herein, the R5saryl group can be a C6to C20, a C6to C15, or a C6to C10aryl group. In any aspect disclosed herein, the R5ssubstituted aryl group can be a C6to C20, a C6to C15, or a C6to C10substituted aryl group. In any aspect disclosed herein, the R5saralkyl group can be a C7to C20, a C7to C15, or a C7to C10aralkyl group. In any aspect disclosed herein, the R5ssubstituted aralkyl group can be a C7to C20, a C7to C15, or a C7to C10substituted aralkyl group. Each substituent of a substituted alkyl group (general or specific), substituted cycloalkyl group (general or specific), substituted aryl group (general or specific), and/or substituted aralkyl group (general or specific) can be a halogen, hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxyl group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently disclosed herein. These substituent halogens, substituent hydrocarbyl groups, and substituent hydrocarboxy groups can be utilized without limitation to further describe a substituted group (general or specific) which can be utilized R5s. In an aspect, R5scan be a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group; alternatively, a methyl group, an ethyl group, an n-propyl (1-propyl) group, an isopropyl (2-propyl) group, an n-butyl (1-butyl) group, a sec-butyl (2-butyl) group, an isobutyl (2-methyl-1-propyl) group, a tert-butyl (2-methyl-2-propyl) group, an n-pentyl (1-pentyl) group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-1-butyl group, a tert-pentyl (2-methyl-2-butyl) group, a 3-methyl-1-butyl group, a 3-methyl-2-butyl group, or a neo-pentyl (2,2-dimethyl-1-propyl) group; or alternatively, a methyl group, an ethyl group, an iso-propyl (2-propyl) group, a tert-butyl (2-methyl-2-propyl) group, or a neopentyl (2,2-dimethyl-1-propyl) group. In some aspects, the R5salkyl groups can be substituted. Each substituent of a R5ssubstituted alkyl group independently can be a halogen or a hydrocarboxy group; alternatively, a halogen; or alternatively, a hydrocarboxy group. Substituent halogens and substituent hydrocarboxy groups (general and specific) are independently described herein and these substituent groups can be utilized without limitation to further describe a substituted alkyl group (general or specific) which can be utilized as R5s. In an aspect, R5scan be a cyclopentyl group, a substituted cyclopentyl group, a cyclohexyl group, a substituted cyclohexyl group; alternatively, a cyclopentyl group or a substituted cyclopentyl group; or alternatively, a cyclohexyl group or a substituted cyclohexyl group. In further aspects, R5scan be a 2-substituted cyclohexyl group, a 2,6-disubstituted cyclohexyl group, a 2-subsituted cyclopentyl group, or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2,6-disubstituted cyclohexyl group; alternatively, a 2-substituted cyclohexyl group or a 2,6-disubstituted cyclohexyl group; alternatively, a 2-subsituted cyclopentyl group or a 2,5-disubstituted cyclopentyl group; alternatively, a 2-substituted cyclohexyl group or a 2-subsituted cyclopentyl group; or alternatively, a 2,6-disubstituted cyclohexyl group or a 2,5-disubstituted cyclopentyl group. In an aspect, one or more substituents of a multi-substituted cycloalkyl group utilized as R5scan be the same or different; alternatively, all the substituents of a multi-substituted cycloalkyl group can be the same; or alternatively, all the substituents of a multi-substituted cycloalkyl group can be different. Each substituent of a cycloalkyl group (general or specific) having a specified number of ring carbon atoms independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently described herein and these substituent groups can be utilized without limitation to further describe a substituted cycloalkyl group (general or specific) which can be utilized as R5s. In a non-limiting aspect, R5scan be a cyclohexyl group, a 2-alkylcyclohexyl group, or a 2,6-dialkylcyclohexyl group; or alternatively, a cyclopentyl group, a 2-alkylcyclopentyl group, or a 2,5-dialkylcyclopentyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe alkylcyclohexyl groups (general or specific), dialkylcyclohexyl groups (general or specific), alkylcyclopentyl groups (general or specific), and/or dialkylcyclopentyl groups (general or specific) which can be utilized as R5s. Generally, the alkyl substituents of a disubstituted cyclohexyl or cyclopentyl group can be the same, or alternatively, the alkyl substituents can be different. In some non-limiting aspects, R5sheteroatomic ligand structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein can be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, a 2-tert-butylcyclohexyl group, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In other non-limiting aspects, R5scan be a 2-methylcyclohexyl group, a 2-ethylcyclohexyl group, a 2-isopropylcyclohexyl group, or a 2-tert-butylcyclohexyl group; or alternatively, a 2,6-dimethylcyclohexyl group, a 2,6-diethylcyclohexyl group, a 2,6-diisopropylcyclohexyl group, or a 2,6-di-tert-butylcyclohexyl group. In an aspect, R5sheteroatomic ligand structure provided herein, and/or any heteroatomic ligand chromium compound complex structure provided herein can be a cyclopentyl group, a 2-methylcyclopentyl group, a cyclohexyl group, or a 2-methylcyclohexyl group; alternatively, a cyclopentyl group or a cyclohexyl group; or alternatively, a 2-methylcyclopentyl group or a 2-methylcyclohexyl group. In an aspect, R5scan be a phenyl group or a substituted phenyl group; alternatively, a phenyl group; or alternatively, a substituted phenyl group. In some aspects, R5scan be a 2-substituted phenyl group, a 4-substituted phenyl group, a 2,4-disubstituted phenyl group, a 2,6-disubstituted phenyl group, or a 2,4,6-trisubstituted phenyl group; alternatively, a 2-substituted phenyl group or a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group, a 2,6-disubstituted phenyl group, or a 2,4,6-trisubstituted phenyl group; alternatively, a 2,4-disubstituted phenyl group or a 2,6-disubstituted phenyl group; alternatively, a 2-substituted phenyl group; alternatively, a 4-substituted phenyl group; alternatively, a 2,4-disubstituted phenyl group; alternatively, a 2,6-disubstituted phenyl group; or alternatively, a 2,4,6-trisubstituted phenyl group. In an aspect, one or more substituents of a multi-substituted phenyl group utilized as R5scan be the same or different; alternatively, all the substituents can be the same, or alternatively, all the substituents can be different. Each substituent of a substituted phenyl group (general or specific) independently can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Substituent halogens, substituent hydrocarbyl groups (general and specific), and substituent hydrocarboxy groups (general and specific) are independently described herein and these substituent groups can be utilized without limitation to further describe a substituted phenyl group (general or specific) which can be utilized as R5s. In a non-limiting aspect, R5scan be a phenyl group, a 2-alkylphenyl group, a 4-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, or a 2,4,6-trialkylphenyl group; alternatively, a 2-alkylphenyl group, a 2,4-dialkylphenyl group, a 2,6-dialkylphenyl group, or a 2,4,6-trialkylphenyl group. Alkyl substituent groups (general and specific) are independently described herein and these alkyl substituent groups can be utilized, without limitation, to further describe any alkyl substituted phenyl group which can be utilized as R5s. Generally, the alkyl substituents of dialkylphenyl groups (general of specific) or trialkylphenyl groups (general or specific) can be the same, or alternatively, the alkyl substituents can be different. In some non-limiting aspects, R5scan be a phenyl group, a 2-methylphenyl group, a 2-ethylphenyl group, a 2-n-propylphenyl group, a 2-isopropylphenyl group, a 2-tert-butylphenyl group, a 2,6-dimethylphenyl group, a 2,6-diethylphenyl group, a 2,6-di-n-propylphenyl group, a 2,6-diisopropylphenyl group, a 2,6-di-tert-butylphenyl group, a 2-isopropyl-6-methylphenyl group, or a 2,4,6-trimethylphenyl group; alternatively, a phenyl group, a 2-methylphenyl group, a 2,6-dimethylphenyl group, or a 2,4,6-trimethylphenyl group. Generally, L2s, of any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup, can be an organylene group; alternatively, an organylene group consisting of inert functional groups; alternatively, a hydrocarbylene group; or alternatively, an alkylene group. In an aspect, the L2sorganylene group can be a C1to C20, a C1to C15, or a C1to C10organylene group. In an aspect, the L2sorganylene group consisting of inert functional groups can be a C1to C20, a C1to C15, or a C1to C10organylene group consisting of inert functional groups. In an aspect, the L2salkylene group can be a C1to C20, C1to C15, or a C1to C10alkylene group. In an aspect, L2sof any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup can be —(CRPRP′)m— where each RPand RP′can independently be hydrogen, methyl, ethyl, propyl, isopropyl, or butyl groups and m can be an integer from 1 to 12. In some aspects, L2sof any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup can be a methylene group (—CH2—), an eth-1,2-ylene group (—CH2CH2—), a prop-1,3-ylene group (—CH2CH2CH2—), a prop-1,2-ylene group (—CH(CH3)CH2—), a prop-2,2-ylene group (—C(CH3)2—), a but-1,4-ylene group (—CH2CH2CH2CH2—), or a 2-methylprop-1,3-ylene group (—CH2CH(CH3)CH2—); or alternatively a methylene group (—CH2—), an eth-1,2-ylene group (—CH2CH2—), or a prop-1,2-ylene group (—CH(CH3)CH2—). In an aspect, L2sof any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup, can be 1,2-cyclohexylene, a substituted 1,2-cyclohexylene, 1,3-cyclohexylene, a substituted 1,3-cyclohexylene, 1,4-cyclohexylene, a substituted 1,4-cyclohexylene, 3,3′-bicyclohexylene, a substituted 3,3′-bicyclohexylene, 4,4′-bicyclohexylene, a substituted 4,4′-bicyclohexylene, bis(3-cyclohexylene)methane, a substituted bis(3-cyclohexylene)methane, bis(4-cyclohexylene)methane, a substituted bis(4-cyclohexylene)methane, 1,2-bis(3-cyclohexylene)ethane, a substituted 1,2-bis(3-cyclohexylene)ethane, 1,2-bis(4-cyclohexylene)ethane, a substituted 1,2-bis(4-cyclohexylene)ethane, 1,2-bis(3-cyclohexylene)-propane, a substituted 1,2-bis(3-cyclohexylene)propane, 1,2-bis(4-cyclohexylene)propane, a substituted 1,2-bis(4-cyclohexylene)propane, 2,2-bis(3-cyclohexylene)propane, a substituted 2,2-bis(3-cyclohexylene)propane, 2,2-bis(4-cyclohexylene)propane, or a substituted 2,2-bis(4-cyclohexylene)propane. In some aspects, L2sof any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup can be a substituted 1,2-cyclohexylene, a substituted 1,3-cyclohexylene, a substituted 1,4-cyclohexylene, a substituted 3,3′-bicyclohexylene, a substituted 4,4′-bicyclohexylene, a substituted bis(3-cyclohexylene)methane, a substituted bis(4-cyclohexylene)methane, a substituted 1,2-bis(3-cyclohexylene)ethane, a substituted 1,2-bis(4-cyclohexylene)ethane, a substituted 1,2-bis(3-cyclohexylene)propane, a substituted 1,2-bis(4-cyclohexylene)propane, a substituted 2,2-bis(3-cyclohexylene)propane, or a substituted 2,2-bis(4-cyclohexylene)propane. In an aspect, each substituent of a substituted cyclohexylene, a substituted bis(cyclohexylene)methane, a substituted bis(cyclohexylene)ethane, or a substituted 1,2-bis(3-cyclohexylene)propane which can be utilized as L2scan be a hydrocarbyl group. Substituent groups (general and specific) are independently disclosed herein and can be utilized without limitation to further describe a substituted cyclohexylene (general or specific), a substituted bis(cyclohexylene)methane (general or specific), a substituted bis(cyclohexylene)ethane (general or specific), or a substituted 1,2-bis(3-cyclohexylene)propane (general or specific) which can be utilized as L2s. In an aspect, L2sof any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup can be 1,2-phenylene, a substituted 1,2-phenylene, 1,3-phenylene, a substituted 1,3-phenylene, 1,4-phenylene, a substituted 1,4-phenylene, 3,3′-biphenylene, a substituted 3,3′-biphenylene, 4,4′-biphenylene, a substituted 4,4′-biphenylene, bis(3-phenylene)methane, a substituted bis(3-phenylene)methane, bis(4-phenylene)methane, a substituted bis(4-phenylene)methane, 1,2-bis(3-phenylene)ethane, a substituted 1,2-bis(3-phenylene)ethane, 1,2-bis(4-phenylene)ethane, a substituted 1,2-bis(4-phenylene)ethane, 1,2-bis(3-phenylene)propane, a substituted 1,2-bis(3-phenylene)propane, 1,2-bis(4-phenylene)propane, a substituted 1,2-bis(4-phenylene)propane, 2,2-bis(3-phenylene)propane, a substituted 2,2-bis(3-phenylene)propane, 2,2-bis(4-phenylene)propane, or a substituted 2,2-bis(4-phenylene)propane. In some aspects, L2sof any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L2sgroup can be a substituted 1,2-phenylene, a substituted 1,3-phenylene, a substituted 1,4-phenylene, a substituted 3,3′-biphenylene, a substituted 4,4′-biphenylene, a substituted bis(3-phenylene)methane, a substituted bis(4-phenylene)methane, a substituted 1,2-bis(3-phenylene)ethane, a substituted 1,2-bis(4-phenylene)ethane, a substituted 1,2-bis(3-phenylene)propane, a substituted 1,2-bis(4-phenylene)propane, a substituted 2,2-bis(3-phenylene)propane, or a substituted 2,2-bis(4-phenylene)propane. In an aspect, each substituent of a substituted phenylene (general or specific), a substituted biphenylene (general or specific), a substituted bis(phenylene)methane (general or specific), a substituted bis(phenylene)ethane (general or specific), and/or a substituted bis(phenylene)propane (general or specific) which can be utilized as L2scan be a hydrocarbyl group. Substituent hydrocarbyl groups (general and specific) are independently disclosed herein and can be utilized without limitation to further describe a substituted phenylene (general or specific), a substituted biphenylene (general or specific), a substituted bis(phenylene)methane (general or specific), a substituted bis(phenylene)ethane (general or specific), and/or a substituted bis(phenylene)propane (general or specific) which can be utilized as L2s. Generally, L3sand/or L4s, of any heteroatomic ligand and/or any heteroatomic ligand chromium compound complex having an L3sand/or L4sgroup, independently can be an organylene group; alternatively, an organylene group consisting of inert functional groups; alternatively, a hydrocarbylene group; alternatively, an alkylene group. In an aspect, the L3sand/or L4sorganylene group independently can be a C1to C20, a C1to C15, or a C1to C10organylene group. In an aspect, the L3Sand/or L4sorganylene group consisting of inert functional groups independently can be a C1to C20, a C1to C15, or a C1to C10organylene group consisting of inert functional groups. In an aspect, the L3sand/or L4shydrocarbylene group independently can be a C1to C20, a C1to C15, or a C1to C10hydrocarbylene group. In an aspect, the L3sand/or L4salkylene group independently can be a C1to C20, C1to C15, or a C1to C10alkylene group. In an aspect, L3sand/or L4sof any heteroatomic ligand structure and/or any heteroatomic ligand chromium compound complex having an L3sand/or L4sgroup independently can be —(CRPRP′)m− where each RPand RP′can independently be hydrogen, methyl, ethyl, propyl, isopropyl, or butyl groups and m can be an integer from 1 to 12. In some aspects, L3sand/or L4sof any heteroatomic ligand structure and/or any heteroatomic ligand chromium compound complex having an L3sand/or L4sgroup independently can be a methylene group (—CH2—), an eth-1,2-ylene group (—CH2CH2—), an ethen-1,2-ylene group (—CH═CH—), a prop-1,3-ylene group (—CH2CH2CH2—), a prop-1,2-ylene group (—CH(CH3)CH2—), a prop-2,2-ylene group (—C(CH3)2—), a 1-methylethen-1,2-ylene group (—C(CH3)═CH—), a but-1,4-ylene group (—CH2CH2CH2—CH2—), a but-1,3-ylene group (—CH2CH2CH(CH3)—), a but-2,3-ylene group (—CH(CH3)CH(CH3)—), a but-2-en-2,3-ylene group (—C(CH3)C(CH3)—), a 3-methylbut-1,3-ylene group (—CH2CH2C(CH3)2—), a 1,2-cyclopentylene group, a 1,2-cyclohexylene group, or a phen-1,2-ylene group; alternatively, a methylene group (—CH2—), an eth-1,2-ylene group (—CH2CH2—), a prop-1,3-ylene group (—CH2CH2CH2—), a prop-1,2-ylene group (—CH(CH3)CH2—), a prop-2,2-ylene group (—C(CH3)2—), a but-1,4-ylene group (—CH2CH2CH2—CH2—), a but-1,3-ylene group (—CH2CH2CH(CH3)—), a but-2,3-ylene group (—CH(CH3)CH(CH3)—), a 1,2-cyclopentylene group, a 1,2-cyclohexylene group, or a phen-1,2-ylene group; or alternatively, an eth-1,2-ylene group (—CH2CH2—), a prop-1,3-ylene group (—CH2CH2CH2—), a prop-1,2-ylene group (—CH(CH3)CH2—), a but-1,3-ylene group (—CH2CH2CH(CH3)—), a but-2,3-ylene group (—CH(CH3)CH(CH3)—), a 1,2-cyclopentylene group, a 1,2-cyclohexylene group, or a phen-1,2-ylene group. Various aspects described herein refer to non-hydrogen substituents such as halogen (or halo, halide), hydrocarbyl, hydrocarboxy, alkyl, and/or alkoxy substituents. In an aspect, each non-hydrogen substituent of any aspect calling for a substituent can be a halogen, a hydrocarbyl group, or a hydrocarboxy group; alternatively, a halogen or a hydrocarbyl group; alternatively, a halogen or a hydrocarboxy group; alternatively, a hydrocarbyl group or a hydrocarboxy group; alternatively, a halogen; alternatively, a hydrocarbyl group; or alternatively, a hydrocarboxy group. Each hydrocarbyl substituent independently can be a C1to C10hydrocarbyl group; or alternatively, a C1to C5hydrocarbyl group. Each hydrocarboxy substituent independently can be a C1to C10hydrocarboxy group; or alternatively, a C1to C5hydrocarboxy group. Each halide substituent independently can be a fluoride, chloride, bromide, or iodide; alternatively, a fluoride or chloride; alternatively, a fluoride; alternatively, a chloride; alternatively, a bromide; or alternatively, an iodide. In an aspect, any hydrocarbyl substituent independently can be an alkyl group, an aryl group, or an aralkyl group; alternatively, an alkyl group; alternatively, an aryl group; or alternatively, an aralkyl group. In an aspect, any alkyl substituent independently can be a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, a 2-pentyl group, a 3-pentyl group, a 2-methyl-1-butyl group, a tert-pentyl group, a 3-methyl-1-butyl group, a 3-methyl-2-butyl group, or a neo-pentyl group; alternatively, a methyl group, an ethyl group, an isopropyl group, a tert-butyl group, or a neo-pentyl group; alternatively, a methyl group; alternatively, an ethyl group; alternatively, an isopropyl group; alternatively, a tert-butyl group; or alternatively, a neo-pentyl group. In an aspect, any aryl substituent independently can be phenyl group, a tolyl group, a xylyl group, or a 2,4,6-trimethylphenyl group; alternatively, a phenyl group; alternatively, a tolyl group, alternatively, a xylyl group; or alternatively, a 2,4,6-trimethylphenyl group. In an aspect, any aralkyl substituent independently can be benzyl group or an ethylphenyl group (2-phenyleth-1-yl or 1-phenyleth-1-yl); alternatively, a benzyl group; alternatively, an ethylphenyl group; alternatively, a 2-phenyleth-1-yl group; or alternatively, a 1-phenyleth-1-yl group. In an aspect, any hydrocarboxy substituent independently can be an alkoxy group, an aryloxy group, or an aralkoxy group; alternatively, an alkoxy group; alternatively, an aryloxy group, or an aralkoxy group. In an aspect, any alkoxy substituent independently can be a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, a sec-butoxy group, an isobutoxy group, a tert-butoxy group, an n-pentoxy group, a 2-pentoxy group, a 3-pentoxy group, a 2-methyl-1-butoxy group, a tert-pentoxy group, a 3-methyl-1-butoxy group, a 3-methyl-2-butoxy group, or a neo-pentoxy group; alternatively, a methoxy group, an ethoxy group, an isopropoxy group, a tert-butoxy group, or a neo-pentoxy group; alternatively, a methoxy group; alternatively, an ethoxy group; alternatively, an isopropoxy group; alternatively, a tert-butoxy group; or alternatively, a neo-pentoxy group. In an aspect, any aryloxy substituent independently can be phenoxy group, a toloxy group, a xyloxy group, or a 2,4,6-trimethylphenoxy group; alternatively, a phenoxy group; alternatively, a toloxy group, alternatively, a xyloxy group; or alternatively, a 2,4,6-trimethylphenoxy group. In an aspect, any aralkoxy substituent independently can be benzoxy group. Generally, the chromium compound or the chromium compound of the heteroatomic ligand chromium compound complexes described herein can have formula CrXpwhere X represents a monoanionic ligand, and p represents the number of monoanionic ligands (and the oxidation state of the chromium in the chromium compound). The monoanionic ligand (X) and p are independent elements of the chromium compound or the chromium compound of the heteroatomic ligand chromium compound complexes described herein and are independently described herein. The independent descriptions of the monoanionic ligand (X) and p can be utilized without limitation, and in any combination, to further describe the chromium compound or the chromium compound of the heteroatomic ligand chromium compound complexes described herein. Generally, the chromium atom of the chromium compound (CrXp) can have any positive oxidation state available to a chromium atom. In an aspect, the chromium atom can have an oxidation state of from +2 to +6; alternatively, from +2 to +4; or alternatively, from +2 to +3. In some aspects, the chromium atom of the chromium compound (CrXp) can have an oxidation state of +1; alternatively, +2; alternatively, +3; or alternatively, +4. The monoanion (X) of the chromium compound can be any monoanion. In an aspect, the monoanion (X) can be a halide, a carboxylate, a β-diketonate, a hydrocarboxide, a nitrate, or a chlorate. In some aspects, the monoanion (X) can be a halide, a carboxylate, a β-diketonate, or a hydrocarboxide. In any aspect, the hydrocarboxide can be an alkoxide, an aryloxide, or an aralkoxide. Generally, hydrocarboxide (and subdivisions of hydrocarboxide) are the anion analogues of the hydrocarboxy group. In other aspects, the monoanion (X) can be a halide, a carboxylate, a β-diketonate, or an alkoxide; or alternatively, a halide or a β-diketonate. In other aspects, the monoanion (X) can be a halide; alternatively, a carboxylate; alternatively, a β-diketonate; alternatively, a hydrocarboxide; alternatively, an alkoxide; or alternatively, an aryloxide. Generally, when the heteroatomic ligand of the heteroatomic ligand chromium compound complex is a neutral heteroatomic ligand the number of monoanions (p) can equal the oxidation state of the chromium atom. When the heteroatomic ligand of the heteroatomic ligand chromium compound complex is an anionic heteroatomic ligand the number of monoanions (p) can equal one less than the oxidation state of the chromium atom. In an aspect, the number of monoanions can be from 2 to 6; alternatively, from 2 to 4; alternatively, from 2 to 3; alternatively, 1; alternatively, 2; alternatively, 3; or alternatively, 4. Generally, each halide of the chromium compound independently can be fluorine, chlorine, bromine, or iodine; or alternatively, chlorine, bromine, or iodine. In an aspect, each halide monoanion of the chromium compound can be chlorine; alternatively, bromine; or alternatively, iodine. Generally, each carboxylate of the chromium compound independently can be a C1to C20carboxylate; or alternatively, a C1to C10carboxylate. In an aspect, each carboxylate of the chromium compound independently can be acetate, a propionate, a butyrate, a pentanoate, a hexanoate, a heptanoate, an octanoate, a nonanoate, a decanoate, an undecanoate, or a dodecanoate; or alternatively, a pentanoate, a hexanoate, a heptanoate, an octanoate, a nonanoate, a decanoate, an undecanoate, or a dodecanoate. In some aspects, each carboxylate of the chromium compound independently can be acetate, propionate, n-butyrate, valerate (n-pentanoate), neo-pentanoate, capronate (n-hexanoate), n-heptanoate, caprylate (n-octanoate), 2-ethylhexanoate, n-nonanoate, caprate (n-decanoate), n-undecanoate, or laurate (n-dodecanoate); alternatively, valerate (n-pentanoate), neo-pentanoate, capronate (n-hexanoate), n-heptanoate, caprylate (n-octanoate), 2-ethylhexanoate, n-nonanoate, caprate (n-decanoate), n-undecanoate, or laurate (n-dodecanoate); alternatively, capronate (n-hexanoate); alternatively, n-heptanoate; alternatively, caprylate (n-octanoate); or alternatively, 2-ethylhexanoate. In some aspects, the carboxylate of the chromium compound can be triflate (trifluoroacetate). Generally, each β-diketonate of the chromium compound independently can be any C1to C20a β-diketonate; or alternatively, any C1to C10β-diketonate. In an aspect, each β-diketonate of the chromium compound independently can be acetylacetonate (i.e., 2,4-pentanedionate), hexafluoroacetylacetonate (i.e., 1,1,1,5,5,5-hexafluoro-2,4-pentanedionate), or benzoylacetonate); alternatively, acetylacetonate; alternatively, hexafluoroacetylacetonate; or alternatively, benzoylacetonate. Generally, each hydrocarboxide of the chromium compound independently can be any C1to C20hydrocarboxide; or alternatively, any C1to C10hydrocarboxide. In an aspect, each hydrocarboxide of the chromium compound independently can be a C1to C20alkoxide; alternatively, a C1to C10alkoxide; alternatively, a C6to C20aryloxide; or alternatively, a C6to C10aryloxide. In an aspect, each alkoxide of the chromium compound independently can be methoxide, ethoxide, a propoxide, or a butoxide; alternatively, methoxide, ethoxide, isopropoxide, or tert-butoxide; alternatively, methoxide; alternatively, an ethoxide; alternatively, an iso-propoxide; or alternatively, a tert-butoxide. In an aspect, the aryloxide can be phenoxide. In some non-limiting aspects, the chromium compound and/or the chromium compound of the heteroatomic ligand chromium compound complex can comprise, can consist essentially of, or consist of, a chromium(II) halide, a chromium(II) carboxylate, or a chromium(II) β-diketonate; or alternatively, a chromium(III) halide, a chromium(III) carboxylate, or a chromium(III) β-diketonate. In other non-limiting aspects, the chromium compound and/or the chromium compound of the heteroatomic ligand chromium compound complex can comprise, can consist essentially of, or consist of, a chromium(II) halide; alternatively, a chromium(III) halide; alternatively, a chromium (II) carboxylate; alternatively, a chromium(III) carboxylate; alternatively, a chromium(II) β-diketonate; or alternatively, a chromium(III) β-diketonate. Halides, carboxylates, β-diketonates are independently described herein and these halides, carboxylates, β-diketonate and these independently described halides, carboxylates, β-diketonates can be utilized without limitation and in any combination to further described the chromium compound and/or the chromium compound of the heteroatomic ligand chromium compound complex. In further non-limiting aspects, the chromium compound and/or the chromium compound of the heteroatomic ligand chromium compound complex can comprise, can consist essentially of, or consist of, chromium(II) chloride, chromium(III) chloride, chromium(II) fluoride, chromium(III) fluoride, chromium(II) bromide, chromium(III) bromide, chromium(II) iodide, chromium(III) iodide, chromium(II) acetate, chromium(III) acetate, chromium(II) 2-ethylhexanoate, chromium(III) 2-ethylhexanoate, chromium(II) triflate, chromium(III) triflate, chromium(II) nitrate, chromium(III) nitrate, chromium(II) acetylacetonate, chromium(III) acetylacetonate, chromium(II) hexafluoracetylacetonate, chromium(III) hexafluoracetylacetonate, chromium(III) benzoylacetonate, or chromium(III) benzoylacetonate; alternatively, chromium(III) chloride, chromium(III) fluoride, chromium(III) bromide, chromium(III) iodide, chromium(III) chloride (THF) complex, chromium(III) acetate, chromium(III) 2-ethylhexanoate, chromium(III) triflate, chromium(III) nitrate, chromium(III) acetylacetonate, chromium(III) hexafluoracetylacetonate, or chromium(III) benzoylacetonate; alternatively, chromium(III) chloride, or chromium(III) acetylacetonate; alternatively, chromium(III) chloride; or alternatively, chromium(III) acetylacetonate. In a non-limiting aspect, the heteroatomic ligand chromium compound complex can be selected from any one or more of a heteroatomic ligand chromium compound complex having i) Structure NPFCr1 where R1is 2,6-dimethylphenyl, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,6-dimethylphenyl, R3is H, R4and R5are phenyl, and X is chlorine; R1is 2,6-dimethylphenyl, R3is H, R4and R5are 4-methoxyphenyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R3is H, R4and R5are phenyl, and X is chlorine; and R1is 2,4,6-trimethylphenyl, R3is H, R4and R5are 4-methoxyphenyl, and X is chlorine: ii) Structure NPACr1 where R1is 2,6-dimethylphenyl, R2is phenyl, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is phenyl, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2is phenyl, R3is H, R4and R5are phenyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is phenyl, R3is H, R4and R5are phenyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2is 4-methylbenzyl, R3is H, R4and R5are phenyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2is phenyl, R3is H, R4and R5are 4-methoxyphenyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2is 4-t-butylphenyl, R3is H, R4and R5are methyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-t-butylphenyl, R3is H, R4and R5are methyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4and R5are phenyl, and X is chlorine; R1is 3,5-dimethylphenyl, R2is phenyl, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4and R5are 4-methoxyphenyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4is t-butyl, R5is phenyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4is methyl, R5is phenyl, and X is chlorine; R1and R2are joined to form a prop-1,3-ylene group, R3is H, R4and R5are isopropyl, and X is chlorine; R1and R2are joined to form a but-1,4-ylene group, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4and R5are joined to form a but-1,4-ylene group, and X is chlorine; R1is 2,4,6-trimethylphenyl, R2is 4-methylbenzyl, R3is H, R4and R5are joined to form a 2,2′-dimethylbiphenylene group, and X is chlorine: iii) Structure GUCr1 where R1is 2-methylphenyl, R2ais 2-methylphenyl, R2bis H, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2ais phenyl, R2bis H, R3is H, R4and R5are isopropyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2ais phenyl, R2bis H, R3is H, R4and R5are phenyl, and X is chlorine; R1is 2,6-dimethylphenyl, R2aand R2bare phenyl, R3is H, R4and R5are isopropyl, and X is chlorine: iv) Structure GUCr4 where L12is prop-1,3-ylene, L23is prop-1, 3-ylene, R4and R5are isopropyl, and X is chlorine; L12is prop-1,3-ylene, L23is prop-1,3-ylene, R4and R5are cyclopentyl, and X is chlorine; L12is prop-1,3-ylene, L23is prop-1,3-ylene, R4and R5are cyclohexyl, and X is chlorine; L12is prop-1,3-ylene, L23is prop-1,3-ylene, R4and R5are phenyl, and X is chlorine; L12is but-1,3-ylene, L23is prop-1,3-ylene, R4and R5are isopropyl, and X is chlorine; L12is but-1,3-ylene, L23is prop-1,3-ylene, R4and R5are cyclopentyl, and X is chlorine; L12is but-1,3-ylene, L23is but-1,3-ylene, R4and R5are isopropyl, and X is chlorine; L12is but-1,3-ylene, L23is but-1,3-ylene, R4and R5are phenyl, and X is chlorine; L12is ethen-1,2-ylene, L23is prop-1,3-ylene, R4and R5are isopropyl, and X is chlorine; L12is ethen-1,2-ylene, L23is prop-1,3-ylene, R4and R5are cyclopentyl, and X is chlorine; L12is ethen-1,2-ylene, L23is prop-1,3-ylene, R4and R5are cyclohexyl, and X is chlorine; L12is phen-1,2-ylene, L23is eth-1,2-ylene, R4and R5are isopropyl, and X is chlorine: and v) Structure HCPACr2 where T is sulfur, L12is ethen-1,2-ylene, R3is H, R4and R5are isopropyl, and X is chlorine; and T is sulfur, L12is phen-1,2-ylene, R3is H, R4and R5are isopropyl, and X is chlorine. In a non-limiting aspect, the heteroatomic ligand can be any one or more of HL 1, HL 2, HL 3, HL 4. HL 5, HL 6, HL 7, HL 7, and HL 9. In some non-limiting aspects, the diphosphino amine chromium compound complex can be a chromium compound complex of any one or more of HLCr 1, HLCr 2, HLCr 3, HLCr 4, HLCr 5, HLCr 6, HLCr 7, HLCr 8, and HLCr 9. In other non-limiting aspects, the diphosphino amine chromium compound complex can be a chromium(III) chloride or chromium(III) acetylacetonate complex of any one or more of HLCr 1, HLCr 2, HLCr 3, HLCr 4, HLCr 5, HLCr 6, HLCr 7, HLCr 8, and HLCr 9. While not shown in all of the chromium compound names and formulas and/or heteroatomic ligand chromium compound complex formulas and structures provided herein, one of ordinary skill in the art will recognize that a neutral ligand, Q, can be associated with the chromium compounds and/or the heteroatomic ligand chromium compound complexes described/depicted herein which do not explicitly disclose/depict a neutral ligand. Consequently, chromium compounds and/or heteroatomic ligand chromium compound complexes having a neutral ligand, Q, can be considered as equivalent to the chromium compounds and/or heteroatomic ligand chromium compound complexes depicted herein not having the neutral ligand, Q. Additionally, it should be understood that while some of the chromium compounds and/or heteroatomic ligand chromium compound complexes described/depicted/provided herein do not formally show the presence of a neutral ligand, the chromium compounds and/or heteroatomic ligand chromium compound complexes having neutral ligands (e.g., nitriles and ethers, among others) are fully contemplated and encompassed herein as potential chromium compounds and/or heteroatomic ligand chromium compound complexes that can be utilized in the catalyst system used in aspects of the present disclosure. Generally, the neutral ligand of any chromium compound and/or heteroatomic ligand chromium compound complex, when present, independently can be any neutral ligand that forms an isolatable compound with the chromium compound and/or heteroatomic ligand chromium compound complex. In an aspect, each neutral ligand independently can be a nitrile or an ether; alternatively, a nitrile; or alternatively, an ether. The number of neutral ligands, q, can be any number that forms an isolatable compound with the chromium compound, and/or heteroatomic ligand chromium compound complex. In an aspect, the number of neutral ligands can be from 0 to 6; alternatively, 0 to 3; alternatively, 0; alternatively, 1; alternatively, 2; alternatively, 3; or alternatively, 4. Generally, each nitrile ligand independently can be a C2to C20nitrile; or alternatively, a C2to C10nitrile. In an aspect, each nitrile ligand independently can be a C2to C20aliphatic nitrile, a C7to C20aromatic nitrile, a C8to C20aralkane nitrile, or any combination thereof; alternatively, a C2to C20aliphatic nitrile; alternatively, a C7to C20aromatic nitrile; or alternatively, a C8to C20aralkane nitrile. In some aspects, each nitrile ligand independently can be a C2to C10aliphatic nitrile, a C7to C10aromatic nitrile, a C8to C10aralkane nitrile, or any combination thereof; alternatively, a C1to C10aliphatic nitrile; alternatively, a C7to C10aromatic nitrile; or alternatively, a C8to C10aralkane nitrile. In an aspect, each aliphatic nitrile independently can be acetonitrile, propionitrile, a butyronitrile, benzonitrile, or any combination thereof; alternatively, acetonitrile; alternatively, propionitrile; alternatively, a butyronitrile; or alternatively, benzonitrile. Generally, each ether ligand independently can be a C2to C40ether; alternatively, a C2to C30ether; or alternatively, a C2to C20ether. In an aspect, each ether ligand independently can be a C2to C40aliphatic ether, a C3to C40aliphatic cyclic ether, a C4to C40aromatic cyclic ether; alternatively, a C2to C40aliphatic acyclic ether or a C3to C40aliphatic cyclic ether; alternatively, a C2to C40aliphatic acyclic ether; alternatively, a C3to C40aliphatic cyclic ether; or alternatively, a C4to C40aromatic cyclic ether. In some aspects, each ether ligand independently can be a C2to C30aliphatic ether, a C3to C30aliphatic cyclic ether, a C4to C30aromatic cyclic ether; alternatively, a C2to C30aliphatic acyclic ether or a C3to C30aliphatic cyclic ether; alternatively, a C2to C30aliphatic acyclic ether; alternatively, a C3to C30aliphatic cyclic ether; or alternatively, a C4to C30aromatic cyclic ether. In other aspects, each ether ligand independently can be a C2to C20aliphatic ether, a C3to C20aliphatic cyclic ether, a C4to C20aromatic cyclic ether; alternatively, a C2to C20aliphatic acyclic ether or a C3to C20aliphatic cyclic ether; alternatively, a C2to C20aliphatic acyclic ether; alternatively, a C3to C20aliphatic cyclic ether; or alternatively, a C4to C20aromatic cyclic ether. In some aspects, each ether ligand independently can be dimethyl ether, diethyl ether, a dipropyl ether, a dibutyl ether, methyl ethyl ether, a methyl propyl ether, a methyl butyl ether, tetrahydrofuran, a dihydrofuran, 1,3-dioxolane, tetrahydropyran, a dihydropyran, a pyran, a dioxane, furan, benzofuran, isobenzofuran, dibenzofuran, diphenyl ether, a ditolyl ether, or any combination thereof; alternatively, dimethyl ether, diethyl ether, a dipropyl ether, a dibutyl ether, methyl ethyl ether, a methyl propyl ether, a methyl butyl ether, or any combination thereof; tetrahydrofuran, a dihydrofuran, 1,3-dioxolane, tetrahydropyran, a dihydropyran, a pyran, a dioxane, or any combination thereof; furan, benzofuran, isobenzofuran, dibenzofuran, or any combination thereof; diphenyl ether, a ditolyl ether, or any combination thereof; alternatively, dimethyl ether; alternatively, diethyl ether; alternatively, a dipropyl ether; alternatively, a dibutyl ether; alternatively, methyl ethyl ether; alternatively, a methyl propyl ether; alternatively, a methyl butyl ether; alternatively, tetrahydrofuran; alternatively, a dihydrofuran; alternatively, 1,3-dioxolane; alternatively, tetrahydropyran; alternatively, a dihydropyran; alternatively, a pyran; alternatively, a dioxane; alternatively, furan; alternatively, benzofuran; alternatively, isobenzofuran; alternatively, dibenzofuran; alternatively, diphenyl ether; or alternatively, a ditolyl ether. While the heteroatomic ligand chromium compound complex formulas and structures provided herein are shown as neutral complexes, one of ordinary skill in the art will recognize that heteroatomic ligand chromium compound complexes can comprise or can exist as “ate” complexes comprising a negatively charged heteroatomic ligand chromium compound complex and an associated positively charged metal or metal complex cation. Additionally, it should be understood that while the heteroatomic ligand chromium compound complexes described/depicted/provided herein are shown as neutral complexes, the “ate” complexes comprising a negatively charged heteroatomic ligand chromium compound complex and an associated positively charged metal or metal complex cation are implicitly and fully contemplated as potential heteroatomic ligand chromium compound complexes that can be utilized in the catalyst system used in aspects of the present disclosure. Generally, the organoaluminum compound utilized in the catalyst systems disclosed herein can be any organoaluminum compound which in conjunction with the heteroatomic ligand chromium compound complex (or the chromium compound and heteroatomic ligand) can catalyze the formation of an oligomer product. In an aspect, the organoaluminum compound can comprise, can consist essentially of, or can be, an aluminoxane, an alkylaluminum compound, or any combination thereof; alternatively, an aluminoxane; or alternatively, an alkylaluminum compound. In an aspect, the alkylaluminum compound can comprise, can consist essentially of, or can be, a trialkylaluminum, an alkylaluminum halide, an alkylaluminum alkoxide, or any combination thereof. In some aspects, the alkylaluminum compound can comprise, can consist essentially of, or can be, a trialkylaluminum, an alkylaluminum halide, or any combination thereof; alternatively, a trialkylaluminum, an alkylaluminum alkoxide, or any combination thereof; or alternatively, a trialkylaluminum. In other aspects, the alkylaluminum compound can be a trialkylaluminum; alternatively, an alkylaluminum halide; or alternatively, an alkylaluminum alkoxide. In an aspect, the aluminoxane utilized in the catalyst systems which are utilized in the processes and systems can comprise, can consist essentially of, or can be, any aluminoxane which in conjunction with the heteroatomic ligand chromium compound complex (or the chromium compound and heteroatomic ligand) can catalyze the formation of an oligomer product. In a non-limiting aspect, the aluminoxane can have a repeating unit characterized by the Formula I: wherein R′ is a linear or branched alkyl group. Alkyl groups of the aluminoxanes and alkylaluminum compounds are independently described herein and can be utilized without limitation to further describe the aluminoxanes having Formula I and/or the alkylaluminum compounds. Generally, n of Formula I can be greater than 1; or alternatively, greater than 2. In an aspect, n can range from 2 to 15; or alternatively, range from 3 to 10. In an aspect, each halide of any alkylaluminum halide disclosed herein can independently be fluoride, chloride, bromide, or iodide; or alternatively, chloride, bromide, or iodide. In an aspect, each halide of any alkylaluminum halide disclosed herein can be fluoride; alternatively, chloride; alternatively, bromide; or alternatively, iodide. In an aspect, each alkyl group of an aluminoxane and/or alkylaluminum compound independently can be a C1to C20alkyl group; alternatively, a C1to C10alkyl group; or alternatively, a C1to C6alkyl group. In an aspect, each alkyl group of an aluminoxane and/or alkylaluminum compound a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, or an octyl group; alternatively, a methyl group, a ethyl group, a butyl group, a hexyl group, or an octyl group. In some aspects, each alkyl group of an aluminoxane and/or alkylaluminum compound can be a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an iso-butyl group, an n-hexyl group, or an n-octyl group; alternatively, a methyl group, an ethyl group, an n-butyl group, or an iso-butyl group; alternatively, a methyl group; alternatively, an ethyl group; alternatively, an n-propyl group; alternatively, an n-butyl group; alternatively, an iso-butyl group; alternatively, an n-hexyl group; or alternatively, an n-octyl group. In an aspect, each alkoxide group of any alkylaluminum alkoxide disclosed herein independently can be a C1to C20alkoxy group, a C1to C10alkoxy group, or a C1to C6alkoxy group. In an aspect, each alkoxide group of any alkylaluminum alkoxide disclosed herein independently can be a methoxy group, an ethoxy group, a propoxy group, a butoxy group, a pentoxy group, a hexoxy group, a heptoxy group, or an octoxy group; alternatively, a methoxy group, an ethoxy group, a butoxy group, a hexoxy group, or an octoxy group. In some aspects, each alkoxide group of any alkylaluminum alkoxide disclosed herein independently can be a methoxy group, an ethoxy group, an n-propoxy group, an n-butoxy group, an iso-butoxy group, an n-hexoxy group, or an n-octoxy group; alternatively, a methoxy group, an ethoxy group, an n-butoxy group, or an iso-butoxy group; alternatively, a methoxy group; alternatively, an ethoxy group; alternatively, an n-propoxy group; alternatively, an n-butoxy group; alternatively, an iso-butoxy group; alternatively, an n-hexoxy group; or alternatively, an n-octoxy group. In a non-limiting aspect, useful trialkylaluminum compounds can include trimethylaluminum, triethylaluminum, tripropylaluminum, tributylaluminum, trihexylaluminum, trioctylaluminum, or mixtures thereof. In some non-limiting aspects, useful trialkylaluminum compounds can include trimethylaluminum, triethylaluminum, tripropylaluminum, tri-n-butylaluminum, tri-isobutylaluminum, trihexylaluminum, tri-n-octylaluminum, or mixtures thereof; alternatively, triethylaluminum, tri-n-butylaluminum, tri-isobutylaluminum, trihexylaluminum, tri-n-octylaluminum, or mixtures thereof; alternatively, triethylaluminum, tri-n-butylaluminum, trihexylaluminum, tri-n-octylaluminum, or mixtures thereof. In other non-limiting aspects, useful trialkylaluminum compounds can include trimethylaluminum; alternatively, triethylaluminum; alternatively, tripropylaluminum; alternatively, tri-n-butylaluminum; alternatively, tri-isobutylaluminum; alternatively, trihexylaluminum; or alternatively, tri-n-octylaluminum. In a non-limiting aspect, useful alkylaluminum halides can include diethylaluminum chloride, diethylaluminum bromide, ethylaluminum dichloride, ethylaluminum sesquichloride, and mixtures thereof. In some non-limiting aspects, useful alkylaluminum halides can include diethylaluminum chloride, ethylaluminum dichloride, ethylaluminum sesquichloride, and mixtures thereof. In other non-limiting aspects, useful alkylaluminum halides can include diethylaluminum chloride; alternatively, diethylaluminum bromide; alternatively, ethylaluminum dichloride; or alternatively, ethylaluminum sesquichloride. In a non-limiting aspect, the aluminoxane can be, comprise, or consist essentially of, methylaluminoxane (MAO), ethylaluminoxane, modified methylaluminoxane (MMAO), n-propylaluminoxane, iso-propyl-aluminoxane, n-butylaluminoxane, sec-butylaluminoxane, iso-butylaluminoxane, t-butylaluminoxane, 1-pentylaluminoxane, 2-entylaluminoxane, 3-pentyl-aluminoxane, iso-pentyl-aluminoxane, neopentylaluminoxane, or mixtures thereof. In some non-limiting aspects, the aluminoxane can be, comprise, or consist essentially of, methylaluminoxane (MAO), modified methylaluminoxane (MMAO), isobutyl aluminoxane, t-butyl aluminoxane, or mixtures thereof. In other non-limiting aspects, the aluminoxane can be, comprise, or consist essentially of, methylaluminoxane (MAO); alternatively, ethylaluminoxane; alternatively, modified methylaluminoxane (MMAO); alternatively, n-propylaluminoxane; alternatively, iso-propyl-aluminoxane; alternatively, n-butylaluminoxane; alternatively, sec-butylaluminoxane; alternatively, iso-butylaluminoxane; alternatively, t-butyl aluminoxane; alternatively, 1-pentyl-aluminoxane; alternatively, 2-pentylaluminoxane; alternatively, 3-pentyl-aluminoxane; alternatively, iso-pentyl-aluminoxane; or alternatively, neopentylaluminoxane. The components of the catalyst system can be combined in any order, in any manner, and for any length of time (e.g., aging) to prepare the catalyst system. The catalyst system mixture can be aged for any suitable period of time (e.g., 5 sec to 48 hr, from 10 sec to 36 hr, from 30 sec to 24 hr, from 1 min to 18 hr, from 5 min to 6 hr, from 10 min to 4 hr, or from 20 min to 2 hr) in the substantial absence of ethylene prior to introducing the catalyst system mixture into the activation vessel. Herein, the substantial absence of ethylene means that the catalyst system mixture contains less than 1 wt. % ethylene, based on the total weight of the catalyst system mixture. In some instance, less than 0.5 wt. %, less than 0.1 wt. %, or less than 0.05 wt. % ethylene, based upon the total weight of the catalyst system mixture, is present in the catalyst system mixture prior to the activation vessel. While not limited thereto, the catalyst system can be formed at a minimum temperature or −40° C., −20° C., 0° C., 10° C., 15° C., or 20° C.; alternatively or additionally, at a maximum temperature of 100° C., 75° C., 60° C., or 40° C. Generally, the catalyst system can be formed at a temperature in a range from any minimum temperature disclosed herein to any maximum temperature disclosed herein. Accordingly, suitable non-limiting temperatures for forming the catalyst system can be in a range from −40° C. to 100° C., from −20° C. to 100° C., from 0° C. to 100° C., from 10° C. to 75° C., from 15° C. to 60° C., or from 20° C. to 40° C. In these and other aspects, these temperature ranges also are meant to encompass circumstances where the catalyst system is formed at a series of different temperatures, instead of at a single fixed temperature, falling within the respective ranges. The Al to Cr molar ratio of the catalyst system (or in which the oligomer product is formed) can be in a range of 10:1 to 5,000:1, from 50:1 to 3,000:1, from 50:1 to 3,000:1, from 75:1 to 2,000:1, from 100:1 to 2,000:1, or from 100:1 to 1,000:1. If more than one complex and/or more than one organoaluminum are employed, the Al to Cr ratio is based on the total moles of chromium and/or aluminum. Oligomerization Processes Aspects of this disclosure are directed to ethylene oligomerization processes and the formation of an oligomer product. A first process can comprise (or consist essentially of, or consist of) a) forming a first mixture in an activation vessel, the first mixture comprising ethylene, a catalyst system comprising i) a heteroatomic ligand chromium compound complex and an organoaluminum compound, or ii) a heteroatomic ligand, a chromium compound, and an organoaluminum compound, optionally, a first organic reaction medium, and optionally, hydrogen; b) maintaining the first mixture in the activation vessel for an average residence time; c) introducing into a reaction zone: ethylene, the first mixture from step b), a second organic reaction medium, and optionally, hydrogen; and forming an oligomer product in the reaction zone. A second process consistent with this disclosure can comprise a) forming a first mixture in an activation vessel, the first mixture comprising 1) a first catalyst system comprising i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound, 2) a first feed comprising ethylene, a second catalyst system comprising i) a second heteroatomic ligand chromium compound complex and a second organoaluminum compound, or ii) a second heteroatomic ligand, a second chromium compound, and a second organoaluminum compound, a second organic reaction medium, a second oligomer product, and optionally hydrogen, 3) optionally, a first organic reaction medium, and 4) optionally, hydrogen; b) maintaining the first mixture in the activation vessel for an average residence time; c) introducing into a reaction zone: ethylene, the first mixture from step b), the second organic reaction medium, and optionally, hydrogen; and d) forming an oligomer product in the reaction zone. In an aspect, the process can further comprise discharging a reaction zone effluent comprising the oligomer product (or the second oligomer product); or alternatively, ethylene, a catalyst system (or a second catalyst system), an organic reaction medium (or a second organic reaction medium), an oligomer product (or a second oligomer product), and optionally hydrogen. In an aspect, a portion of a reaction zone effluent is fed to the activation vessel as the first feed. In these first and second processes, the average residence time of the first mixture in the activation vessel can be in a range from 10 sec up to i) a period of time sufficient to form an amount of a first oligomer product in the activation vessel equal to 5%, 4%, 3%, 2%, or 1% of an amount of the oligomer product formed in the reaction zone; or ii) a conversion of ethylene in the activation vessel of 5 mol %, 4 mol %, 3 mol %, 2 mol %, 1 mol %, or 0.5 mol % of a total ethylene utilized in step a) and step c); or iii) a catalyst system productivity in the activation vessel of 5%, 4%, 3%, 2%, or 1% of a catalyst system productivity in the reaction zone; or iv) a fouling rate in the activation vessel of 0.065 mg/cm2-hr, 0.06 mg/cm2-hr, 0.055 mg/cm2-hr, 0.05 mg/cm2-hr, 0.045 mg/cm2-hr, or 0.04 mg/cm2-hr; or v) an oligomer product discharge rate from the activation vessel of 0.15 lb/gal/hr, 0.125 lb/gal/hr, 0.1 lb/gal/hr, or 0.075 lb/gal/hr, and an oligomer product discharge rate in the reaction zone can be in a range from 0.75 to 6 lb/gal/hr, 1 to 6 lb/gal/hr, 1.2 to 5.5 lb/gal/hr, 1.4 to 5 lb/gal/hr, 1.5 to 4.5 lb/gal/hr, or 1.6 to 4.25 lb/gal/hr; or vi) a ΔT from the inlet of the activation vessel to the outlet of the activation vessel of 5° C., 4° C., 3° C., 2° C., or 1° C.; or vii) a maximum time of 8 hr, 6 hr, 4 hr, 2 hr, 1 hr, 45 min, 30 min, 20 min, or 15 min. In these first and second processes, an amount of ethylene introduced into the activation vessel can be less than 50%, 40%, 30%, or 20% of an amount of ethylene introduced into the reaction zone; or step b) can be performed at an activation temperature and an activation pressure, and the first mixture in the activation vessel can be below the bubble point at the activation temperature and the activation pressure. Generally, the features of the first process and the second process (e.g., the catalyst system, the organic reaction medium, the first mixture and its average residence time in the activation vessel, the oligomerization conditions under which the oligomer product is formed, and the reaction zone, among others) are independently described herein, and these features can be combined without limitation, and in any combination, to further describe the first process and the second process. Moreover, additional process steps can be performed before, during, and/or after any of the steps of any of the processes disclosed herein, unless stated otherwise. Referring first to step a) of the first process, which is directed to forming a first mixture in an activation vessel, the first mixture can comprise ethylene, a catalyst system comprising i) a heteroatomic ligand chromium compound complex and an organoaluminum compound, or ii) a heteroatomic ligand, a chromium compound, and an organoaluminum compound, optionally, a first organic reaction medium, and optionally, hydrogen. Suitable heteroatomic ligand chromium compound complexes, heteroatomic ligands, chromium compounds, and organoaluminum compounds are disclosed herein and can be used without limitation in the first process. While not required, the catalyst system can further comprise a suitable catalyst system organic medium, such as an aromatic hydrocarbon (e.g., benzene, toluene, xylene (including ortho-xylene, meta-xylene, para-xylene, or mixtures thereof), cumene, and ethylbenzene, or a C8and/or C9aromatic stream (Total Atosol 100, ExxonMobil A100, and Shell Solv100, or other streams containing xylenes, cumene, or ethylbenzene, among others), among other aromatic hydrocarbons). While also not required, a first organic reaction medium often can be combined with ethylene and the catalyst system to form the first mixture in the activation vessel. When employed, any suitable organic reaction medium can be used as the first organic reaction medium, such as a hydrocarbon. Hydrocarbons can include, for example, aliphatic hydrocarbons, aromatic hydrocarbons, petroleum distillates, or combinations thereof; alternatively, aliphatic hydrocarbons; or alternatively, aromatic hydrocarbons. Aliphatic hydrocarbons which can be used as the first organic reaction medium include C3to C20aliphatic hydrocarbons; alternatively, C4to C15aliphatic hydrocarbons; or alternatively, C5to C10aliphatic hydrocarbons. The aliphatic hydrocarbons can be cyclic or acyclic and/or can be linear or branched, unless otherwise specified. In some aspects, the aliphatic hydrocarbon which can be utilized as the first organic reaction medium can be a hydrocarbon olefin (linear or branched, or terminal or internal). Non-limiting examples of suitable acyclic aliphatic hydrocarbon reaction medium that can be utilized singly or in any combination include propane, iso-butane, n-butane, butane (n-butane or a mixture of linear and branched C4acyclic aliphatic hydrocarbons), pentane (n-pentane or a mixture of linear and branched C5acyclic aliphatic hydrocarbons), hexane (n-hexane or a mixture of linear and branched C6acyclic aliphatic hydrocarbons), heptane (n-heptane or a mixture of linear and branched C7acyclic aliphatic hydrocarbons), octane (n-octane or a mixture of linear and branched C8acyclic aliphatic hydrocarbons), or combinations thereof; alternatively, iso-butane, n-butane, butane (n-butane or a mixture of linear and branched C4acyclic aliphatic hydrocarbons), pentane (n-pentane or a mixture of linear and branched C5acyclic aliphatic hydrocarbons), hexane (n-hexane or a mixture of linear and branched C6acyclic aliphatic hydrocarbons), heptane (n-heptane or a mixture of linear and branched C7acyclic aliphatic hydrocarbons), octane (n-octane or a mixture of linear and branched C8acyclic aliphatic hydrocarbons), or combinations thereof; alternatively, iso-butane, n-butane, butane (n-butane or a mixture of linear and branched C4acyclic aliphatic hydrocarbons), pentane (n-pentane or a mixture of linear and branched C5acyclic aliphatic hydrocarbons), heptane (n-heptane or a mixture of linear and branched C7acyclic aliphatic hydrocarbons), octane (n-octane or a mixture of linear and branched C8acyclic aliphatic hydrocarbons), or combinations thereof; alternatively, propane; alternatively, iso-butane; alternatively, n-butane; alternatively, butane (n-butane or a mixture of linear and branched C4acyclic aliphatic hydrocarbons); alternatively, pentane (n-pentane or a mixture of linear and branched C5acyclic aliphatic hydrocarbons); alternatively, hexane (n-hexane or a mixture of linear and branched C6acyclic aliphatic hydrocarbons); alternatively, heptane (n-heptane or a mixture of linear and branched C7acyclic aliphatic hydrocarbons); or alternatively, octane (n-octane or a mixture of linear and branched C8acyclic aliphatic hydrocarbons). In other aspects, the acyclic aliphatic reaction medium can be a product of the oligomerization (e.g., 1-hexene and/or 1-octene). Non-limiting examples of suitable cyclic aliphatic hydrocarbon reaction medium include cyclohexane and methyl cyclohexane; alternatively, cyclohexane; or alternatively, methylcyclohexane. Aromatic hydrocarbons which can be useful as an organic reaction medium include C6to C20aromatic hydrocarbons, or alternatively, C6to C10aromatic hydrocarbons. Non-limiting examples of suitable aromatic hydrocarbons that can be utilized singly or in any combination include benzene, toluene, xylene (including ortho-xylene, meta-xylene, para-xylene, or mixtures thereof), cumene, and ethylbenzene, or combinations thereof; alternatively, benzene; alternatively, toluene; alternatively, xylene (including ortho-xylene, meta-xylene, para-xylene or mixtures thereof); alternatively, cumene; or alternatively, ethylbenzene. In a particular aspect of this disclosure, the first organic reaction medium can comprise, or consist essentially of, or consist of, cyclohexane. Ethylene, the catalyst system components, and the first organic reaction medium can be combined in any order. Thus, the first organic reaction medium can be introduced into the activation vessel with any one of the ethylene, the catalyst system organic medium (if used), the heteroatomic ligand chromium compound complex, and the organoaluminum compound (or the heteroatomic ligand, the chromium compound, and the organoaluminum compound), or in any combination. For instance, in one aspect, a first feed stream comprising ethylene and at least a portion of the first organic reaction medium can be introduced into the activation vessel separate from a catalyst system mixture comprising the catalyst system. The catalyst system mixture also can contain a portion of the first organic reaction medium. In some aspects, the heteroatomic ligand chromium compound complex and the organoaluminum compound are separately introduced into the activation vessel, or at least one of the heteroatomic ligand, the chromium compound, and the organoaluminum compound is separately introduced into the activation vessel. In an aspect, the heteroatomic ligand and the chromium compound are separately introduced into the activation vessel; this technique is generally referred to as in-situ formation of the heteroatomic ligand chromium compound complex in the activation vessel. In other aspects, a catalyst system mixture comprising the heteroatomic ligand chromium compound complex and the organoaluminum compound (or the heteroatomic ligand, the chromium compound, and the organoaluminum compound), and optionally at least a portion of the first organic reaction medium, can be formed and then introduced into the activation vessel. The catalyst system mixture can further contain a catalyst system organic medium. Catalyst system organic medium are independently described herein and can utilized without limitation to further describe the processes described herein (e.g., ethylbenzene or other suitable aromatic hydrocarbons described herein). Thus, the catalyst system mixture can be formed prior to entering the activation vessel. The catalyst system mixture can be aged for any suitable period of time (e.g., 5 sec to 48 hr, from 10 sec to 36 hr, from 30 sec to 24 hr, from 1 min to 18 hr, from 5 min to 6 hr, from 10 min to 4 hr, or from 20 min to 2 hr) in the substantial absence of ethylene prior to introducing the catalyst system mixture into the activation vessel. While not required, hydrogen can be combined with ethylene—and the optional first organic reaction medium—to form the first mixture in the activation vessel. Thus, in one aspect of the disclosure, hydrogen is present in step a) of the first process, while in another aspect, hydrogen is not present. In an aspect, step b) of the first process (or the second process) maintains the first mixture of the first process (or the second process) in the activation vessel for an average residence time. Generally, the average residence time can range from a minimum average residence time to a maximum average residence time. Generally, the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min. The maximum average residence time for the average residence time range can be based upon any one or more of several parameters including time, a period of time sufficient to form an amount of a first oligomer product in the activation vessel, a conversion of ethylene in the activation vessel compared to the total ethylene utilized in step a) and step c), a catalyst system productivity in the activation vessel compared to a catalyst system productivity in the reaction zone, a fouling rate in the activation vessel, an oligomer product discharge rate from the activation vessel, or a ΔT from the inlet of the activation vessel to the outlet of the activation vessel. Alternatively or additionally, the activation step b) of the first process (or the second process) can be performed using an amount of ethylene introduced into the activation vessel that is a percentage of the amount of the ethylene introduced into the reaction zone and/or can be performed at an activation temperature and an activation pressure such that the first mixture in the activation vessel can be below the bubble point at the activation temperature and the activation pressure. These features are independently described and can be utilized singly or in any combination to further describe the processes described herein. The appropriate residence time in the activation vessel can vary depending upon the identity of the heteroatomic ligand chromium compound complex (or heteroatomic ligand and chromium compound), the organoaluminum compound, and/or operating conditions in the activation vessel, but often can be on the order of the induction time for the particular catalyst system under oligomerization reaction conditions. Additionally, the components of the catalyst system can be various energetic states which at any given time or temperature lead the catalyst system having a distribution of molecules in various stages of activation. In an ideal situation, the average residence time would exactly match the induction time, such that the first mixture contains fully activated catalyst, but no oligomerization of ethylene has yet to occur. However, one of ordinary skill in the art would recognize, the average residence time can include catalyst system residence times which are greater than the induction time. Thus, some oligomer product can be formed in the activation vessel and the maximum average residence time may be described using parameters which could include features which indicate that a small amount of ethylene oligomerization is occurring in the activation vessel. In an aspect, step b) of the first process (or the second process), the first mixture can be maintained in the activation vessel for an average residence time. In one aspect, the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; additionally or alternatively, the maximum average residence time can be 8 hr, 6 hr, 4 hr, 2 hr, 45 min, 30 min, 20 min, or 15 min. Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to 45 min, from 10 sec to 30 min, from 30 sec to 30 min, from 30 sec to 20 min, from 1 min to 45 min, from 1 min to 30 min, or from 2 min to 15 min. Other appropriate ranges for the average residence time are readily apparent from this disclosure. In an aspect, the first mixture in the first process (or the second process) can be maintained in the activation vessel for an average residence time in which the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; alternatively or additionally, the maximum average residence time can be a period of time sufficient to form an amount of a first oligomer product in the activation vessel equal to 5% (or 4%, or 3%, or 2%, or 1%) of an amount of the oligomer product formed in the reaction zone. Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time, defined by the amount of the first oligomer product formed in the activation vessel, disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to 5%, from 10 sec to 1%, from 30 sec to 4%, from 30 sec to 1%, from 1 min to 3%, or from 2 min to 2% (the upper limit of 1-5% is based on the amount of the first oligomer product formed in the activation vessel divided by the amount of the oligomer product formed in the reaction zone). The appropriate maximum average residence time in the activation vessel can vary. Other appropriate ranges for the average residence time are readily apparent from this disclosure. Once the induction time of the catalyst system has been reached, oligomerization of ethylene can commence. Beneficially, no substantial oligomerization occurs in the activation vessel, but generally, a very small amount (typically less than or equal to 5% of the amount of oligomer product formed in the reaction zone, and lesser amounts such as 4%, 3%, 2%, 1%, and so forth) is permissible. The respective amounts of the first oligomer product formed in the activation vessel and the oligomer product formed in the reaction zone can be quantified using a gas chromatography (GC) analytical technique, or other suitable techniques. In an aspect, the first mixture in the first process (or the second process) can be maintained in the activation vessel for an average residence time in which the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; alternatively or additionally, the maximum average residence time can be characterized by a conversion of ethylene in the activation vessel that is less than or equal to 5 mol % (or 4 mol %, or 3 mol %, or 2 mol %, or 1 mol %, or 0.5 mol %) of a total ethylene utilized in step a) and step c) of the first and second processes. Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time, defined by the conversion of ethylene in the activation vessel, disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to 5 mol %, from 10 sec to 1 mol %, from 30 sec to 4 mol %, from 30 sec to 1 mol %, from 1 min to 3 mol %, or from 2 min to 2 mol % (the upper limit of 0.5-5 mol % is based on the conversion of ethylene in the activation vessel divided by the total ethylene utilized in step a) and step c) of the first process (or the second process)). Thus, it is anticipated that either a very small amount, or effectively none, of the ethylene feed will be converted (e.g., to an oligomer) in the activation vessel. Other appropriate ranges for the average residence time are readily apparent from this disclosure. The amount of ethylene conversion can be quantified using an on-line Raman spectroscopy analytical technique, or other suitable techniques. In an aspect, the first mixture in the first process (or the second process) can be maintained in the activation vessel for an average residence time in which the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; alternatively or additionally, the maximum average residence time can be characterized by a catalyst system productivity in the activation vessel that is equal to 5%, 4%, 3%, 2%, or 1% of a catalyst system productivity in the reaction zone (in units of kg of normal alpha olefin produced per gram of chromium per hour, kg NAO/g Cr per hr). Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time, defined by the catalyst system productivity in the reaction zone, disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to 5%, from 10 sec to 1%, from 30 sec to 4%, from 30 sec to 1%, from 1 min to 3%, or from 2 min to 2% (the upper limit of 1-5% is based on the catalyst system productivity in the activation vessel divided by the catalyst system productivity in the reaction zone). The appropriate maximum average residence time in the activation vessel can vary. Once the induction time of the catalyst system has been reached, oligomerization of ethylene can commence, and the catalyst system productivity can begin to increase. Other appropriate ranges for the average residence time are readily apparent from this disclosure. In some instances, the productivity in the activation vessel can be equal to zero—no normal alpha olefin has been produced. In an aspect, the first mixture in the first process (or the second process) can be maintained in the activation vessel for an average residence time in which the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; alternatively or additionally, the maximum average residence time can be characterized by a fouling rate in the activation vessel of equal to 0.065 mg/cm2-hr, 0.06 mg/cm2-hr, 0.055 mg/cm2-hr, 0.05 mg/cm2-hr, 0.045 mg/cm2-hr, or 0.04 mg/cm2-hr. Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time, defined by the fouling rate in the activation vessel, disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to an activation vessel fouling rate of 0.065 mg/cm2-hr, from 10 sec to an activation vessel fouling rate of 0.04 mg/cm2-hr, from 30 sec to an activation vessel fouling rate of 0.055 mg/cm2-hr, from 30 sec to an activation vessel fouling rate of 0.045 mg/cm2-hr, from 1 min to an activation vessel fouling rate of 0.05 mg/cm2-hr, or from 2 min to an activation vessel fouling rate of 0.045 mg/cm2-hr. The appropriate maximum fouling rate in the activation vessel can vary. Once the induction time of the catalyst system has been reached, oligomerization of ethylene can commence. Other appropriate ranges for the average residence time are readily apparent from this disclosure. In circumstances where substantially no oligomerization takes place in the activation vessel—only activation of the catalyst in the presence of ethylene—it is expected that the fouling rate will be negligible. In an aspect, the first mixture in the first process (or the second process) can be maintained in the activation vessel for an average residence time in which the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; alternatively or additionally, the maximum average residence time can be characterized by an oligomer product discharge rate from the activation vessel that is equal to 0.15 lb/gal/hr, 0.125 lb/gal/hr, 0.1 lb/gal/hr, or 0.075 lb/gal/hr, whereas the oligomer product discharge rate from the reaction zone often can be in a range from 0.75 to 6 lb/gal/hr, from 1 to 6 lb/gal/hr, 1.2 to 5.5 lb/gal/hr, 1.4 to 5 lb/gal/hr, 1.5 to 4.5 lb/gal/hr, or 1.6 to 4.25 lb/gal/hr. Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time, defined by the oligomer product discharge rate from the activation vessel, disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to an activation vessel oligomer product discharge rate equal to 0.15 lb/gal/hr, from 10 sec to an activation vessel oligomer product discharge rate equal to 0.075 lb/gal/hr, from 30 sec to an activation vessel oligomer product discharge rate equal to 0.075 lb/gal/hr, from 1 min to an activation vessel oligomer product discharge rate equal to 0.125 lb/gal/hr, or from 2 min to an activation vessel oligomer product discharge rate equal to 0.1 lb/gal/hr, and where the oligomer product discharge rate from the reaction zone often can be in a range from 0.75 to 6 lb/gal/hr, from 1 to 6 lb/gal/hr, from 1.2 to 5.5 lb/gal/hr, from 1.4 to 5 lb/gal/hr, from 1.5 to 4.5 lb/gal/hr, or from 1.6 to 4.25 lb/gal/hr. Stated another way, the oligomer product discharge rate from the activation vessel can be less than or equal to 7.5% of the oligomer product discharge rate from the reaction zone, such as less than or equal to 5%, less than or equal to 3%, less than or equal to 2%, or less than or equal to 1%. The oligomer product discharge rate from the activation vessel can be effectively zero if no measurable oligomerization occurs in the activation vessel. Other appropriate ranges for the average residence time are readily apparent from this disclosure. In an aspect, the first mixture in the first process (or the second process) can be maintained in the activation vessel for an average residence time in which the minimum average residence time can be 10 sec, 30 sec, 1 min, or 2 min; alternatively or additionally, the maximum average residence time can be characterized by a ΔT from the inlet of the activation vessel to the outlet of the activation vessel of equal to 5° C., 4° C., 3° C., 2° C., or 1° C. (due to the exothermic ethylene oligomerization reaction). Generally, the average residence time can be in a range from any minimum time disclosed herein to any maximum time, defined by the heat generated in the activation vessel, disclosed herein. Accordingly, suitable non-limiting ranges for the average residence time can include the following: from 10 sec to a ΔT from the inlet of the activation vessel to the outlet of the activation vessel of 5° C., from 10 sec to a ΔT of 2° C., from 30 sec to a ΔT of 2° C., from 30 sec to a ΔT of 1° C., from 1 min to a ΔT of 3° C., or from 2 min to a ΔT of 3° C. (the ΔT is measured from the inlet of the activation vessel to the outlet of the activation vessel). The ΔT is based upon the heat generated in the activation vessel without any external cooling being applied to the activation vessel. Thus, it is anticipated that either a very small amount of heat is generated, or effectively no heat is generated (particularly, if no substantial oligomerization of ethylene occurs), in the activation vessel. Other appropriate ranges for the average residence time are readily apparent from this disclosure. In an aspect, the first mixture in the first process (or the second process) can have an amount of ethylene introduced into the activation vessel that is less than 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, or less than or equal to 5% of an amount of ethylene introduced into the reaction zone. Although it is possible to have a majority of the ethylene feed, or even all of the ethylene feed, introduced into the activation vessel, only a minor fraction of ethylene fed to the activation vessel is needed to activate the catalyst system for oligomerization. In an aspect, step b) of the first process (or the second process) can be performed at an activation temperature and an activation pressure such that the first mixture in the activation vessel can be below the bubble point at the activation temperature and the activation pressure. In an aspect, the activation temperature can be any oligomerization temperature suitable for the formation of the oligomer product in step d) of the first process (or the second process). Often, the oligomer product can be formed at (and/or the activation temperature independently can be) a minimum temperature of 0° C., 20° C., 30° C., 40° C., 45° C., or 50° C.; additionally or alternatively, at a maximum temperature of 165° C., 160° C., 150° C., 140° C., 130° C., 115° C., 100° C., or 90° C. Generally, the oligomerization temperature at which the oligomer product is formed (or the activation temperature independently) can be in a range from any minimum temperature disclosed herein to any maximum temperature disclosed herein. Accordingly, suitable non-limiting ranges for the oligomerization temperature and/or activation temperature independently can include the following: from 0° C. to 165° C., from 20° C. to 160° C., from 20° C. to 115° C., from 40° C. to 160° C., from 40° C. to 140° C., from 50° C. to 150° C., from 50° C. to 140° C., from 50° C. to 130° C., from 50° C. to 100° C., from 60° C. to 115° C., from 70° C. to 100° C., or from 75° C. to 95° C. Other appropriate activation and oligomerization temperatures and temperature ranges are readily apparent from this disclosure. The activation pressure can be any oligomerization pressure suitable for the formation of the oligomer product in step d) of the first process (or the second process). For example, the oligomer product can be formed at (or the activation pressure, or activation ethylene partial pressure, independently can be) a minimum pressure (or ethylene partial pressure) of 50 psig (344 kPa), 100 psig (689 kPa), 200 psig (1.4 MPa), or 250 psig (1.5 MPa); additionally or alternatively, at a maximum pressure (or ethylene partial pressure) of 4,000 psig (27.6 MPa), 3,000 psig (20.9 MPa), 2,000 psig (13.8 MPa), or 1,500 psig (10.3 MPa). Generally, the oligomerization pressure (or ethylene partial pressure) at which the oligomer product is formed (or the activation pressure, or activation ethylene partial pressure, independently) can be in a range from any minimum pressure disclosed herein to any maximum pressure disclosed herein. Accordingly, suitable non-limiting oligomerization pressures (or ethylene partial pressures) and/or activation pressures (or activation ethylene partial pressures) ranges independently can include the following: from 50 psig (344 kPa) to 4.000 psig (27.6 MPa), from 100 psig (689 kPa) to 3,000 psig (20.9 MPa), from 100 psig (689 kPa) to 2.000 psig (13.8 MPa), from 200 psig (1.4 MPa) to 2,000 psig (13.8 MPa), from 200 psig (1.4 MPa) to 1,500 psig (10.3 MPa), or from 250 psig (1.5 MPa) to 1,500 psig (10.3 MPa). Other appropriate oligomerization pressures (or ethylene partial pressures) and/or activation pressures (or activation ethylene partial pressures) are readily apparent from this disclosure. Referring now to step c) of the first process (or the second process), ethylene, the first mixture from step b) of the first process (or the second process), a second organic reaction medium, and optionally hydrogen can be introduced into a reaction zone. Thus, in one aspect of the disclosure, hydrogen is present in step c) of the first process (or the second process), while in another aspect, hydrogen is not present. When used, hydrogen can be fed directly to the reaction zone, however, in an aspect, hydrogen can be combined with the ethylene and or second organic reaction medium feed prior to introduction into the reaction zone. In the reaction zone, the hydrogen partial pressure can be at least 1 psig (6.9 kPa), 5 psig (34 kPa), 10 psig (69 kPa), 25 psig (172 kPa), or 50 psig (345 kPa); additionally or alternatively, a maximum hydrogen partial pressure of 2000 psig (13.8 MPa), 1750 psig (12.1 MPa), 1500 psig (10.3 MPa), 1250 psig (8.6 MPa), 1000 psig (6.9 MPa), 750 psig (5.2 MPa), 500 psig (3.4 MPa), or 400 psig (2.8 MPa). Generally, the hydrogen partial pressure can range from any minimum hydrogen partial pressure disclosed herein to any maximum hydrogen partial pressure disclosed herein. Therefore, suitable non-limiting ranges for the hydrogen partial pressure can include the following ranges: from 1 psig (6.9 kPa) to 2000 psig (13.8 MPa), from 1 psig (6.9 kPa) to 1750 psig (12.1 MPa), from 5 psig (34 kPa) to 1500 psig (10.3 MPa), from 5 psig (34 kPa) to 1250 psig (8.6 MPa), from 10 psig (69 kPa) to 1000 psig (6.9 MPa), from 10 psig (69 kPa) to 750 psig (5.2 MPa), from 10 psig (69 kPa) to 500 psig (3.5 MPa), from 25 psig (172 kPa) to 750 psig (5.2 MPa), from 25 psig (172 kPa) to 500 psig (3.4 MPa), from 25 psig (172 kPa) to 400 psig (2.8 MPa), or from 50 psig (345 kPa) to 500 psig (3.4 MPa). Other appropriate hydrogen partial pressures in the reaction zone for the formation of the oligomer product are readily apparent from this disclosure. In some non-limiting aspects, the activation vessel can include hydrogen and the activation vessel can independently have any hydrogen partial pressure utilized for the reaction zone described herein. The second organic reaction medium can be any of the hydrocarbons discussed herein for the first organic reaction medium described herein (e.g., cyclohexane). In some aspects, the second organic reaction medium and the first organic reaction medium are the same hydrocarbon (or mixture of hydrocarbons), although this is not a requirement. Ethylene, the first mixture from step b) (which contains “activated” catalyst system or catalyst system which has had its induction period reduced), the second organic reaction medium, and hydrogen (when used) can be combined in any order. For instance, the ethylene and the first mixture from step b) can be introduced separately into the reaction zone in step c). In a particular non-limiting aspect of step c), a second feed stream is formed that comprises ethylene, at least a portion (and in some cases, all) of the second organic reaction medium, and hydrogen (when used), and this second feed stream is introduced into the reaction zone separately from the first mixture from step b) (which contains the catalyst system). In step d) of the first process (or the second process), an oligomer product is formed in the reaction zone. Suitable temperatures, ethylene partial pressures, and hydrogen partial pressures are independently discussed herein. These independently described features can utilized without limitation and in any combination to further the conditions of the reaction zone and/or the conditions at which the oligomerization product is formed. The reaction zone in which the oligomer product is formed can comprise any suitable reactor. Non-limiting examples of reactor types can include a stirred tank reactor, a plug flow reactor, or any combination thereof; alternatively, a fixed bed reactor, a continuous stirred tank reactor, a loop slurry reactor, a solution reactor, a tubular reactor, a recycle reactor, or any combination thereof. In an aspect, the reaction zone can have more than one reactor in series or in parallel, and including any combination of reactor types and arrangements. Moreover, the oligomerization process used to form the oligomer product can be a continuous process or a batch process, or any reactor or vessel within the oligomerization reaction system can be operated continuously or batchwise. In accordance with the first process (or second process) of this disclosure, the productivity of the catalyst system under oligomerization conditions generally can be greater than 50 kg, greater than 100 kg, greater than 250 kg, greater than 500 kg, greater than 750 kg, greater than 1000 kg, greater than 1250 kg, greater than 1500 kg, or greater than 2000 kg, of normal alpha olefins per gram of chromium per hour. For the purpose of determining the productivity, the conditions under the oligomer product is formed can include MAO, using cyclohexane as the reaction medium and 50 psig hydrogen pressure, and with an oligomerization temperature of 90° C. and an ethylene pressure of 875 psig. The first process (or the second process) can include a step of discharging (from the reaction zone) a reaction zone effluent stream comprising the oligomer product. The reaction zone effluent stream can further comprise, in some aspects, the catalyst system, ethylene, and organic reaction medium. The catalyst system can be deactivated by contacting the reaction zone effluent stream with a suitable catalyst system deactivating agent, or subjecting the oligomer product to suitable process steps to deactivate the catalyst system, or a combination of both. The reaction zone effluent stream wherein the catalyst system has been deactivated can be referred to as a deactivated reaction zone effluent stream. The catalyst system deactivating agent can comprise (or consist essentially of, or consist of) water, an alcohol compound, an amine compound, or any combination thereof; alternatively, water; alternatively, an alcohol compound; or alternatively, an amine compound. In an aspect, the alcohol compound can be a monoalcohol compound, a diol compound, a polyol compound, or any combination thereof. In some aspects, the alcohol compound can comprise, consist essentially of, or consist of, a C1to C20mono alcohol. In some aspects, the alcohol compound can comprise, consist essentially of, or consist of, methanol, ethanol, a propanol, a butanol, a pentanol, a hexanol, a heptanol, an octanol, a nonanol, a decanol, an undecanol, or mixtures thereof. In some aspects, the alcohol compound can comprise, consist essentially of, or consist of, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, iso-butanol, sec-butanol, t-butanol, 1-hexanol, 2-hexanol, 3-hexanol, 1-heptanol, 2-heptanol, 3-heptanol, 4-heptanol, 1-octanol, 2-octanol, 3-octanol, 4-octanol, 2-ethyl-1-hexanol, 2-methyl-3-heptanol, 1-decanol, 2-decanol, 3-decanol, 4-decanol, 5-decanol, 1-undecanol, 2-undecanol, 7-methyl-2-decanol, a 1-docecanol, a 2-dodecanol, 2-ethyl-1-decanol, or mixtures thereof. Additionally or alternatively, the catalyst system can be deactivated by contact with an aqueous base solution or aqueous acid solution (e.g., an aqueous Group 1 metal hydroxide solution or an aqueous mineral acid solution). Such deactivation processes to deactivate the catalyst system can also potentially remove a portion, or substantially all, of the metal catalyst system components from the oligomer product. The first process (or the second process) can further comprise a step of isolating a liquid oligomer product, e.g., from the reaction zone effluent stream, from the deactivated reaction zone effluent stream, from solid polymer product, from the organic reaction medium, etc., using any suitable technique. Various suitable separation techniques can be employed, as would be recognized by those of skill in the art. In an aspect, and not limited thereto, a filtration process, an evaporation process, or a distillation process can be used, as well as combinations of more than one separation technique. The liquid oligomer product can contain C6olefins; alternatively, C8olefins; or alternatively, C6and C8olefins. Based on the weight of the liquid oligomer product, the amount of C6and/or C8olefins (C6olefins, C8olefins, or total C6+C8olefins) typically can fall within a range from 70 to 99.9 wt. %, from 75 to 99.8 wt. %, or from 80 to 99.6 wt. %. Selectivity to α-olefins in the liquid oligomer product can be unexpectedly high. In an aspect, the C6olefins, of a liquid oligomer product comprising at least 10 wt. % C6olefins, can contain 1-hexene in an amount ranging from 80 to 99.99 mol %, from 82 to 99.99 mol %, from 85 to 99.99 mol %, from 90 to 99.99 mol %, or from 95 to 99.9 mol %. Likewise, in an aspect, the C8olefins, of a liquid oligomer product comprising at least 10 wt. % C8olefins, can contain 1-octene in an amount ranging from 80 to 99.99 mol %, from 85 to 99.99 mol %, from 90 to 99.99 mol %, from 95 to 99.9 mol %, or from 97 to 99.99 mol %. Referring now to the second process disclosed herein, which can comprise a) forming a first mixture in an activation vessel, the first mixture comprising 1) a first catalyst system comprising i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound, 2) a first feed comprising ethylene, a second catalyst system comprising i) a second heteroatomic ligand chromium compound complex and a second organoaluminum compound, or ii) a second heteroatomic ligand, a second chromium compound, and a second organoaluminum compound, a second organic reaction medium, a second oligomer product, and optionally hydrogen, 3) optionally, a first organic reaction medium, and 4) optionally, hydrogen; b) maintaining the first mixture in the activation vessel for an average residence time; c) introducing into a reaction zone: ethylene, the first mixture from step b), the second organic reaction medium, and optionally, hydrogen; and d) forming an oligomer product in the reaction zone. In the second process, a portion of a reaction zone effluent is fed to the activation vessel as the first feed. All of the features and characteristics disclosed herein for the first process are likewise applicable to the second process disclosed herein. As such, the average residence time (as a range from a minimum time to a maximum time defined by one or more of time, ethylene conversion in the activation vessel, catalyst system productivity in the activation vessel and the reaction zone, fouling rate, oligomer product discharge rate from the activation vessel, and ΔT from the inlet of the activation vessel to the outlet of the activation vessel), the percentage of overall ethylene fed to the activation vessel, the bubble point, the catalyst system organic medium, the pressure and temperature conditions in the activation vessel and reaction zone, the use of hydrogen, the downstream separations, the liquid oligomer product described herein (among other process features described herein) can be utilized without limitation and in any combination to further describe the second process. In step a) of the second process, a first mixture is formed in the activation vessel and the first mixture comprises 1) a first catalyst system comprising i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound, 2) a first feed comprising ethylene, a second catalyst system comprising i) a second heteroatomic ligand chromium compound complex and a second organoaluminum compound, or ii) a second heteroatomic ligand, a second chromium compound, and a second organoaluminum compound, a second organic reaction medium, a second oligomer product, and optionally hydrogen, 3) optionally, a first organic reaction medium, and 4) optionally, hydrogen. Suitable heteroatomic ligand chromium compound complexes, heteroatomic ligands, chromium compounds, and organoaluminum compounds are disclosed herein and can be used without limitation in the second process. The first and the second heteroatomic ligand chromium compound complexes, heteroatomic ligands, chromium compounds, and organoaluminum compounds can be the same or different. Generally, however, the first and the second catalyst systems can contain the same components. While not required, a first organic reaction medium often can be combined with the first catalyst system and the first feed to form the first mixture in the activation vessel. Likewise, while not required, hydrogen also can be fed to the activation vessel and combined with the first catalyst system and the first feed—and the optional first organic reaction medium—to form the first mixture in the activation vessel. The first feed is a portion of a reaction zone effluent stream from the reaction zone, and is often referred to as a slip stream from the reaction zone. Since the first feed comes from the reaction zone, it contains ethylene, the second catalyst system, the second organic reaction medium, a second oligomer product, and optionally hydrogen. Nominally, the first feed can contain 50-75 wt. % of the second organic reaction medium, 10-30 wt. % of the second oligomer product (e.g., hexenes and/or octenes), and 5-15 wt. % of ethylene. While the first feed contains ethylene, this does not preclude a further ethylene feed to the activation vessel. The first catalyst system components, the first feed comprising ethylene (and other components), and the first organic reaction medium can be combined in any order. For instance, a first feed stream comprising ethylene and at least a portion of the first organic reaction medium can be introduced into the activation vessel separate from the first catalyst system or one or more components of first catalyst system. In another example, the first catalyst system mixture stream can include the first catalyst system and the first organic reaction medium (and optionally, a suitable catalyst system organic medium). In some aspects, the first heteroatomic ligand chromium compound complex and the first organoaluminum compound can be separately introduced into the activation vessel, or at least one of the first heteroatomic ligand, the first chromium compound, and the first organoaluminum compound can be separately introduced into the activation vessel. In an aspect, the heteroatomic ligand and the chromium compound are separately introduced into the activation vessel; this technique is generally referred to as in-situ formation of the heteroatomic ligand chromium compound complex in the activation vessel. The first organic reaction medium can be mixed with any single catalyst system component, or any combination of catalyst system components, prior to the activation vessel. In other aspects, a catalyst system mixture comprising the first heteroatomic ligand chromium compound complex and the first organoaluminum compound (or the first heteroatomic ligand, the first chromium compound, and the first organoaluminum compound), and optionally at least a portion of the first organic reaction medium, can be formed and then introduced into the activation vessel. The catalyst system mixture can further contain a catalyst system organic medium, such as an aromatic hydrocarbon (e.g., benzene, toluene, xylene (including ortho-xylene, meta-xylene, para-xylene, or mixtures thereof), cumene, and ethylbenzene, or a C8and/or C9aromatic stream (Total Atosol 100, ExxonMobil A100, and Shell Solv100, or other streams containing xylenes, cumene, or ethylbenzene, among others), among other aromatic hydrocarbons). Thus, the catalyst system mixture can be formed prior to entering the activation vessel. The catalyst system mixture can be aged for any suitable period of time (e.g., 5 sec to 48 hr, from 10 sec to 36 hr, from 30 sec to 24 hr, from 1 min to 18 hr, from 5 min to 6 hr, from 10 min to 4 hr, or from 20 min to 2 hr) in the substantial absence of ethylene prior to introducing the catalyst system mixture into the activation vessel. In step b) of the second process, the first mixture can be maintained in the activation vessel for an average residence time as discussed herein for step b). In step c) of the second process, ethylene, the first mixture from step b), the second organic reaction medium, and optionally hydrogen can be introduced into a reaction zone, as discussed herein for step c). The ethylene introduced into the reaction zone in step c) often is substantially devoid of any catalyst system or any catalyst system components. In step d) of the second process, an oligomer product is formed in the reaction zone, as discussed herein for step d). Similar to the first process, the second process also can include steps of discharging a reaction zone effluent stream comprising the oligomer product from the reaction zone, deactivating the catalyst system, isolating a liquid oligomer product, and recovering C6and/or C8olefins (e.g., C6and/or C8α-olefins). Oligomerization Reaction Systems A reaction system consistent with aspects of the present disclosure can comprise (a) an activation vessel configured to form a first mixture, wherein the activation vessel is further configured for an average residence time of the first mixture in the activation vessel, (b) one or more activation vessel inlets configured to introduce i) ethylene and a catalyst system mixture, or ii) ethylene and components of a catalyst system mixture, into the activation vessel, (c) an activation vessel outlet configured to withdraw the first mixture from the activation vessel, (d) a reaction zone configured to contact ethylene, the first mixture, a second organic reaction medium, and optionally hydrogen to form an oligomer product, (e) one or more reaction zone inlets configured to introduce ethylene, the second organic reaction medium, and the first mixture from the activation vessel outlet into the reaction zone, and (f) a reaction zone outlet configured to withdraw a reaction zone effluent stream containing the oligomer product from the reaction zone. In the reaction system, the average residence time of the first mixture in the activation vessel can be in a range from 10 sec, 30 sec, 1 min, or 2 min up to a period of time i) sufficient to form an amount of a first oligomer product in the activation vessel equal to 5% (or 4%, or 3%, or 2%, or 1%) of an amount of the oligomer product formed in the reaction zone; or ii) for a conversion of ethylene in the activation vessel of 5 mol % (or 4 mol %, or 3 mol %, or 2 mol %, 1 mol %, or 0.5 mol %) of a total ethylene utilized in step a) and step c); or iii) for a catalyst system productivity in the activation vessel of 5%, 4%, 3%, 2%, or 1% of a catalyst system mixture productivity in the reaction zone (in units of kg of normal alpha olefin produced per gram of chromium per hour, kg NAO/g Cr per hr); or iv) for a fouling rate in the activation vessel of 0.065 mg/cm2-hr, 0.06 mg/cm2-hr, 0.055 mg/cm2-hr, 0.05 mg/cm2-hr, 0.045 mg/cm2-hr, or 0.04 mg/cm2-hr; or v) for an oligomer product discharge rate from the activation vessel of 0.15 lb/gal/hr, 0.125 lb/gal/hr, 0.1 lb/gal/hr, or 0.075 lb/gal/hr, and an oligomer product discharge rate of the reaction zone can be in a range from 0.75 to 6 lb/gal/hr, from 1 to 6 lb/gal/hr, from 1.2 to 5.5 lb/gal/hr, from 1.4 to 5 lb/gal/hr, from 1.5 to 4.5 lb/gal/hr, or from 1.6 to 4.25 lb/gal/hr; or vi) for a ΔT from the inlet of the activation vessel to the outlet of the activation vessel of 5° C., 4° C., 3° C., 2° C., or 1° C.; or vii) of 8 hr, 6, hr, 4 hr, 2 hr, 1 hr, 45 min, 30 min, 20 min, or 15 min. In the reaction system, an amount of ethylene introduced into the activation vessel can be less than 50%, 40%, 30%, or 20% of an amount of ethylene introduced into the reaction zone; or the activation vessel can be operated at an activation temperature and an activation pressure, and the first mixture in the activation vessel can be below the bubble point at the activation temperature and the activation pressure. All of the features and characteristics disclosed herein for the first and second processes (e.g., average residence time, ethylene conversion in the activation vessel, catalyst system productivity in the activation vessel and the reaction zone, fouling rate, oligomer product discharge rate from the activation vessel and the reaction zone, heat generation in the activation vessel, percentage of overall ethylene fed to the activation vessel, bubble point, catalyst system organic medium, pressure and temperature conditions in the activation vessel and reaction zone, use of hydrogen, downstream separations, and so forth) are likewise applicable to the oligomerization reaction system disclosed herein. The (a) activation vessel is configured to form the first mixture, and can be further configured for an average residence time of the first mixture in the activation vessel. The activation vessel can comprise any suitable reactor. Non-limiting examples of reactor types can include a stirred tank reactor (e.g., a CSTR), a plug flow reactor, or any combination thereof; alternatively, a fixed bed reactor, a continuous stirred tank reactor, a loop slurry reactor, a solution reactor, a tubular reactor, a recycle reactor, or any combination thereof. Moreover, the activation vessel can be configured to operate continuously or batchwise. The (b) one or more activation vessel inlets are configured to introduce i) ethylene and a catalyst system mixture, or ii) ethylene and components of a catalyst system mixture, into the activation vessel. Optionally, at least one of the one or more activation vessel inlets can be further configured to introduce a first organic reaction medium and/or hydrogen into the activation vessel. For instance, some or all of the first organic reaction medium can be introduced into the activation vessel along with the catalyst system mixture or the catalyst system mixture components, along with the ethylene, or introduced separately from the catalyst system mixture, catalyst system mixture components, and ethylene. If used, hydrogen can be introduced into the activation vessel with the ethylene feed, the organic reaction medium, or separately from the catalyst system mixture, catalyst system mixture components, ethylene, and organic reaction medium. In an aspect, the one or more activation vessel inlets can be configured to introduce the catalyst system mixture into the activation vessel, wherein the catalyst system mixture comprises (i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or (ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound. The catalyst system mixture can further contain a catalyst system organic medium, such as an aromatic hydrocarbon (e.g., ethylbenzene, xylene (any specific or combination thereof), cumene, or a C8and/or C9aromatic stream (e.g., Total Atosol 100, ExxonMobil A100, and Shell Solv100, or other streams containing xylenes, cumene, or ethylbenzene, among others), among other aromatic hydrocarbons). In another aspect, the one or more activation vessel inlets can be configured to introduce the components of the catalyst system mixture into the activation vessel, wherein the components comprise (i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound (and optional catalyst system organic medium), or (ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound (and optional catalyst system organic medium). The (c) activation vessel outlet can be configured to withdraw the first mixture from the activation vessel, and (e) one or more reaction zone inlets can be configured to introduce ethylene, the second organic reaction medium, and the first mixture from the activation vessel outlet into the reaction zone. Therefore, the activation vessel outlet and at least one of the one or more reaction zone inlets can be fluidly connected. Further, the one or more reaction zone inlets can be further configured to introduce hydrogen into the reaction zone, and hydrogen can be fed directly into the reaction zone or combined with ethylene, the organic reaction medium, (or with ethylene and the second organic reaction medium) prior to entering the reaction zone. The (f) reaction zone outlet can be configured to withdraw a reaction zone effluent stream containing the oligomer product from the reaction zone. Often, the reaction zone effluent stream can further comprise ethylene, the second organic reaction medium, or the catalyst system mixture, or any combination thereof. If desired, the reaction system can further comprise (g) a controller configured to control the average residence time of the first mixture in the activation vessel, and/or an activation temperature in the activation vessel, and/or an activation pressure in the activation vessel, and/or an ethylene conversion in the activation vessel, and/or an ethylene flow rate into the activation vessel. The controller, which can comprise any suitable processing unit or computer system, can be used to analyze the data regarding the reaction system (e.g., the reaction zone and the activation vessel), and adjust the various process parameters based on the prevailing conditions in the overall reaction system. As an example, if fouling and/or ethylene conversion of greater than 1-5% is found in the activation vessel (or any other feature providing an upper limit to the average residence time described herein), the average residence time of the first mixture in the activation vessel can be decreased to decrease the fouling and/or ethylene conversion (or any other feature providing an upper limit to the average residence time described herein) that occurs within the activation vessel. Referring now toFIG.1, which illustrates an oligomerization reaction system100consistent with an aspect of the present disclosure. The system100can include a catalyst preparation vessel120, an activation vessel140, a reaction zone160, and a separations system180. InFIG.1, a heteroatomic ligand chromium compound complex feed stream or heteroatomic ligand and chromium compound feed stream (either separately of combined)102, an organoaluminum feed stream104, and a first organic reaction medium feed stream108enter the catalyst preparation vessel120. After a suitable period of aging (if needed), a catalyst system mixture stream105exits the catalyst preparation vessel, and then enters the activation vessel140. In some aspects, all or part of the first organic reaction medium feed stream108can be fed directly to activation vessel140. While not specifically shown inFIG.1, a catalyst system organic medium (e.g., a suitable aromatic hydrocarbon) can be present in the heteroatomic ligand chromium compound complex feed stream or the heteroatomic ligand and chromium compound feed stream (either one or both)102, and/or the organoaluminum feed stream104, and/or can be fed as a separate feed stream to the catalyst preparation vessel120. In an aspect, the catalyst system organic medium can be combined with the heteroatomic ligand chromium compound complex feed stream or the heteroatomic ligand and chromium compound feed stream (either one or both)102and/or the organoaluminum feed stream104, and the resulting mixture then contacted with the first organic reaction medium feed stream108. It is understood that there are many different methods in which the catalyst system mixture stream105can be prepared, and this disclosure is not limited only to those options described in reference toFIG.1or otherwise disclosed herein. An ethylene feed stream130inFIG.1can be split into two streams, one of which is an activation vessel ethylene feed stream125, which is combined with the catalyst system mixture stream105in the activation vessel140. As described herein, the catalyst system is first contacted with ethylene in the activation vessel140(and prior to the reaction zone160) for a particular residence time to activate the catalyst, such that some or all of the induction period in the oligomerization reaction zone can be eliminated. InFIG.1, the second stream split from the ethylene feed stream130is mixed with an optional hydrogen feed stream115and a second reaction medium feed stream155, thereby forming a combined feed stream135to reaction zone160, which is contacted with a first mixture stream145exiting the activation vessel140. Alternatively, and not shown inFIG.1, all or part of the optional hydrogen feed stream115(and/or all or part of the second reaction medium feed stream155) can be fed directly to reaction zone160. Ethylene oligomerization occurs in the reaction zone160, and exiting the reaction zone160is a reaction zone effluent stream175, which enters a separations system180for isolation of desired oligomers products, such as 1-hexene and 1-octene. The separations system180also can be configured for catalyst system deactivation and/or removal. Referring now toFIG.2, which illustrates another oligomerization reaction system200consistent with an aspect of the present disclosure. The system200can include an activation vessel240, a reaction zone260, a separations system280, an ethylene feed stream230, an activation vessel ethylene feed stream225, an optional hydrogen feed stream215, a second reaction medium feed stream255, a combined feed stream235to the reaction zone260, and a reaction zone effluent stream275, which are generally the same as described for the similarly numbered components inFIG.1. InFIG.2, a catalyst preparation vessel is not present, so a first reaction medium feed stream208is combined with a catalyst system stream206to form a catalyst system mixture stream212. The catalyst system mixture stream212is combined with activation vessel ethylene feed stream225in the activation vessel240to form a first mixture stream245, which exits the activation vessel240and is fed to the reaction zone260. Alternatively, and not shown inFIG.2, the catalyst system mixture (comprising the heteroatomic ligand chromium compound complex, organoaluminum compound, and optionally catalyst system organic medium) or the catalyst system mixture components (the heteroatomic ligand, the chromium compound, the organoaluminum compound, and optionally catalyst system organic medium) can be feed directly (either as a combined stream or as one or more separate feeds) to the activation vessel240to form the first mixture stream245. Referring now toFIG.3, which illustrates yet another oligomerization reaction system300consistent with an aspect of the present disclosure. The system300can include an activation vessel340, a reaction zone360, a separations system380, an optional hydrogen feed stream315, a second reaction medium feed stream355, a combined feed stream335to the reaction zone360, and a reaction zone effluent stream375, which are generally the same as described for the similarly numbered components inFIG.1andFIG.2. Likewise, the system300can include a first reaction medium feed stream308, a catalyst system stream306, and a catalyst system mixture stream312, which are generally the same as described for the similarly numbered components inFIG.2. Alternatively,FIG.3can employ the catalyst preparation vessel and related streams shown inFIG.1(e.g.,102,104,108, and120, with stream105entering the activation vessel140). InFIG.3, ethylene feed stream330is not split and there is no direct feed of only ethylene into the activation vessel340. Instead, a first feed365from the reaction zone360(e.g., it can be a portion of the reaction zone effluent stream375) enters the activation vessel340. The first feed365contains ethylene (and for example, catalyst, reaction medium, oligomer product, and optionally hydrogen), and is combined with the catalyst system mixture stream312in the activation vessel340to form a first mixture stream345, which exits the activation vessel340and is fed to the reaction zone360. Examples The disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations to the scope of this disclosure. Various other aspects, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present disclosure or the scope of the appended claims. All manipulations were carried out using a nitrogen filled drybox and standard Schlenk techniques using oven dried glassware (>1 h at 110° C. under vacuum, −30 mm Hg). Cyclohexane diluent was obtained from Sigma-Aldrich in anhydrous grade and degassed prior to use with N2. MMAO-3A/20 was obtained from AkzoNobel/Nouryon and stored in an N2filled drybox. Methylcyclohexane was obtained from Sigma-Aldrich, degassed under N2, and stored over molecular sieves in the drybox. Ethylbenzene, m-xylene, or xylenes was obtained from Sigma-Aldrich, degassed under N2, and stored over activated 13X molecular sieves in the drybox. Standard Batch Reactor Procedure The catalyst system mixture was prepared by dissolving a N2-phosphinyl guanidine chromium(III) trichloride tetrahydrofuran complex having Structure GuCr4 (5.3 μmoles) in 1 g of ethylbenzene and adding MMAO-3A or MMAO-20 to provide the desired Al:Cr ratio. The catalyst system mixture was stirred for 1-5 hr at 50° C. in a vial containing a magnetic stir bar under a nitrogen atmosphere. The contents of the vial were then introduced into 200 mL of dry, degassed cyclohexane. The catalyst mixture in cyclohexane was charged to an evacuated reactor (500 mL stainless steel ZipperClave) held at 60° C. under vacuum. The reactor was then charged with 50 psig H2and 875 psig ethylene. The reactor temperature was increased to 90° C. and ethylene fed on demand to maintain a pressure of 875 psig. The reactor was maintained at 90° C. using internal cooling coils using tower water and an external water bath if necessary. Once 90° C. was reached, the reaction was allowed to continue for 20-30 min after which reactor was cooled to 35° C. The ethylene was then vented, and the product collected and filtered. Polymer, if observed, was dried and weighed. A sample of the liquid reactor contents was then analyzed by GC on an Agilent 7890-LTM equipped with an Agilent DB-5msUI column (Agilent P/N 222-5532UIL™) with a 30 m length, 0.25 ID, and 0.25 μm film thickness and a flame ionization detector. Table 3 summarizes the catalyst systems, oligomerization conditions, oligomer product properties, and catalyst productivities for Examples 1-4. FIG.4shows the ethylene demand for Examples 1-4 and demonstrates that the catalyst induction period was approximately 6.5 min when MMAO-20 was used and approximately 16.5 min when MMAO-3A was used. After pressure equilibration at the desired 875 psig pressure, the reactor ethylene demand was low until the catalyst system was “fully” activated and initiated the ethylene oligomerization reaction at the respective times (6.5 min or 16.5 min) as shown by the rapid increase in ethylene consumption. Without being limited by theory, the ethylene oligomerization reaction appears to be initiated by the presence of high pressure ethylene. Experiments in batch reactors with low pressure ethylene (15 psig) did not prove viable and led to catalyst decomposition, poor reactor performance, or both. Thus, the catalyst systems of Examples 1-4 could benefit significantly from exposure in an activation vessel to ethylene, as described herein, for a suitable residence time (e.g., up to approximately 6.5 min or 16.5 min, respectively), such that the catalyst system would be fully activated for ethylene oligomerization when it enters the reactor (no induction time needed). Example 5 is a constructive example that demonstrates the impact of adding an activation vessel prior to an oligomerization reactor. Using a simple pre-catalyst to activated catalyst system to dead catalyst system model and simple CSTR kinetics, a theoretical plot for a catalyst system with an induction period of 5 min is illustrated inFIG.5. Surprisingly, less than half of the catalyst system (only 41%) is active during the residence time of the reactor. However, if the activation vessel can provide sufficient conditions and residence time to activate the catalyst system before entering the reactor, an increase in active catalyst system can be achieved. The impact of utilizing a catalyst system activation vessel prior to the oligomerization reactor is shown graphically inFIG.6. Note the significant increase in the amount of active catalyst in the reactor. Elimination of the induction period in the oligomerization reactor results in 65% active catalyst—a significant 59% increase in active catalyst in the reactor. The end result is more productivity (more oligomer product) per amount of chromium (or catalyst), since the catalyst is being used more efficiently, and there is more total active catalyst present in the oligomerization reactor due to the use of an activation vessel. TABLE 3Summary of Examples 1-4.Example1234SolventEthylbenzeneEthylbenzeneEthylbenzeneEthylbenzeneSolvent Mass1111(g)DiluentCyclohexaneCyclohexaneCyclohexaneCyclohexaneDiluent200200200200Volume (mL)Complex0.00530.00530.00530.0053(mmol)Chromium0.27670.27670.27670.2767(mg)Al:Cr ratio623623623623Organo-MMAO-3AMMAO-3AMMAO-20MMAO-20aluminumAging Time1511(hr)MAO (g)1.2751.2751.2751.275Reaction25302020Time (min)H2Pressure50505050(psig)Reaction90909090Temperature(° C.)Ethylene875875875875Pressure(psig)Ethylene159174171167Used (g)Ethylene70576457Conversion(%)Oligomer ProductPolymer1.93.13.32.2Product (g)Liquid NAO1099510793Product (g)Polymer1.73.23.02.3(wt. %)Ethylbenzene0.91.00.91.1(wt. %)C# dist data(wt. %)C640.541.838.140.3C840.649.246.550.1C101.92.62.22.3C1217.06.413.37.3C14+0.00.00.00.0(C6+ C8)81.190.984.590.4C6Purity85.3684.9383.6883.80Methyl-6.426.807.177.17cyclopentaneMethylenecy-6.496.887.107.14clopentaneC8Purity96.1596.1195.9395.931-Octene39.0547.2444.5648.09(wt. %)1-Hexene34.5435.4831.8733.77(wt. %)Productivitieskg(C6+ C8)/319314325304(g Cr)kg(C6+ C8)/767627976912(g Cr)/hrkg(C6+ C8)/35353634(g Complex) The disclosure refers to numerous aspects and specific examples. Many variations will suggest themselves to those skilled in the art in light of the above detailed description. All such obvious variations are within the full intended scope of the appended claims. Other statements of the disclosure can include, but are not limited to, the following (statements are described as “comprising” but, alternatively, can “consist essentially of” or “consist of”): Statement 1. A process comprising: a) forming a first mixture in an activation vessel, the first mixture comprising:1) ethylene,2) a catalyst system comprising i) a heteroatomic ligand chromium compound complex and an organoaluminum compound, or ii) a heteroatomic ligand, a chromium compound, and an organoaluminum compound,3) optionally, a first organic reaction medium, and4) optionally, hydrogen; b) maintaining the first mixture in the activation vessel for an average residence time; c) introducing into a reaction zone:i) ethylene,ii) the first mixture from step b),iii) a second organic reaction medium, andiv) optionally, hydrogen; and d) forming an oligomer product in the reaction zone; wherein the average residence time of the first mixture in the activation vessel is in a range from 10 sec up to a period of timei) sufficient to form an amount of a first oligomer product in the activation vessel equal to 5% of an amount of the oligomer product formed in the reaction zone; orii) where a conversion of ethylene in the activation vessel is equal to 5 mol % of a total ethylene utilized in step a) and step c); oriii) where a catalyst system productivity in the activation vessel is equal to 5% of a catalyst system mixture productivity in the reaction zone; oriv) where a fouling rate in the activation vessel is equal to 0.065 mg/cm2-hr; orv) where an oligomer product discharge rate from the activation vessel is equal to 0.15 lb/gal/hr, and an oligomer product discharge rate from the reaction zone is in a range from 0.75 to 6 lb/gal/hr; orvi) where a ΔT from the inlet of the activation vessel to the outlet of the activation vessel is equal to 5° C.; orvii) of 8 hr; orviii) any combination thereof; or wherein an amount of ethylene introduced into the activation vessel is less than 50% of an amount of ethylene introduced into the reaction zone; or wherein step b) is performed at an activation temperature and an activation pressure, and the first mixture in the activation vessel is below the bubble point at the activation temperature and the activation pressure; or any combination thereof. Statement 2. The process defined in statement 1, wherein, in step a), a first feed stream comprising ethylene and at least a portion of the first organic reaction medium is introduced into the activation vessel separate from a catalyst system mixture comprising the catalyst system. Statement 3. The process defined in statement 1 or 2, wherein i) the heteroatomic ligand chromium compound complex and the organoaluminum compound are, or ii) at least one of the heteroatomic ligand, the chromium compound, and the organoaluminum compound is, separately introduced into the activation vessel. Statement 4. The process defined in statement 1 or 2, wherein a catalyst system mixture comprising i) the heteroatomic ligand chromium compound complex and the organoaluminum compound, or ii) the heteroatomic ligand, the chromium compound, and the organoaluminum compound, and optionally at least a portion of the first organic reaction medium, is formed and then introduced into the activation vessel. Statement 5. The process defined in statement 4, wherein the catalyst system mixture is aged for any suitable period of time in the substantial absence of ethylene prior to introducing the catalyst system mixture into the activation vessel. Statement 6. A process comprising: a) forming a first mixture in an activation vessel, the first mixture comprising:1) a first catalyst system comprising i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound,2) a first feed comprising ethylene, a second catalyst system comprising i) a second heteroatomic ligand chromium compound complex and a second organoaluminum compound, or ii) a second heteroatomic ligand, a second chromium compound, and a second organoaluminum compound, a second organic reaction medium, a second oligomer product, and optionally hydrogen,3) optionally, a first organic reaction medium, and4) optionally, hydrogen; b) maintaining the first mixture in the activation vessel for an average residence time; c) introducing into a reaction zone:i) ethylene,ii) the first mixture from step b),iii) the second organic reaction medium, andiv) optionally, hydrogen; and d) forming an oligomer product in the reaction zone; wherein a portion of a reaction zone effluent is fed to the activation vessel as the first feed, and wherein the average residence time of the first mixture in the activation vessel is in a range from 10 sec up to a period of time i) sufficient to form an amount of a first oligomer product in the activation vessel equal to 5% of an amount of the oligomer product formed in the reaction zone; or ii) where a conversion of ethylene in the activation vessel is equal to 5 mol % of a total ethylene utilized in step a) and step c); or iii) where a catalyst system productivity in the activation vessel is equal to 5% of a catalyst system mixture productivity in the reaction zone; or iv) where a fouling rate in the activation vessel is equal to 0.065 mg/cm2-hr; or v) where an oligomer product discharge rate from the activation vessel is equal to 0.15 lb/gal/hr, and an oligomer product discharge rate from the reaction zone is in a range from 0.75 to 6 lb/gal/hr; or vi) where a ΔT from the inlet of the activation vessel to the outlet of the activation vessel is equal to 5° C.; or vii) of 8 hr; or viii) any combination thereof; or wherein an amount of ethylene introduced into the activation vessel is less than 50% of an amount of ethylene introduced into the reaction zone; or wherein step b) is performed at an activation temperature and an activation pressure, and the first mixture in the activation vessel is below the bubble point at the activation temperature and the activation pressure; or any combination thereof. Statement 7. The process defined in statement 6, wherein, in step a), a first feed stream comprising ethylene and at least a portion of the first organic reaction medium is introduced into the activation vessel separate from a catalyst system mixture comprising the first catalyst system. Statement 8. The process defined in statement 6 or 7, wherein i) the first heteroatomic ligand chromium compound complex and the first organoaluminum compound are, or ii) at least one of the first heteroatomic ligand, the first chromium compound, and the first organoaluminum compound is, separately introduced into the activation vessel. Statement 9. The process defined in statement 6 or 7, wherein a catalyst system mixture comprising i) the first heteroatomic ligand chromium compound complex and the first organoaluminum compound, or ii) the first heteroatomic ligand, the first chromium compound, and the first organoaluminum compound, and optionally at least a portion of the first organic reaction medium, is formed and then introduced into the activation vessel. Statement 10. The process defined in statement 9, wherein the catalyst system mixture is aged for any suitable period of time in the substantial absence of ethylene prior to introducing the catalyst system mixture into the activation vessel. Statement 11. The process defined in any one of statements 6-10, wherein, in step c), the ethylene introduced into the reaction zone is substantially devoid of catalyst system or catalyst system components. Statement 12. The process defined in any one of statements 1-11, wherein, in step c), the ethylene and the first mixture from step b) are introduced separately into the reaction zone. Statement 13. The process defined in any one of statements 1-11, wherein, in step c), a second feed stream comprising ethylene, at least a portion of the second organic reaction medium, and optionally hydrogen is introduced into the reaction zone separate from the first mixture from step b). Statement 14. The process defined in any one of the preceding statements, wherein the amount of a first oligomer product in the activation vessel is less than or equal to 5% (or any or value or range disclosed herein, e.g., less than or equal to 4%, 3%, 2%, or 1%) of the amount of the oligomer product formed in the reaction zone. Statement 15. The process defined in any one of the preceding statements, wherein the conversion of ethylene in the activation vessel is less than or equal to 5 mol % (or in any range of molar ratios disclosed herein, e.g., less than or equal to 4 mol %, less than or equal to 3%, less than or equal to 2%, etc.) of the total ethylene utilized in step a) and step c). Statement 16. The process defined in any one of the preceding statements, wherein the catalyst system productivity in the activation vessel is less than or equal to 5% (or in any range disclosed herein, e.g., less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, etc.) of the catalyst system mixture productivity in the reaction zone. Statement 17. The process defined in any one of the preceding statements, wherein the fouling rate in the activation vessel is less than or equal to 0.065 mg/cm2-hr, or in any range of fouling rates disclosed herein, e.g., less than or equal to 0.06 mg/cm2-hr, less than or equal to 0.055 mg/cm2-hr, less than or equal to 0.05 mg/cm2-hr, etc. Statement 18. The process defined in any one of the preceding statements, wherein the oligomer product discharge rate from the activation vessel is less than or equal to 0.15 lb/gal/hr, or in any range of any oligomer product discharge rate from the activation vessel disclosed herein, e.g., less than or equal to 0.125 lb/gal/hr, less than or equal to 0.1 lb/gal/hr, less than or equal to 0.075 lb/gal/hr, etc. Statement 19. The process defined in any one of the preceding statements, wherein the oligomer product discharge rate from the reaction zone is in a range from 0.75 to 6 lb/gal/hr, or in any range of any oligomer product discharge rate from the reaction zone disclosed herein, e.g., 0.75 to 6 lb/gal/hr, 1 to 6 lb/gal/hr, 1.2 to 5.5 lb/gal/hr, 1.4 to 5 lb/gal/hr, 1.5 to 4.5 lb/gal/hr, or 1.6 to 4.25 lb/gal/hr, etc. Statement 20. The process defined in any one of the preceding statements, wherein a ΔT from the inlet of the activation vessel to the outlet of the activation vessel is less than or equal to 5° C. (or any other value disclosed herein, e.g., less than or equal to 4° C., 3° C., 2° C., or 1° C.). Statement 21. The process defined in any one of the preceding statements, wherein the average residence time is less than or equal to 8 hr (or any time or any range disclosed herein, e.g., less than or equal to 6 hr, 4 hr, 2 hr, 1 hr, less than or equal to 45 min, less than or equal to 30 min, less than or equal to 20 min, or less than or equal to 15 min, or in a range from 10 sec to 45 min, from 10 sec to 30 min, from 30 sec to 20 min, from 1 min to 30 min, or from 2 min to 15 min). Statement 22. The process defined in any one of the preceding statements, wherein the amount of ethylene introduced into the activation vessel is less than or equal to 50% (or in any range disclosed herein, e.g., less than or equal to 40%, less than or equal to 30%, less than or equal to 25%, etc.) of the amount of ethylene introduced into the reaction zone. Statement 23. The process defined in any one of the preceding statements, wherein: step b) is performed at the activation temperature and the activation pressure; and the first mixture in the activation vessel is below the bubble point at the activation temperature and the activation pressure. Statement 24. The process defined in any one of the preceding statements, wherein hydrogen is present in step a). Statement 25. The process defined in any one of the preceding statements, wherein hydrogen is present in step c). Statement 26. A reaction system comprising: (a) an activation vessel configured to form a first mixture, wherein the activation vessel is further configured for an average residence time of the first mixture in the activation vessel; (b) one or more activation vessel inlets configured to introduce i) ethylene and a catalyst system mixture, or ii) ethylene and components of a catalyst system mixture, into the activation vessel; (c) an activation vessel outlet configured to withdraw the first mixture from the activation vessel; (d) a reaction zone configured to contact ethylene, the first mixture, a second organic reaction medium, and optionally hydrogen to form an oligomer product; (e) one or more reaction zone inlets configured to introduce ethylene, the second organic reaction medium, and the first mixture from the activation vessel outlet into the reaction zone; and (f) a reaction zone outlet configured to withdraw a reaction zone effluent stream containing the oligomer product from the reaction zone; wherein the average residence time of the first mixture in the activation vessel is in a range from 10 sec up to a period of time i) sufficient to form an amount of a first oligomer product in the activation vessel equal to 5% of an amount of the oligomer product formed in the reaction zone; or ii) where a conversion of ethylene in the activation vessel is equal to 5 mol % of a total ethylene utilized in step a) and step c); or iii) where a catalyst system productivity in the activation vessel is equal to 5% of a catalyst system mixture productivity in the reaction zone; or iv) where a fouling rate in the activation vessel is equal to 0.065 mg/cm2-hr; or v) where an oligomer product discharge rate from the activation vessel is equal to 0.15 lb/gal/hr and an oligomer product discharge rate from the reaction zone is in a range from 0.75 to 6 lb/gal/hr; or vi) where a ΔT from the inlet of the activation vessel to the outlet of the activation vessel is equal to 5° C.; or vii) of 8 hr; or viii) any combination thereof; or wherein an amount of ethylene introduced into the activation vessel is less than 50% of an amount of ethylene introduced into the reaction zone; or wherein step b) is performed at an activation temperature and an activation pressure, and the first mixture in the activation vessel is below the bubble point at the activation temperature and the activation pressure; or any combination thereof. Statement 27. The system defined in statement 26, wherein the one or more reaction zone inlets are further configured to introduce hydrogen into the reaction zone. Statement 28. The system defined in statement 26 or 27, wherein the activation vessel outlet and at least one of the one or more reaction zone inlets are fluidly connected. Statement 29. The system defined in any one of statements 26-28, wherein the reaction zone effluent stream further comprises ethylene, the second organic reaction medium, the catalyst system mixture, or any combination thereof. Statement 30. The system defined in any one of statements 26-29, wherein the amount of a first oligomer product in the activation vessel is less than or equal to 5% (or any or value or range disclosed herein, e.g., less than or equal to 4%, 3%, 2%, or 1%) of the amount of the oligomer product formed in the reaction zone. Statement 31. The system defined in any one of statements 26-30, wherein the activation vessel is further configured for the conversion of ethylene of less than or equal to 5 mol % (or in any range of molar ratios disclosed herein, e.g., less than or equal to 4 mol %, less than or equal to 3%, less than or equal to 2%, etc.) of the total ethylene utilized in the activation vessel and the reaction zone. Statement 32. The system defined in any one of statements 26-31, wherein the activation vessel is further configured for a catalyst system productivity of less than or equal to 5% (or in any range disclosed herein, e.g., less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, etc.) of a catalyst system mixture productivity in the reaction zone. Statement 33. The system defined in any one of statements 26-32, wherein the fouling rate in the activation vessel is less than or equal to 0.065 mg/cm2-hr, or in any range of fouling rates disclosed herein, e.g., less than or equal to 0.06 mg/cm2-hr, less than or equal to 0.055 mg/cm2-hr, less than or equal to 0.05 mg/cm2-hr, etc. Statement 34. The system defined in any one of statements 26-33, wherein the oligomer product discharge rate from the activation vessel is less than or equal to 0.15 lb/gal/hr, or in any range of any oligomer product discharge rate from the activation vessel disclosed herein, e.g., less than or equal to 0.125 lb/gal/hr, less than or equal to 0.1 lb/gal/hr, less than or equal to 0.075 lb/gal/hr, etc. Statement 35. The system defined in any one of statements 26-34, wherein the oligomer product discharge rate from the reaction zone is in a range from 1 to 6 lb/gal/hr, or in any range of any oligomer product discharge rate from the reaction zone disclosed herein, e.g., 0.75 to 6 lb/gal/hr, 1 to 6 lb/gal/hr, 1.2 to 5.5 lb/gal/hr, 1.4 to 5 lb/gal/hr, 1.5 to 4.5 lb/gal/hr, or 1.6 to 4.25 lb/gal/hr, etc. Statement 36. The system defined in any one of statements 26-35, wherein the activation vessel is further configured for a ΔT from the inlet of the activation vessel to the outlet of the activation vessel of less than or equal to 5° C. (or any other value or range disclosed herein, e.g., less than or equal to 4° C., 3° C., 2° C., or 1° C.). Statement 37. The system defined in any one of statements 26-36, wherein the average residence time is less than or equal to 2 hr (or any time or any range disclosed herein, e.g., less than or equal to 1 hr, less than or equal to 45 min, less than or equal to 30 min, less than or equal to 20 min, or less than or equal to 15 min, or in a range from 10 sec to 45 min, from 10 sec to 30 min, from 30 sec to 20 min, from 1 min to 30 min, or from 2 min to 15 min). Statement 38. The system defined in any one of statements 26-37, wherein the amount of ethylene introduced into the activation vessel is less than or equal to 50% (or in any range disclosed herein, e.g., less than or equal to 40%, less than or equal to 30%, less than or equal to 25%, etc.) of the amount of ethylene introduced into the reaction zone. Statement 39. The system defined in any one of statements 26-38, wherein: the activation vessel is further configured to operate at the activation temperature and the activation pressure; and the first mixture in the activation vessel is below the bubble point at the activation temperature and the activation pressure. Statement 40. The system defined in any one of statements 26-39, wherein at least one of the one or more activation vessel inlets is further configured to introduce a first organic reaction medium and/or hydrogen into the activation vessel. Statement 41. The system defined in any one of statements 26-40, wherein the one or more activation vessel inlets are configured to introduce the catalyst system mixture into the activation vessel, wherein the catalyst system mixture comprises (i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or (ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound. Statement 42. The system defined in any one of statements 26-40, wherein the one or more activation vessel inlets are configured to introduce the components of the catalyst system mixture into the activation vessel, wherein the components comprise (i) a first heteroatomic ligand chromium compound complex and a first organoaluminum compound, or (ii) a first heteroatomic ligand, a first chromium compound, and a first organoaluminum compound. Statement 43. The system defined in any one of statements 26-42, wherein the activation vessel is configured to operate batchwise or continuously. Statement 44. The system defined in any one of statements 26-43, wherein the activation vessel is any suitable vessel or any vessel disclosed herein, e.g., a stirred tank (CSTR), a flow reactor (plug flow), etc. Statement 45. The system defined in any one of statements 26-44, wherein the reaction system further comprises (g) a controller configured to control the average residence time of the first mixture in the activation vessel, and/or temperature in the activation vessel, and/or pressure in the activation vessel, and/or ethylene conversion in the activation vessel, and/or ethylene flow rate into the activation vessel. | 277,824 |
11859026 | DETAILED DESCRIPTION In this application, a monomer composition of functionalized pyranine monomer being “substantially free” of unfunctionalized pyranine compound means that the monomer composition comprises less than 5 mol % of unfunctionalized pyranine compound, or less than 4 mol %, or less than 3 mol %, or less than 2 mol %, or less than 1 mol %, or less than 0.5 mol %, based on the total moles of the unfunctionalized pyranine compound and functionalized pyranine monomer in the monomer composition. In this application, a polymer composition of a water soluble fluorescent tagged polymer being “substantially free” of unpolymerized pyranine means that the composition comprises less than 5 mol %, or less than 4 mol %, or less than 3 mol %, or less than 2 mol %, or less than 1 mol %, or less than 0.5 mol %, of total unpolymerized pyranine, based on the moles of total pyranine in the composition. The “total unpolymerized pyranine” in the polymer composition is the sum of the unfunctionalized pyranine compound and unpolymerized pyranine monomer that is not polymerized into the polymer. The “total pyranine” in the polymer composition is the sum of the total unpolymerized pyranine and the pyranine monomer that is polymerized into the polymer. In this application, the term “water-soluble polymer” means that the polymer has a solubility in water of at least 1 g/L, or preferably at least 10 g/L, or more preferably at least 100 g/L, when measured in an aqueous composition having a pH of 7 at a temperature of 25° C. In this application, the term “water-soluble organic co-solvent” means that the co-solvent has a solubility in water of at least 20 g/L, or preferably at least 50 g/L, or more preferably at least 70 g/L, when measured in an aqueous composition having a pH of 7 at a temperature of 25° C. In this application, the term “dosing” of a reactant into a reaction mixture means that the reactant is added over a period of time during the course of the reaction, as opposed to a single addition of an entire reactant portion. The present application is based upon the discovery that pyranine can be reacted with a functionalizing agent to provide a pyranine monomer having a polymerizable functional group, the reaction taking place in an aqueous system with an excess of the functionalizing agent, under conditions such that the reaction goes substantially to completion, thereby providing a monomer composition of functionalized polymerizable pyranine monomers, the composition containing less than 5 mol % of unfunctionalized pyranine compound based on the total moles of functionalized pyranine monomer and unfunctionalized fluorescent compound. These compositions of functionalized pyranine monomers are useful in the preparation of water soluble fluorescent tagged polymers that can be used as treatment polymers in industrial water systems, and as scale-control polymers in oilfield applications. In one embodiment, a method for making a functionalized pyranine monomer composition comprises the steps ofproviding a starting amount of pyranine in an aqueous solvent system,adding an amount of base to the aqueous solvent system,dosing to the aqueous solvent system an amount of a polymerizable functionalizing agent to form a reaction mixture, thereby initiating the reaction of the pyranine with the functionalizing agent to functionalize the pyranine molecule with a polymerizable functional group, andmaintaining the dosing of the functionalizing agent to the reaction mixture during the reaction of the pyranine with the functionalizing agent until the amount of functionalizing agent dosed to the system exceeds the starting amount of pyranine on a molar basis. In one embodiment, a method for making a functionalized pyranine monomer composition comprises the steps ofproviding a starting amount of pyranine in an aqueous solvent system,adding an amount of base to the aqueous solvent system,dosing to the aqueous solvent system an amount of a polymerizable functionalizing agent to form a reaction mixture, thereby initiating the reaction of the pyranine with the functionalizing agent to functionalize the pyranine molecule with a polymerizable functional group,maintaining the dosing of the functionalizing agent to the reaction mixture during the reaction of the pyranine with the functionalizing agent until the amount of functionalizing agent dosed to the system exceeds the starting amount of pyranine on a molar basis, andcontinuing the reaction of pyranine with the functionalizing agent until at least 95 mol % of the starting amount of pyranine has been functionalized with the functionalizing agent. In one embodiment, the base is added to the solvent system prior to the dosing of the functionalizing agent. In one embodiment the step of adding the base is accomplished by the simultaneous dosing of the base and the functionalizing agent to the aqueously solvent system, with the dosing of both the functionalizing agent and the base being maintained during the reaction of the pyranine with the functionalizing agent until the amount of functionalizing agent dosed to the system exceeds the starting amount of pyranine on a molar basis. In one embodiment, the dosing of base to the reaction mixture continues after the addition of the functionalizing agent is complete and during the continued reaction of the pyranine with the functionalizing agent. In one embodiment the method of the disclosure is carried out over a time period of from about five minutes to about 24 hours; in one embodiment from about 30 minutes to about 18 hours, in one embodiment from about 1 hour to about ten hours. To optimize the conversion of unfunctionalized pyranine compound to functionalized pyranine monomer, it is preferred that the functionalizing agent and the base each be dosed slowly into the reaction mixture. In one embodiment, the functionalizing agent can be added at a rate of no more than 50% of the total dosage amount per hour, or no more than 40% of the total dosage amount per hour, or no more than 30% of the total dosage amount per hour, or no more than 25% of the total dosage amount per hour, or no more than 20% of the total dosage amount per hour, or no more than 15% of the total dosage amount per hour, or no more than 10% of the total dosage amount per hour. The base is dosed to the reaction mixture at a rate no faster than the rate of the dosage of the functionalizing agent, based on the total dosage amount of base. Following the dosing method of the disclosure herein can result in at least 95 mol % of the starting amount of pyranine being functionalized with the functionalizing agent; as compared to a much lower functionalization rate obtained when all the reactants are added to the solvent system in a single shot. The skilled artisan will adjust the dosage rates and time of the reaction to achieve optimum functionalization of the pyranine based on the disclosure herein, taking into consideration the quantity of reactants, and the capacity and features of the reaction vessel and dosing apparatus used for each use of the disclosed method. In one embodiment, the reaction mixture is heated during the step of dosing of the reactants. The heating may be continued during the step of maintaining the reaction until at least 95 mol % of the starting amount of pyranine has been functionalized with the functionalizing agent. In one embodiment the reaction may be terminated by discontinuing the heating of the reaction mixture. In one embodiment, if a co-solvent is used as discussed below, the reaction may be terminated by distilling the co-solvent. The reaction temperature can be at least 50° C., or at least 60° C., or at least 70° C., or at least 80° C. In one embodiment the reaction temperature is in the range of 70-75° C. In one embodiment of the method the aqueous reaction medium optionally comprises one or more water-soluble organic co-solvents. In one embodiment the one or more water-soluble organic co-solvents are selected from the group consisting of C1-C6alcohols. In one embodiment the organic co-solvent is selected from the group consisting of methanol, ethanol, n-propanol and isopropanol. In one embodiment of the method the organic co-solvent is selected from one or more of methanol, n-propanol, and isopropanol. In one embodiment of the method the organic co-solvent is selected from one or more of methanol and n-propanol. In one embodiment the water-soluble organic co-solvent is n-propanol. In some embodiments the alcohol co-solvent has unlimited solubility in water. When a water soluble organic co-solvent is used, the ratio of water to total organic co-solvent on a volume:volume basis is in the range of 20:1-1:20, or in the range of 10:1-1:10, or in the range of 5:1-1:5, or in the range of 4:1-1:4, or in the range of 3:1-1:3. In one embodiment of the method the polymerizable functional group comprises a carbon-carbon double bond, and the functionalizing agent can be a compound containing such a carbon-carbon double bond, which compound reacts with the hydroxyl group on the pyranine to functionalize the pyranine compound with the carbon-carbon double bond, thereby creating a polymerizable pyranine monomer. In one embodiment of the method, the functionalizing agent is a compound of the formula (I) R—C(═CH2)—R1—X (I) wherein R1is selected from optionally substituted —C1-C10alkyl-, -aryl-C1-C10alkyl-, —C(O)—, —CH2NH—C(O)—, —C(CH3)2—NH—C(O)—, R is H or optionally substituted C1-C10alkyl-, and X is a leaving group. In one embodiment of the method, R1is selected from —C1-C10alkyl- and -aryl-C1-C10alkyl-, or —C1-C6alkyl- and -aryl-C1-C6alkyl-, or —C1-C3alkyl- and -aryl-C1-C3alkyl-. In one embodiment R1is methylene. In one embodiment R1is benzyl. In one embodiment R is H or C1-C10alkyl-. In one embodiment R is H or C1-C6alkyl-. In one embodiment R is H. In one embodiment R is C1-3alkyl-. In one embodiment R is methyl. In one embodiment R is H. The leaving group X is a moiety that will chemically separate from the R—C(═CH2)—R1— moiety when the functionalizing agent is present in the functionalization reaction system. In one embodiment X is a halide ion. In one embodiment X is chloride. In one embodiment X is —O—C(O)C(CH3)═CH2. Other suitable leaving groups include —SO2C6H4CH3and —SO2CH3. In one aspect, a monomer composition comprises a pyranine monomer of formula (IIa): wherein M is selected from the group consisting of hydrogen, sodium, potassium, cesium, rubidium, lithium, ammonium, tetralkylammonium, and R1is selected from optionally substituted —C1-C10alkyl-, -aryl-C2-C10alkyl-, —C(O)—, —CH2NH—C(O)—, and —C(CH3)2—NH—C(O)—, and R is H or optionally substituted C1-C10alkyl-; and wherein the monomer composition comprises less than 5 mol % of unfunctionalized pyranine compound based on the total moles of the unfunctionalized pyranine and pyranine monomer in the monomer composition. In one embodiment, where M is tetraalkylammonium, the alkyl groups on the ammonium ion are independently selected from linear or branched C1-C4alkyl. In one embodiment, M is tetramethylammonium. In one aspect the monomer composition comprises less than 4 mol %, or less than 3 mol %, or less than 2 mol %, or less than 1 mol %, or less than 0.5 mol % of unfunctionalized pyranine compound, based on the total moles of the unfunctionalized pyranine and pyranine monomer in the monomer composition. Preferably M is selected from the group consisting of hydrogen, sodium and potassium. More preferably, M is selected from the group consisting of sodium and potassium. In an embodiment wherein R1is —CH2— and R is —CH3, the polymerizable functional group is methallyl, and the methallyl-functionalized pyranine monomers include those selected from the group consisting of compounds of the formula (III): wherein M is selected from the group consisting of hydrogen, sodium, potassium, cesium, rubidium, lithium, ammonium, and tetralkylammonium, and n is 1. In formula (IIa) above, the pyranine molecule is functionalized through the pendant hydroxyl group to form an alkoxylated molecule. In some embodiments, some of the pyranine molecules can be functionalized directly on the pyrene ring structure to form an alkylated molecule, illustrated in formula (IIb): where M is selected from any of hydrogen, sodium, potassium, cesium, rubidium, lithium, ammonium, and tetralkylammonium. Those skilled in the art will recognize that the alkylation can take place at any of the carbon atoms at locations 2, 4, 5, 7, 9 or 10 as illustrated in formula (IIb). Both the alkoxylated reaction product of formula (IIa) and the alkylated reaction product of formula (IIb) will be polymerizable monomers that can be included in a mixture of monomers to be polymerized in a subsequent polymerization reaction to form water soluble fluorescent tagged polymers. Representative suitable fluorescent pyranine monomers made by the method of the disclosure herein include without limitation those selected from the group consisting of 8-(methallyloxy)-1,3,6-pyrene trisulfonic acid, methallyl-8-(hydroxy)-1,3,6-pyrene trisulfonic acid, 8-(allyloxy)-1,3,6-pyrene trisulfonic acid, vinyl benzyl-8-(hydroxy)-1,3,6-pyrene trisulfonic acid, allyl-8-(hydroxy)-1,3,6-pyrene trisulfonic acid, 2-(methallyl)-1,3,6-pyrene trisulfonic acid, 4-(methallyl)-1,3,6-pyrene trisulfonic acid, 5-(methallyl)-1,3,6-pyrene trisulfonic acid, 7-(methallyl)-1,3,6-pyrene trisulfonic acid; 9-(methallyl)-1,3,6-pyrene trisulfonic acid; 10-(methallyl)-1,3,6-pyrene trisulfonic acid; 2-(allyl)-1,3,6-pyrene trisulfonic acid, 4-(allyl)-1,3,6-pyrene trisulfonic acid, 5-(allyl)-1,3,6-pyrene trisulfonic acid, 7-(allyl)-1,3,6-pyrene trisulfonic acid; 9-(allyl)-1,3,6-pyrene trisulfonic acid; 10-(allyl)-1,3,6-pyrene trisulfonic acid, 2-vinyl benzyl-1,3,6-pyrene trisulfonic acid, 4-vinyl benzyl-1,3,6-pyrene trisulfonic acid, 5-vinyl benzyl-1,3,6-pyrene trisulfonic acid, 7-vinyl benzyl-1,3,6-pyrene trisulfonic acid, 9-vinyl benzyl-1,3,6-pyrene trisulfonic acid, 10 benzyl-1,3,6-pyrene trisulfonic acid; 8-(3-vinyl benzyloxy)-1,3,6-pyrene trisulfonic acid; 8-(4-vinyl benzyloxy)-1,3,6-pyrene trisulfonic acid; and the sodium, potassium, cesium, rubidium, lithium, ammonium, and tetralkylammonium salts of any of the foregoing. Representative functionalizing agents suitable for use in the reaction to make fluorescent monomers by the method disclosed herein include without limitation allyl chloride, vinyl benzyl chloride, methacrylic anhydride, allyl isocyanate, 3-isopropenyl-α,α-dimethylbenzyl isocyanate (m-TMI available from Allnex USA Inc., Alpharetta, Ga.), maleic anhydride, itaconic anhydride, (meth) acryloyl chloride, methallyl chloride (or methallyl bromide) for n=1; 4-bromo-1-butene, for n=2; 5-bromo-1-pentene, for n=3; 6-bromo-1-hexene, for n=4; 8-bromo-1-octene, for n=6; and 11-bromo-1-undecene, for n=9. 4-bromo-1-butene-2-methyl, for n=2; 5-bromo-1-pentene-2-methyl, for n=3; 6-bromo-1-hexene-2-methyl, for n=4; 8-bromo-1-octene-2-methyl, for n=6; and 11-bromo-1-undecene-2-methyl, for n=9. At least some of these functionalizing agents are available from Sigma-Aldrich Corp., St. Louis, Mo. The base is a strong base such as sodium hydroxide, and is used in a stoichiometric amount to deprotonate the hydroxyl group on the pyranine molecule. An excess of functionalizing agent is used to ensure substantially complete conversion of the pyranine to the desired functionalized polymerizable monomer. In one embodiment the functionalizing agent is present in at least 10% molar excess, in one embodiment at least 50% molar excess, in one embodiment at least 100% molar excess, relative to the starting amount of pyranine. When an excess of the functionalizing agent of the formula R—C(═CH2)—R1—X is reacted with pyranine in an aqueous medium in the presence of the base, the excess functionalizing agent will react in a side reaction with the base to form an alcohol of the formula R—C(═CH2)—R1—OH as a reaction byproduct. This side reaction also will occur if too great an excess of sodium hydroxide is introduced initially. Slow addition of the functionalizing agent to the reaction mixture optimizes the reaction of the functionalizing agent with the pyranine compound and minimizes the side reactions that produce byproducts, such as the R—C(═CH2)—R1—OH alcohol. Addition of the deprotonating base and the functionalizing agent to the pyranine reaction mixture in concurrent streams over an extended period also minimizes unwanted side reactions. Advantageously, the reaction can be accomplished in an aqueous reaction medium, as discussed above. It is a further advantage that the functionalization reaction can be carried out under ambient atmosphere, and no inert atmosphere or pressurized vessels are required. Optionally, the reaction mixture can be heated to further ensure substantially complete functionalization of the pyranine compound. Higher reaction temperatures can be used to shorten the reaction time and drive the reaction further to completion. Generally the reaction is carried out at a temperature in the range of about 20° C. to about 80° C. for a period of time in the range of about 3 to about 10 hours. For the representative embodiment in which the functionalizing agent is methallyl chloride, the functionalization reaction can be illustrated as Reaction of Pyranine with Methally Chloride While the reaction product is illustrated above as including only the alkoxylated monomer of formula (IIa), it will be understood that the reaction product also may include a portion of the alkylated monomer of formula (IIb). Advantageously, this reaction product, including functionalized pyranine monomers, water, co-solvent, and byproducts can be used directly in the polymerization process without a separate isolation step. It is a feature of the disclosed method that any unreacted functionalizing agent or any alcohol byproduct, both of which contain double bonds, can be polymerized into the water treatment polymer with no adverse effects on the properties or effectiveness thereof. In some instances the presence of the unreacted functionalizing agent and alcohol byproduct as co-monomers in the water treatment polymer can also improve the properties or effectiveness of the water treatment polymer. If no isolation step is used then it is preferred that the co-solvent be selected from either methanol or n-propanol, as these co-solvents will not act as a chain transfer agent in the subsequent polymerization reaction and therefore will not affect the molecular weight of the polymer product. In embodiments in which the subsequent polymerization reaction is conducted in the presence of isopropanol, then isopropanol can be used as a co-solvent in the functionalization reaction of the pyranine monomer. Alternatively, some or all of the water and optional co-solvent can be removed such as by evaporation or distillation, and the desired functionalized monomer reaction product is collected as a solid. It is a feature of the present application that the functionalization reaction is driven substantially to completion, to minimize the amount of unfunctionalized pyranine present in the monomer reaction product composition and ultimately in the water treatment polymer composition. After preparation and optional isolation of the fluorescent monomer, fluorescent-tagged water soluble polymers containing these fluorescent monomers can be prepared by inclusion of the fluorescent monomer reaction product into a water soluble polymer. The amount of fluorescent monomer that is used should be an amount sufficient to allow the water soluble polymer to be detected in the aqueous environment that it is used. The minimum amount of fluorescent moiety that can be used is that amount which gives a signal-to-noise ratio (S/N) of 3 at the desired polymer dosage. The signal-to-noise ratio is that value where the magnitude of the transduced signal (including but not limited to electronic and optical signals) due to the presence of a target analyte in a measurement device is greater than or equal to a level three (3) times the magnitude of a transduced signal where the analyte (species) of interest is not present in the measurement device. The amount of fluorescent monomer in the tagged polymers is in the range of from about 0.01 wt. % to about 10.0 wt. %, preferably from about 0.05 wt. % to about 2 wt. %, and most preferably from about 0.1 wt. % to about 1.0 wt. %. (When mol percentages are given in this patent application it is understood that these are calculated mol percentages, not measured.) The amount of the fluorescent monomer will be sufficient to allow the fluorescence to be detected without hindering the scale-inhibition or other desired function of the polymer. The water soluble polymer further comprises one, two, three or more additional monomers. In one aspect of the disclosure the other monomers of the water soluble polymer can be selected from one or more of the group consisting of acrylic acid and salts, methacrylic acid and salts, maleic acid and salts, maleic anhydride, acrylamide, crotonic acid and salts; in one aspect the water soluble polymer can additionally include one or more monomers selected from the group consisting of methacrylic acid and salts, maleic acid and salts, maleic anhydride, crotonic acid and salts, itaconic acid and salts, acrylamide, methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid and salts, sodium (meth)allyl sulfonate, allyloxybenzene sulfonic acid and its salts, polyethylene glycol monomethacrylate, vinyl phosphoric acid and salts, styrene sulfonic acid and salts, vinyl sulfonic acid and salts, 3-allyloxy-2-hydroxypropane sulfonic acid and salts, N-alkyl (meth)acrylamide, t-butyl (meth)acrylate, N-alkyl (meth)acrylate, N-alkanol-N-alkyl(meth)acrylate, vinyl acetate, 2-N-alkyl(meth)acrylate, alkyl vinyl ether, alkoxyethyl acrylate, N-alkanol (meth)acrylamide, N,N-dialkyl(meth)acrylamide and 1-vinyl-2-pyrrolidinone; in one aspect the water soluble polymer can additionally include one or more monomers selected from the group consisting of sulfomethylacrylamide and sulfoethylacrylamide. In one embodiment, the water soluble polymer comprises the fluorescent monomer; maleate monomer; a monomer selected from the group consisting of acrylic acid, methacrylic acid and 2-ethylacrylicacid, and combinations thereof; and a nitrogen-containing, nonionic comonomer selected from the group consisting of acrylamide, methacrylamide, ethylacrylamide, propylacrylamide, isopropylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N,N-dipropylacrylamide, N-methylolacrylamide and t-butylacrylamide. In one embodiment, the water soluble polymer is a maleate copolymer comprising about 50 mole percent of maleate monomer, about 40 to about 50 mole percent of a monomer selected from acrylic acid, methacrylic acid and 2-ethylacrylicacid, and combinations thereof, about 1 to about 10 mole percent of the nitrogen-containing, nonionic comonomer; and about 0.05 to about 1 mole percent of the fluorescent monomer. In one embodiment the water soluble polymer is a polymer disclosed in U.S. Pat. No. 5,925,610 and references cited therein, incorporated herein by reference in their entirety, with the addition of a fluorescent monomer as described herein. In one embodiment, the water soluble polymer comprises the fluorescent monomer, an allyloxybenzenesulfonic acid monomer, a methallyl sulfonic acid monomer, a copolymerizable olefinically unsaturated carboxylic acid monomer and a copolymerizable nonionic monomer. In one embodiment, the water soluble polymer comprises at least about 2.5 mol % of allyloxybenzenesulfonic acid monomer, at least about 0.5 mol % of a methallyl sulfonic add monomer, about 10-20 mol % of a copolymerizable olefinically unsaturated carboxylic acid monomer, about 60-97 mol % of a copolymerizable nonionic monomer, and about 0.05 to about 1 mole percent of the fluorescent monomer. In one embodiment the water soluble polymer is a polymer disclosed in U.S. Pat. No. 5,698,512 and references cited therein, incorporated herein by reference in their entirety, with the addition of a fluorescent monomer as described herein. In one embodiment, the water-soluble polymer comprises the fluorescent monomer as disclosed herein, a dicarboxylic acid monomer, a monocarboxylic acid monomer, a non-ionic monomer, and sulfonated or sulfated monomer or combinations thereof. In one embodiment the dicarboxylic acid monomer is selected from itaconic acid, maleic acid, maleic anhydride, citraconic acid, mesaconic acid, glutaconic acid, aconitic acid, fumaric acid, tricarboxy ethylene, and mixtures thereof, preferably maleic acid or maleic anhydride; the monocarboxylic acid monomer is selected from acrylic acid, methacrylic acid, 2-ethylacrylicacid, alpha-chloro-acrylic acid, alpha-cyano acrylic acid, alpha-chloro-acrylic acid, alpha-cyano acrylic acid, beta methyl-acrylic acid (crotonic acid), alpha-phenyl acrylic acid, beta-acryloxy propionic acid, sorbic acid, alpha-chloro sorbic acid, angelic acid, cinnamic acid, p-chloro cinnamic acid, beta-styryl acrylic acid (1-carboxy-4-phenyl butadiene-1,3), and mixtures thereof, preferably acrylic acid, methacrylic acid, ethacrylic acid and mixtures thereof; the non-ionic monomer is selected from the group consisting of C1-C6alkyl esters of (meth)acrylic acid and the alkali or alkaline earth metal or ammonium or tetralkylammonium salts thereof, acrylamide and the C1-C6alkyl-substituted acrylamides, the N-alkyl-substituted acrylamides and the N-alkanol-substituted acrylamides, the C1-C6alkyl esters and C1-C6alkyl half-esters of unsaturated vinylic acids, such as maleic acid and itaconic acid, and C1-C6alkyl esters of saturated aliphatic monocarboxylic acids, such as acetic acid, propionic acid and valeric acid, preferably methyl (meth)acrylate, mono- and dimethyl maleate, mono- and di-ethyl itaconate, and (meth)allyl acetates, propionates and valerates; and the sulfonated or sulfated monomer consists of one or more ethylenically unsaturated monomers containing a sulfonate functionality, including but not limited to (meth)acrylamido methyl propane sulfonic add, styrene sulfonic add, acrylamido alkyl or aryl sulfonic add, allyl sulfonic add, methallyl sulfonic add, and salts thereof, preferably (meth)acrylamido methyl propane sulfonic add (AMPS) sodium salt. In one embodiment the water soluble polymer is a polymer disclosed in U.S. Pat. No. 7,087,189 and references cited therein, incorporated herein by reference in their entirety, with the addition of a fluorescent monomer as described herein. In one embodiment the water soluble polymer can further include a naturally derived hydroxy-containing chain transfer agent selected from a monosaccharide, disaccharide, oligosaccharide or polysaccharide, and derivatives of any of the foregoing. Such hydroxy-containing chain transfer agents are described in U.S. Pat. Nos. 7,666,963 and 9,109,068, incorporated herein by reference in its entirety. Other suitable chain transfer agents can include without limitation mercaptans, ferric and cupric salts, bisulfites, and lower secondary alcohols, preferably isopropanol. All molecular weights in this patent application are weight average molecular weights. The weight average molecular weight of these polymers, apart from any optional naturally derived hydroxy-containing chain transfer agent, is from about 500 atomic mass units (hereinafter “a.m.u.”) to about 200,000 a.m.u. Preferably the molecular weight is from about 1000 a.m.u. to about 100,000 a.m.u. Most preferably, the molecular weight is from about 1000 a.m.u. to about 40,000 a.m.u. Labeling of the water soluble polymer through the use of the fluorescent monomers disclosed herein is achieved by synthesizing the water soluble polymer in the presence of the fluorescent monomer. The polymerization is generally carried out in an aqueous medium through the copolymerization of fluorescent monomers with one or more water soluble ethylenically unsaturated monomers. The polymers may be prepared by any number of conventional means well known to those skilled in the art including, for instance, such techniques as bulk, emulsion, suspension, precipitation, or preferably solution polymerization. The polymer compositions are preferably prepared in an aqueous medium in the presence of any initiator or initiator system capable of liberating free radicals under the reaction conditions employed. The free radical initiators are present in an amount ranging from about 0.01% to about 3% by weight based on total monomer weight. In an embodiment, the initiating system is soluble in water to at least 0.1 weight percent at 25° C. Suitable initiators include, but are not limited to, peroxides, azo initiators as well as redox systems, such as tert-butyl hydroperoxide and erythorbic acid, and metal ion based initiating systems. Initiators may also include both inorganic and organic peroxides, such as hydrogen peroxide, benzoyl peroxide, acetyl peroxide, and lauryl peroxide; organic hydroperoxides, such as cumene hydroperoxide and t-butyl hydroperoxide. In an embodiment, the inorganic peroxides, such as sodium persulfate, potassium persulfate and ammonium persulfate, are preferred. In another embodiment, the initiators comprise metal ion based initiating systems including Fe and hydrogen peroxide, as well as Fe in combination with other peroxides. Organic peracids such as peracetic acid can be used. Peroxides and peracids can optionally be activated with reducing agents, such as sodium bisulfite, sodium formaldehyde, or ascorbic acid, transition metals, hydrazine, and the like. A preferred system is the redox system of sodium persulfate and sodium bisulfite. Azo initiators, especially water soluble azo initiators, may also be used. Water soluble azo initiators include, but are not limited to, 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2′-Azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, 2,2′-Azobis(2-methylpropionamidine)dihydrochloride, 2,2′-Azobis[N-(2-carboxyethyl)-2-methylpropionamidine]hydrate, 2,2′-Azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane}dihydrochloride, 2,2′-Azobis[2-(2-imidazolin-2-yl)propane], 2,2′-Azobis(1-imino-1-pyrrolidino-2-ethylpropane)dihydrochloride, 2,2′-Azobis{2-methyl-N-[1,1-bis(hydroxymethyl)-2-hydroxyethl]propionamide}, 2,2′-Azobis[2-methyl-N-(2-hydroxyethyl)propionamide] and others. The polymer compositions may be added to the aqueous systems or may be formulated into various water treatment compositions which may then be added to the aqueous systems. In certain aqueous systems where large volumes of water are continuously treated to maintain low levels of deposited matter, the polymers may be used at levels as low as 0.5 ppm (parts per million). The upper limit on the level of polymer used will be dependent upon the particular aqueous system to be treated. For example, when used to disperse particulate matter the polymer may be used at levels ranging from 0.5 ppm to 2,000 ppm. When used to inhibit the formation or deposition of mineral scale the polymer may be used at levels ranging from 0.5 ppm to 100 ppm, preferably from 3 ppm to 20 ppm, more preferably from 5 ppm to 10 ppm. Once prepared, the water soluble polymers can be incorporated into a water treatment composition comprising about 10-25 wt % of the water soluble polymer and optionally other water treatment chemicals. Such other chemicals include corrosion inhibitors such as orthophosphates, zinc compounds and azoles such as tolyltriazole and benzotriazole. As indicated above, the level of the fluorescent polymer utilized in the water treatment compositions will be determined by the treatment level desired for the particular aqueous system to be treated. Conventional water treatment compositions are known to those skilled in the art and exemplary water treatment compositions are set forth below, in which HEDP is hydroxyethylidene diphosphonic acid and TKPP is tetrapotassium pyrophosphate. Formulation 1Formulation 211.3% Polymer (40% active)11.3% Polymer (40% active)47.7% Water59.6% Water4.2% HEDP4.2% HEDP10.3% NaOH18.4% TKPP24.5% Sodium Molybdate7.2% NaOH2.0% Tolyltriazole2.0% TolyltriazolepH 13.0pH 12.6Formulation 3Formulation 422.6% Polymer (40% active)11.3% Polymer (40% active)51.1% Water59.0% Water8.3% HEDP4.2% HEDP14.0% NaOH19.3% NaOH4.0% Tolyltriazole2.0% Tolyltriazole4.2% ZnCl2pH 12.5pH 13.2Formulation 525.0% Polymer (40% active)65.0% Water (soft or DI)6.0% 2-Phosphonobutane-1,2,4-Tricarboxylic Acid (50% active)1.0% Muriatic Acid3.0% BenzotriazolepH 13.0 Once created, the fluorescent water soluble polymers can be used as scale inhibitors in any industrial water system where a scale inhibitor is needed. This disclosure further relates to a method of inhibiting scale in an industrial water system, the method comprising the steps ofa) providing an industrial water system susceptible to unwanted scaling; andb) adding to the water of said industrial water system from about 0.5 ppm to about 2000 ppm of a scale inhibitor, wherein said scale inhibitor comprises a fluorescent polymer composition comprising fluorescent pyranine monomers and non-fluorescent monomers, said fluorescent polymer composition being substantially free of pyranine, said fluorescent pyranine monomers being selected from monomers of formula (IIa) wherein R1is selected from optionally substituted C1-C10alkyl, aryl-C2-C10alkyl, —C(O)—, —CH2NH—C(O)—, —C(CH3)2—NH—C(O)—, R is optionally substituted C1-C10alkyl, and M is selected from the group consisting of hydrogen, sodium, potassium, cesium, rubidium, lithium, ammonium, and tetralkylammonium, wherein the sum of the mole percent of unfunctionalized pyranine compound and the mole percent of unpolymerized pyranine monomer is no more than 5 mole percent of the total pyranine in the polymer composition. The non-fluorescent monomers are selected from one or more of the group consisting of acrylic add and salts, methacrylic acid and salts, maleic acid and salts, maleic anhydride, acrylamide, crotonic acid and salts; itaconic acid and salts, methacrylamide, 2-acrylamido-2-methylpropanesulfonic acid and salts, sodium (meth)allyl sulfonate, allyloxybenzene sulfonic acid and its salts, polyethylene glycol monomethacrylate, vinyl phosphonic acid and salts, styrene sulfonic acid and salts, vinyl sulfonic add and salts, 3-allyloxy-2-hydroxypropane sulfonic add and salts, N-alkyl (meth)acrylamide, t-butyl (meth)acrylate, N-alkyl (meth)acrylate, N-alkanol-N-alkyl(meth)acrylate, vinyl acetate, 2-Hydroxy N-alkyl(meth)acrylate, alkyl vinyl ether, alkoxyethyl acrylate, N-alkanol (meth)acrylamide, N,N-dialkyl(meth)acrylamide and 1-vinyl-2-pyrrolidinone, sulfomethylacrylamide and sulfoethylacrylamide. Industrial water systems, include, but are not limited to, cooling tower water systems (including open recirculating, closed and once-through systems); petroleum wells, downhole formations, geothermal wells and other oil field applications; boilers and boiler water systems; mineral process waters including mineral washing, flotation and benefaction; paper mill digesters, washers, bleach plants and white water systems; black liquor evaporators in the pulp industry; gas scrubbers and air washers; continuous casting processes in the metallurgical industry; air conditioning and refrigeration systems; industrial and petroleum process water; indirect contact cooling and heating water, such as pasteurization water; water reclamation and purification systems; membrane filtration water systems; food processing streams (meat, vegetable, sugar beets, sugar cane, grain, poultry, fruit and soybean); and waste treatment systems as well as in clarifiers, liquid-solid applications, municipal sewage treatment and industrial or municipal water systems. In addition, the fluorescent water soluble polymers can be used as scale inhibitors in oilfield applications. Scale formation is a major problem in oilfield applications. Subterranean oil recovery operations can involve the injection of an aqueous solution into the oil formation to help move the oil through the formation and to maintain the pressure in the reservoir as fluids are being removed. The injected water, either surface water (lake or river) or seawater (for operations offshore) can contain soluble salts such as sulfates and carbonates. These salts tend to be incompatible with ions already present in the oil-containing reservoir (formation water). The formation water can contain high concentrations of certain ions that are encountered at much lower levels in normal surface water, such as strontium, barium, zinc and calcium. Partially soluble inorganic salts, such as barium sulfate and calcium carbonate, often precipitate from the production water as conditions affecting solubility, such as temperature and pressure, change within the producing well bores and topsides. This is especially prevalent when incompatible waters are encountered such as formation water, seawater, or produced water. Barium sulfate and strontium sulfate form very hard, very insoluble scales that are difficult to prevent. Barium sulfate or other inorganic supersaturated salts can precipitate onto the formation forming scale, thereby clogging the formation and restricting the recovery of oil from the reservoir. The insoluble salts can also precipitate onto production tubing surfaces and associated extraction equipment, limiting productivity, production efficiency and compromising safety. Certain oil-containing formation waters are known to contain high barium concentrations of 400 ppm, and higher. Since barium sulfate forms a particularly insoluble salt, the solubility of which declines rapidly with temperature, it is difficult to inhibit scale formation and to prevent plugging of the oil formation and topside processes and safety equipment. Methods of using water soluble polymers as scale control agents in oilfield applications include but are not limited to continuous injection, squeeze treatment, slow release of solid scale inhibitor. These methods are well known in the art and are described in Chapter 3-scale control, Production chemicals for oil and gas industry, CRC press, 2009, pages 75-84. As stated previously, these fluorescent water soluble polymers function as scale inhibitors. As these water soluble polymers are consumed performing that function, their fluorescent signal will decrease and thus the decrease in the fluorescent signal can be used to indicate that undesired scaling is taking place. Advantageously, the tagged polymer compositions are substantially free of unpolymerized pyranine, which includes both unfunctionalized pyranine and pyranine monomer; if present, the unpolymerized pyranine would emit its own fluorescent signal, so that the decrease in signal from the consumption of the tagged polymer would be difficult or impossible to detect. The water soluble polymer tagged with the fluorescent monomer may be used in the industrial water systems singly or in combination with other polymers, which are not tagged. When used in an industrial water system, the fluorescent signal of the water soluble polymers can be used to determine how much polymer is present in the industrial water system, as is known in the art. An advantage of the fluorescent monomers disclosed herein is that in their use in the formation of a tagged polymer, the fluorescent monomer is not significantly affected by other structures in the polymer or by other ingredients in the system. A further advantage of the disclosed water soluble polymers is that the spectral properties, i.e. both excitation and emission of the polymers are in the visible wavelength region, thus allowing the use of solid state instrumentation and potentially minimizing interferences that generally occur in the UV wavelength region. EXAMPLES The following examples are intended to be illustrative of the present invention and to teach one of ordinary skill in the art to make and use the invention. These examples are not intended to limit the invention in any way. The pyranine used in the following examples was Keystone™ Liquid Pyranine sold as an aqueous solution of 19-23% pyranine by Keystone Aniline Corporation, Chicago, Ill. For purposes of calculating completion of reaction it was assumed that the pyranine solutions used contained 23% pyranine. Methallyl chloride was obtained from Alfa Aesar, Tewksbury, Mass. NMR measurements were conducted using13C analysis on an Agilent DD2 MR 500 MHz NMR Spectrometer, and in DMSO solvent, unless otherwise indicated. Liquid chromatography measurements were conducted using a liquid chromatography/ultra-violet/mass spectroscopy procedure as follows: 10 ul of sample was weighed into an auto-sampling vial and diluted with 1.0 ml of water. The diluted sample was analyzed by LC/UV 400 nm/MS with indicated conditions. ColumnPoroshell C8 50 mm × 4 mm (Agilent, Santa Clara, CA)Mobile phase99% A - 25 mm ammonium acetate1% B - AcetonitrileOven40°C.Flow Rate0.50ml/minInjection2.0ul The mass spectroscopy trace was used to qualitatively confirm the peak, but quantitation was from the ultraviolet trace only. There was no interference in the area of pyranine. Example 1: Methallyl Oxy Pyranine Synthesized in Water and Methanol 548.7 g Keystone™ liquid pyranine solution and 100.1 g of methanol were charged to a 1 L multi-neck round bottom flask equipped with mechanical agitator, thermocouple, methallyl chloride dosing line, NaOH 50% dosing line, and condenser. The mixture was heated to 70° C. and upon reaching the reaction temperature slow additions of methallyl chloride and NaOH 50% were begun. The reaction mixture was refluxed at 70-72° C. during the addition. The methallyl chloride was added over 4 hours while the 50% NaOH was added over 6 hr period, for addition rates of about 7 g/hr and 5 g/hr, respectively. After addition of the NaOH, 50% solution was complete, the reaction mixture was held at 70° C. for 2 more hours. The methanol was removed by distillation at 70-75° C. under nitrogen sparging. Approximately 120 g of distillate was removed. Table 1 summarizes the material balance of the initial reaction mixture. TABLE 1Material balanceEWMaterialWt (g)Wt %(g/eq)molesPyranine solution548.778.012280.000.241(assume 23%)NaOH, 50%25.63.64800.320Methallyl chloride29.04.1290.550.320Methanol100.114.23——Total703.4100.0 Table 2 sets forth the composition of the reaction product after distillation, as determined by NMR. TABLE 2Percentage Composition of Example 1 Reaction ProductReaction ProductComponentMole %Weight %methallyl oxy pyranine82.387.1methallyl pyranine9.810.3Unreacted Pyranine1.41.5Methallyl Alcohol3.30.4Dimethallyl Ether3.20.7 The unfunctionalized pyranine content was 1.4 mol % of the total moles of unfunctionalized pyranine, methallyl oxy pyranine and methallyl pyranine. Example 2: Methallyl Oxy Pyranine Synthesized in Water and 1-Propanol 548.7 g Keystone™ liquid pyranine solution and 100.1 g of 1-propanol were charged to a 1 L multi-neck round bottom flask equipped with mechanical agitator, thermocouple, methallyl chloride dosing line, NaOH, 50% dosing line, and condenser. The mixture was heated to 70° C. and upon reaching the reaction temperature slow additions of methallyl chloride and NaOH, 50% were begun. The reaction mixture was refluxed at 70-72° C. during the addition. The methallyl chloride was added over 4 hours while the 50% NaOH was added over 6 hr period. After completion of the NaOH, 50% addition, the reaction mixture was held at 70° C. for 2 more hours. No distillation or other isolation steps were performed on the reaction product. Table 3 summarizes the material balance of the initial reaction mixture. TABLE 3Material balanceEWMaterialWt (g)Wt %(g/eq)molesPyranine solution548.778.012280.000.241(calculated as 23%)*NaOH, 50%25.63.64800.320Methallyl chloride29.04.1290.550.3201-propanol100.114.23——Total703.4100.0*Pyranine solution as supplied is 19-23% pyranine Table 4 sets forth the composition of the reaction product as determined by NMR. TABLE 4Composition of Example 2 Reaction productReaction ProductComponentMole %Weight %methallyloxy pyranine52.281.5methallyl pyranine6.19.5Methallyl Alcohol34.86.8Dimethallyl Ether2.80.92-methyl-3-propoxyprop-1-ene4.21.3 No unfunctionalized pyranine was detected by NMR. The unfunctionalized pyranine content was 0.11 wt % of the final solution as determined by LC. The unfunctionalized pyranine content was 0.55 wt % (0.61 mol %) of the total of pyranine, methallyl oxy pyranine and methallyl pyranine. Example 3: Methallyl Oxy Pyranine Synthesized in Water and 2-Propanol Example 2 was repeated, but 2-propanol was used instead of 1-propanol. The unfunctionalized pyranine content was 0.50 wt % (0.55 mol %) of the total of unfunctionalized pyranine, methallyl oxy pyranine and methallyl pyranine, as determined by NMR. Example 4: Comparative—Monomer Example II of U.S. Pat. No. 6,312,644 A quantity of the Keystone™ pyranine solution was dried in an oven at 60° C. over a 24 hour period to remove the water. Under a nitrogen atmosphere, the dried pyranine (solid, 2.62 g, 5.0 mmol) was added to dry DMSO (25 mL) along with NaOH, 50% (0.48 g, 6.0 mmol) and stirred at room temperature for a 30 minute period. Not all of the pyranine was dissolved after 30 minutes. However, following Monomer Example II of U.S. Pat. No. 6,312,644, allyl chloride (0.4831 g, 6.31 mmol) was added to the mixture in a single addition. The reaction mixture was stirred for a 6-hr period at room temperature. The next day the reaction mixture was filtered through a glass filter into a 100-mL round bottom flask; the solid filtered material was assumed to be sodium chloride. The majority of DMSO was removed by rotary evaporation (80 C, 7 Torr). The residue was washed with 100 mL of acetone for a 3-hr period which caused an insoluble solid to precipitate. The solid was filtered, collected and dried at room temperature to remove residual acetone. Only 1.0 g of solid was collected from the reaction. Analysis of the solid by NMR (D2O solvent) is reported in Table 5. No alkylation product was detected by NMR. TABLE 5Composition of Example 4 Reaction ProductExample 4ComponentMole %Weight %Allyl oxypyranine91.091.6Unreacted Pyranine9.08.4 It was determined by liquid chromatography that the sample contained unreacted pyranine at a concentration of 80 mg/g, or 8 wt % or 9 mole %. Example 5: Synthesis of Polymer Containing Methallyl Oxy Pyranine Monomer 247 g of water was added to a round bottom flask. Next, 66.1 g of maleic anhydride was added with stirring. 27 g of 50% sodium hydroxide was then added along with 0.0616 g of ferrous ammonium sulfate hexahydrate. The initial charge was heated to 85° C. A monomer mixture containing 125.3 g of acrylic acid, 11.9 g of methyl methacrylate, 74 g of AMPS 2403 from Lubrizol (50% AMPS) and 22.5 g of the liquid reaction product from Example 2, (which contains 1.88 g of methallyl oxy pyranine, 0.1 mole percent of the monomer mixture) was added over 4 hours. Simultaneously, an initiator solution containing 15.3 g of sodium persulfate, 50.9 g of 35% hydrogen peroxide dissolved in 25 g of water was added over the same period of 4 hours. The reaction mixture was held for one hour at 85° C. The reaction mixture was then cooled down to room temperature and 50.4 g of 50% sodium hydroxide was added. The polymer solution contained approximately 40% polymer solids and a pH of 4.5. Example 6: Methallyl Oxy Pyranine Synthesized in Water and 1-Propanol, with all Base Added at Start of Reaction Pyranine solution, 19-23% in water (553.9 g, Milikin), 1-propanol (100.0 g) and sodium hydroxide, 50% (25.88 g) were charged to a 1-L multi-neck round bottom flask equipped with mechanical agitator, thermocouple, methallyl chloride dosing line, and condenser. The mixture was heated to 70° C. and upon reaching the reaction temperature a flow of methallyl chloride begun (0.13 mL/min, 247 minutes, 34.71 g) and the reaction mixture refluxed at 70-72° C. during the addition. After completion of the methallyl chloride addition, the mixture was digested for a 4 hr period at 70° C. The reaction mixture was cooled and discharged (700 g). Table 6 summarizes the material balance and Table 7 the analysis of the sample compared to the sample made by co-dosing the sodium hydroxide and methallyl chloride to the pyranine solution. TABLE 6Material balance of Example 6EWMaterialWt(g)Wt %(g/eq)molesPyranine solution, 19-23%553.978.072280.000.243NaOH, 50%25.93.65800.324Methallyl chloride29.74.1890.550.3281-propanol100.014.09—— TABLE 7NMR analysis of co-dosing processEx. 6Ex. 6(NaOH(NaOHaddedaddedEx. 2Ex. 2upfront)upfront)(co-dosing)(co-dosing)ComponentWeight %Mole %Weight %Mole %methallyl oxy79.148.481.552.2PyranineUnreacted1.61.1NDNDPyranineMethallyl8.039.06.834.8AlcoholDimethallyl0.72.10.92.8Ethermethallyl9.55.89.55.8Pyranine2-methyl-3-1.23.61.34.2propoxyprop-1-ene As seen in Table 7, the co-dosing method gives higher amounts of pyranine reaction product along with reducing the amount of unreacted pyranine to below the detection limit of NMR of ˜1 mol %. A higher rate of methallyl alcohol formation is likely the cause of the lower conversion for the process in which the NaOH is added upfront. This hypothesis is supported by the higher amount of methallyl alcohol seen in the NMR analysis. The method of Example 2 achieved higher conversion of pyranine to polymerizable monomers than the method of Example 6. Example 7: Synthesis of Polymer An initial charge of 248 g deionized water and 66 g of maleic anhydride was added to a 1-liter glass reactor with inlet ports for an agitator, water cooled condenser, thermocouple, and adapters for the addition of monomer and initiator solutions. The reactor contents were heated 85° C. 27 g of 50% sodium hydroxide and 0.0616 g of ferrous ammonium sulfate hexahydrate was added. A mixed monomer solution which consisted of 125.5 g of acrylic acid, 11.9 g of methyl methacrylate, 74.3 g of AMPS 2403 (50% solution of sodium AMPS from Lubrizol) 8.13 g of the monomer solution from Example 2 was fed to the reactor via measured slow-addition with stirring over a period of 4 hours. An initiator solution of 50.9 g of 35% hydrogen peroxide, 15.2 grams sodium persulfate dissolved in 25 grams water was concurrently added, starting at the same time as the monomer solution, for a period of 4 hours. The reaction product was then held at 85° C. for 30 minutes. Next, 0.36 g of erythorbic acid dissolved in 3 g of water was added. Immediately after that, 0.36 g of tertiary butyl hydroperoxide, 70% solution dissolved in 3 g of water was added. The reaction mixture was then heated at 85° C. for 1 hour. The polymers partially neutralized with 50.4 g of 50% sodium hydroxide. The final reaction mixture was an amber colored solution with a solids of about 40%, and a pH of 4.4. Example 8: Synthesis of Polymer An initial charge of 248 g deionized water and 66 g of maleic anhydride was added to a 1-liter glass reactor with inlet ports for an agitator, water cooled condenser, thermocouple, and adapters for the addition of monomer and initiator solutions. The reactor contents were heated 85° C. 27 g of 50% sodium hydroxide and 0.0616 g of ferrous ammonium sulfate hexahydrate was added. A mixed monomer solution which consisted of 125.5 g of acrylic acid, 11.9 g of methyl methacrylate, 74.3 g of AMPS 2403 (50% solution of sodium AMPS from Lubrizol) 22.35 g of the monomer solution from Example 2 was fed to the reactor via measured slow-addition with stirring over a period of 4 hours. An initiator solution of 50.9 g of 35% hydrogen peroxide, 15.2 grams sodium persulfate dissolved in 25 grams water was concurrently added, starting at the same time as the monomer solution, for a period of 4 hours. The reaction product was then held at 85° C. for 30 minutes. Next, 0.36 g of erythorbic acid dissolved in 3 g of water was added. Immediately after that, 0.36 g of tertiary butyl hydroperoxide, 70% solution dissolved in 3 g of water was added. The reaction mixture was then heated at 85° C. for 1 hour. The polymers partially neutralized with 50.4 g of 50% sodium hydroxide. The final reaction mixture was an amber colored solution with a solids of about 40%, and a pH of 4.4. Example 9: Scale Control Various water treatment polymers were evaluated for their ability to prevent the precipitation of calcium carbonate in typical cooling water conditions, a property commonly referred to as the threshold inhibition. Solutions were prepared in which the ratio of calcium concentration to alkalinity was 1.000:1.448 to simulate typical conditions in industrial water systems used for cooling. Generally, water wherein the alkalinity is proportionately less will be able to reach higher levels of calcium, and water containing a proportionally greater amount of alkalinity will reach lower levels of calcium. Since cycle of concentration is a general term, one cycle was chosen, in this case, to be that level at which calcium concentrations equaled 100.0 mg/L Ca as CaCO3(40.0 mg/L as Ca). The complete water conditions at one cycle of concentration (i.e., make-up water conditions) were as follows: Simulated Make-Up Water Conditions:100.00 mg/L Ca as CaCO3(40.0 mg/L as Ca) (one cycle of concentration)49.20 mg/L Mg as CaCO3(12.0 mg/L as Mg)2.88 mg/L Li as CaCO3(0.4 mg/L Li as Li)144.80 M Alkalinity (144.0 mg/L as HCO3)13.40 P Alkalinity (16.0 mg/L as CO3) In dynamic testing, where the pH is about 8.80, bulk water temperature is around 104° F., flow is approximately 3.0 m/s, and heat transfer is approximately 17,000 BTU/hr/ft2, above average threshold inhibitors can reach anywhere from four to five cycles of concentration with this water before significant calcium carbonate precipitation begins. Average threshold inhibitors may only be able to reach three to four cycles of concentration before precipitating, while below average inhibitors may only reach two to three cycles of concentration before precipitation occurs. Polymer performance is generally expressed as percent calcium inhibition. This number is calculated by taking the actual soluble calcium concentration at any given cycle, dividing it by the intended soluble calcium concentration for that same given cycle, and then multiplying the result by 100. Resulting percentage amounts that are below 90% calcium inhibition are considered to be indicators of a significant precipitation of calcium carbonate. However, there are two ways in which an inhibitor can react once the threshold limit is reached. Some lose practically all of their calcium carbonate threshold inhibition properties, falling from 90-100% to below 25% threshold inhibition. Others are able to “hold on” better to their inhibition properties, maintaining anywhere from 50% to 80% threshold inhibition. Testing beyond the threshold limit in order to determine each inhibitor's ability to “hold on” has been found to be a better method of predicting an inhibitor's ability to prevent the formation of calcium carbonate in the dynamic testing units. It also allows for greater differentiation in test results. Therefore, a higher cycle (4.0 cycles) was chosen for this test. At this concentration, above average inhibitors should be expected to give better than 60% threshold inhibition. Poor inhibitors should be expected to give less than 20% threshold inhibition, while average inhibitors should fall somewhere in between. Materials:One incubator/shaker, containing a 125 mL flask platform, with 34 flask capacity34 Screw-cap Erlenmeyer Flasks (125 mL)1 Brinkmann Dispensette (100 mL)Deionized WaterElectronic pipette(s) capable of dispensing between 0.0 mL and 2.5 mL250 Cycle Hardness Solution*10,000 mg/L treatment solutions, prepared using known active solids of the desired treatment*10% and 50% solutions of NaOH250 Cycle Alkalinity Solution*0.2 μm syringe filters or 0.2 μm filter membranes34 Volumetric Flasks (100 mL)Concentrated Nitric Acid * See solution preparations in next section. Solution Preparations: All chemicals used were reagent grade and weighed on an analytical balance to ±0.0005 g of the indicated value. All solutions were made within thirty days of testing. The hardness, alkalinity, and 12% KCl solutions were prepared in a one liter volumetric flask using DI water. The following amounts of chemical were used to prepare these solutions— 250 Cycle Hardness Solution:10,000 mg/L Ca⇒36.6838 g CaCl2·2H2O3,000 mg/L Mg⇒25.0836 g MgCl2·6H2O100 mg/L⇒Li 0.6127 g LiCl 250 Cycle Alkalinity Solution:36,000 mg/L HCO3⇒48.9863 g NaHCO34,000 mg/L CO3⇒7.0659 g Na2CO3 10,000 mg/L Treatment Solutions: Using percentage of active product in the supplied treatment, 250 mL of a 10,000 mg/L active treatment solution was made up for every treatment tested. The pH of the solutions was adjusted to between 8.70 and 8.90 using 50% and 10% NaOH solutions by adding the weighed polymer into a specimen cup or beaker and filling with DI water to approximately 90 mL. The pH of this solution was then adjusted to approximately 8.70 by first adding the 50% NaOH solution until the pH reached 8.00, and then by using the 10% NaOH until the pH equaled 8.70. The solution was then poured into a 250 mL volumetric flask. The specimen cup or beaker was rinsed with DI water and this water was added to the flask until the final 250 mL was reached. The amount of treatment product to be weighed was calculated as follows: Gramsoftreatmentneeded=(10,000mg/L)(0.25L)(decimal%ofactivetreatment)(1000mg) Test Setup Procedure: The incubator shaker was turned on and set for a temperature of 50° C. to preheat. 34 screw cap flasks were set out in groups of three to allow for triplicate testing of each treatment, allowing for testing of eleven different treatments. The one remaining flask was used as an untreated blank. The Brinkmann dispensette was calibrated to deliver 96.6 mL, using DI water, by placing a specimen cup or beaker on an electronic balance and dispensing the water into the container for weighing. The dispensette was adjusted accordingly, until a weight of 96.5-96.7 g DI water was delivered. This weight was recorded, the procedure was repeated for a total of three measurements, and the average determined. Once calibrated, 96.6 mL DI water was dispensed into each flask. Using a 2.5 mL electric pipette, 1.60 mL of hardness solution was added to each flask to simulate four cycles of make-up water. Using a 250 μL electronic pipette, 200 μL of desired treatment solution were added to each flask to achieve a 20 mg/L active treatment dosage. A new tip on the electric pipette was used for each treatment solution so cross contamination did not occur. Using a 2.5 mL electric pipette, 1.60 mL of alkalinity solution was added to each flask to simulate four cycles of make-up water. The addition of alkalinity was done while swirling the flask, so as not to generate premature scale formation from high alkalinity concentration pooling at the addition site. One “blank” solution was prepared in the exact same manner as the above treated solutions, except DI water was added in place of the treatment solution. All 34 flasks uncapped were placed onto the shaker platform and the door closed. The shaker was run at 250 rpm and 50° C. for 17 hours. A “total” solution was prepared in the exact same manner as the above treated solutions were prepared, except that DI water was used in place of both the treatment solution and alkalinity solution. This solution was capped and left overnight outside the shaker. Test Analysis Procedure: Once 17 hours had passed, the 34 flasks were removed from the shaker and allowed to cool for one hour. Each flask solution was filtered through a 0.2 μm filter membrane. The filtrate was analyzed directly for lithium, calcium, and magnesium concentrations by either an Inductively Couple Plasma (ICP) Optical Emission System or Flame Atomic Absorption (AA) system. The “total” solution was analyzed in the same manner. Calculations of Results: Once the lithium, calcium, and magnesium concentrations were known in all 34 shaker samples and in the “total” solution, the percent inhibition was calculated for each treatment. The lithium was used as a tracer of evaporation in each flask (typically about ten percent of the original volume). The lithium concentration found in the “total” solution was assumed to be the starting concentration in all 34 flasks. The concentrations of lithium in the 34 shaker samples were each divided by the lithium concentration found in the “total” sample. These results provided the multiplying factor for increases in concentration, due to evaporation. The calcium and magnesium concentrations found in the “total” solution were also assumed to be the starting concentrations in all 34 flasks. By multiplying these concentrations by each calculated evaporation factor for each shaker sample, the final intended calcium and magnesium concentration for each shaker sample was determined. By subtracting the calcium and magnesium concentrations of the “blank” from both the actual and intended concentrations of calcium and magnesium, then dividing the resulting actual concentration by the resulting intended concentration and multiplying by 100, the percent inhibition for each treated sample was calculated. The triplicate treatments were averaged to provide more accurate results. Example: “Total” concentration analysis results:Li=1.61 mg/LCa=158.0 mg/LMg=50.0 mg/L “Blank” concentration analysis results:Li=1.78 mg/LCa=4.1 mg/LMg=49.1 mg/L Shaker sample concentration analysis results:Li=1.78 mg/LCa=150.0 mg/LMg=54.0 mg/L By taking the Li concentration from the shaker sample and dividing by the Li concentration in the “total” sample, the evaporation factor was determined as— ⇒1.78 mg/L/1.61 mg/L=1.11 By multiplying the Ca and Mg concentrations in the “total” sample by this factor, the final intended concentrations of Ca and Mg in the shaker sample were determined as— Ca⇒1.11×158.0 mg/L=175.4 mg/L Ca Mg⇒1.11×50.0 mg/L=55.5 mg/L Mg Finally, by subtracting the calcium and magnesium concentrations of the “blank” from both the actual and intended concentrations of calcium and magnesium, then dividing the resulting actual concentrations of Ca and Mg in the shaker sample by the resulting final intended concentrations and multiplying by 100, the percent threshold inhibition of calcium and magnesium was calculated as— Ca⇒((150.0 mg/L−4.1 mg/L)/(175.4 mg/L−4.1 mg/L))×100=85.2% Ca inhibition Mg⇒((54.0 mg/L−49.1 mg/L)/(55.5 mg/L−49.1 mg/L))×100=76.6% Mg inhibition The polymers of Example 7 and 8 were tested according to the procedure outlined above. TABLE 8percent calcium carbonate inhibition%%%%inhibitioninhibitioninhibitioninhibitionPolymerat 2 ppmat 3 ppmat 4 ppmat 5 ppmExample 7618792Example 88799100Polymer of567594100Example 7withoutfluorescenttag In the test above, anything above 80% inhibition is considered acceptable. These data in Table 8 indicate that the carbonate inhibition performance of the polymer is the same with the fluorescent tag as it is without the tag, indicating that the presence of the tag does not interfere with the primary purpose of the polymer which is scale minimization. The specific examples herein disclosed are to be considered as being primarily illustrative. Various changes beyond those described will, no doubt, occur to those skilled in the art; and such changes are to be understood as forming a part of this invention insofar as they fall within the spirit and scope of the appended claims. | 63,984 |
11859027 | DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. In the figures, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements components and/or groups or combinations thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups or combinations thereof. As used herein, the term “and/or” includes any and all possible combinations or one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and claims and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity. It will be understood that when an element is referred to as being “on,” “attached” to, “connected” to, “coupled” with, “contacting,” etc., another element, it can be directly on, attached to, connected to, coupled with, and/or contacting the other element or intervening elements can also be present. In contrast, when an element is referred to as being, for example, “directly on,” “directly attached” to, “directly connected” to, “directly coupled” with, or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature can have portions that overlap or underlie the adjacent feature. Spatially relative terms, such as “under,” “below,” “lower,” “over,” “upper,” and the like, may be used herein for ease of description to describe an element's or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus the exemplary term “under” can encompass both an orientation of over and under. The device may otherwise be oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly,” “downwardly,” “vertical,” “horizontal,” and the like are used herein for the purpose of explanation only, unless specifically indicated otherwise. It will be understood that, although the terms first, second, etc., may be used herein to describe various elements, components, regions, layers, and/or sections, these elements, components, regions, layers, and/or sections should not be limited by these terms. Rather, these terms are only used to distinguish one element, component, region, layer, and/or section, from another element, component, region, layer, and/or section. Thus, a first element, component, region, layer, or section discussed herein could be termed a second element, component, region, layer, or section without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise. 1. Stereolithography Apparatus and Resins Resins for additive manufacturing are known and described in, for example, DeSimone et al., U.S. Pat. Nos. 9,211,678; 9,205,601; and 9,216,546. Dual cure resins for additive manufacturing are known and described in, for example, Rolland et al., U.S. Pat. Nos. 9,676,963; 9,598,606; and 9,453,142. Non-limiting examples of dual cure resins include, but are not limited to, resins for producing objects comprised of polymers such as polyurethane, polyurea, and copolymers thereof; objects comprised of epoxy; objects comprised of cyanate ester; objects comprised of silicone, etc. Techniques for additive manufacturing are known. Suitable techniques include bottom-up or top-down additive manufacturing, generally known as stereolithography. Such methods are known and described in, for example, U.S. Pat. No. 5,236,637 to Hull, U.S. Pat. Nos. 5,391,072 and 5,529,473 to Lawton, U.S. Pat. No. 7,438,846 to John, U.S. Pat. No. 7,892,474 to Shkolnik, U.S. Pat. No. 8,110,135 to El-Siblani, U.S. Patent Application Publication No. 2013/0292862 to Joyce, and US Patent Application Publication No. 2013/0295212 to Chen et al. The disclosures of these patents and applications are incorporated by reference herein in their entirety. In some embodiments, the resins characterized by the methods described herein are used to carry out one of the family of methods sometimes referred to as continuous liquid interface production (CLIP). CLIP is known and described in, for example, U.S. Pat. Nos. 9,211,678; 9,205,601; 9,216,546; and others; in J. Tumbleston et al., Continuous liquid interface production of 3D Objects,Science347, 1349-1352 (2015); and in R. Janusziewcz et al., Layerless fabrication with continuous liquid interface production,Proc. Natl. Acad. Sci. USA113, 11703-11708 (Oct. 18, 2016). Other examples of methods and apparatus for carrying out particular embodiments of CLIP include, but are not limited to: Batchelder et al., US Patent Application Pub. No. US 2017/0129169 (May 11, 2017); Sun and Lichkus, US Patent Application Pub. No. US 2016/0288376 (Oct. 6, 2016); Willis et al., US Patent Application Pub. No. US 2015/0360419 (Dec. 17, 2015); Lin et al., US Patent Application Pub. No. US 2015/0331402 (Nov. 19, 2015); D. Castanon, US Patent Application Pub. No. US 2017/0129167 (May 11, 2017); B. Feller, US Patent Application Pub. No. US 2018/0243976 (published Aug. 30, 2018); M. Panzer and J. Tumbleston, US Patent Application Pub. No. US 2018/0126630 (published May 10, 2018); and K. Willis and B. Adzima, US Patent Application Pub. No. US 2018/0290374 (Oct. 11, 2018). 2. Test Cell and Test Apparatus. Apparatus. An embodiment of a test apparatus of the present invention is shown inFIGS.1-3(and schematically inFIG.9) an embodiment of a test cell of the invention is shown inFIGS.4-5, embodiments of a thermal sensor of the present invention is shown inFIGS.6-8. In the illustrative embodiment of the apparatus, the apparatus (seeFIGS.1-3) generally includes a light source (101) positioned in an outer housing (102) or cabinet, an inner test chamber (103), a door (104), optionally, but in some embodiments preferably, with a latch (105) providing access to the inner test chamber103), optionally, but in some embodiments preferably, a gasket such as a silicone gasket (not shown) to seal the door, and a test cell (10) positioned within the test chamber. Gas springs, pneumatic hold-open cylinders (107) or the like are optionally included on the door for ease of operation, but hold open latches, or other alternatives, can be used. A test cell (10) is positioned in the inner chamber, as discussed further below. In the illustrative embodiment, the test chamber includes a chamber floor (130), chamber side walls (131), and an elevated pedestal (132) on which the test cell (10) is placed (seeFIGS.2-3) (though numerous other configurations will be apparent to those skilled in the art). A shutter (not shown) can be included as a part of the light engine to more precisely control the light dose, if desired. A light transmissive spill protection window (134) is included to protect the light source and other apparatus components situated within the cabinet, but outside of the inner test chamber. The light source (101) is secured in place by a set of mounts (136) which are rigidly fixed to the test chamber (132), and hence to the test cell (10), by connecting them to the test chamber floor (130) (where the test chamber floor and pedestal are optionally but, in some embodiments preferably, a single piece, such as machined from a single piece of aluminum). An air inlet duct (121) and an air outlet duct (122) are provided from the exterior of the cabinet into the test chamber, and air (optionally, but in some embodiments preferably, temperature controlled and humidity controlled) can be circulated through the chamber from an external air temperature and humidity control unit, such as available from Orion Machinery North America LLC, 126 Holmes, Liberty Hill, Texas, 78642 USA. Temperature and humidity sensors (seeFIG.9), can be operatively associated with the test chamber, and with the temperature and humidity control unit, in any suitable way, including positioning within the test chamber, within an inlet and/or outlet duct, etc. A barometric pressure sensor (133) can be positioned in any suitable location, including inside the test chamber, outside the test chamber within the cabinet (as shown inFIG.3), on the exterior of the cabinet, etc. Note that ducts, light sources, shutters, various circuit components, and the like are preferably located in a separate compartment (Chamber A ofFIG.9) outside of the inner test chamber (Chamber B ofFIG.9), although located within the outer housing or cabinet of the apparatus. While an apparatus as described above is currently believed desirable for more accurate testing, it will be appreciated that a simplified apparatus, such as a table-top apparatus, may be also be used to carry out the methods of the present invention, particularly where less accuracy is required. Test cell. The test cell (10) (seeFIGS.4-5) includes a support plate (11) having a top surface portion (12), a bottom surface portion (13), and an opening formed therein. A light transmissive base member (14) is directly or indirectly connected to the bottom surface portion so as to define with the opening a well (15) in which well a resin sample can be deposited. At least one, or a plurality of (e.g., two, three, four or more), sensors (20) are connected to or operatively associated with the base member. Suitable sensors include thermal sensors, strain sensors, and combinations thereof, and are discussed further below. The light-transmissive base member (14) can be formed from any suitable polymeric or inorganic material, or inorganic material, including but not limited to quartz, glass (e.g., borosilicate glass), sapphire, an organic polymer, and combination thereof. The support plate (11) can optionally include alignment features or aligners (18) (to align with corresponding or matching alignment features on the pedestal), and can optionally include screws (17) (or other fixation elements) for securing the test cell to the pedestal. In some embodiments, an alignment assembly including a plurality of aligners (18) for kinematic balls may be connected to or formed on the bottom surface portion (13) of the support plate (11). Sensor. One preferred embodiment of a sensor (20) is shown inFIG.6, and a second preferred embodiment of a sensor (20) is shown inFIGS.7-8. Both comprise an array of thin film resistive sensors (30) as the sensor (20), with four separate sensors shown in the embodiment ofFIG.6, and three separate sensors shown in the embodiment ofFIG.7. A plurality of separate sensors are included in some embodiments to provide a back-up if one sensor should fail, and/or provide separate concurrent, simultaneous, or sequential tests (e.g., on the same sample of resin dispensed into the well), the results of which can be combined and/or compared to enhance accuracy. Resistive sensors can sense heat given off by the light polymerizable material during exposure to light due to the exothermic nature of the polymerization reaction, can sense strain exerted on the base as the resin polymerizes, or combinations thereof. In both embodiments, a typically flat, light transmissive, base member has at least one (and indeed a plurality of) thin film resistive sensors formed thereon. Each thin film sensor includes a resistive element (31), a pair of input arms (32,33) electrically connected to the resistive element (31); and optionally, but in some embodiments preferably, one or two sense arms (35,36) electrically connected to the resistive element (while 3-arm sensing can be used, 4 arm sensing, or “Kelvin sensing” is currently preferred). The resistive element, the input arms, and the sense arms when present, are comprised of any suitable conductor, such as platinum, titanium, or indium tin oxide (ITO). The input arms (32,33), and optionally the sense arms (35,36) when present, further comprise a conductive (e.g., gold) upper coat (37) (e.g., configured to reduce parasitic resistance in the sensor array, and/or provide for better electrical contact to the array). A light transmissive protective top coating (38) (e.g., a silicon dioxide layer) can be formed on the sensor over the thin film array, and over the conductive upper coat when present. The illustrated embodiment optionally further includes a secondary well (41) formed in the support plate, the secondary well having an optionally removable, light-transmissive, floor (42) (also referred to herein as a secondary window). The secondary well can be used to conduct an additional or alternate test of photosensitivity. Resin is depicted in the secondary well as a set of six polymerized spots (45) on which a conventional photosensitivity test may be performed, when the spots are of different heights due to different light exposures at each location. In some embodiments a lifting tab (43) may be included to facilitate removal of the secondary window (42). While the test cell is described above primarily with reference to thin film resistive strain sensors, those skilled in the art will appreciate that other approaches can be used. For example, the array of thin film sensors can serve as strain sensors (e.g., sensing strain on the base member that occurs due to shrinkage of photopolymerizable material as it polymerizes, strain caused by heat generated by the exothermic photopolymerization reaction, etc.) in addition to or in combination with their function as thermal sensors. A thermocouple or thermistor can be connected to the base member, in addition to or in place of the thin film sensor (the thermocouple generating a voltage in response to heat, as opposed to a change in resistance in response to heat and/or strain). An infra-red sensor can be directed at the base member, in addition to or as an alternative to any of the foregoing. 3. Test Methods. Because the resins are sensitive to fluctuations in temperature and humidity, temperature and humidity are preferably sensed and controlled, as shown inFIG.9(though a simplified, albeit less accurate, test can be performed simply with a light source, as noted above). Also, since results can vary depending on barometric pressure, the barometric pressure is optionally, but in some embodiments preferably, recorded, along with the sensed heat of reaction and/or strain produced during the test, so that photosensitivity can be calculated with reference to the barometric pressure at which the test was conducted. Thus as noted above, a method of determining the photosensitivity of a photopolymerizable material can be carried out by: (a) depositing a sample of the photopolymerizable material into the well of a test cell or test apparatus as described above; then (b) exposing the photopolymerizable material to a predetermined dose of light through the base member; and (c) sensing heat and/or strain generated by the resin with the sensor in response to the exposure as a measure of the photosensitivity of the photopolymerizable material. In some embodiments, the temperature, humidity, and/or pressure within the test chamber is sensed, concurrently with the step of (c) sensing heat and/or strain. In some embodiments, the method further includes the step of: (d) determining the photosensitivity of the photopolymerizable material from the heat and/or strain sensed in step (c), and optionally from the sensed temperature, humidity, and/or pressure within the test chamber. In some embodiments, as discussed further below, the sensing step is carried out by taking a plurality of heat and/or strain measures (e.g., at least 100, 200, or 400 separate measures) during the exposing step. In some embodiments, as discussed further below, the sensing step includes determining an induction time for the resin in response to the dose of light, with a shorter induction time indicating greater photosensitivity. While the temperature and humidity at which the test is carried out can be set at any convenient point, a temperature set point of 25 degrees centigrade, at 50 percent relative humidity, may typically be used. The choice of wavelength may be based upon the material being tested, but light in the ultraviolet range may be used, typically that emitted from an LED light source having a peak wavelength of 385 to 390 nanometers. The light dose to the material during the test is typically from 0.01 or 0.1 mW/cm2to 5 or 50 mW/cm2, for up to 30 or 60 seconds exposure time, and photosensitivity can be determined from the induction time of the resin in response to the light dose. “Induction time” as used herein refers to the time required, after the onset of the light dose, for the resin to begin releasing heat, and/or exerting strain (as detected by the sensor(s)). For example, in some embodiments, the sensors (20) illustrated inFIGS.4-8may detect the released heat and/or strain. The longer the induction time, the lesser the photosensitivity of the resin (and, conversely, the shorter the induction time, the greater the photosensitivity of the resin). Accuracy of the determination of the induction time can be enhanced by more frequent sampling of the temperature of the resin (and/or strain exerted by the resin), during its exposure to the light dose, and preferably at least 100, 200, 400, or 500 separate temperature and/or strain measures are taken, up to 1000 or more). Additional features of the sensed data, such as the steepness of the rise in temperature/strain, peak signal, and decay after peak may also be utilized as indications of photosensitivity, alone or in combination with induction time. Note also that the entirety of the light-transmissive base member need not be exposed to light during the test. The region exposed to light may be based upon the particular sensor type and configuration employed, but, for a thin film thermal or strain sensor, it can include the region of the base member supporting, or directly beneath, the resistive element. In some embodiments, a curve of recorded strain and/or recorded temperature versus exposure time may be utilized to determine photosensitivity using embodiments of the apparatus of the present invention. Regarding the section of the curve used to determine photosensitivity (such as when expressed as Fc), the initial portion of the curve before the initial temperature and/or strain rise time is preferably used. The rest of the curve can optionally be captured, as it has additional information about the photosensitivity of the resin. For resolution better than 0.5% of the induction time, that region of the curve is preferably sampled at least 1000 separate times (that is, at least 1000 separate temperature and/or strain measures are taken). More specifically, in some embodiments, the exposure time required to initiate curing of the resin at a given exposure irradiance determines the photosensitivity through the relation Photosensitivity=t_cure*I_0 where t_cure is the exposure time required to initiate cure and I_0is the irradiance (energy flux per unit area) incident on the resin at the location of the sensor. The time to cure is determined from critical points in the transient temperature and/or strain signal, S(t), measured by the sensor. These critical points can include, for example, the time to when the signal value first becomes greater than a threshold, (e.g., a minimum t such that S(t)>S_threshold), and/or the time to when the signal's derivative is greater than a threshold value, (e.g., a minimum t such that dS(t)/dt>dS/dt_thresh), and/or the time when the signal first reaches a local maximum derivative (e.g., a minimum t such that argmax_t (dS/dt)), and/or the time when the second derivative of the signal reaches its maximum value (e.g., minimum t such that argmax_t (d2S/dt2)), and/or the time when the tangent lines best-fit to the curve before and after the first initial rise time intersect. Other techniques for determining photosensitivity will be apparent to those skilled in the art, and hence the foregoing are intended as specific examples, and are not to be construed as limiting of the present invention. An optional secondary window can be included on the test cell as noted above, on which different regions of resin can be polymerized with different light doses to different heights, and the heights of each polymerized region measured with a micrometer, to conduct an alternate or comparative test of photosensitivity, if desired. The secondary window in the illustrated embodiment is removable and disposable, and is provided with lifting tabs (43) to facilitate removal and replacement. The alternate photosensitivity test can be conducted in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art. See, e.g., P. Jacobs,Rapid Prototyping&Manufacturing: Fundamentals of StereoLithography, pages 33, 270-274 (1stEd. 1992). FIG.9is a schematic illustration of an apparatus of the present invention. As illustrated inFIG.9, in some embodiments, a test apparatus (e.g., test apparatus (100) discussed herein) may include two chambers (e.g., Chamber A and Chamber B), though the present invention is not limited thereto. A first chamber (e.g., Chamber B) may include the test cell, such as test cell (10) illustrated and discussed with respect toFIGS.1-5. The first chamber may also include sensors, including sensors (20) described herein. The sensors may include, for example, pressure sensors, temperature sensors, and/or humidity sensors, though the present invention is not limited thereto. The first chamber may also include a sample sensor (e.g., sensor (20)), which may, in some embodiments, include thin film resistive sensors (30) such as those illustrated and discussed herein with respect toFIGS.7and8. As discussed herein, a photopolymerizable material (e.g., a resin) may be placed within a test cell of the first chamber to determine a photosensitivity of the photopolymerizable material. The test apparatus may further include a second chamber (e.g., Chamber A) which may include machinery to operate and control the test cell. For example, the second chamber may include an illuminator which may transmit light through one or more optics into the first chamber. In some embodiments, the light may be translated through a transmissive window of a test cell in the first chamber. The second chamber may also include precision sensor drive electronics that are communicatively coupled to the sample sensor, e.g., sample sensor (20), of the first chamber. The sensor drive electronics may be configured to send and/or receive an electronic signal from the sample sensor. In some embodiments, the precision sensor drive electronics may be configured to exchange one or more control signals with the sample sensor so as to assist in the determination of the photosensitivity of the photopolymerizable material. The second chamber may also include data acquisition electronics. The data acquisition electronics may be configured to be communicatively coupled to one or more sensors (e.g., sample sensor (20), pressure, temperature, and/or humidity sensors) within the first chamber and/or second chamber. The data acquisition electronics may receive data from the sensors within the first chamber and/or second chamber to gather data associated with the inventive processes described herein. A temperature controller may be communicatively coupled to the first chamber and/or the second chamber. The temperature controller may be configured to control a temperature and/or relative humidity of the test apparatus. In some embodiments, the temperature controller may be configured to control the temperature and/or humidity based on signals received from one or more sensors in the first and/or the second chamber of the test apparatus. In some embodiments, the temperature controller may be an external air temperature and humidity control unit that may be coupled to the test apparatus, such as through an air inlet duct (121) and/or an air outlet duct (122) as illustrated inFIG.3. The test apparatus may also include a controller and a memory coupled to the controller. The controller may control operation of the test apparatus. For example, the controller may be communicatively coupled to the temperature controller to exchange temperature and/or humidity set points to control temperature and/or humidity of the test apparatus. The controller may be communicatively coupled to the data acquisition electronics to exchange sensor readings and/or timing signals and/or to monitor sensor signals from the first and/or second chambers. The controller may be communicatively coupled to the illuminator to exchange timing signals and/or to provide illumination control (e.g., control of an intensity, wavelength, and/or pattern) for the illuminator. The controller may provide control signals and interpret data responsive thereto, using algorithms described herein, to determine photosensitivity of the photopolymerizable material in the first chamber. The controller may be of any suitable type, such as a general-purpose computer. The memory may include a volatile (or “working”) memory, such as random-access memory, and/or at least one non-volatile or persistent memory, such as a hard drive or a flash drive. The controller may use hardware, software implemented with hardware, firmware, tangible computer-readable storage media having instructions stored thereon, and/or a combination thereof, and may be implemented in one or more computer systems or other processing systems. The controller may also utilize a virtual instance of a computer. As such, the devices and methods described herein may be embodied in any combination of hardware and software that may all generally be referred to herein as a “circuit,” “component,” and/or “system.” Furthermore, aspects of the present invention may take the form of a computer program product embodied in one or more computer readable media having computer readable program code embodied thereon. Any combination of one or more computer readable media may be utilized. The computer readable media may be a computer readable signal medium or a computer readable storage medium. A computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage medium would include the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device. A computer readable signal medium may include a propagated data signal with computer readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such a propagated signal may take any of a variety of forms, including, but not limited to, electro-magnetic, optical, or any suitable combination thereof. A computer readable signal medium may be any computer readable medium that is not a computer readable storage medium and that can communicate, propagate, or transport a program for use by or in connection with an instruction execution system, apparatus, or device. Program code embodied on a computer readable signal medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, RF, etc., or any suitable combination of the foregoing. The controller may be configured to execute computer program code for carrying out operations for aspects of the present invention, which computer program code may be written in any combination of one or more programming languages, including an object oriented programming language such as Java, Scala, Smalltalk, Eiffel, JADE, Emerald, C++, C#, VB.NET, or the like, conventional procedural programming languages, such as the “C” programming language, Visual Basic, Fortran 2003, COBOL 2002, PHP, ABAP, dynamic programming languages such as Python, PERL, Ruby, and Groovy, or other programming languages. The controller may be, or may include, one or more programmable general purpose or special-purpose microprocessors, digital signal processors (DSPs), programmable controllers, application specific integrated circuits (ASICs), programmable logic devices (PLDs), field-programmable gate arrays (FPGAs), trusted platform modules (TPMs), or a combination of such or similar devices, which may be collocated or distributed across one or more data networks. Connections between internal components of the test apparatus are shown only in part and connections between internal components of the test apparatus and external components are not shown for clarity, but are provided by additional components known in the art, such as busses, input/output boards, communication adapters, network adapters, etc. The connections between the internal components of the test apparatus, therefore, may include, for example, a system bus, a Peripheral Component Interconnect (PCI) bus or PCI-Express bus, a HyperTransport or industry standard architecture (ISA) bus, a small computer system interface (SCSI) bus, a universal serial bus (USB), IIC (I2C) bus, an Advanced Technology Attachment (ATA) bus, a Serial ATA (SATA) bus, and/or an Institute of Electrical and Electronics Engineers (IEEE) standard 1394 bus, also called “Firewire.” A user interface may be coupled to the controller. The user interface may be of any suitable type. The user interface may include a display and/or one or more user input devices. The display may be accessible to the controller via the connections between the system components. The display may provide graphical user interfaces for receiving input, displaying intermediate operation/data, and/or exporting output of the methods described herein. The display may include, but is not limited to, a monitor, a touch screen device, etc., including combinations thereof. The input device may include, but is not limited to, a mouse, keyboard, touch screen, stylus, camera, etc., including combinations thereof. The input device may be accessible to the controller via the connections between the system components. The user interface may interface with and/or be operated by computer readable software code instructions resident in the memory that are executed by the controller. ThoughFIG.9illustrates example locations of components within particular chambers with example connections therebetween, it will be understood that other configurations are possible without deviating from the present inventive concepts. For example, component illustrated in one chamber may, in some embodiments, be placed in another chamber, or vice versa. Similarly, though two chambers are illustrated, more, or fewer, chambers are possible. In some embodiments, the controller may be included as part of the test apparatus. In some embodiments, the controller may be remote and may, for example, communicate with the test apparatus with a network or other communication infrastructure (e.g., a wired or wireless network). The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. | 33,828 |
11859028 | DESCRIPTION OF EMBODIMENTS An embodiment of the present invention relates to a method for manufacturing a polymer using a microreactor including a flow path capable of mixing a plurality of liquids to perform radical polymerization of a monomer component containing two or more types of monomers in the presence of a polymerization initiator; wherein the microreactor includes a first inlet port configured to feed the monomer component and an additional inlet port located downstream of the first inlet port; and the method includes feeding the monomer component through the first inlet port and the additional inlet port. Examples of the additional inlet port include an Nth inlet port (N is an integer of two or more), such as a second inlet port, a third inlet port, and a fourth inlet port. In an embodiment of the present invention, the radical polymerization may be performed in the presence of a polymerization initiator and a chain transfer agent. In a flow path located between the first inlet port and the second inlet port, a monomer component fed through the first inlet port undergoes radical polymerization (referred to as a first reaction). In a flow path located downstream of the second inlet port (the flow path located between the second inlet port and the third inlet port in the case that the microreactor includes the third inlet port), a reaction product produced by the first reaction and a monomer component fed through the second inlet port undergo radical polymerization (referred to as a second reaction). In addition, in a flow path located downstream of the third inlet port (the flow path located between the third inlet port and the fourth inlet port in the case that the microreactor includes the fourth inlet port), a reaction product obtained by the second reaction and a monomer component fed through the third inlet port undergo radical polymerization (referred to as a third reaction). In this manner, in a flow path located downstream of the Nth inlet port, a reactant produced in a flow path located between an (N−1)th inlet port and the Nth inlet port and a monomer component fed through the Nth inlet port undergo radical polymerization (referred to as an Nth reaction, where N is the same as described above). The reaction product produced by the first reaction undergoes a polymer chain extension reaction described later, which extends the polymer chain thereof. That is, the reaction product has a role as a nucleus of the polymer. Thus, the first reaction can also be referred to as a “polymer nucleation reaction”. The Nth reaction (the second or later reaction) is mainly a reaction that extends the polymer chain of the reaction product produced by the immediately preceding reaction. Thus, the Nth reaction can also be referred to as a “polymer chain extension reaction”. However, the Nth reaction may include a reaction that forms a new reaction product that can become a polymer nucleus of the polymer, as a reaction other than the reaction that extends the polymer chain of the reaction product produced by the immediately preceding reaction. That is, the monomer component fed through the Nth inlet port need not only be used in the reaction that extends the polymer chain described above but may form a new polymer (polymer nucleus) through radical polymerization with an unreacted monomer component. A polymer is produced by radical polymerization of a monomer component containing two or more types of monomers, but the ratio of the monomer units contained in the polymer varies depending on reaction conditions, such as reactivities and concentrations of the monomers used and time. For example, in a case where the reactivities of the monomers used are greatly different, a polymer formed in the early stage of the polymerization reaction contains high ratio of units derived from a more reactive monomer, and a polymer formed in the late stage of the polymerization reaction contains high ratio of units derived from a less reactive monomer. In this manner, the ratio of the monomer units contained in the polymer differs depending on the reaction conditions, and in turn the ratio of the unreacted monomers also differs. The above situation may also be possible in the first reaction, i.e., the polymer nucleation reaction. Additionally, the polymer chain extension reaction can be controlled such that the polymer of the same composition is produced continuously, by feeding the monomer component in the polymer chain extension reaction to adjust the difference in concentration of each monomer generated in the first reaction. A well-known or commonly used radical polymerization initiator can be used as the polymerization initiator, and examples thereof include a polymerization initiator containing a cyano group and a polymerization initiator containing no cyano group. One type of polymerization initiator may be used alone, or two or more types may be used. Examples of the polymerization initiator containing a cyano group include azo compounds containing a cyano group, such as azobisisobutyronitrile (2,2′-azobis(isobutyronitrile)), 2,2′-azobis(2,4-dimethylvaleronitrile), 2,2′-azobis(2-methylbutyronitrile), and 4,4′-azobis(4-cyanovaleric acid). A well-known or commonly used radical polymerization initiator can be used as the polymerization initiator containing no cyano group, and examples thereof include azo compounds containing no cyano group, such as dimethyl-2,2′-azobisisobutyrate, 2,2′-azobis(2,4,4-trimethylpentane), 2,2′-azobis(2-methylpropane), and dibutyl-2,2′-azobisisobutyrate. Also included are peroxide compounds containing no cyano group; such as ketone peroxides, such as methyl ethyl ketone peroxide and cyclohexanone peroxide; peroxyketals, such as 1,1-bis(tert-hexylperoxy)-3,3,5-trimethylcyclohexane and 1,1-bis(tert-hexylperoxy)cyclohexane; hydroperoxides or dialkyl peroxides, such as p-menthane hydroperoxide and 2,5-dimethyl-2,5-bis(tert-butylperoxy)hexane; diacyl peroxides, such as isobutyryl peroxide and 3,5,5-trimethylhexanoyl peroxide; peroxy esters, such as 1,1,3,3-tetramethylbutylperoxy neodecanate and tert-hexylperoxy neodecanate; and peroxydicarbonates, such as di-n-propyl peroxydicarbonate and diisopropyl peroxydicarbonate. Further included are redox compounds containing no cyano group, such as hydrogen peroxide and ammonium persulfate. A chain transfer agent well known or commonly used in radical polymerization can be used as the chain transfer agent, and examples thereof include chain transfer agents containing a thiocarbonylthio group (chain transfer agents containing a cyano group and a thiocarbonylthio group, and chain transfer agents containing no cyano group and containing a thiocarbonylthio group). One type of chain transfer agent may be used alone, or two or more types may be used. Examples of chain transfer agents containing a cyano group and a thiocarbonylthio group include dithiobenzoate-based chain transfer agents containing a cyano group, such as 2-cyano-2-propyl 4-cyanobenzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 2-cyano-2-propylbenzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester; trithiocarbonate-based chain transfer agents containing a cyano group, such as 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 2-cyano-2-propyl dodecyl trithiocarbonate, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, poly(ethylene glycol)methyl ether 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoate, poly(ethylene glycol)methyl ether(4-cyano-4-pentanoate dodecyl trithiocarbonate), and cyanomethyl dodecyl trithiocarbonate; dithiocarbamate-based chain transfer agents containing a cyano group, such as cyanomethyl methyl(phenyl)carbamodithioate, cyanomethyl diphenylcarbamodithioate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, and cyanomethyl methyl(4-pyridyl)carbamodithioate; and xanthate-based chain transfer agents containing a cyano group. Among them, from viewpoint of molecular weight distribution of the resulting polymer, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 2-cyano-2-propylbenzodithioate, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, and 2-cyano-2-propyl dodecyl trithiocarbonate are preferred. Examples of chain transfer agents containing no cyano group and containing a thiocarbonylthio group include dithiobenzoate-based chain transfer agents containing no cyano group, such as 2-phenyl-2-propyl benzodithioate, 1-(methoxycarbonyl)ethyl benzodithioate, benzyl benzodithioate, ethyl-2-methyl-2-(phenylthiocarbonylthio)propionate, methyl-2-phenyl-2-(phenylcarbonothioylthio)acetate, ethyl-2-(phenylcarbonothioylthio)propionate, and bis(thiobenzoyl)disulfide; trithiocarbonate-based chain transfer agents containing no cyano group, such as 2-(dodecylthiocarbonylthioylthio)propionic acid, 2-(dodecylthiocarbonylthioylthio)-2-methyl propionic acid, methyl-2-(dodecylthiocarbonylthioylthio)-2-methyl propionate, 2-(dodecylthiocarbonylthioylthio)-2-methyl propionic acid N-hydroxy succinimide ester, poly(ethylene glycol)methyl ether(2-methyl-2-propionic acid dodecyl trithiocarbonate), poly(ethylene glycol)bis[2-(dodecylthiocarbonylthioylthio)-2-methyl propionate], 2-(dodecylthiocarbonylthioylthio)-2-methyl propionic acid 3-azido-1-propanol ester, 2-(dodecylthiocarbonylthioylthio)-2-methyl propionic acid pentafluorophenyl ester, poly(ethylene glycol)methyl ether 2-(dodecylthiocarbonylthioylthio)-2-methylpropionate, poly(ethylene glycol)bis[2-(dodecylthiocarbonylthioylthio)-2-methylpropionate], and bis(dodecylsulfanylthiocarbonyl)disulphide; dithiocarbamate-based chain transfer agents containing no cyano group, such as benzyl 1H-pyrrole-1-carbodithioate, methyl 2-propionate methyl(4-pyridinyl)carbamodithioate, and N,N′-dimethyl N,N′-di(4-pyridinyl)thiuram disulfide; and xanthate-based chain transfer agents containing no cyano group. Among them, from the viewpoint of molecular weight distribution of the resulting polymer, ethyl-2-methyl-2-(phenylthiocarbonylthio)propionate is preferred. In an embodiment of the present invention, when a chain transfer agent containing a cyano group and a thiocarbonylthio group is used, a polymer having a cyano group at the terminal (polymer terminal) is obtained. A cyano group exhibits low solubility in solvents (e.g., photoresist solvents), and thus polymers having a cyano group at the terminal tend to have low solubility in solvents. In the polymer, however, the chain transfer agent has a high ability to adjust the degree of polymerization of the polymer, and thus a polymer having a homogeneous copolymer composition and a narrow molecular weight distribution is readily formed. As a result, the polymer readily exhibits high solubility in solvents (e.g., photoresist solvents). On the other hand, when a chain transfer agent containing no cyano group and containing a thiocarbonylthio group is used as the chain transfer agent, and a polymerization initiator containing no cyano group is used as the polymerization initiator, a polymer having no cyano group at the terminal is obtained. Thus, the polymer readily exhibits high solubility in solvents. An embodiment of the present invention may be performed in the absence of a solvent or in the presence of a solvent (polymerization solvent). Examples of the solvent include glycol-based solvents (glycol-based compounds), ester-based solvents, ketone-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, hydrocarbon-based solvents, and mixed solvents thereof. One type of polymerization solvent may be used alone, or two or more types may be used. Examples of the glycol-based solvent include propylene glycol monomethyl ether acetate, ethylene glycol monomethyl ether acetate, ethylene glycol monoethyl ether acetate, and ethylene glycol monobutyl ether acetate. Examples of the ester-based solvent include lactate ester-based solvents, such as ethyl lactate; propionate ester-based solvents, such as methyl 3-methoxypropionate; acetate ester-based solvents, such as methyl acetate, ethyl acetate, propyl acetate, and butyl acetate. Examples of the ketone-based solvent include acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl amyl ketone, cyclopentanone, and cyclohexanone. Examples of the ether-based solvent include linear ethers, such as diethyl ether, diisopropyl ether, dibutyl ether, and dimethoxyethane; and cyclic ethers, such as tetrahydrofuran and dioxane. Examples of the amide-based solvent include N,N-dimethylformamide. Examples of the sulfoxide-based solvent include dimethyl sulfoxide. Examples of the hydrocarbon-based solvent include aliphatic hydrocarbons, such as pentane, hexane, heptane, and octane; alicyclic hydrocarbons, such as cyclohexane and methylcyclohexane; and aromatic hydrocarbons, such as benzene, toluene, and xylene. Among them, glycol-based solvents, such as propylene glycol monomethyl ether and propylene glycol monomethyl ether acetate; ester-based solvents, such as ethyl lactate; ketone-based solvents, such as acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl amyl ketone, cyclopentanone, and cyclohexanone; and mixed solvents thereof are preferably used. Polymer Nucleation Reaction The polymer nucleation reaction, i.e. the first reaction, of an embodiment of the present invention is a reaction in which a monomer component fed through the first inlet port into the microreactor undergoes radical polymerization. The monomer component fed from the first inlet port undergoes a radical polymerization initiated by the polymerization initiator present in the microreactor system to form a reaction product (radical polymer). The method for feeding the monomer component into the microreactor is not particularly limited. Examples thereof include a method for feeding the monomer component into the microreactor via an inlet path (hereinafter, it may be referred to as a “monomer inlet path”). The method for feeding the polymerization initiator into the microreactor is not particularly limited. Examples thereof include method for feeding the polymerization initiator into the microreactor via an inlet path (hereinafter, it may be referred to as a “polymerization initiator inlet path”). The feeding of the polymerization initiator into the microreactor is not particularly limited. Examples thereof include [1] feeding the polymerization initiator before the feeding of the monomer component into the microreactor, [2] feeding of the polymerization initiator simultaneously with the feeding of the monomer component into the microreactor, and [3] feeding of the polymerization initiator after the feeding of the monomer component into the microreactor, and in particular, [2] is preferred in that it enables efficient mixing of the monomer component and the polymerization initiator, and facilitates control of the reaction time and reaction temperature. In the case where the radical polymerization is performed in the presence of a polymerization initiator and a chain transfer agent, the method for feeding the chain transfer agent is the same as that described for the method for feeding the polymerization initiator, and examples thereof include method for feeding the chain transfer agent into the microreactor via an inlet path (hereinafter, it may be referred to as a “chain transfer agent inlet path”). In addition, the inlet path may be the same as the polymerization initiator inlet path. That is, the method may be a method for preparing a solution containing a polymerization initiator and a chain transfer agent in advance, and feeding the solution into the microreactor via an inlet path (hereinafter, it may be referred to as an “inlet path for a polymerization initiator and others”). The use of a chain transfer agent enables control of the polymerization reaction, thereby providing a polymer having a more homogeneous copolymer composition and a narrower molecular weight distribution. The reaction temperature of the present reaction (polymer nucleation reaction), i.e., the temperature of the flow path located between the first inlet port and the second inlet port, is not particularly limited, and can be appropriately selected according to the purpose. For example, it is preferably from 0 to 200° C., more preferably from 20 to 180° C., still more preferably from 40 to 160° C., particularly preferably from 60 to 140° C., and most preferably from 80 to 120° C. The molar concentration of the monomer component (total molar concentration of the monomers) in the present reaction is not particularly limited, and can be appropriately selected according to the purpose, but, for example, it is preferably from 0.01 to 5.0 mol/L, more preferably from 0.05 to 3.0 mol/L, and particularly preferably from 0.1 to 2.0 mol/L. At a concentration within the above range, the reaction readily achieves a good yield of the polymer produced per unit time. On the other hand, at a concentration higher than 5.0 mol/L, the reaction has problems that the viscosity of the reaction solution increases or the monomer component does not dissolve. In addition, at a concentration less than 0.01 mol/L, the reaction has a problem that the reaction rate decreases and the amount of the polymer produced decreases (the reaction does not proceed sufficiently). The concentration above refers to the molar concentration of the monomer component in the flow path immediately after fed into the microreactor. The flow rate of the reaction solution in the present reaction is not particularly limited, and can be appropriately selected according to the purpose, but, for example, it is preferably from 0.001 to 10 mL/min, more preferably from 0.005 to 3 mL/min, and particularly preferably from 0.01 to 1 mL/min. At a flow rate within the above range, the rapid mixing of the monomer component and the polymerization initiator are readily achieved, and further, the pressure loss tends to be prevented. The flow rate above refers to the flow rate of the reaction solution in the flow path immediately after fed into the microreactor. The residence time in the present reaction is not particularly limited, and can be appropriately selected according to the purpose, but, for example, it is preferably from 1 to 180 min, more preferably from 5 to 120 min, and particularly preferably from 8 to 90 min. At a residence time within the above range, the resulting polymer tends to have a narrower molecular weight distribution. The concentration of the polymerization initiator in the present reaction is not particularly limited, and can be appropriately selected according to the composition and concentration of the monomer component, but, for example, it is preferably from 0.0001 to 3.0 mol/L, more preferably from 0.0005 to 1.5 mol/L, still more preferably from 0.001 to 0.5 mol/L, and particularly preferably from 0.005 to 0.2 mol/L. At a concentration within the above range, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentration of the polymerization initiator in the flow path immediately after fed into the microreactor. The concentration of the chain transfer agent in the present reaction is not particularly limited, and can be appropriately selected according to the composition and concentration of the monomer component, but, for example it is preferably from 0.0001 to 3.0 mol/L, more preferably from 0.0005 to 1.5 mol/L, still more preferably from 0.001 to 0.5 mol/L, and particularly preferably from 0.005 to 0.2 mol/L. At a concentration within the above range, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentration of the chain transfer agent in the flow path immediately after fed into the microreactor. The total concentration of the polymerization initiator and the chain transfer agent in the present reaction is not particularly limited, and can be appropriately selected according to the composition and concentration of the monomer component, but, for example, it is preferably from 0.0001 to 3.0 mol/L, more preferably from 0.0005 to 1.5 mol/L, still more preferably from 0.001 to 0.5 mol/L, and particularly preferably from 0.005 to 0.2 mol/L. At a concentration within the above range, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentration in the flow path immediately after the agents are fed into the microreactor. The concentration ratio of the polymerization initiator and the chain transfer agent to the monomers (polymerization initiator and chain transfer agent/monomers) in an embodiment of the present invention is not particularly limited, but, for example, it is preferably from 0.001 to 100.0 mol %, more preferably from 0.01 to 50.0 mol %, still more preferably from 0.05 to 30.0 mol %, and particularly preferably from 0.1 to 20.0 mol %. At a concentration ratio within the above range, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentrations of the polymerization initiator and the chain transfer agent in the flow path immediately after fed into the microreactor. Polymer Chain Extension Reaction The polymer chain extension reaction, i.e., the Nth reaction (N is an integer of 2 or greater), is a reaction in which, in a flow path located downstream of the Nth inlet port, a reaction product produced in a flow path located between an (N−1)th inlet port and the Nth inlet port and a monomer component fed through the Nth inlet port undergo radical polymerization. The method for feeding the monomer component into the microreactor is not particularly limited. Examples thereof include a method for feeding the monomer component into the microreactor via an inlet path. For example, the second reaction is a reaction in which, in a flow path located downstream of the second inlet port (a flow path located between the second inlet port and the third inlet port in the case that the microreactor includes the third inlet port), a reaction product produced in a flow path located between the first inlet port and the second inlet port and a monomer component fed through the second inlet port undergo radical polymerization. In addition, examples of the method for feeding the monomer component into the microreactor include a method for feeding the monomer component into the microreactor via an inlet path. The monomer component to be fed through the first inlet port and the additional inlet port preferably contains the same monomers; more preferably, the same monomers are contained therein and a difference between the contents of each monomer is within ±5%; still more preferably, the same monomers are contained therein and the difference between the contents of each monomer is within ±1%; and particularly preferably, the same monomers are contained therein in the same amount (substantially the same). The “difference between the contents of each monomer” means a difference between a content (wt. %) of a particular monomer contained in a monomer component to be fed through the additional inlet port and a content (wt. %) of the particular monomer contained in a monomer component to be fed through the first inlet port (wt. %). For example, in a case where the content of a monomer A contained in a monomer component to be fed through the first inlet port is 50 wt. %, and the content of the monomer A contained in a monomer component to be fed to the additional inlet port is 51 wt. %, the difference (%) is (51/50−1)×100=2%. In addition, “a difference between the contents of each monomer is within ±5%” means that a difference between the contents of each monomer is within ±5% in all the monomers contained in the monomer component. For example, it means that when the monomer component contains a monomer A and a monomer B, the difference between the contents of the monomer A is within ±5% and the difference between the contents of the monomer B is within ±5%. The reaction temperature of the present reaction (polymer chain extension reaction), i.e., the temperature of the flow path located downstream of the Nth inlet port (e.g., between the second inlet port and the third inlet port), is not particularly limited, and can be appropriately selected according to the purpose. For example, it is preferably from 0 to 200° C., more preferably from 20 to 180° C., still more preferably from 40 to 160° C., particularly preferably from 60 to 140° C., and most preferably from 80 to 120° C. The reaction temperature of the present reaction may be the same as the reaction temperature of the polymer nucleation reaction. The molar concentration of the monomer component (total molar concentration of the monomer) in the present reaction is not particularly limited, and can be appropriately selected according to the purpose, but, for example, it is preferably from 0.01 to 5.0 mol/L, more preferably from 0.05 to 3.0 mol/L, and particularly preferably from 0.1 to 2.0 mol/L. At a concentration within the range described above, the reaction readily achieves a good yield of the polymer produced per unit time. On the other hand, at a concentration higher than 5.0 mol/L, the reaction has problems that the viscosity of the reaction solution increases and the monomer component does not dissolve. Alternatively, at a concentration less than 0.01 mol/L, the reaction has a problem that the reaction rate decreases and the amount of the polymer produced decreases (the reaction does not proceed sufficiently). The molar concentration above refers to the molar concentration of the monomer component in the flow path immediately after fed into the microreactor. The flow rate of the reaction solution in the present reaction is not particularly limited, and can be appropriately selected according to the purpose. For example, it is preferably from 0.001 to 10 mL/min, more preferably from 0.005 to 3 mL/min, and particularly preferably from 0.01 to 1 mL/min. At a flow rate within the range described above, the rapid mixing of the monomer component and the polymerization initiator can be achieved, and further, the pressure loss tends to be prevented. The flow rate described above refers to the flow rate of the reaction solution in the flow path immediately after fed into the microreactor. The residence time in the present reaction is not particularly limited, and can be appropriately selected according to the purpose, but, for example, it is preferably from 1 to 180 min, more preferably from 5 to 120 min, and particularly preferably from 8 to 90 min. At a residence time within the above range, the resulting polymer tends to have a narrower molecular weight distribution. The concentration of the polymerization initiator in the present reaction is not particularly limited, and can be appropriately selected according to the composition and concentration of the monomer component. For example, it is preferably from 0.0001 to 3.0 mol/L, more preferably from 0.0005 to 1.5 mol/L, still more preferably from 0.001 to 0.5 mol/L, and particularly preferably from 0.005 to 0.2 mol/L. At a concentration within the range described above, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentration of the polymerization initiator in the flow path immediately after fed into the microreactor. The concentration of the chain transfer agent in the present reaction is not particularly limited, and can be appropriately selected according to the composition and concentration of the monomer component. For example, it is preferably from 0.0001 to 3.0 mol/L, more preferably from 0.0005 to 1.5 mol/L, still more preferably from 0.001 to 0.5 mol/L, and particularly preferably from 0.005 to 0.2 mol/L. At a concentration within the range described above, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentration of the chain transfer agent in the flow path immediately after fed into the microreactor. The total concentration of the polymerization initiator and the chain transfer agent in the present reaction is not particularly limited, and can be appropriately selected according to the composition and concentration of the monomer component. For example, it is preferably from 0.0001 to 1.0 mol/L, more preferably from 0.0003 to 0.5 mol/L, still more preferably from 0.0005 to 0.3 mol/L, and particularly preferably from 0.001 to 0.1 mol/L. At a concentration within the range described above, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration above refers to the concentrations of the polymerization initiator and the chain transfer agent in the flow path immediately after fed into the microreactor. The concentration ratio of the polymerization initiator and the chain transfer agent to the monomers (polymerization initiator and chain transfer agent/monomers) in the present reaction is not particularly limited. For example, it is preferably from 0.001 to 100.0 mol %, more preferably from 0.01 to 50.0 mol %, still more preferably from 0.05 to 30.0 mol %, and particularly preferably from 0.1 to 20.0 mol %. At a concentration ratio within the range described above, blockage of the flow path of the microreactor can be prevented, and the resulting polymer tends to have a narrower molecular weight distribution. The concentration ratio above refers to the concentration ratio of the polymerization initiator and the chain transfer agent to the monomers in the flow path immediately after fed into the microreactor. The method for collecting the polymer is not particularly limited. Examples thereof include a method using precipitation (including re-precipitation). For example, the target polymer can be obtained by adding the reaction solution to a solvent (precipitation solvent) to precipitate the polymer; or by dissolving the polymer again in an appropriate solvent, and adding the solution to a solvent (a re-precipitation solvent) to re-precipitate the polymer; or alternatively by adding a solvent (a re-precipitation solvent or a polymerization solvent) to the reaction solution to dilute the reaction solution. The precipitation or re-precipitation solvent may be either an organic solvent or water and may be a mixed solvent. The precipitation or re-precipitation solvent is not particularly limited, and may be the same solvent as the polymerization solvent or a different solvent. Examples of the precipitation or re-precipitation solvent include organic solvents exemplified as polymerization solvents (glycol-based solvents, ester-based solvents, ketone-based solvents, ether-based solvents, amide-based solvents, sulfoxide-based solvents, and hydrocarbon-based solvents); halogenated hydrocarbons (halogenated aliphatic hydrocarbons, such as methylene chloride, chloroform, and carbon tetrachloride; halogenated aromatic hydrocarbons, such as chlorobenzene and dichlorobenzene); nitro compounds (such as nitromethane and nitroethane); nitriles (such as acetonitrile and benzonitrile); carbonates (such as dimethyl carbonate, diethyl carbonate, ethylene carbonate, and propylene carbonate); carboxylic acids (such as acetic acid); and mixed solvents containing these solvents. Among them, the precipitation or re-precipitation solvent is preferably a solvent containing at least a hydrocarbon (in particular, an aliphatic hydrocarbon, such as hexane and heptane) or an alcohol (in particular, such as methanol, ethanol, propanol, isopropyl alcohol, and butanol). In such a solvent containing at least a hydrocarbon, as a ratio of a hydrocarbon (e.g., an aliphatic hydrocarbon, such as hexane and heptane) and an additional solvent (e.g., esters, such as ethyl acetate), the hydrocarbon/the additional solvent (volume ratio at 25° C.), is from 10/90 to 99/1, preferably the hydrocarbon/the additional solvent (volume ratio at 25° C.) is from 30/70 to 98/2, more preferably the hydrocarbon/the additional solvent (volume ratio at 25° C.) is 50/50 to 97/3. Also preferred as the precipitation or re-precipitation solvent are a mixed solvent of an alcohol (in particular, methanol) and water, and a mixed solvent of a glycol-based solvent (in particular, polyethylene glycol) and water. In such a solvent, as a ratio (volume ratio at 25° C.) of the organic solvent (an alcohol or a glycol-based solvent) to water, for example, the organic solvent/water (volume ratio at 25° C.) is from 10/90 to 99/1, preferably the organic solvent/water (volume ratio at 25° C.) is from 30/70 to 98/2, and more preferably the organic solvent/water (volume ratio at 25° C.) is from 50/50 to 97/3. The polymer obtained by precipitation (including re-precipitation) is subjected to rinsing or a process of washing by adding the polymer to a solvent under stirring to disperse the polymer in the solvent (sometimes referred to as “repulping”), as necessary. The polymer may be subjected to rinsing after repulping. Materials adhered to the polymer, such as a residual monomer and a low molecular weight oligomer, can be efficiently removed by repulping or rinsing the polymer produced by polymerization with a solvent. In an embodiment of the present invention, a repulping or rinsing solvent is preferably a solvent containing, among others, at least a hydrocarbon (in particular, an aliphatic hydrocarbon, such as hexane and heptane), an alcohol (in particular, such as methanol, ethanol, propanol, isopropyl alcohol, and butanol), or esters (in particular, such as ethyl acetate). After the precipitation (including re-precipitation), repulping, or rinsing, for example, the solvent may be removed as necessary by decantation, filtration, or the like, and drying treatment may be performed. Microreactor As the microreactor, a microreactor including a flow path capable of mixing a plurality of liquids can be used. The microreactor includes a plurality of inlet ports at different locations in the flow path along the flow, and includes at least an inlet port (a first inlet port) configured to feed a monomer component into the microreactor, and an additional inlet port (an Nth inlet port) configured to feed a monomer component into the microreactor, the additional inlet port being located downstream of the first inlet port. The microreactor may include one or two or more of the additional inlet ports. In addition, as necessary, the microreactor may include an inlet path, that communicates with the flow path, configured to feed a liquid into the flow path through the first inlet port or the additional inlet port. Examples of the inlet path include the polymerization initiator inlet path, the chain transfer agent inlet path, the inlet path for polymerization initiator and others, and the monomer inlet path, as described above. When the microreactor includes an inlet path, the inlet port (e.g., the first inlet port and the second inlet port) method a confluence section of the inlet path and the flow path. In addition, as necessary, the microreactor may further include a configuration other than the flow path, the inlet port, and the inlet path. The cross-sectional shape of the flow path is not particularly limited, and can be appropriately selected according to the purpose. Examples thereof include a circle, a rectangle, a semicircle, and a triangle. The microreactor may include an inlet port configured to feed the polymerization initiator into the microreactor at a location upstream of the first inlet port, at the same location as the first inlet port, or at a location downstream of the first inlet port (between the first inlet port and the second inlet port). From the viewpoint of keeping the concentration of the polymerization initiator in the flow path constant, the microreactor includes an inlet port configured to feed the polymerization initiator into the microreactor preferably at the same location as the first inlet port. Similarly, when using the chain transfer agent, the microreactor may include an inlet port configured to feed the chain transfer agent into the microreactor at a location upstream of the first inlet port, at the same location as the first inlet port, or at a location downstream of the first inlet port, and the microreactor includes an inlet port configured to feed the chain transfer agent into the microreactor preferably at the same location as the first inlet port. The microreactor may or may not include an inlet port configured to feed the polymerization initiator into the microreactor at the same location as the Nth inlet port (e.g., the second inlet port) or at a location downstream of the Nth inlet port. When feeding the polymerization initiator into the microreactor, from the viewpoint of keeping the concentration of the polymerization initiator in the flow path constant, the microreactor includes an inlet port configured to feed the polymerization initiator into the microreactor preferably at the same location as the Nth inlet port. Similarly, when using the chain transfer agent, the microreactor includes an inlet port configured to feed the chain transfer agent into the microreactor at the same location as the Nth inlet port or at a location downstream of the Nth inlet port, and the microreactor includes an inlet port configured to feed the chain transfer agent into the microreactor preferably at the same location as the Nth inlet port. The microreactor is not particularly limited as long as it includes a flow path capable of mixing a plurality of liquids, and can be appropriately selected according to the purpose, and examples thereof include micromixers (such as substrate type micromixers and pipe joint type micromixers) and branched tubes. A substrate type micromixer includes a substrate having a flow path formed in the interior or on the surface of the substrate and sometimes may be referred to as a microchannel. The substrate type micromixer is not particularly limited as long as it does not impair the effect of an embodiment of the present invention, and can be appropriately selected according to the purpose. Examples thereof include a mixer having a fine flow path for mixing described in Pamphlet of WO 96/30113; and a mixer described in a literature “Microreactors” Chapter 3, by W. Ehrfeld, V. Hessel, and H. Lowe, published by Wiley-VCH. In addition to the flow path, an inlet path, in communication with the flow path, configured to feed a plurality of liquids into the flow path is preferably formed in the substrate type micromixer. That is, the substrate type micromixer is preferably configured to branch the flow path at the upstream side according to the number of the inlet path. The number of the inlet path is not particularly limited, and can be appropriately selected according to the purpose. Preferably, a plurality of liquids desired to be mixed are fed from separate inlet paths and combined and mixed in a flow path. In addition, the substrate type micromixer may be configured such that one liquid is charged in the flow path in advance and an additional liquid is fed thereto through the inlet path. The pipe joint type micromixer includes a flow path formed in the interior thereof, and, as necessary, a connecting part for connecting the flow path formed in the interior of the pipe joint type micromixer and a tube. The connection method of the connecting part is not particularly limited, and can be appropriately selected, according to the purpose, from well-known tube connection methods. Examples thereof include screw-in type, union type, butt welding type, slip-on welding type, socket welding type, flange type, flareless type, flare type, and mechanical type. In addition to the flow path, an inlet path, in communication with the flow path, configured to feed a plurality of liquids into the flow path is preferably formed in the interior of the pipe joint type micromixer. That is, the pipe joint type micromixer is preferably configured to branch the flow path at the upstream side according to the number of the inlet path. In a case where the microreactor includes two inlet paths, for example, a T-shaped or Y-shaped pipe joint configuration can be used in the pipe joint type micromixer, and in a case where the microreactor includes three inlet paths, for example, a cross-shaped pipe joint configuration can be used. Alternatively, the pipe joint type micromixer may be configured such that one liquid is charged in the flow path in advance and an additional liquid is fed thereto through the inlet path. The material of the micromixer (e.g., the flow path) is not particularly limited, and can be appropriately selected according to requirements, such as heat resistance, pressure resistance, solvent resistance, and ease of processing. Examples thereof include stainless steel, titanium, copper, nickel, aluminum, and silicon; and fluororesins, such as Teflon (trade name) and perfluoroalkoxy resin (PFA); trifluoroacetamide (TFAA), and polytetrafluoroethylene (PTFE). The micromixer precisely controls the flow of the reaction solution by its microstructure, and thus it is preferably fabricated by a microfabrication technique. The microfabrication technique is not particularly limited, and can be selected appropriately according to the purpose. Examples thereof include (a) LIGA technology, which combines X-ray lithography and electroplating, (b) high aspect ratio photolithography method using EPON SUB, (c) mechanical micromachining processing (such as micro-drilling process employing a high-speed drilling machine equipped with a micrometer-order drill bit), (d) high aspect ratio processing of silicon by Deep RIE, (e) Hot Emboss processing, (f) optical fabrication method, (g) laser processing method, and (h) ion beam method. A commercially available product can be used as the micromixer, and examples thereof include a microreactor equipped with an interdigital channel structure; a single mixer and a caterpillar mixer available from Institut für Mikrotechnik Mainz (IMM); a microglass reactor available from Microglass Inc.; Cytos available from CPC Systems, Inc.; a YM-1 type mixer and a YM-2 type mixer available from Yamatake Corporation; a mixing tee and a tee (T-connector) available from Shimadzu GLC Ltd.; an IMT chip reactor available from Institute of Microchemical Technology Co., Ltd., Micro High Mixer developed by Toray Engineering Co., Ltd.; and a union tee available from Swagelok Company. The microreactor may be configured such that the micromixer is used alone, or a tube reactor is further connected downstream of the micromixer to extend the flow path. The length of the flow path can be adjusted by connecting the tube reactor downstream of the micromixer. The residence time (reaction time) of the mixed liquid is proportional to the length of the flow path. The tube reactor is configured to precisely control the time (control the residence time) required for a subsequent reaction of the solution that has been rapidly mixed by the micromixer to be performed. The tube reactor is not particularly limited, and, for example, the configuration such as the tube inner diameter, outer diameter, length, and material thereof, can be appropriately selected according to a desired reaction. A commercially available product can be used as the tube reactor. The material of the tube reactor is not particularly limited, and materials exemplified as the material of the micromixer can be suitably used. The flow path has a function of mixing a plurality of liquids by diffusion and a function of removing the heat of reaction. The mixing method of the liquid in the flow path is not particularly limited, and can be appropriately selected according to the purpose. Examples thereof include laminar flow mixing and turbulent flow mixing. Among them, laminar flow mixing (static mixing) can preferably facilitate the efficient reaction control and heat removal. Note that the flow path of the microreactor is so minute that the plurality of liquids fed from the inlet paths naturally and readily form a laminar flow-dominant flow, and diffuse in a direction orthogonal to the flow to be mixed. The laminar flow mixing may be configured such that the laminar flow cross-section of the flowing liquid is divided by further providing a branching point and a confluence point in the flow path, and may be configured to increase the mixing speed. On the other hand, in a case where turbulent flow mixing (dynamic mixing) is performed in the flow path of the microreactor, the flow can be changed from laminar flow to turbulent flow by adjusting the flow rate and the shape of the flow path (the three-dimensional shape of the liquid contact portion; shapes, such as the bending, of the flow path; roughness of the wall surface; and the like). The turbulent flow mixing has an advantage of good mixing efficiency and high mixing speed compared to the laminar flow mixing. Here, a flow path having a smaller inner diameter can shorten the diffusion distance of the molecule, and in turn reduce the time required for mixing and improve the mixing efficiency. Furthermore, the ratio of the surface area to the volume becomes large, and, for example, this facilitates temperature control, such as removal of the heat of reaction. On the other hand, a flow path with too small inner diameter increases pressure drop in the liquid flow, and requires a special high pressure-resistant pump to be used for pumping liquid, and this may result in a high manufacturing cost. This also increases the tendency for blockage of the flow path due to the reaction product. Furthermore, the pumping flow rate is limited, and thus the structure of the micromixer may be limited. The inner diameter of the flow path is not particularly limited as long as it does not impair the effect of an embodiment of the present invention, and can be appropriately selected according to the purpose. For example, it is preferably from 50 μm to 15 mm, more preferably from 100 μm to 10 mm, still more preferably from 200 μm to 5 mm, and particularly preferably from 500 μm to 3 mm. A flow path with an inner diameter less than 50 μm may increase the pressure drop. A flow path with an inner diameter greater than 15 mm has a smaller surface area per unit volume, and as a result, may have difficulties in rapid mixing and removal of the heat of reaction. On the other hand, a flow path with an inner diameter within the above range achieves rapid mixing of a monomer component and a polymerization initiator (and a chain transfer agent) fed thereto, and can efficiently remove the heat of reaction, and thus tends to facilitate control of the heat of reaction. The cross-sectional area of the flow path is not particularly limited, and can be appropriately selected according to the purpose. For example, it is preferably from 5000 μm2to 800 mm2, and more preferably from 0.75 mm2to 30 mm2. A flow path with a cross-sectional area within the range described above achieves rapid mixing of a monomer component and a polymerization initiator (and a chain transfer agent) fed thereto, and can efficiently remove the heat of reaction, and thus tends to facilitate control of the heat of reaction. The length (total length) of the flow path in the microreactor is not particularly limited, and can be appropriately adjusted according to the optimal reaction time. For example, it is preferably from 0.5 to 500 m, and more preferably from 1 to 400 m. The length of the flow path in the polymer nucleation reaction (the first reaction) is, for example, preferably from 0.1 to 125 m, more preferably from 0.3 to 100 m, and still more preferably from 0.5 to 80 m. The length of the flow path in the polymer chain extension reaction (e.g., the second reaction) is, for example, preferably from 0.1 to 125 m, more preferably from 0.3 to 100 m, and still more preferably from 0.5 to 80 m. The inlet path is in communication with the flow path and has a function of feeding a plurality of liquids into the flow path. Another end in the inlet path opposite from the side in communication with the flow path is usually connected to a container containing a liquid desired to be mixed. The inner diameter of the inlet path is not particularly limited as long as it does not impair the effect of an embodiment of the present invention, and can be appropriately selected according to the purpose. For example, it is preferably from 50 μm to 15 mm, more preferably from 100 μm to 10 mm, still more preferably from 200 μm to 5 mm, and particularly preferably from 500 μm to 3 mm. When the microreactor has a plurality of inlet paths, the inner diameter of each inlet path may be the same or different from each other. The configuration other than the flow path and the inlet path is not particularly limited, and can be appropriately selected according to the purpose. Examples thereof include a pump used for pumping liquid, a temperature control means, a reaction promoting means, a sensor, and a tank for storing a manufactured polymer. The pump is not particularly limited, and can be appropriately selected from those that can be used industrially, and it is preferably a pump that does not cause pulsation during pumping liquid, and examples thereof include a plunger pump, a gear pump, a rotary pump, and a diaphragm pump. The temperature control method is not particularly limited, and can be appropriately selected according to the reaction temperature. Examples thereof include a constant temperature bath, a circulator, and a heat exchanger. For example, in a case where the reaction temperature is 80° C., an oil bath is preferably used. In addition, in a case where cooling is performed to collect the obtained polymer, a constant temperature layer filled with water or ice water is preferably used. The reaction promoting method can be appropriately selected according to a liquid to be mixed and a desired reaction, and examples thereof include a method for imparting vibration energy, a heating method, a light irradiation method, and a voltage application method. Examples of the microreactor including a voltage application method include a microflow electrochemical reactor disclosed in JP 2006-104538 A. The sensor is not particularly limited, and examples thereof include a temperature sensor, a flow rate sensor, and a pressure sensor for measuring pressure in the flow path. Monomer Component The monomer component of an embodiment of the present invention contains two or more types of monomers. Examples of the monomer constituting the monomer component include (meth)acrylic-based monomers, aromatic vinyl monomers, carboxylic acid vinyl esters, conjugated diene-based monomers, olefin-based monomers, vinyl halides, and vinylidene halides, and from the viewpoint of reactivity thereof, the monomer is preferably a (meth)acrylic-based monomer. That is, the monomer component to be fed through the first inlet port and the additional inlet port preferably contains two or more types of (meth)acrylic-based monomers. Examples of the (meth)acrylic-based monomer include (meth)acrylic acid; alkyl (meth)acrylates, such as methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, butyl (meth)acrylate, t-butyl (meth)acrylate, pentyl (meth)acrylate, and hexyl (meth)acrylate; cycloalkyl (meth)acrylates, such as 1-methylcyclopentyl (meth)acrylate, 1-ethylcyclopentyl (meth)acrylate, 1-isopropylcyclopentyl (meth)acrylate, 1-propylcyclopentyl (meth)acrylate, 1-methylcyclohexyl (meth)acrylate, 1-ethylcyclohexyl (meth)acrylate, 1-isopropylcyclohexyl (meth)acrylate, and 1-propylcyclohexyl (meth)acrylate; (meth)acrylates having a cyclic ester group, such as γ-butyrolactone (meth)acrylate; (meth)acrylates having a cyclic ether group, such as 3,4-epoxycyclohexyl (meth)acrylate, glycidyl (meth)acrylate, β-methylglycidyl acrylate, and oxetanyl (meth)acrylate; (meth)acrylic esters having a hydroxyl group, such as 2-hydroxyethyl (meth)acrylate, hydroxypropyl (meth)acrylate, hydroxybutyl (meth)acrylate, and caprolactone-modified 2-hydroxyethyl (meth)acrylate; and ethylene glycol (meth)acrylates, such as methoxy diethylene glycol (meth)acrylate, ethoxy diethylene glycol (meth)acrylate, isooctyloxy diethylene glycol (meth)acrylate, phenoxy triethylene glycol (meth)acrylate, methoxy triethylene glycol (meth)acrylate, and methoxy polyethylene glycol (meth)acrylate. In addition to the above, monomers explained for the photoresist resin described later are exemplified. Examples of the aromatic vinyl monomer include styrene; alkyl styrenes (such as vinyltoluenes, such as o-, m-, and p-methylstyrenes; vinylxylenes, such as 2,4-dimethylstyrene; p-ethylstyrene; p-isopropylstyrene; p-butylstyrene; and p-t-butylstyrene); α-alkylstyrene (such as α-methylstyrene and α-methyl-p-methylstyrene); alkoxystyrene (such as o-, m-, and p-methoxystyrene, and p-t-butoxystyrene); halostyrene (such as o-, m-, and p-chlorostyrene, and p-bromostyrene); and styrenesulfonic acid and alkali metal salts thereof. Examples of the carboxylic acid vinyl ester include C1-10carboxylic acid vinyl esters, such as vinyl formate, vinyl acetate, vinyl propionate, and vinyl pivalate. Examples of the conjugated diene-based monomer include C4-16dienes, such as butadiene, isoprene, chloroprene, neoprene, 1,3-pentadiene, 2,3-dimethyl-1,3-butadiene, piperine, 3-butyl-1,3-octadiene, and phenyl-1,3-butadiene. Examples of the olefin monomer include C2-10alkenes, such as ethylene, propylene, and butene (such as isobutene). Examples of the vinyl halide include vinyl fluoride, vinyl chloride, and vinyl bromide. Examples of the vinylidene halide include vinylidene fluoride, vinylidene chloride, and vinylidene bromide. An embodiment of the present invention can provide a polymer having a homogeneous copolymer composition and a narrow molecular weight distribution, and thus is particularly suited for manufacturing a photoresist resin. Thus, the monomer component preferably contains a monomer having a group whose portion is eliminated by the action of an acid to form a polar group (it may be referred to as an “acid-degradable group”). As a result, the polymer (photoresist resin) exhibits increased polarity due to the action of the acid, and in turn has increased solubility in an alkaline developer. Examples of the polar group include acidic groups, such as phenolic hydroxyl groups, carboxy groups, fluorinated alcohol groups (preferably a hexafluoroisopropanol group), sulfonic acid groups, sulfonamido groups, sulfonylimide groups, (alkylsulfonyl)(alkylcarbonyl)methylene groups, (alkylsulfonyl)(alkylcarbonyl)imide groups, bis(alkylcarbonyl)methylene groups, bis(alkylcarbonyl)imide groups, bis(alkylsulfonyl)methylene groups, bis(alkylsulfonyl)imide groups, tris(alkylcarbonyl)methylene groups, and tris(alkylsulfonyl)methylene groups; and alcoholic hydroxyl groups. Among them, carboxy groups, fluorinated alcohol group (preferably a hexafluoroisopropanol group), and sulfonate groups are preferred. The acid-degradable group is preferably a group in which a hydrogen atom of the polar group has been substituted with a group to be eliminated by an acid. Examples of the acid-degradable group include —C(RI)(RII)(RIII) and —C(RIV)(RV)(ORVI). In the formula, RIto RIII, and RVIeach independently represent an alkyl group, a cycloalkyl group, an aryl group, an aralkyl group, or an alkenyl group. RIVand RVeach independently represent a hydrogen atom, an alkyl group, a cycloalkyl group, an aryl group, an aralkyl group, or an alkenyl group. At least two groups of RIto RIIImay be bonded to each other to form a ring. In addition, RIVand RVmay be bonded to each other to form a ring. The number of carbon atoms in the acid-degradable group is not particularly limited, but is preferably 4 or greater, and more preferably 5 or greater. The upper limit of the number of carbon atoms is not particularly limited, but is preferably 20. The alkyl group of the RIto RVIis preferably an alkyl group having from 1 to 8 carbons, and examples thereof include a methyl group, an ethyl group, a propyl group, a n-butyl group, a s-butyl group, a t-butyl group, a hexyl group, and an octyl group. The cycloalkyl group of the RIto RVImay be a monocyclic hydrocarbon group or a polycyclic (bridged cyclic) hydrocarbon group. The monocyclic hydrocarbon group is preferably a cycloalkyl group having from 3 to 8 carbon atoms, and examples thereof include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, and a cyclooctyl group. The polycyclic hydrocarbon group is preferably a cycloalkyl group having from 6 to 20 carbon atoms, and examples thereof include an adamantyl group, a norbornyl group, an isoboronyl group, a camphanyl group, a dicyclopentyl group, an α-pinel group, a tricyclodecanyl group, a tetracyclododecyl group, and an androstanyl group. At least one carbon atom in the cycloalkyl group may be substituted with a hetero atom, such as an oxygen atom. The aryl group of the RIto RVIis preferably an aryl group having from 6 to 14 carbons, and examples thereof include a phenyl group, a naphthyl group, and an anthryl group. The aralkyl group of the RIto RVIis preferably an aralkyl group having from 7 to 12 carbons, and examples thereof include a benzyl group, a phenethyl group, and a naphthylmethyl group. The alkenyl group of the RIto RVIis preferably an alkenyl group having from 2 to 8 carbons, and examples thereof include a vinyl group, an allyl group, a butenyl group, and a cyclohexenyl group. A ring formed by the at least two groups of the RIto RIIIbonded to each other, and a ring formed by RIVand RVbonded to each other are preferably a cycloalkane ring. Examples of the cycloalkane ring include monocyclic cycloalkane rings, such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, and a cyclohexane ring; and polycyclic cycloalkane rings, such as a norbornane ring, a tricyclodecane ring, a tetracyclododecane ring, and an adamantane ring. The alkyl group, the cycloalkyl group, the aryl group, the aralkyl group, the alkenyl group, and the cycloalkane ring in RIto RVImay each have a substituent. The acid-degradable group is preferably, among others, a t-butyl group, t-amyl group, and groups represented by Formulas (I) to (IV) below. R2to R7, Ra, n, p, and a ring Z1in Formulas (I) to (IV) above are respectively the same as R2to R7, Ra, n, p, and a ring Z1in Formulas (a1) to (a4) described later. The acid-degradable group may be provided via a spacer. The spacer is the same as a linking group exemplified and described as A in Formula (1) described later. Examples of the monomer having an acid-degradable group include monomers represented by Formula (1) below. In Formula (1) above, R1represents the acid-degradable group. In Formula (1) above, R represents a hydrogen atom, a halogen atom, or an alkyl group that has from 1 to 6 carbons and may have a halogen atom. Examples of the halogen atom include a chlorine atom, a bromine atom, and an iodine atom. Examples of the alkyl group having from 1 to 6 carbons include a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, an iso-butyl group, a s-butyl group, a t-butyl group, a pentyl group, an isoamyl group, a s-amyl group, a t-amyl group, a hexyl group. Examples of the alkyl group having from 1 to 6 carbons and having a halogen atom include a group (halo (C1-6) alkyl group) in which one or two or more of hydrogen atoms constituting the alkyl group have been substituted with halogen atoms, such as trifluoromethyl and 2,2,2-trifluoroethyl groups. In Formula (1) above, A represents a single bond or a linking group. Examples of the linking group include a carbonyl group (—C(═O)—), an ether bond (—O—), an ester bond (—C(═O)—O—), an amide bond (—C(═O)—NH—), a carbonate bond (—O—C(═O)—O—), a group in which a plurality of these groups are linked, and a group in which an alkylene group and these groups are bonded. Examples of the alkylene group include linear or branched alkylene groups, such as a methylene group, a methylmethylene group, a dimethylmethylene group, an ethylene group, a propylene group, and a trimethylene group; and divalent alicyclic hydrocarbon groups (in particular, divalent cycloalkylene groups), such as a 1,2-cyclopentylene group, a 1,3-cyclopentylene group, a cyclopentylidene group, a 1,2-cyclohexylene group, a 1,3-cyclohexylene group, a 1,4-cyclohexylene group, and a cyclohexylidene group. The monomer represented by Formula (1) above is preferably, among others, at least one type of monomer selected from the group consisting of monomers represented by Formulas (a1) to (a4) below. “At least one type of monomer selected from the group consisting of monomers represented by Formulas (a1) to (a4)” may be referred to as a “monomer a”. In the monomers represented by Formulas (a1) to (a4) above, R represents a hydrogen atom, a halogen atom, or an alkyl group that has from 1 to 6 carbons and may have a halogen atom, similarly to R in Formula (1), and A represents a single bond or a linking group. A in Formulas (a1) to (a4) above is preferably, among others, a single bond, and a group in which an alkylene group and a carbonyloxy group are bonded (alkylene-carbonyloxy group). R2to R4are the same or different and represent an alkyl group that has from 1 to 6 carbons and may have a substituent. R2and R3may be bonded to each other to form a ring. R5and R6are the same or different and represent an alkyl group that has from 1 to 6 carbons and may have a hydrogen atom or a substituent. R7represents a —COORcgroup, and the Rcrepresents a tertiary hydrocarbon group, a tetrahydrofuranyl group, a tetrahydropyranyl group, or an oxepanyl group that has a substituent. n represents an integer of 1 to 3, wherein, when n is 2 or 3, two or three R7s may each be the same or different. Rarepresents a substituent bonded to the ring Z1, the substituents being the same or different and are an oxo group, an alkyl group, a hydroxy group that may be protected with a protecting group, a hydroxyalkyl group that may be protected with a protecting group, or a carboxy group that may be protected with a protecting group; p represents an integer of 0 to 3; and the ring Z1represents an alicyclic hydrocarbon ring having from 3 to 20 carbons; wherein, when p is 2 or 3, two or three Ras may each be the same or different. Examples of the alkyl group in the Rainclude alkyl groups having from 1 to 6 carbons, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a s-butyl group, a t-butyl group, a pentyl group, an isoamyl group, a s-amyl group, a t-amyl group, and n-hexyl group. Examples of the hydroxyalkyl group in the Rainclude hydroxy C1-6alkyl groups, such as a hydroxymethyl group, a 2-hydroxyethyl group, a 1-hydroxyethyl group, a 3-hydroxypropyl group, a 2-hydroxypropyl group, a 4-hydroxybutyl group, and a 6-hydroxyhexyl group. Examples of the protecting group that the hydroxy group and the hydroxyalkyl group in the Ramay have include C1-4alkyl groups, such as a methyl group, an ethyl group, and a t-butyl group; a group that forms an acetal bond together with an oxygen atom constituting the hydroxy group (e.g., C1-4alkyl-O—C1-4alkyl groups, such as a methoxymethyl group); a group that forms an ester bond together with the oxygen atom constituting the hydroxy group (e.g., such as an acetyl group and a benzoyl group). Examples of the protecting group of the carboxy group in the Rainclude C1-6alkyl groups, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, a s-butyl group, a t-butyl group, a pentyl group, an isoamyl group, a s-amyl group, a t-amyl group, and a hexyl group; a 2-tetrahydrofuranyl group; a 2-tetrahydropyranyl group; and a 2-oxepanyl group. Examples of the alkyl group having from 1 to 6 carbons in the R2to R6include linear or branched alkyl groups, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a n-butyl group, an iso-butyl group, a s-butyl group, a t-butyl group, a pentyl group, an isoamyl group, a s-amyl group, a t-amyl group, and a hexyl group. In an embodiment of the present invention, among them, C1-4alkyl groups are preferable, C1-3alkyl groups are more preferable, and C1-2alkyl groups are still more preferable. Examples of the substituent that the alkyl group having 1 to 6 carbons in the R2to R6may have include a halogen atom, a hydroxy group, a substituted hydroxy group (e.g., such as a C1-4alkoxy group, such as a methoxy group, an ethoxy group, and a propoxy group), and a cyano group. Examples of the alkyl group having from 1 to 6 carbons and having a substituent include a halo (C1-6) alkyl group in which one or two or more of hydrogen atoms constituting the alkyl group have been substituted with halogen atoms, such as a trifluoromethyl group and a 2,2,2-trifluoroethyl group; a hydroxymethyl group, a 2-hydroxyethyl group, a methoxymethyl group, a 2-methoxyethyl group, an ethoxymethyl group, a 2-ethoxy ethyl group, a cyanomethyl group, and a 2-cyanoethyl group. In the case where R2and R3are bonded to each other to form a ring, examples of the ring include alicyclic hydrocarbon rings that have 3 to 12 carbons and may have a substituent. Examples of the tertiary hydrocarbon group in the Rcinclude a t-butyl group and a t-amyl group. Examples of the substituent that the tertiary hydrocarbon group in the Rcmay have include a halogen atom, a hydroxy group, a substituted hydroxy group (e.g., such as a C1-4alkoxy group, such as a methoxy group, an ethoxy group, and a propoxy group), and a cyano group. Examples of the alicyclic hydrocarbon ring having from 3 to 20 carbons in the ring Z1include monocyclic alicyclic hydrocarbon rings; such as approximately 3- to 20-membered (preferably 3- to 15-membered and particularly preferably 5- to 12-membered) cycloalkane rings, such as a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, a cyclohexane ring, and a cyclooctane ring; and approximately 3- to 20-membered (preferably 3- to 15-membered and particularly preferably 5- to 10-membered) cycloalkene rings, such as a cyclopropene ring, a cyclobutene ring, a cyclopentene ring, and a cyclohexene ring; an adamantane ring; rings containing a norbornane ring or a norbornene ring, such as a norbornane ring, a norbornene ring, a bornane ring, an isobornane ring, a tricyclo[5.2.1.02,6] decane ring, and a tetracyclo [4.4.0.12,5.17,10] dodecane ring; a ring in which a polycyclic aromatic fused ring has been hydrogenated (preferably a fully hydrogenated ring), such as a perhydroindene ring, a decalin ring (perhydronaphthalene ring), a perhydrofluorene ring (a tricyclo [7.4.0.03,8] tridecane ring), and a perhydroanthracene ring; approximately from bicyclic to hexacyclic bridged hydrocarbon rings, such as bridged hydrocarbon rings of bicyclic system, tricyclic system, tetracyclic system, etc. (e.g., bridged cyclic hydrocarbon rings having approximately from 6 to 20 carbons), such as a tricyclo[4.2.2.12,5]undecane ring. The monomer component preferably contains a monomer having an alicyclic skeleton at least having —C(═O)—O—, —S(═O)2—O—, or —C(═O)—O—C(═O)—. The use of monomers having an alicyclic skeleton can impart better substrate adhesion and etching resistance to polymers (photoresist resins). The monomer having an alicyclic skeleton at least having —C(═O)—O—, —S(═O)2—O—, or —C(═O)—O—C(═O)— may be referred to as a “monomer b”. The monomer b is preferably, among others, at least one type of monomer selected from the group consisting of monomers represented by Formulas (b1) to (b5) below. In Formulas (b1) to (b5) below, R represents a hydrogen atom, a halogen atom, or an alkyl group that has from 1 to 6 carbons and may have a halogen atom, and A represents a single bond or a linking group. X represents a non-bond, a methylene group, an ethylene group, an oxygen atom, or a sulfur atom. Y represents a methylene group or a carbonyl group. Z represents a divalent organic group (e.g., an alkylene group exemplified and described as an alkylene group that may be contained in A in the monomers represented by Formulas (a1) to (a4) (in particular, a linear alkylene group having from 1 to 3 carbons)). V1to V3are the same or different and represent —CH2—, —C(═O)—, or —C(═O)—O— with the proviso that at least one of V1to V3is —C(═O)—O—. R8to R14are the same or different and represent a hydrogen atom, a fluorine atom, an alkyl group that may have a fluorine atom, a hydroxy group that may be protected with a protecting group, a hydroxyalkyl group that may be protected with a protecting group, a carboxy group that may be protected with a protecting group, or a cyano group. Examples of R and A in the monomers represented by Formulas (b1) to (b5) include the same examples as R and A in the monomers represented by Formulas (a1) to (a4). Examples of the alkyl group, the hydroxy group that may be protected with a protecting group, the hydroxyalkyl group that may be protected with a protecting group, and a carboxy group that may be protected with a protecting group in R8to R14in the monomers represented by Formulas (b1) to (b5) include the same examples as those for Rain the monomers represented by Formulas (a1) to (a4). Examples of the alkyl group in the R8to R14include a group [fluoro (C1-6) alkyl group] in which one or two or more of hydrogen atoms constituting the alkyl group have been substituted with fluorine atoms, such as trifluoromethyl and 2,2,2-trifluoroethyl groups. The monomers represented by Formulas (b1) to (b4) above may have one or two or more R8to R11therein, respectively, and the monomers have preferably one to three R8to R11therein, respectively. In addition, in a case where the monomers represented by Formulas (b1) to (b4) above have two or more R8to R11therein, two or more R8to RH may be the same or different, respectively. Among monomers b, a monomer represented by Formula (b1) where R8is an electron-withdrawing group, such as a cyano group, a group having an amide group, a group having an imide group, or a fluoro (C1-6) alkyl group; a monomer represented by Formula (b2); a monomer represented by Formula (b3) where Y is a carbonyl group; a monomer represented by Formula (b4); and a monomer represented by Formula (b5) are preferred in that they can impart excellent substrate adhesion and etching resistance to polymers (photoresist resins), has excellent solubility in an alkaline developer, and can form a fine pattern with high precision. In Formula (b1) above, in a case where R8is an electron-withdrawing group, such as a cyano group, a group having an amide group, a group having an imide group, or a fluoro (C1-6) alkyl group, the R8is particularly preferably at least bonded to a carbon atom marked by * in Formula (b1). The monomer component may further contain a monomer c. The monomer c is a monomer represented by Formula (c1) below. In a case where the monomer component contains the monomer c, the monomer component can impart higher transparency and etching resistance to polymers (photoresist resins). In the formula, R represents a hydrogen atom, a halogen atom, or an alkyl group that has from 1 to 6 carbons and may have a halogen atom. A represents a single bond or a linking group. Rbrepresents a hydroxy group that may be protected with a protecting group, a hydroxyalkyl group that may be protected with a protecting group, a carboxy group that may be protected with a protecting group, or a cyano group, and among them, a hydroxy group and a cyano group are preferred. q represents an integer of 1 to 5. The ring Z2represents an alicyclic hydrocarbon ring having from 6 to 20 carbons; wherein, when q is an integer of 2 to 5, two to five Rbs may each be the same or different. Examples of R and A in the monomers represented by Formula (c1) include the same examples as R and A in the monomers represented by Formulas (a1) to (a4). In addition, examples of the hydroxy group that may be protected with a protecting group, the hydroxyalkyl group that may be protected with a protecting group, and a carboxy group that may be protected with a protecting group in Rbin the monomers represented by Formula (c1) include the same examples as those for Rain the monomers represented by Formulas (a1) to (a4). The ring Z2in the monomers represented by Formula (c1) represents an alicyclic hydrocarbon ring having from 6 to 20 carbons, and examples thereof include monocyclic alicyclic hydrocarbon rings; such as approximately 6- to 20-membered (preferably 6- to 15-membered and particularly preferably 6- to 12-membered) cycloalkane rings, such as a cyclohexane ring and a cyclooctane ring; and approximately 6- to 20-membered (preferably 6- to 15-membered and particularly preferably 6- to 10-membered) cycloalkene rings, such as a cyclohexene ring; an adamantane ring; rings containing a norbornane ring or a norbornene ring, such as a norbornane ring, a norbornene ring, a bornane ring, an isobornane ring, a tricyclo[5.2.1.02,6] decane ring, and a tetracyclo [4.4.0.12,5.17,10] dodecane ring; a ring in which a polycyclic aromatic fused ring has been hydrogenated (preferably a fully hydrogenated ring), such as a perhydroindene ring, a decalin ring (perhydronaphthalene ring), a perhydrofluorene ring (a tricyclo [7.4.0.03,8] tridecane ring), and a perhydroanthracene ring; approximately from bicyclic to hexacyclic bridged hydrocarbon rings, such as bridged hydrocarbon rings of bicyclic system, tricyclic system, tetracyclic system, etc. (e.g., bridged cyclic hydrocarbon rings having approximately from 6 to 20 carbons), such as a tricyclo [4.2.2.12,5] undecane ring. Among them, the ring Z2is preferably a ring containing a norbornane ring or a norbornene ring; and an adamantane ring. Polymer The polymer obtained in an embodiment of the present invention has a homogeneous copolymer composition and a narrow molecular weight distribution, and thus it has, for example, a characteristic that its solubility in solvents is extremely high. Therefore, the polymer can be suitably used as a photoresist resin or the like. The weight average molecular weight (Mw) of the polymer is not particularly limited. It is, for example, preferably from 1000 to 50000, more preferably from 1500 to 40000, still more preferably from 2000 to 20000, particularly preferably from 2500 to 10000, and most preferably from 3000 to 6000. The molecular weight distribution (Mw/Mn) of the polymer is not particularly limited. It is, for example, preferably 2.00 or less, more preferably 1.45 or less, and still more preferably 1.20 or less. When the polymer obtained in an embodiment of the present invention is used as a photoresist resin, the polymer, which has a molecular weight distribution (Mw/Mn) of 1.45 or less, preferably exhibits excellent solubility in an alkaline developer and can form a fine pattern with a high precision. The weight average molecular weight (Mw) and the number average molecular weight (Mn) in the present specification can be measured, for example, by GPC using polystyrene as a reference material. Photoresist Resin Composition The polymer obtained in an embodiment of the present invention can be used as a photoresist resin as described above. That is, a composition containing the polymer obtained in an embodiment of the present invention and a radiation-sensitive acid generator can be used as a photoresist resin composition. A commonly used or well-known compound that efficiently generates an acid by exposure to a radiation, such as a visible light, an ultraviolet light, a far-ultraviolet light, an electron beam, or an X-ray, can be used as the radiation-sensitive acid generator. Such a compound is composed of a nucleus and an acid to be generated. Examples of the nucleus include onium salt compounds, such as iodonium salts, sulfonium salts (including tetrahydrothiophenium salts), phosphonium salts, diazonium salts, and pyridinium salts; sulfonimide compounds; sulfone compounds; sulfonate compounds; disulfonyldiazomethane compounds; disulfonylmethane compounds; oxime sulfonate compounds; and hydrazine sulfonate compounds. In addition, examples of the acid to be generated by the exposure include alkyl or fluorinated alkyl sulfonic acids, alkyl or fluorinated alkyl carboxylic acids, and alkyl or fluorinated alkyl sulfonyl imide acids. One type of them may be used alone, or two or more types may be used. The radiation-sensitive acid generator can be used in an amount appropriately selected according to the strength of the acid to be produced by the irradiation with the radiation, the ratio of each repeating unit in the photoresist resin, and the like. For example, the amount can be selected from a range from 0.1 to 30 parts by weight, preferably from 1 to 25 parts by weight, and still more preferably from 2 to 20 parts by weight per 100 parts by weight of the photoresist resin. The photoresist resin composition can be prepared, for example, by mixing the photoresist resin and the radiation-sensitive acid generator in a solvent for a resist. Solvents such as a glycol-based solvent, an ester-based solvent, a ketone-based solvent, and a mixed solvent thereof exemplified as the polymerization solvent can be used as the solvent for a resist. The concentration of the photoresist resin in the photoresist resin composition is not particularly limited, and is, for example, from 3 to 40 wt. %. The photoresist resin composition may contain an alkali-soluble component, such as an alkali-soluble resin (e.g., a novolac resin, a phenol resin, an imide resin, and a carboxy group-containing resin); a coloring agent (e.g., a dye); or the like. Pattern Forming Method The photoresist resin composition is coated onto a base material or a substrate and dried, and then the coating film (resist film) is exposed via a predetermined mask (or further baked after the exposure) to form a latent image pattern, then subjected to an alkaline dissolution, and thus a fine pattern can be formed with high precision. Examples of the base material or the substrate include silicon wafers, metals, plastics, glasses, and ceramics. The coating of the photoresist resin composition can be performed using a commonly used coating method, such as a spin coater, a dip coater, and a roller coater. The thickness of the coating film is, for example, from 0.05 to 20 μm and preferably from 0.1 to 2 μm. A radiation, such as a visible light, an ultraviolet light, a far-ultraviolet light, an electron beam, and an X-ray can be used for the exposure. An acid is generated from the radiation-sensitive acid generator by the exposure, and a protecting group (acid-degradable group), such as a carboxy group, of a polymerized unit (repeating unit having an acid-degradable group) that becomes alkali soluble by the action of the acid of the photoresist resin composition is quickly eliminated by this acid to form a carboxy group and the like that contribute to solubilization. As a result, the predetermined pattern can be formed with high accuracy by development with an alkaline developer. EXAMPLES Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited by these examples. The weight average molecular weight (Mw) and the number average molecular weight (Mn) of the resin were determined by gel permeation chromatography (GPC) measurement using a tetrahydrofuran solvent. Polystyrene was used for a reference sample and a refractometer (refractive index detector; RI detector) was used as a detector. In addition, the GPC measurement was performed using three columns connected in series, available from Showa Denko K.K. (trade name “KF-806L”), under conditions of a column temperature of 40° C., a RI temperature of 40° C., and a tetrahydrofuran flow rate of 0.8 mL/min. The molecular weight distribution (Mw/Mn) was calculated from the measurements. The microreactor used in the present examples is the microreactor illustrated in the FIGURE including a micromixer composed of a T-shaped pipe joint and a tube reactor connected downstream of the micromixer. Specifically, the microreactor includes an inlet path for polymerization initiator and others1, which is an inlet path for a polymerization initiator, or a polymerization initiator and a chain transfer agent; a first monomer inlet path2; a micromixer3, which is a confluence section (mixing section) of these inlet paths; a tube reactor5, which is a flow path connected downstream of the micromixer3; a second monomer inlet path4; a micromixer6, which is a confluence section (mixing section) of the tube reactor5and the second monomer inlet path4, and a tube reactor7, which is a flow path connected downstream of the micromixer6. The microreactor used in the present examples further includes pumps for pumping liquid, which are provided upstream of the inlet path for polymerization initiator and others1, upstream of the first monomer inlet path2, and upstream of the second monomer inlet path4. These pumps are omitted in the FIGURE. In addition, the microreactor includes collection sections of the reaction solution at the junction between the tube reactor5and the micromixer6, and at the downstream end of the tube reactor7, but the collection sections are omitted in the FIGURE. A custom-made product available from Sanko Seiki K.K. was used as the micromixer (an equivalent micromixer can be obtained upon request for manufacture based on the description of the present examples). The mixer was made of stainless steel, T-shaped, and had an inner diameter of 400 μm. Stainless steel tubes available from GL Sciences Inc. were used as the tube reactors5and7. A syringe pump Model 11 Plus available from Harvard Corporation was used as the pump for pumping liquid. The reaction temperature was controlled by immersing the entire microreactor in a constant temperature bath. Examples 1 and 2 First Stage Reaction Azobisisobutyronitrile (AIBN) as a polymerization initiator, and a mixture of equimolar amounts of γ-butyrolactone (meth)acrylate (GBLMA) and 1-methylcyclopentyl (meth)acrylate (MCPMA) as a first monomer component were fed into the microreactor via the inlet path for polymerization initiator and others, and the first monomer inlet path, respectively. Thereafter, the reaction solution was collected, and amounts of the remaining monomers, weight average molecular weight of the reaction product (polymer), and the like were measured. Conversion ratios of the monomers in the first stage reaction, the weight average molecular weight and molecular weight distribution of the polymer, and conditions, such as the flow rate of the microreactor, were as listed in Table 1. Second Stage Reaction After the first stage reaction, a mixture of equimolar amounts of GBLMA and MCPMA was further fed as a second monomer component into the microreactor via the second monomer inlet path. Thereafter, the reaction solution was collected, and amounts of the remaining monomers, weight average molecular weight of the reaction product (polymer), and the like were measured. Conversion ratios of the monomers in the second stage reaction, the weight average molecular weight and molecular weight distribution of the polymer, and conditions, such as the flow rate of the microreactor, were as listed in Table 2. Example 3 First Stage Reaction A mixture of azobisisobutyronitrile (AIBN) as a polymerization initiator and 2-cyano-2-propyl 4-cyanobenzodithioate (RAFT-A) as a chain transfer agent, and a mixture of equimolar amounts of γ-butyrolactone (meth)acrylate (GBLMA) and 1-methylcyclopentyl (meth)acrylate (MCPMA) as a first monomer component were fed into the microreactor via the inlet path for polymerization initiator and others, and the first monomer inlet path, respectively. Thereafter, the reaction solution was collected, and amounts of the remaining monomers, weight average molecular weight of the reaction product (polymer), and the like were measured. Conversion ratios of the monomers in the first stage reaction, the weight average molecular weight and molecular weight distribution of the polymer, and conditions, such as the flow rate of the microreactor, were as listed in Table 1. Second Stage Reaction After the first stage reaction, a mixture of equimolar amounts of GBLMA and MCPMA was further fed as a second monomer component into the microreactor via the second monomer inlet path. Thereafter, the reaction solution was collected, and amounts of the remaining monomers, weight average molecular weight of the reaction product (polymer), and the like were measured. Conversion ratios of the monomers in the second stage reaction, the weight average molecular weight and molecular weight distribution of the polymer, and conditions, such as the flow rate of the microreactor, were as listed in Table 2. Examples 4 to 24 Examples 4 to 24 were performed in the same manner as in Example 3 with the exception that the polymerization initiator, the chain transfer agent, and the reaction conditions were changed to those listed in Tables 1 and 2. Weight average molecular weight s and the like of the polymers obtained in the first and second stage reactions were measured and listed in Tables 1 and 2, respectively. TABLE 1First stage reactionConcentrations ofPolymerizationpolymerizationinitiator andChainMonomerinitiator andchain transfer agent/First monomerPolymerizationtransferFlow rateconcentrationchain transfer agentmonomersTemperatureExamplescomponentinitiatoragent(mL/min )(mol/L)(mol/L)(mol %)(° C.)Example 1GBLMA/MCPMAAIBN—0.060.810.068.080Example 2GBLMA/MCPMAAIBN—0.060.810.067.280Example 3GBLMA/MCPMAAIBNRAFT-A0.060.810.068.080Example 4GBLMA/MCPMAAIBNRAFT-A0.060.810.034.080Example 5GBLMA/MCPMAAIBNRAFT-A0.060.810.034.090Example 6GBLMA/MCPMAAIBNRAFT-A0.030.810.067.480Example 7GBLMA/MCPMAAIBNRAFT-A0.060.810.067.180Example 8GBLMA/MCPMAAIBNRAFT-A0.060.810.010.780Example 9GBLMA/MCPMAAIBNRAFT-A0.060.810.011.880Example 10GBLMA/MCPMAAIBNRAFT-A0.060.810.010.980Example 11GBLMA/MCPMAAIBNRAFT-A0.060.810.011.880Example 12GBLMA/MCPMAAIBNRAFT-A0.060.810.011.890Example 13GBLMA/MCPMAAIBNRAFT-A0.060.810.011.880Example 14GBLMA/MCPMAAIBNRAFT-A0.060.810.011.890Example 15GBLMA/MCPMAAIBNRAFT-A0.060.810.011.8100Example 16GBLMA/MCPMAAIBNRAFT-A0.060.810.011.8110Example 17GBLMA/MCPMAAIBNRAFT-A0.060.810.011.8120Example 18GBLMA/MCPMAAIBNRAFT-B0.060.810.011.880Example 19GBLMA/MCPMAV-601RAFT-A0.060.810.011.880Example 20GBLMA/MCPMAV-601RAFT-B0.060.810.011.880Example 21GBLMA/MCPMAV-601RAFT-B0.060.810.033.680Example 22GBLMA/MCPMAV-601RAFT-B0.060.810.033.680Example 23GBLMA/MCPMAV-601RAFT-B0.060.810.033.680Example 24GBLMA/MCPMAV-601RAFT-B0.060.810.033.680First stage reactionWeightaverageMolecularFlow pathGBLMAMCPMAAverageGBLMAMCPMAmolecularweightFlow pathinnerFlow pathResidenceconversionconversionconversionproportionproportionweightdistributionlengthdiametervolumetimeratioratioratioin systemin systemof polymerof polymerExamples(m)(mm)(mL)(min)(%)(%)(%)(%)(%)(Mw)(Mw/Mn)Example 14.50.50.8814.760.835.148.063.436.613,8601.77Example 21.21.00.9415.765.731.648.767.532.511,4371.75Example 34.50.50.8814.725.117.721.458.641.47571.25Example 44.50.50.8814.723.79.516.671.428.61,0091.10Example 54.50.50.8814.747.631.939.859.840.29561.32Example 64.50.50.8829.474.538.356.466.133.99091.10Example 71.21.00.9415.752.119.635.972.727.37471.09Example 81.21.00.9415.736.10.918.597.62.41,5391.17Example 92.31.01.8130.126.919.523.258.042.01,3481.20Example 102.31.01.8130.116.316.216.350.249.81,3371.15Example 114.61.03.6160.252.939.246.157.442.61,8701.18Example 124.61.03.6160.281.468.174.754.545.52,1491.19Example 131.21.00.9415.79.83.96.971.528.51,1061.12Example 141.21.00.9415.727.020.523.856.843.21,9561.17Example 151.21.00.9415.741.632.136.956.443.62,1131.18Example 161.21.00.9415.743.233.038.156.743.31,9651.19Example 171.21.00.9415.727.518.823.159.440.61,5341.17Example 184.61.03.6160.270.855.563.256.143.95,1341.45Example 194.61.03.6160.249.542.045.854.145.91,8361.17Example 204.61.03.6160.265.650.958.256.343.73,8151.34Example 214.61.03.6160.276.763.970.354.645.42,9531.28Example 224.61.03.6160.276.662.769.755.045.02,8111.28Example 234.61.03.6160.272.260.666.454.445.62,9741.29Example 244.61.03.6160.275.978.477.249.250.82,8041.29 TABLE 2Second stage reactionConcentrations ofPolymerizationFlow ratepolymerizationinitiator andduringMonomerinitiator andchain transfer agent/Flow pathSecond monomerfeedingFlow rateconcentrationchain transfer agentmonomersTemperaturelengthExamplescomponent(mL/min)(mL/min)(mol/L)(mol/L)(mol %)(° C.)(m)Example 1GBLMA/MCPMA0.040.100.720.0344.7807.7Example 2GBLMA/MCPMA0.040.100.720.0314.3802.0Example 3GBLMA/MCPMA0.040.100.850.0344.0807.7Example 4GBLMA/MCPMA0.040.100.870.0171.9807.7Example 5GBLMA/MCPMA0.040.100.760.0172.2907.7Example 6GBLMA/MCPMA0.020.050.680.0314.6807.7Example 7GBLMA/MCPMA0.040.100.780.0303.8802.0Example 8GBLMA/MCPMA0.040.100.870.0030.3802.0Example 9GBLMA/MCPMA0.040.100.840.0080.9804.0Example 10GBLMA/MCPMA0.040.100.880.0040.4804.0Example 11GBLMA/MCPMA0.040.100.730.0081.0808.0Example 12GBLMA/MCPMA0.040.100.600.0081.3908.0Example 13GBLMA/MCPMA0.040.100.920.0080.8802.0Example 14GBLMA/MCPMA0.040.100.840.0080.9902.0Example 15GBLMA/MCPMA0.040.100.780.0081.01002.0Example 16GBLMA/MCPMA0.040.100.770.0081.01102.0Example 17GBLMA/MCPMA0.040.100.840.0080.91202.0Example 18GBLMA/MCPMA0.040.100.650.0081.2808.0Example 19GBLMA/MCPMA0.040.100.740.0081.0808.0Example 20GBLMA/MCPMA0.040.100.680.0081.1808.0Example 21GBLMA/MCPMA0.040.100.620.0152.5808.0Example 22GBLMA/MCPMA0.040.100.620.0152.5808.0Example 23GBLMA/MCPMA0.040.100.640.0152.4808.0Example 24GBLMA/MCPMA0.040.100.590.0152.6808.0Second stage reactionPolymerweightPolymerFlow pathGBLMAMCPMAAverageGBLMAMCPMAaveragemolecularinnerFlow pathResidenceconversionconversionconversionproportionproportionmolecularweightdiametervolumetimeratioratioratioin systemin systemweightdistributionExamples(mm)(mL)(min)(%)(%)(%)(%)(%)(Mw)(Mw/Mn)Example 10.51.5114.967.747.057.459.041.016,0431.82Example 21.01.5715.478.043.660.864.135.914,7411.91Example 30.51.5114.936.126.231.257.942.11,1841.27Example 40.51.5114.935.220.127.763.736.31,4371.14Example 50.51.5114.956.941.249.158.042.01,4871.16Example 60.51.5129.685.247.866.564.135.91,1951.18Example 71.01.5715.458.829.744.366.433.61,0691.16Example 81.01.5715.442.412.027.277.922.12,3681.22Example 91.03.1430.838.428.333.457.642.41,9861.22Example 101.03.1430.822.019.720.952.847.21,9151.18Example 111.06.2861.663.349.156.256.343.72,8241.20Example 121.06.2861.669.957.363.654.945.12,4361.20Example 131.01.5715.429.922.026.057.642.41,7071.17Example 141.01.5715.444.033.638.856.743.33,1911.19Example 151.01.5715.448.737.142.956.843.23,1551.18Example 161.01.5715.426.518.222.359.240.82,1791.20Example 171.01.5715.415.910.513.260.339.71,5461.16Example 181.06.2861.671.158.164.655.045.06,2401.45Example 191.06.2861.654.946.950.953.946.12,6721.19Example 201.06.2861.666.853.260.055.744.34,9531.31Example 211.06.2861.677.465.771.654.145.93,6731.31Example 221.06.2861.685.373.579.453.746.33,7001.32Example 231.06.2861.685.476.681.052.747.34,0061.34Example 241.06.2861.689.693.791.648.951.13,8511.35 REFERENCE SIGNS LIST 1Inlet path for polymerization initiator and others2First monomer inlet path3Micromixer4Second monomer inlet path5Tube reactor6Micromixer7Tube reactor | 92,376 |
11859029 | DETAILED DESCRIPTION Before explaining at least one aspect of the disclosed and/or claimed inventive concept(s) in detail, it is to be understood that the disclosed and/or claimed inventive concept(s) is not limited in its application to the details of construction and the arrangement of the components or steps or methodologies set forth in the following description or illustrated in the drawings. The disclosed and/or claimed inventive concept(s) is capable of other aspects or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. Unless otherwise defined herein, technical terms used in connection with the disclosed and/or claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference herein their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference. All of the articles and/or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of the disclosed and/or claimed inventive concept(s) have been described in terms of aspects, it will be apparent to those of ordinary skill in the art that variations may be applied to the articles and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosed and/or claimed inventive concept(s). All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosed and/or claimed inventive concept(s). As utilized in accordance with the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings. The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” is used to mean “and/or” unless explicitly indicated to refer to alternatives only if the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the quantifying device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term “about” is utilized, the designated value may vary by plus or minus twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent. The use of the term “at least one” will be understood to include one as well as any quantity more than one, including but not limited to, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000 or more depending on the term to which it is attached. In addition, the quantities of 100/1000 are not to be considered limiting as lower or higher limits may also produce satisfactory results. In addition, the use of the term “at least one of X, Y, and Z” will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., “first”, “second”, “third”, “fourth”, etc.) is solely for the purpose of differentiating between two or more items and, unless otherwise stated, is not meant to imply any sequence or order or importance to one item over another or any order of addition. As used herein, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, BXn, BXn+1, or combinations thereof” is intended to include at least one of: A, BXn, BXn+1, ABXn, A BXn+1, BXnBXn+1, or ABXnBXn+1and, if order is important in a particular context, also BXnA, BXn+1A, BXn+1BXn, BXn+1BXnA, BXnBXn+1A, ABXn+1BXn, BXnABXn+1, or BXn+1ABXn. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BXnBXn, AAA, MBXn, BXnBXnBXn+1, AAABXnBXn+1BXn+1BXn+1BXn+1, BXn+1BXnBXnAAA, BXn+1A BXnABXnBXn, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context. The term “each independently selected from the group consisting of” means when a group appears more than once in a structure, that group may be selected independently each time it appears. The term “hydrocarbyl” includes straight-chain and branched-chain alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl groups, and combinations thereof with optional heteroatom(s). A hydrocarbyl group may be mono-, di- or polyvalent. The term “alkyl” refers to a functionalized or unfunctionalized, monovalent, straight-chain, branched-chain, or cyclic C1-C60hydrocarbyl group optionally having one or more heteroatoms. In one non-limiting embodiment, an alkyl is a C1-C45hydrocarbyl group. In another non-limiting embodiment, an alkyl is a C1-C30hydrocarbyl group. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, tert-octyl, iso-norbornyl, n-dodecyl, tert-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The definition of “alkyl” also includes groups obtained by combinations of straight-chain, branched-chain and/or cyclic structures. The term “aryl” refers to a functionalized or unfunctionalized, monovalent, aromatic hydrocarbyl group optionally having one or more heteroatoms. The definition of aryl includes carbocyclic and heterocyclic aromatic groups. Non-limiting examples of aryl groups include phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, anthracenyl, furyl, thienyl, pyridyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzothiophenyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxyazinyl, pyrazolotriazinyl, and the like. The term “aralkyl” refers to an alkyl group comprising one or more aryl substituent(s) wherein “aryl” and “alkyl” are as defined above. Non-limiting examples of aralkyl groups include benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. The term “alkylene” refers to a functionalized or unfunctionalized, divalent, straight-chain, branched-chain, or cyclic C1-C40hydrocarbyl group optionally having one or more heteroatoms. In one non-limiting embodiment, an alkylene is a C1-C30group. In another non-limiting embodiment, an alkylene is a C1-C20group. Non-limiting examples of alkylene groups include: The term “arylene” refers to a functionalized or unfunctionalized, divalent, aromatic hydrocarbyl group optionally having one or more heteroatoms. The definition of arylene includes carbocyclic and heterocyclic groups. Non-limiting examples of arylene groups include phenylene, naphthylene, pyridinylene, and the like. The term “heteroatom” refers to oxygen, nitrogen, sulfur, silicon, phosphorous, or halogen. The heteroatom(s) may be present as a part of one or more heteroatom-containing functional groups. Non-limiting examples of heteroatom-containing functional groups include ether, hydroxy, epoxy, carbonyl, carboxamide, carboxylic ester, carboxylic acid, imine, imide, amine, sulfonic, sulfonamide, phosphonic, and silane groups. The heteroatom(s) may also be present as a part of a ring such as in heteroaryl and heteroarylene groups. The term “halogen” or “halo” refers to Cl, Br, I, or F. The term “ammonium” includes protonated NH3and protonated primary, secondary, and tertiary organic amines. The term “functionalized” with reference to any moiety refers to the presence of one or more functional groups in the moiety. Various functional groups may be introduced in a moiety by way of one or more functionalization reactions known to a person having ordinary skill in the art. Non-limiting examples of functionalization reactions include: alkylation, epoxidation, sulfonation, hydrolysis, amidation, esterification, hydroxylation, dihydroxylation, amination, ammonolysis, acylation, nitration, oxidation, dehydration, elimination, hydration, dehydrogenation, hydrogenation, acetalization, halogenation, dehydrohalogenation, Michael addition, aldol condensation, Canizzaro reaction, Mannich reaction, Clasien condensation, Suzuki coupling, and the like. In one non-limiting embodiment, the term “functionalized” with reference to any moiety refers to the presence of one more functional groups selected from the group consisting of alkyl, alkenyl, hydroxyl, carboxyl, halogen, alkoxy, amino, imino, and combinations thereof, in the moiety. The term “monomer” refers to a small molecule that chemically bonds during polymerization to one or more monomers of the same or different kind to form a polymer. The term “polymer” refers to a large molecule comprising one or more types of monomer residues (repeating units) connected by covalent chemical bonds. By this definition, polymer encompasses compounds wherein the number of monomer units may range from very few, which more commonly may be called as oligomers, to very many. Non-limiting examples of polymers include homopolymers, and non-homopolymers such as copolymers, terpolymers, tetrapolymers and the higher analogues. The polymer may have a random, block, and/or alternating architecture. The term “homopolymer” refers to a polymer that consists essentially of a single monomer type. The term “non-homopolymer” refers to a polymer that comprises more than one monomer types. The term “copolymer” refers to a non-homopolymer that comprises two different monomer types. The term “terpolymer” refers to a non-homopolymer that comprises three different monomer types. The term “branched” refers to any non-linear molecular structure. The term includes both branched and hyper-branched structures. The term “block copolymer” refers to a polymer comprising at least two blocks of polymerized monomers. Any block may be derived from either a single monomer resulting in a homopolymeric subunit, or two or more monomers resulting in a copolymeric (or non-homopolymeric) subunit in the block copolymer. The block copolymers may be diblock copolymers (i.e., polymers comprising two blocks of monomers), triblock copolymers (i.e., polymers comprising three blocks of monomers), multiblock copolymers (i.e., polymers comprising more than three blocks of monomers), or combinations thereof. The block copolymers may be linear, branched, star or comb like, and have structures such as [A][B], [A][B][A], [A][B][C], [A][B][A][B], [A][B][C][B], etc. An exemplary representation of block copolymer is [A]x[B]yor [A]x[B]y[C]z, wherein x, y and z are the degrees of polymerization (DP) of the corresponding blocks [A], [B], and [C]. Additional insight into the chemistry, characterization and applications of block copolymers may be found in the book ‘Block Copolymers: Synthetic Strategies, Physical Properties, and Applications’, by Nikos Hadjichristidis, Stergios Pispas, and George Floudas, John Wiley and Sons (2003), the contents of which are herein incorporated in its entirety by reference. The term “controlled radical polymerization” refers to a specific radical polymerization process, also denoted by the term of “living radical polymerization”, in which use is made of control agents, such that the polymer chains being formed are functionalized by end groups capable of being reactivated in the form of free radicals by reversible transfer or reversible termination reactions. The control agents used in controlled radical polymerization reactions are termed “chain transfer agents”. These agents can be in the form of (A) low molecular weight compounds that are typically added as molecular weight modifiers during the preparation of homopolymers, or (B) macromolecular chain transfer agents that are formed by end group functionalization of the polymer chains formed during homopolymerization reactions that are typically added as molecular weight modifiers during the preparation of non-homopolymers such as, for example, diblock copolymers and multiblock polymers. The term “addition-fragmentation” refers to a two-step chain transfer mechanism during polymerization of block copolymers wherein a radical addition is followed by fragmentation to generate a new radical species. The term “free radical addition polymerization initiator” refers to a compound used in a catalytic amount to initiate a free radical addition polymerization. The choice of initiator depends mainly upon its solubility and its decomposition temperature. The term “non-radiation initiator” refers to a free radical polymerization initiator that can generate free radicals when subjected to a non-radiation source of energy. In the context of the disclosed and/or claimed inventive concepts, the method of preparation of homopolymers and non-homopolymers involving contact with at least one non-radiation initiator is meant to imply that the method does not involve exposure to radiation as a source of energy for generating free radicals to activate the reaction. The non-radiation method according to the disclosed and/or claimed inventive concepts provides superior reaction yields and polymer purity as well as excellent control over distribution of molecular weights of the resulting homopolymers and non-homopolymers. These attributes are much superior to what is known in related art. The term “thermochemical initiator” refers to a free radical polymerization initiator that generates free radicals when subjected to thermal energy. The term “non-aqueous solvent” refers to non-polar organic solvents and polar organic solvents. The term “alkyl acrylate” refers to an alkyl ester of an acrylic acid or an alkyl acrylic acid. The term “alkyl acrylamide” refers to an alkyl amide of an acrylic acid or an alkyl acrylic acid. The term “moiety” refers to a part or a functional group of a molecule. In the block copolymer structures, the notation—b—on the polymer backbone is meant to denote block configuration of repeating units. An exemplary block copolymer structure is shown below: The term “colloidal” refers to the state of matter having nanometer dimensions. The terms “personal care composition” and “cosmetics” refer to compositions intended for use on or in the human body, such as skin, sun, hair, oral, cosmetic, and preservative compositions, including those to alter the color and appearance of skin and hair. The term “pharmaceutical composition” refers to any composition comprising at least one pharmaceutically active ingredient, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. The term “coating composition” refers to an aqueous-based or solvent-based liquid composition that may be applied to a substrate and thereafter solidified (for example, by radiation, air curing, post-crosslinking or ambient temperature drying) to form a hardened coating on the substrate. The term “oilfield composition” refers to a composition that may be used in the exploration, extraction, recovery, and/or completion of any hydrocarbon. Non-limiting examples of oilfield compositions include drilling fluids, cementing fluids, anti-agglomerants, kinetic hydrate inhibitors, shale swelling inhibitors, drilling muds, servicing fluids, gravel packing muds, friction reducers, fracturing fluids, completion fluids, and work over fluids. The term “hydrophilic monomer” refers to a monomer having solubility in water of greater than about 10 percent by weight at 25° C. The term “hydrophobic monomer” refers to a monomer having solubility in water of lesser than about 10 percent by weight at 25° C. All percentages, ratio, and proportions used herein are based on a weight basis unless other specified. In a first aspect, the disclosed and/or claimed inventive concept(s) provides a method for preparation of a homopolymer comprising contacting in an aqueous medium: (1) a monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety, (2) at least one chain transfer agent, and (3) at least one non-radiation initiator. In one non-limiting embodiment, the chain transfer agent is a reversible addition-fragmentation chain transfer agent. In one non-limiting embodiment, the non-radiation initiator is a thermochemical initiator. In one non-limiting embodiment, the aqueous medium further comprises at least one non-aqueous solvent. Non-limiting examples of non-aqueous solvents include functionalized or unfunctionalized alcohols, acids, ethers, ketones, nitriles, lactones, esters, amines, amides, carbonates, carbamates, halocarbons, hydrocarbons, and combinations thereof. In one non-limiting embodiment, the monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety has a structure: wherein each R1and R2is independently selected from the group consisting of hydrogen, halogens, functionalized and unfunctionalized C1-C4alkyl, and each X is independently selected from the group consisting of OR3, OM, halogen, N(R4)(R5), and combinations thereof; each Y is independently oxygen, NR6or sulfur; each R3, R4, R5, and R6is independently selected from the group consisting of hydrogen, functionalized and unfunctionalized alkyl, and combinations thereof; each M is independently selected from the group consisting of metal ions, ammonium ions, organic ammonium cations, and combinations thereof; and each Q1, Q2, Q3, and Q4is independently a functionalized or unfunctionalized alkylene. In one non-limiting embodiment, each Q1, Q2, Q3, and Q4is independently selected from the group consisting of functionalized and unfunctionalized C1-C12alkylene. Non-limiting examples of such alkylene groups include —CH2—. —CH2—CH2—, —CH(CH3)—CH2—, —CH2—CH(CH3)—, —C(CH3)2—CH2—, —CH2—C(CH3)2—, —CH(CH3)—CH(CH3)—, —C(CH3)2—C(CH3)2—, —CH2—CH2—CH2—, —CH(CH3)—CH2—CH2—, —CH2—CH(CH3)—CH2—, —CH2—CH2—CH(CH3)—, —CH2—CH2—CH2—CH2—, —CH2—CH2—CH2—CH2—CH2—, —CH2—CH2—CH2—CH2—CH2—CH2—, and —CH2—CH2—CH2—CH2—CH2—CH2—CH2—. In another non-limiting embodiment, each Q1, Q2, Q3, and Q4is independently selected from the group consisting of functionalized and unfunctionalized C2-C6alkylene. Non-limiting examples of such alkylene groups include: In one non-limiting embodiment, each R1and R2is independently selected from the group consisting of hydrogen, methyl and combinations thereof. In another non-limiting embodiment, R1and R2are hydrogens. In another non-limiting embodiment, R1is independently hydrogen or methyl; R2is X is selected from the group consisting of OR3, OM, halogens, and N(R4)(R5); each R3, R4, and R5is independently selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl; and each M is independently selected from the group consisting of metal ions, ammonium ions, organic ammonium cations, and combinations thereof. In another non-limiting embodiment, R1is independently hydrogen; R2is X is selected from the group consisting of OR3, OM, halogens, and N(R4)(R5); each R3, R4, and R5is independently selected from the group consisting of hydrogen and functionalized and unfunctionalized alkyl; and each M is independently selected from the group consisting of metal ions, ammonium ions, organic ammonium cations, and combinations thereof. The monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety maybe be synthesized using methods described in the art, e.g., by reaction of an N-hydroxylalkyl lactam with carboxylic acids such as (meth)acrylic acid, esters such as (meth)acrylate esters, amides such as (meth)acrylamides, anhydrides such as (meth)acrylic anhydride, or similar compounds. Methods of synthesis include those described in patents: U.S. Pat. Nos. 2,882,262; 5,523,340; 6,369,163; U.S. Pat. Application Publication 2007/123673; GB924623; GB930668; GB1404989; WO03/006569; and EP385918. Each of the aforementioned patents is herein incorporated by reference in its entirety. Non-limiting examples of N-hydroxyalkyl lactams include N-hydroxymethyl pyrrolidone, N-hydroxymethyl caprolactam, N-hydroxyethyl pyrrolidone, N-hydroxyethyl caprolactam, N-hydroxypropyl pyrrolidone, and N-hydroxypropyl caprolactam. Non-limiting examples of carboxylic acids include: acrylic acid, methacrylic acid, itaconic acid, crotonic acid, fumaric acid, succinic acid, and maleic acid. Non-limiting examples of acrylates and (meth)acrylates include methyl, ethyl, butyl, n-octyl, 2-ethylhexyl acrylates and their (meth)acrylate analogues. Non-limiting examples of anhydrides include (meth)acrylic anhydride, formic anhydride, succinic anhydride, and maleic anhydride. In one non-limiting embodiment, the monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety used in the preparation of a homopolymer according to the claimed and/or disclosed inventive concept(s) has a structure: In a second aspect, the disclosed and/or claimed inventive concept(s) provides a method for preparation of a non-homopolymer comprising contacting in an aqueous medium: (1) a macromolecular chain transfer agent derived from at least one monomer A, (2) at least one monomer B, and (3) at least one non-radiation initiator, with the proviso that at least one of said monomer A and monomer B, but not both, is a monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety and other said monomer A or monomer B is a dissimilar comonomer. In one non-limiting embodiment, monomer A is a monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety and monomer B is a dissimilar comonomer. In another non-limiting embodiment, monomer B is a monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety and monomer A is a dissimilar comonomer. In one non-limiting embodiment, the dissimilar comonomer is selected from the group consisting of hydrophilic comonomers, hydrophobic comonomers, and combinations thereof. In one non-limiting embodiment, the aqueous medium is a homogeneous medium and said dissimilar comonomer is a hydrophilic comonomer. In one non-limiting embodiment, the hydrophilic comonomer is selected from the group consisting of functionalized or unfunctionalized hydroxyalkyl (meth)acrylates, glyceryl (meth)acrylates, epoxyalkyl (meth)acrylates, N-alkylaminoalkyl (meth)acrylates, N,N-dialkylaminoalkyl (meth)acrylates, oligoethyleneglycol (meth)acrylates, etherified oligoethyleneglycol (meth)acrylates, polyalkyleneglycol (meth)acrylates, etherified polyalkyleneglycol (meth)acrylates, (meth)acrylamides, N-alkyl(meth)acrylamides, N,N-dialkyl(meth)acryl amides, N-hydroxyalkyl (meth)acrylamides, N-epoxyalkyl (meth)acrylamides, N-aminoalkyl (meth)acrylamides, N,N-dialkylaminoalkyl (meth)acrylamides, (meth)acrylates and (meth)acrylamides comprising sulfonic acid moieties and salts thereof, alkenyl sulfonic acids and salts thereof, (meth)acrylates and (meth)acrylamides comprising quaternary ammonium moieties, N-vinyl lactams, N-vinyl pyrrolidone, vinyl alcohol, N-alkenyl carboxamides, alpha-beta unsaturated dicarboxylic acids and salts thereof, alpha-beta unsaturated dicarboxylic anhydrides, amic acids, ester acids and salts thereof, diesters, diamides, esteramides, and combinations thereof. In one non-limiting embodiment, the aqueous medium is a heterogeneous medium and said dissimilar comonomer is a hydrophobic comonomer. In one non-limiting embodiment, the hydrophobic comonomer is selected from the group consisting of functionalized or unfunctionalized styrene, vinyl chloride, vinyl 2-ethylhexanoate, vinyl laurate, vinyl stearate, vinyl neo-pentanoate, vinyl 2-ethylhexanoate, vinyl neo-nonanoate, vinyl neo-decanoate, vinyl neo-undecanoate, vinyl neo-dodecanoate, isobutyl vinyl ether, 2-chloroethyl vinyl ether, stearyl vinyl ether, n-butyl acrylate, isobutyl acrylate, sec-butyl acrylate, tert-butyl acrylate, n-pentyl acrylate, neopentyl acrylate, n-hexyl acrylate, n-heptyl acrylate, n-octyl acrylate, isooctyl acrylate, isononyl acrylate, n-decyl acrylate, isodecyl acrylate, 2-ethylhexyl acrylate, benzyl acrylate, oleyl acrylate, palmityl acrylate, stearyl acrylate, n-butyl methacrylate, isobutyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, n-pentyl methacrylate, neopentyl methacrylate, n-hexyl methacrylate, n-heptyl methacrylate, n-octyl methacrylate, isooctyl methacrylate, isononyl methacrylate, n-decyl methacrylate, isodecyl methacrylate, 2-ethylhexyl methacrylate, benzyl methacrylate, oleyl methacrylate, palmityl methacrylate, stearyl methacrylate, unsaturated vinyl esters of (meth)acrylic acid such as those derived from fatty acids and fatty alcohols, monomers derived from cholesterol, vinyl chloride, 1-butene, 2-butene, 1-pentene, 1-hexene, 1-octene, isobutylene, isoprene, and combinations thereof. Further non-limiting examples of hydrophilic and hydrophobic monomers can be found in the Research Disclosure, the disclosure of which is herein incorporated by reference in its entirety. In one non-limiting embodiment, the monomer comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety used in the preparation of a non-homopolymer according to the claimed and/or disclosed inventive concept(s) has a structure: and combinations thereof. Further non-limiting examples of monomers comprising at least one acryloyl moiety and at least one functionalized or unfunctionalized lactam moiety can be found in WO2011/063208, the disclosure of which is herein incorporated by reference in its entirety. In one non-limiting embodiment, the reversible addition-fragmentation chain transfer agent is selected from the group consisting of dithioesters, trithiocarbonates, dithiocarbamates, xanthates, and combinations thereof. Non-limiting examples of reversible addition-fragmentation chain transfer agents can be found in the following publications, each of which is herein incorporated by reference in its entirety: (1)Polymer, volume 49(5), 2008, 1079-1131; (2)Macromolecules, volume 50, 2017, 7433-7447; (3) U.S. Pat. No. 7,230,063; and (4) U.S. Pat. No. 7,632,966. In one non-limiting embodiment, the non-homopolymer according to the claimed and/or disclosed inventive concept(s) is a diblock copolymer or a multiblock polymer. In one non-limiting embodiment, the non-homopolymer according to the claimed and/or disclosed inventive concept(s) is a diblock copolymer. In another non-limiting embodiment, the non-homopolymer according to the claimed and/or disclosed inventive concept(s) is a multiblock polymer. In one non-limiting embodiment, the non-homopolymer according to the claimed and/or disclosed inventive concept(s) has a structure selected from the group consisting of: wherein each x and y is independently an integer having a value from about 10 to about 50000, and R is selected from the group consisting of hydrogen, methyl, and combinations thereof. In another non-limiting embodiment, the non-homopolymer according to the claimed and/or disclosed inventive concept(s) has a structure selected from the group consisting of: wherein each x, y and z is independently an integer having a value from about 10 to about 50000, and R8, R9and R10is selected from the group consisting of hydrogen, methyl, and combinations thereof. Reversible addition-fragmentation chain transfer (RAFT) polymerization is one of the most robust and versatile methods for providing living characteristics to radical polymerization. With appropriate selection of the RAFT agent for the monomers and reaction conditions, it is applicable to the majority of monomers subject to radical polymerization. The process can be used in the synthesis of well-defined homo-, gradient, diblock, triblock, and star polymers and more complex architectures, which include microgels and polymer brushes. When preparing, for example, a block copolymer in the presence of the control agent, the end of the growing block is provided with a specific functionality that controls the growth of the block by means of reversible free radical deactivation. The functionality at the end of the block is of such a nature that it can reactivate the growth of the block in a second and/or third stage of the polymerization process with other ethylenically unsaturated monomers providing a covalent bond between, for example, a first and second block [A] and [B] and with any further optional blocks. Further details on the chemistry of synthesis of block copolymers by RAFT processes may be found in the following publications, each of which is herein incorporated in its entirety by reference:Polymer,2008, volume 49, 1079-1131; Chemical Society Reviews,2014, volume 43, 496-505; Macromolecules,1998, volume 31, 5559-5562; andPolymer,2013, volume 54, 2011-2019. Further examples on methods of synthesis of block polymers according to the disclosed and/or claimed inventive concepts may be found in the Research Disclosure, the disclosure of which is herein incorporated by reference in its entirety. The block copolymers according to the disclosed and/or claimed inventive concept(s) may be prepared according to the examples set out below. These examples are presented herein for purposes of illustration of the disclosed and/or claimed inventive conept(s) and are not intended to be limiting, for example, the preparations of the polymers. In the examples, the following abbreviations are used:NAEP: N-2-(acryloyloxy)ethyl pyrrolidonePNAEP: Poly(N-2-(acryloyloxy)ethyl pyrrolidone)NMEP: N-2-(methacryloyloxy)ethyl pyrrolidoneHEA: 2-hydroxyethyl acrylatePHEA: poly(2-hydroxyethyl acrylate)GMA: Glycerol monomethacrylateOEGA: Oligo(ethylene glycol) methyl ether acrylatePOEGA: Poly(oligo(ethylene glycol) methyl ether acrylate)DEA: 2-(diethylamino)ethyl methacrylatePDEA: Poly(-(diethylamino)ethyl methacrylate)NIPAM: N-isopropylacrylamidePNIPAM: Poly(N-isopropylacrylamide)AscAc: Ascorbic acidKPS: Potassium persulfateSMA: Stearyl (meth)acrylateBzMA: Benzyl (meth)acrylateS: StyrenePS: PolystyrenenBA: n-butyl acrylatePnBA: Poly(n-butyl acrylate)P(S-stat-nBA): Statistical copolymer of S and nBAAIBN: α,α′-azoisobutyronitrileAVCA: 4,4′-azobis(4-cyanopentanoic acid)DDMAT: 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acidPETTC: 4-cyano-4-(2-phenylethanesulfanylthiocarbonyl)sulfanylpentanoic acidMPETTC: 4-(2-aminoethylmorpholine) amide of PETTCCPDB: cyano-2-propyl benzodithioateDMF: Dimethyl formamideVA-044: 2,2-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochlorideCTA: Chain transfer agentDP: Degree of polymerizationGPC: Gel permeation chromatographyNMR: Nuclear magnetic resonanceMn: Number-average molecular weightMw: Weight-average molecular weight EXAMPLES Synthesis of PNAEP Homopolymer Example 1: RAFT Solution Homopolymerization of NAEP in Water at 30° C. A typical protocol for the synthesis of a PNAEP80(mean DP=80) homopolymer is as follows: NAEP (1.00 g, 5.46 mmol), DDMAT RAFT agent (24.9 mg, 68.2 μmol; target DP=80) and AscAc (2.4 mg, 13.6 μmol; DDMAT/AscAc molar ratio=5.0) were weighed into a 14 mL vial charged with a magnetic flea and degassed with nitrogen in an ice bath for 30 min (reaction solution 1). Deionized water (0.6873 g, corresponding to a 60% w/w solution), and KPS (3.7 mg, 13.6 μmol; DDMAT/KPS molar ratio=5.0) were weighed into a separate 14 mL vial (reaction solution 2), sealed using a rubber septum and degassed with nitrogen in an ice bath for 30 min. After 30 min, the vial containing reaction solution 1 was immersed in an oil bath set at 30° C. Following this, reaction solution 2 was added to this vial via a degassed syringe and needle to reaction solution 1 under nitrogen. The polymerization was monitored for 5 min, resulting in a final monomer conversion of 99% as judged by1H NMR spectroscopy. DMF GPC analysis indicated an Mnof 12,300 g mol−1and an Mw/Mnof 1.15. Targeting mean DPs of above 150 required reaction times of up to 60 min for high conversion. Example 2: RAFT Solution Homopolymerization of NAEP in Water at 70° C. NAEP (1.00 g, 5.46 mmol), DDMAT RAFT agent (24.9 mg, 68.2 μmol; target DP=80), deionized water (0.6847 g, corresponding to a 60% w/w solution), and AIBN (2.2 mg, 13.6 μmol; DDMAT/AIBN molar ratio=5.0) were weighed into a 14 mL vial charged with a magnetic flea. This reaction vial was then placed in an ice bath and degassed with nitrogen for 30 min. Following this, the vial was then immersed in an oil bath set at 70° C. and the reaction solution was stirred for 50 min, resulting in a final monomer conversion of 99% as judged by1H NMR spectroscopy. DMF GPC analysis indicated an Mnof 13,300 g mol−1and an Mw/Mnof 1.14. Glass transition temperatures (Tg) for four PNAEPxhomopolymers prepared via RAFT aqueous solution polymerization of NAEP utilizing the low-temperature redox initiator were determined using differential scanning calorimetry (DSC) for DPs ranging between 50 and 400. This technique indicated Tgvalues below room temperature for mean DPs of less than 400. A Tgof approximately 19.6° C. was obtained for a PNAEP400homopolymer. A series of PNAEP homopolymers were prepared targeting a range of DPs using either AIBN or the low-temperature redox initiator. The results are presented in Table 1. A DDMAT/initiator molar ratio of 5.0 was used for all these homopolymerizations. DMF GPC analysis was used to determine the Mnand Mw/Mnvalues in each case.1H NMR analysis indicated that high NAEP conversions (≥98%) were achieved using either AIBN at 70° C. or the redox initiator at 30° C. when targeting PNAEP DPs of up to 120 or 400, respectively. For PNAEP syntheses targeting a DP of 200, the faster rate of polymerization achieved at 30° C. did not adversely affect RAFT control over these polymerizations, with Mw/Mnremaining less than 1.20 up to DP 400. Thus, the low-temperature redox initiator route was adopted for all subsequent RAFT syntheses. When targeting DPs above 400, reaction solutions became very viscous when using 60% w/w NAEP, which led to significantly lower conversions (<80%). TABLE 1Summary of Target PNAEP DP, conversions, molecular Weights(Mn), and dispersities (Mw/Mn) obtained for PNAEP homopolymersPrepared by RAFT Aqueous Solution Polymerization ofNAEP at either 30° C. (low-temperature redox initiator)or 70° C. (AIBN) at 60% w/w SolidsTargetConversionTemperatureMnExamplePNAEP DP(%)° C.g/molMw/Mn340997076001.134609970112001.135809970133001.1461009970157001.1971209970193001.15840993074001.199609930100001.1510809930123001.15111009830152001.15121209830171001.16131509930215001.15142009930276001.16154009930414001.18167507530746001.2617100070301154001.26 Example 18: Preparation of PNAEPX Macro-CTA The typical protocol for the synthesis of a PNAEP62macro-CTA by RAFT aqueous solution polymerization is as follows: NAEP (10.00 g, 54.6 mmol), DDMAT RAFT agent (199.0 mg, 0.5458 mmol; target DP=100) and AscAc (1.0 mg, 5.5 μmmol) were weighed into a 14 mL vial charged with a magnetic flea (reaction solution 1). This reaction solution was then placed in an ice bath and degassed with nitrogen for 30 min. Deionized water (6.8010 g, 60% w/w), and KPS (1.5 mg, 5.5 μmol; DDMAT/KPS molar ratio=100) were weighed into a second 14 mL vial (reaction solution 2) and degassed with nitrogen in an ice bath for 30 min. After 30 min, the vial containing reaction solution 1 was immersed in an oil bath set at 30° C. Reaction solution 2 was then added via a degassed syringe and needle to reaction solution 1 under nitrogen. The polymerization proceeded for 8 min before being quenched via exposure to air and immersed in an ice bath to quench the polymerization.1H NMR analysis of the disappearance of vinyl signals assigned to PNAEP indicated a monomer conversion of 60%. The crude homopolymer was purified by precipitating into a ten-fold excess of diethyl ether. This purification protocol was repeated twice to give a PNAEP macro-CTA containing less than 1% residual monomer. The mean degree of polymerization was calculated to be 62 as judged by1H NMR spectroscopy. DMF GPC analysis indicated an Mnof 9,800 g mol−1and an Mw/Mnof 1.25. Other PNAEPxmacro-CTAs were obtained by adjusting the NAEP/DDMAT molar ratio. Synthesis of PNAEP62-PHEAyDiblock Copolymers Example 19: PNAEP62-PHEAyDiblock Copolymers with Target DP=100 PNAEP62macro-CTA (0.250 g, 21.3 μmol), HEA (0.2476 g, 2.1324 mmol; target DP=100) and AscAc (0.8 mg, 4.3 μmol; PNAEP62macro-CTA) were weighed into a 14 mL vial charged with a magnetic flea (reaction solution 1). This vial was immersed in an ice bath and the solution was degassed with nitrogen for 30 min. Deionized water (2.2306 g, corresponding to a 15% w/w solution) and KPS (1.2 mg, 4.3 μmol; PNAEP62macro-CTA/KPS molar ratio=5.0) were weighed into a separate 14 mL vial (reaction solution 2) and degassed with nitrogen in an ice bath for 30 min. Reaction solution 1 was then immersed in an oil bath set at 30° C. Reaction solution 2 was added to this vial via a degassed syringe and needle under nitrogen. The polymerization proceed for 18 h before being quenched by exposing the reaction solution to air and immersing the reaction vial in an ice bath.1H NMR studies indicated more than 99% conversion while DMF GPC analysis indicated a Mnof 29,400 g mol−1and an Mw/M. of 1.22. Example 20: PNAEP62-PHEAyDiblock Copolymers with Target DP Upto 400 PNAEP62macro-CTA (obtained as per Example 3) was used to prepare a series of PNAEP62-PHEAxdiblock copolymers via RAFT aqueous solution polymerization of HEA targeting PHEA DPs of between 50 and 400. A DDMAT/KPS molar ratio of 5.0 was used in all cases.1H NMR studies indicated that high HEA conversions (>99%) were achieved within 18 h. Furthermore, DMF GPC analysis of the resulting PNAEP62-PHEAxdiblock copolymers indicated a linear increase in Mnwith increasing PHEA DP. Relatively low dispersities (Mw/Mn<1.35) were obtained for all PNAEP62-PHEAxdiblock copolymers. Moreover, comparison with the GPC trace recorded for the PNAEP62macro-CTA confirmed high blocking efficiencies in each case. Synthesis of PNAEP71-POEGAyDiblock Copolymers Example 21: PNAEP71-POEGAyDiblock Copolymers with Target DP=100 PNAEP71macro-CTA (0.250 g, 21.3 μmol), OEGA (Mn≈454 g mol−1, 0.3872 g, 853 μmol; target DP=40) and AscAc (0.8 mg, 4.3 μmol) were weighed into a 14 mL vial charged with a magnetic flea (reaction solution 1). This vial was placed in an ice bath and the solution was degassed with nitrogen for 30 min. Deionized water (2.3066 g, corresponding to a 20% w/w solution), and KPS (1.2 mg, 4.3 μmol; PNAEP71macro-CTA/KPS molar ratio=5.0) were weighed into a separate 14 mL vial (reaction solution 2) and degassed with nitrogen using an ice bath for 30 min. Reaction solution 1 was immersed in an oil bath set at 30° C. Reaction solution 2 was then added to this vial via a degassed syringe and needle under nitrogen.1H NMR studies indicated more than 99% conversion while DMF GPC analysis indicated an Mnof 20,400 g mol−1and an Mw/Mnof 1.27. Example 22: PNAEP71-POEGAyDiblock Copolymers with Target DP Upto 400 PNAEP71macro-CTA was utilized for the RAFT aqueous solution polymerization of OEGA, targeting POEGA DPs of between 50 and 400 and using a DDMAT/KPS molar ratio of 5.0. OEGA conversions of at least 99% were achieved for all PNAEP71-POEGAxdiblock copolymers within 18 h at 30° C., as judged by1H NMR. DMF GPC analyses of this series of PNAEP71-POEGAxdiblock copolymers indicated a monotonic increase in Mnwith increasing POEGA DP, as expected. Relatively low dispersities (Mw/Mn<1.30) were achieved for this PNAEP71-POEGAxdiblock copolymer series, which suggests good RAFT control. Moreover, comparison of the GPC traces obtained for these PNAEP71-POEGAxdiblock copolymers with that of the precursor PNAEP71macro-CTA indicated high blocking efficiencies. Synthesis of PNAEP95-PNIPAMxDiblock Copolymers Example 23: PNAEP95-PNIPAMyDiblock Copolymers with Target DP=100 to 300 The RAFT polymerization of NIPAM was conducted in an oil bath set to 22° C., which is below the LCST of PNIPAM homopolymer (˜32° C.). NIPAM conversions of at least 99% were achieved for all PNAEP95-PNIPAMydiblock copolymers within 1 h at this temperature, as judged by1H NMR studies conducted in D2O. DMF GPC analysis of this series of PNAEP95-PNIPAMydiblock copolymers indicated a monotonic increase in Mnwith increasing PNIPAM DP. Relatively low dispersities (Mw/Mn<1.40) were observed in all cases, indicating reasonably good RAFT control. Moreover, comparison of the GPC traces obtained for these PNAEP95-PNIPAMydiblock copolymers with that of the precursor PNAEP95macro-CTA indicated relatively high blocking efficiencies. Example 24: Preparation of PDEAX Macro-CTA DEA (10.00 g, 54.0 mmol), MPETTC RAFT agent (244.1 mg, 0.540 mmol; target DP=100), ACVA (50.4 mg, 180 μmol; MPETTC/ACVA molar ratio=3.0) and THF (6.86 g, corresponding to a 60% w/w solution) were weighed into a 50 mL round-bottom flask charged with a magnetic flea. This flask was placed in an ice bath and degassed with nitrogen for 30 min before being immersed in an oil bath set at 70° C. The polymerization proceed for 190 min, affording a monomer conversion of 95% as judged by 11-1 NMR. The crude homopolymer was purified by precipitation into a ten-fold excess of mildly alkaline water (pH 10). This neutral PDEA homopolymer was then dried under vacuum before being protonated using an aqueous solution of 1.0 M HCl. The fully protonated PDEA homopolymer was isolated in its HCl salt via precipitation into a ten-fold excess of acetone. This homopolymer was then dried in a vacuum oven to afford a PDEA macro-CTA containing less than 1% residual monomer. Its mean degree of polymerization was determined to be 99 by1H NMR spectroscopy. Chloroform GPC analysis indicated an Mnof 10,800 g mol−1and an Mw/Mnof 1.24. Example 25: Synthesis of PDEA100-PNAEPyDiblock Copolymers PDEA100macro-CTA (200 mg, 10.5 NAEP (190 mg, 1.054 mmol; target DP=100) and AscAc (0.37 mg, 2.1 μmol) were weighed into a 14 mL vial charged with a magnetic flea (reaction solution 1). This vial was immersed in an ice bath and degassed with nitrogen for 30 min. Dilute aqueous HCl (0.001 M, 1.12 g), and KPS (57 mg, 2.1 μmol; PDEA100macro-CTA/KPS molar ratio=3.0) were weighed into a separate 14 mL vial (reaction solution 2; final pH 2), which was immersed in an ice bath and degassed with nitrogen for 30 min. The vial containing reaction solution 1 was then immersed in an oil bath set at 30° C. Reaction solution 2 was added to this vial using a degassed syringe/needle under nitrogen to afford a final solution at pH 2 targeting 30% w/w solids.1H NMR studies indicated that an NAEP conversion of 99% was achieved after 120 min. Other diblock copolymer compositions were obtained by adjusting the NAEP/PDEA100macro-CTA molar ratio to give target PNAEP DPs ranging from 50 to 100. Example 26: Preparation of PNAEP67Macro-CTA NAEP (10.00 g, 54.6 mmol), DDMAT RAFT agent (199.0 mg, 0.546 mmol; target DP=100), and AscAc (1.0 mg, 5.5 μmol) were weighed into a 14 mL vial charged with a magnetic flea (reaction solution 1). This reaction solution was then placed in an ice bath and degassed with nitrogen for 30 min. Deionised water (4.372 g, 70% w/w) and KPS (1.5 mg, 5.5 μmol; DDMAT/KPS molar ratio=100) were weighed into a second 14 mL vial (reaction solution 2) and degassed with nitrogen in an ice bath for 30 min. After 30 min, the vial containing reaction solution 1 was immersed in an oil bath set at 30° C. Reaction solution 2 was then added using a degassed syringe and needle to reaction solution 1 under nitrogen. The NAEP polymerisation proceeded for 8 min before being quenched via exposure to air and immersed in an ice bath.1H NMR analysis of the disappearance of vinyl signals indicated a monomer conversion of 62%. The crude PNAEP homopolymer was purified by dialysis against water (72 h) using a 3500 MWCO dialysis membrane (Fisher Scientific) to give a PNAEP macro-CTA containing less than 1% residual monomer. Its mean DP was calculated to be 67 as judged by1H NMR spectroscopy analysis in CDCl3. Chloroform GPC analysis indicated an Mnof 19.2 kg mol−1and an Mw/M, of 1.19. Example 27: Preparation of PNAEP67-PSyDiblock Copolymer A typical protocol used for the synthesis of the PNAEP67-PS350diblock copolymer was as follows: PNAEP67macro-CTA (0.185 g, 14.6 μmol), deionised water (2.880 g, corresponding to a 20% w/w solution) and VA-044 (1.580 mg, 4.9 μmol; PNAEP67/VA-044=3.0) were weighed into a 10 mL round-bottom flask charged with a magnetic flea. NaOH (20 μL, 1 M) was added to raise the pH to 7.0. This flask was then immersed in an ice bath and the solution was degassed with nitrogen for 30 min. Styrene (1.0 g) was weighed into a separate 14 mL vial and degassed with nitrogen in an ice bath for 30 min. After 30 min, styrene (0.59 ml, 5.12 mmol; target DP=350) was added to the flask using a degassed syringe and needle under nitrogen. The contents of the flask were then stirred vigorously to ensure thorough mixing and degassed for a further 5 min before being immersed in an oil bath set at 80° C. The styrene polymerisation was allowed to proceed for 2 h before being quenched by exposing the reaction solution to air and immersing the reaction vial in an ice bath.1H NMR spectroscopy analysis of the disappearance of vinyl signals indicated a final styrene conversion of 99%. Chloroform GPC analysis indicated a Mnof 46.6 kg mol−1and an Mw/Mnof 1.28. Other target diblock copolymer compositions were obtained by adjusting the styrene/PNAEP67molar ratio. Example 28: Preparation of PNAEP67-PnBAyDiblock Copolymer A typical protocol used for the synthesis of the PNAEP67-PnBA500diblock copolymer was as follows: PNAEP67macro-CTA (0.185 g, 14.6 μmol), deionised water (4.501 g, corresponding to a 20% w/w solution) and KPS (1.320 mg, 4.9 μmol; PNAEP67/KPS=3.0) were weighed into a 10 mL round-bottom flask charged with a magnetic flea. HCl (10 μL, 0.2 M) was added to reduce the pH to 3.0. This flask was then immersed in an ice bath, and the solution was degassed with nitrogen for 30 min. nBA (1.500 g) was weighed into a separate 14 mL vial and degassed with nitrogen in an ice bath for 30 min. An AsAc stock solution (0.01% w/w) was weighed into a second 14 mL vial and degassed with nitrogen in an ice bath for 30 min. After 30 min nBA (1.05 ml, 7.32 mmol; target DP=500) was added to the flask using a degassed syringe and needle under nitrogen. The flask contents were then stirred vigorously to ensure thorough mixing and degassed for 5 min before being immersed in an oil bath set at 30° C. After 1 min, AsAc (0.09 ml, 4.9 μmol; KPS/AscAc molar ratio=1.0) was added to the flask. The nBA polymerisation was allowed to proceed for 1 h before being quenched by exposing the reaction solution to air and immersing the reaction vial in an ice bath.1H NMR spectroscopy analysis of the disappearance of vinyl signals indicated a final nBA conversion of 99%. Chloroform GPC analysis of this copolymer indicated a Mnof 86.6 kg mol−1and an Mw/Mnof 1.56. Other diblock copolymer compositions were obtained by adjusting the nBA/PNAEP67molar ratio. Example 29: Preparation of PNAEP67-P(S-Stat-nBA)yDiblock Copolymer A typical protocol used for the synthesis of the PNAEP67-P(S-stat-nBA)400diblock copolymer was as follows: PNAEP67macro-CTA (0.185 g, 14.6 μmol), deionised water (3.472 g, corresponding to a 20% w/w solution) and KPS (1.320 mg, 4.9 μmol; PNAEP67/KPS=3.0) were weighed into a 10 mL round-bottom flask charged with a magnetic flea. HCl (10 μL, 0.2 M) was added to reduce the pH to 3.0. This flask was then immersed in an ice bath, and the solution was degassed with nitrogen for 30 min. nBA and styrene (1.500 g) were weighed into separate 14 mL vials and degassed with nitrogen in an ice bath for 30 min. An AsAc stock solution (0.01% w/w) was weighed into a second 14 mL vial and degassed with nitrogen in an ice bath for 30 min. After 30 min styrene (0.34 ml, 2.97 mmol) and nBA (0.41 ml, 2.89 mmol; overall copolymer DP=400, nBA content=55% by mass) was added to the flask using a degassed syringe and needle under nitrogen. The flask contents were then stirred vigorously to ensure thorough mixing and degassed for 5 min before being immersed in an oil bath set at 30° C. After 1 min, AscAc (0.09 ml, 4.9 μmol; KPS/AscAc molar ratio=1.0) was added to the flask. The polymerisation was allowed to proceed for 3 h before being quenched by exposing the reaction solution to air and immersing the reaction vial in an ice bath.1H NMR spectroscopy analysis of the disappearance of vinyl signals indicated a final comonomer conversion of 99% conversion. Chloroform GPC analysis of this copolymer indicated a Mnof 86.6 kg mol−1and an Mw/M, of 1.56. Other diblock copolymer compositions were obtained by adjusting the (styrene+nBA)/PNAEP67molar ratio. Example 30: Preparation of PNMEP28Macro-CTA The typical protocol for the preparation of PNMEP28macro-CTA is described below. NMEP (9.37 g, 47.4 mmol), CPDB RAFT agent (0.30 g, 1.36 mmol; target DP=35), ACVA (76.0 mg, 0.27 mmol; CPDB/ACVA molar ratio=5.0) and ethanol (14.59 g, 40% w/w solids) were weighed into a 50 mL round-bottom flask immersed in an ice bath and degassed with continuous stirring for 30 min. The reaction was allowed to proceed for 270 min in an oil bath set to 70° C., resulting in a monomer conversion of 90% as judged by1H NMR spectroscopy. The polymerization was then quenched by exposing the hot reaction solution to air and cooling to 20° C. The crude polymer was precipitated into excess diethyl ether to remove residual monomer before freeze-drying in the minimum amount of water to afford a dry pink powder. The mean DP was calculated to be 28 by comparing the integrated aromatic protons arising from the CPDB RAFT agent. GPC analysis using chloroform eluent indicated an Mnof 5000 g mol−1and Mw/Mnof 1.23 against a series of ten near-monodisperse poly(methyl methacrylate) calibration standards. Example 31: Preparation of PNMEP28-PLMAyDiblock Copolymer A typical protocol for the synthesis of PNMEP28-PLMA87(LMA/NMEP mass ratio=4:1) is described as follows: PNMEP28macro-CTA (0.15 g, 26.10 μmol), LMA (0.58 g, 2.27 mmol; target DP=87 and ACVA (1.50 mg, 5.22 μmol; 0.19 mL of a 7.89 g dm−3ethanolic stock solution; PNMEP28/ACVA molar ratio=5.0) were dissolved in an 80:20 w/w ethanol-water mixture (2.92 g). The reaction vial was sealed and degassed under N2for 30 min before placing in a pre-heated oil bath set at 70° C. for 16 h. The polymerization was quenched by exposing the hot reaction solution to air and cooling to 20° C. The resulting diblock copolymer nanoparticles were characterized by1H NMR spectroscopy, DLS and TEM with 0.01% w/w dispersions being prepared via dilution using an 80:20 w/w ethanol-water mixture. Chloroform GPC analysis indicated an Mnof 19 800 g mol−1and an Mw/Mnof 1.28. Other diblock compositions were synthesized by adjusting the amount of LMA monomer to target LMA/NMEP mass ratios ranging between 2:1 and 7:1. Table 2 shows the corresponding DPs of the PLMA blocks.1H NMR analysis indicated that more than 98% monomer conversion was achieved in all cases. TABLE 2Summary of the target diblock copolymer compositions. LMA monomer conversions,residual levels of NMEP and LMA monomer, molecular weight data and glass transitiontemperature (Tg) values.TargetdiblockLMAResidualResidualPLMAPNMEPcopolymerConversionaNMEPbLMAcMndMw/TgeTgExamplecomposition(%)ppmppmg/molMnd° C.° C.32PNMEPn.dn.dn.d5,0001.23n.d65macro-CTA33PNMEP28->99136—13,3001.22−4848PLMA4334PNMEP28->99309—17,1001.22−4845PLMA6535PNMEP28->9919998319,8001.28−4646PLMA8736PNMEP28-99132103722,1001.29−47—PLMA10937PNMEP28-99155115625,0001.34−48—PLMA13038PNMEP28-99168115326,1001.40−47—PLMA152n.d = not determinedaLMA conversion determined by1H NMR spectroscopy using a 10:1 d1-chlorofom:d6-acetone mixture.bDetermined by HPLC—Agilent Poroshell EC-C18 100 × 4.6 mm × 3.5 μm. 0.1% (v/v) aqueous orthophosphoric acid (A)/acetonitrile (B) (5% B to 100% B in 20 minutes, 2-minute hold at 100% B, re-equilibrate at 5% for 5 minutes), flow rate of 0.40 mL min−1.cDetermined by gas chromatography—Restek Rxi-624Sil-MS capillary column (30 m × 0.32 mm, Dt= 1.8 μm), H2carrier gas, 45.5 cm s−1. Oven programme- 2 min hold at 100° C., 10° C. min−1ramp to 300° C., 4-minute hold.dDetermined by chloroform GPC using a refractive index detector and expressed relative to a series of poly(methyl methacrylate) calibration standards.eDetermined by DSC at a rate of 10° C. min1 Copolymer Characterization 1H NMR Spectroscopy: 1H NMR spectra were recorded at 25° C. in d4-methanol using a 400 MHz Bruker Avance-400 spectrometer (64 scans averaged per spectrum). GPC: The molecular weights and dispersities of the homopolymers series and diblock copolymers were determined by using an Agilent 1260 infinity set-up comprising two Polymer Laboratories PL gel 5 μm Mixed-C columns and a refractive index detector operating at 60° C. The mobile phase was HPLC-grade DMF containing 10 mmol LiBr at a flow rate of 1.0 mL min−1. Ten near-monodisperse poly(methyl methacrylate) standards (PMMA; Mn=625 to 618,000 g mol−1) were used for calibration. The molecular weights and dispersities of the PDEA homopolymers were determined by using an Agilent 1260 infinity set-up comprising two Polymer Laboratories PL gel 5 μm Mixed-C columns and a refractive index detector operating at 35° C. The mobile phase was HPLC-grade chloroform containing 0.25% v/v TEA at a flow rate of 1.0 mL min−1. Ten near-monodisperse poly(methyl methacrylate) standards (PMMA; Mn=625 to 618,000 g mol−1) were used for calibration. Visible Absorption Spectroscopy. Spectra were recorded from 400 to 800 nm for 1.0% w/w aqueous solutions of various PNAEP and PNMEP homopolymers between 20 and 80° C. at 5° C. increments using a Shimadzu UV-1800 spectrometer. An increase in turbidity at 600 nm indicated the lower critical solution temperature (LCST) of the polymer. Spin-coated copolymer films were prepared by depositing a 200 μL aliquot of a 20% w/w aqueous dispersion onto a glass slide mounted on a vacuum-free Ossila Spin Coater (initially rotating at 250 rpm, followed by rapid acceleration up to 3000 rpm for 15 min). For transmittance studies, films were prepared as described above and their transparency was assessed by visible absorption spectroscopy using a Perkin-Elmer Lambda 25 spectrometer. Spectra were recorded from 200 to 800 nm at 2 nm intervals at a scan speed of 960 nm/min. Copolymer film thicknesses were measured using a micrometer screw gauge. Dynamic Light Scattering (DLS). DLS studies were conducted using a Malvern Instruments Zetasizer Nano series instrument equipped with a 4 mW He—Ne laser (λ=633 nm) and an avalanche photodiode detector. Scattered light was detected at 173°. Intensity-average hydrodynamic diameters were calculated via the Stokes-Einstein equation, while zeta potentials were determined via the Henry equation using the Smoluchowski approximation Transmission Electron Microscopy (TEM). As-prepared 20% w/w copolymer dispersions were diluted at 20° C. to generate 0.10% w/w aqueous dispersions. Copper/palladium TEM grids (Agar Scientific, UK) were coated in-house to produce thin films of amorphous carbon. These grids were then treated with a plasma glow discharge for 30 s to create a hydrophilic surface. One droplet of aqueous diblock copolymer dispersion (20 μL; 0.10% w/w) was placed on a freshly-treated grid for 1 min and then blotted with filter paper to remove excess solution. To stain the deposited nanoparticles, an aqueous solution of uranyl formate (10 μL; 0.75% w/w) was placed on the sample-loaded grid via micropipet for 20 s and then carefully blotted to remove excess stain. Each grid was then dried using a vacuum hose. Imaging was performed using a Philips CM100 instrument operating at 100 kV and equipped with a Gatan 1 k CCD camera. Small-Angle X-Ray Scattering (SAXS). SAXS patterns were recorded at a national synchrotron facility (station 122, Diamond Light Source, Didcot, Oxfordshire, UK) using monochromatic X-ray radiation (λ=0.124 nm with q ranging from 0.01 to 2.00 nm−1where q=4π sin θ/λ is the length of the scattering vector and θ is one-half of the scattering angle) and a 2D Pilatus 2M pixel detector (Dectris, Switzerland). A glass capillary of 2 mm diameter was used as a sample holder and all measurements were conducted on 1.0% w/w copolymer dispersions in 80:20 w/w ethanol-water. X-ray scattering data were reduced and normalized using standard routines by the beamline and were further analyzed using Irena SAS macros for Igor Pro. Reversed Phase High Performance Liquid Chromatography (Reversed Phase HPLC). HPLC analysis was performed on an HP 1100 series LC equipped with a quadratic pump, an autosampler and a diode array detector. An Agilent Poroshell EC-C18 100×4.6 mm column with a particle size of 3.5 μm was used at 40° C. The mobile phase consisted of water with 0.1% (v/v) orthophosphoric acid (A) and acetonitrile (B) and was run under gradient conditions (5% B to 100% B in 20 minutes, 2-minute hold at 100% B, re-equilibrate at 5% for 5 minutes) at a flow rate of 0.40 mL min−1, a run time of 27 min and an injection volume of 5 μL. The analyte was detected at a wavelength of 210 nm, against a 360 nm reference wavelength. Nanoparticle dispersions were diluted to 2.0% w/w using deionized water. The resulting dispersions were shaken for 20 minutes and decanted into centrifugal cut-off filters (Merck Amicon Ultra-4, 3 KDa nominal molecular weight) to remove high molecular weight polymeric material. These were centrifuged at an RCF of 8422 g (9000 rpm; rotor radius=9.3 cm) for 20 min to produce 4 ml approx. aqueous filtrate for evaluation and quantitation of residual NMEP monomer. Concentration was measured based the detector response of external NMEP standards of known concentration. Differential Scanning Calorimetry (DSC). DSC studies were performed using a TA Instruments Discovery DSC instrument equipped with TZero low-mass aluminium pans and hermetically-sealed lids. Copolymers (and homopolymers) were equilibrated above their glass transition temperatures for 10 min before performing two consecutive thermal cycles at a rate of 10° C. min−1. Two cycles were performed to minimise the thermal history of each sample. Molecular weight data for both the PNMEP homopolymer precursor and the series of PNMEP28-PLMAydiblock copolymers were obtained using chloroform GPC at 35° C., with the eluent containing 0.25% TEA by volume. Two Polymer Laboratories PL gel 5 μm Mixed C columns were connected in series to a Varian 390 multidetector suite (refractive index detector) and a Varian 290 LC pump injection module using a flow rate of 1.0 mL min−1. Ten near-monodisperse poly(methyl methacrylate) standards (PMMA; Mn=625-618000 g mol−1) were used for calibration and data were analysed using Varian Cirrus GPC software. UV GPC chromatograms were obtained simultaneously by detection at a fixed wavelength of 308 nm, which corresponds to the absorption maximum of the dithiobenzoate RAFT end-groups. Glass transition temperatures for six PNMEP28-PLMAydiblock copolymers were determined using a Pyris 1 Perkin-Elmer differential scanning calorimeter operating over a temperature range from −90 to 100° C. at a heating/cooling rate of 10° C. min−1. Each copolymer (10 mg) was dried for at least 24 h in a vacuum oven at 70° C. prior to analysis. Dried samples were hermetically sealed in a vented aluminum pan, and the instrument was calibrated for heat flow and temperature using both indium and zinc standards. Samples were annealed at 100° C. for 5 min before cooling to −90° C. and maintaining this temperature for 1 min. The glass transition temperature (Tg) was then determined by heating the copolymer up to 100° C. | 62,253 |
11859030 | EXAMPLES 1. Measuring Methods The following definitions of terms and determination methods apply for the above general description of the invention as well as to the below examples unless otherwise defined. Calculation of comonomer content of the second propylene copolymer fraction (R-PP2): C(PP)-w(PP1)×C(PP1)w(PP2)=C(PP2)(I)whereinw(PP1) is the weight fraction [in wt.-%] of the first propylene copolymer fraction (R-PP1),w(PP2) is the weight fraction [in wt.-%] of second propylene copolymer fraction (R-PP2),C(PP1) is the comonomer content [in mol-%] of the first propylene copolymer fraction (R-PP1),C(PP) is the comonomer content [in mol-%] of the random propylene copolymer (R-PP),C(PP2) is the calculated comonomer content [in mol-%] of the second propylene copolymer fraction (R-PP2). Calculation of the xylene cold soluble (XCS) content of the second propylene copolymer fraction (R-PP2): XS(PP)-w(PP1)×XS(PP1)w(PP2)=XS(PP2)(II)whereinw(PP1) is the weight fraction [in wt.-%] of the first propylene copolymer fraction (R-PP1),w(PP2) is the weight fraction [in wt.-%] of second propylene copolymer fraction (R-PP2),XS(PP1) is the xylene cold soluble (XCS) content [in wt.-%] of the first propylene copolymer fraction (R-PP1),XS(PP) is the xylene cold soluble (XCS) content [in wt.-%] of the random propylene copolymer (R-PP),XS(PP2) is the calculated xylene cold soluble (XCS) content [in wt.-%] of the second propylene copolymer fraction (R-PP2), respectively. Calculation of melt flow rate MFR2(230° C./2.16 kg) of the second propylene copolymer fraction (R-PP2): MFR(PP2)=10[log(MFR(PP))-w(PP1)×log(MFR(PP1))w(PP2)](III)whereinw(PP1) is the weight fraction [in wt.-%] of the first propylene copolymer fraction (R-PP1),w(PP2) is the weight fraction [in wt.-%] of second propylene copolymer fraction (R-PP2),MFR(PP1) is the melt flow rate MFR2(230° C./2.16 kg) [in g/10 min] of the first propylene copolymer fraction (R-PP1),MFR(PP) is the melt flow rate MFR2(230° C./2.16 kg) [in g/10 min] of the random propylene copolymer (R-PP),MFR(PP2) is the calculated melt flow rate MFR2(230° C./2.16 kg) [in g/10 min] of the second propylene copolymer fraction (R-PP2). Calculation of comonomer content of the elastomeric propylene copolymer (E), respectively: C(RAHECO)-w(PP)×C(PP)w(E)=C(E)(IV)whereinw(PP) is the weight fraction [in wt.-%] of the random propylene copolymer (R-PP), i.e. polymer produced in the first and second reactor (R1+R2),w(E) is the weight fraction [in wt.-%] of the elastomeric propylene copolymer (E), i.e. polymer produced in the third reactor (R3)C(PP) is the comonomer content [in mol-%] of the random propylene copolymer (R-PP), i.e. comonomer content [in mol-%] of the polymer produced in the first and second reactor (R1+R2),C(RAHECO) is the comonomer content [in mol-%] of the propylene copolymer, i.e. is the comonomer content [in mol-%] of the polymer obtained after polymerization in the third reactor (R3),C(E) is the calculated comonomer content [in mol-%] of elastomeric propylene copolymer (E), i.e. of the polymer produced in the third reactor (R3). MFR2(230° C./2.16 kg) is measured according to ISO 1133 at 230° C. and 2.16 kg load. Quantification of Microstructure by NMR Spectroscopy Quantitative nuclear-magnetic resonance (NMR) spectroscopy was used to quantify the comonomer content and comonomer sequence distribution of the polymers. Quantitative13C{1H} NMR spectra were recorded in the solution-state using a Bruker Advance III 400 NMR spectrometer operating at 400.15 and 100.62 MHz for1H and13C respectively. All spectra were recorded using a13C optimised 10 mm extended temperature probehead at 125° C. using nitrogen gas for all pneumatics. Approximately 200 mg of material was dissolved in 3 ml of 1,2-tetrachloroethane-d2(TCE-d2) along with chromium-(III)-acetylacetonate (Cr(acac)3) resulting in a 65 mM solution of relaxation agent in solvent (Singh, G., Kothari, A., Gupta, V., Polymer Testing 28 5 (2009), 475). To ensure a homogenous solution, after initial sample preparation in a heat block, the NMR tube was further heated in a rotatary oven for at least 1 hour. Upon insertion into the magnet the tube was spun at 10 Hz. This setup was chosen primarily for the high resolution and quantitatively needed for accurate ethylene content quantification. Standard single-pulse excitation was employed without NOE, using an optimised tip angle, 1 s recycle delay and a bi-level WALTZ16 decoupling scheme (Zhou, Z., Kuemmerle, R., Qiu, X., Redwine, D., Cong, R., Taha, A., Baugh, D. Winniford, B., J. Mag. Reson. 187 (2007) 225; Busico, V., Carbonniere, P., Cipullo, R., Pellecchia, R., Severn, J., Talarico, G., Macromol. Rapid Commun. 2007, 28, 1128). A total of 6144 (6k) transients were acquired per spectra. Quantitative13C{1H} NMR spectra were processed, integrated and relevant quantitative properties determined from the integrals using proprietary computer programs. All chemical shifts were indirectly referenced to the central methylene group of the ethylene block (EEE) at 30.00 ppm using the chemical shift of the solvent. This approach allowed comparable referencing even when this structural unit was not present. Characteristic signals corresponding to the incorporation of ethylene were observed Cheng, H. N., Macromolecules 17 (1984), 1950). With characteristic signals corresponding to 2,1 erythro regio defects observed (as described in L. Resconi, L. Cavallo, A. Fait, F. Piemontesi, Chem. Rev. 2000, 100 (4), 1253, in Cheng, H. N., Macromolecules 1984, 17, 1950, and in W-J. Wang and S. Zhu, Macromolecules 2000, 33 1157) the correction for the influence of the regio defects on determined properties was required. Characteristic signals corresponding to other types of regio defects were not observed. The comonomer fraction was quantified using the method of Wang et. al. (Wang, W-J., Zhu, S., Macromolecules 33 (2000), 1157) through integration of multiple signals across the whole spectral region in the13C{1H} spectra. This method was chosen for its robust nature and ability to account for the presence of regio-defects when needed. Integral regions were slightly adjusted to increase applicability across the whole range of encountered comonomer contents. For systems where only isolated ethylene in PPEPP sequences was observed the method of Wang et. al. was modified to reduce the influence of non-zero integrals of sites that are known to not be present. This approach reduced the overestimation of ethylene content for such systems and was achieved by reduction of the number of sites used to determine the absolute ethylene content to: E=0.5(Sββ+Sβγ+Sβδ+0.5(Sαβ+Sαγ)) Through the use of this set of sites the corresponding integral equation becomes: E=0.5(IH+IG+0.5(IC+ID)) using the same notation used in the article of Wang et. al. (Wang, W-J., Zhu, S., Macromolecules 33 (2000), 1157). Equations used for absolute propylene content were not modified. The mole percent comonomer incorporation was calculated from the mole fraction: E[mol %]=100*fE The weight percent comonomer incorporation was calculated from the mole fraction: E[wt %]=100*(fE*28.06)/((fE*28.06)+((1−fE)*42.08)) The comonomer sequence distribution at the triad level was determined using the analysis method of Kakugo et al. (Kakugo, M., Naito, Y., Mizunuma, K., Miyatake, T. Macromolecules 15 (1982) 1150). This method was chosen for its robust nature and integration regions slightly adjusted to increase applicability to a wider range of comonomer contents. The relative content of isolated to block ethylene incorporation was calculated from the triad sequence distribution using the following relationship (equation (I)): I(E)=fPEP(fEEE+fPEE+fPEP)×100(I)whereinI(E) is the relative content of isolated to block ethylene sequences [in %];fPEP is the mol fraction of propylene/ethylene/propylene sequences (PEP) in the sample;fPEE is the mol fraction of propylene/ethylene/ethylene sequences (PEE) and of ethylene/ethylene/propylene sequences (EEP) in the sample;fEEE is the mol fraction of ethylene/ethylene/ethylene sequences (EEE) in the sample. Intrinsic viscosity is measured according to DIN ISO 1628/1, October 1999 (in Decalin at 135° C.). The xylene cold solubles (XCS, wt.-%): Content of xylene cold solubles (XCS) is determined at 25° C. according ISO 16152; first edition; 2005 Jul. 1. The part which remains insoluble is the xylene cold insoluble (XCI) fraction. 2. Examples The catalysts used in the polymerization processes for the heterophasic propylene copolymers of the present invention were prepared as follows: Reference Catalyst Preparation of the solid catalyst component Used Chemicals:TiCl4(CAS 7550-45-90) was supplied by commercial source.20% solution in toluene of butyl ethyl magnesium (Mg(Bu)(Et)), provided by Crompton2-ethylhexanol, provided by Merck Chemicals3-Butoxy-2-propanol, provided by Sigma-Aldrichbis(2-ethylhexyl)citraconate, provided by Contract ChemicalsViscoplex® 1-254, provided by EvonikHeptane, provided by Chevron Preparation of Mg Complex 3.4 litre of 2-ethylhexanol and 810 ml of propylene glycol butyl monoether in a molar ratio 4/1) were added to a 20% reactor. Then, 7.8 litre of a 20% solution in toluene of BEM (butyl ethyl magnesium) provided by Crompton GmbH was slowly added to the well stirred alcohol mixture. During the addition, the temperature was kept at 10° C. After addition, the temperature of the reaction mixture was raised to 60° C. and mixing was continued at this temperature for 30 minutes. Finally, after cooling to room temperature the obtained Mg-alkoxide was transferred to a storage vessel. 21.2 g of Mg alkoxide prepared above was mixed with 4.0 ml bis(2-ethylhexyl) citraconate for 5 min. After mixing the obtained Mg complex was used immediately in the preparation of catalyst component. Preparation of the Solid Catalyst Component 19.5 ml titanium tetrachloride was placed in a 300 ml reactor equipped with a mechanical stirrer at 25° C. Mixing speed was adjusted to 170 rpm. 26.0 of Mg-complex prepared above was added within 30 minutes keeping the temperature at 25° C. 3.0 ml of Viscoplex 1-254 and 24.0 ml of heptane were added to form an emulsion. Mixing was continued for 30 minutes at 25° C. Then, the reactor temperature was raised to 90° C. within 30 minutes. The reaction mixture was stirred for further 30 minutes at 90° C. Afterwards, stirring was stopped and the reaction mixture was allowed to settle for 15 minutes at 90° C. The solid material was washed with 100 ml of toluene, with of 30 ml of TiCl4, with 100 ml of toluene and two times with 60 ml of heptane. 1 ml of donor was added to the two first washings. Washings were made at 80° C. under stirring 30 min with 170 rpm. After stirring was stopped, the reaction mixture was allowed to settle for 20-30 minutes and followed by siphoning. Afterwards, stirring was stopped and the reaction mixture was allowed to settle for 10 minutes decreasing the temperature to 70° C. with subsequent siphoning, and followed by N2sparging for 20 minutes to yield an air sensitive powder. Catalyst has a surface area measured by BET method below 5 m2/g, i.e. below the detection limit. Ti content was 2.6 wt-%. Inventive Catalyst The catalyst of the Reference Catalyst was batch-mode prepolymerised with 1-butene in the presence of dicyclopentyl dimethoxy silane as the external donor (ED) and cocatalyst (TEAL) in a catalyst vessel under nitrogen blanket at a temperature of 20-30° C. with Al/Ti molar ratio of 1 and Al/ED molar ratio of 0.75. The weight ratio of 1-butene/catalyst in the vessel was 2/1 resulting in a batch-mode prepolymerized catalyst with a polymerization degree of 2 g polymer/1 g catalyst (100% conversion). Comparative Example 1 The catalyst prepared according to the Reference catalyst was used as such (=comparative catalyst) along with triethyl-aluminium (TEAL) as co-catalyst and dicyclopentyl dimethoxy silane as external donor (ED) in the polymerization process. Polymerisation conditions and results are disclosed in Table 1. Reference Example—Propylene Homopolymerization The inventive catalyst was used in a process for producing a propylene homopolymer in a process comprising a process prepolymerization step, polymerization in a loop reactor followed by polymerization in a gas phase reactor. No comonomers were added into the process and no external donor was used. Conditions and results are disclosed in Table 1. Inventive Example 1 The actual polymerization of propylene with ethylene to produce the heterophasic propylene copolymer was carried out using the Inventive catalyst without an external donor. Conditions and results are disclosed in Table 1. Based on the XCS of reference example (homoPP), it has been simulated how much the XCS would increase if there is no external donor present in the polymerisation process for producing heterophasic polymerisation using Inventive catalyst. TABLE 1Conditions and resultsComparativeInventiveReferenceExample 1Example 1exampleExampleheterophasic PPheterophasic PPhomoPPPrepolymerizationCatalystComparativeInventive catalystInventivecatalystcatalystCatalyst feed g/h21.82.3Cocatalyst, TEAL feed180180150g/t C3ED feed g/t C320No feedNo feedB1 Temperature (° C.)303030B1 Residence time (h)0.330.330.33LoopB2 Temperature (° C.)707080B2 H2/C3 ratio0.90.90.5(mol/kmol)B2 C2/C3 ratio4.24.00(mol/kmol)B2 Split %373565B2 MFR2 (g/10 min)6.97.56.9B2 XCS (%)6.885.0B2 C2 content (%)2.42.150GPR1B3 Temperature (° C.)808080B3 H2/C3 ratio2.72.68.0(mol/kmol)B3 C2/C3 ratio45.346.60(mol/kmol)B3 Split %333535B3 MFR2 (g/10 min)1.81.99.6B3 XCS (%)21.327.04.2B3 Ethene content (%)6.97.40GPR2B4 Temperature (° C.)7070—B4 C2/C3 ratio329.2303.2—(mol/kmol)B4 H2/C2 ratio79.385.7—(mol/kmol)B4 Split %3030—B4 MFR2 (g/10 min)1.01.0—B4 XCS (%)38.952.0—B4 Ethene content (%)14.914.4—B4 Viscosity of AM3.12.9—(dl/g)B4 Ethene of AM (%)30.633—FinalPP Mixer MFR2 (g/101.01.09.8productmin)PP Mixer viscosity of3.03.0AM (dl/g)PP Mixer ethene14.814.60content (%)PP Mixer XCS (%)38.552.03.8PP Mixer BD376361425PP Mixer PSD avg0.70.80.8 As can be seen from the examples, using the batch-prepolymerized catalyst of the invention it is possible to produce heterophasic propylene-ethylene polymers with a similar ethylene content, but still having a higher XCS. Because no external donor is needed in the actual polymerization stage (comprising also the process prepolymerization step) the polymer has higher purity as the compounds used as external donor, which is desired in some applications such as in food and in the mechanical field. | 14,680 |
11859031 | DETAILED DESCRIPTION The Summary, Claims, and Abstract are incorporated here by reference. Certain embodiments are described below as numbered aspects for easy cross-referencing. Embodiments of the invention provide an alternative non-MCN precatalyst composition, which has two silicon-free organic solubilizing groups. The composition beneficially has a significantly increased solubility in alkanes and/or a significantly increased catalyst light-off, both compared to those of HN5Zr dibenzyl. Aspect 1. A compound of formula (1), drawn above, wherein M is Zr or Hf and each R independently is methyl, an unsubstituted (C2-C4)alkyl group, an unsubstituted (C5-C12)alkyl group (e.g., an unsubstituted (C5-C9)alkyl group or an unsubstituted (C10-C12)alkyl group), an unsubstituted or substituted quaternary-arylalkyl group, or both R groups are bonded together to give R′-R′, wherein R′-R′ is an unsubstituted or substituted (aryl)alkylene. Each R group and R′-R′ group is free of a cyclopentadienyl group, a silicon atom, a carbon-carbon double bond, and a carbon-carbon triple bond. Each substituent independently may be selected from unsubstituted (C1-C5)alkyl, halogen, —Oalkyl, and —N(alkyl)2. Each quaternary-arylalkyl group sequentially contains a quaternary alkyl, a phenylene, and a (C1-C3)alkylene linker. The quaternary alkyl is bonded to the phenylene, which is bonded to the (C1-C3)alkylene linker, which is bonded to the metal M. The (C1-C3)alkylene linker and R′-R′ groups are divalent. The quaternary alkyl contains a quaternary carbon atom, which may be directly or indirectly bonded to the phenylene. A quaternary carbon atom is an element having atomic number 6 in the Periodic Table of the Elements that is bonded to four other carbon atoms. Aspect 2. The compound of aspect 1 wherein each R independently is a quaternary-(aryl)alkyl group of formula —(C(RA)2)mQCR1R2R3, wherein subscript m is 1, 2, or 3; wherein each RAindependently is H or (C1-C3)alkyl; wherein each Q independently is absent, a (C1-C3)alkylene, or an unsubstituted or substituted phenylene; wherein each R1, R2, and R3is independently H or a (C1-C15)alkyl that independently is unsubstituted or substituted; wherein each substituted group independently has one or more substituents independently selected from unsubstituted (C1-C5)alkyl, halogen, —Oalkyl, and —N(alkyl)2. In some aspects at least one, alternatively two, alternatively each of R1, R2, and R3is independently a (C1-C15)alkyl. In some aspects, with the proviso that when subscript m is 2, the resulting (C(RA)2)mis not C(RA)2CH(RA) or C(RA)2CH2; and when subscript m is 3, the resulting (C(RA)2)mis not C(RA)2CH(RA)C(RA)2or C(RA)2CH2C(RA)2. The optional proviso is intended to exclude compounds that may be prone to undergoing beta-hydride elimination. In some aspects subscript m is 2, alternatively 1. In some aspects each RAindependently is H or unsubstituted (C1-C2)alkyl, alternatively H or methyl, alternatively H. In some aspects each Q is absent. In some aspects at least one, alternatively each Q is present. When each Q is present, each Q independently may be a (C1-C3)alkylene, alternatively CH2, alternatively CH2CH2, alternatively CH2CH2CH2, alternatively CH2CH(CH3). Alternatively each Q independently may be unsubstituted 1,4-phenylene, unsubstituted 1,3-phenylene, or 1,2-phenylene; alternatively unsubstituted 1,2-phenylene; alternatively unsubstituted 1,3-phenylene; alternatively unsubstituted 1,4-phenylene. The 1,2-phenylene is benzene-1,2-diyl; 1,3-phenylene is benzene-1,3-diyl; and 1,4-phenylene is benzene-1,4-diyl. The “unsubstituted phenylene” means the phenylene is of formula C6H4. In some aspects each R group is unsubstituted. Aspect 3. The compound of aspect 1 or 2 wherein at least one, alternatively each R is independently —CH2QCR1R2R3; wherein each Q independently is unsubstituted phenylene; wherein each R1, R2, and R3is independently an unsubstituted (C1-C15)alkyl. Aspect 4. The compound of aspect 2 or 3 wherein at least one, alternatively each R is —CH2-(unsubstituted phenylene)-CR1R2R3; wherein each unsubstituted phenylene is unsubstituted 1,4-phenylene, unsubstituted 1,3-phenylene, or unsubstituted 1,2-phenylene; wherein each R1, R2, and R3is independently unsubstituted (C1-C15)alkyl, alternatively (C1-C3)alkyl, alternatively methyl. In some aspects one R is the —CH2CR1R2R3and the other R is an unsubstituted (C1-C15)alkyl. In some aspects the phenylene is (i) unsubstituted 1,4-phenylene; (ii) unsubstituted 1,3-phenylene; or (iii) unsubstituted 1,2-phenylene. In some aspects the phenylene is unsubstituted 1,4-phenylene. Aspect 5. The compound of aspect 1 wherein each R independently is methyl, an unsubstituted (C2-C4)alkyl group, or an unsubstituted (C5-C12)alkyl group (e.g., an unsubstituted (C5-C9)alkyl group). In some aspects each R is methyl, alternatively each R is an unsubstituted (C2-C4)alkyl group, alternatively each R is an unsubstituted (C5-C9)alkyl group, alternatively one R is methyl and the other R is an unsubstituted (C5-C9)alkyl group. In some aspects the unsubstituted (C5-C9)alkyl group is 2,2-dimethylpropyl (neopentyl). Aspect 6. The compound of aspect 1 wherein both R groups are bonded together to give R′-R′, wherein R′-R′ is an unsubstituted or substituted alkylene, alternatively a substituted (C4-C5)alkylene. In some aspects R′-R′ is —(CH2)3C(H)(R4)CH2— or —CH2(C(R4)))2CH2—, wherein each R4independently is an unsubstituted (C1-C5)alkyl. The R′-R′ may be 2,2,3,3-tetramethylbutane-1,4-diyl or 2-(2′,2′-dimethylpropyl)-pentane-1,5-diyl. Aspect 7. The compound of aspect 1 wherein both R groups are bonded together to give R′-R′, wherein R′-R′ is a substituted arylalkylene, alternatively a 4-(unsubstituted (C1-C5)alkyl)-1,2-bezenedimethylene. The 4-(unsubstituted (C1-C5)alkyl)-1,2-bezenedimethylene is —CH2-[4-(unsubstituted (C1-C5)alkyl-(1,2-phenylene)]-CH2—. In some aspects the 4-(unsubstituted (C1-C5)alkyl)-1,2-bezenedimethylene is 4-(2,2-dimethylpropyl)-1,2-benzenedimethylene (i.e., —CH2-[4-(CH3C(CH3)2CH2)-(1,2-phenylene)]-CH2—). Aspect 8. The compound of any one of aspects 1 to 7 wherein M is Zr. In other aspects M is Hf. Aspect 9. The compound of any one of aspects 1 to 8 characterized by solubility in hexanes containing at least 60 weight percent n-hexane (CH3(CH2)4CH3) of at least 0.10 weight percent based on total weight of the compound and hexanes. Aspect 10. A compound of formula (1A), (1B), (1C), or (1D): Aspect 11. A method of synthesizing the compound of formula (1) of any one of aspects 1 to 10, the method comprising contacting a compound of formula (2) wherein M is as defined for compound (1) and each X independently is C1, Br, or I, with an organometallic reagent of formula X1MgR or M1Rn; wherein R is as defined for compound (1) according to any one of aspects 1 to 10; X1is Cl, Br, or I; M1is selected from Li, Zn, Sn, and Cu; and subscript n is an integer from 1 to 4 and is equal to the formal oxidation state of M1; in an aprotic solvent under effective reaction conditions, thereby synthesizing the compound of formula (1). In some aspects the organometallic reagent X1MgR is used and is X1MgC((RA)2)mQCR1R2R3, alternatively X1MgCH2QCR1R2R3, alternatively X1MgCH2C(CH3)3, alternatively 2,2-dimethylpropylmagnesium chloride or 4-tert-butylbenzylmagnesium chloride; wherein X1is Cl or Br, alternatively Cl, alternatively Br. In some aspects the organometallic reagent M1Rnis used and is M1(C((RA)2)mQCR1R2R3)n, alternatively M1(CH2QCR1R2R3)n, alternatively M1(CH2QC(CH3)3)n, alternatively 2,2-dimethylpropyl lithium or 4-tert-butylbenzyl lithium; wherein M1is Li. In some aspects the molar ratio of moles of compound (2) to moles of the organometallic halide reagent is from 1:2 to 1:10. Aspect 12. The method of aspect 11 further comprising a preliminary step of contacting a compound of formula (3): wherein each R10independently is (C1-C15)alkyl, alternatively (C1-C6)alkyl, with a reagent of formula X—C(CH3)3, wherein X is as defined for the compound (2), in an aprotic solvent under effective reaction conditions to synthesize the compound (2). In some aspects reagent X—C(CH3)3is tert-butyl chloride, tert-butyl bromide or tert-butyl iodide; alternatively tert-butyl chloride (also known as 1-chloro-2,2-dimethylpropane). Aspect 13. The method of aspect 12, further comprising a preliminary step of contacting a compound of formula (4): with a reagent of formula M(N(R10)2)4, wherein M is as defined for compound (1) and each R10independently is (C1-C15)alkyl, in an aprotic solvent under effective reaction conditions to synthesize the compound (3). In some aspects each R10independently is alternatively (C1-C6)alkyl, alternatively methyl or ethyl, alternatively methyl. In some aspects the compound being synthesized in aspects 11 to 13 is the compound of any one of aspects 1 to 10. The molar ratio of compound (4) to M(N(R10)2)4may be from 1:10 to 10:1, alternatively from 1:5 to 5:1, alternatively from 1:2 to 2:1, alternatively 1:1. In the Examples described later, compounds (1A) to (1C) were synthesized according to the method of aspect 11. Compound (1 D) was synthesized directly from compound (4). Aspect 14. A solution of the compound of any one of aspects 1 to 10 in an alkane, wherein the solution is a liquid at 25 degrees Celsius and 101 kilopascals and the concentration of the compound in the solution is at least 0.10 weight percent based on weight of the solution. The alkane may be hexanes, isopentane, a mineral oil, or a combination of any two or more thereof. The alkane may be hexanes and/or isopentane, alternatively hexanes and/or a mineral oil, alternatively isopentane and/or a mineral oil. Aspect 15. A catalyst system comprising, or made from, a compound of any one of aspects 1 to 10, an activator, optionally a hydrocarbon solvent, and optionally a support material. The catalyst system may be a homogeneous catalyst system (one phase) or a heterogeneous catalyst system (two phase). The activator may be an alkylaluminoxane or a trialkylaluminum compound. In some aspects the catalyst system comprises the support material, and the support material is an untreated silica, alternatively a calcined untreated silica, alternatively a hydrophobing agent-treated silica, alternatively a calcined and hydrophobing agent-treated silica. In some aspects the hydrophobing agent is dichlorodimethylsilane. The catalyst system is useful as an olefin polymerization catalyst system in solution phase, slurry phase, and gas phase polymerization reactions, such as may be used for making polyethylene polymers or polypropylene polymers. In some aspects the formulation is free of Cr, Ti, Mg, or an unsubstituted or substituted cyclopentadienyl group; alternatively Cr, Ti, and Mg; alternatively an unsubstituted or substituted cyclopentadienyl group. Aspect 16. The catalyst system of aspect 15 further comprising a metallocene precatalyst, or a product of an activation reaction of the metallocene precatalyst and an activator. Examples of such metallocene precatalysts are described later. The activator contacting the metallocene precatalyst may be the same as, alternatively different than the activator contacting the compound (1). In some aspects the metallocene precatalyst or product of activation thereof further comprises a support material, which may be the same as or different than the optional support material for compound (1). Aspect 17. A method of making a polyolefin polymer, the method comprising contacting the catalyst system of aspect 15 or 16 with at least one olefin monomer selected from ethylene, propylene, a (C4-C20)alpha-olefin, and 1,3-butadiene in a polymerization reactor under effective polymerization conditions, thereby making the polyolefin polymer. In some aspects the at least one olefin monomer is ethylene and optionally a (C4, C6, or C8)alpha-olefin. The polymerization reactor may be a reactor configured for solution phase polymerization, slurry phase polymerization, or gas phase polymerization of the at least one olefin monomer. The reactors and effective polymerization conditions for solution phase polymerization, slurry phase polymerization, or gas phase polymerization are well known. Without wishing to be bound by theory, it is believed that the quaternary-hydrocarbyl groups, R, impart enhanced solubility of compound (1) in alkanes. The enhanced solubility may be characterized for comparison purposes as solubility of compound (1) in hexanes containing at least 60 weight percent n-hexane (CH3(CH2)4CH3) as measured using the Solubility Test Method, described below. Advantageously, compound (1) has a solubility in hexanes containing at least 60 weight percent n-hexane of at least 0.10 wt % in an alkane solvent. In some aspects, the solubility of compound (1) in hexanes containing at least 60 weight percent n-hexane is from 0.10 to 25 wt %, alternatively from 0.5 wt % to 25 wt %, alternatively from 1 wt % to 25 wt %, alternatively from 2 wt % to 25 wt %, alternatively from 3 wt % to 25 wt %, alternatively from 5 wt % to 25 wt %, alternatively from 10.0 wt % to 25 wt %, alternatively from 15 wt % to 25 wt %, alternatively from 20.0 wt % to 25 wt %, alternatively from 0.10 to 20.0 wt %, alternatively from 0.5 wt % to 20.0 wt %, alternatively from 1 wt % to 15 wt %, alternatively from 2 wt % to 15 wt %, alternatively from 3 wt % to 15 wt %, alternatively from 5 wt % to 15 wt %, alternatively from 1.0 wt % to 15 wt %, alternatively from 1.0 wt % to 10.0 wt %, as measured using the Solubility Test Method. Advantageously, the solubility in hexanes containing at least 60 weight percent n-hexane of compound (1) is surprisingly better than that of HN5Zr dibenzyl, which has solubility of just 0.03 wt % in hexanes containing at least 60 weight percent n-hexane. Compound (1) may be employed either in a first part (a main catalyst) or in a second part (as trim catalyst) of the catalyst system. Compound (1) is useful in the combining-the-parts feed method described in the INTRODUCTION. Additionally, compound (1) may be combined with an activator and the combination fed to an in-line mixer or a polymerization reactor independently from feeding of a combination of the metallocene precatalyst and activator to the same in-line mixer or polymerization reactor. This so-called “separate-the-parts” feed method beneficially avoids the aforementioned transition complexity of transitions between catalyst systems and enables greater operational flexibility for olefin polymerization processes in a single polymerization reactor. Compound (1) has sufficient solubility in alkanes such that it may be employed as a HMW precatalyst, with or without a LMW precatalyst, in the catalyst system. The increased solubility of compound (1) in alkanes also enables greater flexibility in a polymerization processes run in a single polymerization reactor and for making a bimodal polyethylene composition comprising LMW and HMW polyethylene components. Compound (1) solves the instability problem of prior alkanes-insoluble non-MCN precatalysts because compound (1) may be stored as a solution in alkanes free of activator. The catalyst system made from compound (1) and activator has faster light-off than a comparative catalyst system made from HN5Zr dibenzyl and the same activator. And yet compound (1) may make a polyethylene having same MWD as MWD of a polyethylene made by the comparative catalyst system. The faster light-off of the catalyst system made from compound (1) and the activator may beneficially result in reduced distributor plate fouling in a gas phase polymerization reactor containing a recycle loop, whereby some polymer particles with active catalyst are entrained back to the reactor where they can grow and foul the distributor plate. The faster light-off of the catalyst system may be characterized as a shorter time to maximum temperature as measured in vitro using 1-octene as monomer according to the Light-off Test Method, described later. The catalyst system made from compound (1) and activator enables making of polyethylene resins having a lesser proportion of particles characterized as “fines”, which is defined later. There are many well-known reasons why fines can cause problems in operating a gas phase polymerization reactor having a recycle line and/or an expanded upper section, such as UNIPOL™ reactor from Univation Technologies, LLC or other reactors. Fines are known to lead to an increased tendency for static and sheeting in such reactor. Fines can increase particle carry-over from the reactor into the recycle line and result in fouling inside the recycle loop, such as in a heat exchanger, compressor, and/or distributor plate. Fines can also build up in the reactor's expanded section because, it is believed, fines are more prone and/or susceptible to electrostatic forces. Fines can also cause problems with polyethylene polymers made by gas phase polymerization in such a reactor. Fines may continue to polymerize in cold zones of the reactor, either in the recycle loop or expanded section, and produce a polyethylene having a molecular weight that is higher than that targeted in the bulk fluidized bed. Fines can eventually make their way back from the recycle loop into the fluidized bed, and then into the polyethylene product, leading to higher level of gels in the polyethylene product. The polyethylene resins made by the catalyst system made from compound (1) and an activator have reduced wt % of fines. The catalyst system made from compound (1) and activator enables making of polyethylene resins having larger particle sizes than those of polyethylene resins made by the comparative catalyst system made from the HN5Zr dibenzyl and the same activator. The larger particle sizes of polyethylene resins made by the inventive catalyst system may be useful for decreasing settled bulk densities of the resin. Resins with a higher proportion of fines can have a higher settled bulk density because the smaller particles of the fines can shift downward and fill in spaces between larger particles. If the settled bulk density is too high, the resin can be difficult to fluidize, causing localized overheating and forming resin chunks in certain regions of the reactor process such as near edges of a distributor plate or in a product discharge system. A polyethylene resin may be made using a bimodal catalyst system, wherein an alkanes solution of compound (1) is used as trim catalyst (second part) and a combination of all of an MCN precatalyst, activator, and a remainder of compound (1) are used as the first part, all of a combining-the-parts feed method, may have reduced gel content compared to a polyethylene resin made using the same bimodal catalyst system except wherein a supported HN5Zr dibenzyl is used as trim catalyst and a remainder of HN5Zr dibenzyl and the same MCN precatalyst are used as the first part. Because the compound (1) has significantly greater solubility in hexanes containing at least 60 weight percent n-hexane, than does HN5Zr dibenzyl, compound (1) has significantly greater solubility in alkanes solvents such as mineral oil than does HN5Zr dibenzyl. This means compound (1) may be fed as an alkanes solution (e.g., typically a solution in mineral oil) as a trim catalyst in the “combining-the-parts” feed method described earlier, whereby it can be mixed with a remainder of compound (1) and all of the MCN precatalyst of a first part in an in-line mixer to give a bimodal catalyst system that may make a bimodal polyethylene composition without the increased gel content found for HN5Zr dibenzyl for the reasons described above, and to solve the earlier gel problem. Without being bound by theory, it is believed that if in a comparative precatalyst of formula (1) wherein the subscript m would be 0, and thus the quaternary carbon atom of the quaternary-hydrocarbyl groups would be directly bonded to metal M, a synthesis of such a comparative precatalyst may be difficult. Alternatively, if in a comparative precatalyst of formula (1) wherein the subscript m would be 4 or greater, and thus the quaternary carbon atom of the quaternary-hydrocarbyl groups would be spaced apart from the metal M by additional carbon atoms, a steric effect of the closer inventive quaternary arylalkyl functional group on metal M could be lost. Compound (1) Compound (1) is a non-metallocene precatalyst of molecular formula (C26H39N3)MR2, wherein M and R groups are as defined for compound (1). Compound (1) contains two N-substituted pentamethyl-phenyl groups and may have general chemical name bis(quaternary-hydrocarbyl)[N′-(2,3,4,5,6-pentamethylphenyl)-N-[2-(2,3,4,5,6-pentamethylphenyl)amino-κN]ethyl]-1,2-ethane-diaminato(2-)κN,κN′](zirconium or hafnium). For example, when M is Zr and each R is 4-tertiary-butylbenzyl, compound (1) may have chemical name bis(4-tert-butylbenzyl)[N′-(2,3,4,5,6-pentamethylphenyl)-N-[2-(2,3,4,5,6-pentamethylphenyl)amino-κN]ethyl]-1,2-ethane-diaminato(2-)κN,κN′]zirconium. In compound (1) each R independently may be the unsubstituted or substituted quaternary-hydrocarbyl group of formula —C((RA)2)mQCR1R2R3, wherein subscript m, RA, R1, R2, and R3 are as defined for compound (1) of any one of aspects 2 to 6. In some aspects, each R is the same or different and is independently selected from: methyl; 2,2-dimethylpropyl; 2,2-dimethylhexyl; 2,2-dimethyloctyl; 2-ethylhexyl; 2-ethyloctyl; 2-tert-butylphenylmethyl; 3-tert-butylphenylmethyl; 4-tert-butylphenylmethyl; 2-ethylphenylmethyl; 3-n-butylphenylmethyl; 4-n-butylphenylmethyl; 2-n-butylphenylmethyl; 3-ethylphenylmethyl; 4-ethylphenylmethyl; 2-n-octylphenylmethyl; 3-n-octylphenylmethyl; and 4-n-octylphenylmethyl. In some aspects each R is the same. In some aspects compound (1) is selected from: (i) compound (1A); (ii) compound (1B); (iii) compound (1) wherein each R is 2-tert-butylphenylmethyl; (iv) compound (1) wherein each R is 3-tert-butylphenylmethyl; (v) compound (1) wherein one R is 4-tert-butylphenylmethyl and the other R is methyl; (vi) compound (1) wherein one R is 2,2-dimethylpropyl (i.e., CH2C(CH3)3) and the other R is methyl; (vii) compound (1) wherein each R is 2-ethylhexyl; (viii) compound (1) wherein each R is 2,2-dimethylpropyl I; (ix) compound (1) wherein each R is 2,2-dimethylhexyl; (x) compound (1) wherein each R is hexyl; (xi) compound (1) wherein both R groups are bonded together to form 4-(2,2-dimethylpropyl)-1,2-benzenedimethylene; (xii) compound (1) wherein both R groups are bonded together to form 2-(2′,2′-dimethylpropyl)-pentane-1,5-diyl; (xiii) compound (1) wherein both R groups are bonded together to form 2,2,3,3-tetramethylbutane-1,4-diyl; and (xiv) a combination of any two or more of (i) to (xiii) (e.g., (i) and (ii)). In some aspects compound (1) is any one of compounds (1A) to (1 D), alternatively compound (1) is selected from any three of compounds (1A) to (1 D), alternatively compound (1) is compound (1A) or (1B), alternatively compound (1) is compound (1C) or (1D), alternatively compound (1) is compound (1A), alternatively compound (1) is compound (1B), alternatively compound (1) is compound (1C), alternatively compound (1) is compound (1D). Compound (1) includes solvates and solvent-free embodiments thereof. The substituted quaternary-hydrocarbyl group is formally derived by replacing from 1 to 4 hydrogen atoms (i.e., carbon-bonded hydrogen atoms, H—C, independently chosen) of the unsubstituted hydrocarbon with a substituent group. In some aspects each unsubstituted quaternary-hydrocarbyl group has from 4 to 50 carbon atoms, alternatively from 4 to 20 carbon atoms, alternatively from 4 to 10 carbon atoms, alternatively from 5 to 6 carbon atoms. Compound (1), after being activated with an activator, makes a catalyst system that is effective for polymerizing one or more olefin monomers, thereby making a polyolefin polymer. Each olefin monomer is independently selected from ethylene, propylene, a (C4-C20)alpha-olefin, and 1,3-butadiene. Each (C4-C20)alpha-olefin independently may be 1-butene, 1-hexene, or 1-octene; alternatively 1-butene or 1-hexene; alternatively 1-butene; alternatively 1-hexene. In some aspects the olefin monomer is selected from ethylene, a (C4-C20)alpha-olefin, and 1,3-butadiene; alternatively ethylene and a (C4-C20)alpha-olefin; alternatively ethylene and 1-hexene; alternatively ethylene and 1-octene; alternatively ethylene. Compound (1) may be used with a metallocene catalyst to make a bimodal catalyst system for making a bimodal polyethylene composition. In some aspects, compound (1) is combined with a metallocene precatalyst or catalyst, at least one activator, and optionally a support, to make a catalyst system comprising, or made from, the metallocene precatalyst, compound (1), the at least one activator, and optionally the support (solid, particulate material). Compound (1) is useful for making a HMW polyethylene component of a bimodal polyethylene composition. The metallocene precatalyst is useful for making a LMW polyethylene component of the bimodal polyethylene composition. The bimodal polyethylene composition is made by polymerizing one or more olefin monomers. In some aspects the bimodal polyethylene composition is made from ethylene only; alternatively from a combination of ethylene and one (C4-C8)alpha-olefin comonomer. Compound (1) may also be interchangeably referred to as a precatalyst, a catalyst component, or a HMW catalyst. Also contemplated is a derivative of compound (1) wherein compound (4) is covalently bonded to a carrier polymer. In an embodiment, the middle nitrogen atom (bonded to two ethylene groups) in compound (4) may bonded to the carrier polymer. Alternatively a methyl group of one of the pentamethylcyclopentadienyl groups of compound (4) might be replaced with an alkylene group that is bonded to the carrier polymer. Ligand-bound polymers are generally described in U.S. Pat. Nos. 5,473,202 and 5,770,755. Synthesis In the method of synthesizing compound (1), including the preliminary steps, an aprotic solvent may be used in any one or more of the contacting steps. The aprotic solvent independently may be a hydrocarbon solvent such as an alkylarene (e.g., toluene, xylene), an alkane, a chlorinated aromatic hydrocarbon (e.g., chlorobenzene), a chlorinated alkane (e.g., dichloromethane), a dialkyl ether (e.g., diethyl ether), or a mixture of any two or more thereof. The aprotic solvent may be any one of those used later in the synthesis Examples. Each of the contacting steps in the method of synthesizing compound (1) independently may be conducted under effective reaction conditions. Effective reaction conditions may comprise techniques for manipulating air-sensitive and/or moisture-sensitive reagents and reactants such as Schlenk-line techniques and an inert gas atmosphere (e.g., nitrogen, helium, or argon). Effective reaction conditions may also comprise a sufficient reaction time, a sufficient reaction temperature, and a sufficient reaction pressure. Each reaction temperature independently may be from −78° to 120° C., alternatively from −30° to 30° C. Each reaction pressure independently may be from 95 to 105 kPa, alternatively from 99 to 103 kPa. Progress of any particular reaction step may be monitored by analytical methods such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry to determine a reaction time that is effective for maximizing yield of intended product. Alternatively, each reaction time independently may be from 30 minutes to 48 hours. Solvent “Hydrocarbon solvent” means a liquid material at 25° C. that consists of carbon and hydrogen atoms, and optionally one or more halogen atoms, and is free of carbon-carbon double bonds and carbon-carbon triple bonds. The hydrocarbon solvent may be an alkane, an arene, or an alkylarene (i.e., arylalkane). Examples of hydrocarbon solvents are alkanes such as mineral oil, pentanes, hexanes, heptanes, octanes, nonanes, decanes, undecanes, dodecanes, etc., and toluene, and xylenes. In one embodiment, the hydrocarbon solvent is an alkane, or a mixture of alkanes, wherein each alkane independently has from 5 to 20 carbon atoms, alternatively from 5 to 12 carbon atoms, alternatively from 5 to 10 carbon atoms. Each alkane independently may be acyclic or cyclic. Each acyclic alkane independently may be straight chain or branched chain. The acyclic alkane may be pentane, 1-methylbutane (isopentane), hexane, 1-methylpentane (isohexane), heptane, 1-methylhexane (isoheptane), octane, nonane, decane, or a mixture of any two or more thereof. The cyclic alkane may be cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, methycyclopentane, methylcyclohexane, dimethylcyclopentane, or a mixture of any two or more thereof. Additional examples of suitable alkanes include Isopar-C, Isopar-E, and mineral oil such as white mineral oil. In some aspects the hydrocarbon solvent is free of mineral oil. The hydrocarbon solvent may consist of one or more (C5-C12)alkanes. Catalyst System The catalyst system comprises a combination of compound (1) and an activator; alternatively the catalyst system comprises an activation reaction product of an activation reaction of compound (1) and the activator. The catalyst system may be made under effective activation conditions. Effective activation conditions may comprise techniques for manipulating catalysts such as in-line mixers, catalyst preparation reactors, and polymerization reactors. The activation may be performed in an inert gas atmosphere (e.g., nitrogen, helium, or argon). Effective activation conditions may also comprise a sufficient activation time and a sufficient activation temperature. Each activation temperature independently may be from 20° to 800° C., alternatively from 300° to 650° C. Each activation time independently may be from 10 seconds to 2 hours. “Activator”, also known as a cocatalyst, is a compound or a composition comprising a combination of reagents, wherein the compound or composition increases the rate at which a transition metal compound (e.g., compound (1) or metallocene precatalyst) oligomerizes or polymerizes unsaturated monomers, such as olefins, such as ethylene or 1-octene. An activator may also affect the molecular weight, degree of branching, comonomer content, or other properties of the oligomer or polymer (e.g., polyolefin). The transition metal compound (e.g., compound (1) or metallocene precatalyst) may be activated for oligomerization and/or polymerization catalysis in any manner sufficient to allow coordination or cationic oligomerization and or polymerization. Typically, the activator contains aluminum and/or boron, alternatively aluminum. Examples of suitable activators are alkylaluminoxanes and trialkylaluminum compounds. Aluminoxane (also known as alumoxane) activators may be utilized as an activator for one or more of the precatalyst compositions including compound (1) or metallocene precatalyst. Aluminoxane(s) are generally oligomeric compounds containing —Al(R)—O— subunits, where R is an alkyl group; which are called alkylaluminoxanes (alkylaluminoxanes). The alkylaluminoxane may be unmodified or modified. Examples of alkylaluminoxanes include methylaluminoxane (MAO), modified methylaluminoxane (MMAO), ethylaluminoxane, and isobutylaluminoxane. Unmodified alkylaluminoxanes and modified alkylaluminoxanes are suitable as activators for precatalysts such as compound (1). Mixtures of different aluminoxanes and/or different modified aluminoxanes may also be used. For further descriptions, see U.S. Pat. Nos. 4,665,208; 4,952,540; 5,041,584; 5,091,352; 5,206,199; 5,204,419; 4,874,734; 4,924,018; 4,908,463; 4,968,827; 5,329,032; 5,248,801; 5,235,081; 5,157,137; 5,103,031; and EP 0 561 476; EP 0 279 586; EP 0 516 476; EP 0 594 218; and PCT Publication WO 94/10180. When the activator is an aluminoxane (modified or unmodified), the maximum amount of activator may be selected to be a 5,000-fold molar excess over the precursor based on the molar ratio of moles of Al metal atoms in the aluminoxane to moles of metal atoms M (e.g., Zr or Hf) in the precatalyst (e.g., compound (1)). Alternatively or additionally the minimum amount of activator-to-precatalyst-precursor may be a 1:1 molar ratio (Al/M). Trialkylaluminum compounds may be utilized as activators for precatalyst (e.g., compound (1) or metallocene precatalyst) or as scavengers to remove residual water from polymerization reactor prior to start-up thereof. Examples of suitable alkylaluminum compounds are trimethylaluminum, triethylaluminum, triisobutylaluminum, tri-n-hexylaluminum, and tri-n-octylaluminum. The catalyst system may include a support or carrier material. A support material is a particulate solid that may be nonporous, semi-porous, or porous. A carrier material is a porous support material. Examples of support materials are talc, inorganic oxides, inorganic chloride, zeolites, clays, resins, and mixtures of any two or more thereof. Examples of suitable resins are polystyrene, functionalized or crosslinked organic supports, such as polystyrene divinyl benzene polyolefins. Inorganic oxide support materials include Group 2, 3, 4, 5, 13 or 14 metal oxides. The preferred supports include silica, which may or may not be dehydrated, fumed silica, alumina (see, for example, PCT Publication WO 99/60033), silica-alumina and mixtures thereof. Other useful supports include magnesia, titania, zirconia, magnesium chloride (U.S. Pat. No. 5,965,477), montmorillonite (EP 0 511 665), phyllosilicate, zeolites, talc, clays (U.S. Pat. No. 6,034,187), and the like. Also, combinations of these support materials may be used, for example, silica-chromium, silica-alumina, silica-titania and the like. Additional support materials may include those porous acrylic polymers described in EP 0 767 184, which is incorporated herein by reference. Other support materials include nanocomposites as disclosed in PCT Publication WO 99/47598; aerogels as disclosed in PCT Publication WO 99/48605; spherulites as disclosed in U.S. Pat. No. 5,972,510; and polymeric beads as disclosed in PCT Publication WO 99/50311. The support material may have a surface area in the range of from about 10 m2/g to about 700 m2/g, a pore volume in the range of from about 0.1 cm3/g to about 4.0 cm3/g, and average particle size in the range of from about 5 microns to about 500 microns. The support material may be a silica (e.g., fumed silica), alumina, a clay, or talc. The fumed silica may be hydrophilic (untreated), alternatively hydrophobic (treated). In some aspects the support is a hydrophobic fumed silica, which may be prepared by treating an untreated fumed silica with a hydrophobing agent such as dimethyldichlorosilane, a polydimethylsiloxane fluid, or hexamethyldisilazane. In some aspects the treating agent is dimethyldichlorosilane. In one embodiment, the support is Cabosil™ TS-610. One or more compound(s) (1) and/or one or more activators, and optionally other precatalyst (e.g., a metallocene or Ziegler-Natta precatalyst), may be deposited on, contacted with, vaporized with, bonded to, incorporated within, adsorbed or absorbed in, or on, one or more support or carrier materials. Such a supported catalyst system comprises the inventive catalyst (compound (1) and activator), optional other catalyst (e.g., metallocene precatalyst or Ziegler-Natta precatalyst and activator) is/are in a supported form deposited on, contacted with, or incorporated within, adsorbed or absorbed in, or on, the material. The compound (1) and/or other precatalysts may be spray dried according to the general methods described in U.S. Pat. No. 5,648,310. The support used with compound (1), and any other precatalysts, may be functionalized, as generally described in EP 0 802 203, or at least one substituent or leaving group is selected as described in U.S. Pat. No. 5,688,880. The metallocene precatalyst may be any one of the metallocene catalyst components described in U.S. Pat. No. 7,873,112B2, column 11, line 17, to column 22, line 21. In some aspects the metallocene precatalyst is selected from the metallocene precatalyst species named in U.S. Pat. No. 7,873,112B2, column 18, line 51, to column 22, line 5. In some aspects the metallocene precatalyst is selected from bis(η5-tetramethylcyclopentadienyl)zirconium dichloride; bis(η5-tetramethylcyclopentadienyl)zirconium dimethyl; bis(η5-pentamethylcyclopentadienyl)zirconium dichloride; bis(η5-pentamethylcyclopentadienyl)zirconium dimethyl; (1,3-dimethyl-4,5,6,7-tetrahydroindenyl)(1-methylcyclopentadienyl)zirconium dimethyl; bis(1-methyl-3-n-butylcyclopentadienyl)zirconium dichloride; bis(1-methyl-3-n-butylcyclopentadienyl)zirconium dimethyl; bis(n-propylcyclopentadienyl)hafnium dichloride; bis(n-propylcyclopentadienyl)hafnium dimethyl; bis(n-butylcyclopentadienyl)zirconium dichloride; and bis(n-butylcyclopentadienyl)zirconium dimethyl. In some aspects the metallocene catalyst is a product of an activation reaction of an activator and any one of the aforementioned metallocene precatalysts. Polymerization Reactor and Method Solution phase polymerization and/or slurry phase polymerization of olefin monomer(s) are well-known. See for example U.S. Pat. No. 8,291,115B2. An aspect of the polymerization method uses a gas-phase polymerization (GPP) reactor, such as a stirred-bed gas phase polymerization reactor (SB-GPP reactor) or a fluidized-bed gas-phase polymerization reactor (FB-GPP reactor), to make the polyolefin polymer. Such reactors and methods are generally well-known. For example, the FB-GPP reactor/method may be as described in U.S. Pat. Nos. 3,709,853; 4,003,712; 4,011,382; 4,302,566; 4,543,399; 4,882,400; 5,352,749; 5,541,270; EP-A-0 802 202; and Belgian Patent No. 839,380. These SB-GPP and FB-GPP polymerization reactors and processes either mechanically agitate or fluidize by continuous flow of gaseous monomer and diluent the polymerization medium inside the reactor, respectively. Other useful reactors/processes contemplated include series or multistage polymerization processes such as described in U.S. Pat. Nos. 5,627,242; 5,665,818; 5,677,375; EP-A-0 794 200; EP-B1-0 649 992; EP-A-0 802 202; and EP-B-634421. Polymerization operating conditions are any variable or combination of variables that may affect a polymerization reaction in the GPP reactor or a composition or property of a bimodal ethylene-co-1-hexene copolymer composition product made thereby. The variables may include reactor design and size; compound (1) composition and amount; reactant composition and amount; molar ratio of two different reactants; presence or absence of feed gases such as H2and/or O2, molar ratio of feed gases versus reactants, absence or concentration of interfering materials (e.g., H2O), absence or presence of an induced condensing agent (ICA), average polymer residence time in the reactor, partial pressures of constituents, feed rates of monomers, reactor bed temperature (e.g., fluidized bed temperature), nature or sequence of process steps, time periods for transitioning between steps. Variables other than that/those being described or changed by the method or use may be kept constant. In operating the polymerization method, control individual flow rates of ethylene (“C2”), hydrogen (“H2”) and 1-hexene (“C6” or “Cx” wherein x is 6) to maintain a fixed comonomer to ethylene monomer gas molar ratio (Cx/C2, e.g., C6/C2) equal to a described value (e.g., 0.00560 or 0.00703), a constant hydrogen to ethylene gas molar ratio (“H2/C2”) equal to a described value (e.g., 0.00229 or 0.00280), and a constant ethylene (“C2”) partial pressure equal to a described value (e.g., 1,000 kPa). Measure concentrations of gases by an in-line gas chromatograph to understand and maintain composition in the recycle gas stream. Maintain a reacting bed of growing polymer particles in a fluidized state by continuously flowing a make-up feed and recycle gas through the reaction zone. Use a superficial gas velocity of 0.49 to 0.67 meter per second (m/sec) (1.6 to 2.2 feet per second (ft/sec)). Operate the FB-GPP reactor at a total pressure of about 2344 to about 2413 kilopascals (kPa) (about 340 to about 350 pounds per square inch-gauge (psig)) and at a described first reactor bed temperature RBT. Maintain the fluidized bed at a constant height by withdrawing a portion of the bed at a rate equal to the rate of production of particulate form of the bimodal ethylene-co-1-hexene copolymer composition, which production rate may be from 10 to 20 kilograms per hour (kg/hour). Remove the product bimodal ethylene-co-1-hexene copolymer composition semi-continuously via a series of valves into a fixed volume chamber, wherein this removed bimodal ethylene-co-1-hexene copolymer composition is purged to remove entrained hydrocarbons and treated with a stream of humidified nitrogen (N2) gas to deactivate any trace quantities of residual catalyst. See polymerization method described herein. The catalyst system may be fed into the polymerization reactor(s) in “dry mode” or “wet mode”, alternatively dry mode, alternatively wet mode. The dry mode is a dry powder or granules. The wet mode is a suspension in an inert liquid such as mineral oil. Induced condensing agent (ICA). An inert liquid useful for cooling materials in gas phase polymerization reactor(s). Its use is optional. The ICA may be a (C5-C20)alkane, e.g., 2-methylbutane (i.e., isopentane). The methods that use the ICA may be referred to as being an induced condensing mode operation (ICMO). ICMO is described in U.S. Pat. Nos. 4,453,399; 4,588,790; 4,994,534; 5,352,749; 5,462,999; and 6,489,408. Measure concentration of ICA in gas phase using gas chromatography by calibrating peak area percent to mole percent (mol %) with a gas mixture standard of known concentrations of ad rem gas phase components. Concentration of ICA may be from 1 to 10 mol %. The polymerization conditions may further include one or more additives such as a chain transfer agent or a promoter. The chain transfer agents are well known and may be alkyl metal such as diethyl zinc. Promoters are known such as in U.S. Pat. No. 4,988,783 and may include chloroform, CFCI3, trichloroethane, and difluorotetrachloroethane. Prior to reactor start up, a scavenging agent may be used to react with moisture and during reactor transitions a scavenging agent may be used to react with excess activator. Scavenging agents may be a trialkylaluminum. Gas phase polymerizations may be operated free of (not deliberately added) scavenging agents. The polymerization conditions for gas phase polymerization reactor/method may further include an amount (e.g., 0.5 to 200 ppm based on all feeds into reactor) of a static control agent and/or a continuity additive such as aluminum stearate or polyethyleneimine. The static control agent may be added to the FB-GPP reactor to inhibit formation or buildup of static charge therein. In an embodiment the method uses a pilot scale fluidized bed gas phase polymerization reactor (Pilot Reactor) that comprises a reactor vessel containing a fluidized bed of a powder of the bimodal ethylene-co-1-hexene copolymer composition, and a distributor plate disposed above a bottom head, and defining a bottom gas inlet, and having an expanded section, or cyclone system, at the top of the reactor vessel to decrease amount of resin fines that may escape from the fluidized bed. The expanded section defines a gas outlet. The Pilot Reactor further comprises a compressor blower of sufficient power to continuously cycle or loop gas around from out of the gas outlet in the expanded section in the top of the reactor vessel down to and into the bottom gas inlet of the Pilot Reactor and through the distributor plate and fluidized bed. The Pilot Reactor further comprises a cooling system to remove heat of polymerization and maintain the fluidized bed at a target temperature. Compositions of gases such as ethylene, alpha-olefin (e.g., 1-hexene), and hydrogen being fed into the Pilot Reactor are monitored by an in-line gas chromatograph in the cycle loop in order to maintain specific concentrations that define and enable control of polymer properties. The catalyst system may be fed as a slurry or dry powder into the Pilot Reactor from high pressure devices, wherein the slurry is fed via a syringe pump and the dry powder is fed via a metered disk. The catalyst system typically enters the fluidized bed in the lower ⅓ of its bed height. The Pilot Reactor further comprises a way of weighing the fluidized bed and isolation ports (Product Discharge System) for discharging the powder of bimodal ethylene-co-1-hexene copolymer composition from the reactor vessel in response to an increase of the fluidized bed weight as polymerization reaction proceeds. In some embodiments the FB-GPP reactor is a commercial scale reactor such as a UNIPOL™ reactor or UNIPOL™ II reactor, which are available from Univation Technologies, LLC, a subsidiary of The Dow Chemical Company, Midland, Michigan, USA. In some aspects any compound, composition, formulation, material, mixture, or reaction product herein may be free of any one of the chemical elements selected from the group consisting of: H, Li, Be, B, C, N, O, F, Na, Mg, Al, Si, P, S, Cl, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, As, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, I, Cs, Ba, Hf, Ta, W, Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, lanthanoids, and actinoids; with the proviso that chemical elements required by the compound, composition, formulation, material, mixture, or reaction product (e.g., Zr required by a zirconium compound, or C and H required by a polyethylene, or C, H, and O required by an alcohol) are not counted. Bimodal. Having (only) two maxima in a frequency distribution. Bimodal in reference to a polymer composition means the polymer composition consists essentially of a higher molecular weight (HMW) component and a lower molecular weight (LMW) component. Bimodal polymer compositions include post-reactor blends (wherein the LMW and HMW components are synthesized in different reactors or in a same reactor at different times separately and later blended together such as by melt extrusion) and reactor blends (wherein the LMW and HMW components are synthesized in the same reactor). The bimodal copolymer composition may be characterized by two peaks separated by a distinguishable local minimum therebetween in a plot of dW/d Log(MW) on the y-axis versus Log(MW) on the x-axis to give a Gel Permeation Chromatograph (GPC) chromatogram, wherein Log(MW) and dW/d Log(MW) are as defined herein and are measured by Gel Permeation Chromatograph (GPC) Test Method described herein. Bimodal referring to a catalyst system means a catalyst system that contains two different catalysts for catalyzing a same polymerization process (e.g., olefin polymerization) and producing a bimodal polymer composition. Two catalysts are different if they differ from each other in at least one of the following characteristics: (a) their catalytic metals are different (Ti versus Zr, Zr versus Hf, Ti versus Hf; not activator metals such as Al); (b) one catalyst has a functional ligand covalently bonded to its catalytic metal and the other catalyst is free of functional ligands bonded to its catalytic metal; (c) both catalysts have functional ligands covalently bonded to their catalytic metal and the structures of at least one of functional ligand of one of the catalysts is different than the structure of each of the functional ligand(s) of the other catalyst (e.g., cyclopentadienyl versus propylcyclopentadienyl or butylcyclopentadienyl versus (pentamethylphenylamido)ethyl)-amine); and (d) for catalysts disposed on a support material, the compositions of the support materials are different. Functional ligands do not include leaving groups X as defined later. Two catalysts of a bimodal catalyst system may be disposed on the same support material, either on the same particles of the same support material or each on different particles of the same support material. The same catalyst in terms of catalytic metal and ligands wherein a portion thereof is disposed on a support material and a different portion thereof is dissolved in an inert solvent, the different portions do not by themselves constitute a bimodal catalyst system. Catalyst system. A reaction product of an activation reaction of a precatalyst and an activator (i.e., a catalyst per se) and, optionally, one or more compatible companion materials such as a different catalyst for making a component of a bimodal polymer, a hydrocarbon solvent for conveying the catalyst, a modifier compound for attenuating reactivity of the catalyst, a support material on which the catalyst is disposed, a carrier material in which the catalyst is disposed, or a combination of any two or more thereof, or a reaction product of a reaction thereof. Consisting essentially of, consist(s) essentially of, and the like. Partially-closed ended expressions that exclude anything that would affect the basic and novel characteristics of that which they describe, but otherwise allow anything else. In some aspects any one, alternatively each “comprising” or “comprises” may be replaced by “consisting essentially of” or “consists essentially of”, respectively; alternatively by “consisting of” or “consists of”, respectively. Consisting of and consists of. Closed ended expressions that exclude anything that is not specifically described by the limitation that it modifies. In some aspects any one, alternatively each expression “consisting essentially of” or “consists essentially of” may be replaced by the expression “consisting of” or “consists of”, respectively. Dry. Generally, a moisture content from 0 to less than 5 parts per million based on total parts by weight. Materials fed to the reactor(s) during a polymerization reaction are dry. Feed. Quantity of reactant or reagent that is added or “fed” into a reactor. In continuous polymerization operation, each feed independently may be continuous or intermittent. The quantities or “feeds” may be measured, e.g., by metering, to control amounts and relative amounts of the various reactants and reagents in the reactor at any given time. Feed line. A pipe or conduit structure for transporting a feed. Higher molecular weight (HMW) component. A subgroup of macromolecules having a peak in the GPC plot of dW/d Log(MW) on the y-axis versus Log(MW) on the x-axis that is at a higher molecular weight. Hydrocarbyl. A monovalent radical formally derived by removing a H atom from a hydrocarbon compound consisting of C and H atoms. Hydrocarbylene. A divalent radical formally derived by removing two H atoms from a hydrocarbon compound consisting of C and H atoms, wherein the two H atoms are removed from different carbon atoms of the hydrocarbon compound. Inert. Generally, not (appreciably) reactive or not (appreciably) interfering therewith in the inventive polymerization reaction. The term “inert” as applied to the purge gas or ethylene feed means a molecular oxygen (O2) content from 0 to less than 5 parts per million based on total parts by weight of the purge gas or ethylene feed. Lower molecular weight (LMW) component. A subgroup of macromolecules having a peak in the GPC plot of dW/d Log(MW) on the y-axis versus Log(MW) on the x-axis that is at a lower molecular weight. Metallocene catalyst. Homogeneous or heterogeneous material that contains a cyclopentadienyl ligand-metal complex and enhances olefin polymerization reaction rates. Substantially single site or dual site. Each metal is a transition metal Ti, Zr, or Hf. Each cyclopentadienyl ligand independently is an unsubstituted cyclopentadienyl group or a hydrocarbyl-substituted cyclopentadienyl group. In some aspects the metallocene catalyst has two cyclopentadienyl ligands, and at least one, alternatively both of the cyclopentenyl ligands independently is a hydrocarbyl-substituted cyclopentadienyl group. Each hydrocarbyl-substituted cyclopentadienyl group may independently have 1, 2, 3, 4, or 5 hydrocarbyl substituents. Each hydrocarbyl substituent may independently be a (C1-C4)alkyl. Two or more substituents may be bonded together to form a divalent substituent, which with carbon atoms of the cyclopentadienyl group may form a ring. Multimodal. Having two or more maxima in a frequency distribution. Ziegler-Natta catalysts. Heterogeneous materials that enhance olefin polymerization reaction rates and are prepared by contacting inorganic titanium compounds, such as titanium halides supported on a magnesium chloride support, with an activator. Alternatively precedes a distinct embodiment. ASTM means the standards organization, ASTM International, West Conshohocken, Pennsylvania, USA. Any comparative example is used for illustration purposes only and shall not be prior art. Free of or lacks means a complete absence of; alternatively not detectable. Terms used herein have their IUPAC meanings unless defined otherwise. For example, see Compendium of Chemical Terminology. Gold Book, version 2.3.3, Feb. 24, 2014. IUPAC is International Union of Pure and Applied Chemistry (IUPAC Secretariat, Research Triangle Park, North Carolina, USA). Periodic Table of the Elements is the IUPAC version of May 1, 2018. May confers a permitted choice, not an imperative. Operative means functionally capable or effective. Optional(ly) means is absent (or excluded), alternatively is present (or included). Properties may be measured using standard test methods and conditions. Ranges include endpoints, subranges, and whole and/or fractional values subsumed therein, except a range of integers does not include fractional values. Room temperature: 23° C.±1° C. “HN5” is not pentazole. EXAMPLES Isoparaffin fluid: ISOPAR-C from ExxonMobil. Mineral oil: HYDROBRITE 380 PO White mineral oil from Sonneborn. Preparation 1A: preparation of an activator formulation comprising spray-dried methylaluminoxane/treated fumed silica (sdMAO) in hexanes/mineral oil. Slurry 1.6 kg of treated fumed silica (CABOSIL TS-610) in 16.8 kg of toluene, then add a 10 wt % solution (11.6 kg) MAO in toluene to give a mixture. Using a spray dryer set at 160° C. and with an outlet temperature at 70° to 80° C., introduce the mixture into an atomizing device of the spray dryer to produce droplets of the mixture, which are then contacted with a hot nitrogen gas stream to evaporate the liquid from the mixture to give a powder. Separate the powder from the gas mixture in a cyclone separator, and discharge the separated powder into a container to give the sdMAO as a fine powder. Preparation 1B: preparation of a slurry of the activator formulation of Preparation 1A. Slurry the sdMAO powder of Preparation 1A in a mixture of 10 wt % n-hexane and 78 wt % mineral oil to give the activator formulation having 12 wt % sdMAO/treated fumed silica solids in the hexane/mineral oil. Preparation 2: preparation of a spray-dried metallocene with activator formulation. Replicate Preparations 1A and 1B except prepare an activator formulation by slurrying 1.5 kg of treated fumed silica (CABOSIL TS-610) in 16.8 kg of toluene, followed by adding a 10 wt % solution (11.1 kg) of MAO in toluene and (MeCp)(1,3-dimethyl-4,5,6,7-tetrahydroindenyl)ZrMe2, wherein Me is methyl, Cp is cyclopentadienyl, and MeCp is methylcyclopentadienyl, in an amount sufficient to provide a loading of 40 micromoles Zr per gram of solid. Slurry the resultant powder to give an activator formulation of 22 wt % solids in 10 wt % isoparaffin fluid and 68 wt % mineral oil. Advantageously, the activator formulation does not include a HMW precatalyst, and can be employed to produce polymer compositions with very low ratios of HMW/LMW components. Further, transitions to other catalyst systems are simplified compared to the combining-the-parts feed method of the Introduction. Preparation 3: synthesis of compound (4) {(HN(CH2CH2NHC6(CH3)5)2)}. Replicate Procedure 2 of U.S. Pat. No. 6,967,184B2, column 33, line 53, to column 34, line 9, to give compound (4), as drawn above. Preparation 4: synthesis of 4-tert-butylbenzylmagnesium chloride. Under an atmosphere of nitrogen in a glovebox having a freezer component, charge a first oven-dried 120 mL glass jar with three small, PTFE-coated magnetic stir bars and 1.33 g (54.7 mmol) of magnesium turnings. Seal the jar with a PTFE-lined cap, and stir contents vigorously for 40 hours. PTFE is poly(tetrafluoroethylene). Then add 40 mL anhydrous, degassed diethyl ether. Place the jar in the glovebox freezer for 15 minutes to cool the contents of the jar to −30° C. In a second oven-dried 120 mL glass jar, prepare a solution of 4-(1,1,-dimethylethyl)benzyl chloride (2.0 g, 10.9 mmol) in 60 mL of anhydrous, degassed diethyl ether. Seal the jar with a PTFE-lined cap, and place the second glass jar in the glovebox freezer for 15 minutes to cool its contents to −30° C. Add the solution of the second jar to an addition funnel, and add dropwise the contents of the addition funnel to the contents of in the first glass jar over 45 minutes. Use 10 mL of diethyl ether to rinse the residual contents of the addition funnel into the reaction mixture of the first glass jar. Stir the resulting mixture and allow it to come to room temperature for 2.5 hours. Filter the mixture through a PTFE frit into a clean vial to give a solution of 4-tert-butylbenzylmagnesium chloride in diethyl ether. Titrate a portion of the filtrate with iodine/LiCl to determine the concentration of the 4-tert-butylbenzylmagnesium chloride in the solution. Preparation 5: synthesis of 3-n-butylbenzyl alcohol. Under an atmosphere of nitrogen in a glove box, charge an oven dried round bottom flask with a PTFE-coated magnetic stir bar and a reflux condenser with 3-n-butylbenzoic acid (2.0 g, 11.2 mmol) and 10 mL of dry, degassed THF. Add a solution of borane in tetrahydrofuran (22.4 mL, 22.4 mmol), attach a reflux condenser to the flask, and heat the mixture to reflux for 4 hours. Remove the flask from the glove box, and place under an atmosphere of nitrogen on a Schlenk line, then cool to 0° C. in an ice bath. Slowly add 5 mL of ethanol, then pour the resulting mixture into 30 mL of water, and extract with three 30 mL portions of diethyl ether. Combine and dry the diethyl ether extracts over anhydrous magnesium sulfate, filter through diatomaceous earth, and concentrate under reduced pressure to give a pale orange oil. Dissolve the oil in a minimal amount of hexane, and pass the solution through a plug of silica eluting with a 1:1 volume/volume (v/v) mixture of ethyl acetate and hexane. Concentrate the filtrate under reduced pressure to obtain the 3-n-butylbenzyl alcohol as a pale orange oil.1H NMR (400 MHz, Chloroform-d) δ 7.28-7.23 (m, 1H), 7.19-7.14 (m, 3H), 7.10 (dd, J=7.5, 1.5 Hz, 1H), 4.65 (s, 2H), 2.63-2.55 (m, 2H), 1.64 (d, J=11.9 Hz, 2H), 1.64-1.54 (m, 2H), 1.41-1.28 (m, 2H), 0.91 (t, J=7.3 Hz, 4H).13C NMR (101 MHz, Chloroform-d) δ 143.31, 140.76, 128.44, 127.77, 127.08, 124.26, 65.49, 35.60, 33.63, 22.38, 13.94. Preparation 6: synthesis of 3-n-butylbenzyl chloride. Under an atmosphere of nitrogen on a Schlenk line, charge a 100 mL round bottom flask with 3-n-butylbenzyl alcohol made in Preparation 5 (1.57 g, 9.6 mmol) and add 12 mL of dry degassed dichloromethane. Cool the flask to 0° C. in an ice bath, and add 0.1 mL of triethylamine (0.8 mmol) and add thionyl chloride (1.39 mL, 19.1 mmol) slowly via syringe. Stir the mixture under an atmosphere of nitrogen and allow to come to room temperature over 22 hours. Carefully pour the mixture into 50 mL of ice water and extract with three 30 mL portions of dichloromethane. Wash the combined dichloromethane layers with two 50 mL portions of saturated aqueous sodium bicarbonate and two 50 mL portions saturated aqueous sodium chloride, then dry over magnesium sulfate and concentrate under reduced pressure. The 3-n-butylbenzyl chloride is obtained as a pale yellow liquid.1H NMR (400 MHz, Chloroform-d) δ 7.25 (dd, J=8.3, 7.4 Hz, 1H), 7.21-7.16 (m, 2H), 7.12 (dt, J=7.4, 1.6 Hz, 1H), 4.56 (s, 2H), 2.64-2.56 (m, 2H), 1.65-1.50 (m, 3H), 1.34 (dq, J=14.6, 7.3 Hz, 2H), 0.92 (t, J=7.3 Hz, 3H).13C NMR (101 MHz, Chloroform-d) δ 143.51, 137.32, 128.63, 128.59, 128.51, 125.83, 46.43, 35.50, 33.53, 22.37, 13.93. Preparation 7: synthesis of 3-n-butylbenzylmagnesium chloride. Under an atmosphere of nitrogen in a glovebox having a freezer component, charge a first oven-dried 40 mL glass vial with three small, PTFE-coated magnetic stir bars and 330 mg (13.7 mmol) of magnesium turnings. Seal the vial with a PTFE-lined septum cap, and stir contents vigorously for 40 hours. Then add 10 mL anhydrous, degassed diethyl ether. Place the jar in the glovebox freezer for 15 minutes to cool the contents of the jar to −30° C. In a second oven-dried 40 mL glass vial, prepare a solution of 3-(n-butyl)benzyl chloride of Preparation 6 (0.5 g, 10.9 mmol) in15 mL of anhydrous, degassed diethyl ether. Seal the jar with a PTFE-lined septum cap, and place the second glass vial in the glovebox freezer for 15 minutes to cool its contents to −30° C. Add the solution of the second jar to an addition funnel, and add dropwise the contents of the addition funnel to the contents of in the first glass jar over 10 minutes. Use 2 mL of diethyl ether to rinse the residual contents of the addition funnel into the reaction mixture of the first glass jar. Stir the resulting mixture and allow it to come to room temperature for 1.5 hours. Filter the mixture through a PTFE frit into a clean vial to give a solution of 3-n-butylbenzylmagnesium chloride in diethyl ether. Titrate a portion of the filtrate with iodine/LiCl to determine the concentration of the 3-n-butylbenzylmagnesium chloride in the solution. Preparation 8: synthesis of tetra(3-methylbenzyl)zirconium. Under an atmosphere of nitrogen in a glovebox having a freezer component, charge a 40 mL oven-dried vial with a PTFE-coated stir bar with zirconium(IV) chloride (0.25 g, 0.6 mmol) and 10 mL of toluene. Seal the vial with a PTFE-lined septum cap and place the vial in the glovebox freezer for 15 minutes to cool the contents of the jar to −30° C. Slowly add a solution of 3-methylbenzylmagnesium chloride (7.35 mL, 2.6 mmol) of Preparation 7, then cover the vial with aluminum foil and stir the mixture while allowing to come to room temperature in the dark for 16 hours. Add 15 mL of diethyl ether and filter the mixture through diatomaceous earth, then concentrate the mixture to a volume of about 2 mL. Add a 10 mL portion of pentane place the vial in the glovebox freezer overnight. Collect the resulting yellow precipitate by filtration then triturate the resulting solid in 5 mL of hexane and dry under vacuum three times to remove the residual THF. Add 5 mL of toluene to the resulting solid and filter through a 0.45 μM PTFE syringe filter. Concentrate the filtrate under reduced pressure, then triturate in 5 mL of hexane and dry under vacuum three times. Add 5 mL of pentane and place the vial in the glove box freezer for 72 hours. Filter the mixture through diatomaceous earth and wash the filter cake with 10 mL of hexane. Concentrate the filtrate under reduced pressure to give the tetra(3-methylbenzyl)zirconium as a yellow-brown oil.1H NMR (400 MHz, Benzene-d6) δ 7.03 (t, J=7.6 Hz, 1H), 6.82 (ddt, J=7.5, 1.8, 0.9 Hz, 1H), 6.34 (dt, J=8.0, 1.4 Hz, 1H), 6.11 (d, J=1.9 Hz, 1H), 2.06 (s, 3H), 1.52 (s, 2H).13C NMR (101 MHz, Benzene-d6) δ 140.90, 140.02, 130.97, 128.68, 125.92, 124.99, 71.42, 21.68. Bimodality Test Method: determine presence or absence of resolved bimodality by plotting dWf/d Log M (mass detector response) on y-axis versus Log M on the x-axis to obtain a GPC chromatogram curve containing local maxima log(MW) values for LMW and HMW polyethylene component peaks, and observing the presence or absence of a local minimum between the LMW and HMW polyethylene component peaks. The dWf is change in weight fraction, d Log M is also referred to as d Log (MW) and is change in logarithm of molecular weight, and Log M is also referred to as Log (MW) and is logarithm of molecular weight. Deconvoluting Test Method: segment the chromatogram obtained using the Bimodality Test Method into nine (9) Schulz-Flory molecular weight distributions. Such deconvolution method is described in U.S. Pat. No. 6,534,604. Assign the lowest four MW distributions to the LMW polyethylene component and the five highest MW distributions to the HMW polyethylene component. Determine the respective weight percents (wt %) for each of the LMW and HMW polyethylene components in the bimodal ethylene-co-1-hexene copolymer composition by using summed values of the weight fractions (Wf) of the LMW and HMW polyethylene components and the respective number average molecular weights (Mn) and weight average molecular weights (Mw) by known mathematical treatment of aggregated Schulz-Flory MW distributions. Density is measured according to ASTM D792-13, Standard Test Methods for Density and Specific Gravity (Relative Density) of Plastics by Displacement, Method B (for testing solid plastics in liquids other than water, e.g., in liquid 2-propanol). Report results in units of grams per cubic centimeter (g/cm3). Gel permeation chromatography (GPC) Test Method: Weight-Average Molecular Weight Test Method: determine Mw, number-average molecular weight (Mn), and Mw/Mnusing chromatograms obtained on a High Temperature Gel Permeation Chromatography instrument (HTGPC, Polymer Laboratories). The HTGPC is equipped with transfer lines, a differential refractive index detector (DRI), and three Polymer Laboratories PLgel 10 μm Mixed-B columns, all contained in an oven maintained at 160° C. Method uses a solvent composed of BHT-treated TCB at nominal flow rate of 1.0 milliliter per minute (mL/min.) and a nominal injection volume of 300 microliters (μL). Prepare the solvent by dissolving 6 grams of butylated hydroxytoluene (BHT, antioxidant) in 4 liters (L) of reagent grade 1,2,4-trichlorobenzene (TCB), and filtering the resulting solution through a 0.1 micrometer (μm) PTFE filter to give the solvent. Degas the solvent with an inline degasser before it enters the HTGPC instrument. Calibrate the columns with a series of monodispersed polystyrene (PS) standards. Separately, prepare known concentrations of test polymer dissolved in solvent by heating known amounts thereof in known volumes of solvent at 160° C. with continuous shaking for 2 hours to give solutions. (Measure all quantities gravimetrically.) Target solution concentrations, c, of test polymer of from 0.5 to 2.0 milligrams polymer per milliliter solution (mg/mL), with lower concentrations, c, being used for higher molecular weight polymers. Prior to running each sample, purge the DRI detector. Then increase flow rate in the apparatus to 1.0 mL/min/, and allow the DRI detector to stabilize for 8 hours before injecting the first sample. Calculate Mwand Mnusing universal calibration relationships with the column calibrations. Calculate MW at each elution volume with following equation: logMX=log(KX/KPS)aX+1+aPS+1aX+1logMPS, where subscript “X” stands for the test sample, subscript “PS” stands for PS standards, aPS=0.67, KPS=0.000175, and aXand KXare obtained from published literature. For polyethylenes, aX/KX=0.695/0.000579. For polypropylenes aX/KX=0.705/0.0002288. At each point in the resulting chromatogram, calculate concentration, c, from a baseline-subtracted DRI signal, IDRI, using the following equation: c=KDRIIDRI/(dn/dc), wherein KDRIis a constant determined by calibrating the DRI, /indicates division, and dn/dc is the refractive index increment for the polymer. For polyethylene, dn/dc=0.109. Calculate mass recovery of polymer from the ratio of the integrated area of the chromatogram of concentration chromatography over elution volume and the injection mass which is equal to the pre-determined concentration multiplied by injection loop volume. Report all molecular weights in grams per mole (g/mol) unless otherwise noted. Further details regarding methods of determining Mw, Mn, MWD are described in US 2006/0173123 page 24-25, paragraphs [0334] to [0341]. Plot of dW/d Log(MW) on the y-axis versus Log(MW) on the x-axis to give a GPC chromatogram, wherein Log(MW) and dW/d Log(MW) are as defined above. High Load Melt Index (HLMI) I21Test Method: use ASTM D1238-13, Standard Test Method for Melt Flow Rates of Thermoplastics by Extrusion Platometer, 190° C./21.6 kilograms (kg). Report results in units of grams eluted per 10 minutes (g/10 min.). Light-off Test Method: Under an atmosphere of nitrogen in a glovebox, charge a 40 mL glass vial with a PTFE-coated, magnetic stir bar and 0.16 g of spray dried methylaluminoxane powder of Preparation 1A. To the charged vial add 11 mL of 1-octene, and then insert the vial into an insulated sleeve mounted on a magnetic stir plate turning at approximately 300 rotations per minute (rpm). To the insulated vial add 8 micromoles (μmol) of precatalyst (e.g., compound (1) or HN5Zr dibenzyl). Cap the vial with a rubber septum. Insert a thermocouple probe through the rubber septum into the vial such that the tip of the thermocouple probe is below the liquid level. Record the temperature of the contents of the vial at 5 second intervals, continuing until after the maximum temperature is reached. Download the temperature and time data to a spreadsheet, and plot thermo-kinetic profiles for analysis. The Light-off Test Method may be adapted to qualify organometallic precatalysts and borate activators; assess aging of Ziegler-Natta, molecular catalysts, or pre-polymerized catalysts; characterize unsupported methylaluminoxanes and methylaluminoxanes chemisorbed on porous silica; assess effects of catalyst poisons; measure activation kinetics of leaving group modifications on organometallic precatalysts; measure effect of reversible coordinating compounds on kinetic profiles of molecular and Ziegler-Natta catalysts; screen activity of new catalysts, activators, co-catalysts, catalyst modifiers, activator modifiers, scavengers, chain transfer agents, or chain shuttling agents; assess effects of contaminants in catalysts; characterize Ziegler-Natta catalysts; and assess olefin monomer purity. Melt Index I5(“I5”) Test Method: use ASTM D1238-13, using conditions of 190° C./5.0 kg. Report results in units of grams eluted per 10 minutes (g/10 min.). Melt Flow Ratio MFR5: (“I21/I5”) Test Method: calculated by dividing the value from the HLMI I21Test Method by the value from the Melt Index 15 Test Method. Solubility Test Method: to a 20-mL vial is added, at room temperature and ambient pressure, a known mass of test precatalyst (e.g., compound (1)) and a known volume of hexanes containing at least 60 weight percent n-hexane. A PTFE-coated magnetic stir bar is added and the mixture is allowed to stir for 1 hour before the vial is removed from the stir plate, and the mixture is allowed to sit overnight. The next day the suspension is filtered through a 0.4 μm PTFE syringe filter into a tared vial, giving a known mass of supernatant, and the hexanes are removed under reduced pressure, leaving a measurable mass of the compound of formula (1) from which wt. % solubility is calculated. Comparative Example 1 (CE1): synthesis of [N′-(2,3,4,5,6-pentamethylphenyl)-N-[2-(2,3,4,5,6-pentamethylphenyl)amino-κN]ethyl]-1,2-ethane-diaminato(2-)κN,κN′]zirconium dichloride (abbreviated herein as “HN5Zr dichloride”) is described in U.S. Pat. No. 6,967,184B2. Measure the light-off performance according to the Light-Off Test Method. Time to maximum temperature result is reported later in Table 1. Comparative Example 2 (CE2): synthesis of bis(phenylmethyl)[N′-(2,3,4,5,6-pentamethylphenyl)-N-[2-(2,3,4,5,6-pentamethylphenyl)amino-κN]ethyl]-1,2-ethane-diaminato(2-)κN,κN′]zirconium (abbreviated herein as “HN5Zr dibenzyl”) may be accomplished by reacting HN5Zr dichloride of CE1 with two molar equivalents of benzylmagnesium chloride in anhydrous tetrahydrofuran. Measure the light-off performance according to the Light-Off Test Method and measure the according to the Solubility Test Method. Solubility and time to maximum temperature results are reported later in Table 1. Inventive Example 1 (IE1): synthesis of compound (3a) (compound (3) wherein each R10is methyl) from compound (4), which is prepared according to Preparation 3. Under a nitrogen atmosphere in a glovebox, charge an oven-dried 400 mL glass jar with a PTFE-coated magnetic stir bar, compound (4) (10 g, 25.3 mmol), and 200 mL of dry, degassed n-pentane. Then add solid tetrakis(dimethylamino)zirconium(IV) (6.76 g, 25.3 mmol) in small portions, and stir the resulting mixture at 25° C. for 16 hours. Cool the mixture in a freezer in the glove-box for 1 hour. Filter off precipitated (3a), and wash the filtercake with cold n-pentane. Dry the washed compound (3a) under reduced pressure to give 12.62 g (87.1% yield) of compound (3a) as a white powder.1H NMR (400 MHz, Benzene-d6) δ 3.37 (dt, 2H), 3.10 (d, 6H), 3.02 (dd, 3H), 2.68 (dq, 4H), 2.51 (d, 12H), 2.20 (q, 18H), 2.14 (s, 7H), 1.84 (s, 1H);13C NMR (101 MHz, Benzene-d6) δ 149.77, 132.34, 132.14, 130.04, 129.98, 129.32, 56.29, 48.86, 44.35, 40.91, 17.31, 17.27, 16.72, 16.65, 16.09. Inventive Example 2 (IE2): synthesis of compound (2a) (compound (2) wherein M is Zr and each X is Cl) from compound (3a) Under a nitrogen atmosphere in a glovebox, charged an oven-dried 400 mL glass jar with a PTFE-coated, magnetic stir bar, compound (3a) (12.62 g, 22.0 mmol), and 250 mL of dry, degassed diethyl ether. Add chlorotrimethylsilane (6.2 mL, 48.5 mmol), and stir the mixture at 25° C. for 24 hours. Cool the mixture in the glove box freezer for 1 hour. Filter off precipitated (2a), and wash the filtercake with cold n-pentane. Dry the washed (2a) under reduced pressure to give 10.77 g (88.0% yield) of compound (2a), i.e., bis(2-(pentamethylphenylamido)ethyl)-amine zirconium(IV) dichloride, as a white powder.1H NMR (400 MHz, Benzene-d6) δ 3.40 (dt, 1H), 2.95 (dt, 1H), 2.59 (dp, 2H), 2.49 (s, 3H), 2.46 (s, 3H), 2.43-2.34 (m, 1H), 2.13 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H).13C NMR (101 MHz, Benzene-d6) δ 145.64, 133.37, 133.20, 132.61, 129.84, 129.57, 57.69, 48.97, 17.03, 17.01, 16.70, 16.47. Inventive Example 3 (IE3): synthesis of compound (1A) (compound (1) wherein M is Zr and each R is CH2-(1,4-phenylene)-C(CH3)3) from compound (2a) Charge a clean oven dried jar with a PTFE-coated magnetic stir bar, the compound (2a) (1.5 g, 2.69 mmol), and 100 mL of dry, degassed toluene to make a solution of compound (2a) in toluene. Place the jar in a glovebox freezer along with a separate bottle containing the solution of 4-tert-butylbenzylmagnesium chloride of Preparation 4 for 15 minutes to cool to −30° C. Then add the solution of 4-tert-butylbenzylmagnesium chloride to an addition funnel, and add the contents of the addition funnel dropwise to the solution of the compound (2a). Stir the mixture and allow it to come to room temperature (r.t.) over 1 hour. Then add 0.5 mL of 1,4-dioxane, and filter the resulting mixture through diatomaceous earth. Concentrate the filtrate under reduced pressure, and take up the resulting residue in 30 mL of toluene. Again filter through and concentrate under reduced pressure to give a twice filtered/concentrated residue. Triturate the residue with three 10 mL portions of hexane, and dry the triturated residue under reduced pressure to ensure complete removal of toluene. Add 20 mL of pentane to the residue, and place the resulting mixture in the glovebox freezer for 72 hours to give a yellow precipitate, which is collected by filtration through a chilled PTFE frit and dried under reduced pressure to give 0.95 g of compound (1A) (45% yield).1H NMR (400 MHz, Benzene-d6) δ 7.31-7.23 (m, 2H), 7.18-7.07 (m, 4H), 5.73-5.66 (m, 2H), 3.45 (dt, J=11.8, 5.5 Hz, 2H), 3.25 (dd, J=9.8, 4.5 Hz, 1H), 3.15 (dt, J=12.0, 5.7 Hz, 2H), 2.76-2.65 (m, 2H), 2.49 (d, J=4.4 Hz, 13H), 2.28 (s, 6H), 2.14 (d, J=18.8 Hz, 11H), 1.77 (s, 2H), 1.33 (s, 8H), 1.21 (s, 8H), 0.87 (s, 2H).13C NMR (101 MHz, Benzene-d6) δ 152.70, 148.52, 147.67, 142.21, 136.97, 133.69, 132.32, 131.19, 130.57, 130.41, 129.41, 126.93, 125.50, 124.38, 63.41, 58.04, 53.38, 49.37, 34.13, 34.08, 31.90, 31.88, 17.18, 17.14, 17.06, 16.68, 16.61. Measure the light-off performance according to the Light-Off Test Method and measure the according to the Solubility Test Method. Solubility and time to maximum temperature results are reported later in Table 1. Inventive Example 4 (IE4) (prophetic): preparation of solutions of compound (1A) (compound (1) wherein M is Zr and each R is CH2-(1,4-phenylene)-C(CH3)3) in hexane. Dissolve measured quantities of compound (1A) in separate aliquots of hexane to give 700 mL of 0.91 wt % compound (1A) in hexane, 700 mL of 1.18 wt % compound (1A) in hexane, and 550 mL of 0.91 wt % compound (1A) in hexane, respectively. The solutions do not need to be chilled but may be transported or stored at 25° C. Inventive Example 5 (IE5) (prophetic): preparation of a precatalyst formulation of compound (1A) in alkanes. Charge the three solutions of compound (1A) of IE4 to a 106 liter (L) capacity cylinder. Add 11.3 kilograms (kg) of high purity isopentane to the cylinder to give a precatalyst formulation of 0.10 wt % solution of compound (1A) in hexane/isopentane mixture. The precatalyst formulation of compound (1A) does not need to be chilled, but may be transported or stored at 25° C. Inventive Example 6 (IE6) (prophetic): making unimodal catalyst system from compound (1A) and activator. Separately feed the activator formulation of Preparation 1B through a catalyst injection tube and feed freshly-prepared precatalyst system of IE5 through a different catalyst injection tube into an in-line mixer, wherein the contact each other to give the unimodal catalyst system, which then flows through an injection tube into the reactor. Inventive Example 7 (IE7) (prophetic): making a bimodal catalyst system comprising a non-metallocene catalyst made from compound (1A) and a metallocene catalyst made from (MeCp)(1,3-dimethyl-4,5,6,7-tetrahydroindenyl)ZrMe2, wherein Me is methyl, Cp is cyclopentadienyl, and MeCp is methylcyclopentadienyl. Separately feed the spray-dried metallocene with activator formulation of Preparation 2 through a catalyst injection tube and feed the precatalyst formulation of compound (1A) of IE5 through a different catalyst injection tube into an in-line mixer, wherein the feeds contact each other to form the catalyst system, which then flows through an injection tube into the reactor. Inventive Example 8 (IE8) (prophetic): copolymerization of ethylene and 1-hexene using a unimodal catalyst system prepared from compound (1A) to make a unimodal poly(ethylene-co-1-hexene) copolymer. For each run, use a gas phase fluidized bed reactor that has a 0.35 m internal diameter and 2.3 m bed height and a fluidized bed primarily composed of polymer granules. Pass fluidization gas through the bed at a velocity of from 0.51 meter per second (m/s) to 0.58 m/s. Exit the fluidization gas from the top of the reactor, and pass the exited gas through a recycle gas line having a recycle gas compressor and heat exchanger before re-entering it into the reactor below a distribution grid. Maintain a constant fluidized bed temperature of 105° C. by continuously adjusting the temperature and/or flow rate of cooling water used for temperature control. Introduce gaseous feed streams of ethylene, nitrogen and hydrogen together with 1-hexene comonomer into the recycle gas line. Operate the reactor at a total pressure of 2410 kilopascals gauge (kPa gauge). Vent the reactor to a flare to control the total pressure. Adjust individual flow rates of ethylene, nitrogen, hydrogen and 1-hexene to maintain gas composition targets. Set ethylene partial pressure at 1.52 megapascal (MPa). Set the 1-hexene/ethylene (C6/C2) molar ratio to 0.0050 and the hydrogen/ethylene (H2/C2) molar ratio to 0.0020. Maintain ICA (isopentane) concentration at 8.5 to 9.5 mol %. Measure concentrations of all gasses using an on-line gas chromatograph. Feed freshly-prepared unimodal catalyst system of IE6 into the polymerization reactor at a rate sufficient to maintain a production rate of about 13 to 16 kg/hour poly(ethylene-co-1-hexene) copolymer, while also controlling feed rate to achieve a loading of 50 micromoles of zirconium per gram of spray dried solids. The poly(ethylene-co-1-hexene) copolymer (“resin”) is characterized as unimodal molecular weight distribution, and by a high load melt index (HLMI or I21) in g/10 minutes, a density of in g/cm3, a number-average molecular weight (Mn), a weight-average molecular weight (Mw), a z-average molecular weight (Mz), and a molecular weight distribution (Mw/Mn). IE8 makes a unimodal high molecular weight copolymer using a unimodal catalyst system comprising an activator formulation that does not comprise a precatalyst, and a precatalyst formulation comprising precatalyst (1) that does not contain activator. Expected resin particle size and particle size distribution data are shown later in Table 2. Inventive Example 9 (IE9) (prophetic): copolymerization of ethylene and 1-hexene using a bimodal catalyst system prepared from compound (1A) and a metallocene to make a bimodal poly(ethylene-co-1-hexene) copolymer. Replicate the polymerization procedure of IE8 except instead of feeding the unimodal catalyst system of IE6 feed the bimodal catalyst system of IE7 into the reactor. Adjust the ratio of compound (1A) feed to spray-dried metallocene slurry to adjust the high load melt index (121) of the bimodal poly(ethylene-co-1-hexene) copolymer in the reactor to approximately 6 g/10 minutes. Increase the C6/C2 molar ratio to 0.0060 to reduce the density of bimodal poly(ethylene-co-1-hexene) copolymer. Adjust the feed rate of the spray dried metallocene slurry and compound (1A) solution at a rate sufficient to maintain a production rate of about 13 to 16 kg/hour of the bimodal poly(ethylene-co-1-hexene) copolymer. The bimodal poly(ethylene-co-1-hexene) copolymer produced is bimodal, has an 121 of 6 g/10 minutes, a melt flow ratio (121/15), a density in g/cm3, Mn, Mw, Mz, and Mw/Mn. The bimodality of the bimodal poly(ethylene-co-1-hexene) copolymer of IE9 is illustrated by the prophetic GPC plot shown inFIG.1. Expected resin particle size and particle size distribution data are given later in Table 2. Inventive Example 10 (IE10): synthesis of compound (1B) (compound (1) wherein M is Zr and each R is CH3) from compound (2a). Under an atmosphere of nitrogen in a glovebox, charge an oven-dried 100 mL glass jar with a PTFE-coated magnetic stir bar, compound (2a) (0.5 g, 0.9 mmol), and 25 mL of dry, degassed dichloromethane. Place the mixture in the glove box freezer for 1 hour to cool to −30° C. Slowly add a 3.0 M solution of methylmagnesium bromide in diethyl ether (0.6 mL, 1.8 mmol) with stirring, then allow the mixture to warm to room temperature with stirring for 30 minutes. Quench the mixture with 0.2 mL of 1,4-dioxane, then filter it through PTFE, and concentrate the filtrate under reduced pressure. Triturate the residue in 20 mL of n-pentane, and filter the resulting solid. Dry the solid under reduced pressure to give 0.32 g (69% yield) of compound (1B) as a pale orange powder.1H NMR (400 MHz, Benzene-d6) δ 3.40 (ddd, J=12.3, 8.9, 5.5 Hz, 3H), 3.11 (ddd, J=12.3, 5.2, 3.3 Hz, 2H), 2.51 (s, 7H), 2.49 (s, 7H), 2.47-2.42 (m, 5H), 2.21 (s, 6H), 2.18 (s, 7H), 2.11 (s, 7H), 0.17 (s, 3H), 0.07 (s, 3H). Measure the light-off performance according to the Light-Off Test Method and measure the according to the Solubility Test Method. Solubility and time to maximum temperature results are reported later in Table 1. Inventive Example 11 (IE11): synthesis of compound (1C) (compound (1) wherein M is Zr and each R is CH2-(1,3-phenylene)-C4H9) from compound (2a) Charge a clean oven dried jar with a PTFE-coated magnetic stir bar with the compound (2a) (0.4 g, 0.7 mmol), and 20 mL of dry, degassed toluene to make a solution of compound (2a) in toluene. Place the jar in a glovebox freezer along with a separate bottle containing the solution of 3-n-butylbenzylmagnesium chloride of Preparation 7 for 15 minutes to cool to −30° C. Then add the solution of 3-n-butylbenzylmagnesium chloride to an addition funnel, and add the contents of the addition funnel dropwise to the solution of the compound (2a). Stir the mixture and allow it to come to room temperature (r.t.) over 16 hours. Then add 20 mL of diethyl ether, and filter the resulting mixture through diatomaceous earth. Concentrate the filtrate under reduced pressure, and take up the resulting residue in 30 mL of toluene. Again filter through diatomaceous earth and concentrate under reduced pressure to give a twice filtered/concentrated residue. Triturate the residue with three 10 mL portions of hexane, and dry the triturated residue under reduced pressure to ensure complete removal of toluene. Add 20 mL of pentane to the residue, and place the resulting mixture in the glovebox freezer for 72 hours to give a yellow precipitate, which is collected by filtration through a chilled PTFE frit and dried under reduced pressure to give 0.12 g of compound (1C) (22% yield).1H NMR (400 MHz, Benzene-d6) δ 7.21 (t, J=7.4 Hz, 1H), 7.08-7.01 (m, 2H), 6.88 (t, J=7.5 Hz, 1H), 6.81 (dt, J=7.6, 1.4 Hz, 1H), 6.76-6.71 (m, 1H), 5.58-5.51 (m, 2H), 3.48 (dt, J=11.8, 5.6 Hz, 2H), 3.34 (s, 1H), 3.19 (dt, J=12.1, 5.8 Hz, 2H), 2.73 (dq, J=12.2, 6.0 Hz, 3H), 2.61 (td, J=7.5, 6.9, 4.0 Hz, 5H), 2.48 (d, J=5.8 Hz, 10H), 2.27 (s, 6H), 2.15 (s, 7H), 2.11 (s, 7H), 1.83 (s, 2H), 1.72-1.61 (m, 3H), 1.44-1.35 (m, 3H), 1.31 (dd, J=14.8, 7.4 Hz, 3H), 0.93 (s, 2H), 0.93-0.86 (m, 3H).13C NMR (101 MHz, Benzene-d6) δ 147.35, 146.46, 142.43, 133.37, 132.09, 131.93, 130.96, 130.25, 130.11, 124.83, 123.77, 121.68, 119.94, 63.63, 57.68, 53.33, 49.12, 36.11, 36.07, 32.67, 22.32, 16.82, 16.78, 16.70, 16.35, 16.29, 13.79. Measure the light-off performance according to the Light-Off Test Method and measure the according to the Solubility Test Method. Solubility and time to maximum temperature results are reported later in Table 1. Inventive Example 12 (IE12): synthesis of compound (1D) (compound (1D) wherein M is Zr and each R is CH2-(1,3-phenylene)-CH3) from compound (4) Charge a clean oven dried 40 mL vial with a PTFE-coated magnetic stir bar with tetra(3-methylbenzyl)zirconium of Preparation 8 (0.12 g, 0.2 mmol) and 5 mL of dry, degassed toluene. Add the compound 4 as a solid to the vial and stir the mixture at room temperature for 2 hours. Add 30 mL of pentane to the mixture and collect a beige solid by filtration, then wash the solid with 10 mL of cold pentane to give 88 mg of the desired product (53.4% yield).1H NMR (400 MHz, Benzene-d6) δ 7.25-7.10 (m, 1H), 7.05-6.98 (m, 2H), 6.86-6.70 (m, 3H), 5.50 (d, J=7.8 Hz, 1H), 5.44 (s, 1H), 3.53-3.40 (m, 2H), 3.29-3.20 (m, 1H), 3.15 (dt, J=12.0, 5.8 Hz, 2H), 2.69 (q, J=6.1, 5.5 Hz, 3H), 2.57 (td, J=10.9, 5.3 Hz, 2H), 2.47 (s, 6H), 2.42 (s, 6H), 2.29 (s, 3H), 2.24 (s, 7H), 2.15 (s, 7H), 2.10 (s, 7H), 1.98 (s, 3H), 1.78 (s, 2H), 0.91-0.83 (m, OH), 0.87 (s, 2H).13C NMR (101 MHz, Benzene-d6) δ 147.27, 141.46, 137.28, 133.33, 132.11, 131.90, 130.95, 130.22, 130.14, 125.71, 124.35, 121.30, 120.39, 63.48, 57.66, 53.13, 49.13, 21.59, 16.77, 16.71, 16.34, 16.27. Measure the light-off performance according to the Light-Off Test Method and measure the according to the Solubility Test Method. Solubility and time to maximum temperature results are reported later in Table 1. Comparative Example 3 (CE3): copolymerization of ethylene and 1-hexene using a comparative unimodal catalyst system made with HN5Zr dibenzyl of CE2 in a spray-dried formulation with hydrophobic fumed silica and MAO to make a comparative unimodal poly(ethylene-co-1-hexene) copolymer. Replicate the procedure of IE8 except using the comparative unimodal catalyst system instead of the unimodal catalyst system of IE6. The comparative poly(ethylene-co-1-hexene) copolymer is characterized as unimodal molecular weight distribution, an high load melt index (HLMI or I21) of 0.20 g/10 minutes and a density of 0.9312 g/cm3. Resin particle size and particle size distribution are shown later in Table 2. TABLE 1solubility in hexanes containing at least 60 wt % n-hexaneand light-off performance in polymerization of 1-octene.Solubility inLight-off Performance (TimePrecatalystHexanes (wt %)to Maximum (minutes)HN5Zr dichlorideNot measured5.2(CE1)HN5Zr dibenzyl0.0378.6(CE2)Compound (1A)2.30.8Compound (1B)0.61.6Compound (1C)0.138.7Compound (1D)0.56.3 Compound (1A) has a solubility of 2.3 weight percent in hexanes containing at least 60 weight percent n-hexane measured according to the Solubility Test Method. Unpredictably, the solubility of compound (1A) in hexanes is 76 times greater than the solubility of HN5Zr dibenzyl (CE2) in hexanes. Compound (1A) has a time to maximum temperature of 0.8 minute in the Light-Off Test Method. Unpredictably, the time to maximum temperature of compound (1A) is 6 times better than HN5Zr dichloride (CE1) and 99 times better than HN5Zr dibenzyl (CE2). In Table 1, compound (1) has significantly increased solubility in alkanes, which enables reduced complexity of transitions between catalyst systems, and has significantly greater light-off performance than those of comparative precatalyst HN5Zr dibenzyl, which can decrease distributor plate fouling in gas phase polymerization reactors. Thus, compound (1) solves the aforementioned problems of prior non-MCN precatalysts. TABLE 2resin average particle size and particle size distributionof CE3 and expected values for IE8 and IE9.CE3IE8IE9Particle Property(measured)(expected)(expected)APS (mm)0.071212.00 mm (10 mesh) screen (wt %)41.260101.00 mm (18 mesh) screen (wt %)35.530300.500 mm (35 mesh) screen (wt %)15.32300.250 mm (60 mesh) screen (wt %)6.00.2200.125 mm (120 mesh) screen1.70.14(wt %)0.074 mm (200 mesh) screen0.30.10.5(wt %)Bottom Catch Pan (wt %)0.10.000.00Fines (wt % of total)0.40.10.5 In Table 2, APS (mm) is average particle size in millimeters. The expected average particle size of the particles of the prophetic inventive unimodal poly(ethylene-co-1-hexene) copolymer of IE8 is larger than the measured APS of the comparative unimodal poly(ethylene-co-1-hexene) copolymer of CE3. The bottom catch pan collects any particles that pass through the 0.074 mm (200 mesh) screen. The percent fines is equal to the sum of the wt % of particles that are trapped by the 0.074 mm (200 mesh) screen plus the wt % of particles that pass through the 0.074 mm (200 mesh) screen and are collected in the bottom catch pan. In Table 2, the measured percent fines of the comparative unimodal poly(ethylene-co-1-hexene) copolymer of CE3 is greater than the expected percent fines of the prophetic inventive unimodal poly(ethylene-co-1-hexene) copolymer of IE8. | 91,551 |
11859032 | DETAILED DESCRIPTION The pyridyldiamido and quinolinyldiamido transition metal complexes described herein were tested for terpolymerization capability at increasing VNB feed rates and a target ethylene content of approximately 60%. Analytical techniques such as gel permeation chromatograph (GPC), dynamic shear rheology, and extensional viscosity analyses of the resulting polymers were all consistent with higher long-chain branching (LCB) levels as the VNB content was increased. Performing the same experiments with ENB as the diene resulted in, for instance, 0.8 wt % ENB incorporation, indicating that approximately 75% of the vinyl group of VNB in the polymer was reacted to form long chain branches. GPC-4D analysis of the VNB-EPDM samples resulted in 100% recovery, which suggested negligible gel content in the material. However, in-reactor gel was obtained upon opening the reactor, albeit at a lower amount compared with those observed using metallocene-type polymerization catalysts. Thus, the inventors have found an improved method of forming ethylene-α-olefin diene elastomers, as set forth more particularly herein. In any embodiment, the “dual-polymerizable dienes” are diene monomers selected from vinyl substituted strained bicyclic and unconjugated dienes, and alpha-omega linear dienes where both sites of unsaturation are polymerizable by a polymerization catalyst (e.g., Ziegler-Natta, vanadium, metallocene, etc.); and more preferably from vinyl norbornenes and C7 to C12 alpha-omega linear dienes (e.g., 1,7-heptadiene and 1,9-decadiene), and is most preferably 5-vinyl-2-norbornene (VNB). The b-EDE formed therefrom comprises “dual-polymerizable diene derived monomer units”. In any embodiment, the “singly-polymerizable dienes” are diene monomers in which only one of the double bonds is activated by a polymerization catalyst and is selected from cyclic and linear alkylenes, non-limiting examples of which include an unconjugated diene (and other structures where each double bond is two carbons away from the other), 5-ethylidene-2-norbornene, 4-vinylcyclohexene and other strained bicyclic and unconjugated dienes, and dicyclopentadiene. More preferably, the singly-polymerizable diene is selected from C7 to C30 cyclic singly-polymerizable dienes. Most preferably the singly-polymerizable diene is 5-ethylidene-2-norbornene (ENB). The b-EDE formed therefrom comprises “singly-polymerizable diene derived monomer units”. In any embodiment, a “branched” ethylene-α-olefin diene elastomer (b-EDE) has a branching index value at a molecular weight of 1×106g/mol, g′1000, of less than or equal to 0.860 as calculated using the output of the GPC-IR5-LS-VIS method as follows. The average intrinsic viscosity, [ηavg], of the sample is calculated by: [η]avg=∑ci[η]i∑ci, where the summations are over the chromatographic slices, “i”, between the integration limits. The branching index g′avgis defined as: gavg′=[η]avgkMvα. In any embodiment, the branched polymer has minimal gel content. As used herein, the “gel content” refers to an insoluble portion (in hydrocarbon solvent) of polymer determined by extraction of a sample of the b-EDE in a hydrocarbon solvent such as cyclohexane, toluene or isohexane, which are typically used to dissolve b-EDE. In any embodiment, the gel content of the inventive b-EDE is less than 5, or 1, or 0.1 wt %. In any embodiment, the “pyridyldiamido and quinolinyldiamido transition metal complexes” include organometallic complexes of a transition metal ion, especially titanium, zirconium or hafnium, with one or more ligands that include at least one pyridyl and/or quinolinyl group and at least two other alkylamine ligands, and at least one leaving group, preferably a halogen or alkyl group, that is reactive towards the appropriate boron and/or aluminum-based activator. In any embodiment, the pyridyldiamido and quinolinyldiamido transition metal complexes are selected from one of the following structures: wherein M is titanium, hafnium or zirconium, most preferably hafnium;R1and R10are independently selected from the group consisting of hydrocarbyls (such as alkyls, aryls), substituted hydrocarbyls (substituents pendant to the hydrocarbyl), heterohydrocarbyls (non-carbon atoms within the hydrocarbyl), and silyl groups; most preferably R1and R10comprise an aniline structure that may be substituted with C1 to C5 alkyls;R2and R9are each, independently, divalent hydrocarbyls or a chemical bond;R3, R4, R5, R6, R7, and R8are independently selected from the group consisting of hydrogen, hydrocarbyls (e.g., alkyls and aryls), substituted hydrocarbyls (e.g., heteroaryl), alkoxy, aryloxy, halogen, amino, and silyl, and wherein adjacent R groups may be joined to form a substituted or unsubstituted hydrocarbyl or heterocyclic ring, where the ring has 5, 6, 7, or 8 ring atoms and where substitutions on the ring can join to form additional rings;X is an anionic leaving group, where the X groups may be the same or different and any two X groups may be linked to form a dianionic leaving group; andZ is —(R11)pQJ(R12)qwherein Q is carbon, oxygen, nitrogen, or silicon (preferably nitrogen), and where J is carbon or silicon (preferably carbon), p is 1 or 2; and q is 1 or 2; and R11and R12are independently selected from the group consisting of hydrogen, hydrocarbyls (preferably alkyls), and substituted hydrocarbyls, and wherein adjacent R11and R12groups may be joined to form an aromatic or saturated, substituted or unsubstituted hydrocarbyl ring, where the ring has 5, 6, 7, or 8 ring carbon atoms and where substitutions on the ring can join to form additional rings; most preferably Z forms a bicyclic hydrocarbyl comprising a C6 cyclic portion and a C4 to C6 cyclic portion, where an example of Z is a divalent tetrahydroindenyl or divalent tetrahydronaphthalene. In any embodiment, the pyridyldiamido and quinolinyldiamido transition metal complexes are selected from one of the following structures: wherein the “Me” represents “methyl” and “iPr” represents “iso-propyl”, and wherein these groups could also variously be any C1 to C10 alkyl (normal, iso, and/or tertiary), and the saturated ring may variously be a 4 to 6 membered ring, interchangeably between the two structures. Thus in any embodiment is a process to produce a branched ethylene-α-olefin diene elastomer (b-EDE) comprising (or consisting essentially of, or consisting of) combining a catalyst precursor and an activator with a feed comprising ethylene, C3 to C12 α-olefins, and a dual-polymerizable diene to obtain a b-EDE; where the catalyst precursor is selected from pyridyldiamide and quinolinyldiamido transition metal complexes. The catalyst or catalyst precursor must also be combined with at least one “activator” to effect polymerization of the cyclic olefin monomers and ethylene, wherein the activator preferably comprises a non-coordinating borate anion and a bulky organic cation. In any embodiment, the non-coordinating borate anion comprises a tetra(perfluorinated C6 to C14 aryl)borate anion and substituted versions thereof; most preferably the non-coordinating borate anion comprises a tetra(pentafluorophenyl)borate anion or tetra(perfluoronaphthyl)borate anion. Preferably the bulky organic cation is selected from the following structures (a) and (b): wherein each R group is independently hydrogen, a C6 to C14 aryl (e.g., phenyl, naphthyl, etc.), a C1 to C10 or C20 alkyl, or substituted versions thereof; and more preferably at least one R group is an C6 to C14 aryl or substituted versions thereof. In any embodiment, the bulky organic cation is a reducible Lewis Acid, especially a trityl-type cation (wherein each “R” group in (a) is aryl) capable of extracting a ligand from the catalyst precursor, where each “R” group is an C6 to C14 aryl group (phenyl, naphthyl, etc.) or substituted C6 to C14 aryl, and preferably the reducible Lewis acid is triphenyl carbenium and substituted versions thereof. Also, in any embodiment, the bulky organic cation is a Brønsted acid capable of donating a proton to the catalyst precursor, wherein at least one “R” group in (b) is hydrogen. Exemplary bulky organic cations of this type in general include ammoniums, oxoniums, phosphoniums, silyliums, and mixtures thereof; preferably ammoniums of methylamine, aniline, dimethylamine, diethylamine, N-methylaniline, diphenylamine, trimethylamine, triethylamine, N,N-dimethyl aniline, methyldiphenylamine, pyridine, p-bromo-N,N-dimethylaniline, and p-nitro-N,N-dimethyl aniline; phosphoniums from triethylphosphine, triphenylphosphine, and diphenylphosphine; oxoniums from ethers, such as dimethyl ether diethyl ether, tetrahydrofuran, and dioxane; and sulfoniums from thioethers, such as diethyl thioethers and tetrahydrothiophene, and mixtures thereof. The catalyst precursor preferably reacts with the activator upon their combination to form a “catalyst” or “activated catalyst” that can then effect the polymerization of monomers. The catalyst may be formed before combining with monomers, after combining with monomers, or simultaneous therewith. In any embodiment, the combining takes place at a temperature within a range from 80, or 90° C. to 120, or 140, or 160° C. and a pressure within a range from 0.5 MPa to 4, or 6, or 8 MPa. Most preferably the combining in a solution process, meaning that all components in the polymerization are soluble in the medium (diluent and/or monomers) or at least 80, or 90 wt % of the components are soluble and dissolved in the medium. In any embodiment, the solution process can be carried out in one or more single-phase, liquid-filled, stirred tank reactor with continuous flow of feeds to the system and continuous withdrawal of products under steady state conditions. When more than one reactor is used, the reactors may be operated in a serial or parallel configuration making essentially the same or different polymer components. Advantageously, the reactors would produce polymers with different properties, such as different molecular weights, or different monomer compositions, or different levels of long-chain branching, or any combinations thereof. All polymerizations can be performed in a system with a solvent comprising any one or more of C4 to C12 alkanes, using soluble metallocene catalysts or other single-site catalysts and discrete, non-coordinating borate anion as co-catalysts. A homogeneous dilute solution of tri-n-octyl aluminum or other aluminum alkyl in a suitable solvent may be used as a scavenger in concentrations appropriate to maintain reaction. Chain transfer agents, such as hydrogen, can be added to control molecular weight. Polymerizations can be at high temperatures described above and high conversions to maximize macromer re-insertions that create long chain branching, if so desired. This combination of a homogeneous, continuous, solution process helped to ensure that the products had narrow composition and sequence distributions. In any embodiment the process also comprises further combining a singly-polymerizable diene. Also in any embodiment the α-olefins comprise (or consist of) propylene. Finally, hydrogen is preferably present to less than 5, or 1, or 0.8, or 0.4, or 0.2 sccm (standard cubic centimeter per min.) from the feed; and most preferably hydrogen is absent from the feed. When referring to the “feed”, this components that are combined include only those substances in the feed. Produced from the process is a branched ethylene-α-olefin diene elastomer (b-EDE) comprising (or consisting of, or consisting essentially of) within a range from 40, or 45 to 65, or 70, or 75, or 80 wt % of ethylene-derived units by weight of the b-EDE, 0.1 to 0.8, or 1, or 1.4, or 1.8, or 2 wt % of singly-polymerizable diene derived units by weight of the b-EDE, within a range from 0.1 to 0.5, or 0.8, or 1, or 1.4, or 1.8, or 2 wt % of a singly-polymerizable diene derived units by weight of the b-EDE, and the remainder comprising C3 to C12 α-olefin derived units (preferably propylene derived units), wherein the b-EDE has a weight average molecular weight (Mw) within a range from 100 kg/mole to 200, or 240, or 280, or 300, or 400, or 600, or 750 kg/mole, and wherein the b-EDE has a g′avgof 0.9 or more, and a g′1000of less than 0.9, or 0.85, or 0.8 (or within a range from 0.4, or 0.6, or 0.65 to 0.8 or 0.85 or 0.9). The inventive b-EDEs may be useful in any number of applications such as rubber profiles (like automotive solid and sponge profiles, building profiles), hoses, mechanical goods, films (cast and/or blown) and sheets of material, such as for roofing applications, as well as thermoformed articles, blow molded articles, rotomolded articles, and injection molded articles. Particularly desirable end uses include automotive components and gaskets. Any of these articles may be foamed articles which are formed by means known in the art. Foamed or not, some specific uses of the inventive b-EDEs include weather stripping, heat insulation, opening trim, and car trunk or car hood seals. The various descriptive elements and numerical ranges disclosed herein for the inventive process and b-EDE therefrom can be combined with other descriptive elements and numerical ranges to describe the invention(s); further, for a given element, any upper numerical limit can be combined with any lower numerical limit described herein, including the examples in jurisdictions that allow such combinations. The features of the inventions are demonstrated in the following non-limiting examples. EXAMPLES The synthesis of the catalyst precursor is described here, as well as the polymerization examples. Proton (1H) Nuclear Magnetic Resonance Catalyst characterization was accomplished using proton NMR, wherein the1H NMR data was collected at 23° C. in a 5 mm probe using a Varian spectrometer with a1H frequency of at least 400 MHz. Data was recorded using a maximum pulse width of 45°, 8 sec between pulses and signal averaging 120 transients. All NMR spectra were referenced using the peak corresponding to the deuterated solvent. Starting Reagents Sodium hydride (NaH), 8-bromoquinolin-2(1H)-one, t-butyldimethylsilylchloride, n-butyllithium, t-butyllithium, Pd2(dba)3, 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos), K2CO3, dichloromethane, methanol, POCl3, n-hexane, 1,2,3,4-tetrahydronaphthalen-1-ol, N,N,N′,N′-tetramethyl ethylene diamine (TMEDA), pentane, 1,2-dibromotetrafluoroethane, Na2SO4, triethylamine, acetic anhydride, 4-(dimethylamino)pydridine (DMAP), ethyl acetate, Na2CO3, potassium hydroxide (KOH), pyridinium chlorochromate (PCC), aniline, toluene, TiCl4, NaBH3CN, acetic acid, CDCl3, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, 1,4-dioxane, cesium carbonate, Pd(PPh3)4, benzene, Hf(NMe2)4, Me3Al, 6-bromopyridine-2-carboxaldehyde, 2,6-diisopropylaniline, indan-1-ol and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-di oxaborolane were purchased from commercial sources and used as received. Hf(NMe2)2Cl2, 1,2-dimethoxyethane (DME), and dimethylmagnesium were prepared following published procedures (Erker et al., in 19 ORGANOMETALLICS 127 (2000); Coates and Heslop, in J. CHEM. SOC. A 514 (1968); Andersen et al., in J. CHEM. SOC., DALTONTRANS. 809 (1977)). Tetrahydrofuran (THF, Merck) and diethyl ether (Merck) were freshly distilled from benzophenone ketyl were used for organometallic synthesis and catalysis. 8-(2,6-Diisopropylphenylamino)quinolin-2(1H)-one To a suspension of NaH (5.63 g of 60 wt % in mineral oil, 140 mmol) in tetrahydrofuran (1000 mL) was added 8-bromoquinolin-2(1H)-one (30.0 g, 134 mmol) in small portions at 0° C. The obtained reaction mixture was warmed to 23° C. (room temperature), stirred for 30 min, then cooled to 0° C. Then t-butyldimethylsilylchloride (20.2 g, 134 mmol) was added in one portion. This mixture was stirred for 30 min at 23° C. and then poured into water (1 L). The protected 8-bromoquinolin-2(1H)-one was extracted with diethyl ether (3×400 mL). The combined extracts were dried over Na2SO4and then evaporated to dryness. Yield 45.2 g (quant., 99% purity by GC/MS) of a dark red oil. To a solution of 2,6-diisopropylaniline (27.7 mL, 147 mmol) and toluene (1.5 L) was added n-butyllithium (60.5 mL, 147 mmol, 2.5 M in hexanes) at 23° C. The obtained suspension was heated briefly to 100° C. and then cooled to 23° C. To the reaction mixture was added Pd2(dba)3(dba=dibenzylideneacetone) (2.45 g, 2.68 mmol) and XPhos (2.55 g, 5.36 mmol) followed by the addition of the protected 8-bromoquinolin-2(1H)-one (45.2 g, 134 mmol). The obtained dark brown suspension was heated at 60° C. until lithium salt precipitate disappeared (ca. 30 min). The resulting dark red solution was quenched by addition of water (100 mL), and the organic layer was separated, dried over Na2SO4and then evaporated to dryness. The obtained oil was dissolved in a mixture of dichloromethane (1000 mL) and methanol (500 mL), followed by an addition of 12 M HCl (50 mL). The reaction mixture was stirred at 23° C. for 3 hr., then poured into 5% K2CO3(2 L). The product was extracted with dichloromethane (3×700 mL). The combined extracts were dried over Na2SO4, filtered, and then evaporated to dryness. The resulting solid was triturated with n-hexane (300 mL), and the obtained suspension collected on a glass frit. The precipitate was dried in vacuum. Yield 29.0 g (67%) of a marsh-green solid. Anal. calc. for C21H24N2O: C, 78.71; H, 7.55; N, 8.74. Found: C, 79.00; H, 7.78; N, 8.50.1H NMR (CDCl3): δ 13.29 (br.s, 1H), 7.80-7.81 (d, 1H, J=9.5 Hz), 7.35-7.38 (m, 1H), 7.29-7.30 (m, 3H), 6.91-6.95 (m, 2H), 6.58-6.60 (d, 1H, J=9.5 Hz), 6.27-6.29 (m, 1H), 3.21 (sept, 2H, J=6.9 Hz), 1.25-1.26 (d, 6H, J=6.9 Hz), 1.11-1.12 (d, 6H, J=6.9 Hz). 2-Chloro-N-(2,6-diisopropylphenyl)quinolin-8-amine 29.0 g (90.6 mmol) of 8-(2,6-diisopropylphenylamino)quinolin-2(1H)-one was added to 400 mL of POCl3in one portion. The resulting suspension was heated for 40 hrs. at 105° C., then cooled to 23° C., and poured into 4000 cm3of a crushed ice. The crude product was extracted with 3×400 mL of diethyl ether. The combined extract was dried over K2CO3and then evaporated to dryness. The resulting solid was triturated with 30 mL of cold n-hexane, and the formed suspension was collected on a glass frit. The obtained solid was dried in vacuum. Yield 29.0 g (95%) of a yellow-green solid. Anal. calc. for C21H23N2Cl: C, 74.43; H, 6.84; N, 8.27. Found: C, 74.68; H, 7.02; N, 7.99.1H NMR (CDCl3): δ 8.04-8.05 (d, 1H, J=8.6 Hz), 7.38-7.39 (d, 1H, J=8.5 Hz), 7.33-7.36 (m, 1H), 7.22-7.27 (m, 4H), 7.04-7.06 (d, 1H, J=8.1 Hz), 6.27-6.29 (d, 1H, J=7.8 Hz), 3.20 (sept, 2H, J=6.9 Hz), 1.19-1.20 (d, 6H, J=6.9 Hz), 1.10-1.11 (d, 6H, J=6.9 Hz). 8-Bromo-1,2,3,4-tetrahydronaphthalen-1-ol To a mixture of 78.5 g (530 mmol) of 1,2,3,4-tetrahydronaphthalen-1-ol, 160 mL (1.06 mol) of N,N,N′,N′-tetramethylethylenediamine, and 3000 mL of pentane cooled to −20° C. 435 mL (1.09 mol) of 2.5 MnBuLi in hexanes was added dropwise. The obtained mixture was refluxed for 12 hrs. then cooled to −80° C., and 160 mL (1.33 mol) of 1,2-dibromotetrafluoroethane was added. The obtained mixture was allowed to warm to 23° C. and then stirred for 12 hrs. at this temperature. After that, 100 mL of water was added. The resulting mixture was diluted with 2000 mL of water, and the organic layer was separated. The aqueous layer was extracted with 3×400 mL of toluene. The combined organic extract was dried over Na2SO4and then evaporated to dryness. The residue was distilled using the Kugelrohr apparatus, b.p. 150-160° C./1 mbar. The obtained yellow oil was dissolved in 100 mL of triethylamine, and the formed solution was added dropwise to a stirred solution of 71.0 mL (750 mmol) of acetic anhydride and 3.00 g (25.0 mmol) of 4-dimethylaminopyridine in 105 mL of triethylamine. The formed mixture was stirred for 5 min, then 1000 mL of water was added, and the obtained mixture was stirred for 12 hrs. After that, the reaction mixture was extracted with 3×200 mL of ethyl acetate. The combined organic extract was washed with aqueous Na2CO3, dried over Na2SO4, and then evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 μm, eluent: hexane-ethyl acetate=30:1, vol.). The isolated ester was dissolved in 1500 mL of methanol, 81.0 g (1.45 mol) of KOH was added, and the obtained mixture was heated to reflux for 3 hrs. The reaction mixture was then cooled to 23° C. and poured into 4000 mL of water. The title product was extracted with 3×300 mL of dichloromethane. The combined organic extract was dried over Na2SO4and then evaporated to dryness. Yield 56.0 g (47%) of a white crystalline solid.1H NMR (CDCl3): δ 7.38-7.41 (m, 1H, 7-H); 7.03-7.10 (m, 2H, 5,6-H); 5.00 (m, 1H, 1-H), 2.81-2.87 (m, 1H, 4/4′-H), 2.70-2.74 (m, 1H, 4′/4-H), 2.56 (br.s., 1H, OH), 2.17-2.21 (m, 2H, 2,2′-H), 1.74-1.79 (m, 2H, 3,3′-H). 8-Bromo-3,4-dihydronaphthalen-1(2H)-one To a solution of 56.0 g (250 mmol) of 8-bromo-1,2,3,4-tetrahydronaphthalen-1-ol in 3500 mL of dichloromethane was added 265 g (1.23 mol) of pyridinium chlorochromate (PCC). The resulting mixture was stirred for 5 hrs. at 23° C., then passed through a pad of silica gel 60 (500 mL; 40-63 μm), and finally evaporated to dryness. Yield 47.6 g (88%) of a colorless solid.1H NMR (CDCl3): δ 7.53 (m, 1H, 7-H); 7.18-7.22 (m, 2H, 5,6-H); 2.95 (t, J=6.1 Hz, 2H, 4,4′-H); 2.67 (t, J=6.6 Hz, 2H, 2,2′-H); 2.08 (quint, J=6.1 Hz, J=6.6 Hz, 2H, 3,3′-H). (8-Bromo-1,2,3,4-tetrahydronaphthalen-1-yl)phenylamine To a stirred solution of 21.6 g (232 mmol) of aniline in 140 mL of toluene was added 10.93 g (57.6 mmol) of TiCl4over 30 min at 23° C. under argon atmosphere. The resulting mixture was stirred for 30 min at 90° C. followed by an addition of 13.1 g (57.6 mmol) of 8-bromo-3,4-dihydronaphthalen-1(2H)-one. This mixture was stirred for 10 min at 90° C., then cooled to 23° C., and poured into 500 mL of water. The product was extracted with 3×50 mL of ethyl acetate. The combined organic extract was dried over Na2SO4, evaporated to dryness, and the residue was re-crystallized from 10 mL of ethyl acetate. The obtained crystalline solid was dissolved in 200 mL of methanol, 7.43 g (118 mmol) of NaBH3CN and 3 mL of acetic acid were added in argon atmosphere. This mixture was heated to reflux for 3 h, then cooled to 23° C., and evaporated to dryness. The residue was diluted with 200 mL of water, and crude product was extracted with 3×100 mL of ethyl acetate. The combined organic extract was dried over Na2SO4and evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 μm, eluent: hexane-ethyl acetate-triethylamine=100:10:1, vol.). Yield 13.0 g (75%) of a yellow oil. Anal. Calc. for C16H16BrN: C, 63.59; H, 5.34; N, 4.63. Found: C, 63.82; H, 5.59; N, 4.49.1H NMR (CDCl3): δ 7.44 (m, 1H), 7.21 (m, 2H), 7.05-7.11 (m, 2H), 6.68-6.73 (m, 3H), 4.74 (m, 1H), 3.68 (br.s, 1H, NH), 2.84-2.89 (m, 1H), 2.70-2.79 (m, 1H), 2.28-2.32 (m, 1H), 1.85-1.96 (m, 1H), 1.76-1.80 (m, 1H), 1.58-1.66 (m, 1H). N-Phenyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydronaphthalen-1-amine To a solution of 13.0 g (43.2 mmol) of (8-bromo-1,2,3,4-tetrahydronaphthalen-1-yl)phenylamine in 250 mL tetrahydrofuran (THF) was added 17.2 mL (43.0 mmol) of 2.5 MnBuLi at −80° C. Further on, this mixture was stirred for 1 hr. at this temperature, and 56.0 mL (90.3 mmol) of 1.6 MtBuLi in pentane was added. The resulting mixture was stirred for 1 hr. at the same temperature. Then, 16.7 g (90.0 mmol) of 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was added. After that the cooling bath was removed, and the resulting mixture was stirred for 1 hr. at 23° C. Finally, 10 mL of water was added, and the obtained mixture was evaporated to dryness. The residue was diluted with 200 mL of water, and crude product was extracted with 3×100 mL of ethyl acetate. The combined organic extract was dried over Na2SO4and then evaporated to dryness. Yield 15.0 g (98%) of a yellow oil. Anal. Calc. for C22H28BNO2: C, 75.65; H, 8.08; N, 4.01. Found: C, 75.99; H, 8.32; N, 3.79.1H NMR (CDCl3): δ 7.59 (m, 1H), 7.18-7.23 (m, 4H), 6.71-6.74 (m, 3H), 5.25 (m, 1H), 3.87 (br.s, 1H, NH), 2.76-2.90 (m, 2H), 2.12-2.16 (m, 1H), 1.75-1.92 (m, 3H), 1.16 (s, 6H), 1.10 (s, 6H). 2-(8-Anilino-5,6,7,8-tetrahydronaphthalen-1-yl)-N-(2,6-diisopropylphenyl)quinolin-8-amine To a solution of 13.8 g (41.0 mmol) of 2-chloro-N-(2,6-diisopropylphenyl)quinolin-8-amine in 700 mL of 1,4-dioxane were added 15.0 g (43.0 mmol) of N-phenyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydronaphthalen-1-amine, 35.0 g (107 mmol) of cesium carbonate and 400 mL of water. The obtained mixture was purged with argon for 10 min followed by an addition of 2.48 g (2.15 mmol) of Pd(PPh3)4. The formed mixture was stirred for 2 hrs. at 90° C., then cooled to 23° C. To the obtained two-phase mixture 700 mL of n-hexane was added. The organic layer was separated, washed with brine, dried over Na2SO4, and then evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 eluent: hexane-ethyl acetate-triethylamine=100:5:1, vol.) and then re-crystallized from 150 mL of n-hexane. Yield 15.1 g (70%) of a yellow powder. Anal. calc. for C37H39N3: C, 84.53; H, 7.48; N, 7.99. Found: C, 84.60; H, 7.56; N, 7.84.1H NMR (CDCl3): δ 7.85-7.87 (d, J=7.98 Hz, 1H), 7.56 (br.s, 1H), 7.43-7.45 (d, J=8.43 Hz, 1H), 7.21-7.38 (m, 6H), 7.12 (t, J=7.77 Hz, 1H), 6.87-6.89 (d, J=7.99 Hz, 1H), 6.74 (t, J=7.99 Hz, 1H), 6.36 (t, J=7.32 Hz, 1H), 6.14-6.21 (m, 3H), 5.35 (br.s, 1H), 3.56 (br.s, 1H), 3.20-3.41 (m, 2H), 2.83-2.99 (m, 2H), 2.10-2.13 (m, 1H), 1.77-1.92 (m, 3H), 1.13-1.32 (m, 12H). Complex of quinolinyldiamido (QDA) Benzene (50 mL) was added to 2-(8-anilino-5,6,7,8-tetrahydronaphthalen-1-yl)-N-(2,6-diisopropylphenyl)quinolin-8-amine (2.21 g, 4.20 mmol) and Hf(NMe2)4(1.58 g, 4.45 mmol) to form a clear orange solution. The mixture was heated to reflux for 16 hrs. to form a clear red-orange solution. Most of the volatiles were removed by evaporation under a stream of nitrogen to afford a concentrated red solution (ca. 5 mL) that was warmed to 40° C. Then hexane (30 mL) was added and the mixture was stirred to cause orange crystalline solid to form. This slurry was cooled to −40° C. for 30 min. then the solid was collected by filtration and washed with additional cold hexane (2×10 mL). The resulting quinolinyldiamide hafnium diamide was isolated as an orange solid and dried under reduced pressure (2.90 g, 3.67 mmol, 87.4% yield). This solid was dissolved in toluene (25 mL) and Me3Al (12.8 mL, 25.6 mmol) was added. The mixture was warmed to 40° C. for 1 hr. then evaporated under a stream of nitrogen. The crude product (2.54 g) was about 90% pure by1H NMR spectroscopy. The solid was purified by recrystallization from CH2Cl2-hexanes (20 mL-20 mL) by slow evaporation to give pure product as orange crystals (1.33 g, 43.2% from ligand). The mother liquor was further concentrated for a second crop (0.291 g, 9.5% from ligand). (Solvent: CD2Cl2(ca. 10 mg sample/mL solvent))(Reference peak=CHDCl2δ 5.32 ppm). Preparation of N-[(6-bromopyridin-2-yl)methyl]-2,6-diisopropylaniline A solution of 85.0 g (457 mmol) of 6-bromopyridine-2-carbaldehyde and 80.9 g (457 mmol) of 2,6-diisopropylaniline in 1000 mL of ethanol was refluxed for 8 hrs. The obtained solution was evaporated to dryness, and the residue was re-crystallized from 200 mL of methanol. In argon atmosphere, to thus obtained 113.5 g (329 mmol) of N-[(1E)-(6-bromopyridin-2-yl)methylene]-2,6-diisopropylaniline were added 33.16 g (526 mmol) of NaBH3CN, 9 mL of acetic acid and 1000 mL of methanol. This mixture was refluxed for 12 h, then cooled to 23° C., poured into 1000 mL of water, and crude product was extracted with 3×200 mL of ethyl acetate. The combined extract was dried over sodium sulfate and evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 μm, eluent: hexane-ethyl acetate=10:1, vol.). Yield 104.4 g (66%) of a yellow oil. Anal. calc. for C18H23BrN2: C, 62.25; H, 6.68; N, 8.07. Found: C, 62.40; H, 6.87; N, 7.90.1H NMR (CDCl3): δ 7.50 (m, 1H, 4-H in Py), 7.38 (m, 1H, 5-H in Py), 7.29 (m, 1H, 3-H in Py), 7.05-7.12 (m, 3H, 3,4,5-H in 2,6-iPr2C6H3), 4.18 (s, 2H, CH2NH), 3.94 (br.s, 1H, NH), 3.33 (sept, J=6.8 Hz, 2H, CHMe2), 1.23 (d, J=6.8 Hz, 12H, CHMe2). Preparation of 7-bromoindan-1-ol To a mixture of 100 g (746 mmol) of indan-1-ol, 250 mL (1.64 mol) of N,N,N′,N′-tetramethylethylenediamine, and 3000 mL of pentane cooled to −20° C., 655 mL (1.64 mol) of 2.5M nBuLi in hexanes was added. The reaction mixture was then refluxed for 12 hrs. and then cooled to −80° C. Then, 225 mL (1.87 mol) of 1,2-dibromotetrafluoroethane was added, and the resulting mixture was allowed to warm to 23° C. This mixture was stirred for 12 h, and then 100 mL of water was added. The resulting mixture was diluted with 2000 mL of water, and the organic layer was separated. The aqueous layer was extracted with 3×400 mL of toluene. The combined organic extract was dried over Na2SO4and evaporated to dryness. The residue was distilled using a Kugelrohr apparatus, b.p. 120-140° C./1 mbar. The resulting yellow oil was dissolved in 50 mL of triethylamine, and the obtained solution added dropwise to a stirred solution of 49.0 mL (519 mmol) of acetic anhydride and 4.21 g (34.5 mmol) of 4-(dimethylamino)pyridine in 70 mL of triethylamine. The resulting mixture was stirred for 5 min, then 1000 mL of water was added, and stirring was continued for 12 hrs. Then, the reaction mixture was extracted with 3×200 mL of ethyl acetate. The combined organic extract was washed with aqueous Na2CO3, dried over Na2SO4, and evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 μm, eluent: hexane-ethyl acetate=30:1, vol.). The resulting ester was dissolved in 1000 mL of methanol, 50.5 g (900 mmol) of KOH was added, and this mixture was refluxed for 3 hrs. The reaction mixture was then cooled to 23° C. and poured into 4000 mL of water. Crude product was extracted with 3×300 mL of dichloromethane. The combined organic extract was dried over Na2SO4and evaporated to dryness. Yield 41.3 g (26%) of a white crystalline solid. Anal. Calc for C9H9BrO: C, 50.73; H, 4.26. Found: C, 50.85; H, 4.48.1H NMR (CDCl3): δ 7.34 (d, J=7.6 Hz, 1H, 6-H); 7.19 (d, J=7.4 Hz, 1H, 4-H); 7.12 (dd, J=7.6 Hz, J=7.4 Hz, 1H, 5-H); 5.33 (dd, J=2.6 Hz, J=6.9 Hz, 1H, 1-H), 3.18-3.26 (m, 1H, 3- or 3′-H), 3.09 (m, 2H, 3,3′-H); 2.73 (m, 2H, 2,2′-H). Preparation of 7-bromoindan-1-one to a solution of 37.9 g (177 mmol) of 7-bromoindan-1-ol in 3500 mL of dichloromethane 194 g (900 mmol) of pyridinium chlorochromate was added. The resulting mixture was stirred at 23° C. for 5 hrs., then passed through a silica gel pad (500 mL), and the elute was evaporated to dryness. Yield 27.6 g (74%) of a white crystalline solid. Anal. Calc for C9H7BrO: C, 51.22; H, 3.34. Found: C, 51.35; H, 3.41.1H NMR (CDCl3): δ 7.51 (m, 1H, 6-H); 7.36-7.42 (m, 2H, 4,5-H); 3.09 (m, 2H, 3,3′-H); 2.73 (m, 2H, 2,2′-H). Preparation of 7-bromo-N-phenyl-2,3-dihydro-1H-inden-1-amine To a stirred solution of 10.4 g (112 mmol) of aniline in 60 mL of toluene 5.31 g (28.0 mmol) of TiCl4was added for 30 min at 23° C. in argon atmosphere. The resulting mixture was stirred at 90° C. for 30 min followed by an addition of 6.00 g (28.0 mmol) of 7-bromoindan-1-one. The resulting mixture was stirred for 10 min at 90° C., poured into 500 mL of water, and crude product was extracted with 3×100 mL of ethyl acetate. The organic layer was separated, dried over Na2SO4, and then evaporated to dryness. The residue was crystallized from 10 mL of ethyl acetate at −30° C. The resulting solid was separated and dried in vacuum. After that it was dissolved in 100 mL of methanol, 2.70 g (42.9 mmol) of NaBH3CN and 0.5 mL of glacial acetic acid was added. The resulting mixture was refluxed for 3 hrs. in argon atmosphere. The resulting mixture was cooled to 23° C. and then evaporated to dryness. The residue was diluted with 200 mL of water, and crude product was extracted with 3×50 mL of ethyl acetate. The combined organic extract was dried over Na2SO4and evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 μm, eluent: hexane-ethyl acetate-triethylamine=100:10:1, vol.). Yield 5.50 g (68%) of a yellow oil. Anal. calc. for C15H14BrN: C, 62.52; H, 4.90; N, 4.86. Found: C, 62.37; H, 5.05; N, 4.62.1H NMR (CDCl3): δ 7.38 (m, 1H, 6-H in indane); 7.22 (m, 3H, 3,5-H in phenyl and 4-H in indane); 7.15 (m, 1H, 5-H in indane); 6.75 (m, 1H, 4-H in indane); 6.69 (m, 2H, 2,6-H in phenyl); 4.94 (m, 1H, 1-H in indane); 3.82 (br.s, 1H, NH); 3.17-3.26 (m, 1H, 3- or 3′-H in indane); 2.92-2.99 (m, 2H, 3′- or 3-H in indane); 2.22-2.37 (m, 2H, 2,2′-H in indane). Preparation of N-phenyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-amine To a solution of 2.50 g (8.70 mmol) of 7-bromo-N-phenyl-2,3-dihydro-1H-inden-1-amine in 50 mL THF 3.50 mL (8.70 mmol) of 2.5MnBuLi in hexanes was added at −80° C. in argon atmosphere. The reaction mixture was then stirred for 1 hr. at this temperature. Then, 11.1 mL (17.8 mmol) of 1.7MtBuLi in pentane was added, and the reaction mixture was stirred for 1 hr. Then, 3.23 g (17.4 mmol) of 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane was added. Then, the cooling bath was removed, and the resulting mixture was stirred for 1 hr. at 23° C. To the formed mixture 10 mL of water was added, and the resulting mixture was evaporated to dryness. The residue was diluted with 200 mL of water, and the title product was extracted with 3×50 mL of ethyl acetate. The combined organic extract was dried over Na2SO4and evaporated to dryness. Yield 2.80 g (96%) of a light yellow oil. Anal. calc. For C21H26BNO2: C, 75.24; H, 7.82; N, 4.18. Found: C, 75.40; H, 8.09; N, 4.02.1H NMR (CDCl3): δ 7.63 (m, 1H, 6-H in indane); 7.37-7.38 (m, 1H, 4-H in indane); 7.27-7.30 (m, 1H, 5-H in indane); 7.18 (m, 2H, 3,5-H in phenyl); 6.65-6.74 (m, 3H, 2,4,6-H in phenyl); 5.20-5.21 (m, 1H, 1-H in indane); 3.09-3.17 (m, 1H, 3- or 3′-H in indane); 2.85-2.92 (m, 1H, 3′- or 3-H in indane); 2.28-2.37 (m, 1H, 2- or 2′-H in indane); 2.13-2.19 (m, 1H, 2′- or 2-H in indane); 1.20 (s, 6-H, 4,5-Me in BPin); 1.12 (s, 6H, 4′,5′-Me in BPin). Preparation of 7-(6-(((2,6-diisopropylphenyl)amino)methyl)pyridin-2-yl)-N-phenyl-2,3-dihydro-1H-inden-1-amine A solution of 2.21 g (21.0 mmol) of Na2CO3in a mixture of 80 mL of water and 25 mL of methanol was purged with argon for 30 min. The obtained solution was added to a mixture of 2.80 g (8.40 mmol) of N-phenyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1-amine, 2.90 g (8.40 mmol) of N-[(6-bromopyridin-2-yl)methyl]-2,6-diisopropylaniline, 0.48 g (0.40 mmol) of Pd(PPh3)4, and 120 mL of toluene. This mixture was stirred for 12 hrs. (h) at 70° C., then cooled to 23° C. The organic layer was separated, the aqueous layer was extracted with 3×50 mL of ethyl acetate. The combined organic extract was washed with brine, dried over Na2SO4and evaporated to dryness. The residue was purified by flash chromatography on silica gel 60 (40-63 μm, eluent: hexane-ethyl acetate-triethylamine=100:5:1, vol.). Yield 2.00 g (50%) of a yellow oil. Anal. calc. For C33H37N3: C, 83.33; H, 7.84; N, 8.83. Found: C, 83.49; H, 7.66; N, 8.65.1H NMR (CDCl3): δ 7.56-7.61 (m, 3H, 6-H in indane and 4.5-H in Py); 7.46-7.51 (m, 2H, 3,5-H in phenyl); 7.14-7.16 (m, 1H, 4-H in indane); 7.08-7.12 (m, 5H, 3-H in Py, 3,4,5-H in 2,6-diisopropylphenyl and 5-H in indane); 6.65 (m, 1H, 4-H in phenyl); 6.53 (m, 2H, 2,6-H in phenyl); 5.21-5.22 (m, 1H, 1-H in indane); 3.95-4.15 (m, 4H, CH2NH and NH-phenyl and NH-2,6-diisopropylphenyl); 3.31 (sept, J=6.8 Hz, 2H, CH in 2,6-diisopropylphenyl); 3.16-3.24 (m, 1H, 3- or 3′-H in indane); 2.91-2.97 (m, 1H, 3′- or 3-H in indane); 2.21-2.37 (m, 2H, 2,2′-H in indane); 1.19-2.21 (m, 12H, CH3in 2,6-diisopropylaniline). Preparation of pyridyldiamide catalyst precursor (PDA) Toluene (5 mL) was added to 7-(6-(((2,6-diisopropylphenyl)amino)methyl)pyridin-2-yl)-N-phenyl-2,3-dihydro-1H-inden-1-amine (0.296 g, 0.623 mmol) and Hf(NMe2)2Cl2(dme) (0.267 g, 0.623 mmol) to form a clear colorless solution. The mixture was loosely capped with aluminum foil and heated to 95° C. for 3 hrs. The mixture was then evaporated to a solid and washed with Et2O (5 mL) to afford 0.432 g of the presumed (pyridyldiamide) HfCl2complex. This was dissolved in CH2Cl2(5 mL) and cooled to −50° C. An Et2O solution of dimethylmagnesium (3.39 mL, 0.747 mmol) was added dropwise and the mixture was allowed to warm to ambient temperature. After 30 min. the volatiles were removed by evaporation and the residue was extracted with CH2Cl2(10 mL) and filtered. The solution was concentrated to 2 mL and pentane (4 mL) was added. Cooling to −10° C. overnight afforded colorless crystals that were isolated and dried under reduced pressure. Yield=0.41 g, 92%.1H NMR (CD2Cl2, 400 MHz): 8.00 (t, 1H), 6.85-7.65 (13H), 5.06 (d, 1H), 4.91 (dd, 1H), 4.50 (d, 1H), 3.68 (sept, 1H), 3.41 (m, 1H), 2.85 (m, 1H), 2.61 (sept, 1H), 2.03 (m, 1H), 1.85 (m, 1H), 1.30 (m, 2H), 1.14 (d, 3H), 1.06 (d, 3H), 0.96 (d, 3H), 0.68 (3, 3H), −0.48 (s, 3H), −0.84 (s, 3H). Polymerization In particular, all examples were produced using a solution polymerization process in a 1.0-liter continuous stirred-tank reactor (autoclave reactor). The autoclave reactor was equipped with a stirrer, a water-cooling/steam-heating element with a temperature controller, and a pressure controller. Solvents and monomers were purified by passing through purification columns packed with mol sieves. Isohexane (solvent) was passed through four columns in series whereas ethylene, propylene, and toluene were each purified by passing through two columns in series. Purification columns are regenerated periodically (about twice/year) or whenever there is evidence of low catalyst activity. 5-ethylidene-2-norbornene (ENB) was purified in a glove box by passing through a bed of basic alumina under a steady nitrogen gas purge. 5-vinyl-2-norbornene (VNB) was purified by stirring the diene with sodium-potassium alloy (NaK) then filtering through a bed of basic or neutral alumina. Tri-n-octylaluminum (TNOAL, available from Sigma Aldrich, Milwaukee, Wis.) solution was diluted to a concentration of 1.84×10−6using isohexane. Catalyst used for examples 1-12 was the PDA catalyst described above (MW 720.0 g/mol). The activator used was N,N-dimethylanilinium tetrakis(pentafluorophenyl) borate. Catalyst solution was prepared daily and used on the same day. The solution was prepared by dissolving 40.0 mg of the catalyst and 45.4 to 47.9 mg of the activator in 450 mL toluene (catalyst concentration=1.24 to 1.30×10−07mol/mL, catalyst/activator (molar ratio) about 0.98). This solution was pumped into the reactor through a designated dip-tube at a desired rate using an Isco pump. The PDA catalyst precursor was fed at a rate of 9.26×10−8mol/min for samples 1-6; and 9.77×10−8mol/min for samples 7-12; and activator was fed at a rate of 9.45×10−8mol/min for samples 1-6; and 9.97×10−8mol/min for samples 7-12. TNOAL was fed at a rate of 7.37×10−6mol/min. For examples 13-20, the catalyst used was the QDA catalyst described above (732.2 g/mol). The QDA catalyst precursor was fed at a rate of 1.82×10−7mol/min; and the activator (N,N-dimethylanilinium tetrakis(pentafluorophenyl) borate) was fed at a rate of 1.86×10−7mol/min. TNOAL was fed at a rate of 7.37×10−6mol/min. Composition was controlled by adjusting the feed ratio of the monomers. Ethylene and propylene feed rates were held constant for all examples listed in Table 1 while the diene feed rate was varied. No hydrogen was added. All the reactions were carried out at a gauge pressure of about 2.2 MPa and a temperature of 110° C. The collected samples were first placed on a boiling-water steam table in a hood to evaporate a large fraction of the solvent and unreacted monomers, and then, dried in a vacuum oven at a temperature of about 90° C. for about 12 hours. The vacuum oven dried samples were weighed to obtain yields. Ethylene, ENB, and VNB (VNB incorporated only through the endocyclic double bond) content of the polymers were determined by FTIR (ASTM D3900, ASTM D6047). Monomer conversions were calculated using the polymer yield, composition and the amount of monomers fed into the reactor. Catalyst activity (also referred as to catalyst productivity) was calculated based upon the yield and the feed rate of catalyst. Mooney measurements were made to gauge molecular weight and long-chain branching of the EPDM terpolymers. Samples were later analyzed using GPC as described below to determine the molecular weight distribution as well as g′ values. In Table 1, the reactor conditions for polymerization are set forth, and additionally include a reactor temperature of 110° C.; reactor pressure of 320 psig; where the activator was N,N-dimethylanilinium tetrakis(pentafluorophenyl)borate; the catalyst feed was 9.2-9.8×10−8mol/min for PDA and 1.8×10−7for QDA; an tri-n-octylaluminum feed was 7.4×10−6mol/min; and an isohexane feed of 60-65 g/min. TABLE 1Reactor Conditions for polymerizationReactor InputFT-IR dataEthylenePropyleneENBVNBEthyleneFeedFeedFeedFeedH2(wt %)ENBVNBSampleCatalyst(g/min)(g/min)(g/min)(g/min)(SCCM)uncorrected(wt %)(wt %)1PDA2.08.0—0.00—51.1%——2PDA2.08.0—0.03—57.2%—0.1%3PDA2.08.0—0.04—58.4%—0.2%4PDA2.08.0—0.00—56.6%—0.0%5PDA2.08.0—0.03—64.5%—0.1%6PDA2.08.0—0.04—63.6%—0.2%7PDA2.08.00.00—45.2%——8PDA2.08.00.04—54.4%0.8%—9PDA2.08.0—0.00—48.2%——10PDA2.08.0—0.04—58.3%—0.2%11PDA2.08.0—0.07—64.3%—0.4%12PDA2.08.0—0.11—65.0%—0.6%13QDA2.55.00.04—41.6%0.6%—14QDA2.55.00.042.043.9%0.5%—15QDA2.55.00.072.049.3%0.8%—16QDA2.55.00.112.057.0%1.0%—17QDA2.55.00.142.058.0%1.3%—18QDA2.55.0—0.042.048.1%—0.2%19QDA2.55.0—0.072.055.0%—0.4%20QDA2.55.0—0.112.055.3%—0.7% TABLE 2Polymer Collection and Mooney DataCatSampleCollectionEfficiencyquantityTime(g poly/SampleMLMLRA(g)(min)g cat)1——37.315373002——24.515245003——20.715207004——29.415294005——24.020180006——39.33019650756.148.931.51047250853.055.769.13034550932.555.228.610429001023.150.844.230221001120.862.933.530167501216.677.126.130130501370.5159.978.020292501442.360.966.515332501554.858.758.115290501664.985.744.115220501754.159.453.520200631848.9250.859.315296501953.9339.648.215241002027.2154.745.61522800 TABLE 3Gel Permeation Chromatography DataMn (LS),Mw (LS),wt %Samplekg/molkg/molg′Avgg′1000C2 units1641311.032—51.092511210.9990.86060.993481200.9810.82362.674641281.021—57.755471040.9950.78564.466481180.9900.81063.2971052201.029—42.858871970.9900.87052.879831751.028—46.0010541420.9920.88160.4411421450.9340.75863.8512411450.7810.68665.99131302581.0291.00940.2914961931.0211.05743.73151022011.0391.04849.39161082071.0320.91257.9117961881.0270.89759.3918822300.9060.74647.9519752220.8740.70955.3620521650.8260.60256.20 IR Spectrometry Total ethylene content of the elastomers was determined using a Nicolet 6700 FTIR (ASTM D3900, ASTM D6047). The granular elastomers from the reactor was first extruded and pelletized. Pellet samples were compression molded into a 10 mil thick pad. The pressed pad was placed in the instrument such that the IR beam passes through the pad and then measures the remaining signal on the other side. Methyl groups from the propylene affect the absorption, so the machine was calibrated to a range of ethylene content. Mooney Viscosity and Mooney Relaxation Area Mooney viscosity is a property used to monitor the quality of both natural and synthetic rubbers. It measures the resistance of rubber to flow at a relatively low shear rate. The highly branched polymers have a Mooney viscosity ML (1+4) at 125° C. of 30 to 100 MU (preferably 40 to 80, preferably 45 to 70, preferably 50 to 65), where MU is Mooney Units. While the Mooney viscosity indicates the plasticity of the rubber, the Mooney relaxation area (MLRA) provides a certain indication of the effects of molecular weight distribution and elasticity of the rubber. The highly branched compositions also have a MLRA of 30 to 100 MU (preferably 40 to 80, preferably 45 to 70). Another indication of melt elasticity is the ratio of MLRA/ML (1+4). This ratio has the dimension of time and can be considered as a “relaxation time.” A higher number signifies a higher degree of melt elasticity. Long chain branching will slow down the relaxation of the polymer chain, hence increasing the value of MLRA/ML (1+4). In the present compositions, the MLRA/ML is 1 or less, and as low as 0.9, or 0.8. Mooney viscosity and Mooney relaxation area are measured using a Mooney viscometer, operated at an average shear rate of about 2 s−1according to the following modified ASTM D1646: A square of sample is placed on either side of the rotor. The cavity is filled by pneumatically lowering the upper platen. The upper and lower platens are electrically heated and controlled at 125° C. The torque to turn the rotor at 2 rpm is measured by a torque transducer. The sample is preheated for 1 min. after the platens was closed. The motor is then started and the torque is recorded for a period of 4 min. Results are reported as ML (1+4) at 125° C., where M is Mooney viscosity number, L denotes the large rotor, “1” is the sample preheat time in min., “4” is the sample run time in min. after the motor starts, and 125° C. is the test temperature. The MLRA data is obtained from the Mooney viscosity measurement when the rubber relaxed after the rotor is stopped. The MLRA is the integrated area under the Mooney torque-relaxation time curve from 1 to 100 secs. The MLRA can be regarded as a stored energy term which suggests that, after the removal of an applied strain, the longer or branched polymer chains can store more energy and require longer time to relax. Therefore, the MLRA value of a bimodal rubber (the presence of a discrete polymeric fraction with very high molecular weight and distinct composition) or a long chain branched rubber are larger than a broad or a narrow molecular weight rubber when compared at the same Mooney viscosity values. Mooney viscosity values greater than about 100 cannot generally be measured using ML (1+4) at 125° C. In this event, a higher temperature is used (e.g., 150° C.), with eventual longer shearing time (i.e., 1+8 at 125° C. or 150° C.), but more preferably, the Mooney measurement is carried out using a non-standard small rotor as described below. The non-standard rotor design is employed with a change in Mooney scale that allows the same instrumentation on the Mooney machine to be used with higher Mooney rubbers. This rotor is termed MST, Mooney Small Thin, in contrast with ML. Molecular Weight Determinations The distribution and the moments of molecular weight (Mw, Mn, Mz, and Mw/Mn) in Table 1 were determined by using a high temperature Gel Permeation Chromatography (Polymer Char GPC-IR) equipped with a multiple-channel band-filter based Infrared detector IR5, an 18-angle light scattering detector and a viscometer (not used here). The GPC trace and mass balance traces are for Sample 3 are shown inFIG.1. Three Agilent PLgel 10 μm Mixed-B LS columns were used to provide polyolefin separation through size exclusion. Aldrich reagent grade 1,2,4-trichlorobenzene (TCB) with 300 ppm of the antioxidant butylated hydroxytoluene was used as the mobile phase. The TCB mixture was filtered through a 0.1 μm polytetrafluoroethylene filter and degassed with an online degasser before entering the GPC instrument. The nominal flow rate was 1.0 mL/min and the nominal injection volume was 200 μL. The whole system including transfer lines, columns, detectors were contained in an oven maintained at 145° C. A given amount of polyolefin sample was weighed and sealed in a standard vial with 80 μL flow marker (heptane) added to it. After loading the vial in the auto-sampler, polyolefin was automatically dissolved in the instrument with 8 mL added TCB solvent. The polyolefin was dissolved at 160° C. with continuous shaking for about 1 hr. for most polyethylene samples or 2 hrs. for polypropylene samples. The TCB densities used in concentration calculation were 1.463 g/mL at 23° C. and 1.284 g/mL at 145° C. The sample solution concentration was from 0.2 to 2.0 mg/mL, with lower concentrations being used for higher molecular weight samples. The concentration “c” at each point in the chromatogram was calculated from the baseline-subtracted IR5 broadband signal intensity “I” using the following equation: c=βI, where β is the mass constant determined with polyethylene or polypropylene standards. The mass recovery was calculated from the ratio of the integrated area of the concentration chromatography over elution volume and the injection mass which is equal to the pre-determined concentration multiplied by injection loop volume. The conventional molecular weight (IR molecular weight “M”) was determined by combining universal calibration relationship with the column calibration which was performed with a series of mono-dispersed polystyrene (PS) standards ranging from 700 g/mole to 10,000,000 g/mole. The molecular weight “M” at each elution volume was calculated with following equation: logM=log(KPS/K)a+1+aPS+1a+1logMPS, where the variables with subscript “PS” stands for “polystyrene” while those without a subscript are for the test samples. In this method, aps=0.67 and KPS=0.000175 while “a” and “K” are calculated from a series of empirical formula established in the literature (T. Sun, P. Brant, R. R. Chance, and W. W. Graessley, 34(19) MACROMOLECULES6812-6820 (2001)). Specifically, the value of a/K is 0.695/0.000579 for polyethylene and 0.705/0.0002288 for polypropylene. Molecular weight is expressed in g/mole or kg/mole. The values for Mw are determined ±500 g/mole, and for Mn±100 g/mole. The comonomer composition is determined by the ratio of the IR5 detector intensity corresponding to CH2and CH3channel calibrated with a series of PE and PP homo/copolymer standards whose nominal value are predetermined by NMR or FTIR such as an ExxonMobil Chemical Company commercial grade of LLDPE, polypropylene, etc. The LS detector is the 18-angle Wyatt Technology High Temperature Dawn Heleosii™. The LS molecular weight “M” at each point in the chromatogram is determined by analyzing the LS output using the Zimm model for static light scattering (W. Burchard & W. Ritchering, “Dynamic Light Scattering from Polymer Solutions,” in 80 PROGRESS INCOLLOID& POLYMERSCIENCE, 151-163 (Steinkopff, 1989)): KocΔR(θ)=1MP(θ)+2A2c, here, ΔR(θ) is the measured excess Rayleigh scattering intensity at scattering angle θ, “c” is the polymer concentration determined from the infrared analysis, A2 is the second virial coefficient. P(θ) is the form factor for a monodisperse random coil, and Kois the optical constant for the system: Ko=4π2n2(dn/dc)2λ4NA, where NAis Avogadro's number, and (dn/dc) is the refractive index increment for the system. The refractive index, n=1.500 for TCB at 145° C. and λ=665 nm. A high temperature Agilent (or Viscotek Corporation) viscometer, which has four capillaries arranged in a Wheatstone bridge configuration with two pressure transducers, is used to determine specific viscosity and branching. One transducer measures the total pressure drop across the detector, and the other, positioned between the two sides of the bridge, measures a differential pressure. The specific viscosity, ηS, for the solution flowing through the viscometer is calculated from their outputs. The intrinsic viscosity, [η], at each point in the chromatogram is calculated from the following equation: [η]=ηS/c where c is concentration and was determined from the infrared (IR5) broadband channel output. The viscosity “M” at each point is calculated from the below equation: M=KPSMαPS+1/[η]. The branching index (g′avg) is calculated using the output of the GPC-IR5-LS-VIS method as follows. The average intrinsic viscosity, [ηavg], of the sample is calculated by: [η]avg=∑ci[η]i∑ci, where the summations are over the chromatographic slices, “i”, between the integration limits. The branching index g′avgis defined as: gavg′=[η]avgkMVα. The Mvis the viscosity-average molecular weight based on molecular weights determined by LS analysis. Also, as used herein the g′1000is the value of g′ at a molecular weight of 1,000,000 g/mole, thus a measure of the amount of branching on the high molecular weight component of the polymer. Branching data for inventive Sample 3 is shown inFIG.1. Phase Angle Dynamic shear melt rheological data was measured with an Advanced Rheometrics Expansion System (ARES) using parallel plates (diameter=25 mm) in a dynamic mode under nitrogen atmosphere. For all experiments, the rheometer was thermally stable at 190° C. for at least 30 min. before inserting compression-molded sample of resin onto the parallel plates. To determine the samples viscoelastic behavior, frequency sweeps in the range from 0.01 to 385 rad/s were carried out at a temperature of 190° C. under constant strain. Depending on the molecular weight and temperature, strains of 10% and 15% were used and linearity of the response was verified. A nitrogen stream was circulated through the sample oven to minimize chain extension or cross-linking during the experiments. All the samples were compression molded at 190° C. and no stabilizers were added. A sinusoidal shear strain is applied to the material. If the strain amplitude is sufficiently small the material behaves linearly. It can be shown that the resulting steady-state stress will also oscillate sinusoidally at the same frequency but will be shifted by a phase angle Δ with respect to the strain wave. The stress leads the strain by Δ. For purely elastic materials Δ=0° (stress is in phase with strain) and for purely viscous materials, Δ=90° (stress leads the strain by 90° although the stress is in phase with the strain rate). For viscoelastic materials, 0<Δ<90. The shear thinning slope (STS) was measured using plots of the logarithm (base ten) of the dynamic viscosity versus logarithm (base ten) of the frequency. The slope is the difference in the log(dynamic viscosity) at a frequency of 100 s−1and the log(dynamic viscosity) at a frequency of 0.01 s−1divided by 4. Dynamic viscosity is also referred to as complex viscosity or dynamic shear viscosity. Rheological data may be presented by plotting the phase angle versus the absolute value of the complex modulus (G*) to produce a van Gurp-Palmen plot. The plot of conventional polyethylene polymers shows monotonic behavior and a negative slope toward higher G* values. Conventional LLDPE polymer without long chain branches exhibit a negative slope on the van Gurp-Palmen plot. For branched modifiers, the phase angels shift to a lower value as compared with the phase angle of a conventional ethylene polymer without long chain branches at the same value of G*. The van Gurp-Palmen plots of some embodiments of the branched modifier polymers described in the present disclosure exhibit two slopes—a positive slope at lower G* values and a negative slope at higher G* values. Such a plot is presented inFIG.2aandFIG.2b. Sentmanat Extensional Rheology Extensional Rheometry was performed on an Anton-Paar MCR 501 or TA Instruments DHR-3 using a SER Universal Testing Platform (Xpansion Instruments, LLC), model SER2-P or SER3-G. The SER (Sentmanat Extensional Rheometer) Testing Platform is described in U.S. Pat. Nos. 6,578,413 and 6,691,569. A general description of transient uniaxial extensional viscosity measurements is provided, for example, in “Strain hardening of various polyolefins in uniaxial elongational flow,” 47(3) THESOCIETY OFRHEOLOGY, INC., J. RHEOL., 619-630 (2003); and “Measuring the transient extensional rheology of polyethylene melts using the SER universal testing platform,” 49(3) THESOCIETY OFRHEOLOGY, INC., J. RHEOL., 585-606 (2005). Strain hardening occurs when a polymer is subjected to uniaxial extension and the transient extensional viscosity increases more than what is predicted from linear viscoelastic theory. Strain hardening is observed as abrupt upswing of the extensional viscosity in the transient extensional viscosity versus time plot. A strain hardening ratio (SHR) is used to characterize the upswing in extensional viscosity and is defined as the ratio of the maximum transient extensional viscosity over three times the value of the transient zero-shear-rate viscosity at the same strain. Strain hardening is present in the material when the ratio is greater than 1. The SER instrument consists of paired master and slave windup drums mounted on bearings housed within a chassis and mechanically coupled via intermeshing gears. Rotation of the drive shaft results in a rotation of the affixed master drum and an equal but opposite rotation of the slave drum which causes the ends of the polymer sample to be sound up onto the drums resulting in the sample stretched. The sample is mounted to the drums via securing clamps in most cases. In addition to the extensional test, samples are also tested using transient steady shear conditions and matched to the extensional data using a correlation factor of three. This provides the linear viscoelastic envelope (LVE). Rectangular sample specimens with dimensions approximately 18.0 mm long×12.70 mm wide are mounted on the SER fixture. Samples are generally tested at three Hencky strain rates: 0.01 s−1, 0.1 s−1and 1 s−1. The testing temperature is 150° C. The polymer samples were prepared as follows: the sample specimens were hot pressed at 190° C., mounted to the fixture, and equilibrated at 150° C. Such plots are presented inFIG.3for a comparative elastomer andFIG.4for an inventive Sample 3 elastomer. With respect to a composition or polyolefin, “consisting essentially of” means that the claimed polyolefin, composition and/or article includes the named components and no additional components that will alter its measured properties by any more than ±1, 2, 5, or 10%, and most preferably means that “additives” are present, if at all, to a level of less than 5, or 4, or 3, or 2 wt % by weight of the composition. Such additional additives can include, for example, inorganic fillers (such as talc, glass, and other minerals), carbon black, nucleators, clarifiers, colorants (soluble and insoluble), foaming agents, antioxidants, alkyl-radical scavengers (preferably vitamin E or other tocopherols and/or tocotrienols), anti-ultraviolet light agents, acid scavengers, curatives and cross-linking agents, mineral and synthetic oils, aliphatic and/or cyclic containing oligomers or polymers (and other “hydrocarbon resins”), and other additives well known in the art. With respect to a process or apparatus, the phrase “consisting essentially of” means that the claimed process does not include any other process steps (or apparatus features/means) that change the nature of the overall claimed process, such as an additional polymerization step, or additional olefin/polyolefin separation step, or additional re-directing of polymerization medium flow, heating, cooling, pressurizing, and/or depressurizing that impart a change in the final polyolefin product by any more than ±1, 2, or 5% from a measured chemical properties. For all jurisdictions in which the doctrine of “incorporation by reference” applies, all of the test methods, patent publications, patents and reference articles are hereby incorporated by reference either in their entirety or for the relevant portion for which they are referenced. | 59,965 |
11859033 | EXAMPLE 1 In an AISI 316 steel vertical autoclave, equipped with baffles and a stirrer working at 570 rpm, 3.5 liter of demineralized water were introduced. The temperature was then brought to reaction temperature of 80° C. and the selected amount of 34% w/w aqueous solution of cyclic surfactant of formula (VI) as defined above, with Xa=NH4, was added. VDF and ethane were introduced to the selected pressure variation reported in Table 1. A gaseous mixture of TFE-VDF in the molar nominal ratio reported in Table 1 was subsequently added via a compressor until reaching a pressure of 20 bar. Then, the selected amount of a 3% by weight water solution of sodium persulfate (NaPS) as initiator was fed. The polymerization pressure was maintained constant by feeding the above mentioned TFE-VDF while adding the PPVE monomer at regular intervals until reaching the total amount indicated in the table 1. When 1000 g of the mixture were fed, the reactor was cooled at room temperature, the latex was discharged, frozen for 48 hours and, once unfrozen, the coagulated polymer was washed with demineralized water and dried at 160° C. for 24 hours. The composition of the obtained polymer F-1, as measured by NMR, was Polymer (F-1)(693/99): TFE (69.6% mol)—VDF (27.3% mol)—PPVE (2.1% mol), having melting point Tm=218° C. and MFI=5 g/10′. EXAMPLE 2 The procedure of example 1 was repeated, by introducing the amount of ingredients indicated in the second column of Table 1. The composition of the obtained polymer F-2, as measured by NMR, was Polymer (F-1)(693/100): TFE (68% mol)—VDF (29.8% mol)—PPVE (2.2% mol), having melting point Tm=219° C. and MFI=1.5 g/10′. COMPARATIVE EXAMPLE 1 The procedure of example 1 was repeated, by introducing the amount of ingredients indicated in the third column of Table 1. The composition of the obtained polymer P-1, as measured by NMR, was Polymer (C-1)(693/67): TFE (71% mol)—VDF (28.5% mol)—PPVE (0.5% mol), having melting point Tm=249° C. and MFI=5 g/10′. COMPARATIVE EXAMPLE 2 In an AISI 316 steel horizontal reactor, equipped with a stirrer working at 42 rpm, 56 liter of demineralized water were introduced. The temperature was then brought to reaction temperature of 65° C. and the selected amount of 40% w/w aqueous solution of cyclic surfactant of formula (VI) as defined above, with X1=NH4, was added. VDF and ethane were introduced to the selected pressure variation reported in Table 1. A gaseous mixture of TFE-VDF in the molar nominal ratio reported in Table 1 was subsequently added via a compressor until reaching a pressure of 20 bar. Then, the selected amount of a 0.25% by weight water solution of sodium persulfate (NaPS) as initiator was fed. The polymerization pressure was maintained constant by feeding the above mentioned TFE-VDF while adding the PPVE monomer at regular intervals until reaching the total amount indicated in the table 1. When 16000 g of the mixture were fed, the reactor was cooled at room temperature, the latex was discharged, frozen for 48 hours and, once unfrozen, the coagulated polymer was washed with demineralized water and dried at 160° C. for 24 hours. The composition of the obtained polymer C-2, as measured by NMR, was Polymer (C-2)(SA1100): TFE (70.4% mol)—VDF (29.2% mol)—PPVE (0.4% mol), having melting point Tm=232° C. and MFI=8 g/10′. COMPARATIVE EXAMPLE 3 The procedure of Comparative Example 2 was repeated, by introducing the following changes:demineralized water introduced into the reactor: 66 litres;polymerization temperature of 80° C.polymerization pressure: 12 abs barInitiator solution concentration of 6% by weightMVE introduced in the amount indicated in table 1Overall amount of monomers mixture fed in the reactor: 10 000 g, with molar ratio TFE/VDF as indicated in Table 1. All the amount of ingredients are indicated in the fifth column of Table 1. The composition of the obtained polymer (C-3), as measured by NMR, was Polymer (C-3)(693/22): TFE (72.1% mol)—VDF (26% mol)—PMVE (1.9% mol), having melting point Tm=226° C. and MFI=8 g/10′. TABLE 1(F-1)(F-2)(C-1)(C-2)(C-3)Surfactant solution [g]505050740800Surfactant [g/l]4.854.854.855.284.12Initiator solution [ml]1001001002500600Initiator [g/kg]3.03.03.00.396.0VDF [bar]1.81.801.81.8TFE/VDF mixture70/3070/3070/3070/3069/301[molar ratio]FPVE [g]1221223166002Ethane [bar]0.60.30.2520.11gaseous mixture containing 1% moles of perfluoromethylvinylether (FMVE);2initial partial pressure of FMVE 0.35 bar. The results regarding polymers (F-1), (F-2) of the invention, and comparative (C-1), (C-2) and (C-3) are set forth in Table 2 here below TABLE 2693/99693/100693/67SA1100693/14(F-1)(F-2)(C-1)(C-2)(C-3)Elongation at5777392904035break [%, 200° C.]Tensile modulus425374484594500[MPa, 23° C.]Tensile yield stress11.611.414.015.512.5[MPa, 23° C.]Tensile modulus29385676—[MPa, 170° C.]Tensile modulus1210484723[MPa, 200° C.]SHI [MPa, 23° C.]3.65.11.91.61.7ESR as yieldingNoNoYieldingYieldingYielding[time, 23° C.]YieldingYieldingafter 1after 1after 1minminmin In particular, the polymer (F) of the present invention as notably represented by the polymers (F-1), (F-2), surprisingly exhibits a higher elongation at break at 200° C. as compared to the polymers (C-1) and (C-2) of the prior art. Also, the polymer (F) of the present invention as notably represented by the polymers (F-1), (F-2), despite its lower tensile modulus, which remains nevertheless in a range perfectly acceptable for various fields of use, surprisingly exhibits a higher strain hardening rate by plastic deformation as compared to the polymers (C-1) and (C-2) of the prior art. Finally, the polymer (F) of the present invention as notably represented by the polymers (F-1) and (F-2) surprisingly exhibits higher environmental stress resistance when immersed in fuels as compared to the polymers (C-1) and (C-2) of the prior art. Yet, comparison of polymer (F) according to the present invention with performances of polymer (C-3) comprising perfluoromethylvinylether (FMVE) as modifying monomer shows the criticality of selecting perfluoropropylvinylether: indeed, FMVE is shown producing at similar monomer amounts, copolymer possessing too high stiffness, and hence low elongation at break, unsuitable for being used e.g. in O&G applications. | 6,302 |
11859034 | DETAILED DESCRIPTION In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of various exemplary embodiments or implementations of the invention. As used herein “embodiments” and “implementations” are interchangeable words that are non-limiting examples of devices or methods employing one or more of the inventive concepts disclosed herein. It is apparent, however, that various exemplary embodiments may be practiced without these specific details or with one or more equivalent arrangements. In other instances, well-known structures and devices are shown in block diagram form in order to avoid unnecessarily obscuring various exemplary embodiments. Further, various exemplary embodiments may be different, but do not have to be exclusive. For example, specific shapes, configurations, and characteristics of an exemplary embodiment may be used or implemented in another exemplary embodiment without departing from the inventive concepts. Unless otherwise specified, the illustrated exemplary embodiments are to be understood as providing exemplary features of varying detail of some ways in which the inventive concepts may be implemented in practice. Therefore, unless otherwise specified, the features, components, modules, layers, films, panels, regions, and/or aspects, etc. (hereinafter individually or collectively referred to as “elements”), of the various embodiments may be otherwise combined, separated, interchanged, and/or rearranged without departing from the inventive concepts. The use of cross-hatching and/or shading in the accompanying drawings is generally provided to clarify boundaries between adjacent elements. As such, neither the presence nor the absence of cross-hatching or shading conveys or indicates any preference or requirement for particular materials, material properties, dimensions, proportions, commonalities between illustrated elements, and/or any other characteristic, attribute, property, etc., of the elements, unless specified. Further, in the accompanying drawings, the size and relative sizes of elements may be exaggerated for clarity and/or descriptive purposes. When an exemplary embodiment may be implemented differently, a specific process order may be performed differently from the described order. For example, two consecutively described processes may be performed substantially at the same time or performed in an order opposite to the described order. Also, like reference numerals denote like elements. When an element, such as a layer, is referred to as being “on,” “connected to,” or “coupled to” another element or layer, it may be directly on, connected to, or coupled to the other element or layer or intervening elements or layers may be present. When, however, an element or layer is referred to as being “directly on,” “directly connected to,” or “directly coupled to” another element or layer, there are no intervening elements or layers present. To this end, the term “connected” may refer to physical, electrical, and/or fluid connection, with or without intervening elements. Further, the D1-axis, the D2-axis, and the D3-axis are not limited to three axes of a rectangular coordinate system, such as the x, y, and z-axes, and may be interpreted in a broader sense. For example, the D1-axis, the D2-axis, and the D3-axis may be perpendicular to one another, or may represent different directions that are not perpendicular to one another. For the purposes of this disclosure, “at least one of X, Y, and Z” and “at least one selected from the group consisting of X, Y, and Z” may be construed as X only, Y only, Z only, or any combination of two or more of X, Y, and Z, such as, for instance, XYZ, XYY, YZ, and ZZ. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Although the terms “first,” “second,” etc. may be used herein to describe various types of elements, these elements should not be limited by these terms. These terms are used to distinguish one element from another element. Thus, a first element discussed below could be termed a second element without departing from the teachings of the disclosure. Spatially relative terms, such as “beneath,” “below,” “under,” “lower,” “above,” “upper,” “over,” “higher,” “side” (e.g., as in “sidewall”), and the like, may be used herein for descriptive purposes, and, thereby, to describe one elements relationship to another element(s) as illustrated in the drawings. Spatially relative terms are intended to encompass different orientations of an apparatus in use, operation, and/or manufacture in addition to the orientation depicted in the drawings. For example, if the apparatus in the drawings is turned over, elements described as “below” or “beneath” other elements or features would then be oriented “above” the other elements or features. Thus, the exemplary term “below” can encompass both an orientation of above and below. Furthermore, the apparatus may be otherwise oriented (e.g., rotated 90 degrees or at other orientations), and, as such, the spatially relative descriptors used herein interpreted accordingly. The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. As used herein, the singular forms, “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Moreover, the terms “comprises,” “comprising,” “includes,” and/or “including,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, components, and/or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It is also noted that, as used herein, the terms “substantially,” “about,” and other similar terms, are used as terms of approximation and not as terms of degree, and, as such, are utilized to account for inherent deviations in measured, calculated, and/or provided values that would be recognized by one of ordinary skill in the art. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is a part. Terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and should not be interpreted in an idealized or overly formal sense, unless expressly so defined herein. As used herein, the term “nanometer” may be abbreviated “nm”. As used herein, the term “micrometers” may be abbreviated “μm”. As used herein, the term “percent” may be abbreviated “%”. The terms “hydrogen” and “deuterium” refer to their respective atoms and corresponding radicals, and the terms “—F, —Cl, —Br, and —I” are radicals of, respectively, fluorine, chlorine, bromine, and iodine. As used herein, the term “atom” may mean an element or its corresponding radical bonded to one or more other atoms. As used herein, a substituent for a monovalent group, e.g., alkyl, may also be, independently, a substituent for a corresponding divalent group, e.g., alkylene. FIG.1is a schematic diagram of an exemplary embodiment of a curable composition made according to principles of the invention.FIG.2is a schematic diagram of an exemplary embodiment of an oxide-containing complex ofFIG.1.FIG.3is a schematic diagram of an exemplary embodiment of a method of preparing an oxide-containing complex according to principles of the invention.FIG.4is a schematic diagram of another exemplary embodiment of an oxide-containing complex constructed according to principles of the invention.FIG.5is a schematic diagram of an exemplary embodiment of a cured material made according to principles of the invention. A curable composition100shown inFIG.1may include a curable material120and an oxide-containing complex110. The oxide-containing complex110shown inFIGS.2to4may include i) an oxide core111and ii) an organic group113chemically bound to an atom111A on a surface of the oxide core111. The oxide-containing complex110may include at least one organic group113. The organic group113in the oxide-containing complex110shown inFIGS.2to4may include a) a curable group117reactable with the curable material120and b) a linking group115linking the atom111A on a surface of the oxide core111to the curable group117. By curing the curable composition100shown inFIG.1, a cured material200shown inFIG.5may be prepared. The cured material200ofFIG.5may include a matrix material220derived from the curable material120included in the curable composition100. The atom111A on a surface of the oxide-containing complex110included in the curable composition100may be “chemically bound” to the matrix material220via i) a residue group118derived from a reaction between the curable group117in the oxide-containing complex110and the curable material120; and ii) the linking group115in the oxide-containing complex110. Accordingly, the cured material200ofFIG.5is different from a cured material in which a matrix material described above and an oxide core (e.g., a spherical oxide particle) are not “chemically bound to each other” and are “physically mixed” with each other (e.g., Film B). As described above, the atom111A on a surface of the oxide core111in the cured material200may be “firmly and chemically bound to” the matrix material220. Thus, even when external force, such as bending and/or distortion, is removed after application of the external force to the cured material200, the oxide core111may effectively serve as a structural supporter for restoring the initial shape of the cured material200. Therefore, the cured material200may have excellent resilience. Oxide-Containing Complex110in Curable Composition100 The curable composition100ofFIG.1may include the oxide-containing complex110. The oxide core111in the oxide-containing complex110may include an aluminum oxide (e.g., Al2O3), a silicon oxide (e.g., SiO2), or a combination thereof. Because the oxide core111includes an aluminum oxide, a silicon oxide, or a combination thereof, the cured material200ofFIG.5may have excellent optical properties (e.g., light transmittance, refractive index, or the like). For example, the oxide core111may be an aluminum oxide or a silicon oxide. In some exemplary embodiments, the oxide core111may be a silicon oxide. In one or more exemplary embodiments, the atom111A on a surface of the oxide core111may be oxygen. The oxide core111may be a spherical particle. For example, the diameter D15 of the oxide core111may be in a range of about 1 nm to about 50 nm, about 1 nm to about 30 nm, or about 15 nm to about 25 nm. The refractive index of the oxide core111may be in a range of about 1.2 to about 2.5, about 1.2 to about 2.0, about 1.2 to about 1.8, or about 1.3 to about 1.8. Because the diameter D15 and/or the refractive index of the oxide core111is within any of these ranges described above, the cured material200ofFIG.5may have excellent optical properties (e.g., light transmittance, refractive index, or the like). The diameter D15 may be measured by evaluating a particle size distribution curve using any suitable commercially available particle size analyzer and evaluating the diameter corresponding to 15% of a passing mass percentage. The oxide-containing complex110inFIGS.2to4may include at least one organic group113. In an exemplary embodiment, the organic group113may be represented by Formula 1: wherein, in Formula 1,L1may be:*—N(R11)—*′, *—O—*′, *—S—*′, or *—C(═O)—*′; ora C1-C60alkylene group, a C1-C60oxyalkylene group, a C6-C60arylene group, or a C6-C60oxyarylene group, each unsubstituted or substituted with deuterium, a hydroxyl group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, a biphenyl group, or any combination thereof,* and *′ may each indicate a binding site to an adjacent atom,R11may be hydrogen, deuterium, a hydroxyl group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, or a biphenyl group,a1 may be an integer from 1 to 10,000 (for example, an integer from 1 to 5,000 or an integer from 1 to 1,000), and when a1 is 2 or greater, at least two L1(s) may be identical to or different from each other,T20may be the curable group117,a2 may be an integer from 1 to 10, and when a2 is 2 or greater, at least two T20(s) may be identical to or different from each other, andT1and T2may each independently be hydrogen, deuterium, a hydroxyl group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, a biphenyl group, oxygen bound to silicon in an adjacent linking group, or a binding site to oxygen disposed between the linking group and an adjacent linking group,wherein in Formula 1, * indicates a binding site to the atom111A on a surface of the oxide core111. The curable group117in the oxide-containing complex110and T20in Formula 1 may be any suitable group that may react with the curable material120in the curable composition100upon curation. For example, the curable group117in the oxide-containing complex110and T20in Formula 1 may comprise a vinyl-based group, an acrylate-based group, an acrylamide-based group, an epoxy-based group, or any combination thereof. In an exemplary embodiment, the curable group117in the oxide-containing complex110and T20in Formula 1 may be a group represented by one of Formulae 1-2(1) to 1-2(8): wherein, in Formulae 1-2(1) to 1-2(8),R1to R5may each independently be hydrogen, deuterium, a hydroxyl group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, or a biphenyl group, and* may indicate a binding site to the linking group115. The organic group113in the oxide-containing complex110may include the linking group115. Accordingly, the cured material200inFIG.5may have improved elastic force, flexibility, and/or resilience. The linking group115may be any suitable group that links the atom111A on a surface of the oxide core111to the curable group117. For example, referring to Formula 1, the linking group115may be a group represented by Formula 1-1: wherein, in Formula 1-1, L1, a1, T1, T2, and * may respectively be understood by referring to the descriptions of L1, a1, T1, T2, and * provided herein, and *′ may indicate a binding site to the curable group117in the oxide-containing complex110or T20in Formula 1. For example, in the oxide-containing complex110shown inFIG.4, the atom111A on a surface of the oxide core111is oxygen. In addition, the linking group115in the oxide-containing complex110shown inFIG.4may be a group represented by Formula 1-1 in which T1may be a binding site to oxygen disposed between the linking group and an adjacent linking group, T2may be oxygen bound to silicon in an adjacent linking group, L1may be a propylene group, and a1 may be 1, and the curable group117(or T20in Formula 1) may be a group represented by Formula 1-2(2) in which R1may be a methyl group, and R2and R3may each be hydrogen. In some exemplary embodiments, although not seen inFIG.4, the linking group115in the oxide-containing complex110shown inFIG.4may be a group represented by Formula 1-1 in which T1may be a binding site to oxygen disposed between the linking group and an adjacent linking group, T2may be oxygen bound to silicon in an adjacent linking group, L1may be an oxypropylene group, and a1 may be 1, and the curable group117(or T20in Formula 1) may be a group represented by Formula 1-2(5) in which R1may be a methyl group, and R2and R3may each be hydrogen. The weight ratio between the oxide core111and the organic group113in the oxide-containing complex110may be in a range of about 10:1 to about 1:10, for example, about 7:1 to about 1:7. In some exemplary embodiments, the weight ratio between the oxide core111and the organic group113in the oxide-containing complex110may be in a range of about 1:1 to about 1:5, for example, about 1:1 to about 1:3. When the weight ratio between the oxide core111and the organic group113in the oxide-containing complex110is within any of these ranges described above, the cured material200ofFIG.5may have excellent elastic force, flexibility, and/or resilience as well as excellent optical properties. Curable Material120in Curable Composition100 The curable material120in the curable composition100shown inFIG.1may be any suitable material that may change to the matrix material220in the cured material200shown inFIG.5through a curing process. The curable material120may consist of one type of compound or may be a mixture of at least two different types of compounds. The refractive index of the curable material120may be in a range of about 1.2 to about 2.0, about 1.2 to about 1.7, about 1.2 to about 1.5, or about 1.3 to about 1.5. Because the refractive index of the oxide material120is within any of these ranges described above, the cured material200ofFIG.5may have excellent optical properties (e.g., light transmittance, refractive index, or the like). For example, the curable material120may be photopolymerizable monomer(s). In an exemplary embodiment, the curable material120may be acrylic monomer(s). When the curable material120is acrylic monomer(s), the matrix material220in the cured material200ofFIG.5may be a polymer polymerized through a curing process (e.g., a photopolymerization process) of the acrylic monomer(s). Examples of the acrylic monomer include monofunctional (meth)acrylate monomers such as 2-ethylphenoxy(meth)acrylate, 2-ethylthiophenyl(meth)acrylate, phenyl(meth)acrylate, biphenylmethyl(meth)acrylate, benzyl(meth)acrylate, 2-phenylethyl(meth)acrylate, 3-phenylpropyl(meth)acrylate, 4-phenylbutyl(meth)acrylate, 2-2-methylphenylethyl(meth)acrylate, 2-3-methylphenylethyl(meth)acrylate, 2-4-methylphenylethyl(meth)acrylate, 2-(4-propylphenyl)ethyl(meth)acrylate, 2-(4-(1-methylethyl)phenyl)ethyl(meth)acrylate, 2-(4-methoxyphenyl)ethyl(meth)acrylate, 2-(4-cyclohexylphenyl)ethyl(meth)acrylate, 2-(2-chlorophenyl)ethyl(meth)acrylate, 2-(3-chlorophenyl)ethyl(meth)acrylate, 2-(4-chlorophenyl)ethyl(meth)acrylate, 2-(4-bromophenyl)ethyl(meth)acrylate, 2-(3-phenylphenyl)ethylmeth)acrylate, 2-(4-benzylphenyl)ethyl(meth)acrylate, o-phenylphenoxyethylacrylate, and isobornyl acrylate; difunctional (meth)acrylate monomers such as dicyclopentanyl di(meth)acrylate, caprolactone-modified dicyclopentenyl di(meth)acrylate, allylated cyclohexyl di(meth)acrylate, tricyclodecandimethanol(meth)acrylate, dimethylol dicyclopentane di(meth)acrylate, tricyclodecan dimethanol(meth)acrylate, 9,9-bis[4-(2-acryloyloxyethoxy)phenyl]fluorene, bisfluorene diacrylate, bisphenol-modified fluorene diacrylate, phenyl-modified urethane diacrylate, and bisfluorene-modified urethane diacrylate; trifunctional or polyfunctional (meth)acrylate monomers such as trimethylol propane tri(meth)acrylate, ethoxylated-trimethylolpropane tri(meth)acrylate, propoxylated-trimethylolpropane tri(meth)acrylate, tris2-hydroxyethylisocyanurate tri(meth)acrylate, glycerin tri(meth)acrylate, pentaerythritol tri(meth)acrylate, dipentaerythritol tri(meth)acrylate, ditrimethylolpropane tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, ditrimethylolpropane tetra(meth)acrylate, dipentaerythritol tetra(meth)acrylate, dipentaerythritol penta(meth)acrylate, ditrimethylolpropane penta(meth)acrylate, dipentaerythritol hexa(meth)acrylate, and ditrimethylolpropane hexa(meth)acrylate; or any combination thereof. In some exemplary embodiments, examples of the acrylic monomer include Compound 1 (2-ethylhexyl acrylate), Compound 2 (butyl acrylate), Compound 3 (vinyl acetate), Compound 4 (methyl methacrylate), Compound 5 (ethyl acrylate), Compound 6 (methyl acrylate), Compound 7 (benzyl acrylate), Compound 8 (phenoxyethyl acrylate), Compound 9 (acrylic acid), Compound 10 (hydroxyethyl methacrylate), Compound 11 (glycidyl methacrylate), Compound 12 (acetoacetoxyethyl methacrylate), and Compound 13 (2-hydroxyethyl acrylate): The amount of the curable material120in the curable composition100shown inFIG.1may be in a range of about 10 parts to about 99.5 parts by weight, about 50 parts to about 99 parts by weight, or about 70 parts to about 98 parts by weight, based on 100 parts by weight of the curable composition100. When the amount of the curable material120is within any of these ranges described above, an exposed portion may be effectively formed during exposure for curing the curable composition100to form the cured material200having excellent strength. Curable Composition100 The curable composition100ofFIG.1may include the oxide-containing complex110described above and the curable material120. The amount of the oxide-containing complex110in the curable composition100ofFIG.1may be in a range of about 0.01 parts to about 20 parts by weight, about 0.1 parts to about 10 parts by weight, or about 0.5 parts to about 5 parts by weight, based on 100 parts by weight of the curable material120. When the amount of the oxide-containing complex110is within any of these ranges described above, the cured material200ofFIG.5may have excellent elastic force, flexibility, and/or resilience as well as excellent optical properties. In an exemplary embodiment, the difference (an absolute value) in the refractive index between the curable material120and the oxide core111may be in a range of about 0.01 to about 0.5, about 0.01 to about 0.4, or about 0.02 to about 0.3. In one or more exemplary embodiments, the refractive index of the curable material120may be smaller than a refractive index of the oxide core111. When the refractive index of the curable material120and the refractive index of the oxide core111are within any of these ranges described above, multiple reflection and/or scattering of light incident on the cured material200ofFIG.5may be effectively induced, thereby allowing manufacture of the cured material200having excellent optical properties. The term “refractive index” as used herein refers to an absolute refractive index with respect to D-line (λ=589 nm, yellow) of sodium (Na). For example, the term “refractive index” may be an absolute refractive index measured at a temperature of 25° C. and in a relative humidity of 50% with light of the wavelength of 589 nm by using a refractive index measurer (e.g., ellipsometer (sold under the trade designation M-2000 Ellipsometer by J. A. Woollam of Lincoln, Nebraska)), for example, according to the Cauchy Film Model. The term “refractive index of the curable material120” refers to 1) when the curable material120consists of one type of compound, the refractive index of the one type of compound or 2) when the curable material120is a mixture of at least two different types of compounds, a refractive index of a compound having a greatest amount from the at least two different types of compounds. For example, i) when the curable material120is a mixture of Compounds A, B, and C, ii) when Compounds A, B, and C respectively have an amount (parts by weight) of a, b, and c, and iii) when a>b>c, a refractive index of the curable material120may be a refractive index of Compound A. When the curable material120is a mixture of at least two different types of compounds, and two or more compounds have the greatest amount from among the at least two different types of compounds, the refractive index of the curable material120may be an average value of refractive indexes of the compounds having the greatest amount. For example, i) when the curable material120is a mixture of Compounds A, B, and C, ii) when Compounds A, B, and C respectively have an amount (parts by weight) of a, b, and c, and iii) when a=b>c, the refractive index of the curable material120may be an average value of the refractive index of Compound A and the refractive index of Compound B. Table 1 shows refractive indexes of 2-ethylhexyl acrylate, isobornyl acrylate, acrylic acid, methyl methacrylic acid, and 2-hydroxyethylacrylate, which are exemplary embodiments of the curable material120, respectively; Al2O3and SiO2, which are exemplary embodiments of the oxide core111, respectively; and TiO2used in Film A described herein. TABLE 1MaterialRefractive IndexCurable2-Ethylhexyl acrylate (Compound 1)1.43MaterialIsobornyl acrylate1.47120Acrylic acid (Compound 9)1.39Methyl methacrylate (Compound 4)1.492-Hydroxyethyl acrylate (Compound 13)1.45OxideAl2O31.76Core 111SiO21.45TiO22.61 For example, as shown in Table 1, since SiO2, i.e., the oxide core111, has a refractive index of 1.45, and 2-ethylhexyl acrylate having the greatest amount (60 parts by weight based on 100 parts by weight of “Curable Composition 1”) among the curable materials120has a refractive index of 1.43 in “Curable Composition 1”, the difference between a refractive index of the curable material120and the refractive index of the oxide core111in “Curable Composition 1” is 0.02, and the refractive index of the curable material120is smaller than the refractive index of the oxide core111in “Curable Composition 1”. The curable composition100may further include any suitable polymerization initiator that may polymerize the curable material120described above, e.g., any suitable photopolymerization initiator. From the viewpoint of polymerization characteristics, initiation efficiency, absorption wavelength, availability, and price, the photopolymerization initiator may be an acetophenone-based compound, a benzophenone-based compound, a triazine-based compound, a biimidazole-based compound, an oxime-based compound, a thioxanthone-based compound, or any combination thereof. Examples of the acetophenone-based compound include diethoxyacetophenone, 2-hydroxy-2-methyl-1-phenylpropane-1-one, benzyldimethyl ketal, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropane-1-one, 1-hydroxycyclohexylphenylketone, 2-methyl-1-(4-methylthiophenyl)-2-morpholinopropane-1-one, 2-benzyl-2-dimethylamino-1-(4-morpholinophenyl)butane-1-one, 2-hydroxy-2-methyl-1-[4-(1-methylvinyl)phenyl]propane-1-one, 2-(4-methylbenzyl)-2-(dimethylamino)-1-(4-morpholinophenyl)butane-1-one, hydroxydimethyl acetophenone, and the like. Examples of the benzophenone-based compound include benzophenone, o-benzoyl benzoic acid methyl, 4-phenyl benzophenone, 4-benzoyl-4′-methyldiphenylsulfide, 3,3′,4,4′-tetra(tert-butylperoxycarbonyl)benzophenone, 2,4,6-trimethylbenzophenone, and the like. Examples of the triazine-based compound include 2,4-bis(trichloromethyl)-6-(4-methoxyphenyl)-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-(4-methoxynaphthyl)-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-piperonyl-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-(4-methoxystyryl)-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-[2-(5-methylfuran-2-yl)ethenyl]-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-[2-(furan-2-yl)ethenyl]-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-[2-(4-diethylamino-2-methylphenyl)ethenyl]-1,3,5-triazine, 2,4-bis(trichloromethyl)-6-[2-(3,4-dimethoxyphenyl)ethenyl]-1,3,5-triazine, and the like. Examples of the biimidazole-based compound include 2,2′-bis(2-chlorophenyl)-4,4′,5,5′-tetraphenylbiimidazole, 2,2′-bis(2,3-dichlorophenyl)-4,4′,5,5′-tetraphenylbiimidazole, 2,2′-bis(2-chlorophenyl)-4,4′,5,5′-tetra(alkoxyphenyl)biimidazole, 2,2′-bis(2-chlorophenyl)-4,4′,5,5′-tetra(tri alkoxyphenyl)biimidazole, 2,2-bis(2,6-dichlorophenyl)-4,4′,5,5′-tetraphenyl-1,2′-biimidazole, an imidazole compound in which phenyl groups at positions of 4,4′,5,5′ are substituted with carboalkoxy groups, and the like. Examples of the oxime-based compound include o-ethoxycarbonyl-α-oxyimino-1-phenylpropane-1-one and the like. Examples of the thioxanthone-based compound include 2-isopropylthioxanthone, 2,4-diethylthioxanthone, 2,4-dichlorothioxanthone, 1-chloro-4-propoxythioxanthone, and the like. Examples of the commercially available photopolymerization initiators include those sold under trade designations Irgacure-907, Irgacure 184, Irgacure 819, Irgacure 250, Darocur 1173, Irgacure OXE 01, and Irgacure OXE 02 (available from BASF of Ludwigshafen, Germany), sold under trade designations WPI-113, WPI-116, WPI-169, WPI-170, WPI-124, WPAG-638, WPAG-469, WPAG-370, WPAG-367, and WPAG-336 (available from Wako Pure Chemical Industries, Ltd. of Osaka, Japan), sold under trade designations B2380, B2381, C1390, D2238, D2248, D2253, 10591, T1608, T1609, T2041, and T2042 (available from Tokyo Kasei Kogyo Co., Ltd. of Yamatokoriyama, Japan), sold under trade designations AT-6992 and At-6976 (available from ACETO of Port Washington, New York), sold under trade designations CPI-100, CPI-100P, CPI101A, CPI-200K, and CPI-210S (available from San-Apro Ltd. of Kyoto, Japan), sold under trade designations SP-056, SP-066, SP-130, SP-140, SP-150, SP-170, SP-171, and SP-172 (available from ADEKA Corporation, of Tokyo, Japan), sold under trade designations CD-1010, CD-1011, and CD-1012 (available from Sartomer Company, Inc. of Exton, Pennsylvania), sold under trade designations San Aid SI-60, SI-80, SI-100, SI-60L, SI-80L, SI-100L, SI-L145, SI-L150, SI-L160, SI-L110, and SI-L147 (available from Sanshin Kagaku Kogyo Co., Ltd. of Nagoya, Japan), sold under the trade designation P12074 (available from Rhodia S. A. of La Defense, France), and hydroxydimethyl acetophenone available from Millipore Sigma with majority ownership by Merck KGaA, Darmstadt, Germany. The amount of the photopolymerization initiator may be in a range of about 0.1 parts to 10 parts by weight, 0.5 parts to 5 parts by weight, or 0.5 parts to 3 parts by weight, based on 100 parts by weight of the curable composition100. When the amount of the photopolymerization initiator is within any of these ranges described above, an exposed portion may be effectively formed during exposure for curing the curable composition100to form the cured material200having excellent strength. The curable composition100may consist of the curable material120, the oxide-containing complex110, and the photopolymerization initiator described above. In some exemplary embodiments, the curable composition100may further include any suitable solvent that is miscible with the curable material120and the oxide-containing complex110described above. Examples of the curable composition100include solvents including alkyleneglycol alkylethers such as ethyleneglycol monomethylether, ethyleneglycol monoethylether, ethyleneglycol monopropylether, ethyleneglycol monobutylether, propyleneglycol monomethylether, propyleneglycol methylethylether, and the like; diethyleneglycol dialkylethers such as diethyleneglycol dimethylether, diethyleneglycol diethylether, diethyleneglycol dipropylether, diethyleneglycol dibutylether, and the like; alkyleneglycol alkyletheracetates such as methylcellosolveacetate, ethylcellosolveacetate, propyleneglycol monomethyletheracetate, propyleneglycol monoethyletheracetate, propyleneglycol monopropyletheracetate, and the like; alkoxy alkylacetates such as methoxybutylacetate, methoxypentylacetate, and the like; aromatic hydrocarbons such as benzene, toluene, xylene, mesitylene, and the like; ketones such as methylethylketone, acetone, methylamylketone, methylisobutylketone, cyclohexanone, and the like; alcohols such as ethanol, propanol, butanol, hexanol, cyclohexanol, ethyleneglycol, glycerin, and the like; esters such as 3-ethoxypropionate ethyl ester, 3-methoxypropionate methyl ester, 3-phenyl-propionate ethyl ester, and the like; cyclic esters such as γ-butyrolactone and the like; or any combination thereof. The amount of the solvent may be in a range of about 20 parts to about 70 parts by weight, for example, about 30 parts to about 60 parts by weight, based on 100 parts by weight of the curable composition100. When the amount of the solvent is within any of these ranges described above, the curable composition100may have an excellent viscosity, while maintaining high dispersibility of solid in the curable composition100. The curable composition100may further include an alkali soluble resin, a dispersant, or any combination thereof, in addition to the curable material120, the oxide-containing complex110, the polymerization initiator, and the solvent described above. The alkali soluble resin may serve to alkali-solubilize an unexposed portion to enable removal of the unexposed portion and to remain an exposed portion after exposure of the curable composition100, and to uniformly disperse the oxide-containing complex110in the curable composition100. The alkali soluble resin may be selected from those having an acid value in a range of about 50 to about 200 (KOH mg/g). The term “acid value” as used herein refers to a measured value of a needed amount, typically milligrams (mg), of potassium hydroxide required for neutralizing 1 gram (g) of polymers and involves solubility. When the alkali soluble resin has an acid value within the range described above, excellent developing velocity, adhesiveness to substrate, and storage stability of the curable composition100may be achieved. The alkali soluble resin may be a polymer derived from a carboxyl group-containing unsaturated monomer, or a copolymer of a monomer having an unsaturated bond copolymerizable with the monomer, or any combination thereof. Examples of the carboxyl group-containing unsaturated monomer may include unsaturated monocarboxylic acid, unsaturated dicarboxylic acid, unsaturated tricarboxylic acid, or any combination thereof. Examples of the unsaturated monocarboxylic acid may include acrylic acid, methacrylic acid, crotonic acid, α-chloroacrylic acid, cinnamic acid, and the like. Examples of the unsaturated dicarboxylic acid may include maleic acid, fumaric acid, itaconic acid, citraconic acid, mesaconic acid, and the like. Examples of the unsaturated dicarboxylic acid include acid anhydrides (e.g., maleic anhydride, itaconic anhydride, citraconic anhydride, and the like). Further, the unsaturated dicarboxylic acid may be mono(2-(meth)acryloyloxy alkyl)ester thereof, e.g., mono(2-acryloyloxyethyl) succinate, mono(2-methacryloyloxyethyl) succinate, mono(2-acryloyloxyethyl) phtalate, mono(2-methacryloyloxyethyl) phtalate, or the like. The unsaturated dicarboxylic acid may be, for example, ω-carboxypolycaprolactone monoacrylate, ω-carboxypolycaprolactone monomethacrylate, or the like. The carboxyl group-containing monomer may be used alone or in combination of at least two types thereof. In addition, the monomer copolymerizable with the carboxyl group-containing unsaturated monomer may include an aromatic vinyl compound, an unsaturated carboxylic acid ester compound, an unsaturated carboxylic acid amino alkylester compound, an unsaturated carboxylic acid glycidylester compound, a carboxylic acid vinylester compound, an unsaturated ether compound, a vinyl cyanide compound, an unsaturated amide compound, an unsaturated imide compound, an aliphatic conjugated diene compound, a macromonomer having a monoacryloyl group or a monomethacryloyl group at a terminus of a molecular chain, a bulky monomer, or any combination thereof. The amount of the alkali soluble resin may be in a range of about 5 parts to about 80 parts by weight, for example, about 10 parts to about 70 parts by weight, based on 100 parts by weight of the curable composition100. When the amount of the alkali soluble resin is within any of these ranges described above, film reduction of a pixel portion of an exposed portion upon development may be prevented, thus obtaining satisfactory omission of a non-pixel portion. The dispersant may be used to enhance deagglomeration effect of the oxide-containing complex110in the curable composition100. The dispersant may be a resin type dispersant, such as a phosphoric acid ester-based dispersant, a urethane-based dispersant, an acrylic dispersant, or the like. In particular, the commercially available dispersant may include sold under trade designations DISPER BYK-103, DISPER BYK-110, DISPER BYK-111, DISPER BYK-2000, DISPER BYK-2001, DISPER BYK-2011, DISPER BYK-2070, DISPER BYK-2150, DISPER BYK-160, DISPER BYK-161, DISPER BYK-162, DISPER BYK-163, DISPER BYK-164, and DISPER BYK-166 available from Byk-Chemie GmbH of Wesel, Germany. The amount of the dispersant may be in a range of about 0.1 parts to about 15 parts by weight, for example, about 1 part to about 10 parts by weight, based on 100 parts by weight of the curable composition100. When the amount of the dispersant is within any of these ranges described above, agglomeration of the oxide-containing complex110in the curable composition100may be substantially prevented. In addition, the curable composition100may further include an adhesion promoter for increasing adhesion to a substrate, a surfactant for improving coating properties, an antioxidant, an ultraviolet absorber, or any combination. Other Exemplary Embodiments of Curable Composition According to some exemplary embodiments, a curable composition may include: a curable material; and an oxide-containing complex, wherein the oxide-containing complex may include i) an oxide core and ii) an organic group chemically bound to an atom on a surface of the oxide core, the organic group may include a) a curable group reactable with the curable material and b) a linking group linking the atom on a surface of the oxide core to the curable group, and the difference (an absolute value) between a refractive index of the curable material and a refractive index of the oxide core may be in a range of about 0.01 to about 0.5. In some exemplary embodiments, the oxide-containing complex is as described herein. In some exemplary embodiments, the oxide included in the oxide core of the oxide-containing complex may be selected from any suitable oxides satisfying a refractive index difference range between a refractive index of the curable material and a refractive index of the oxide core. In some exemplary embodiments, the curable material is as described herein. Method of Preparing Curable Composition100 A method of preparing the curable composition100described above may include: providing the oxide-containing complex110including i) the oxide core111and ii) the organic group113chemically bound to the atom111A on a surface of the oxide core111, and mixing the oxide-containing complex110with the curable material120to provide the curable composition100including the oxide-containing complex110and the curable material120. The oxide-containing complex110and the curable material120may respectively be understood by referring to the descriptions of the oxide-containing complex110and the curable material120provided herein. In an exemplary embodiment, providing of the oxide-containing complex110may include reacting an oxide precursor110A with a compound represented by Formula 2: wherein in Formula 2, L1, a1, T20, and a2 may respectively be understood by referring to the descriptions of L1, a1, T20, and a2, and T11to T13may each independently be a hydroxyl group or a C1-C20alkoxy group. The oxide precursor110A shown inFIG.3may be understood by referring to the description of the oxide core111described herein, except that the organic group113is not bound to an atom on a surface of the oxide precursor110A, unlike the oxide core111described herein. At least one hydroxyl group may be present on a surface of the oxide precursor110A, as shown inFIG.3. T11to T13in Formula 2 may be hydrolyzed to thereby be a silanol (*—Si—OH) group. As a result of a dehydration condensation reaction between the silanol group and a hydroxyl group on a surface of the oxide precursor110A, the organic group113(e.g., the group represented by Formula 1) may be chemically bound to the atom111A on a surface of the oxide core111, as described herein. For example, the compound represented by Formula 2 may be:i) 3-(trimethoxysilyl)propyl methacrylate; orii) a compound in which 3-(glycidyloxypropyl)trimethoxylan and poly(ethyleneglycol)methacrylate are bound to each other by an epoxy ring-opening reaction. The reacting of the oxide precursor110A with the compound represented by Formula 2 may be carried out in the presence of an acid catalyst. For example, the acid catalyst may include hydrochloric acid (HCl), sulfuric acid (H2SO4), acetic acid (CH3COOH), nitric acid (HNO3), or any combination thereof. The solvent that may be used in reaction between the oxide precursor110A and the compound represented by Formula 2 may be any suitable solvent that may be mixed with the oxide precursor110A and the compound represented by Formula 2. For example, the solvent may include water, methanol, ethanol, ethylene glycol, glycerol, or any combination thereof. Cured Material200 According to another aspect, the curable composition100described above may be cured to prepare the cured material200of the curable composition100. The cured material200ofFIG.5may include the matrix material220derived from the curable material120included in the curable composition100ofFIG.4. The “matrix material220derived from the curable material120” may be, for example, the matrix material220(e.g., a polymer) that is modified due to polymerization of the curable material120(e.g., a photopolymerizable monomer). As shown inFIGS.3to5, the atom111A on a surface of the oxide-containing complex110included in the curable composition100may be “chemically bound” to the matrix material220via i) the residue group118derived from a reaction between the curable group117in the oxide-containing complex110and the curable material120; and ii) the linking group115in the oxide-containing complex110. In some exemplary embodiments, the residue group118shown inFIG.5may be represented by Formula 3: *-(L11)a11-*′ Formula 3 wherein, in Formula 3,L11may be:*—N(R21)—*′, *—O—*′, *—S—*′, or *—C(═O)—*′; ora C1-C60alkylene group, a C1-C60oxyalkylene group, a C6-C60arylene group, or a C6-C60oxyarylene group, each unsubstituted or substituted with deuterium, a hydroxyl group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, a biphenyl group, or any combination thereof,* and *′ may each indicate a binding site to an adjacent atom,R21may be hydrogen, deuterium, a hydroxyl group, a C1-C20alkyl group, a C1-C20alkoxy group, a phenyl group, or a biphenyl group,a11 may be an integer from 1 to 10,000 (for example, an integer from 1 to 5,000 or an integer from 1 to 1,000), and when a11 is 2 or greater, at least two Lu(s) may be identical to or different from each other,* in Formula 3 may indicate a binding site to the linking group115shown inFIG.5, and*′ in Formula 3 may indicate a binding site to the matrix material220shown inFIG.5. The matrix material220in cured material200shown inFIG.5may be chemically bound to the oxide core111via i) the residue group118and ii) the linking group115, thereby forming a network structure. As such, the oxide core111in the cured material200ofFIG.5is chemically bound to the matrix material220via i) the residue group118and ii) the linking group115, the cured material200may have excellent resilience upon application and/or removal of external force, as compared with a cured material in which a matrix material described above and an oxide core (e.g., a spherical oxide particle) are not “chemically bound to each other” and are “physically mixed” with each other (e.g., Film B). The cured material200may be used in various applications, such as an adhesive member, an insulating member, an optical member, a protective member, a release member, and the like, all of which may be especially adapted for use in electronic devices and articles, depending on the matrix material220. For example, the matrix material220in the cured material200may be an adhesive material. When the matrix material220in the cured material200is an adhesive material, the cured material200may be an adhesive member. The cured material200may be in the form of a film. For example, a thickness of the film may be in a range of about 0.1 μm to about 700 μm, about 1 μm to about 600 μm, or about 5 μm to about 500 μm (see, e.g., Films 1 to 3 in the Examples described herein). The cured material200may have an excellent light transmittance. For example, a light transmittance of the cured material200with respect to light having the maximum emission wavelength of 600 nm is 96 percent (%) or higher, for example, about 97% to about 100%. Thus, the cured material200may be useful in various devices (e.g., an organic light-emitting device, a quantum dot light-emitting device, or the like) having a light-emitting member. Method of Preparing Cured Material200 A method of preparing the cured material200may include: providing the curable composition100on a substrate; and curing the curable composition100. Upon the curing of the curable composition100, a chemical reaction between the curable group117in the oxide-containing complex110and the curable material120may occur. As a result, the residue group118as shown inFIG.5may be formed. Therefore, the atom111A on a surface of the oxide core111may be chemically bound to the matrix material220via i) the residue group118and ii) the linking group115in the oxide-containing complex110. A substrate onto which the curable composition100may be provided may be used in various manners depending on the application field. For example, when the cured material200is used as an adhesive member that adheres an electronic device member to a cover window, the substrate may be a top of the electronic device member. The curing of the curable composition100may be performed by photopolymerization, and exposure may be carried out for photopolymerization. The exposure may be exposure to ultraviolet rays. The curing of the curable composition100may further include baking before and/or after the exposure for removing a portion of a solvent in the curable composition100. Article or Device Including Cured Material200 According to some exemplary embodiments, an article or a device may include the cured material200. The article may be a film laminate including the cured material200. For example, the film laminate may be an antistatic film laminate including a substrate, the cured material200, and an antistatic film, which are sequentially stacked in any of the devices disclosed herein. The device may be various electronic devices, for example, an electronic device, a cellular phone, a lighting, and the like. For example, the electronic device may be a light-emitting diode (LED) such as an organic light-emitting device (OLED), a quantum dot light-emitting device (QLED) for use in an electronic device. In an exemplary embodiment, the device may be a flexible device, a foldable device, or a rollable device, in which application and removal of external force is relatively frequent. Hereinafter, the curable composition100, the cured material200, and a method of preparing the same will be described in more detail through Examples and/or Comparative Examples. EXAMPLES Synthesis of Oxide-Containing Complex 1 An amount of 50 milliliters (mL) of ethanol and 2.5 g of SiO2particles (having a diameter D15 of 20 nm) were mixed together, followed by stirring for 30 minutes. The mixture was homogenized using a homogenizer in an ice water bath for 30 minutes. Then, a mixture of 2.5 g of 3-(trimethoxysilyl)propyl methacrylate (MEMO) and 1 mL of water were added dropwise thereto, followed by stirring for 2 hours. The pH of the resulting mixture was adjusted to 2 using hydrochloric acid aqueous solution (having a concentration of 38 mole %). Then, a reaction was carried out at a stirring rate of 800 rotations per minute (rpm) and at a temperature of 60° C. under reflux. The resulting mixture was washed with ethanol and water, and then, centrifuge (at 4,000 rpm) was performed for 30 minutes, followed by drying at a temperature of 60° C. for 4 hours, thereby preparing Oxide-containing Complex 1. Synthesis of Oxide-Containing Complex 2 Oxide-containing Complex 2 was synthesized in substantially the same manner as in Synthesis of Oxide-containing Complex 1, except that MEMO was used in an amount of 3.375 g. Synthesis of Oxide-Containing Complex 3 Oxide-containing Complex 3 was synthesized in substantially the same manner as in Synthesis of Oxide-containing Complex 1, except that MEMO was used in an amount of 6.75 g. Synthesis of Oxide-Containing Complex A Oxide-containing Complex A was synthesized in substantially the same manner as in Synthesis of Oxide-containing Complex 2, except that 2.5 g of TiO2particles (having a diameter D15 of 20 nm) was used instead of SiO2particles. Preparation of Curable Composition 1 Oxide-containing Complex 1 (1 parts by weight), Curable Monomer 1 (60 parts by weight), Curable Monomer 2 (20 parts by weight), Curable Monomer 3 (4 parts by weight), Curable Monomer 4 (4 parts by weight), Curable Monomer 5 (10 parts by weight), and a photopolymerization initiator (1 parts by weight) were mixed together, followed by stirring for 30 minutes by using a paste mixer, to prepare Curable Composition 1. Curable Monomers 1 to 5 and the photopolymerization initiator may respectively be understood by referring to the descriptions of Curable Monomers 1 to 5 and the photopolymerization initiator provided herein.Curable Monomer 1: 2-ethylhexyl acrylate (Aldrich Company)Curable Monomer 2: isobornyl acrylate (Aldrich Company)Curable Monomer 3: acrylic acid (Aldrich Company)Curable Monomer 4: methyl methacrylate (Aldrich Company)Curable Monomer 5: 2-hydroxyethyl acrylate (Aldrich Company)Photopolymerization initiator: hydroxydimethyl acetophenone (Aldrich Company) Preparation of Curable Compositions 2, 3, and A Curable compositions 2, 3, and A were prepared in substantially the same manner as in Preparation of Curable Composition 1, except that Oxide-containing Complexes 2, 3, and A were respectively used instead of Oxide-containing Complex 1. Preparation of Curable Composition B Curable composition B was prepared in substantially the same manner as in preparation of Curable Composition 1, except that 2.5 g of SiO2particles (having a diameter D15 of 20 nm) was used instead of Oxide-containing Complex 1. Preparation of Curable Composition C Curable composition C was prepared in substantially the same manner as in Preparation of Curable Composition 1, except that Oxide-containing Complex 1 was not used. TABLE 2Compound UsedCurableUsed Oxide-for Introducing aCompositionContaining ComplexCurable Group toNo.or Oxide ParticlesOxide Corethe Oxide Core1Oxide-ContainingSiO2ParticlesMEMOComplex 1(2.5 g)(2.5 g)2Oxide-ContainingSiO2ParticlesMEMOComplex 2(2.5 g)(3.375 g)3Oxide-ContainingSiO2ParticlesMEMOComplex 3(2.5 g)(6.7 g)AOxide-ContainingTiO2ParticlesMEMOComplex A(2.5 g)(3.375 g)BOxide ParticlesSiO2Particles—(2.5 g)C——— Preparation of Film 1 Curable Composition 1 was provided to a space between two polyethylene terephthalate (PET) films using a roll-to-roll coater. Then, pre-baking was performed using a hotplate at a temperature of 100° C. for 1 minute, and UV light (365 nm) was incident at an exposure amount of 200 millijoules per square centimeter (mJ/cm2) using an exposure device. Thereafter, in a nitrogen atmosphere, post-baking was performed in a heating oven at a temperature of 180° C. for 30 minutes, thereby preparing Film 1 having a thickness of 50 μm. Preparation of Films 2, 3, A, B, and C Films 2, 3, A, B, and C were prepared in substantially the same manner as in Preparation of Film 1, except that Curable Compositions 2, 3, A, B, and C were used instead of Curable Composition 1. Evaluation Example 1 (Measurement of Light Transmittance) Light transmittance (%) of Films 1, 2, 3, A, B, and C with respect to light having a maximum emission wavelength of 600 nm was measured using a UV-vis spectrometer. The results thereof are shown in Table 3. The light transmittance of each film is shown in a value (%) relative to the light transmittance of Film C. TABLE 3Light TransmittanceUsed Oxide-Compound Usedto Light Having aContainingfor IntroducingMaximum EmissionComplex ora Curable GroupWavelength ofFilmOxideOxideto the Oxide600 nm (RelativeNo.ParticlesCoreCoreValue, %)1Oxide-SiO2MEMO97.0ContainingParticles(2.5 g)Complex 1(2.5 g)2Oxide-SiO2MEMO99.1ContainingParticles(3.375 g)Complex 2(2.5 g)3Oxide-SiO2MEMO99.5ContainingParticles(6.7 g)Complex 3(2.5 g)AOxide-TiO2MEMO89.5ContainingParticles(3.375 g)Complex A(2.5 g)BOxideSiO2—95.4ParticlesParticles(2.5 g)C———100 Referring to the results of Table 3, it was found that Films 1 to 3 had an unexpected and surprisingly excellent light transmittance, as compared with Films A and B. Evaluation Example 2 (Measurement of Peel Strength) Samples were prepared according to the ASTM D3359-17 standard for a 180° peeling test method on Films 1, 2, 3, A, B, and C. Then, the peel strength (N/25 mm) was measured at a velocity of 300 mm/min. The results thereof are shown in Table 4. The peel strength of each film is shown in a value (%) relative to the peel strength of Film C. TABLE 4Used Oxide-Compound UsedContainingfor IntroducingPeelComplex ora Curable GroupStrengthFilmOxideOxideto the Oxide(RelativeNo.ParticlesCoreCoreValue, %)1Oxide-SiO2MEMO100containingParticles(2.5 g)Complex 1(2.5 g)2Oxide-SiO2MEMO96.6containingParticles(3.375 g)Complex 2(2.5 g)3Oxide-SiO2MEMO99.9containingParticles(6.7 g)Complex 3(2.5 g)AOxide-TiO2MEMO92.0containingParticles(3.375 g)Complex A(2.5 g)BOxideSiO2—94.9particlesParticles(2.5 g)C———100 Referring to the results of Table 4, it was found that Films 1 to 3 had an unexpected and surprisingly excellent peel strength, as compared with Films A and B. Evaluation Example 3 (Measurement of Modulus in Break Point) Samples were prepared according to the ASTM D882-18 standard for a tensile test method on Films 1, 2, 3, A, B, and C. Then, the modulus in break point in kiloPascals (kPa) was measured using a stress-strain diagram at a velocity of 50 mm/min. The results thereof are shown in Table 5. The modulus in break point of each film is shown in a value (%) relative to the modulus in break point of Film C. TABLE 5Used Oxide-Compound UsedContainingfor IntroducingModulus inComplex ora Curable GroupBreak PointFilmOxideOxideto the Oxide(RelativeNo.ParticlesCoreCoreValue, %)1Oxide-SiO2MEMO115.6ContainingParticles(2.5 g)Complex 1(2.5 g)2Oxide-SiO2MEMO133.3ContainingParticles(3.375 g)Complex 2(2.5 g)3Oxide-SiO2MEMO141.7ContainingParticles(6.7 g)Complex 3(2.5 g)AOxide-TiO2MEMO83.2ContainingParticles(3.375 g)Complex A(2.5 g)BOxideSiO2—136.0ParticlesParticles(2.5 g)C———100.0 Referring to the results of Table 5, Films 1 to 3 were found to have a modulus in break point better than Film A and equal to or better than Film B. Evaluation Example 4 (Measurement of Relaxation Ratio) Samples were prepared according to the ASTM D882-18 standard for a stress-relaxation test method on Films 1, 2, 3, A, B, and C. Next, the relaxation ratio (%) was measured under a given condition of a velocity of 100 mm/min and 200% strain. The results thereof are shown in Table 6. The relaxation ratio of each film is shown in a value (%) relative to the relaxation ratio of Film C. TABLE 6Used Oxide-Compound UsedContainingfor IntroducingRelaxationComplex ora Curable GroupRatioFilmOxideOxideto the Oxide(RelativeNo.ParticlesCoreCoreValue, %)1Oxide-SiO2MEMO59.48containingParticles(2.5 g)Complex 1(2.5 g)2Oxide-SiO2MEMO55.64containingParticles(3.375 g)Complex 2(2.5 g)3Oxide-SiO2MEMO44.36ContainingParticles(6.7 g)Complex 3(2.5 g)AOxide-TiO2MEMO97.51ContainingParticles(3.375 g)Complex A(2.5 g)BOxideSiO2—124.93ParticlesParticles(2.5 g)C———100 Referring to the results of Table 6, it was found that Films 1 to 3 had an unexpected and surprisingly excellent relaxation ratio, i.e., excellent elasticity, as compared with Films A, B, and C. Although certain exemplary embodiments and implementations have been described herein, other embodiments and modifications will be apparent from this description. Accordingly, the inventive concepts are not limited to such embodiments, but rather to the broader scope of the appended claims and various obvious modifications and equivalent arrangements as would be apparent to a person of ordinary skill in the art. | 56,322 |
11859035 | EMBODIMENTS TO CARRY OUT THE INVENTION [Polymer Used as Base Film for Cell Culture] The polymer used as a base film for cell culture of the present application can be obtained bypolymerizing a cationic monomer represented by the following formula (I): [wherein Ua1, Ua2each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, Ra1represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and Ra2represents a linear or branched alkylene group having 1 to 5 carbon atoms]. The above-mentioned polymer is preferably a copolymer obtained by polymerizing the cationic monomer represented by the above-mentioned formula (I) together with an anionic monomer represented by the following formula (II): [wherein Rbrepresents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. In the present specification, otherwise specifically defined, the “linear or branched alkyl group having 1 to 5 carbon atoms” may be mentioned, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group or a 1-ethylpropyl group. Ra1and Rbare, each independently, preferably selected from a hydrogen atom and a methyl group. Ua1and Ua2are preferably, each independently, selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group and an n-butyl group, a methyl group or an ethyl group, and most preferably a methyl group. In the present specification, otherwise specifically defined, the “linear or branched alkylene group having 1 to 5 carbon atoms” may be mentioned, for example, a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, a 1-ethyl-trimethylene group and the like. Among these, as Ra2, it is preferably selected from an ethylene group and a propylene group. Accordingly, as the cationic monomer represented by the above-mentioned formula (I) may be mentioned 2-N,N-dimethylaminoethyl methacrylate, N-isopropyl acrylamide and the like, and 2-N,N-dimethylaminoethyl methacrylate is preferable. As the anionic monomer represented by the above-mentioned formula (II), there may be mentioned acrylic acid, methacrylic acid or the like, and methacrylic acid is preferable. A molar ratio of the unit derived from a monomer represented by the formula (I)/the unit derived from a monomer represented by the formula (II) in the above-mentioned polymer is 100/0 to 50/50. It is preferably 98/2 to 50/50. It is more preferably 98/2 to 60/40, and particularly preferably 98/2 to 70/30. This is because if the molar ratio of the formula (II) is 51 or more, the anionic property of the polymer becomes excessive and the adhesive force of the cells decreases. (Monomer Having Two or More Carbon-Carbon Unsaturated Bonds) The above-mentioned polymer may be a polymer obtained by polymerizing monomers represented by the formula (I)/the formula (II), and further with a monomer having two or more carbon-carbon unsaturated bonds. The monomer having two or more carbon-carbon unsaturated bonds specifically means a monomer having two or more carbon-carbon double bonds, and for example, there may be mentioned a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, an isoprene compound or the like. As the preferred specific examples, monomers represented by the following formulae (III) to (V) are mentioned. In the formulae, Rcand Rdeach independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, Rerepresents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number of 1 to 50. Among these, it is preferably a monomer represented by the formula (III). A molar ratio of the monomers represented by the formulae (III) to (V) to the above-mentioned entire polymer is preferably 0 to 5.0%. It is further preferably 0 to 3.0%. This is because if the molar ratio of the formulae (III) to (V) is 5.0% or more, there is a fear of gelation during production due to increased molecular weight by excessive crosslinking, whereby the production becomes difficult. Rcand Rdare preferably, each independently, selected from a hydrogen atom and a methyl group. Reis preferably selected from a methylene group, an ethylene group and a propylene group, and most preferably an ethylene group. n is a number of 1 to 50, n is preferably a number of 1 to 30, and n is preferably a number of 1 to 10. The difference between the occupied value of mol % of the monomer represented by the formula (II) based on the entire polymer mentioned above and the occupied value of mol % the monomer represented by the formula (II) based on the total amount of the monomer stocked during the above-mentioned preparation step is 0 to 10 mol %. The polymer of the present application is small in the difference of the monomer stocked ratio and the measured value of the produced polymer according to the producing method mentioned later and it is 0 to 10 mol %. It is further preferably 0 to 8 mol %. The number average molecular weight (Mn) of the above-mentioned polymer is 20,000 to 1,000,000, and further preferably 50,000 to 800,000. The ratio (Mw/Mn) of the weight average molecular weight (Mw) and the above-mentioned number average molecular weight (Mn) of the above-mentioned polymer is 1.01 to 10.00, preferably 1.2 to 8.0, preferably 1.4 to 6.0, preferably 1.5 to 5.0, and preferably 1.6 to 4.5. The above-mentioned number average molecular weight (Mn) and the number average molecular weight (Mn) can be obtained by, for example, Gel Filtration Chromatography described in Examples. By using the polymer of the present application, it is possible to form cell aggregates by adhering cells and then detaching them. Incidentally, the cell aggregates designate a structure formed as a result of aggregation of cells, and the shape is not limited such as a spherical shape, a ring shape and the like. As compared with the conventional cell aggregates produced by non-adhesive culture on a cell low-adhesion plate, there are merits in the points of adjustment of the size (cell aggregates with an arbitrary size can be produced) of the cell aggregates by regulation of an adhesion area and the like. [Method for Producing Polymer Used as Base Film-Forming Agent for Cell Culture] The polymer of the present application can be produced by thermal polymerization method. For example, a polymerized product (polymer) can be obtained by dissolving the monomer of the above-mentioned formula (I) in an organic solvent, adding a radical polymerization initiator, then, if necessary, adding the above-mentioned formula (II), and further, depending on the necessity, adding a monomer having two or more carbon-carbon unsaturated bonds (the monomer represented by the formulae (III) to (V) and the like) to prepare a mixture, and after sufficiently stirring to make the mixture uniform, while flowing nitrogen, for example, at 51° C. or higher, for example, at 51 to 180° C., at 51 to 150° C., at 51 to 130° C., at 51 to 100° C., for example, heating to a reflux temperature (for example, at 66 to 85° C. in tetrahydrofuran) of the solvent, and, for example, stirring for 1 to 48 hours. The obtained polymer may be purified by reprecipitation and dialysis. In one embodiment, it can be prepared by a producing method including a step of dissolving the monomer of the above-mentioned formula (I) in a solvent, adding a polymerization initiator, then, reacting (polymerizing) with, if necessary, the monomer of the above-mentioned formula (II) in a solvent with a total concentration of the both compounds of 0.01% by mass to 40% by mass. As the organic solvent used for the above-mentioned polymerization, there may be mentioned, for example, an ether solvent such as tetrahydrofuran, 1,4-dioxane, etc., an aliphatic alcohol solvent having 1 to 4 carbon atoms such as methanol, ethanol, isopropanol, etc., an aromatic hydrocarbon solvent such as toluene, etc., and a mixed solvent thereof. In order to proceed the polymerization reaction efficiently, it is desirable to use a radical polymerization initiator. Examples of the radical polymerization initiator may be mentioned an azo polymerization initiator such as dimethyl 1,1′-azobis(1-cyclohexanecarboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation), 2,2′-azobis(2,4-dimethylvaleronitrile) (V-65, available from FUJIFILM Wako Pure Chemical Corporation), 2,2′-azobis(isobutyronitrile) (AIBN, available from FUJIFILM Wako Pure Chemical Corporation), 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] n hydrate (VA-057, available from FUJIFILM Wako Pure Chemical Corporation), 2,2′-(N-butyl-2-methylpropionamide) (VAm-110, available from FUJIFILM Wako Pure Chemical Corporation) and the like. An amount of the polymerization initiator to be added is 0.05% by mass to 5% by mass based on the total weight of the monomers to be used for the polymerization. Use of the polymerization initiator not only improves efficiency of the polymerization reaction, but also makes it possible to adjust the physical properties of the polymer by modification of the terminal functional group. [Method for Producing Base Film-Forming Agent for Cell Culture] By mixing the above-mentioned polymer with a water-containing solution by a method known per se, a base film-forming agent for cell culture can be produced. The water-containing solution may be mentioned water, a salt-containing aqueous solution such as physiological saline, a phosphate buffer solution or the like, or a mixed solvent in which water or a salt-containing aqueous solution and an alcohol are combined. As the alcohol, there may be mentioned an alcohol having 2 to 6 carbon atoms, for example, ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (=neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (=t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol and cyclohexanol, which may be used alone or a mixed solvent of combination thereof. Further, to the base film-forming agent, in addition to the above-mentioned copolymer and solvent, other substances may be added, if necessary, within the range that does not impair the performance of the obtained base film. As the other substances, there may be mentioned pH adjusting agents, crosslinking agents, preservatives, surfactants, primers enhancing adhesiveness with the container or the substrate, antifungal agents, sugars and the like. [Method for Producing Base Film for Cell Culture, Method for Manufacturing Cell Culture Container and Cell Culture Container] By coating the above-mentioned base film-forming agent for cell culture onto the surface of a container or a substrate and drying the same, a base film for cell culture and a cell culture container containing the base film can be produced. Here, the “surface” refers to a surface which is in contact with the contents such as cells, cell culture liquid or the like. The container or the substrate may be mentioned, for example, schale or dishes generally used for cell culture such as petri dishes, dishes for tissue culture, multi well plates, etc., flasks such as a cell culture flask, a spinner flask, etc., bags such as plastic bags, Teflon (Registered Trademark) bags, culture bags, etc., plates such as microplates, microwell plates, multi plates, multiwell plates, etc., and bottles such as chamber slide, tubes, trays, roller bottles and the like. It is preferably mentioned schale or dishes, plates and trays. Also, the material of the container or the substrate may be mentioned, for example, glass, a metal, a metal containing compound or a semi-metal containing compound, activated charcoal or a resin. The metal may be mentioned a typical metal: (an alkali metal: Li, Na, K, Rb, Cs; an alkaline earth metal: Ca, Sr, Ba, Ra), a magnesium group element: Be, Mg, Zn, Cd, Hg; an aluminum group element: Al, Ga, In; a rare earth element: Y, La, Ce, Pr, Nd, Sm, Eu; a tin group element: Ti, Zr, Sn, Hf, Pb, Th; an iron group element: Fe, Co, Ni; a vanadium group element: V, Nb, Ta, a chromium group element: Cr, Mo, W, U; a manganese group element: Mn, Re; a noble metal: Cu, Ag, Au; and a platinum group element: Ru, Rh, Pd, Os, Ir, Pt, etc. The metal containing compound or the semi-metal containing compound may be mentioned, for example, ceramics comprising a metal oxide as a basic component, which are a sintered body baked by a heat treatment at a high temperature, a semiconductor such as silicon, an inorganic solid material including a molded product of an inorganic compound such as a metal oxide or a semi-metal oxide (silicon oxide, alumina, etc.), a metal carbide or a semi-metal carbide, a metal nitride or a semi-metal nitride (silicon nitride, etc.), a metal boride or a semi-metal boride, etc., aluminum, nickel-titanium and stainless (SUS304, SUS316, SUS316L, etc.). As the resin, it may be either of a natural resin or a derivative thereof, or a synthetic resin, as the natural resin or a derivative thereof, there may be mentioned cellulose, cellulose triacetate (CTA), nitrocellulose (NC), cellulose to which dextran sulfate has been fixed, etc., and as the synthetic resin, there may be preferably used polyacrylonitrile (PAN), polyester-based polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene(PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyether sulfone (PES), polycarbonate (PC), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS) or Teflon (registered trademark). In the manufacture of the cell culture container of the present invention, at the time of coating the polymer to exist at least a part of the surface of a container or a substrate, it is not necessary to treat it at a high temperature, so that a resin having low heat resistance, etc., can be also applied. A material(s) of the container or the substrate may be one kind or a combination of two or more kinds. Among these materials, it is preferably glass, silicon, silicon oxide, polystyrene (PS), polypropylene (PP), polyether sulfone (PES), polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC), Teflon (registered trademark), cycloolefin polymer (COP), polydimethylsiloxane (PDMS) or stainless (SUS304, SUS316, SUS316L, etc.) alone, or a combination selected from these, and particularly preferably glass, polystyrene (PS), polypropylene (PP), stainless (SUS304, SUS316, SUS316L, etc.) or polydimethylsiloxane (PDMS). As a method for coating the base film-forming agent for cell culture of the present application, for example, an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like can be used, and it is preferably carried out by the printing technique of an inkjet method, a screen printing method, or the like. As another coating method, for example, there may be used methods such as immersing the container in the above-mentioned base film-forming agent, adding the base film-forming agent to the container and allowing to stand for a predetermined time, or coating a coating agent to the surface of the container or the substrate, etc., in the case of a container, or in the cell culture container as one embodiment, it is carried out by the method in which the base film-forming agent is added to the container and allowed it to stand for a predetermined time. Addition can be carried out, for example, by adding the base film-forming agent with 0.5 to 1-fold amount of the whole volume of the container using a syringe, etc. Standing is carried out by appropriately selecting a time and a temperature depending on the material of the container or the substrate and the kind of the base film-forming agent for cell culture and carried out, for example, for 1 minute to 24 hours, preferably for 5 minutes to 3 hours, at 10 to 80° C. According to the procedure, a cell culture container having a base film for cell culture onto at least a part of the surface of the container, preferably over the whole surface thereof can be manufactured. Also, the base film for cell culture of a surface of a container or a substrate obtained by such a method can be used as a cell culture container, after the step of contacting with at least a part of the surface of the above-mentioned container or substrate, preferably after the step of adding the base film-forming agent for cell culture and allowing to stand for a predetermined time, as it is without subjecting to the drying step, or after washing using water or a medium (for example, water, buffer solution, medium, etc.) of a sample applied to cell culture. That is, it can be used as the cell culture container after the step of contacting with at least a part of the surface of the above-mentioned container or substrate, preferably after the step of adding the base film-forming agent for cell culture and allowing to stand for a predetermined time, as it is without subjecting to the drying step within 48 hours, preferably within 24 hours, further preferably within 12 hours, further preferably within 6 hours, further preferably within 3 hours, further preferably within 1 hour, or after washing using water or a medium water or cell culture (for example, water, buffer solution, medium, etc., particularly preferably medium (for example, DMEM medium (Dulbecco's Modified Eagle's Medium)) of a sample applied to cell culture. The container may be applied to a drying step. The drying step is carried out under atmosphere or under vacuum, preferably at a temperature in the range of −200° C. to 200° C. By removing the solvent in the above-mentioned base film-forming agent according to the drying step, the film is completely adhered to the base substrate. The base film can be formed by drying, for example, at room temperature (10° C. to 35° C., preferably 20° C. to 30° C., for example, 25° C.), and in order to form the base film more quickly, it may be dried, for example, at 40° C. to 50° C. If the drying temperature is lower than −200° C., not a usual refrigerant must be used, which lacks versatility, and it takes a long time to dry by sublimation of the solvent, which is not efficient. If the drying temperature exceeds 200° C., pyrolysis of the polymer occurs. More preferable drying temperature is 10° C. to 180° C., and more preferable drying temperature is 20° C. to 150° C. The base film for cell culture of the present application can be produced through the above simple and convenient steps. In addition, in order to eliminate impurities, unreacted monomer, etc., remained in the base film for cell culture, a step of washing with at least one kind of a solvent selected from water and aqueous solution containing an electrolyte(s) may be carried out. The washing is desirably running water washing or ultrasonic wave washing, etc. The above-mentioned water and aqueous solution containing an electrolyte(s) may be heated, for example, in the range of 40° C. to 95° C. The aqueous solution containing an electrolyte(s) is preferably PBS, physiological saline (a material containing sodium chloride alone), Dulbecco's phosphate buffered physiological saline, Tris buffered physiological saline, HEPES buffered physiological saline and Veronal buffered physiological saline, and particularly preferably PBS. After adherence, the coating film does not dissolve even when washed with water, PBS, an alcohol, and the like, and remains firmly adhered to the base substrate. A film thickness of the base film for cell culture of the present application has a maximum film thickness and a minimum film thickness in the range of 1 to 1,000 nm, and preferably in the range of 5 to 500 nm. Before coating and drying steps of the above-mentioned base film, the container or the substrate may be subjected to a cell-adhesion inhibiting treatment. The container or the substrate having a cell-adhesion inhibiting ability can be produced by, for example, through a step of coating a known composition for forming a coating film having a cell-adhesion inhibiting ability. As the composition for forming a coating film having a cell-adhesion inhibiting ability, it is preferable to contain a step of coating a composition for forming a coating film containing a copolymer having a recurring unit containing an organic group represented by the following formula (a) and a recurring unit containing an organic group represented by the following formula (b): [wherein Ua11, Ua12, Ub11, Ub12and Ub13each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, An−represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion and an isothiocyanate ion], and a solvent onto a surface of a container or a substrate and drying the same. The linear or branched alkyl group having 1 to 5 carbon atoms are the same as that defined above. As the above-mentioned composition for forming a coating film coating film, for example, the composition for forming a coating film described in WO 2014/196650 can be used. The coating method of the above-mentioned composition for forming a coating film is not particularly limited, and a usual coating method such as spin coating, dip coating, a solvent casting method, etc., is used. The above-mentioned drying step of the coating film is carried out under atmosphere or under vacuum at a temperature in the range of −200° C. to 180° C. By the drying step, the solvent in the above-mentioned composition for forming a coating film is removed and the formula (a) and the formula (b) of the above-mentioned copolymer forms ionic bonds to completely adhere to the base substrate. The above-mentioned coating film can be formed by drying, for example, at room temperature (10° C. to 35° C., for example, 25° C.), and in order to form the coating film more quickly, it may be dried, for example, at 40° C. to 50° C. In addition, a drying step at an extremely low temperature to low temperature (around −200° C. to −30° C.) by the freeze-drying method may be used. Freeze-drying is called as vacuum freeze-drying, and is a method in which a material to be dried is normally cooled by a refrigerant and the solvent is removed by sublimation in a vacuum state. A general refrigerant used in freeze-drying may be mentioned a mixed medium of dry ice and methanol (−78° C.), liquid nitrogen (−196° C.), and the like. If the drying temperature is −200° C. or lower, not a usual refrigerant must be used, which lacks versatility, and it takes a long time to dry by sublimation of the solvent, which is not efficient. If the drying temperature is 200° C. or higher, ionic bonding reaction at the surface of the coating film proceeds excessively to lose hydrophilicity of the surface, and an adhesion inhibiting ability of biological substances is not exhibited. More preferable drying temperature is 10° C. to 180° C., and more preferable drying temperature is 20° C. to 150° C. After drying, in order to eliminate impurities, unreacted monomer, etc., remained in the coating film, and further to adjust ion balance of the copolymer in the film, it is desirable to wash with one or more solvents selected from water and aqueous solution containing an electrolyte(s) by running water washing or ultrasonic wave washing, etc. The above-mentioned water and aqueous solution containing an electrolyte(s) may be heated, for example, in the range of 40° C. to 95° C. The aqueous solution containing an electrolyte(s) is preferably PBS, physiological saline (a material containing sodium chloride alone), Dulbecco's phosphate buffered physiological saline, Tris buffered physiological saline, HEPES buffered physiological saline and Veronal buffered physiological saline, and particularly preferably PBS. After adherence, the coating film does not dissolve even when washed with water, PBS, an alcohol, and the like, and remains firmly adhered to the base substrate. In the formed coating film, even when biological substances are adhered, these can be easily removed thereafter by washing with water, etc., and the surface of the base substrate onto which the above-mentioned coating film had been formed has an adhesion inhibiting ability of biological substances. A film thickness of the above-mentioned coating film is preferably 5 to 1,000 nm, and further preferably 5 to 500 nm. Also, as the above-mentioned cell culture container, a commercially available cell culture dish subjected to a cell low-adhesion treatment or a cell incubator having a cell-adhesion inhibiting ability may be used and, for example, the cell culture container described in JP 2008-61609A can be used, but the invention is not limited to this. To have cell-adhesion inhibiting ability means that, for example, the relative absorbance (WST O.D. 450 nm) (%) ((absorbance of Example (WST O.D. 450 nm))/−(absorbance of Comparative Example (WST O.D. 450 nm))) compared with no coating film or no treatment of cell low adsorption treatment measured by a fluorescent microscope carried out by the method described in Example of WO 2016/093293 is 50% or lower, preferably 30% or less, and further preferably 20% or less. [Method for Producing Cell Aggregates] The method for producing cell aggregates of the present application is a method for producing cell aggregates which comprises carrying out on a base film for cell culture obtained from a polymer having a unit derived from a monomer represented by the following formula (I): [wherein Ua1, Ua2each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, Ra1represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and Ra2represents a linear or branched alkylene group having 1 to 5 carbon atoms], by the known method per se, for example, by the method described in Examples. The above-mentioned base film for cell culture is preferably a base film obtained from a copolymer having a unit derived from a monomer represented by the formula (I), together with a unit derived from a monomer represented by the following formula (II): [wherein Rbrepresents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. EXAMPLES Hereinafter, the present invention will be explained in more detail by referring to Examples, but the present invention is not limited to these. <Measurement Method of Molecular Weight> The weight average molecular weight shown in the following Synthetic Examples is a result by Gel Filtration Chromatography (hereinafter abbreviated to as GFC). (Measurement Conditions) Apparatus: HLC-8320GPC (manufactured by Tosoh Corporation)GFC column: TSKgel G6000+3000PWXL-CPFlow rate: 1.0 ml/minEluent: Water containing salt/organic mixed solventColumn temperature: 40° C.Detector: RIInjection concentration: polymer solid content 0.05% by massInjection amount: 100 μLCalibration curve: Cubic approximation curveStandard sample: polyethylene oxide (available from Agilent)×10 kinds <Synthetic Example 1> Production (1) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 9.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 27.04 g of tetrahydrofuran and the mixture was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) was added to the above-mentioned tetrahydrofuran solution while maintaining the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible homopolymer (Yielded amount: 6.4 g, Yield: 71%). The weight average molecular weight of this polymer by GFC was 250,000, and the polydispersity was 2.0 (Synthetic Example Polymer 1). <Synthetic Example 2> Production (2) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 9.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 37.10 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.02 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.26 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer (Yielded amount: 6.6 g, Yield: 71%). The weight average molecular weight of this polymer by GFC was 24,000, and the polydispersity was 2.0 (Synthetic Example Polymer 2). <Synthetic Example 3> Production (3) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 41.94 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.48 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer (Yielded amount: 7.3 g, Yield: 69%). The weight average molecular weight of this polymer by GFC was 290,000, and the polydispersity was 1.9 (Synthetic Example Polymer 3). <Synthetic Example 4> Production (4) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 9.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 38.26 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.02 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.55 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer (Yielded amount: 7.2 g, Yield: 75%). The weight average molecular weight of this polymer by GFC was 250,000, and the polydispersity was 1.9 (Synthetic Example Polymer 4). <Synthetic Example 5> Production (5) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 9.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 41.00 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.02 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 1.23 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer (Yielded amount: 5.8 g, Yield: 57%). The weight average molecular weight of this polymer by GFC was 270,000, and the polydispersity was 2.1 (Synthetic Example Polymer 5). <Synthetic Example 6> Production (6) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 9.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 44.58 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.03 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 2.11 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer (Yielded amount: 9.6 g, Yield: 86%). The weight average molecular weight of this polymer by GFC was 270,000, and the polydispersity was 2.4 (Synthetic Example Polymer 6). <Synthetic Example 7> Production (7) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 41.94 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation), 0.48 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) and 0.21 g of polyethylene glycol dimethacrylate (n=about 4) (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer. The weight average molecular weight of this polymer by GFC was 660,000, and the polydispersity was 3.8 (Synthetic Example Polymer 7). <Synthetic Example 8> Production (8) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 43.30 g of ethanol and the monomer was sufficiently dissolved. Then, 0.31 g of dimethyl 1,1′-azobis(1-cyclohexanecarboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.48 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned ethanol solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer. The weight average molecular weight of this polymer by GFC was 88,000, and the polydispersity was 2.4 (Synthetic Example Polymer 8). <Synthetic Example 9> Production (9) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 41.94 g of tetrahydrofuran and the monomer was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.48 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned tetrahydrofuran solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to 60° C. while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer. The weight average molecular weight of this polymer by GFC was 140,000, and the polydispersity was 2.5 (Synthetic Example Polymer 9. <Synthetic Example 10> Production (10) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 31.46 g of ethanol and the monomer was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.48 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned ethanol solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer. The weight average molecular weight of this polymer by GFC was 770,000, and the polydispersity was 4.1 (Synthetic Example Polymer 10). <Synthetic Example 11> Production (11) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 43.90 g of ethanol and the monomer was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 0.97 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned ethanol solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer. The weight average molecular weight of this polymer by GFC was 660,000, and the polydispersity was 3.6 (Synthetic Example Polymer 11). <Synthetic Example 12> Production (10) of Polymer Used as Base Film-Forming Agent for Cell Culture by Thermal Polymerization To 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) was added 70.00 g of ethanol and the monomer was sufficiently dissolved. Then, 0.01 g of dimethyl 1,1′-azobis(1-cyclohexane-carboxylate) (VE-073, available from FUJIFILM Wako Pure Chemical Corporation) and 2.35 g of methacrylic acid (available from Tokyo Chemical Industry Co., Ltd.) were added to the above-mentioned ethanol solution while maintaining the temperature of the mixture to 20° C. or lower. The mixture in which the above-mentioned all the materials were contained which became uniform by sufficient stirring was added to a three-necked flask equipped with a cooling tube and subjected to nitrogen flow, and the temperature of the mixture was raised to reflux temperature while stirring. By stirring under heating in the state of maintaining the above-mentioned circumstance for 24 hours, a polymer was obtained as a reaction product. The reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitates were recovered by filtration and dried under reduced pressure. The obtained powder was dissolved in pure water, and the solution was transferred to a dialysis tube. Dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain a temperature-responsible polymer. The weight average molecular weight of this polymer by GFC was 570,000, and the polydispersity was 3.6 (Synthetic Example Polymer 12). <Comparative Synthetic Example 1> Production (1) of Polymer by Photopolymerization To a transparent vial bottle made of soft glass having a volume of 30 mL were added 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) and 500 μL of water, and the mixture was sufficiently stirred to make it uniform. Then, the mixture was deoxidized by purging the mixture (liquid) with nitrogen gas for 15 minutes. Thereafter, ultraviolet rays were irradiated to this reaction product at about 25° C. for 19 hours using a high-pressure mercury lamp (manufactured by USHIO, Model No.: UM-102) and adjusting the distance that the illuminance at 365 nm was to be 0.1 mW/cm2with an illuminometer, the above-mentioned reaction material was polymerized. The reaction material became viscous after 5 hours and solidified (gelled) after 18 hours to obtain a polymer as a reaction product. This reaction product was difficultly soluble in 2-propanol, and only the partially dissolved portion was transferred to a dialysis tube. Incidentally, the dissolved liquid was a stringy viscous material and was difficult to handle. Then, dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain Comparative Synthetic Example Polymer 1 (Yielded amount: 1.5 g, Yield: 15%). <Comparative Synthetic Example 2> Production (2) of Polymer by Photopolymerization To a transparent vial bottle made of soft glass having a volume of 30 mL were added 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) and 1,000 μL of water, and the mixture was sufficiently stirred to make it uniform. Then, the mixture was deoxidized by purging the mixture (liquid) with nitrogen gas for 15 minutes. Thereafter, ultraviolet rays were irradiated to this reaction product at about 25° C. for 19 hours using a high-pressure mercury lamp (manufactured by USHIO, Model No.: UM-102) and adjusting the distance that the illuminance at 365 nm was to be 0.1 mW/cm2with an illuminometer, the above-mentioned reaction product was polymerized. The reaction material became viscous after 5 hours and solidified after 18 hours to obtain a polymer as a reaction product. This reaction product was difficultly soluble in 2-propanol, and only the partially dissolved solution was transferred to a dialysis tube. Incidentally, the dissolved liquid was a stringy viscous material and was difficult to handle. Then, dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain Comparative Synthetic Example Polymer 2 (Yielded amount: 2.6 g, Yield: 26%). <Comparative Synthetic Example 3> Production (3) of Polymer by Photopolymerization To a transparent vial bottle made of soft glass having a volume of 30 mL were added 10.00 g of 2-(dimethylamino)ethyl methacrylate (available from Tokyo Chemical Industry Co., Ltd.) and 500 μL of water, and the mixture was sufficiently stirred to make it uniform. Then, the mixture was deoxidized by purging the mixture (liquid) with nitrogen gas for 15 minutes. Thereafter, ultraviolet rays were irradiated to this reaction product at about 25° C. for 19 hours using a high-pressure mercury lamp (manufactured by USHIO, Model No.: UM-102) and adjusting the distance that the illuminance at 365 nm was to be 0.1 mW/cm2with an illuminometer, the above-mentioned reaction product was polymerized. The reaction material became viscous after 5 hours and solidified after 18 hours to obtain a polymer as a reaction product. This reaction product was difficultly soluble in 2-propanol, and only the partially dissolved solution was transferred to a dialysis tube. Incidentally, the dissolved liquid was a stringy viscous material and was difficult to handle. Then, dialysis was carried out for 72 hours to purify the reaction product. The solution containing the reaction product was filtered through 1.0 m filter (available from AS ONE Corporation, Model No.: SYGF0605MNXX104) made of glass fiber, and the obtained filtrate was lyophilized to obtain Comparative Synthetic Example Polymer 3 (Yielded amount: 3.3 g, Yield: 33%). <Example 1> (Compositional Analysis by Measurement of 1H-NMR of Polymer) The nuclear magnetic resonance spectra (NMR) of Synthetic Example Polymers 1 to 12 and Comparative Synthetic Example Polymers 1 to 3 were measured using a nuclear magnetic resonance apparatus (manufactured by BRUKER, Model No.: ASCEnd500) and heavy water (D20) as a standard substance. In the following, representative peaks common to Synthetic Example Polymer 1 to Synthetic Example Polymer 6 are shown. 1H-NMR (in D2O) δ 0.8-1.2 (br, —CH2-C(CH3)-), 1.6-2.0 (br, —CH2-C(CH3)-), 2.2-2.4 (br, —N(CH3)2), 2.5-2.7 (br, —CH2-N(CH3)2), 4.0-4.2 (br, —O—CH2-). Here, from the number of protons (in the case of homopolymer of DMAEMA, 3 in monomer one molecule) A of the methyl group —CH2-C(CH3)- (δ 0.8-1.2) at the main chain, and the number of methyl protons (in the case of homopolymer of DMAEMA, 2 in monomer one molecule) B of the —O—CH2- group (δ 4.0-4.2) at the side chain, a ratio of the number of the functional group of the amino group possessed by the side chain and the number of the functional group of the carboxyl group at the side chain was calculated. As a result, with regard to the compositional ratio of 2-(dimethylamino)ethyl methacrylate (hereinafter abbreviated to as “DM”)/methacrylic acid (hereinafter abbreviated to as “MA”) of Synthetic Examples 1 to 12 synthesized by thermal polymerization, in the case of Synthetic Example 1, it was 100/0, in the case of Synthetic Example 2, it was 95/5, in the case of Synthetic Example 3, it was 95/5, in the case of Synthetic Example 4, it was 88/12, in the case of Synthetic Example 5, it was 82/18, in the case of Synthetic Example 6, it was 76/24, in the case of Synthetic Example 7, it was 89/11, in the case of Synthetic Examples 8 to 10, these were 92/8, in the case of Synthetic Example 11, it was 85/15, and in the case of Synthetic Example 12, it was 70/30. In the case of Comparative Synthetic Examples synthesized by photopolymerization, in all of Comparative Synthetic Examples 1, 2 and 3, the ratio was 99/1 so that control of the ratio of DM and MA was difficult. From the above-mentioned results, in the polymers synthesized by thermal polymerization, as compared with the polymers synthesized by photopolymerization, it was confirmed that the ratio of DM and MA could be controlled and further the polymers could be obtained with high yield. Detailed results are shown in Table 1. With regard to the polymers synthesized in Synthetic Examples 1 to 12, the range of the weight average molecular weight Mw was 88,000 to 770,000 so that it was possible to prepare the polymers in a wide range. In addition, the range of the molecular weight distribution (PDI) was 1.9 to 4.1 so that it was possible to prepare the polymers from a small distribution to a relatively large distribution. Moreover, either of the polymers could be synthesized without gelation. On the other hand, in the case where the present polymer was synthesized by photopolymerization, it is difficult to control the molecular weight or to control the molecular weight distribution with a small region. For example, in Patent Gazette (JP Patent No. 5,746,240), when synthesis is carried out in the same method as in Comparative Synthetic Example 1, it is described that PDI=3.0, when synthesis is carried out in the same method as in Comparative Synthetic Example 2, PDI=4.3, and when synthesis is carried out in the same method as in Comparative Synthetic Example 3, PDI=7.4. Further, in the photopolymerization, all the polymers are gelled at the time of synthesis, and it is difficult to control not only the molecular weight but also the reaction itself. When the molecular weight distribution becomes extremely large, there are concerns about dissolution of the low molecular weight components and precipitation of the high molecular weight components. When these are taking into account, by synthesizing a polymer using thermal polymerization, as compared with the case where it is synthesized by photopolymerization, a polymer can be produced stably while controlling the molecular weight and the molecular weight distribution. TABLE 1CompositionChargeratio (NMRratioin D2O)MwProducingDM/MADM/MA(×10{circumflex over ( )}4)Mw/MnmethodSynthetic100/0100/0252.0ThermalExample 1polymer-Synthetic95/595/5242.0izationExample 2Synthetic92/895/5291.9Example 3Synthetic90/1088/12251.9Example 4Synthetic80/2082/18272.1Example 5Synthetic70/3076/24272.4Example 6Synthetic92/889/11663.8Example 7Synthetic92/892/892.4Example 8Synthetic92/892/8142.5Example 9Synthetic92/890/10774.1Example 10Synthetic85/1581/19663.6Example 11Synthetic70/3074/26573.6Example 12ComparativeDM + Pure99/1——Photo-Syntheticwaterpolymer-Example 1500 uLizationComparativeDM + Pure99/1——SyntheticwaterExample 21000 uLComparativeDM + Pure99/1——SyntheticwaterExample 35000 uL <Example 2> Measurement of Surface Profile of Coating Film Example Polymers 3 and 8 having different molecular weights were each dissolved in sterilized water so that the concentration became 0.5 mg/mL to produce base film-forming agents for cell culture. By using an inkjet device (manufactured by MICROJET, Model No.: LaboJet-600), each 200 nL was coated onto a silicon substrate to which hexamethyldisilazane treatment had been done. After curing the film by drying at room temperature for 5 minutes, the surface profile of the coating film was measured by using a surface profiler (manufactured by Kosaka Laboratory Ltd., Model No.: ET-4000A). The measurement conditions were made a measurement force of 100 μN and a feeding rate of 0.05 mm/sec. In Synthetic Example Polymer 3 (Mw=290,000), the peripheral part of the coating film was raised, and the inside became a flat shape. On the other hand, in Synthetic Example Polymer 8 (Mw=88,000), a swelling in the central part was observed in addition to the swelling in the peripheral part of the coating film. It was found that the cross-sectional shape of the coating film changed depending on the molecular weight and the molecular weight distribution of the polymer. <Example 3> Production of Polymer Aqueous Solution for Producing Base Film-Forming Agent for Cell Culture Synthetic Example Polymer 1 to Synthetic Example Polymer 12 were each dissolved in sterilized water to be a concentration of 1 mg/mL to produce polymer aqueous solutions 1 to 12. Example 4: Production Test of Cell Aggregates (4-1. Preparation of Cell Low Adhesion Plate) According to the producing method described in Example 30 of WO 2014/196650, a coating solution was prepared from a copolymer-containing varnish. The prepared coating solution was added to the wells of a 12-well cell culture plate (manufactured by BD Bioscience, #351143) so as to have a solid content of 500 μL (solid content 0.5% by mass)/well, and after allowing to stand at room temperature for one hour, excess coating solution was removed. It was dried overnight at 50° C. using an oven (manufactured by Advantech Toyo Kaisha Ltd., Dryer FC-612). Thereafter, after adding 500 μL of sterilized water per well, it was removed and washing is carried out. In the same manner, washing was further carried out twice, and dried at 50° C. overnight to obtain a cell low-adhesion plate. (Production of Base Film-Forming Agent for Cell Culture, and Preparation of Polymer Coating Plate Used as Base Film for Cell Culture) Each 1 mg/mL of the polymer aqueous solution obtained from Synthetic Example Polymer 3 and Synthetic Example Polymer 4 was diluted with sterilized water so as to be 100 μg/mL to produce base film-forming agents 3 and 4 for cell culture. The produced polymer aqueous solution was dropped onto the above-mentioned cell low-adhesion plate with 1 μL drop by drop, and dried at room temperature for 30 minutes to obtain a polymer coating plate to be used as a base film for cell culture used for the test. (4-2. Preparation of Cells) As the cells, human bone marrow-derived mesenchymal stem cells (available from PromoCell Inc.) were used. As the medium used for culturing the cells, mesenchymal stem cell growth medium Mesenchymal Stem Cell Growth Medium 2 (available from PromoCell Inc.) was used. The cells were statically cultured in a petri dish (medium 10 mL) having a diameter of 10 cm for 2 days or longer while maintaining a 5% carbon dioxide concentration in a 37° C./CO2incubator. Subsequently, after washing these cells with 4 ml of HepesBSS solution (available from PromoCell Inc.), 4 mL of trypsin-EDTA solution (available from PromoCell Inc.) was added thereto and the cells were allowed to stand at room temperature for 5 minutes. 4 mL of Trypsin Neutralizing Solution (available from PromoCell Inc.) was added thereto, and the cells were peeled off and recovered. After centrifuging (manufactured by Tomy Seiko Co., Ltd., Model No. LC-230, 200×g/3 min, room temperature) this suspension, the supernatant was removed and the above-mentioned medium was added to prepare a cell suspension. (4-3. Cell Adhesion Experiment) To the plate prepared as mentioned above, 500 μL of the cell suspension was each added so as to be 5.9×105cells/well (1.75×105cells/cm2). Thereafter, it was allowed to stand in a 37° C./CO2incubator for 3.5 hours in the state of maintaining 5% carbon dioxide concentration. After allowing to stand, non-adherent cells and the medium were removed and washed with PBS to leave only the adherent cells on the wells. After washing, 500 μL/well of a new medium was added thereto, and the state of adherent cells was observed and photographed using an inverted research microscope IX73 (manufactured by Olympus Corporation). As a result, as shown inFIG.2, adhesion of the cells to the portion at which the base film-forming agent 3 for cell culture or the base film-forming agent 4 for cell culture had been coated was confirmed. (4-4. Observation of cell aggregates) The plate tested as mentioned above was allowed to stand for further one day in a 37° C./CO2incubator. After allowing to stand, the state of the cells was observed using an inverted research microscope IX73 (manufactured by Olympus Corporation). As a result, as shown inFIG.3, it was confirmed that the cells adhered onto the base film-forming agent for cell culture 3 and the base film-forming agent for cell culture 4 were peeled off from the plate and aggregated to form cell aggregates (spheroids). From this, it was shown that the base film containing the polymer of the present application was useful as a base film for the cell culture container. Example 5: Production Test of Cell Aggregates (5-1. Production of Base Film-Forming Agent for Cell Culture and Preparation of Polymer Coating Plate Used as Base Film for Cell Culture) Each 1 mg/mL of polymer aqueous solutions obtained from Synthetic Example 1, Synthetic Example 10, Synthetic Example 11 and Synthetic Example 12 was diluted with sterilized water so as to become 50 μg/mL to produce base film-forming agents 5, 6, 7 and 8 for cell culture. The produced polymer aqueous solution was dropped onto a cell low-adhesion dish (manufactured by Sumitomo Bakelite Co., Ltd., #MS-9035X) with 1 μL drop by drop, and dried at 50° C. for 30 minutes to obtain a polymer coating dish to be used as a base film for cell culture used for the test. (5-2. Preparation of Cells) As the cells, human bone marrow-derived mesenchymal stem cells (available from PromoCell Inc.) were used. As the medium used for culturing the cells, mesenchymal stem cell growth medium Mesenchymal Stem Cell Growth Medium 2 (available from PromoCell Inc.) was used. The cells were statically cultured in a petri dish (medium 10 mL) having a diameter of 10 cm for 2 days or longer while maintaining a 5% carbon dioxide concentration in a 37° C./CO2incubator. Subsequently, after washing these cells with 4 ml of HepesBSS solution (available from PromoCell Inc.), 4 mL of trypsin-EDTA solution (available from PromoCell Inc.) was added thereto and the cells were allowed to stand at room temperature for 5 minutes. 4 mL of Trypsin Neutralizing Solution (available from PromoCell Inc.) was added thereto, and the cells were peeled off and recovered. After centrifuging (manufactured by Tomy Seiko Co., Ltd., Model No. LC-230, 200×g/3 min, room temperature) this suspension, the supernatant was removed and the above-mentioned medium was added to prepare a cell suspension. (5-3. Cell Adhesion Experiment) To the plate prepared as mentioned above, the cell suspension was each added so as to be 2.7×106cells/3 mL/dish (3×105cells/cm2). Thereafter, it was allowed to stand in a 37° C./CO2incubator for 2 hours in the state of maintaining 5% carbon dioxide concentration. After allowing to stand, non-adherent cells and the medium were removed and washed with PBS to leave only the adherent cells on the wells. After washing, 2 mL/dish of a new medium was added thereto, and the state of adherent cells was observed and photographed using EVOS FL Auto (manufactured by Thermo Fisher Scientific). As a result, as shown inFIG.4, adhesion of the cells to the portion at which the base film-forming agent 5 for cell culture, the base film-forming agent 6 for cell culture, the base film-forming agent 7 for cell culture and the base film-forming agent 8 for cell culture had been coated was confirmed. (5-4. Observation of Cell Aggregates) The plate tested as mentioned above was continuously time-lapse photographed using EVOS FL Auto. In the time-lapse photography, the same field of view was photographed everyone hour under the conditions of 37° C./5% CO2. As a result, as shown inFIG.5, it was confirmed that the cells adhered onto the base film-forming agent 5 for cell culture, the base film-forming agent 6 for cell culture, the base film-forming agent 7 and the base film-forming agent 8 were peeled off from the plate and aggregated to form cell aggregates (spheroids). From this, it was shown that the base film containing the polymer of the present application was useful as a base film for the cell culture container. UTILIZABILITY IN INDUSTRY The polymer obtained by the producing method of the present invention can be used as a base film-forming agent for cell culture. By using the base film-forming agent of the present invention, a base film for cell culture, and a cell culture container containing the same can be produced. | 69,191 |
11859036 | DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENT(S) A process of treating polystyrene material, such as waste polystyrene material, within a reactor of a system is described below. Suitable waste polystyrene material includes, but it not limited to, expanded, and/or extruded polystyrene foam, and/or rigid products. Virgin polystyrene can also be used. FIG.1Aillustrates Process 10 for treating polystyrene material. Process 10 can be run in batches, but more preferably is a continuous process. The parameters of Process 10, including but not limited to temperature, flow rate of polystyrene, monomers/copolymers grafted during the reaction and/or modification stages, and total number of pre-heat, reaction, or cooling segments, can be modified to create end products of varying molecular weights, such as macromonomers, or polyaromatic products. For example, raising the temperature and/or decreasing the flow rate through the reaction sections or changing the number of reaction sections will result in the product of a lower molecular weight. In Material Selection Stage 1, polystyrene feed is sorted/selected and/or prepared for treatment. In some embodiments, the feed can contain up to 25% polyolefins, PET, EVA, EVOH, and lower levels of undesirable additives or polymers, such as nylon, rubber, PVC, ash, filler, pigments, stabilizers, grit or other unknown particles. In some embodiments, the polystyrene feed has an average molecular weight between 150000 amu and 400000 amu. In some of these embodiments, the polystyrene feed has an average molecular weight between 200000 amu and 250000 amu. In some embodiments, the material selected in Material Selection Stage 1 comprises recycled polystyrene. In other or the same embodiments, the material selected in Material Selection Stage 1 comprises recycled polystyrene and/or virgin polystyrene. In some embodiments, the material selected in Material Selection Stage 1 is can be heated in Heat Stage 2 an extruder and undergoes Pre-Filtration Process 3. In some embodiments, the extruder is used to increase the temperature and/or pressure of the incoming polystyrene and is used to control the flow rates of the polystyrene. In some embodiments, the extruder is complimented by or replaced entirely by pump/heater exchanger combination. Pre-Filtration Process 3 can employ both screen changers and filter beds, along with other filtering techniques/devices to remove contaminants from and purify the heated material. The resulting filtered material is then moved into an optional Pre-Heat Stage 4 which brings the filtered material to a higher temperature before it enters Reaction Stage 5. Pre-Heat Stage 4 can employ, among other devices and techniques, static and/or dynamic mixers and heat exchangers such as internal fins and heat pipes. Material in Reaction Stage 5 undergoes depolymerization. This depolymerization can be a purely thermal reaction and/or it can employ catalysts. Depending on the starting material and the desired end product, depolymerization might be used for a slight or extreme reduction of the molecular weight of the starting material. In some embodiments, the catalyst used is a zeolite or alumina supported system or a combination of the two. In some embodiments, the catalyst is [Fe—Cu—Mo—P]/Al2O3prepared by binding a ferrous-copper complex to an alumina or zeolite support and reacting it with an acid comprising metals and non-metals. Reaction Stage 5 can employ a variety of techniques/devices including, among other things, fixed beds, horizontal and/or vertical reactors, and/or static mixers. In some embodiments, Reaction Stage 5 employs multiple reactors and/or reactors divided into multiple sections. Reaction Stage 5 can also involve grafting various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene onto the depolymerized product. After Reaction Stage 5, the depolymerized material enters optional Modification Stage 6. As in Reaction Stage 5, Modification Stage 6 involves grafting various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene onto the depolymerized product. Cooling Stage 7 can employ heat exchangers, along with other techniques/devices to bring the depolymerized material down to a workable temperature before it enters optional Purification Stage 8. In some embodiments, cleaning/purification of the material via such methods such as nitrogen stripping occurs before Cooling Stage 7. Optional Purification Stage 8 involves the refinement and/or decontamination of the depolymerized material. Techniques/devices that can used in Purification Stage 8 include, but are not limited to, flash separation, absorbent beds, clay polishing, distillation, vacuum distillation and filtration to remove solvents, oils, color bodies, ash, inorganics, and coke. In some embodiments, a thin or wiped film evaporator is used to remove gas, oil, and/or grease from the depolymerized material. In some embodiments, the oil, gas and grease can in turn be burned to help run various Stages of Process 10. Process 10 ends at Finished Product Stage 9 in which the initial starting material selected in Material Selection Stage 1 has been turned into a lower molecular weight polymer. In at least some embodiments, the lower molecular weight polymer at Finished Product Stage 9 is commercially viable and does not need additional processing and/or refining. In other embodiments, the plastic created at Finished Product Stage, needs additional modifications. FIG.1Billustrates Process 20 for treating polystyrene material. Process 20 can be run in batches, but more preferably is a continuous process. The parameters of Process 20, including but not limited to temperature, flow rate of polystyrene, monomers/copolymers grafted during the reaction and/or modification stages, and total number of pre-heat, reaction, or cooling segments, can be modified to create end products of varying molecular weights, such as macromonomers, or polyaromatic products. For example, raising the temperature and/or decreasing the flow rate through the reaction sections or changing the number of reaction sections will result in the product of a lower molecular weight. In Material Selection Stage 21, polystyrene feed is sorted/selected and/or prepared for treatment. In some embodiments the feed can contain up to 25% polyolefins, PET, EVA, EVOH, and lower levels of undesirable additives or polymers, such as nylon, rubber, PVC, ash, filler, pigments, stabilizers, grit or other unknown particles. In some embodiments the material selected in Material Selection Stage 21 comprises recycled polystyrene. In other or the same embodiments, the material selected in Material Selection Stage 21 comprises recycled polystyrene and/or virgin polystyrene. In Solvent Addition Stage 22, solvents, such as toluene, xylenes, cymenes, or terpinenes, are used to dissolve the polystyrene before it undergoes depolymerisation within the reactor bed/vessels. In certain embodiments, the desired product can be isolated via separation or extraction and the solvent can be recycled. In some embodiments, the material selected in Material Selection Stage 21 is heated in an extruder during Heat Stage 23 and undergoes Pre-Filtration Process 24. In some embodiments the extruder is used to increase the temperature and/or pressure of the incoming polystyrene and is used to control the flow rates of the polystyrene. In some embodiments the extruder is complimented by or replaced entirely by pump/heater exchanger combination. Pre-Filtration Process 24 can employ both screen changers and filter beds, along with other filtering techniques/devices to remove contaminants from and purify the heated material. The resulting filtered material is then moved into an optional Pre-Heat Stage 25 which brings the filtered material to a higher temperature before it enters Reaction Stage 26. Pre-Heat Stage 25 can employ, among other devices and techniques, static and/or dynamic mixers and heat exchangers such as internal fins and heat pipes. Material in Reaction Stage 26 undergoes depolymerization. This depolymerization can be a purely thermal reaction and/or it can employ catalysts. Depending on the starting material and the desired end product, depolymerization might be used for a slight or extreme reduction of the molecular weight of the starting material. In some embodiments the catalyst used is a zeolite or alumina supported system or a combination of the two. In some embodiments the catalyst is [Fe—Cu—Mo—P]/Al2O3prepared by binding a ferrous-copper complex to an alumina or zeolite support and reacting it with an acid comprising metals and non-metals. Reaction Stage 26 can employ a variety of techniques/devices including, among other things, fixed beds, horizontal and/or vertical reactors, and/or static mixers. In some embodiments, Reaction Stage 26 employs multiple reactors and/or reactors divided into multiple sections. Reaction Stage 26 can also involve grafting various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene onto the depolymerized product. After Reaction Stage 26, the depolymerized material enters optional Modification Stage 27. As in Reaction Stage 26, Modification Stage 27 involves grafting various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene onto the depolymerized product. Cooling Stage 28 can employ heat exchangers, along with other techniques/devices to bring the depolymerized material down to a workable temperature before it enters optional Purification Stage 29. In some embodiments, cleaning/purification of the material via such methods such as nitrogen stripping occurs before Cooling Stage 28. Purification Stage 29 involves the refinement and/or decontamination of the depolymerized material. Techniques/devices that can used in Purification Stage 8 include, but are not limited to, flash separation, absorbent beds, clay polishing, distillation, vacuum distillation and filtration to remove solvents, oils, color bodies, ash, inorganics, and coke. In some embodiments, a thin or wiped film evaporator is used to remove gas, oil, and/or grease from the depolymerized material. In some embodiments, the oil, gas and grease can in turn be burned to help run various Stages of Process 20. In certain embodiments, the desired product can be isolated via separation or extraction and the solvent can be recycled. Process 20 ends at Finished Product Stage 30 in which the initial starting material selected in Material Selection Stage 1 has been turned into a lower molecular weight polymer. In at least some embodiments, the lower molecular weight polymer at Finished Product Stage 30 is commercially viable and does not need additional processing and/or refining. In other embodiments, the plastic created at Finished Product Stage 30, needs additional modifications. In some embodiments, the finished product has an average molecular weight between 40000 amu and 200000 amu, a melt flow index equal to/greater than 0.5 at 190° C. w/2.16 kg, and/or a glass transition temperature between 50° C. and 110° C. In some of these embodiments, the finished product has an average molecular weight between 55000 amu and 146000 amu, a melt flow index greater than 3.20 at 190° C. w/2.16 kg, and/or a glass transition temperature between 75° C. and 105° C. Referring toFIG.2, system1000includes reactor100with five reactor modules102athrough102e. Reactor modules102can vary in dimensions and/or be connected in parallel and/or series. In other embodiments various numbers of reactor modules102can be used. For example,FIG.3shows system1000with four reactor modules,102athrough102d. Similarly,FIG.4shows system1000with six reactor modules102athrough102f. The ability to customize the number of reactor modules102allows for greater control of the amount of depolymerization. System1000can include hopper111for receiving polystyrene material and/or directing the supply of the polystyrene material to optional extruder106. In some embodiments, extruder106processes the polystyrene material received from hopper111by generating a molten polystyrene material. The temperature of the polystyrene material being processed by extruder106is controlled by modulating the level of shear and/or the heat being applied to the polystyrene material by extruder heater(s)105. Extruder heaters can use a variety of heat sources including, but not limited to, electric, thermal fluids, and/or combustion gases. The heat is modulated by a controller, in response to temperatures sensed by temperature sensor(s)107. In some embodiments, pressure sensor109measures the pressure of the molten polystyrene material being discharged from extruder106, to prevent, or at least reduce, risk of pressure spikes. The discharged molten polystyrene material is pressurized by pump110to affect its flow through heating zone108and reactor100. While flowing through reactor100, the reactor-disposed molten polystyrene material contacts a catalyst material which impacts rate and mechanism for depolymerization. In at least some embodiments, the system operates at a moderate temperature and/or around atmospheric pressure. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in reactor100. Pressure sensor(s)109and/or temperature sensor(s)107can also be used to measure temperature and/or pressure, respectively, of the reactor-disposed molten polystyrene material as it flows through reactor100. Pressure sensor(s)109can monitor for plugs before and/or after each reaction zones. Pressure sensor(s)109can also maintain system pressure below a maximum pressure such as the maximum pressure of reactor100is designed for. Over-pressure can be controlled by feedback from pressure transmitter109to a controller which transmits a command signal to shut down extruder106and pump110, and thereby prevent the pressure from further increasing. In cases when shutdown of extruder106does not relieve the over pressure, dump valve117can be opened into a container to remove material from system1000and avoid an over pressure situation. During shutdown dump valve117can be opened to purge system1000with nitrogen to remove leftover material to avoid clogs and degraded material during the next start up. System1000can also include a pressure relief device, such as a relief valve or a rupture disk, disposed at the outlet of extruder106, to relieve pressure from system1000, in case of over-pressure. Temperature sensor(s)107can facilitate control of the temperature of the reactor-disposed molten polystyrene material being flowed through reactor100. This allows more precise control of the chemical reaction and the resulting depolymerization. Temperature sensor(s)107also aid in maintaining the temperature below a predetermined maximum temperature, for example the maximum design temperature of reactor100. The temperature is controlled by a controller (not shown), which modulates the heat being applied by heaters118disposed in heat transfer communication with the reaction zones102athrough102eof reactor100, in response to the temperatures sensed by temperature sensor(s)119. Flow control can also be provided for within system1000. In some embodiments, system1000includes valve115, disposed at the discharge of extruder106, for controlling flow from extruder106to other unit operations within system1000. Valve116facilitates recirculation. Valve117enables collection of product. During operation, valve115can be closed in order to recirculate the molten polystyrene material and increase the temperature of the molten polystyrene material to a desired temperature. In this case valve116would be open, valve117would be closed, extruder106would be “OFF”, and pump110would be recirculating. Generated molten product material112is cooled within heat exchanger114, which can be, among other ways, water jacketed, air cooled, and/or cooled by a refrigerant. A fraction of the cooled generated molten product material can be recirculated (in which case valve116would be open), for reprocessing and/or for energy conservation. In some embodiments, system1000is configured for purging by nitrogen to mitigate oxidation of the molten product material and the creation of explosive conditions. In another embodiment illustrated inFIG.5, System2000includes reactor600. Reactor600has two reactor modules, namely, inlet reactor module300and outlet reactor module400. System2000also includes extruder606for receiving polystyrene material. Extruder606processes polystyrene material by generating a molten polystyrene material. The temperature of the polystyrene material being processed through reactor600is controlled by modulating the heat being applied to the polystyrene material by process heaters620. Temperature sensors630are provided to measure the temperature of the molten material within reactor600. Temperature controllers632are provided to monitor and control the temperature of process heaters620. Flange heaters622are also provided to mitigate heat losses through the flanged connections. The discharged molten polystyrene feed material is conducted through heater608and reactor600, in series. While flowing through reactor600, the reactor-disposed molten polystyrene material is contacted with the catalyst material to affect the depolymerization. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in reactor600. The generated molten product material is cooled within heat exchanger614, which can be, among other things, water jacketed, air cooled, or cooled by a refrigerant. In some embodiments the waste heat from the cooling molten product can be used to run other processes. A cooling section heater624can be provided to melt wax that solidifies in cooling section. In both System1000and System2000reactors100and600include one or more reactor modules. Each reactor modules includes a respective module reaction zone in which the reactor-disposed molten polystyrene material is brought into contact with a catalyst material over a module-defined residence time, thereby causing depolymerization of the flowing reactor-disposed molten polystyrene material. In some of these embodiments, the module-defined residence time of at least two of the reactor modules is the same or substantially the same. In some of these embodiments, as between at least some of the plurality of module-defined residence times are different. In some of these embodiments the catalyst material of at least two of the reactor modules are the same or substantially the same. In some of these embodiments the catalyst material of at least two of the reactor modules are different. In some embodiments, each of the reactor modules includes a reactor-disposed molten polystyrene material-permeable container that contains the catalyst material. The container is configured to receive molten polystyrene material such that at least partial depolymerization of at least a fraction of the received molten polystyrene material is effected by the catalyst material, and to discharge a molten product material that includes depolymerization reaction products (and can also include unreacted molten polystyrene material and intermediate reaction products, or both). Flowing of the reactor-disposed molten polystyrene material through the reactor-disposed molten polystyrene material-permeable container effects contacting between the catalyst material and the reactor-disposed molten polystyrene material, for effecting the at least partial depolymerization of at least a fraction of the reactor-disposed molten polystyrene material. In this respect, the flowing reactor-disposed molten polystyrene material permeates through the catalyst material within the container, and while permeating through the catalyst material, contacts the catalyst material contained within the container, for effecting the at least partial depolymerization of at least a fraction of the reactor-disposed molten polystyrene material. In both System1000and System2000a first reactor is assembled from the reactor modules. The first reactor has a first reaction zone and includes a total number of “P” reactor modules from “N” reactor modules, wherein “N” is a whole number that is greater than or equal to one. Each one of the “N” reactor modules defines a respective module reaction zone including a catalyst material disposed therein, and is configured for conducting a flow of reactor-disposed molten polystyrene material through the respective module reaction zone, such that, flowing of the reactor-disposed molten polystyrene material through the respective module reaction zone brings it into contact with the catalyst material, thereby causing at least partial depolymerization of at least a fraction of the flowing reactor-disposed molten polystyrene material. In this respect, the first reaction zone includes “P” module reaction zones. When “N” is a whole number that is greater than or equal to two, each one of the “N” reactor modules is configured for connection, in series, to one or more of the other “N” reactor modules such that a plurality of reactor modules are connected to one another, in series, and includes a plurality of module reaction zones that are disposed in fluid communication within one another, in series, such that the total number of module reaction zones correspond to the total number of connected reactor modules. The plurality of connected reactor modules is configured for conducting a flow of reactor-disposed molten polystyrene material through the plurality of module reaction zones, such that it comes into contact with the catalyst material, thereby effecting at least partial depolymerization of at least a fraction of the flowing reactor-disposed molten polystyrene material. When “P” is a whole number that is greater than or equal to two, the assembling of the first reactor includes connecting the “P” reactor modules to one another, in series, such that “P” reaction zones are disposed in fluid communication with one another in series. In the embodiment illustrated inFIG.2, “P” is equal to five, such that reactor100includes five reactor modules102athrough102e, the reaction zone consisting of five module reaction zones104athrough104e, each one respective to a one of the five reactor modules. It is understood that “P” can be more or less than five. In another embodiment illustrated inFIG.5, “P” is equal to two, such that reactor600includes two reactor modules: inlet reactor module300and outlet reactor module400. Molten polystyrene material, for supplying to the constructed reactor, is generated by heating a polystyrene material. In some embodiments, the heating is caused by a heater. InFIG.2the heating is caused by a combination of extruder106and separate heater108. InFIG.5the heating is caused by a combination of extruder606and separate heater608. In such embodiments, the generated molten polystyrene material is forced from the extruder, flowed through a separate heater, and then supplied to the module reaction zone. In some embodiments, the extruders are configured to supply sufficient heat to the polystyrene material such that the generated molten polystyrene material is at a sufficiently high temperature for supply to the reactor, and a separate heater is not required. InFIG.2, pump110receives molten polystyrene material from extruder106and effects transport (or “flowing”) of the molten polystyrene material through heater108, and then through the first reaction zone. In some embodiments, extruder106is configured to impart sufficient force to affect the desired flow of the generated molten polystyrene material, such that pump110is optional.FIG.5shows an example without a pump. In some embodiments, the molten polystyrene material is derived from a polystyrene material feed that is heated to effected generation of the molten polystyrene material. In some embodiments, the polystyrene material feed includes primary virgin granules of polystyrene. The virgin granules can include various molecular weights and melt flows. In some embodiments, the polystyrene material feed includes waste polystyrene material feed. Suitable waste polystyrene material feeds include mixed polystyrene waste such as expanded or extruded foam, and ridged products. e.g. foam food containers, or packaging products. The mixed polystyrene waste can include various melt flows and molecular weights. In some embodiments, the waste polystyrene material feed includes up to 25% of material that is other than polystyrene material, based on the total weight of the waste polystyrene material feed. The molten polystyrene material is supplied to the reactor, and the molten polystyrene material is flowed through the first reaction zone (i.e. including the “P” reaction zones) as reactor-disposed molten polystyrene material. The flowing of the reactor-disposed molten polystyrene material through the first reaction zone brings it into contact with the catalyst material generating a molten product material, including a depolymerization product material (and, in some embodiments, also includes unreacted molten polystyrene material and/or intermediate reaction products). The molten product material is collected. In some embodiments, the catalyst material includes [Fe—Cu—Mo—P]/Al2O3. The catalyst is prepared by binding a ferrous-copper complex to an alumina support and reacting it with an acid comprising metals and non-metals to obtain the catalyst material. Other suitable catalyst materials include zeolite, mesoporous silica, H-mordenite and alumina. The system can also be run in the absence of a catalyst and produces lower molecular weight polymer through thermal degradation. The generated molten product material is discharged from and collected/recovered from the reactor. In some embodiments, the collection of the molten product material is effected by discharging a flow of the molten product material from the reactor. In those embodiments with a plurality of reactor modules, the molten product material is discharged from the first reactor module and supplied to the next reactor module in the series for effecting further depolymerization within the next reactor module in the series, and this continues as-between each adjacent pair of reactor modules in the series. In some embodiments, the generated depolymerization product material includes solvent or monomer (Styrene), polyaromatic solvents, oils and/or greases, and/or lower molecular weight functionalized polymer i.e. increased olefin content. Commercially available greases are generally made by mixing grease base stocks with small amounts of specific additives to provide them with desired physical properties. Generally, greases include four types: (a) admixture of mineral oils and solid lubricants; (b) blends of residuum (residual material that remains after the distillation of petroleum hydrocarbons), uncombined fats, rosin oils, and pitches; (c) soap thickened mineral oils; and (d) synthetic greases, such as poly-alpha olefins and silicones. In some embodiments, the polymeric feed material is one of, or a combination of, virgin polystyrene and/or any one of, or combinations of post-industrial and/or post-consumer waste polystyrene. It is desirable to convert such polymeric feed material into a lower molecular weight polymers, with increased melt flow and olefin content using an embodiment of the system disclosed herein. In each case, the conversion is effected by heating the polystyrene feed material so as to generate molten polystyrene material, and then contacting the molten polystyrene material with the catalyst material within a reaction zone disposed at a temperature of between 200 degrees Celsius and 400 degrees Celsius, preferable 250-370 degrees Celsius. The molecular weight, polydispersity, glass transition, melt flow, and olefin content that is generated depends on the residence time of the molten polystyrene material within the reaction zone. When operating in a continuous system depending on the flowrate of the extruder or gear pump residence times vary from 5-180 minutes, preferably 20-90 minutes, with more than one reactor modules attached in series. In some of these embodiments, the supply and heating of the polystyrene feed material is effected by a combination of an extruder and a pump, wherein the material discharged from the extruder is supplied to the pump. In some of these embodiments, extruder106is a 10 HP, 1.5-inch (3.81 cm) Cincinnati Milacron Pedestal Extruder, Model Apex 1.5, and the pump110is sized at 1.5 HP for a 1.5-inch (3.81 cm) line. Pressure transducer640monitors for plugs within the extruder (as well as prior to pressure transducer642, see below) for maintaining system pressure below a maximum pressure (for example, maximum design pressure of the reactor100). Likewise, pressure transducer642monitors for plugs elsewhere within the system. Over-pressure is controlled by feedback from the pressure transmitted by640and642to a controller which transmits a command signal to shut down the extruder106and the pump110, and thereby prevent the pressure from further increasing. In some embodiments, reactor100is first reactor100, and the reaction zone of the first reactor is a first reaction zone, and the flowing of the molten polystyrene material, through the first reaction zone, is suspended (such as, for example, discontinued). After the suspending, the first reactor is modified When “P” is equal to one, the modifying includes connecting a total number of “R” of the “N−1” reactor modules, which have not been used in the assembly of the first reactor, to the first reactor, wherein “R” is a whole number from 1 to “N−1”, such that another reactor is created and includes a total number of “R+1” reactor modules that are connected to one another, in series, and such that the another reactor includes a second reaction zone including “R+1” module reaction zones. Another reactor is configured to conduct a flow of molten polystyrene material, such that flowing of the reactor-disposed molten polystyrene material through the second reaction zone effects generation of another depolymerization product material and its discharge from another reactor; When “P” is a whole number that is greater than or equal to two, but less than or equal to “N−1”, the modifying includes either one of:(a) removing a total number of “Q” of the “P” reactor modules from the first reactor, wherein “Q” is a whole number from one to “P−1”, such that another reactor is created and includes a total number of “P−Q” reactor modules that are connected to one another, in series, and such that the another reactor includes a second reaction zone including “P−Q” module reaction zones, wherein the another reactor is configured to conduct a flow of molten polystyrene material, such that flowing of the reactor-disposed molten polystyrene material through the second reaction zone effects of generation of another depolymerization product material and its discharge from the another reactor, or(b) connecting a total number of “R” of the “N−P” reactor modules, which have not been used in the assembly of the first reactor, to the first reactor, wherein “R” is a whole number from 1 to “N−P”, such that another reactor is created and includes a total number of “P+R” reactor modules that are connected to one another, in series, and also includes a second reaction zone including “P+R” module reaction zones, wherein another reactor is configured to conduct a flow of molten polystyrene material, such that flowing of the reactor-disposed molten polystyrene material through the second reaction zone effects generation of another depolymerization product material and its discharge from another reactor; When “P” is equal to “N”, the modifying includes removing a total number of “Q” of the “P” reactor modules from the first reactor, wherein “Q” is a whole number from one to “P−1”, such that another reactor is created and includes a total number of “P−Q” reactor modules that are connected to one another, in series, and such that another reactor includes a second reaction zone, including “P−Q” module reaction zones. Another reactor is configured to conduct a flow of molten polystyrene material, such that flowing of the reactor-disposed molten polystyrene material through the second reaction zone effects generation of another depolymerization product material and its discharge from another reactor. In some embodiments, after the modifying of the first reactor to effect creation of another reactor (by either one of connecting/adding or removing reactor modules), another reactor is used to generate a second depolymerization product material. In this respect, polystyrene material is heated to generate a molten polystyrene material, and the molten polystyrene material is flowed through the second reaction zone, to effect generation of a second depolymerization product material. The second depolymerization product material is then collected from the reactor. In some embodiments, the same catalyst material is disposed within each one of the “N” reactor modules. In some embodiments, the reaction zone of each one of the “N” reactor modules is the same or substantially the same. Referring toFIGS.6-14, in at least some embodiments, each reactor modules200includes pipe spool201. In some embodiments, reactor module200includes pipe spool201with opposite first and second ends (only one is shown in the illustrated embodiment), with flanges230at each end, for facilitating connection with other reactor module(s)200. Reactor module200includes inlet202A at a first end of the spool, outlet202B at the opposite second end of the spool, and fluid passage206extending between inlet202A and outlet202B. Fluid passage206includes a catalyst material-containing space that is disposed within the reactor-disposed molten polystyrene material-permeable container, with catalyst material204disposed within catalyst material-containing space216. Catalyst material-containing space216defines module reaction zone205of reactor module200. Reactor module200is configured for receiving reactor-disposed molten polystyrene material by inlet202A and conducting the received molten polystyrene material through fluid passage206such that it is brought into contact with catalyst material204. This causes at least partial depolymerization of at least a fraction of the molten polystyrene material such that molten product material, including depolymerization reaction products (and, in some embodiments, unreacted molten polystyrene material and/or intermediate reaction products (such as partially depolymerized material)), are produced. Reactor module200then discharges the molten product material from outlet202B. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in Reactor module200. Grating can take place, among other places, in the reactor, in line with the stream after cooling, and/or in a separate vessel. Relatively unobstructed fluid passage portion218of fluid passage206extends between inlet202A and outlet202B, and is disposed in fluid communication with catalyst material-containing space216via a wire screen. Wire screen208is disposed within pipe spool201, segmenting fluid passage206into relatively unobstructed fluid passage portion218and catalyst material comprising space204. Wire screen208contains catalyst material204within catalyst material-containing space216, and thereby defines molten polystyrene material-permeable container203. Wire screen208is disposed in spaced apart relationship relative to fluid passage-defining internal wall210of pipe spool201, and extends longitudinally through the length of pipe spool201. The space between wire screen208and internal wall210defines relatively unobstructed fluid passage portion218of fluid passage206. Fluid communication between fluid passage portion218and catalyst material-containing space216is made possible via spaces within wire screen208. Thus wire screen208permits permeation of the molten polystyrene material from relatively unobstructed fluid passage portion218to catalyst material-containing space216(and thereby facilitates contact of the molten polystyrene material with catalyst material204within the reaction zone), and also from catalyst material-containing space216to relatively unobstructed fluid passage portion218(for discharging the molten product material including the depolymerization reaction products and unreacted molten polystyrene material and/or intermediate reaction products), while preventing, or substantially preventing, egress of catalyst material204from catalyst material-containing space216to relatively unobstructed fluid passage portion218. In some embodiments, pipe spool201is cylindrical, and wire screen208is also cylindrical and is nested within pipe spool201, such that relatively unobstructed fluid passage portion218is defined within the annular space between internal wall210of pipe spool201and wire screen208, and catalyst material-containing space216is disposed within wire screen208. In these embodiments, the catalyst material-containing fluid passage portion216is radially spaced outwardly, relative to relatively unobstructed fluid passage portion218, from the axis of pipe spool201. In some embodiments, spacer tube214extends through the space defined by wire screen208and encourages flow of the reactor-disposed molten polystyrene material to the portions of pipe spool201that are in close disposition to a heat transfer element (see below). This embodiment helps maintain the reactor-disposed molten polystyrene material at a desired temperature. Also, by occupying space, spacer tube214effectively reduces the volume of module reaction zone205, thereby increasing the velocity of the flowing reactor-disposed molten polystyrene material. In some embodiments, spacer tube214extends longitudinally through the length of pipe spool201. In some embodiments, catalyst material-containing space216is defined within the annular space between spacer tube214and wire screen208. Reactor-disposed molten polystyrene material is received by inlet202A at the first end of pipe spool201, and, while traversing pipe spool201, via fluid passage206, to the opposite end, is conductible, across wire screen208, between relatively unobstructed fluid passage portion218and catalyst material-containing space216. This produces a molten product material, including depolymerization reaction products (and, in some embodiments, unreacted molten polystyrene material and/or intermediate reaction products), that is discharged via outlet202B at the opposite second end of pipe spool201. While being conducted through catalyst-material containing space216, the reactor-disposed molten polystyrene material is brought into contact with catalyst material204causing at least partial depolymerization of at least a fraction of the reactor-disposed molten polystyrene material. Referring toFIGS.6and14, in some embodiments, baffles222,223are disposed within relatively unobstructed fluid passage portion218. In some embodiments, baffle222is welded to end cap212aand is in the form of a resilient wire that is wrapped about wire screen208. In some embodiments, baffle223is in the form of a resilient wire that is pressed through the space between pipe spool201and wire screen208, welded to end cap212a, and biased against interior wall210of spool201. Baffles222,223encourage mixing of the flowing reactor-disposed molten polystyrene material and promote uniform distribution of heat and mitigate charring of the reactor-disposed molten polystyrene material, which could result in depositing of solid organic material on the structures defining fluid passage206and contribute to fouling. Baffles222,223also encourages flow of reactor-disposed molten polystyrene material from the relatively unobstructed fluid passage portion218towards catalyst material-containing space216and increase contact between the reactor-disposed molten polystyrene material and catalyst material204. Referring toFIGS.9-13, end cap assembly211is provided, and mounted within the interior space of pipe spool201. End cap assembly211includes rigid end caps212aand212b, wire screen208, and spacer tube214. End cap212ais disposed proximate to one end of pipe spool201, and end cap212bis disposed proximate to an opposite end of pipe spool201. In some embodiments, end caps212aand212bare also permeable to flow of reactor-disposed molten polystyrene material. Wire screen208is disposed between end caps212aand212b, and its axial positioning within pipe spool201, relative to pipe spool201, is determined by end caps212aand212b. One end of wire screen208is welded to end cap212a, while the opposite end of wire screen208is disposed within a recess formed in end cap212b, such that catalyst material-containing space216, within which catalyst material204is contained, is defined within the space bounded by wire screen208and end caps212aand212b. Spacer tube214is disposed between end caps212aand212b. One end of spacer tube214is welded to end cap212a, while the opposite end of spacer tube214is disposed within a recess formed in end cap212b. Referring toFIGS.11and12, end cap212ais welded to pipe spool201, for effecting connection of end cap assembly211to pipe spool201. In this respect, end cap212aincludes a plurality of rigid end cap spacers2120ato2120c, projecting radially outwardly from end cap integrator2122(to which wire screen208and spacer tube214are welded). End cap spacers2120atocare received within corresponding recess provided within end cap integrator2122. End cap spacers2120ato2120care spaced-apart from one another such that fluid communication allowed between reactor modules200that are connected to one another, and, specifically between reaction zones of connected reactor modules200. End cap spacers2120ato2120ccan be welded to the interior of pipe spool201, thereby determining the position of end cap212arelative to pipe spool201, and also determining the axial position of spacer tube214relative to pipe spool201(which is welded to end cap212a). Referring toFIGS.9to11, positioning of end cap212brelative to pipe spool201is determined by disposing of end cap212bin contact engagement with pipe spool201, spacer tube214and by an adjacent piping structure, such as welded end cap212aof another reactor module200, or a conduit. Each one of spacer tube214, and the adjacent piping structure are relatively rigid structures, such that the substantially fixed axial positioning of each one of spacer tube214and the adjacent piping structure, relative to pipe spool201, determines the axial positioning of end cap212brelative to pipe spool201. When reactor module200is assembled, end cap212bis pressed between spacer tube214and the adjacent piping structure (in the embodiment illustrated inFIG.8, the adjacent piping structure is end cap212bof another reactor module200), such that axial positioning of end cap212b, relative to pipe spool201(and, therefore, end cap212ais determined by spacer tube214and the adjacent piping structure. End cap212balso includes rigid end cap spacers2124ato2124c, disposed within corresponding recesses within an end cap integrator2126. The end cap integrator includes recesses which receive spacer tube214and wire screen208. End cap spacers2124ato2124care disposed in contact engagement with the interior wall of pipe spool201. End cap spacers2124ato2124cproject radially outwardly from end cap integrator2126. End cap spacers2124ato2124care spaced apart from one another such that fluid can flow between reactor modules200that are connected to one another, and, specifically between reaction zones of connected reactor modules200. When disposed in contact engagement with the interior wall of pipe spool201, and in co-operation with spacer tube214and the adjacent piping structure, end cap spacers2124ato2124cfunction to substantially fix vertical positioning of end cap212brelative to pipe spool201. By configuring end cap212bsuch that end cap212bis removable from end cap assembly211, repairs and maintenance within the reaction zone including the replacement of catalyst material204, is made easier. Heaters220are disposed in heat transfer communication with fluid passage206so as to effect heating of the reactor-disposed molten polystyrene material that is flowing through fluid passage206. Maintaining the flowing reactor-disposed molten polystyrene material at a sufficient temperature leads to at least partial depolymerization. In some embodiments, heaters220include electric heating bands that are mounted to the external wall of pipe spool201and are configured to supply heat to fluid passage206by heat transfer through pipe spool201. Referring toFIGS.16to18, in some embodiments, reactor includes inlet reactor module300, outlet reactor module400, and, optionally, one or more intermediate reactor modules500. In some embodiments, inlet reactor module300includes pipe spool301, having opposite ends, with respective flange330A,330B provided at each one of the opposite ends, for facilitating connection with an outlet reactor module400, and, in some embodiments, an intermediate reactor module500. Inlet reactor module300includes inlet302A at a first end of pipe spool301, outlet302B at the opposite second end of the spool, and fluid passage306extending between inlet302A and outlet302B. Fluid passage306includes catalyst material-containing space316that is disposed within reactor-disposed molten polystyrene material-permeable container303, with catalyst material304disposed within catalyst material-containing space316. Catalyst material-containing space316defines module reaction zone305of reactor module300. Inlet reactor module300is configured for receiving reactor-disposed molten polystyrene material by inlet302A, conducting the received molten polystyrene material through fluid passage306, and while such conducting is being effected, contacting the molten polystyrene material being conducted with catalyst material304such that at least partial depolymerization of at least a fraction of the molten polystyrene material is effected and such that a molten product material is produced that includes depolymerization reaction products (and, in some embodiments, includes unreacted molten polystyrene material and intermediate reaction products, or both), and discharging the molten product material from outlet302B. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in inlet reactor module300. Fluid passage306includes relatively unobstructed fluid passage portion318and catalyst material-containing fluid passage portion315that includes catalyst material-containing space316. Relatively unobstructed fluid passage portion318extends form inlet302A, and is disposed in fluid communication with catalyst material-containing fluid passage portion315via wire screen308. Catalyst material-containing fluid passage portion315extends into outlet302B. Wire screen308is disposed within pipe spool301, segmenting fluid passage306into relatively unobstructed fluid passage portion318and catalyst material-containing fluid passage portion316. Wire screen308is mounted at one end to, and extends from, the first end of pipe spool301and, in some embodiments, is mounted at an opposite end to spacer tube314(see below). Wire screen308contains catalyst material304within catalyst material-containing space316. Wire screen308is disposed in spaced apart relationship relative to fluid passage-defining internal wall310of pipe spool301, and extends longitudinally through a portion of pipe spool301. The space between wire screen308and internal wall310defines a portion of catalyst material-containing fluid passage portion315and extends longitudinally across a portion of pipe spool301to define a portion of catalyst material-containing space316. In this respect, the relatively unobstructed fluid passage portion318extends longitudinally along, or proximate to, an axis of pipe spool301. In some embodiments, wire screen308is cylindrical in shape, and is nested within pipe spool301. In this respect, in some embodiments, catalyst material-containing fluid passage portion315is radially spaced outwardly, relative to relatively unobstructed fluid passage portion318, from the axis of pipe spool301. Fluid communication between relatively unobstructed fluid passage portion318and catalyst material-containing fluid passage portion315is effected via spaces within the wire screen. In this respect, wire screen308is configured to permit permeation of the molten polystyrene material from relatively unobstructed fluid passage portion318to catalyst material-containing fluid passage portion315(and thereby facilitate contact of the molten polystyrene material with catalyst material304within the reaction zone), while preventing, or substantially preventing, egress of catalyst material304from catalyst material-containing space316to relatively unobstructed fluid passage portion318. In some embodiments, at a downstream end of relatively unobstructed fluid passage portion318, an end wall is tapered to encourage flow of the molten polystyrene material towards the catalyst-material containing space via wire screen308, thereby mitigating pooling of the molten polystyrene material. The catalyst material-containing fluid passage portion315extends into an annular space defined between spacer tube314, which is mounted within pipe spool301, and internal wall310of pipe spool301. By occupying this space, spacer tube314encourages flow of the reactor-disposed molten polystyrene material within catalyst material-containing fluid passage portion315to the portions of pipe spool301that are in close disposition to a heat transfer element, and thereby maintaining the reactor-disposed molten polystyrene material at a desired temperature. Also, by occupying space, spacer tube314effectively reduces the volume of module reaction zone305, thereby increasing the velocity of the flowing reactor-disposed molten polystyrene material. Reactor-disposed molten polystyrene material is received within relatively unobstructed fluid passage portion318via inlet302A at the first end of pipe spool301, and conducted across wire screen308to catalyst material-containing space316of catalyst material-containing fluid passage portion315(see directional arrows340). While being conducted through catalyst material-containing fluid passage portion315(see directional arrows342), the molten polystyrene material becomes contacted with catalyst material304such that depolymerization reaction products are produced, and a molten product material, that includes depolymerization reaction products that are produced within catalyst material-containing fluid passage portion315(and, in some embodiments, also includes unreacted molten polystyrene material and intermediate reaction products, or both), is then subsequently discharged via outlet302B at the second opposite end of pipe spool301. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in catalyst material-containing fluid passage portion315. In some embodiments, outlet reactor module400includes pipe spool401, having opposite ends, with flanges provided at each one of the opposite ends, for facilitating connection with an inlet reactor module300, and, in some embodiments, one an intermediate reactor module disposed between inlet and outlet reactor modules300,400. The outlet reactor module400includes an inlet402A at a first end of pipe spool401, an outlet402B at the opposite second end of the spool, and fluid passage406extending between inlet402A and outlet402B. Fluid passage406includes catalyst material-containing space416that is disposed within reactor-disposed molten polystyrene material-permeable container403, with catalyst material404disposed within catalyst material-containing space416. Catalyst material-containing space416defines module reaction zone405of reactor module400. The outlet reactor module400is configured for receiving reactor-disposed molten polystyrene material by inlet402A, conducting the received molten polystyrene material through fluid passage406, and while such conducting is being effected, contacting the molten polystyrene material being conducted with catalyst material404such that at least partial depolymerization of at least a fraction of the molten polystyrene material is effected and such that a molten product material is produced that includes depolymerization reaction products (and, in some embodiments, also includes unreacted molten polystyrene material and intermediate reaction products, or both), and discharging the molten product material from outlet402B. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in outlet reactor module400. The fluid passage406includes catalyst material-containing fluid passage portion415, which includes catalyst material-containing space416, and a relatively unobstructed fluid passage portion418. Catalyst material-containing fluid passage portion415extends from inlet402A, and is disposed in fluid communication with the relatively unobstructed fluid passage portion418via wire screen408. The relatively unobstructed fluid passage portion418extends into outlet402B. In some embodiments, spacer tube414is mounted within pipe spool401at a first end of pipe spool401, such that the space (such as, for example, the annulus) between pipe spool401and spacer tube414defines a portion of catalyst material-containing fluid passage portion415that is extending from inlet402A. By occupying this space, spacer tube414encourages flow of the reactor-disposed molten polystyrene material within the catalyst material-containing fluid passage portion415to the portions of pipe spool401that are in close disposition to a heat transfer element (see below), and thereby maintaining the reactor-disposed molten polystyrene material at a desired temperature. Also, by occupying space, spacer tube414effectively reduces the volume of module reaction zone405, thereby increasing the velocity of the flowing reactor-disposed molten polystyrene material. The catalyst material-containing fluid passage portion415extends into an annular space defined between internal wall410of pipe spool401and wire screen408. Wire screen408is disposed within pipe spool401, segmenting fluid passage406into catalyst material-containing fluid passage portion415and the relatively unobstructed fluid passage portion418. Wire screen408is mounted at one end to, and extends from, the second end of pipe spool401and is mounted at an opposite end to spacer tube414. Wire screen408contains catalyst material404within catalyst material-containing space416. Wire screen408is disposed in spaced apart relationship relative to fluid passage-defining internal wall410of pipe spool401, and extends longitudinally through a portion of pipe spool401. The space between wire screen408and internal wall410defines a portion of catalyst material-containing fluid passage portion415and extends longitudinally across a portion of pipe spool401. In this respect, the relatively unobstructed fluid passage portion418extends longitudinally along, or proximate to, an axis of pipe spool401, and into outlet402B. In some embodiments, wire screen408is cylindrical in shape, and is nested within pipe spool401. In this respect, in some embodiments, catalyst material-containing fluid passage portion415is radially spaced outwardly, relative to the relatively unobstructed fluid passage portion418, from the axis of pipe spool401. Fluid communication between catalyst material-containing fluid passage portion415and the relatively unobstructed fluid passage portion418is effected via spaces within the wire screen. In this respect, wire screen408is configured to permit permeation of the molten polystyrene material from the relatively unobstructed fluid passage portion418to catalyst material-containing fluid passage portion415(and thereby facilitate the contacting of the molten polystyrene material with catalyst material404within the reaction zone), while preventing, or substantially preventing, egress of catalyst material404from catalyst material-containing space416to the relatively unobstructed fluid passage portion418. Reactor-disposed molten polystyrene material is received within catalyst material-containing fluid passage portion415via inlet402A at the first end of pipe spool401(such as, for example, from outlet302B of reactor module300, or such as, for example, from the outlet of intermediate reactor module500, see below), conducted through catalyst material-containing fluid passage portion415(see directional arrows440). While being conducted through catalyst material-containing fluid passage portion415, the molten polystyrene material becomes contacted with catalyst material404such that a molten product material, that includes depolymerization reaction products (and, in some embodiments, also includes unreacted molten polystyrene material and intermediate reaction products, or both), is produced. The molten product material, including the depolymerization products that are produced within catalyst material-containing fluid passage portion415, are conducted across wire screen408to relatively unobstructed fluid passage portion418(see directional arrows442) and subsequently discharged via outlet402B at the second opposite end of pipe spool401. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in catalyst material-containing fluid passage portion415. In some embodiments, the reactor includes one or more intermediate reactor modules500disposed between inlet and outlet reactor modules300,400. In some embodiments, intermediate reactor module500includes pipe spool501, having opposite ends, with flanges530A,530B provided at each one of the opposite ends, for facilitating connection with a reactor module. The flange at a first end is provided for facilitating connection with either one of inlet reactor module300, or another intermediate reactor module500. The flange at the second end is provided for facilitating connect with either one of outlet reactor module400or another intermediate reactor module500. Pipe spool501includes inlet502A at a first end of pipe spool501, outlet502B at an opposite second end of pipe spool501, and fluid passage506extending between inlet502A and outlet502B. Fluid passage506includes catalyst material-containing space516. Catalyst material-containing space516is disposed within reactor-disposed molten polystyrene material-permeable container503, and catalyst material504is disposed within catalyst material-containing space516. Catalyst material-containing space516defines module reaction zone505of reactor module500. Intermediate reactor module500is configured for receiving reactor-disposed molten polystyrene material by inlet502A, conducting the received molten polystyrene material through fluid passage506, and while such conducting is being effected, contacting the molten polystyrene material being conducted with catalyst material504such that at least partial depolymerization of at least a fraction of the molten polystyrene material is effected and such that a molten product material is produced that includes depolymerization reaction products (and, in some embodiments, also includes unreacted molten polystyrene material and intermediate reaction products, or both), and discharging the molten product material from outlet502B. In some embodiments, various monomers and/or copolymers such as, but not limited to, acids, alcohols, acetates, and alkenes such as hexene can be grafted onto the depolymerized product in intermediate reactor module500. Fluid passage506includes catalyst material-containing fluid passage portion515that includes catalyst material-containing space516. In some embodiments, spacer tube514is mounted within pipe spool501at a first end of pipe spool501, such that the space between pipe spool501and spacer tube514defines catalyst material-containing space516. By occupying this space, the spacer tube encourages flow of the reactor-disposed molten polystyrene material within catalyst material-containing fluid passage portion515to the portions of pipe spool501that are in close disposition to a heat transfer element (see below), and thereby maintaining the reactor-disposed molten polystyrene material at a desired temperature. Also, by occupying space, spacer tube514effectively reduces the volume of module reaction zone505, thereby increasing the velocity of the flowing reactor-disposed molten polystyrene material. FIG.19shows a cross-section side-elevation view of catalytic reactor700awith removable static mixer710configured to be heated via thermal fluid and/or molten salt. Static mixer710provides greater mixing in catalytic reactor700aand can result in the need of a lower operating temperature. In some embodiments static mixer710is removable which allows for easier cleaning and maintenance of reactor700a. Removable static mixer710also allows for repurposing of reactor segments. For example, jacketed reactors can be converted to pre-heat or cooling segments. Thermal fluid and/or molten salt can be heated, among other ways, by natural gas, electric, waste process heats, and coal. In some embodiments thermal fluid and/or molten salt reduces the costs of having to use electric. The tubular configuration of catalytic reactor700aoffers several advantages in addition to those already mentioned above. In particular, use of tubular reactors connected in series allows for dependable and consistent parameters, which allows for a consistent product. Specifically, a consistent flow through the tubular sections creates a much more predictable and narrow range of end products than using a continuous stirred reactor, as the surface area of the catalyst and heat input is maximized. One advantage over continuous stirred reactors is elimination of shortcutting, flow in tubular section hypothetically moves as a plug. Each hypothetical plug spends the same amount of time in the reactor. Tubular catalytic reactors can be operated vertically, horizontally, or at any angle in between. Tubular catalytic reactors (the reactor sections) and the corresponding pre-heat sections and cooling sections (seeFIGS.28-30) can be a universal size (or one of several standard sizes). This allows not only for a consistent flow of the material, but also allows for tubular elements to be designed to be interchangeable among the various section and easily added, removed, cleaned, and repaired. In at least some embodiments the inner faces of the tubular sections are made of 304 or 316 steel. The thermal fluid and/or molten salt can enter jacket720via inlet/outlets730. In some embodiments catalytic reactor700ais configured to be mounted with a thermocouple/pressure transducer (not shown) and includes relevant notches735. Notches735are used to bring the thermocouple/pressure transducer in physical contact with the fluid. In some embodiments the thermocouple/pressure transducer will be mounted in a well, which reduces the material in-between the fluid and the sensor. In some embodiments catalytic reactor700aincludes removable screen760that can hold the catalyst. Removable screen760can be easily replaced overcoming disadvantages associated with packed bed reactors challenging maintenance requirements and resulting downtime. In some embodiments, the standardization of removable screen760results in a consistent product leaving each section and/or allows for standardization across multiple reactors. In other or the same embodiments, catalytic reactor700acan include removable adaptor740with cut-outs for static mixer supports. Static mixer supports reduce the force on static mixers710allowing for more forceful/rapid removal. The cut-outs of adaptor740improve the seal between the adapter and the screens. Catalytic reactor700acan include flanges750on one or both ends to connect catalytic reactor700ato other reactors, extruders or the like. FIG.20is a cross-section side-elevation view of catalytic reactor700bwith removable static mixer710configured to use electric heating. In some embodiments electric heating is preferred over using thermal oil/molten salt as it can be more convenient, requires reduced ancillary equipment such as boilers, heating vessels, high temperature pumps, valves, and fittings, and is easier to operate. Further, in some embodiments, reduce of electric heating reduces the overall footprint of the system. In some embodiments catalytic reactor700bis configured to be mounted with a thermocouple/pressure transducer (not shown) and includes relevant notches735. In some embodiments catalytic reactor700bincludes removable screen760that can hold the catalyst. In other or the same embodiments, catalytic reactor700bcan include removable adaptor740with cut-outs for static mixer supports. Catalytic reactor700bcan include flanges750on one or both ends to connect catalytic reactor700bto other reactors, extruders or the like. FIG.21is a cross-section side-elevation view of catalytic reactor700cwith removable annular insert712configured to be heated via thermal fluid and/or molten salt. Annular insert712can create an annular flow which can lead to improved heat transfer, increases in superficial velocity and can be easier to maintain than an empty reactor. The thermal fluid and/or molten salt can enter jacket720via inlet/outlets730. In some embodiments catalytic reactor700cis configured to be mounted with a thermocouple/pressure transducer (not shown) and includes relevant notches735. In some embodiments catalytic reactor700cincludes removable screen760that can hold the catalyst. In other or the same embodiments, catalytic reactor700ccan include removable adaptor740with cut-outs for removable annular and screen support. Catalytic reactor700ccan include flanges750on one or both ends to connect catalytic reactor700cto other reactors, extruders or the like. FIG.22is a cross-section side-elevation view of catalytic reactor700dwith removable annular712insert configured to use electric heating. In some embodiments catalytic reactor700dis configured to be mounted with a thermocouple/pressure transducer (not shown) and includes relevant notches735. In some embodiments catalytic reactor700dincludes removable screen760that can hold the catalyst. In other or the same embodiments, catalytic reactor700dcan include removable adaptor740with cut-outs for removable annular and screen support. Catalytic reactor700dcan include flanges750on one or both ends to connect catalytic reactor700dto other reactors, extruders or the like. FIG.23is a cross-section side-elevation view of a catalytic reactor700ewith empty internals configured to be heated via thermal fluid and/or molten salt. Having a reactor with empty internals can increases the residence time of a given material spends in reactor700ewhich reduces the number of reactors needed to make a specific product along with the volume of the catalyst that can be used. Reactors with empty internals can also be more economic to manufacture when compared to reactors with static mixers. The thermal fluid and/or molten salt can enter jacket720via inlet/outlets730. In some embodiments catalytic reactor700eis configured to be mounted with a thermocouple/pressure transducer (not shown) and includes relevant notches735. In some embodiments catalytic reactor700eincludes removable screen760that can hold the catalyst. In other or the same embodiments, catalytic reactor700ecan include removable adaptor740with cut-outs for removable screen support. Catalytic reactor700ecan include flanges750on one or both ends to connect catalytic reactor700eto other reactors, extruders or the like. FIG.24is a cross-section side-elevation view of catalytic700freactor with empty internals configured to use electric heating. In some embodiments catalytic reactor700fis configured to be mounted with a thermocouple/pressure transducer (not shown) and includes relevant notches735. In some embodiments catalytic reactor700fincludes removable screen760that can hold the catalyst. In other or the same embodiments, catalytic reactor700fcan include removable adaptor740with cut-outs for screen support. Catalytic reactor700fcan include flanges750on one or both ends to connect catalytic reactor700fto other reactors, extruders or the like. FIG.25is a cross-section front-elevation view of a group of catalytic reactors700like the one shown inFIG.19arranged in parallel. Parallel arrangements such as that shown inFIG.25allow for the total rate of production to be more readily increase/decreased as desired with minimal changes to the overall arrangement and allow multiple different levels of depolymerisation to occur at once. Housing800allows catalytic reactors700to be bathed in thermal oil/molten salt which is often more effective than electric. The thermal oil/molten salt is held in chamber780. In some embodiments flange770allows for multiple housings to be joined together. FIG.26is a cross-section side-elevation view of the parallel catalytic reactor arrangement ofFIG.25show in a horizontal configuration. Parallel arrangement allows for higher flowrate units to be built with smaller pressure drops that could cause issues as compared to single tube arrangements. Horizontal configurations are often more convenient to operate/maintain. FIG.27is a cross-section side-elevation view of the parallel catalytic reactor arrangement ofFIG.25show in a vertical configuration. Vertical configurations can reduce stratification of liquid/gas products, and can eliminate need for static mixers. FIG.28is a cross-section side-elevation view of vertical helical internal catalytic reactor arrangement900A with two reactors700alike the one shown inFIG.19connected in series. Horizontal helical mixer pre-heat section820is connected to one reactor700a. Helical mixers can lead to better mixing by avoiding stagnancies and hot spots. Helical mixer cooling segment830is shown connected to the other reactor700aat a 45-degree decline. The decline allows for the product to flow via a gravity, while the 45-degree angle allows for sufficient contact between the cooling medium and the product. In the embodiments shown, vertical helical internal catalytic reactor arrangement900A has several inlet/outlets to allow for the use of thermal fluid/molten salt mixtures however other warming techniques (such as, but not limited to, electric heating) can be used as well. FIG.29is a cross-section side-elevation view of a vertical annular catalytic reactor arrangement900B with two reactors700clike the one shown inFIG.21connected in series. FIG.30is a cross-section side-elevation view of a vertical catalytic reactor arrangement900C with two empty reactors700flike the one shown inFIG.23connected in series. FIG.31is a perspective view of horizontal reactor configuration910with internal helical reactor700bconfigured to use electric heaters870like the one shown inFIG.20. InFIG.31the reactor shell has been removed from part of horizontal reactor configuration910to aid in visualizing the location of internal helical reactor700b. While particular elements, embodiments and applications of the present invention have been shown and described, it will be understood, that the invention is not limited thereto since modifications can be made without departing from the scope of the present disclosure, particularly in light of the foregoing teachings. For example, the numerous embodiments demonstrate that different combinations of components are possible within the scope of the claimed invention, and these described embodiments are demonstrative and other combinations of the same or similar components can be employed to achieve substantially the same result in substantially the same way. Further, all of the claims are hereby incorporated by reference into the description of the preferred embodiments. | 73,056 |
11859037 | DETAILED DESCRIPTION OF THE EMBODIMENTS <<Reactive Silicon Group-Containing Polymer>> A reactive silicon group-containing polymer has a reactive silicon group represented by General Formula (1): —Si(R1)3-a(X)a(1) in which R1each independently is a hydrocarbon group having 1 to 20 carbon atoms, the hydrocarbon group as R1may be substituted and may have a hetero-containing group, X is a hydroxyl group or a hydrolyzable group, and a is 1, 2, or 3. Furthermore, in the reactive silicon group-containing polymer, an atom adjacent to the reactive silicon group has an unsaturated bond. In the reactive silicon group-containing polymer, when the atom adjacent to the reactive silicon group has the unsaturated bond, condensation reactivity is significantly increased. Therefore, the reactive silicon group-containing polymer meeting the above-described requirements exhibits excellent rapid curability even when a low-activity catalyst is added. <Reactive Silicon Group> The reactive silicon group in the reactive silicon group-containing polymer is represented by General Formula (1): —Si(R1)3-a(X)a(1) in which R1each independently is a hydrocarbon group having 1 to 20 carbon atoms, the hydrocarbon group as R1may be substituted and may have a hetero-containing group, X is a hydroxyl group or a hydrolyzable group, and a is 1, 2, or 3. R1is a hydrocarbon group having 1 to 20 carbon atoms. The hydrocarbon group as R1has preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, and particularly preferably 1 to 4 carbon atoms. The hetero-containing group that the hydrocarbon group as R1may have as a substituent is a group including a heteroatom. Here, the heteroatom refers to an atom other than a carbon atom and a hydrogen atom. Suitable examples of the heteroatom include N, O, S, P, Si, and a halogen atom. Regarding the hetero-containing group, the total number of carbon atoms and hetero atoms is preferably 1 to 10, more preferably 1 to 6, and further preferably 1 to 4. Suitable examples of the hetero-containing group include a hydroxyl group; a mercapto group; a halogen atom such as Cl, Br, I, and F; a nitro group; a cyano group; an alkoxy group such as a methoxy group, an ethoxy group, an n-propyloxy group, and an isopropyloxy group; an alkylthio group such as a methylthio group, an ethylthio group, an n-propylthio group, and an isopropylthio group; an acyl group such as an acetyl group, a propionyl group, and a butanoyl group; an acyloxy group such as an acetyloxy group, a propionyloxy group, and a butanoyloxy group; a substituted or unsubstituted amino group such as an amino group, a methylamino group, an ethylamino group, a dimethylamino group, and a diethylamino group; a substituted or unsubstituted aminocarbonyl group such as an aminocarbonyl group, a methylaminocarbonyl group, an ethylaminocarbonyl group, a dimethylaminocarbonyl group, and a diethylaminocarbonyl group; a cyano group, and the like. When R1is a hydrocarbon group substituted with a hetero-containing group, R1has the total number of carbon atoms and hetero atoms of preferably 2 to 30, more preferably 2 to 18, further preferably 2 to 10, and particularly preferably 2 to 6. Specific Examples of the hydrocarbon group having 1 to 20 carbon atoms as R1include an alkyl group such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, an n-octyl group, a 2-ethyl-n-hexyl group, an n-nonyl group, an n-decyl group, an n-undecyl group, an n-dodecyl group, an n-tridecyl group, an n-tetradecyl group, an n-pentadecyl group, an n-hexadecyl group, an n-octadecyl group, an n-nonadecyl group, and an n-icosyl group; an alkenyl group such as a vinyl group, a 2-propenyl group, a 3-butenyl group, and a 4-pentenyl group; a cycloalkyl group such as a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group; an aryl group such as a phenyl group, a naphthalen-1-yl group, a naphthalen-2-yl group, a o-phenylphenyl group, a m-phenylphenyl group, and a p-phenylphenyl group; an aralkyl group such as benzyl group, a phenethyl group, a naphthalen-1-ylmethyl group, and a naphthalen-2-ylmethyl group. Groups in which these hydrocarbon groups are substituted with the above-described hetero-containing groups are also preferred as R1. Suitable examples of R1include a hydrogen atom; an alkyl group such as a methyl group and an ethyl group; an alkyl group having a hetero-containing group such as a chloromethyl group and a methoxymethyl group; a cycloalkyl group such as a cyclohexyl group; an aryl group such as a phenyl group; an aralkyl group such as a benzyl group, and the like. R1is preferably a hydrogen atom, a methyl group, a methoxymethyl group, and a chloromethyl group, more preferably a methyl group and a methoxymethyl group, and further preferably a methoxymethyl group. Examples of X include a hydroxyl group, hydrogen, halogen, an alkoxy group, an acyloxy group, a ketoximate group, an amino group, an amide group, an acid amide group, an aminooxy group, a mercapto group, and an alkenyloxy group. Among them, from the viewpoint of being mildly hydrolyzable and easily handleable, an alkoxy group such as a methoxy group and an ethoxy group is more preferred, and a methoxy group and an ethoxy group are particularly preferred. a is 1, 2, or 3. a is preferably 2 or 3. The reactive silicon group is not particularly limited, as long as it is a group represented by the Formula (1). The reactive silicon group represented by Formula (1) is preferably a group represented by General Formula (1-1) below: —Si(R10)3-b(OR11)b(1-1) in which R10each independently is an alkyl group having 1 to 6 carbon atoms, a haloalkyl group having 1 to 6 carbon atoms, an alkoxyalkyl group having 2 to 6 carbon atoms, or an N,N-dialkylaminoalkyl group represented by —R12N(R13)2, R12is a methyl group or an ethyl group; R13is a methyl group or an ethyl group, R11is an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an acyl group having 2 to 6 carbon atoms, and b is 2 or 3. Specific examples of the alkyl group having 1 to 6 carbon atoms as R10include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, and an n-hexyl group. Among them, a methyl group and an ethyl group are preferred. Specific examples of the haloalkyl group having 1 to 6 carbon atoms as R10include a chloromethyl group, a dichloromethyl group, a trichloromethyl group, a bromomethyl group, a dibromomethyl group, a tribromomethyl group, a 2-chloroethyl group, and a 2-bromoethyl group. Among them, a chloromethyl group and a bromomethyl group are preferred, and a chloromethyl group is more preferred. Specific examples of the alkoxyalkyl group having 2 to 6 carbon atoms as R10include a methoxymethyl group, a 2-methoxyethyl group, a 1-methoxyethyl group, an ethoxymethyl group, a 2-ethoxyethyl group, an n-propyloxymethyl group, and a 2-n-propyloxyethyl group. Among them, a methoxymethyl group, a 2-methoxyethyl group, and an ethoxymethyl group are preferred, and a methoxymethyl group is more preferred. Specific examples of the N,N-dialkylaminoalkyl group represented by —R12N(R13)2as R10include an N, N-dimethylaminomethyl group, an N,N-diethylaminomethyl group, a 2-N,N-dimethylaminoethyl group, and a 2-N,N-diethylaminoethyl group. Among them, an N,N-dimethylaminomethyl group and an N,N-diethylaminomethyl group are preferred, and an N,N-diethylaminomethyl group is more preferred. Specific examples of the alkyl group having 1 to 6 carbon atoms as R11include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, and an n-hexyl group. Among them, a methyl group and an ethyl group are preferred. Specific examples of the alkenyl group having 2 to 6 carbon atoms as R11include a vinyl group, a 2-propenyl group, a 3-butenyl group, and a 4-pentenyl group. Among them, a vinyl group and a 2-propenyl group are preferred. Specific examples of the acyl group having 2 to 6 carbon atoms as R11include an acetyl group, a propionyl group, a butanoyl group, and a pentanoyl group. Among them, an acetyl group is preferred. Specific examples of the reactive silicon group include, but are not limited to a trimethoxysilyl group, a triethoxysilyl group, a tris(2-propenyloxy)silyl group, a triacetoxysilyl group, a dimethoxymethylsilyl group, a diethoxymethylsilyl group, a dimethoxyethylsilyl group, a (chloromethyl)dimethoxysilyl group, a (chloromethyl)diethoxysilyl group, a (methoxymethyl)dimethoxysilyl group, a (methoxymethyl)diethoxysilyl group, an (N,N-diethylaminomethyl)dimethoxysilyl group, and an (N,N-diethylaminomethyl)diethoxysilyl group. Among them, a dimethoxymethylsilyl group, a trimethoxysilyl group, a triethoxysilyl group, and a (methoxymethyl)dimethoxysilyl group are preferred since a cured product having a satisfactory mechanical physical property is obtained. From the viewpoint of activity, a trimethoxysilyl group, a (chloromethyl)dimethoxysilyl group, and a (methoxymethyl)dimethoxysilyl group are more preferred, and trimethoxysilyl group and a (methoxymethyl)dimethoxysilyl group are particularly preferred. From the viewpoint of stability, a dimethoxymethylsilyl group and a triethoxysilyl group are more preferred, and a dimethoxymethylsilyl group is particularly preferred. The average number of the reactive silicon groups included per molecule is preferably 0.5 or more, more preferably 1.0 or more, and further preferably 1.2 or more. The upper limit thereof is preferably 4 or less, and more preferably 3 or less. Furthermore, the reactive silicon group-containing polymer has preferably the above-described reactive silicon groups in an average number of more than 0.8 per end, since the desired effects are easily obtained. This enables a sufficient number of the reactive silicon groups to be introduced at the ends, resulting in a cured product having a sufficient cross-linking density and improved strength. Meanwhile, the reactive silicon group may exist in an average number of more than 1.0 per end, but this is not preferred due to reduced flexibility. Therefore, the reactive silicon group preferably exists in an average number of more than 0.8 but 1.0 or less per end. Herein, the term “end” includes a chain end and an adjacent structure thereof in a polymer molecular chain. More specifically, the term may be defined as a group substituted on bonded atoms corresponding to 20% by number, more preferably 10% by number from the end among the bonded atoms constituting the polymer molecular chain. Furthermore, when expressed in terms of the number of the bonded atoms, the term “end site” may be defined as 50 atoms, more preferably 30 atoms from the end of the polymer molecular chain. A method for obtaining the reactive silicon group-containing polymer having the reactive silicon group in the average number of more than 0.8 per end is not particularly limited. Examples of the method include (1) a method in which a polymer having a methallyl group at the end and hydrosilane undergo a hydrosilylation reaction, (2) a method in which a polymer having a hydroxyl group at the end is allowed to react with isocyanatesilane, and (3) a method in which a polymer having an isocyanate group at the end is allowed to react with aminosilane. However, the method (1), in terms of a reduced curing rate and productivity, and methods (2) and (3), in terms of heat resistance, are not able to be used when a complete tin-free system is required because these methods need to use a tin compound. In the reactive silicon group-containing polymer, the atom adjacent to the reactive silicon group has the unsaturated bond. The atom adjacent to the reactive silicon is not particularly limited, but carbon is preferred. The unsaturated bond is not particularly limited, but a carbon-carbon double bond is preferred. A structure of the end site in the reactive silicon group-containing polymer is preferably at least one structure represented by General Formulas (2) to (4) below: in which R4is a divalent linking group, each of two bonds that R4has is bonded to a carbon atom, an oxygen atom, a nitrogen atom, or a sulfur atom in the linking group, and R2and R3each independently is hydrogen, an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 20 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, or a silyl group. R1, X, and a are the same as described above. R4is the divalent linking group. Each of two bonds that R4has is bonded to a carbon atom, an oxygen atom, a nitrogen atom, or a sulfur atom in the linking group. Here, the phrase “each of two bonds that R4has is bonded to a carbon atom, an oxygen atom, a nitrogen atom, or a sulfur atom in the linking group” means that each of two bonds that R4has exists on a carbon atom, an oxygen atom, a nitrogen atom, or a sulfur atom in the linking group. Specific examples of the divalent linking group include —(CH2)n—, —O—(CH2)n—, —S—(CH2)n—, —NR5—(CH2)n—, —O—C(═O)—NR5—(CH2)n—, and —NR5—C(═O)—NR5—(CH2)n—. Among them, —O—(CH2)n—, —O—C(═O)—NR5—(CH2)n—, and —NR5—C(═O)—NR5—(CH2)n— are preferred, and —O—CH2— is more preferred from the viewpoint of availability of raw materials. R5is a hydrogen atom or a hydrocarbon group having 1 to 10 carbon atoms. Examples of the hydrocarbon group as R5include an alkyl group such as a methyl group, an ethyl group, an n-propyl group, and an isopropyl group; an aryl group such as a phenyl group and a naphthyl group; an aralkyl group such as a benzyl group, n is preferably an integer of 0 to 10, more preferably an integer of 0 to 5, further preferably an integer of 0 to 2, particularly preferably 0 or 1, and most preferably 1. R2and R3each independently is hydrogen, an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 20 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, or a silyl group. The alkyl group has preferably 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, and particularly preferably 1 to 4 carbon atoms. The aryl group has preferably 6 to 12 carbon atoms and more preferably 6 to 10 carbon atoms. The aralkyl group has preferably 7 to 12 carbon atoms. Specific examples of R2and R3include hydrogen; an alkyl group such as a methyl group, an ethyl group, and a cyclohexyl; an aryl group such as a phenyl group and a tolyl group; an aralkyl group such as a benzyl group and a phenethyl group; a silyl group such as a trimethylsilyl group. Among them, hydrogen, a methyl group, and a trimethylsilyl group are preferred, and hydrogen and a methyl group are more preferred, and hydrogen is further preferred. The end site of the reactive silicon group-containing polymer is more preferably at least one structure represented by General Formulas (5) to (7) below: In General Formulas (5) to (7) above, R1, X, and a are the same as described above. The end site of the reactive silicon group-containing polymer is more preferably at least one structure represented by General Formulas (5-1) to (7-1) below: In General Formulas (5-1) to (7-1) above, R10, R11, and b are the same as described above. <Main Chain Structure> A main chain structure of the reactive silicon group-containing polymer may be linear or have a branched chain. The main chain backbone of the reactive silicon group-containing polymer is not particularly limited. Polymers having various main chain backbones can be used as the reactive silicon group-containing polymer. Examples of the main chain backbone of the reactive silicon group-containing polymer include an organic polymer, for example, a polyoxyalkylene-based polymer such as polyoxyethylene, polyoxypropylene, polyoxybutylene, polyoxytetramethylene, a polyoxyethylene-polyoxypropylene copolymer, and a polyoxypropylene-polyoxybutylene copolymer; a saturated hydrocarbon-based polymer such as an ethylene-propylene-based copolymer, a copolymer of polyisobutylene, isobutylene with isoprene, etc., a copolymer of polychloroprene, polyisoprene, isoprene or butadiene with acrylonitrile and/or styrene, etc., a copolymer of polybutadiene, isoprene or butadiene with acrylonitrile and styrene, etc., and a hydrogenated polyolefin-based polymer that is obtained by hydrogenating these polyolefin-based polymers; a polyester-based polymer; a (meth)acrylic acid ester-based polymer that is obtained by subjecting a (meth)acrylic acid ester-based monomer to radical polymerization such as ethyl(meth)acrylate, butyl(meth)acrylate, etc., and a vinyl-based polymer such as a polymer that is obtained by subjecting a monomer such as a (meth)acrylic acid-based monomer, vinyl acetate, acrylonitrile, and styrene to radical polymerization; a graft polymer that is obtained by polymerizing a vinyl monomer in the above-described polymer; a polysulfide-based polymer; a polyamide-based polymer; a polycarbonate-based polymer; a diallyl phthalate-based polymer. Each of the above-described polymers may be a polymer in which blocked regions and grafted regions are mixed. Among them, a saturated hydrocarbon-based polymer, a polyoxyalkylene-based polymer, and a (meth)acrylic acid ester-based polymer are preferred from the viewpoints of a relatively low glass transition temperature and excellent cold resistance of a cured product obtained therefrom, and a polyoxyalkylene-based polymer is more preferred. The reactive silicon group-containing polymer may be a polymer having any one main chain backbone of the above-described various main chain backbones or a mixture of polymers having different main chain backbones. Furthermore, the mixture may be a mixture of polymers that are produced separately or a mixture of polymers that are produced simultaneously so as to give any mixed composition. The reactive silicon group-containing polymer has preferably a number average molecular weight in terms of polystyrene as measured by GPC of 3,000 to 100,000, more preferably 3,000 to 50,000, and particularly preferably 3,000 to 30,000. When the number average molecular weight falls within the above-described range, the reactive silicon group is introduced in an appropriate amount. Therefore, a reactive silicon group-containing polymer having an easily handleable viscosity and excellent workability is easily obtained, while keeping production costs within an appropriate range. A molecular weight of the reactive silicon group-containing polymer can also be expressed in an end group-based molecular weight which is determined by directly measuring an end group concentration of a polymer precursor prior to introduction of the reactive silicon group thereinto using a method for measuring a hydroxyl value according to JIS K 1557 and a titration analysis based on the principle of a method for measuring an iodine value as defined in JIS K 0070 in view of the structure of the polymer (the degree of branching defined by a polymerization initiator used). The end group-based molecular weight of the reactive silicon group-containing polymer may also be determined by creating a calibration curve between the number average molecular weight determined by a standard GPC measurement and the end group-based molecular weight of the polymer precursor, and converting the number average molecular weight determined by GPC of the reactive silicon group-containing polymer into the end group-based molecular weight. A molecular weight distribution (Mw/Mn) of the reactive silicon group-containing polymer is not particularly limited. The molecular weight distribution is preferably narrow, preferably less than 2.0, more preferably 1.6 or less, further preferably 1.5 or less, even more preferably 1.4 or less, particularly preferably 1.3 or less, and most preferably 1.2 or less. The molecular weight distribution of the reactive silicon group-containing polymer can be determined from a number average molecular weight and a weight average molecular weight obtained by GPC measurement. <Method for Synthesizing Reactive Silicon Group-Containing Polymer> A method for synthesizing the reactive silicon group-containing polymer will now be described. (Polyoxyalkylene-Based Polymer) When the polyoxyalkylene-based polymer is used as a main chain of the reactive silicon group-containing polymer, a hydroxyl group-terminated polyoxyalkylene-based polymer is obtained by a method in which a hydroxyl group-containing initiator is polymerized with an epoxy compound using a composite metal cyanide complex catalyst such as a zinc hexacyanocobaltate-glyme complex, and then (i) a method in which a carbon-carbon triple bond is introduced into a hydroxyl group in the resultant hydroxyl group-terminated polyoxyalkylene-based polymer and then a silane compound is added to the carbon-carbon triple bond through a hydrosilylation reaction; (ii) a method in which the resultant hydroxyl group-terminated polyoxyalkylene-based polymer is reacted with a compound having a hydroxyl group-reactive group, a reactive silicon group, and a carbon-carbon double bond; and (iii) a method in which the hydroxyl group-terminated polyoxyalkylene-based polymer is reacted with an excess of a polyisocyanate compound to thereby form an isocyanate group-terminated polymer and then reacted with a compound having an isocyanate group-reactive group, a reactive silicon group, and a carbon-carbon double bond are preferably used. Among these methods, the method (i) is more preferred from the viewpoints of simplicity of the reaction, adjustment of an amount of introduction of the reactive silicon group, and stable physical properties of the resultant reactive silicon group-containing polymer. These methods can be used to obtain a reactive silicon group-containing polymer having an introduction rate of a silyl group into an end of the polymer of 80% or more, which is difficult to achieve through hydroxylation of a polymer having an allyl group, i.e., a carbon-carbon double bond at the end. An example of the hydroxyl group-containing initiator includes a compound having one or more hydroxyl groups such as ethylene glycol, propylene glycol, glycerin, pentaerythritol, low-molecular weight polypropylene glycol, polyoxypropylene triol, allyl alcohol, polypropylene monoallylether, and polypropylene monoalkylether. Examples of the epoxy compound include alkylene oxides such as ethylene oxide and propylene oxide; glycidyl ethers such as methyl glycidyl ether and allyl glycidyl ether, and the like. Among them, propylene oxide is preferred. An example of a group having a carbon-carbon triple bond includes an alkynyl group. Furthermore, other unsaturated groups such as a vinyl group, an allyl group, a methallyl group, or the like may be introduced into a terminal hydroxyl group concomitantly with the alkynyl group. As a method for introducing the carbon-carbon triple bond into the terminal hydroxyl group in the method (i), a method in which a terminal hydroxyl group-containing polymer is treated with an alkali metal salt and then is reacted with a halogenated hydrocarbon compound having a carbon-carbon triple bond is preferably used. Examples of the alkali metal salt include sodium hydroxide, sodium alkoxide, potassium hydroxide, potassium alkoxide, lithium hydroxide, lithium alkoxide, cesium hydroxide, and cesium alkoxide. From the viewpoints of easiness of handling and solubility, sodium hydroxide, sodium methoxide, sodium ethoxide, potassium hydroxide, potassium methoxide, and potassium ethoxide are preferred, and sodium methoxide and potassium methoxide are more preferred. From the viewpoint of availability, sodium methoxide is particularly preferred. The alkali metal salt may be used in a dissolved state in a solvent. Examples of the halogenated hydrocarbon compound having a carbon-carbon triple bond used in the method (i) include propargyl chloride, 1-chloro-2-butyne, 4-chloro-1-butyne, 1-chloro-2-octyne, 1-chloro-2-pentyne, 1,4-dichloro-2-butyne, 5-chloro-1-pentyne, 6-chloro-1-hexyne, propargyl bromide, 1-bromo-2-butyne, 4-bromo-1-butyne, 1-bromo-2-octyne, 1-bromo-2-pentyne, 1,4-dibromo-2-butyne, 5-bromo-1-pentyne, 6-bromo-1-hexyne, propargyl iodide, 1-iodo-2-butyne, 4-iodo-1-butyne, 1-iodo-2-octyne, 1-iodo-2-pentyne, 1,4-diiodo-2-butyne, 5-iodo-1-pentyne, and 6-iodo-1-hexyne. Among them, propargyl chloride, propargyl bromide, and propargyl iodide are more preferred. Furthermore, a halogenated hydrocarbon compound having an unsaturated bond other than the halogenated hydrocarbon compound having a carbon-carbon triple bond such as vinyl chloride, allyl chloride, methallyl chloride, vinyl bromide, allyl bromide, methallyl bromide, vinyl iodide, allyl iodide, and methallyl iodide may be used concomitantly with the halogenated hydrocarbon compound having a carbon-carbon triple bond. Examples of a hydrosilane compound used in the method (i) include halogenated silanes such as trichlorosilane, dichloromethylsilane, chlorodimethylsilane, and dichlorophenylsilane; alkoxy silanes such as trimethoxysilane, triethoxysilane, dimethoxymethylsilane, diethoxymethylsilane, dimethoxyphenylsilane, ethyldimethoxysilane, methoxydimethylsilane, ethoxydimethylsilane, (chloromethyl)dimethoxysilane, (chloromethyl)diethoxysilane, (methoxymethyl)dimethoxysilane, (methoxymethyl)diethoxysilane, (N,N-diethylaminomethyl)dimethoxysilane, and (N, N-diethylaminomethyl)diethoxysilane; acyloxy silanes such as diacetoxymethylsilane and diacetoxyphenylsilane; ketoximate silanes such as bis(dimethylketoximate)methylsilane and bis(cyclohexylketoximate)methylsilane; isopropenyloxy silanes (de-acetone form) such as triisopropenyloxy silane. ((Meth)Acrylic Acid Ester-Based Polymer) When the (meth)acrylic acid ester-based polymer is used as a main chain of the reactive silicon group-containing polymer, examples of a method for producing the reactive silicon group-containing polymer include (I) a method in which a compound having a polymerizable unsaturated group and a reactive functional group (e.g., acrylic acid, 2-hydroxyethyl acrylate) is copolymerized with a monomer having (meth)acrylic structure to thereby obtain a polymer, a carbon-carbon triple bond is introduced into any position in the resultant polymer (preferably, an end of a molecular chain thereof), and then a silane compound is added to the carbon-carbon triple bond through a hydrosilylation reaction to give a reaction silicon group; and (II) a method in which a monomer having (meth)acrylic structure is polymerized through a living radical polymerization method such as an atom transfer radical polymerization to thereby obtain a polymer, a carbon-carbon triple bond is introduced into any position in the resultant polymer (preferably, an end of a molecular chain thereof), and then a silane compound is added to the carbon-carbon triple bond through a hydrosilylation reaction to give a reaction silicon group. (Saturated Hydrocarbon-Based Polymer) When the saturated hydrocarbon-based polymer is used as a main chain of the reactive silicon group-containing polymer, an example of a method for producing the reactive silicon group-containing polymer includes a method in which, as a main monomer, an olefin-based compound having 2 to 6 carbon atoms such as ethylene, propylene, 1-butene, and isobutylene is polymerized to thereby obtain a polymer, a carbon-carbon triple bond is introduced into any position in the resultant polymer (preferably, an end of its molecular chain), and then a silane compound is added to the carbon-carbon triple bond through a hydrosilylation reaction to give a reaction silicon group. <<Curable Compositions>> The (A) reactive silicon group-containing polymer as described above (hereinafter, also referred to as an (A) component) is mixed with various additives, as necessary, to thereby obtain a curable composition. The curable composition typically includes a combination of the (A) reactive silicon group-containing polymer and a (B) curing catalyst (hereinafter, also referred to as a (B) component). Examples of additives other than the (B) curing catalyst include a filler, an adhesiveness imparting agent, a plasticizer, an anti-sagging agent, an antioxidant, a photostabilizer, an ultraviolet absorber, a physical property-adjusting agent, an epoxy group-containing compound, a photocurable material, an oxygen-curable material, and other resins than the reactive silicon group-containing polymer. Furthermore, for the purpose of adjustment of physical properties of the curable composition or the cured product, the curable composition may be added with other additives than those described above, as necessary. Examples of such other additives include a tackifying resin, a solvent, a diluent, an epoxy resin, a surface modifier, a blowing agent, a curability adjusting agent, a flame retardant, silicate, a radical inhibitor, a metal deactivator, an antiozonant, a phosphorus-based peroxide decomposer, a lubricant, a pigment, a fungicide, and the like. Each of representative additives will now be described. <(B) Curing Catalyst> For the curable composition, the (B) curing catalyst acting as a silanol condensation catalyst may be used for the purpose of facilitating a hydrolysis/condensation reaction of the reactive silicon group in the reactive silicon group-containing polymer and subjecting the polymer to chain extension or cross-linking. Examples of the (B) curing catalyst include an organotin compound, a metal carboxylate, an amine compound, a carboxylic acid, and an alkoxy metal. Specific examples of the organotin compound include dibutyltin dilaurate, dibutyltin dioctanoate, dibutyltin bis(butyl maleate), dibutyltin diacetate, dibutyltin oxide, dibutyltin bis(acetylacetonate), dioctyltin bis(acetylacetonate), a reaction product of dibutyltin oxide and a silicate compound, a reaction product of dioctyltin oxide and a silicate compound, and a reaction product of dibutyltin oxide and phthalic acid ester. Specific examples of the metal carboxylate include tin carboxylate, bismuth carboxylate, titanium carboxylate, zirconium carboxylate, and iron carboxylate. Furthermore, a salt formed by combining the below-described carboxylic acid and various metals may be used as the metal carboxylate. Specific examples of the amine compound include amines such as octyl amine, 2-ethylhexyl amine, lauryl amine, and stearyl amine; a nitrogen-containing heterocyclic compound such as pyridine, 1,8-diazabicyclo[5,4,0]undecene-7 (DBU) and 1,5-diazabicyclo[4,3,0]nonene-5 (DBN); guanidines such as guanidine, phenylguanidine, and diphenylguanidine; biguanides such as butylbiguanide, 1-o-tolylbiguanide, and 1-phenylbiguanide; an amino group-containing silane coupling agent; a ketimine compound, and the like. Specific examples of the carboxylic acid include acetic acid, propionic acid, butyric acid, 2-ethylhexanoic acid, lauric acid, stearic acid, oleic acid, linoleic acid, neodecanoic acid, and versatic acid. Specific examples of the alkoxy metal include titanium compounds such as tetrabutyl titanate, titanium tetrakis(acetylacetonate), and diisopropoxytitanium bis(ethylacetoacetate); aluminum compounds such as aluminum tris(acetylacetonate) and diisopropoxyaluminium ethyl acetoacetate; and zirconium compounds such as zirconium tetrakis(acetylacetonate). As other (B) curing catalysts, a fluorine anion-containing compound, a photoacid generator, and a photobase generator may be also used. Two or more kinds of different catalysts may also be used in combination as the (B) curing catalyst. The reactive silicon group included in the (A) reactive silicon group-containing polymer is highly active. Therefore, for the curable composition, an amount of the (B) curing catalyst may be decreased, a catalyst having a lower activity may be used as the (B) curing catalyst, or aminosilane, which is an amino group-containing silane coupling agent, may be used as the (B) curing catalyst. Usually, the aminosilane is often added as an adhesiveness imparting agent. Therefore, when the aminosilane is used as the (B) curing catalyst, the curable composition can be produced without using a routinely-used curing catalyst. Therefore, it is more preferred that no other curing catalysts be added. Especially when the reactive silicon group is a trimethoxysilyl group or a methoxymethyldimethoxysilyl group, excellent curability can be achieved even with the use of only aminosilane as the (B) curing catalyst. A typical amount of use of the (B) curing catalyst is preferably 0.001 to 20 parts by weight, more preferably 0.01 to 15 parts by weight, and particularly preferably 0.01 to 10 parts by weight, with respect to 100 parts by weight of the (A) reactive silicon group-containing polymer. When the organotin compound, the metal carboxylate, the amine compound, the carboxylic acid, the alkoxy metal, and an inorganic acid, etc. are used as the (B) curing catalyst, an amount of use of the (B) curing catalyst is preferably 0.001 to 10 parts by weight, more preferably 0.001 to 5 parts by weight, further preferably 0.001 to 1 part by weight, and particularly preferably 0.001 to 0.5 parts by weight, with respect to 100 parts by weight of the (A) reactive silicon group-containing polymer. When the aminosilane, which is an amino group-containing silane coupling agent, is used as the (B) curing catalyst, the amount of use of the (B) curing catalyst is preferably 0.001 to 10 parts by weight and particularly preferably 0.001 to 5 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. An amount of incorporation of the (B) curing catalyst falling within the above-described range enables curing to progress at a sufficiently high rate while maintaining a curing rate at which operation can be easily performed, and achieves satisfactory storage stability of the curing composition. Generally, the reactive silicon group-containing polymer having a trialkoxysilyl group as the reactive silicon group exhibits satisfactory curability when the organotin compound is used as the (B) curing catalyst, while the curability may be deteriorated when the metal carboxylate, the amine compound, the carboxylic acid, the alkoxy metal, and the inorganic acid, etc. are used as the (B) curing catalyst. Furthermore, in general, the reactive silicon group-containing polymer having a methoxymethyldimethoxysilyl group as the reactive silicon group exhibits satisfactory curability when the amine compound is used as the (B) curing catalyst, while the curability is deteriorated when using a less amount of the amine compound. Furthermore, when the organotin compound, the carboxylic acid, the alkoxy metal, and the inorganic acid, etc. are used as the (B) curing catalyst, the curability may be deteriorated. However, in the (A) reactive silicon group-containing polymer which has the reactive silicon group represented by General Formula (1) and in which the atom adjacent to the reactive silicon group has the unsaturated bond, in particular, the (A) reactive silicon group-containing polymer having any of the structures represented by General Formulas (2) to (4), any combination of the reactive silicon group and the (B) curing catalyst is highly active and exhibits satisfactory curability. <Filler> Various fillers may be incorporated into the curing composition. Examples of the filler include heavy calcium carbonate, colloidal calcium carbonate, magnesium carbonate, diatomaceous earth, clay, talc, titanium oxide, fumed silica, precipitated silica, crystalline silica, molten silica, silicic anhydride, hydrated silicic acid, carbon black, ferric oxide, aluminum powder, zinc oxide, active zinc oxide, PVC powder, PMMA powder, glass fibers, and filaments. An amount of use of the filler is preferably 1 to 300 parts by weight, and particularly preferably 10 to 250 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. For the purpose of decreasing weight (density) of the cured product formed using the curing composition, a balloon (hollow filler) such as an organic balloon and an inorganic balloon may be added. The balloon is a spherical filler of which inside is hollow. Examples of a material of the balloon include an inorganic material such as glass, shirasu, and silica, and an organic material such as a phenolic resin, a urea resin, polystyrene, and saran. An amount of use of the balloon is preferably 0.1 to 100 parts by weight, and particularly preferably 1 to 20 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Adhesiveness Imparting Agent> The adhesiveness imparting agent may be added to the curing composition. A silane coupling agent, a reaction product of the silane coupling agent may be added as the adhesiveness imparting agent. Specific examples of the silane coupling agent include amino group-containing silanes such as γ-aminopropyltrimethoxysilane, γ-aminopropylmethyldimethoxysilane, N-β-aminoethyl-γ-aminopropyltrimethoxysilane, N-β-aminoethyl-γ-aminopropylmethyldimethoxysilane, N-phenyl-γ-aminopropyltrimethoxysilane, and (2-aminoethyl)aminomethyltrimethoxysilane; isocyanate group-containing silanes such as γ-isocyanatepropyltrimethoxysilane, γ-isocyanatepropyltriethoxysilane, γ-isocyanatepropylmethyldimetboxysilane, α-isocyanatemethyltrimethoxysilane, and α-isocyanatemethyldimethoxymethylsilane; mercapto group-containing silanes such as γ-mercaptopropyltrimethoxysilane, γ-mercaptopropyltriethoxysilane, and γ-mercaptopropylmethyldimethoxysilane; epoxy group-containing silanes such as γ-glycidoxypropyltrimethoxysilane and β-(3,4-epoxycyclohexyl)ethyltrimethoxysilane. Only one kind of the adhesiveness imparting agent may be used, or two or more kinds thereof may be used as a mixture. Furthermore, reaction products of various silane coupling agents may also be used as the adhesiveness imparting agent. An amount of use of the silane coupling agent is preferably 0.1 to 20 parts by weight, and particularly preferably 0.5 to 10 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. In the curable composition including the (A) reactive silicon group-containing polymer which has the reactive silicon group represented by General Formula (1) and in which the atom adjacent to the reactive silicon group has the unsaturated bond, in particular, the (A) reactive silicon group-containing polymer having any of the structures represented by General Formulas (2) to (4), the amino group-containing silanes (aminosilane) among the above-described silane coupling agents may also be used as the curing catalyst. In this case, the aminosilane is an additive with both functions as the curing catalyst and the adhesiveness imparting agent. <Plasticizer> The plasticizer may be added to the curing composition. Specific examples of the plasticizer include a phthalic acid ester compound such as dibutyl phthalate, diisononyl phthalate (DINP), diheptyl phthalate, di(2-ethylhexyl)phthalate, diisodecyl phthalate (DIDP), and butylbenzyl phthalate; a terephthalic acid ester compound such as bis(2-ethylhexyl)-1,4-benzene dicarboxylate; a non-phthalic acid ester compound such as diisononyl 1,2-cyclohexanedicarboxylate; an aliphatic polyhydric carboxylic acid ester compound such as dioctyl adipate, dioctyl sebacate, dibutyl sebacate, diisodecyl succinate, and acetyl tributyl citrate; an unsaturated fatty acid ester compound such as butyl oleate and methyl acetylricinolate; alkylsulfonic acid phenyl ester; a phosphoric acid ester compound; a trimellitic acid ester compound; chlorinated paraffin; a hydrocarbon-based oil such as alkyldiphenyl and partially hydrogenated terphenyl; a processing oil; an epoxy plasticizer such as an epoxidized soybean oil and benzyl epoxystearate, and the like. A polymeric plasticizer may also be used. Specific examples of the polymeric plasticizer include a vinyl-based polymer; a polyester-based plasticizer; polyethers, for example, polyether polyol having a number average molecular weight of 500 or more such as polyethylene glycol, polypropylene glycol, and the like, derivatives of these polyether polyols in which a hydroxy group is converted into an ester group, an ether group, or the like; polystyrenes; polybutadiene, polybutene, polyisobutylene, butadiene-acrylonitrile, and polychloroprene. An amount of use of the plasticizer is preferably 5 to 150 parts by weight, more preferably 10 to 120 parts by weight, and particularly preferably 20 to 100 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. The use of the plasticizer falling within the above-described range makes it easy to obtain a curable composition from which a cured product having excellent mechanical strength can be formed while providing the desired effect as the plasticizer. The plasticizer may be used singly, or two or more kinds thereof may be used in combination. <Anti-Sagging Agent> For the purpose of preventing sagging and improving workability, the anti-sagging agent may be added to the curing composition, as necessary. The anti-sagging agent is not particularly limited. Examples of the anti-sagging agent include polyamide waxes; hydrogenated castor oil derivatives; metallic soaps such as calcium stearate, aluminum stearate, and barium stearate. The anti-sagging agent may be used singly, or two or more kinds thereof may be used in combination. An amount of use of the anti-sagging agent is preferably 0.1 to 20 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Antioxidant> The antioxidant (age resister) may be used for the curing composition. The antioxidant can be used to increase weather resistance of the cured product. As the antioxidant, a hindered phenolic system, a monophenolic system, a bisphenolic system, and a polyphenolic system may be exemplified. Specific examples of the antioxidant are described in, for example, Japanese Unexamined Patent Application, Publication Nos. H04-283259 and H09-194731. An amount of use of the antioxidant is preferably 0.1 to 10 parts by weight, and particularly preferably 0.2 to 5 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Photostabilizer> The photostabilizer may be used for the curing composition. The photostabilizer can be used to prevent photooxidative degradation of the cured product. As the photostabilizer, a benzotriazole system, a hindered amine system, and a benzoate-based compound, etc. may be exemplified. The hindered amine system is a particularly preferred photostabilizer. An amount of use of the photostabilizer is preferably 0.1 to 10 parts by weight, and particularly preferably 0.2 to 5 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Ultraviolet Absorber> The ultraviolet absorber may be used for the curing composition. The ultraviolet absorber can be used to increase weather resistance on a surface of the cured product. As the ultraviolet absorber, a benzophenone system, a benzotriazole system, a salicylate system, a substituted tolyl system, and a metal chelate-based compound may be exemplified. The benzotriazole system is a particularly preferred ultraviolet absorber. Suitable specific examples of the benzotriazole system ultraviolet absorber include those sold under the trade names TINUVIN P, TINUVIN 213, TINUVIN 234, TINUVIN 326, TINUVIN 327, TINUVIN 328, TINUVIN 329, and TINUVIN 571 (all manufactured by BASF). An amount of use of the ultraviolet absorber is preferably 0.1 to 10 parts by weight, and particularly preferably 0.2 to 5 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Physical Property-Adjusting Agent> The physical property-adjusting agent may be added to the curable composition for adjusting a tensile property of the cured product produced therefrom, as necessary. The physical property-adjusting agent is not particularly limited. Examples of the physical property-adjusting agent include alkylalkoxysilanes such as phenoxytrimethylsilane, methyltrimethoxysilane, dimethyldimethoxysilane, trimethylmethoxysilane, and n-propyltrimethoxysilane; arylalkoxysilanes such as diphenyldimethoxysilane and phenyltrimethoxysilane; alkylisopropenoxysilane such as dimethyldiisopropenoxysilane, methyltriisopropenoxysilane, and γ-glycidoxypropylmethyldiisopropenoxysilane; trialkylsilylborates such as tris(trimethylsilyl)borate and tris(triethylsilyl)borate; silicone varnish; polysiloxanes, and the like. The physical property-adjusting agent can be used to increase hardness, or, conversely, decrease hardness to produce elongation at break of the cured product of the curable composition. The physical property-adjusting agent may be used singly, or two or more kinds thereof may be used in combination. In particular, a compound from which a compound having a monovalent silanol group in a molecule is produced through hydrolysis has an action of decreasing a modulus of the cured product without deteriorating surface tackiness of the cured product. A compound from which trimethylsilanol is produced is particularly preferred. Examples of the compound from which a compound having a monovalent silanol group in a molecule is produced through hydrolysis include a silicon compound from which silane monool is produced through hydrolysis, which is a derivative of alcohol such as hexanol, octanol, phenol, trimethylolpropane, glycerin, pentaerythritol, and sorbitol. An amount of use of the physical property-adjusting agent is preferably 0.1 to 10 parts by weight, and particularly preferably 0.5 to 5 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Epoxy Group-Containing Compound> The epoxy group-containing compound may be used in the curable composition. The epoxy group-containing compound can be used to increase resiliency of the cured product. As the epoxy group-containing compound, epoxidized unsaturated oils, epoxidized unsaturated fatty acid esters, alicyclic epoxy compounds, and compounds represented by epichlorohydrin derivatives, and mixtures thereof may be exemplified. Specific examples thereof include an epoxidized soybean oil, an epoxidized linseed oil, bis(2-ethylhexyl)-4,5-epoxycyclohexan-1,2-dicarboxylate (E-PS), epoxyoctyl stearate, and epoxybutyl stearate. An amount of use of the epoxy compound is preferably 0.5 to 50 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <Photocurable Material> The photocurable material may be used for the curing composition. The photocurable material can be used to form a film of the photocurable material on a surface of the cured product to thereby improve tackiness and weather resistance of the cured product. Many materials such as organic monomers, oligomers, resins, or compositions containing them are known as this kind of material. As representative photocurable materials, an unsaturated acrylic system compound, which is a monomer, oligomer, or mixtures thereof having one or several acrylic-based or methacrylic-based unsaturated groups; poly(vinyl cinnamate); or an azide resin can be used. An amount of use of the photocurable material is preferably 0.1 to 20 parts by weight, and more preferably 0.5 to 10 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. The use of the photocurable material falling within the above-described range makes it easy to obtain a curable composition from which a cured product having excellent weather resistance, being flexible, and being difficult to crack can be formed. <Oxygen-Curable Material> The oxygen-curable material may be used for the curing composition. As the oxygen-curable material, an unsaturated compound which can react with oxygen in the air may be exemplified. The oxygen-curable material is reacted with oxygen in the air to thereby form a cured film in the vicinity of a surface of the cured product, resulting in actions of, for example, preventing the surface from being tacky and dirt or dust from adhering onto the surface of the cured product. Specific examples of the oxygen-curable material include a drying oil as represented by a tung oil and a linseed oil, and various alkyd resins that are obtained by modifying the compound; those obtained by modifying a resin such as an acryl-based polymer, an epoxy-based resin, and a silicon resin with the drying oil; a liquid polymer such as 1,2-polybutadiene, 1,4-polybutadiene, and a C5 to C8 diene polymer obtained by polymerizing or copolymerizing a diene-based compound such as butadiene, chloroprene, isoprene, and 1,3-pentadiene, and the like. These may be used singly, or two or more kinds thereof may be used in combination. An amount of use of the oxygen-curable material is preferably 0.1 to 20 parts by weight, and more preferably 0.5 to 10 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. The amount of use of the oxygen-curable material falling within the above-described range makes it easy to obtain a sufficient contamination-improving effect and difficult to impair, for example, a tensile property of the cured product. As described in Japanese Unexamined Patent Application, Publication No. H03-160053, the oxygen-curable material is preferably used in combination with the photocurable material. <<Preparation of Curable Compositions>> The above-described curable composition can be prepared as one-component type of which components are all combined in advance, sealed, and stored and which is cured with moisture in the air after construction. Furthermore, the curable composition can also be prepared as two-component type which includes a combined material, serving as a curing agent, and a polymer composition to be mixed with each other before use, the combined material being prepared separately from the polymer composition by combining components such as the (B) curing catalyst, the filler, the plasticizer, and water. From the viewpoint of workability, the one-component type is preferred. In the case of the one-component type curable composition, since components thereof are all combined in advance, it is preferable that a moisture-containing component be previously dehydrated and dried before use or dehydrated under reduced pressure during combining and kneading. Furthermore, in addition to the dehydration and drying method, addition of, as a dehydrating agent, a silicon compound which may be reactive with water such as n-propyltrimethoxysilane, vinyltrimethoxysilane, vinylmethyldimethoxysilane, γ-mercaptopropylmethyldimethoxysilane, γ-mercaptopropylmethyldiethoxysilane, and γ-glycidoxypropyltrimethoxysilane further improves storage stability. An amount of use of the dehydrating agent, in particular, the silicon compound which may be reactive with water such as vinyltrimethoxysilane is preferably 0.1 to 20 parts by weight and more preferably 0.5 to 10 parts by weight, with respect to 100 parts by weight of the reactive silicon group-containing polymer. <<Method for Producing Cured Product>> The above-described curable composition is shaped into the desired shape prior to curing, by means of a method such as application, casting, or filling. The curable composition which has been shaped into the desired shape through application, casting, or filling is cured under the desired environment, for example, normal temperature and normal humidity. The above-described curable composition including the (A) reactive silicon group-containing polymer which has the reactive silicon group represented by General Formula (1) and in which the atom adjacent to the reactive silicon group has the unsaturated bond, in particular, the (A) reactive silicon group-containing polymer having any of the structures represented by General Formulas (2) to (4) is able to be cured in a much shorter time than that for a curable composition including a conventionally known reactive silicon group-containing polymer. <<Application>> The above-described curable composition can be used for a sticker; a sealing material for sealing work on buildings, ships, automobiles, roads, or the like; a molding agent; an adhesive; paint; and a spraying agent. Furthermore, the cured product of the above-described curable composition is suitably used as, for example, a waterproofing material, a coating film waterproofing material, a vibration-proofing material, a damping material, an acoustic insulating material, and a foamed material. Since the cured product obtained by curing the curable composition has excellent flexibility and adhesiveness, the curable composition is more preferably used as the sealing material or the adhesive, among the above-described applications. EXAMPLES One or more embodiments will now be specifically described with reference to Examples. One or more embodiments are not limited to Examples in any way. A number average molecular weight in Examples is a GPC molecular weight measured under the following conditions.Liquid feeding system: HLC-8120GPC manufactured by Tosoh Corporation.Column: TSK-GEL H type manufactured by Tosoh Corporation.Solvent: THEMolecular weight: in terms of polystyreneMeasurement temperature: 40° C. An end group-based molecular weight in Examples is a molecular weight determined using a hydroxyl value determined by the measurement method according to JIS K 1557 and an iodine value determined by the measurement method according to JIS K 0070, in view of the structure of an organic polymer (the degree of branching determined by a polymerization initiator used). An average introduction number of carbon-carbon unsaturated bonds per end of a polymer (Q) described in Examples was calculated according to the following expression: (Average introduction number)=[Iodine value of Polymer (Q)−Iodine value of Polymer precursor (P)]/[Hydroxyl value of Polymer precursor (P)]. An average introduction number of silyl groups per end of polymers (A) and (B) described in Examples was calculated by NMR measurement. Synthesis Example 1 Propylene oxide was polymerized using polyoxypropylene glycol having a number average molecular weight of about 2,000, serving as an initiator, and a zinc hexacyanocobaltate-glyme complex catalyst to thereby obtain polyoxypropylene (P-I) having hydroxyl groups at both ends and having a number average molecular weight of 27,900 (end group-based molecular weight: 17,700) and a molecular weight distribution Mw/Mn of 1.21. To the hydroxyl groups in the resultant hydroxyl group-terminated polyoxypropylene (P-I), was added 1.05 mol equivalents of sodium methoxide as a 28% methanol solution. The methanol was distilled off by vacuum devolatilization, and then 1.16 mol equivalents of propargyl bromide was further added to the hydroxyl groups in the polymer (P-I) to thereby convert the terminal hydroxyl groups into propargyl groups. Unreacted propargyl bromide was removed by devolatilization under reduced pressure. The resultant crude propargyl group-terminated polyoxypropylene was mixed and stirred with n-hexane and water, and then the water was removed by centrifugation and the hexane was devolatilized under reduced pressure from the resultant hexane solution to thereby remove a metal salt in the polymer. Thus, polyoxypropylene (Q-I) having propargyl groups at end sites was obtained. To 500 g of the polymer (Q-I), were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 7.5 g of dimethoxymethylsilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted dimethoxymethylsilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (A-I) having dimethoxymethylsilyl groups at ends and having a number average molecular weight of 28,500. The polymer (A-I) was found to have on average 1.0 dimethoxymethylsilyl group per end and on average 2.0 dimethoxymethylsilyl groups per molecule. Synthesis Example 2 To the hydroxyl groups in the hydroxyl group-terminated polyoxypropylene (P-I) obtained from Synthesis Example 1, was added 1.2 mol equivalents of sodium methoxide as a 28% methanol solution. The methanol was distilled off by vacuum devolatilization, and then 1.5 mol equivalents of allyl chloride was further added to hydroxyl groups in a polymer (P-I) to thereby convert the terminal hydroxyl groups into allyl groups. Hereafter, purification manipulation was performed in the same manner as in Synthesis Example 1. Thus, polyoxypropylene (Q-II) having allyl groups at end sites was obtained. To 500 g of the polymer (Q-II), were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 4.8 g of dimethoxymethylsilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted dimethoxymethylsilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (B-II) having dimethoxymethylsilyl groups at ends and having a number average molecular weight of 28,500. The polymer (B-II) was found to have on average 0.8 dimethoxymethylsilyl group per end and on average 1.6 dimethoxymethylsilyl groups per molecule. Synthesis Example 3 To the hydroxyl groups in the hydroxyl group-terminated polyoxypropylene (P-I) obtained from Synthesis Example 1, was added 1.0 mol equivalent of sodium methoxide as a 28% methanol solution. The methanol was distilled off by vacuum devolatilization, and then 1.0 mol equivalent of allyl glycidyl ether was added to the hydroxyl groups in the polymer (P-I) to thereby allow them to react. Thereafter, 0.28 mol equivalents of a sodium methoxide solution in methanol was added thereto, methanol was removed, and 1.79 mol equivalents of allyl chloride was further added thereto to thereby convert the terminal hydroxyl groups into allyl groups. Hereafter, purification manipulation was performed in the same manner as in Synthesis Example 1. Thus, polyoxypropylene (Q-III) having two or more carbon-carbon unsaturated bonds at an end structure thereof was obtained. In the polymer (Q-III), it was found that on average 2.0 carbon-carbon unsaturated bonds per end site were introduced. To 500 g of the resultant polyoxypropylene (Q-III) having on average 2.0 carbon-carbon unsaturated bonds per end site, were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 9.6 g of dimethoxymethylsilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted dimethoxymethylsilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (B-II) having two or more dimethoxymethylsilyl groups at an end structure thereof and having a number average molecular weight of about 28,500. The polymer (B-II) was found to have on average 1.7 dimethoxymethylsilyl group per end and on average 3.4 dimethoxymethylsilyl groups per molecule. Example 1, and Comparative Examples 1 to 2 One hundred parts by weight of the polymer (A-I), (B-I), or (B-II) described in Table 1 was mixed with 55 parts by weight of DINP (manufactured by J-PLUS Co., Ltd.: diisononyl phthalate), 120 parts by weight of HAKUENKA CCR (manufactured by SHIRAISHI CALCIUM KAISHA, LTD.: precipitated calcium carbonate), 20 parts by weight of TIPAQUE R820 (manufactured by ISHIHARA SANGYO KAISHA, LTD.: titanium oxide), 2 parts by weight of DISPARLON 6500 (manufactured by Kusumoto Chemicals, Ltd.: fatty acid amide wax), 1 part by weight of TINUVIN 770 (manufactured by BASF: bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate), and 1 part by weight of TINUVIN 326 (manufactured by BASF: 2-(3-tert-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole), uniformly dispersed with three rolls, and dehydrated under reduced pressure at 120° C. for 2 hours. Thereafter, the resultant was cooled to 50° C., and then added and mixed with 2 parts by weight of A-171 (manufactured by Momentive: vinyltrimethoxysilane), 3 parts by weight of A-1120 (manufactured by Momentive: N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane), and 2 parts by weight of U-220H (manufactured by Nitto Kasei Co., Ltd.: dibutyltin bisacetylacetonate), and then sealed in a damp-proof cartridge under a condition substantially free from moisture. (Dumbbell Tensile Physical Property) The resultant composition was filled into a mold and cured at 23° C. and 50% RH for 3 days and further at 50° C. for 4 days to thereby produce a sheet-shaped cured product having a thickness of about 3 mm. The sheet-shaped cured product was punched out into the Dumbbell type No. 3, which was subjected to a tensile strength test at 23° C. and 50% RH to thereby measure a modulus at 100% elongation and a strength and elongation at break. The measurement was performed at a tension rate of 200 mm/min with AUTOGRAPH (AGS-J, manufactured by SHIMADZU CORPORATION). The results are presented in Table 1. (Tear Strength) The resultant composition was filled into a mold and cured at 23° C. and 50% RH for 3 days and further at 50° C. for 4 days to thereby produce a sheet-shaped cured product having a thickness of about 3 mm. The sheet-shaped cured product was punched out into the Dumbbell (JIS A type) for the tear strength, which was subjected to a tear test at 23° C. and 50% RH. The measurement was performed at a tension rate of 200 mm/min with AUTOGRAPH (AGS-J, manufactured by SHIMADZU CORPORATION). The results are presented in Table 1. TABLE 1Comp.Comp.Composition (Parts by weight)Ex. 1Ex. 1Ex. 2PolymerA-I100——B-I—100—B-II——100PlasticizerDINP555555Calcium CarbonateCCR120120120Titanium OxideTIPAQUE R820202020Anti-sagging agentDISPARLON 6500222PhotostabilizerTINUVIN 770111Ultraviolet absorberTINUVIN 326111Dehydrating agentA-71222AdhesivenessA-120333imparting agentCondensation catalystU-20H222Dumbbell tensileM50MPa0.370.280.59physical propertyM100MPa0.590.450.88TBMPa3.693.093.14EB%11041133676Tear strengthTBMPa19.216.214.7 The curable composition of Example 1 including the reactive silicon group-containing polymer (A-I) of which end groups have structure meeting the predetermined requirements described above is more excellent in strength (TB) of the resultant cured product than the curable composition including the reactive silicon group-containing polymer (B-I) in which an atom adjacent to a silicon group does not have an unsaturated bond and which has on average no more than 0.8 reactive silicon groups per end. Furthermore, the curable composition of Example 1 including the reactive silicon group-containing polymer (A-I) is more excellent in flexibility (low modulus and high elongation) and strength (TB) of the resultant cured product than the curable composition including the reactive silicon group-containing polymer (B-II) in which an atom adjacent to a silicon group does not have an unsaturated bond and which has on average more than 1.0 reactive silicon group per end. Synthesis Example 4 To 500 g of the polymer (Q-I) obtained in Synthesis Example 1, were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 10.34 g of methoxymethyldimethoxysilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted methoxymethyldimethoxysilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (A-1) having methoxymethyldimethoxysilyl groups at ends and having a number average molecular weight of 28,500. The polymer (A-1) was found to have on average 2.0 methoxymethyldimethoxysilyl groups per molecule. Synthesis Example 5 Propylene oxide was polymerized using polyoxypropylene triol having a number average molecular weight of about 3,000, serving as an initiator, and a zinc hexacyanocobaltate-glyme complex catalyst to thereby obtain polyoxypropylene (P-1) having hydroxyl groups at ends and having a number average molecular weight of 24,600 (end group-based molecular weight: 17,400) and a molecular weight distribution Mw/Mn of 1.31. To the hydroxyl groups in the resultant hydroxyl group-terminated polyoxypropylene (P-1), was added 1.05 mol equivalents of sodium methoxide as a 28% methanol solution. The methanol was distilled off by vacuum devolatilization, and then 1.16 mol equivalents of propargyl bromide was further added to the hydroxyl groups in the polymer (P-1) to thereby convert the terminal hydroxyl groups into propargyl groups. Unreacted propargyl bromide was removed by devolatilization under reduced pressure. The resultant crude propargyl group-terminated polyoxypropylene was mixed and stirred with n-hexane and water, and then the water was removed by centrifugation and the hexane was devolatilized under reduced pressure from the resultant hexane solution to thereby remove a metal salt in the polymer. Thus, polyoxypropylene (Q-1) having propargyl groups at end sites was obtained. To 500 g of the polymer (Q-1), were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 11.49 g of methoxymethyldimethoxysilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted methoxymethyldimethoxysilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (A-2) having methoxymethyldimethoxysilyl groups at ends and having a number average molecular weight of 26,200. The polymer (A-2) was found to have on average 3.0 methoxymethyldimethoxysilyl groups per molecule. Synthesis Example 6 To 500 g of the polymer (Q-I) obtained in Synthesis Example 1, were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 8.37 g of trimethoxysilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted trimethoxysilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (A-3) having trimethoxysilyl groups at ends and having a number average molecular weight of 28,500. The polymer (A-3) was found to have on average 2.0 trimethoxysilyl groups per molecule. Synthesis Example 7 To the hydroxyl groups in the hydroxyl group-terminated polyoxypropylene (P-I) obtained in Synthesis Example 1, was added 1.2 mol equivalents of sodium methoxide as a 28% methanol solution. The methanol was distilled off by vacuum devolatilization, and then 1.5 mol equivalents of allyl chloride was further added to the hydroxyl groups in the polymer (P-I) to thereby convert the terminal hydroxyl groups into allyl groups. Unreacted allyl chloride was removed by devolatilization under reduced pressure. The resultant crude allyl group-terminated polyoxypropylene was mixed and stirred with n-hexane and water, and then the water was removed by centrifugation and the hexane was devolatilized under reduced pressure from the resultant hexane solution to thereby remove a metal salt in the polymer. Thus, a polyoxypropylene polymer (Q-2) having allyl groups at end sites was obtained. To 500 g of the polymer (Q-2), were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 6.5 g of methoxymethyldimethoxysilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted methoxymethyldimethoxysilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (E-1) having methoxymethyldimethoxysilyl groups at ends and having a number average molecular weight of about 28,200. The polymer (E-1) was found to have on average 1.6 methoxymethyldimethoxysilyl groups per molecule. Synthesis Example 8 To 500 g of the polymer (Q-2) obtained in Synthesis Example 7, were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 5.5 g of trimethoxysilane, to thereby effect a hydrosilylation reaction. After the mixed solution was reacted at 90° C. for 2 hours, unreacted trimethoxysilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (E-2) having trimethoxysilyl groups at ends and having a number average molecular weight of 28,500. The polymer (E-2) was found to have on average 1.6 trimethoxysilyl groups per molecule. Synthesis Example 9 To 500 g of the polymer (Q-2) obtained in Synthesis Example 5, were added 150 μL of a platinum-divinyldisiloxane complex (3 wt % isopropanol solution in terms of platinum) and 4.8 g of dimethoxymethylsilane, to thereby effect a hydrosilylation reaction. After reacting at 90° C. for 2 hours, unreacted dimethoxymethylsilane was distilled off under reduced pressure to thereby obtain polyoxypropylene (E-3) having dimethoxymethylsilyl groups at ends and having a number average molecular weight of 28,500. The polymer (E-3) was found to have on average 1.6 dimethoxymethylsilyl groups per molecule. Examples 2 to 16, Comparative Examples 3 to 10 The polymers described in Synthesis Examples 1 to 7 and commercial products were used according to formulas presented in Tables 2 to 4 to examine for their curability. Each of the polymers weighed into a minicup was added with a condensation catalyst, kneaded and stirred together, and left to stand under a constant temperature and constant humidity condition of 23° C. and 50% RH. This time point was determined as a curing initiating time. A surface of the mixture was touched with a spatula every 1 minute for the first 20 minutes and then every 10 minutes, and the time taken until the mixture no longer adhered to the spatula was measured as a skinning time. The results are presented in Tables 2 to 4. The following condensation catalysts were used in Examples 2 to 16 and Comparative Examples 3 to 10.DBU: 1,8-diazabicyclo[5,4,0]undecene-7 (manufactured by Tokyo Chemical Industry Co., Ltd.)PhGu: 45% 1-phenylguanidine solution in N-n-butylbenzenesulfonamide (manufactured by NIPPON CARBIDE INDUSTRIES CO., INC.)VA/DEAPA: versatic acid/3-diethylaminopropylamine=2.5/0.5U-810: NEOSTANN U-810 (manufactured by Nitto Kasei Co., Ltd.)A-1120: N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane (manufactured by Momentive) In Comparative Examples 10, the following polymer that is a reaction product of hydroxyl group-terminated polyether and (dimethoxymethylsilyl)methylisocyanate was used. STP-E30: GENIPSIL STP-E30 (manufactured by Wacker) TABLE 2PolymerEx.Comp. Ex,(composition ratio)2345678910111213345PolymerA-110010010010050A-2100A-31001001001005050E-15010050E-2505010050CondensationDBU0.20.10.20.20.10.20.20.20.20.20.2catalystPhGu0.20.2VA/1.01.0DEAPACurabilitySkinning587121.510171913813165616682time(min.) TABLE 3PolymerEx.Comp. Ex.(composition ratio)1467PolymerA-3100E-1100E-2100CondensationU-810111catalystCurabilitySkinning34156116time(min.) TABLE 4PolymerEx.Comp. Ex.(composition ratio)15168910PolymerA-1100A-2100E-1100E-2100STP-E10100Silane coupling agentA-11203.03.03.03.03.0CurabilitySkinning6637UncuredUncured73time(min.) As is clear from Tables 2 to 4, the curable compositions including the reactive silicon group-containing polymer (A-1), (A-2), or (A-3) which had the reactive silicon group represented by General Formula (1) and in which the atom adjacent to the reactive silicon group had the unsaturated bond exhibited more rapid curability using any condensation catalyst as compared with the curable composition including the organic polymer (E-1) or (E-2), which did not correspond to the reactive silicon group-containing polymer which had the reactive silicon group represented by General Formula (1) and in which the atom adjacent to the reactive silicon group had the unsaturated bond, or a commercially available organic polymer STP-E10. Furthermore, when using a condensation catalyst having a very low activity such as aminosilane, Comparative organic polymer (E-1) or (E-2) had an insufficient activity and was uncured, while the curable composition including the organic polymer (A-1) or (A-2) exhibited satisfactory curability. Moreover, the curable composition including the organic polymer (A-1) or (A-2) also exhibited more rapid curability as compared with the curable composition including the commercially available organic polymer STP-E10. Examples 17, 18, Comparative Example 11 One hundred parts by weight of the polymer of a kind described in Table 5 was mixed with 55 parts by weight of DINP (manufactured by J-PLUS Co., Ltd.: diisononyl phthalate), 120 parts by weight of HAKUENKA CCR (manufactured by SHIRAISHI CALCIUM KAISHA, LTD.: precipitated calcium carbonate), 20 parts by weight of TIPAQUE R820 (manufactured by ISHIHARA SANGYO KAISHA, LTD.: titanium oxide), 2 parts by weight of DISPARLON 6500 (manufactured by Kusumoto Chemicals, Ltd.: fatty acid amide wax), 1 part by weight of TINUVIN 770 (manufactured by BASF: bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate), and 1 part by weight of TINUVIN 326 (manufactured by BASF: 2-(3-tert-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole), uniformly dispersed with three rolls, and dehydrated under reduced pressure at 120° C. for 2 hours. Thereafter, the resultant was cooled to 50° C., and then added and mixed with 2 parts by weight of A-171 (manufactured by Momentive: vinyltrimethoxysilane), 3 parts by weight of A-1120 (manufactured by Momentive: N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane), and 0.3 parts by weight of DBU (1,8-diazabicyclo[5,4,0]undecene-7), and then sealed in a damp-proof cartridge under a condition substantially free from moisture. Examples 19, 20, Comparative Example 12 One hundred parts by weight of the polymer of a kind described in Table 5 was mixed with 55 parts by weight of DINP (manufactured by J-PLUS Co., Ltd.: diisononyl phthalate), 120 parts by weight of HAKUENKA CCR (manufactured by SHIRAISHI CALCIUM KAISHA, LTD.: precipitated calcium carbonate), 20 parts by weight of TIPAQUE R820 (manufactured by ISHIHARA SANGYO KAISHA, LTD.: titanium oxide), 2 parts by weight of DISPARLON 6500 (manufactured by Kusumoto Chemicals, Ltd.: fatty acid amide wax), 1 part by weight of TINUVIN 770 (manufactured by BASF: bis(2,2,6,6-tetramethyl-4-piperidyl) sebacate), and 1 part by weight of TINUVIN 326 (manufactured by BASF: 2-(3-tert-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole), uniformly dispersed with three rolls, and dehydrated under reduced pressure at 120° C. for 2 hours. Thereafter, the resultant was cooled to 50° C., and then added and mixed with 3 parts by weight of A-1120 (manufactured by Momentive: N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane), and then sealed in a damp-proof cartridge under a condition substantially free from moisture. (Skinning Time) The resultant composition was filled into a mold having a thickness of about 5 mm using a spatula and the time at which the surface thereof became flat was determined to be a curing initiating time. The surface was touched with the spatula, and the time taken until a composition to be evaluated no longer adhered to the spatula was measured as a skinning time. The results are presented in Table 5. (Dumbbell Tensile Physical Property) The dumbbell tensile physical property was measured in the same manner as in Example 1. The results are presented in Table 5. TABLE 5Comp.Comp.Ex.Ex.Ex.Ex.Composition171811192012PolymerA-1100100A-3100100E-1100100PlasticizerDINP555555555555Calcium carbonateCCR120120120120120120Titanium oxideTIPAQUE R820202020202020Ant-sagging agentDISPARLON 6500222222PhotostabilizerTINUVIN 770111111Ultraviolet absorberTINUVIN 326111111Dehydrating agentA-171222AdhesivenessA-1120333333imparting agentCondensationDBU0.30.30.3catalystCurabilitySkinning time151611166148Uncured(min.)Dumbbell tensileM50MPa0.370.430.280.430.460.43physical propertyM100MPa0.600.720.460.720.820.72TBMPa2.352.162.142.382.182.38EB%803501900655440655 As is clear from Table 5, in the case where a non-tin condensation catalyst such as DBU is used in a small amount or is not used, the curable composition including the organic polymer (A-1) or (A-3) exhibited more satisfactory curability as compared with the curable composition including the organic polymer (E-1). Example 21, Comparative Example 13 One hundred parts by weight of the polymer of a kind described in Table 6 was mixed with 55 parts by weight of DINP (manufactured by J-PLUS Co., Ltd.: diisononyl phthalate), 120 parts by weight of HAKUENKA CCR (manufactured by SHIRAISHI CALCIUM KAISHA, LTD.: precipitated calcium carbonate), 20 parts by weight of TIPAQUE R820 (manufactured by ISHIHARA SANGYO KAISHA, LTD.: titanium oxide), 2 parts by weight of DISPARLON 6500 (manufactured by Kusumoto Chemicals, Ltd.: fatty acid amide wax), 1 part by weight of TINUVIN 770 (manufactured by BASF: bis(2,2,6,6-tetramethyl-4-piperidyl)sebacate), and 1 part by weight of TINUVIN 326 (manufactured by BASF: 2-(3-tert-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole), uniformly dispersed with three rolls, and dehydrated under reduced pressure at 120° C. for 2 hours. Thereafter, the resultant was cooled to 50° C., and then added and mixed with 2 parts by weight of A-171 (manufactured by Momentive: vinyltrimethoxysilane), 3 parts by weight of A-1120 (manufactured by Momentive: N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane), and 2 parts by weight of U-220H (dibutyltin diacetylacetonate), and then sealed in a damp-proof cartridge under a condition substantially free from moisture. Examples 22, Comparative Example 14 One hundred parts by weight of the polymer of a kind described in Table 6 was mixed with 55 parts by weight of DINP (manufactured by J-PLUS Co., Ltd.: diisononyl phthalate), 120 parts by weight of HAKUENKA CCR (manufactured by SHIRAISHI CALCIUM KAISHA, LTD.: precipitated calcium carbonate), 20 parts by weight of TIPAQUE R820 (manufactured by ISHIHARA SANGYO KAISHA, LTD.: titanium oxide), 2 parts by weight of DISPARLON 6500 (manufactured by Kusumoto Chemicals, Ltd.: fatty acid amide wax), 1 part by weight of TINUVIN 770 (manufactured by BASF: bis(2,2,6,6-tetramethyl-4-piperidyl)sebacate), and 1 part by weight of TINUVIN 326 (manufactured by BASF: 2-(3-tert-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriasole), uniformly dispersed with three rolls, and dehydrated under reduced pressure at 120° C. for 2 hours. Thereafter, the resultant was cooled to 50° C., and then added and mixed with 2 parts by weight of A-171 (manufactured by Momentive: vinyltrimethoxysilane), 3 parts by weight of A-1120 (manufactured by Momentive: N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane), and 1 part by weight of DBU (1,8-diazabicyclo[5,4,0]undecene-7), and then sealed in a damp-proof cartridge under a condition substantially free from moisture. (Skinning Time) The skinning time was measured in the same manner as in Example 17. The results are presented in Table 6. TABLE 6Ex.Comp. Ex.Composition21221314PolymerA-1100100E-3100100PlasticizerDINP55555555CalciumCCR120120120120carbonateTitanium oxideTIPAQUE R82020202020Anti-saggingDISPARLON 65002222agentPhotostabilizerTINUVIN 7701111UltravioletTINUVIN 3261111absorberDehydratingA-1712222agentAdhesivenessA-11203333imparting agentCondensationU-220H22catalystDBU11CurabilitySkinning time2830053Uncured(min.) As is clear from Table 6, under the condition in which a tin catalyst such as U-220H or a non-tin condensation catalyst such as DBU is added, the curable composition including the organic polymer (A-4) exhibits more satisfactory curability as compared with the curable composition including the organic polymer (E-3). Although the disclosure has been described with respect to only a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that various other embodiments may be devised without departing from the scope of the present invention. Accordingly, the scope of the invention should be limited only by the attached claims. | 80,561 |
11859038 | DETAILED DESCRIPTION It will be appreciated that for simplicity and clarity of illustration, where appropriate, reference numerals have been repeated among the different figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the embodiments described herein. However, it will be understood by those of ordinary skill in the art that the embodiments described herein can be practiced without these specific details. In other instances, methods, procedures, and components have not been described in detail so as not to obscure the related relevant feature being described. Also, the description is not to be considered as limiting the scope of the embodiments described herein. The drawings are not necessarily to scale, and the proportions of certain parts may be exaggerated to better illustrate details and features of the present disclosure. The term “comprising,” when utilized, means “including, but not necessarily limited to”; it specifically indicates open-ended inclusion or membership in the so-described combination, group, series, and the like. FIG.1illustrates a flowchart of a method in accordance with an embodiment. The method for manufacturing a block copolymer of polyamide acid is provided by way of embodiments, as there are a variety of ways to carry out the method. Each block shown inFIG.1represents one or more processes, methods, or subroutines carried out in the method. Furthermore, the illustrated order of blocks can be changed. Additional blocks may be added or fewer blocks may be utilized, without departing from this disclosure. The method can begin at block101. At block101, first dianhydride monomers and second dianhydride monomers are provided. Each dianhydride monomer has a chemical structural formula of or includes a first liquid crystal structure between two phthalic anhydride groups. A main chain of the first liquid crystal structure includes at least one first cyclic group and at least two first intermediate groups. Each first intermediate group is provided between the first cyclic group and the phthalic anhydride group adjacent to the first cyclic group, or between two adjacent first cyclic groups. Each first intermediate group is selected from a chemical structural formula of Each first cyclic group is selected from a chemical structural formula of Each second dianhydride monomer includes a first flexible structure between two phthalic anhydride groups. A main chain of the first flexible structure includes a group selected from a group consist of ether bond ketone group sulphone group aliphatic hydrocarbon group, and any combination thereof. In at least one embodiment, each first dianhydride monomer may be 3,3′,4,4′-biphenyltetracarboxylic dianhydride having a chemical structural formula of p-phenylene bis(trimellitate) dianhydride having a chemical structural formula of or cyclohexane-1,4-diylbis(methylene)bis(1,3-dioxo-1,3-dihy-droisobenzofuran-5-carboxylate) having a chemical structural formula of In at least one embodiment, each second dianhydride monomer may be bis-(3-phthalyl anhydride) ether having a chemical structural formula of 4-[(1,3-dihydro-1,3-dioxo-5-isobenzofuranyl)oxy]-1,3-isobenzofurandione having a chemical structural formula of 3,3′,4,4′-benzophenonetetracarboxylic dianhydride having a chemical structural formula of 3,3′,4,4′-diphenyl sulfonetetracarboxylic anhydride having a chemical structural formula of 4,4′-(hexafluoroisoproylidene)diphthalic anhydride having a chemical structural formula of or 4,4′-(4,4′-isopropylidenediphenoxybis(phthalic anhydride) having a chemical structural formula of At block102, first diamine monomers and second diamine monomers are provided. Each first diamine monomer includes a second liquid crystal structure between two phenyl groups with amino. A main chain of the second liquid crystal structure includes a second intermediate group or includes at least one second cyclic group and at least two second intermediate groups. Each second intermediate group is provided between the phenyl groups with amino, between the second cyclic group and the phenyl groups with amino adjacent to the second cyclic group, or between two adjacent second cyclic groups. Each second intermediate group is selected from a chemical structural formula of Each second cyclic group is selected from a chemical structural formula of Each second diamine monomer includes a second flexible structure between two phenyl groups with amino. A main chain of the second flexible structure includes a group selected from a group consist of ether bond ketone group sulphone group aliphatic hydrocarbon group, and any combination thereof. In at least one embodiment, each first diamine monomer may be 4-aminophenyl-4-aminobenzoate having a chemical structural formula of bis(4-aminophenyl)terephthalate having a chemical structural formula of or [4-(4-aminobenzoyl)oxyphenyl] 4-aminobenzoate having a chemical structural formula of In at least one embodiment, each second diamine monomer may be 4,4′-oxydianiline having a chemical structural formula of 4,4′-(4,4′-isopropylidenediphenyl-1,1′-diyldioxy)daniline having a chemical structural formula of 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane having a chemical structural formula of 4,4′-(1,3-phenylenedioxy)dianiline having a chemical structural formula of or 1,3-bis(3-aminophenoxy)benzene having a chemical structural formula of At block103, the first dianhydride monomers and the second diamine monomers are mixed and polymerized to form a first polyamide acid, and the second dianhydride monomers and the first diamine monomers are mixed and polymerized to form a second polyamide acid. In at least one embodiment, the first liquid crystal structure included in the first dianhydride monomer and the second liquid crystal structure included in the first diamine monomer may be the same or be different. The first flexible structure in the second dianhydride monomer and the second flexible structure included in the second diamine monomer may be the same or be different. In at least one embodiment, the first dianhydride monomers, the second diamine monomers and a first solvent are mixed to obtain a first mixture, and the first dianhydride monomers and the second diamine monomers are dissolved in the first solvent. The first solvent is a bipolar aprotic solvent. In at least one embodiment, the first solvent may be selected from a group consist of dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), dimethylsulfoxide, and any combination thereof. In at least one embodiment, in the first mixture, a mass percentage of the first solvent is in a range of 75% to 85%. In at least one embodiment, the second dianhydride monomers, the first diamine monomers and a second solvent are mixed to obtain a second mixture, and the second dianhydride monomers and the first diamine monomers are dissolved in the second solvent. The second solvent is a bipolar aprotic solvent. In at least one embodiment, the second solvent may be selected from a group consist of dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), dimethylsulfoxide, and any combination thereof. In at least one embodiment, in the second mixture, a mass percentage of the second solvent is in a range of 75% to 85%. In at least one embodiment, the first dianhydride monomers and the second diamine monomers may be in a molar ratio of 9:10 to 11:10. In at least one embodiment, the second dianhydride monomers and the first diamine monomers may be in a molar ratio of 9:10 to 11:10. At block104, the first polyamide acid and the second polyamide acid undergo a polymerization reaction to obtain the block copolymer of polyamide acid. In the block copolymer of polyamide acid, the first polyamide acid and the second polyamide acid are alternately connected. In the first polyamide acid, the first liquid crystal structure and the second flexible structure are alternately arranged. In the second polyamide acid, the first flexible structure and the second liquid crystal structure are alternately arranged. FIG.2illustrates a flowchart of a method in accordance with another embodiment. The method for manufacturing a block copolymer of polyamide acid is provided by way of embodiments, as there are a variety of ways to carry out the method. Each block shown inFIG.2represents one or more processes, methods, or subroutines carried out in the method. Furthermore, the illustrated order of blocks can be changed. Additional blocks may be added or fewer blocks may be utilized, without departing from this disclosure. The method can begin at block201. At block201, first dianhydride monomers and second dianhydride monomers are provided. Each dianhydride monomer has a chemical structural formula of or includes a first liquid crystal structure between two phthalic anhydride groups. A main chain of the first liquid crystal structure includes at least one first cyclic group and at least two first intermediate groups. Each first intermediate group is provided between the first cyclic group and the phthalic anhydride group adjacent to the first cyclic group, or between two adjacent first cyclic groups. Each first intermediate group is selected from a chemical structural formula of Each first cyclic group is selected from a chemical structural formula of Each second dianhydride monomer includes a first flexible structure between two phthalic anhydride groups. A main chain of the first flexible structure includes a group selected from a group consist of ether bond ketone group sulphone group aliphatic hydrocarbon group, and any combination thereof. In at least one embodiment, each first dianhydride monomer may be 3,3′,4,4′-Biphenyltetracarboxylic dianhydride having a chemical structural formula of p-Phenylene bis(trimellitate) dianhydride having a chemical structural formula of or cyclohexane-1,4-diylbis(methylene)bis(1,3-dioxo-1,3-dihy-droisobenzofuran-5-carboxy late) having a chemical structural formula of In at least one embodiment, each second dianhydride monomer may be bis-(3-phthalyl anhydride) ether having a chemical structural formula of 4-[(1,3-Dihydro-1,3-dioxo-5-isobenzofuranyl)oxy]-1,3-isobenzofurandione having a chemical structural formula of 3,3′,4,4′-Benzophenonetetracarboxylic dianhydride having a chemical structural formula of 3,3′,4,4′-Diphenyl sulfonetetracarboxylic anhydride having a chemical structural formula of 4,4′-(Hexafluoroisoproylidene)diphthalic anhydride having a chemical structural formula of or 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) having a chemical structural formula of At block202, first diamine monomers and second diamine monomers are provided. Each first diamine monomer includes a second liquid crystal structure between two phenyl groups with amino. A main chain of the second liquid crystal structure includes a second intermediate group or includes at least one second cyclic group and at least two second intermediate groups. Each second intermediate group is provided between the phenyl groups with amino, between the second cyclic group and the phenyl groups with amino adjacent to the second cyclic group, or between two adjacent second cyclic groups. Each second intermediate group is selected from a chemical structural formula of Each cyclic group is selected from a chemical structural formula of Each second diamine monomer includes a second flexible structure between two phenyl groups with amino. A main chain of the second flexible structure includes a group selected from a group consist of ether bond ketone group sulphone group aliphatic hydrocarbon group, and any combination thereof. In at least one embodiment, each first diamine monomer may be 4-Aminobenzoic acid 4-aminophenyl ester having a chemical structural formula of Bis(4-aminophenyl)terephthalate having a chemical structural formula of or [4-(4-aminobenzoyl)oxyphenyl] 4-aminobenzoate having a chemical structural formula of In at least one embodiment, each second diamine monomer may be 4,4′-Oxydianiline having a chemical structural formula of 4,4′-(4,4′-Isopropylidenediphenyl-1,1′-diyldioxy)dianiline having a chemical structural formula of 2,2-Bis[4-(4-aminophenoxy)phenyl]hexafluoropropane having a chemical structural formula of 4,4′-(1,3-Phenylenedioxy)dianiline having a chemical structural formula of or 1,3-Bis(3-aminophenoxy)benzene having a chemical structural formula of At block203, the first dianhydride monomers and the first diamine monomers are mixed and polymerized to form a first polyamide acid, and the second dianhydride monomers and the second diamine monomers are mixed and polymerized to form a second polyamide acid. In at least one embodiment, the first liquid crystal structure included in the first dianhydride monomer and the second liquid crystal structure included in the first diamine monomer may be the same or be different. The first flexible structure in the second dianhydride monomer and the second flexible structure included in the second diamine monomer may be the same or be different. In at least one embodiment, the first dianhydride monomers, the first diamine monomers and a first solvent are mixed to obtain a first mixture, and the first dianhydride monomers and the first diamine monomers are dissolved in the first solvent. The first solvent is a bipolar aprotic solvent. In at least one embodiment, the first solvent may be selected from a group consist of dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), dimethylsulfoxide, and any combination thereof. In at least one embodiment, in the first mixture, a mass percentage of the first solvent is in a range of 75% to 85%. In at least one embodiment, the second dianhydride monomers, the second diamine monomers and a second solvent are mixed to obtain a second mixture, and the second dianhydride monomers and the second diamine monomers are dissolved in the second solvent. The second solvent is a bipolar aprotic solvent. In at least one embodiment, the second solvent may be selected from a group consist of dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), dimethylsulfoxide, and any combination thereof. In at least one embodiment, in the second mixture, a mass percentage of the second solvent is in a range of 75% to 85%. In at least one embodiment, the first dianhydride monomers and the first diamine monomers may be in a molar ratio of 9:10 to 11:10. In at least one embodiment, the second dianhydride monomers and the second diamine monomers may be in a molar ratio of 9:10 to 11:10. At block204, the first polyamide acid and the second polyamide acid are mixed and undergo a polymerization reaction to obtain the block copolymer of polyamide acid. In the block copolymer of polyamide acid, the first polyamide acid and the second polyamide acid are alternately connected. In the first polyamide acid, the first liquid crystal structure and the second liquid crystal structure are alternately arranged. In the second polyamide acid, the first flexible structure and the second flexible structure are alternately arranged. FIG.3illustrates an embodiment of a metal clad laminate100. The metal clad laminate100may be applied to a circuit board200(shown inFIG.4). The metal clad laminate100includes a metal foil10and a polyimide film20bonded to a surface of the metal foil10. The polyimide film20is formed by coating the above block copolymer of polyamide acid on the surface of the metal foil10and then cyclizing the coated block copolymer of polyamide acid by heating. In at least one embodiment, the polyimide film made by the following step: Wherein Ar1 represents a group, and Ar2 represents a group. In at least one embodiment, a temperature of cyclizing the coated block copolymer of polyamide acid is in a range of 300 degrees Celsius to 400 degrees Celsius. In at least one embodiment, a surface roughness of the metal foil10is in a range of 0.12 μm to 2.1 μm. When the block copolymer of polyamide acid includes nitrogen heterocycle, nitrogen atoms of the nitrogen heterocycle may coordinate with the metal foil10, thereby improving a binding force between the polyimide film20and the metal foil10. So that when the metal foil100with a low surface roughness is used to make metal clad laminate100, the polyimide film20formed on the metal foil100may be flat, and a transmittance of the polyimide film20may be improved. The circuit board200may be applied to electronic devices (not shown), such as computers, e-readers, pads, or smart watches. In at least one embodiment, the circuit board200includes a circuit substrate201and a covering film202on the circuit substrate201. The circuit board201includes a polyimide film20and a conductive wiring layer2011bonded to the polyimide film20. Example 1 211.10 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester (APAB) were added into a container and stirred until dissolved. 15.51 g or 0.05 mol of bis-(3-phthalyl anhydride) ether (ODPA) was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene (TPE-M) was added into the container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride (TAHQ) was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Example 2 209.14 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred to be dissolved. 7.76 g or 0.025 mol of bis-(3-phthalyl anhydride) ether and 13.01 g or 0.025 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride)(BPADA) were added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 48 hours to react thereby obtaining a block copolymer of polyamide acid. Example 3 224.91 g of N-methylpyrrolidone, 1.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred until dissolved. 26.02 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Example 4 233.92 g of N-methylpyrrolidone, 5.71 g or 0.025 mol of 4-Aminobenzoic acid 4-aminophenyl ester, and 8.71 g or 0.025 mol of Bis(4-aminophenyl)terephthalate (BPTP) were added into a container and stirred until dissolved. 26.02 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the second container and stirred for 48 hour to react, thereby obtaining a block copolymer of polyamide acid. Example 5 246.68 g of N-methylpyrrolidone, 5.71 g or 0.025 mol of 4-Aminobenzoic acid 4-aminophenyl ester, and 12.96 g or 0.025 mol of 2,2-Bis[4-(4-aminophenoxy)phenyl]hexafluoropropane (HFBAPP) were added into a container and stirred until dissolved. 26.02 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 1 260.76 g of N-methylpyrrolidone, 41.05 g or 0.1 mol of 4,4′-(4,4′-Isopropylidenediphenyl-1,1′-diyldioxy)dianiline (BAPP) were added into a container and stirred until dissolved. 45.87 g or 0.1 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 48 hour to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 2 227.29 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 25.92 g or 0.05 mol of 2,2-Bis[4-(4-aminophenoxy)phenyl]hexafluoropropane was added into the container and stirred until dissolved. 15.51 g or 0.05 mol of bis-(3-phthalyl anhydride) ether was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 3 211.10 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 20.53 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenyl-1,1′-diyldioxy)dianiline was added into the container and stirred until dissolved. 15.51 g or 0.05 mol of bis-(3-phthalyl anhydride) ether was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 4 211.10 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 15.51 g or 0.05 mol of bis-(3-phthalyl anhydride) ether was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 5 209.14 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred to be dissolved. 7.76 g or 0.025 mol of bis-(3-phthalyl anhydride) ether and 13.01 g or 0.025 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) were added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 6 224.91 g of N-methylpyrrolidone, 11.41 g or 0.05 mol of 4-Aminobenzoic acid 4-aminophenyl ester were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 26.02 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 7 233.92 g of N-methylpyrrolidone, 5.71 g or 0.025 mol of 4-Aminobenzoic acid 4-aminophenyl ester, and 8.71 g or 0.025 mol of Bis(4-aminophenyl)terephthalate were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 26.02 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) was added into the second container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Comparative Example 8 246.68 g of N-methylpyrrolidone, 5.71 g or 0.025 mol of 4-Aminobenzoic acid 4-aminophenyl ester, and 12.96 g or 0.025 mol of 2,2-Bis[4-(4-aminophenoxy)phenyl]hexafluoropropane were added into a container and stirred until dissolved. 22.93 g or 0.05 mol of p-Phenylene bis(trimellitate) dianhydride was added into the container and stirred for 1 hour to react. 14.62 g or 0.05 mol of 1,3-Bis(3-aminophenoxy)benzene was added into the container and stirred until dissolved. 26.02 g or 0.05 mol of 4,4′-(4,4′-Isopropylidenediphenoxybis(phthalic anhydride) was added into the container and stirred for 48 hours to react, thereby obtaining a block copolymer of polyamide acid. Fourteen test samples were made. Each of the fourteen test samples comprises a copper foil and a polyimide film made by cyclizing the block copolymer of polyamide acid of the examples 1, 2, 3, 4, and 5, and the comparative examples 1, 2, 3, 4, 5, 6, 7, and 8, respectively. The block copolymer of polyamide acid of the examples 1 to 5 and the comparative examples 1 to 8 were cyclized at a same temperature. The dianhydride monomers and the first diamine monomers of each block copolymer of polyamide acid of the examples (short to Ex) 1 to 5 and the comparative examples (short to Co-ex) 1 to 8 were shown in the following Table 1. Dielectric constant Dk, and dielectric dissipation factor Dfof each of the polyimide film formed by the block copolymer of polyamide acid of examples 1 to 5 and the block copolymer of polyamide acid of the comparative examples 1 to 8 were tested. Thermal resistance, thermal performance, and copper peeling strength of the fourteen test samples were tested. The test results were shown in the following Table 2. The thermal resistance was tested at a temperature of 288 degrees centigrade for 10 seconds, if the polyimide film did not blister and peel off, the result of the thermal resistance test is considered passing, otherwise, the result fails. The thermal performance was tested by the softening temperature (Ts) of the polyimide film. TABLE 1component/molThe 1th diamineThe 2th diamineThe 1th dianhydrideThe 2th dianhydridemonomermonomermonomermonomerAPABBPTPHFBAPPBAPPTPE-MTAHQODPABPADAEx 10.050.050.050.05Ex 20.050.050.050.0250.025Ex 30.050.050.050.05Ex 40.0250.0250.050.050.05Ex 50.0250.0250.050.050.05Co-ex 10.10.1Co-ex 20.050.050.050.05Co-ex 30.050.050.050.05Co-ex 40.050.050.050.05Co-ex 50.050.050.050.0250.025Co-ex 60.050.050.050.05Co-ex 70.0250.0250.050.050.05Co-ex 80.0250.0250.050.050.05 TABLE 2Test resultscopperthermalpeelingperformancestrength(288° C./TsDkDf(kgf/cm)10 sec)(° C.)(10 GHz)(10 GHz)Ex 10.93PASS2913.10.003Ex 21.01PASS2873.10.003Ex 31.33PASS26930.003Ex 41.12PASS27430.002Ex 51.11PASS2822.70.004Co-ex 11.21NG2113.40.008Co-ex 20.53PASS3632.90.006Co-ex 30.67PASS3363.30.006Co-ex 40.72PASS3253.30.007Co-ex 50.78PASS3173.20.006Co-ex 60.75PASS3273.20.006Co-ex 70.74PASS3303.20.006Co-ex 80.67PASS3363.30.006 According to the Table 1 and Table 2, the copper peeling strength of the polyimide films formed by the block copolymer of polyamide acid of examples 1, 2, 3, 4, and 5 are higher than the copper peeling strength of the polyimide films formed by the block copolymer of polyamide acid of comparative examples 2, 3, 4, 5, 6, 7, and 8. The softening temperature of the polyimide films formed by the block copolymer of polyamide acid of examples 1, 2, 3, 4, and 5 are lower than the softening temperature of the polyimide films formed by the block copolymer of polyamide acid of comparative examples 2, 3, 4, 5, 6, 7, and 8. The dielectric dissipation factor of the polyimide films formed by the block copolymer of polyamide acid of examples 1, 2, 3, 4, and 5 are lower than the dielectric dissipation factor of the polyimide films formed by the block copolymer of polyamide acid of comparative examples 2, 3, 4, 5, 6, 7, and 8. That is, by changing the order of the polymerization reaction between the monomers with the liquid crystal structure and the diamine monomers with the flexible structure, the peel strength, the processability, and the dielectric properties of the polyimide film may be improved. According to comparative example 1 and comparative examples 2 to 8, it can be found that the peel strength, the softening temperature and the dielectric properties of the polyimide film may be adjusted by changing the composition and ratio of the monomers. It is to be understood, even though information and advantages of the present embodiments have been set forth in the foregoing description, together with details of the structures and functions of the present embodiments, the disclosure is illustrative only; changes may be made in detail, especially in matters of shape, size, and arrangement of parts within the principles of the present embodiments to the full extent indicated by the plain meaning of the terms in which the appended claims are expressed. | 29,437 |
11859039 | EXAMPLES Polymer molecular weights (called “weight average molecular weight” or “GPC molecular weight” herein) were determined by HMW-GPC (gel permeation chromatography) using polystyrene calibration standards. Median particle size of the latices was determined by laser diffraction using a Beckman Coulter LS13320. Solids content was measured with a Sartorius MA35 moisture balance. Coupling efficiency for the reaction with the multifunctional coupling agent was determined by GPC from the peak integration ratios of the uncoupled polymer relative to the higher molecular weight coupled polymer. Coagulant solution/dispersion was prepared by dissolving/dispersing Ca(NO3)2·4H2O (˜14%) and CaCO3(˜5%) in water at 60° C. while stirring. All the latexes, including the reference sample, were compounded with 5 phr of a commercially available masterbatch composition (containing sulphur, accelerators and anti-oxidant) and stabilized with a commercially available surfactant. The compounds were diluted with demineralized water to obtain the required solid content of 30%. The pH was adjusted to 11-11.5 by addition of a 1M KOH solution and the compounds were stored at ambient temperature while stirring. For the coagulant dipping exercise, metal plates were used and the standard dipping procedure was applied. Films were dipped after 1, 3 and 6 days of maturation. Mechanical properties of the dipped films were determined according to ASTM D412/ISO37 (Die C) using an Instron 3365 tensile bench equipped with a 500N load cell and a long-range travelling extensometer. The tensile properties are shown in Table 5. Examples 1-5. Preparation of Branched Polyisoprene Homopolymers Polymerizations were conducted under a nitrogen atmosphere in a 10 L stainless steel reactor equipped with agitator, cooling, temperature probe, pressure probe and auxiliaries for addition/withdrawal of solvent, monomers and other reagents. Solvents and monomers were purified over alcoa prior to use. As a general procedure, the reactor was charged with 5 L of dry solvent and 450 g of isoprene and heated to 60° C. A dilute s-BuLi solution (0.04 M) was added to the mixture to initiate the polymerization. After conversion of the required amount of monomer to the required molecular weight, the reaction was terminated by addition of the coupling agent (BTMSE or BTESE) in a molar ratio of living chains/coupling agent of 4/1. In some cases, this procedure was repeated once or twice until full conversion of monomer. BTMSE and BTESE were evaluated in isoprene polymerizations at different living chain concentrations. The results are summarized in Table 1. The data shows that BTMSE and BTSE have comparable coupling performance. TABLE 1Coupling reactions of BTMSE and BTESE with differentconcentrations of living polyisoprene chains.LivingchainsUncoupledUncoupledExampleCoupling agent(mmol)after 1 h (%)final (%)1BTMSE0.823222BTMSE120173BTMSE214144BTESE0.5533295BTESE0.82723 Examples 6-8: Preparation of Branched Polyisoprene Homopolymers These were prepared as rubber cements using the procedure described above. Results are shown in Table 2. The control sample was a linear (unbranched) polyisoprene made without using a coupling agent. TABLE 2Branched Polyisoprenes prepared.NumberCouplingGPC Mwcis contentExampleof runsagent(kg/mol)(%)Control-11Not used30008861BTMSE16607872BTESE10837582BTMSE126082 Examples 9-11: Preparation of Branched Polyisoprene Rubber Latex The rubber cement sample was transferred to a bench scale latex production facility equipped with an in-line high shear mixing device with dosing pumps for polymer and soap solutions Emulsions were produced under standardized emulsification conditions from a fixed ratio of cement and aqueous solution of a potassium salt of disproportionated rosin acid. The emulsion was subsequently solvent stripped to obtain a dilute latex, which was concentrated by creaming after addition of sodium alginate. The results are shown in Table 3. Control-2 is a reference polyisoprene rubber latex prepared from the Control-1 rubber cement described above. TABLE 3Branched polyisoprene latices prepared.CementDilute latexConcentrated latexsolidsSoapR/SSolidsSolventPSmedSolidsViscosityPSmedExample(wt %)(wt %)ratiopH(wt %)(ppmr)(μ)pH(wt %)(cPs)(μ)Control-29112.311.5122501.111.364801.39201.211.511.815.34951.811.45346210150.812.111.68.7255111.5611061.211160.612.811.483000.711.7712080.9R/S ratio: Rubber/Soap ratio.PSmed: Median particle size TABLE 4Tensile Properties of Cured Films.TensileYoung'sTearFilmstrengthmodulusstrengthExample(MPa)Elongation(MPa)(kN/m)Control-3287800.132812 (latex ex. 10)246630.222713 (latex ex. 11)237800.2521 Table 4 shows that the tensile properties of the dipped films made from the branched polyisoprenes (Examples 12 and 13) made using the multifunctional coupling agents are very good and comparable to those seen with the control sample film (Control-3). Example 14. Preparation of Branched Styrene-Isoprene Block Copolymers The polymerization was conducted under a nitrogen atmosphere in a 10 L stainless steel reactor equipped with agitator, cooling, temperature probe, pressure probe and auxiliaries for addition/withdrawal of solvent, monomers and other reagents. Solvent and monomers were purified over alcoa prior to use. The reactor was charged with 5 L of dry solvent and 400 g of styrene and heated to 60° C. A dilute s-BuLi solution (0.04 M) was added to the mixture to initiate the polymerization. After conversion of the styrene, 3 kg of isoprene monomer was charged. After conversion of the second monomer to the required molecular weight, the reaction was terminated by addition of the BTMSE coupling agent in a molar ratio of living chains/coupling agent of 4/1. The results are shown in Table 5. Control-4 is a commercial lot sample of polyisoprene made using GPTS (glycidylpropyl trimethoxysilane) as the coupling agent. TABLE 5CE means coupling efficiency. DoB means degree of branching.PS blockSI diblockGPC MWGPC MWCouplingExample(kg/mol)(kg/mol)agentCEDoBControl-410.8159GPTS952.81410.9157BTMSE894.1 Example 15. Preparation of Branched Styrene-Isoprene Block Copolymer Latex The rubber cement sample from Example 14 was transferred to a bench scale latex production facility equipped with an in-line high-shear mixing device with dosing pumps for polymer and soap solutions. The emulsion was produced under standardized emulsification conditions from a fixed ratio of cement and aqueous solution of a potassium salt of disproportionated rosin acid. The emulsion was subsequently solvent-stripped to obtain a dilute latex, which after addition of sodium alginate was concentrated by creaming. The results are shown in Table 6. TABLE 6CementDilute latexConcentrated latexsolidsSoapR/SSolidsPsmedSolidsViscosityPsmedExample(wt %)(wt %)ratio(wt %)(μ)pH(wt %)(cPs)(μ)1515.30.917.014.41.2211.260.6621.20R/S ratio: Rubber/Soap ratio.PSmed: Median particle size. Example 16 represents a dipped film made from the branched styrene-isoprene block copolymer latex of Example 15. Control 5 represents a dipped film made from the branched styrene-isoprene block copolymer latex derived from the block copolymer of Control 4. Table 7 shows that the tensile properties of the dipped films made from the branched styrene-isoprene block copolymer made using the multifunctional coupling agent (Example 16) are very good and comparable to those seen with the control sample film (Control-5). TABLE 7TensileModulusTearExamplestrength500%ElongationstrengthControl 521.41.62108924.71620.41.43111327.3 For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained. It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” include plural references unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items. As used herein, the term “comprising” means including elements or steps that are identified following that term, but any such elements or steps are not exhaustive, and an embodiment can include other elements or steps. Although the terms “comprising” and “including” have been used herein to describe various aspects, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific aspects of the disclosure and are also disclosed. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed disclosure belongs. the recitation of a genus of elements, materials or other components, from which an individual component or mixture of components can be selected, is intended to include all possible sub-generic combinations of the listed components and mixtures thereof. The patentable scope is defined by the claims, and can include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. To an extent not inconsistent herewith, all citations referred to herein are hereby incorporated by reference. Embodiments herein include: Embodiment 1. A branched block copolymer comprising one or more polymer blocks A and one or more polymer blocks B and having a formula (A-B)m(R1O)3-mSi—Y—Si(R2O)3-n(B-A)n, wherein each A block independently comprises at least 90 mole percent of an alkenyl aromatic hydrocarbon; wherein the one or more A blocks form 8-13 weight percent of the total weight of the branched block copolymer; each B block is independently a poly(1,3-diene) block comprising at least 90 mole percent of one or more 1,3-dienes; each A block independently has a weight average molecular weight from 9-15 kg/mol, and each B block independently has a weight average molecular weight from 75-150 kg/mol; R1and R2are independently H, or C1-C6alkyl groups; Y is a C2-C8alkylene group; m and n are integers independently having values from 1-3; and the branched block copolymer has a coupling efficiency (CE) of at least 40%. Embodiment 2. The branched block copolymer of embodiment 1, wherein (m+n) has a value from 3-6. Embodiment 3. The branched block copolymer of embodiment 1, wherein Y is —CH2CH2—, and R1and R2are hydrogen, methyl groups or ethyl groups. Embodiment 4. The branched block copolymer of any of embodiments 1-3, wherein the 1,3-diene is isoprene, and the alkenyl aromatic hydrocarbon is styrene. Embodiment 5. A branched polyisoprene homopolymer having a formula (C)m(R1O)3-mSi—Y—Si(R2O)3-n(C)n, wherein each C block is independently a polyisoprene block having a weight average molecular weight from 400-1000 kg/mol; R1and R2are independently H, or C1-C6alkyl groups; Y is a C2-C8alkylene group; m and n are integers independently having values from 1-3; and the homopolymer has a coupling efficiency (CE) of at least 40%. Embodiment 6. The branched polyisoprene homopolymer of embodiment 5, wherein (m+n) has a value from 3-6. Embodiment 7. The branched polyisoprene homopolymer of any of embodiments 5-6, wherein Y is —CH2CH2— and R1and R2are hydrogen, methyl groups or ethyl groups. Embodiment 8. The branched polyisoprene homopolymer of any of embodiments 5-6, wherein the C block has a weight average molecular weight from 500-700 kg/mol. Embodiment 9. A process for preparing a branched block copolymer, the process comprising: polymerizing a first monomer comprising at least 90 mole percent of an alkenyl aromatic hydrocarbon, at a temperature from 0° C. to 100° C., to form a polymer block A; adding a second monomer comprising at least 90 mole percent of one or more 1,3-dienes and polymerizing at a temperature from 0° C. to 100° C., to form a polymer block B; adding a multifunctional coupling agent of formula (R1O)3Si—Y—Si(OR2)3; and forming the branched block copolymer having a formula (A-B)m(R1O)3-mSi—Y—Si(R2O)3-n(B-A)n; wherein each A block independently comprises at least 90 mole percent of the alkenyl aromatic hydrocarbon, and the one or more A blocks form 8-13 weight percent of the total weight of the branched block copolymer; each B block is independently a poly(1,3-diene) block comprising at least 90 mole percent of one or more 1,3-dienes; each A block independently has a weight average molecular weight from 9-15 kg/mol, and each B block independently has a weight average molecular weight from 75-150 kg/mol; R1and R2are independently H, or C1-C6alkyl groups; Y is a C2-C8alkylene group; m and n are integers independently having values from 1-3; and the branched block copolymer has a coupling efficiency (CE) of at least 40%. Embodiment 10. The process of embodiment 9, wherein the branched block copolymer is formed as a rubber cement having a solids content from 5 wt. % to 35 wt. %. Embodiment 11. The process of embodiment 10, wherein the rubber cement has a zero shear viscosity of less than 25,000 mPas in a solids content range from 15 wt. % to 25 wt. %; a cis content from 70% to 95%; and a weight average molecular weight from 350 to 700 kg/mol. Embodiment 12. A process for preparing a branched polyisoprene homopolymer, the process comprising: polymerizing isoprene, in the presence of an anionic initiator, at a temperature from 0° C. to 100° C., to form a polymer block C, adding a multifunctional coupling agent of formula (R1O)3Si—Y—Si(OR2)3; and forming the branched polyisoprene homopolymer having a formula: (C)m(R1O)3-mSi—Y—Si(R2O)3-n(C)n, wherein each C block is a polyisoprene block independently having a weight average molecular weight from 400-1000 kg/mol; R1and R2are independently H, or C1-C6alkyl groups; Y is a C2-C8alkylene group; m and n are integers independently having values from 1-3; and the homopolymer has a coupling efficiency (CE) of at least 40%. Embodiment 13. The process of embodiment 12, wherein the branched polyisoprene homopolymer is formed as a rubber cement having a solids content from 5 wt. % to 35 wt. %. Embodiment 14. The process of embodiment 13, wherein the rubber cement has a zero shear viscosity of less than 80,000 mPas in a solids content range from 15 wt. % to 25 wt. %; a cis content from 70% to 95%; and a weight average molecular weight from 1,000 to 3500 kg/mol. Embodiment 15. A branched block copolymer of formula (A-B)m(R1O)3-mSi—Y—Si(R2O)3-n(B-A)n, prepared by the method of embodiment 9. Embodiment 16. A branched polyisoprene homopolymer of formula (C)m(R1O)3-mSi—Y—Si(R2O)3-n(C)n, prepared by the method of embodiment 12. | 15,391 |
11859040 | BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. Catalyst for Olefin Polymerization The catalyst for olefin polymerization according to an embodiment of the present invention comprises a first transition metal compound represented by Formula A and a second transition metal compound represented by Formula B. In Formulae A and B, n and o are each an integer of 0 to 2, provided that at least one of them is not 0, and m and 1 are each an integer of 0 to 4. Specifically, n and o may each be 1 or 2, and m and 1 may each be 1. M is titanium (Ti), zirconium (Zr), or hafnium (Hf). Specifically, M may be zirconium (Zr). X is each independently halogen, C1-20alkyl, C2-20alkenyl, C2-20alkynyl, C6-20aryl, C1-20alkyl C6-20aryl, C6-20aryl C1-20alkyl, C1-20alkylamido, C6-20arylamido, or C1-20alkylidene. Specifically, X may each be halogen. More specifically, X may each be chlorine (Cl). Q is carbon (C), silicon (Si), germanium (Ge), or tin (Sn). Specifically, Q may be carbon (C). R1to R7are each independently substituted or unsubstituted C1-20alkyl, substituted or unsubstituted C2-20alkenyl, substituted or unsubstituted C6-20aryl, substituted or unsubstituted C1-20alkyl C6-20aryl, substituted or unsubstituted C6-20aryl C1-20alkyl, substituted or unsubstituted C1-20heteroalkyl, substituted or unsubstituted C3-20heteroaryl, substituted or unsubstituted C1-20alkylamido, substituted or unsubstituted C6-20arylamido, substituted or unsubstituted C1-20alkylidene, or substituted or unsubstituted C1-20silyl. Here, at least one of R1and R2is independently linked to adjacent groups to form a substituted or unsubstituted saturated or unsaturated C4-20ring. In addition, R3to R7may each independently be linked to adjacent groups to form a substituted or unsubstituted saturated or unsaturated C4-20ring. Specifically, at least one of R1and R2may independently be linked to adjacent groups to form a substituted or unsubstituted saturated or unsaturated C4-20ring. More specifically, R1and R2are each linked to adjacent groups to form an unsubstituted unsaturated C4ring. Specifically, R3may be C1-20alkyl. More specifically, R3may be C1-6alkyl. Preferably, R3is n-butyl. Specifically, R4and R5may each be C1-20alkyl. More specifically, R4and R5may each be C1-6alkyl. Preferably, R4and R5are each t-butyl. Specifically, R6and R7may each be C6-20aryl. More specifically, R6and R7may each be phenyl. In a preferred embodiment of the present invention, the compound represented by Formula A may be a compound represented by Formula A-1. In addition, the compound represented by Formula B may be a compound represented by Formula B-1. The catalyst for olefin polymerization according to an embodiment of the present invention may comprise the first transition metal compound and the second transition metal compound at a weight ratio of 20:1 to 1:20. Preferably, the catalyst for olefin polymerization may comprise the first transition metal compound and the second transition metal compound at a weight ratio of 10:1 to 1:10. More preferably, the catalyst for olefin polymerization may comprise the first transition metal compound and the second transition metal compound at a weight ratio of 6:4 to 4:6. When the content ratio of the first transition metal compound and the second transition metal compound is within the above range, an appropriate activity of the supported catalyst may be exhibited, which may be advantageous from the viewpoint of maintaining the activity of the catalyst and economical efficiency. Further, a polyolefin prepared in the presence of the catalyst for polymerizing olefin, which satisfies the above range, has excellent processability, and a film prepared therefrom may have excellent strength and haze. In general, it is known that polyolefins containing a small amount of short chain branches (SCBs) have poor optical properties, and polyolefins containing a large amount of long chain branches (LCBs) have excessively high elasticity, thereby having poor mechanical properties. Polyolefins prepared by the first transition metal compound alone contain a small amount of short chain branches and are relatively poor in optical properties. Polyolefins prepared by the second transition metal compound alone have a large amount of short chain branches and long chain branches, whereby they are excellent in optical properties, whereas they are relatively poor in mechanical properties. That is, it is confirmed in experimental ways that it is difficult to satisfy both optical and mechanical properties when any of the first transition metal compound and the second transition metal compound is used alone, or when the ratio of either the first transition metal compound or the second transition metal compound is excessively high. In contrast, a catalyst for olefin polymerization, which comprises the first transition metal compound and the second transition metal compound at a weight ratio of 0.4:1 to 2.5:1 can produce a polyolefin having excellent strength and haze. As a preferred example, the catalyst for olefin polymerization according to an embodiment of the present invention may further comprise a cocatalyst compound. Here, the cocatalyst compound may comprise at least one of a compound represented by Formula 1, a compound represented by Formula 2, and a compound represented by Formula 3. In Formula 1, n is an integer of 2 or more, and Ramay each independently be halogen, C1-20hydrocarbon, or C1-20hydrocarbon substituted with halogen. Specifically, Ramay be methyl, ethyl, n-butyl, or isobutyl. In Formula 2, D is aluminum (Al) or boron (B), and Rb, Rc, and Rdare each independently halogen, C1-20hydrocarbon, C1-20hydrocarbon substituted with halogen, or C1-20alkoxy. Specifically, when D is aluminum (Al), Rb, Rc, and Rdmay each independently be methyl or isobutyl, and when D is boron (B), Rb, Rc, and Rdmay each be pentafluorophenyl. [L-H]+[Z(A)4]−or [L]+[Z(A)4]−[Formula 3] In Formula 3, L is a neutral or cationic Lewis acid, [L-H]+and [L]+a Brönsted acid, Z is a group 13 element, and A is each independently substituted or unsubstituted C6-20aryl or substituted or unsubstituted C1-20alkyl. Specifically, [LH]+may be a dimethylanilinium cation, [Z(A)4]−may be [B(C6F5)4]−, and [L]+may be [(C6H5)3C]+. Examples of the compound represented by Formula 1 include methylaluminoxane, ethylaluminoxane, isobutylaluminoxane, butylaluminoxane, and the like. Preferred is methylaluminoxane. But it is not limited thereto. Examples of the compound represented by Formula 2 include trimethylaluminum, triethylaluminum, triisobutylaluminum, tripropylaluminum, tributylaluminum, dimethylchloroaluminum, triisopropylaluminum, tri-s-butylaluminum, tricyclopentylaluminum, tripentylaluminum, triisopentyaluminum, trihexyaluminum, trioctyaluminum, ethyldimethylaluminum, methyldiethylaluminum, triphenylaluminum, tri-p-tolylaluminum, dimethylaluminummethoxide, dimethylaluminumethoxide, trimethylboron, triethylboron, triisobutylboron, tripropylboron, and tributylboron. Preferred are trimethylaluminum, triethylaluminum, and triisobutylaluminum. But it is not limited thereto. Examples of the compound represented by Formula 3 include triethylammonium tetraphenylborate, tributylammonium tetraphenylborate, trimethylammonium tetraphenylborate, tripropylammonium tetraphenylborate, trimethylammonium tetra(p-tolyl)borate, trimethylammonium tetra(o,p-dimethylphenyl)borate, tributylammonium tetra(p-trifluoromethylphenyl)borate, trimethylammonium tetra(p-trifluoromethylphenyl)borate, tributylammonium tetrapentafluorophenylborate, N,N-diethylanilinium tetraphenylborate, N,N-diethylanilinium tetrapentafluorophenylborate, diethylammonium tetrapentafluorophenylborate, triphenylphosphonium tetraphenylborate, trimethylphosphonium tetraphenylborate, triethylammonium tetraphenylaluminate, tributylammonium tetraphenylaluminate, trimethylammonium tetraphenylaluminate, tripropylammonium tetraphenylaluminate, trimethylammonium tetra(p-tolyl)aluminate, tripropylammonium tetra(p-tolyl)aluminate, triethylammonium tetra(o,p-dimethylphenyl)aluminate, tributylammonium tetra(p-trifluoromethylphenyl)aluminate, trimethylammonium tetra(p-trifluoromethylphenyl)aluminate, tributylammonium tetrapentafluorophenylaluminate, N,N-diethylanilinium tetraphenylaluminate, N,N-diethylanilinium tetrapentafluorophenylaluminate, diethylammonium tetrapentatetraphenylaluminate, triphenylphosphonium tetraphenylaluminate, trimethylphosphonium tetraphenylaluminate, tripropylammonium tetra(p-tolyl)borate, triethylammonium tetra(o,p-dimethylphenyl)borate, tributylammonium tetra(p-trifluoromethylphenyl)borate, triphenylcarbonium tetra(p-trifluoromethylphenyl)borate, and triphenylcarbonium tetrapentafluorophenylborate. As a preferred example, the catalyst for olefin polymerization according to an embodiment of the present invention may further comprise a carrier for supporting the first transition metal compound, the second transition metal compound, or both. Preferably, the catalyst for olefin polymerization may further comprise a carrier for supporting all of the first transition metal compound, the second transition metal compound, and the cocatalyst compound. In such an event, the carrier may comprise a material containing a hydroxyl group on its surface. Preferably, a material that has been dried to remove moisture from its surface and has a highly reactive hydroxyl group and a siloxane group may be used. For example, the carrier may comprise at least one selected from the group consisting of silica, alumina, and magnesia. Specifically, silica, silica-alumina, and silica-magnesia dried at high temperatures may be used as a carrier. They usually contain oxides, carbonates, sulfates, and nitrates components such as Na2O, K2CO3, BaSO4, and Mg(NO3)2. In addition, they may comprise carbon, zeolite, magnesium chloride, and the like. However, the carrier is not limited thereto. It is not particularly limited as long as it can support the first and second transition metal compounds and the cocatalyst compound. As a method of supporting the transition metal compounds and the cocatalyst compound employed in a catalyst for olefin polymerization on the carrier, a physical adsorption method or a chemical adsorption method may be used. For example, the physical adsorption method may be a method of contacting a solution in which a transition metal compound has been dissolved with a carrier and then drying the same; a method of contacting a solution in which a transition metal compound and a cocatalyst compound have been dissolved with a carrier and then drying the same; or a method of contacting a solution in which a transition metal compound has been dissolved with a carrier and then drying the same to prepare the carrier that supports the transition metal compound, separately contacting a solution in which a cocatalyst compound has been dissolved with a carrier and then drying the same to prepare the carrier that supports the cocatalyst compound, and then mixing them. The chemical adsorption method may be a method of supporting a cocatalyst compound on the surface of a carrier, and then supporting a transition metal compound on the cocatalyst compound; or a method of covalently bonding a functional group on the surface of a carrier (e.g., a hydroxy group (—OH) on the silica surface in the case of silica) with a catalyst compound. The total amount of the first transition metal compound and the second transition metal compound supported on a carrier may be 0.001 mmole to 1 mmole based on 1 g of the carrier. When the content ratio of the transition metal compounds and the carrier satisfies the above range, an appropriate activity of the supported catalyst may be exhibited, which is advantageous from the viewpoint of maintaining the activity of the catalyst and economical efficiency. The amount of the cocatalyst compound supported on a carrier may be 2 mmoles to 15 mmoles based on the 1 g of the carrier. When the content ratio of the cocatalyst compound and the carrier satisfies the above range, it is advantageous from the viewpoint of maintaining the activity of the catalyst and economical efficiency. One or two or more types of a carrier may be used. For example, both the first transition metal compound and the second transition metal compound may be supported on one type of a carrier, or the first transition metal compound and the second transition metal compound may be supported on two or more types of a carrier, respectively. In addition, either one of the first transition metal compound and the second transition metal compound may be supported on a carrier. Preferably, the catalyst for olefin polymerization may a hybrid supported catalyst in which the first transition metal compound and the second transition metal compound are supported together. More preferably, it may a hybrid supported catalyst in which the first transition metal compound and the second transition metal compound are supported together on a single carrier. For example, the catalyst for olefin polymerization may be a hybrid supported catalyst in which the first transition metal compound, the second transition metal compound, and the cocatalyst compound are supported together on silica. However, the examples of the present invention are not limited thereto. According to another embodiment of the present invention, there is provided a polyolefin prepared by polymerizing an olefinic monomer in the presence of the catalyst for olefin polymerization described above. Here, the polyolefin may be a homopolymer of an olefinic monomer or a copolymer of an olefinic monomer and an olefinic comonomer. The olefinic monomer is at least one selected from the group consisting of a C2-20alpha-olefin, a C1-20diolefin, a C3-20cycloolefin, and a C3-20cyclodiolefin. For example, the olefinic monomer may be ethylene, propylene, 1-butene, 1-pentene, 4-methyl-1-pentene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-undecene, 1-dodecene, 1-tetradecene, 1-hexadecene, or the like, and the polyolefin may be a homopolymer comprising only one olefinic monomer or a copolymer comprising two or more olefinic monomers exemplified above. As an exemplary example, the polyolefin may be a copolymer in which ethylene and a C3-20alpha-olefin are copolymerized. Preferred is a copolymer in which ethylene and 1-hexene are copolymerized. But it is not limited thereto. In such an event, the content of ethylene is preferably 55 to 99.9% by weight, more preferably 90 to 99.9% by weight. The content of the alpha-olefinic comonomer is preferably 0.1 to 45% by weight, more preferably 0.1 to 10% by weight. The polyolefin according to an embodiment of the present invention may be prepared by polymerization reaction such as free radical, cationic, coordination, condensation, and addition, but it is not limited thereto. As a preferred example, the polyolefin may be prepared by a gas phase polymerization method, a solution polymerization method, a slurry polymerization method, or the like. When the polyolefin is prepared by a solution polymerization method or a slurry polymerization method, examples of a solvent that may be used include C5-12aliphatic hydrocarbon solvents such as pentane, hexane, heptane, nonane, decane, and isomers thereof; aromatic hydrocarbon solvents such as toluene and benzene; hydrocarbon solvents substituted with chlorine atoms such as dichloromethane and chlorobenzene; and mixtures thereof, but it is not limited thereto. Polyolefin The polyolefin according to an embodiment of the present invention satisfies (1) a molecular weight distribution represented as a polydispersity index (Mw/Mn) of 5 to 20, (2) a density of 0.910 to 0.930 g/cm3, (3) a melt index of 0.5 to 2.0 g/10 minutes when measured at 190° C. under a load of 2.16 kg, and (4) a melt index ratio (MI21.6/MI2.16) of 20 to 30. The polyolefin is prepared in the presence of the hybrid supported catalyst for olefin polymerization as described above and has a relatively wide molecular weight distribution. Specifically, the polyolefin has a molecular weight distribution represented as a polydispersity index (Mw/Mn) of 5 to 20. Preferably, the molecular weight distribution represented as a polydispersity index (Mw/Mn) of the polyolefin may be 6 to 15. Since the polyolefin has a relatively wide molecular weight distribution, the polyolefin exhibits excellent processability, whereby a film obtained therefrom may have good impact resistance. The polyolefin is a low-density polyethylene copolymer having a density in the range of 0.910 to 0.930 g/cm3. Preferably, the density of the polyolefin is in the range of 0.915 to 0.925 g/cm3. If the density of the polyolefin is within the above range, a film obtained from the polyolefin may have good impact resistance. In the preparation of the polyolefin according to an embodiment of the present invention, the density of the polyolefin may be adjusted by the content of alpha-olefin, preferably 1-hexene, relative to the content of ethylene. For example, the lower the content of alpha-olefin relative to ethylene, the higher the density. The higher the content of alpha-olefin, the lower the density. Thus, a polyolefin having a density within the above range may be prepared by adjusting the content of alpha-olefin relative to the content of ethylene in the polyolefin. The polyolefin of the present invention has a melt index of 0.5 to 2.0 g/10 minutes when measured at 190° C. under a load of 2.16 kg according to ASTM D1238. Preferably, the melt index of the polyolefin is in the range of 0.5 to 1.5 g/10 minutes when measured at 190° C. under a load of 2.16 kg. If the melt index of the polyolefin is within the above range, it is possible to balance the processability of the polyolefin and the mechanical properties of a film obtained therefrom. The polyolefin of the present invention has a melt flow ratio (MFR) of 20 to 30, which is a value obtained by dividing the melt index measured at 190° C. under a load of 21.6 kg by the melt index measured at 190° C. under a load of 2.16 kg according to ASTM D1238. Preferably, the MFR of the polyolefin is in the range of 22 to 26. If the MFR of the polyolefin is within the above range, it exhibits excellent processability and is particularly suitable for preparing a blown film. The polyolefin according to an embodiment of the present invention may have a weight average molecular weight (Mw) of 50,000 to 250,000 g/mole. Preferably, the weight average molecular weight (Mw) may be 70,000 to 150,000 g/mole. Here, the weight average molecular weight is a value measured using gel permeation chromatography (GPC) and converted based on standard polystyrene. If the weight average molecular weight of the polyolefin is within the above range, the mechanical properties of a film produced therefrom may be good. The polyolefin according to an embodiment of the present invention may have a BOCD index of 0 to 3.0. Here, the BOCD index refers to a measure of how many short chain branches having 2 to 6 carbon atoms attached to the main chain of a polymer are present in a relatively high molecular weight component. If the BOCD index is 0 or less, it is not a polymer having a BOCD structure. If it is greater than 0, it may be regarded as a polymer having a BOCD structure. The molecular weight, molecular weight distribution, and content of short chain branches of a polymer may be measured simultaneously and continuously using a GPC-FTIR device. The BOCD index may be calculated by the following Equation 1 by measuring the content of short chain branches (unit: number/1000 C) in the 30% range of left and right (60% in total) in the molecular weight distribution (MWD) based on weight average molecular weight (Mw). BOCD index=(content of short chain branches in the high-molecular weight component−content of short chain branches in the low-molecular weight component)/(content of short chain branches in the low-molecular weight component) [Equation 1] In a polymer having a BOCD structure, tie molecules such as short chain branches are more present in the high molecular weight component that is relatively responsible for physical properties than the low molecular weight component, whereby it may have excellent physical properties such as impact strength. The polyolefin according to an embodiment of the present invention may have a content of long chain branches of 0.01 to 0.1 per 10,000 carbon atoms. A long chain branch refers to a long branch having 7 or more carbon atoms attached to the main chain of a polyolefin. It is usually formed when such an alpha-olefin as 1-butene, 1-hexene, and 1-octene is used as a comonomer. Since long chain branches give rise to the physical effect of filling the voids between polymers, they are known to affect the viscosity and elasticity of a molten polymer in general. If long chain branches increase in the polymer chain, causing an increase in the entanglement of the polymer chain, the intrinsic viscosity at the same molecular weight is lowered, which lowers the load on the screw during extrusion and injection, resulting in better workability. In the present invention, long chain branches of the polyolefin may be measured by the method described in Macromolecules, Vol. 33, No. 29, pp. 7481-7488 (2000). The molecular weight distribution (MWD) value is fitted through the complex viscosity measured using MCR702 of Anton Parr, and the maximum peak value is taken. The maximum value of MWD through 3D-GPC is taken. It is then determined from the ratio thereof whether long chain branching is or not. If the ratio is less than 1, the long chain branching value is 0 (Relationship 1a below). If it exceeds 1, the calculated value of Relationship 1b below is taken. LCB104C=GPCpeakviscositypeak<1,0[Relationship1a]LCB104C=GPCpeakviscositypeak<1,1.125log(GPCpeakviscositypeak)[Relationship1b] The polyolefin according to an embodiment of the present invention is excellent in melt strength. When a film is prepared by blowing air into a molten polyolefin to mold the polyolefin into a blown film, bubble stability refers to a feature that the film thus prepared maintains its shape without tearing. The bubble stability is associated with the melt strength. Melt strength refers to the strength to withstand tension when a polymer in a molten or softened state is processed such as blowing or stretching. The polyolefin of the present invention can exhibit high melt strength since a relatively large number of short chain branches are present in the high molecular weight component, and long chain branches are also attached to the main chain of the polymer. The polyolefin according to an embodiment of the present invention has a c2value of −0.3 to −0.2 when a graph of the complex index (Pa·s) with respect to the frequency (rad/s) is fitted with the power law of the following Equation 2. y=c1xc2[Equation 2] A polymer in a molten state has properties that are in between a fully elastic material and a viscous liquid, which is called viscoelasticity. That is, when a polymer in a molten state is subjected to shear stress, the deformation is not proportional to the shear stress, and the viscosity changes according to the shear stress. These properties are understood to be attributable to the large molecular size and complex intermolecular structure of the polymer. In particular, when a polymer is used to prepare a molded article, the shear thinning phenomenon is of importance. The shear thinning phenomenon refers to a phenomenon in which the viscosity of a polymer decreases as the shear rate increases. Such shear thinning characteristics have a great impact on the molding method of a polymer. Equation 2 above is a model for quantitatively evaluating the shear thinning characteristics of a polyolefin and also for predicting the complex viscosity at a high frequency by applying complex viscosity data with respect to the frequency. In Equation 2, x denotes a frequency, y denotes a complex viscosity, and the two variables c1denotes a consistency index, and c2denotes a CV index, which represents the slope of the graph. The higher the complex viscosity at a low frequency, the better the physical properties, and the lower the complex viscosity at a higher frequency, the better the processability. Thus, the smaller the value of c2, that is, the larger the negative slope of the graph, the more preferable. The complex viscosity with respect to the frequency may be measured using, for example, MCR702 of Anton Parr in a frequency range of 0.1 to 500 rad/s and a strain condition of 5% at 190° C. The polyolefin according to an embodiment of the present invention has a shear thinning index of 10 to 15 as defined by the following Equation 3. Shear thinning index=η0/η500[Equation 3] In Equation 3, η0is the complex viscosity at a frequency of 0.1 rad/s, and η500is the complex viscosity at a frequency of 500 rad/s. The larger the shear thinning index, the higher the complex viscosity at a low frequency and the lower the complex viscosity at a higher frequency. Thus, the physical properties and processability of the polymer may be excellent. Film According to still another embodiment of the present invention, there is provided a film molded from the polyolefin. The film according to an embodiment of the present invention comprises the polyolefin of the present invention, Thus, it is excellent in optical properties such as haze and in mechanical properties such as impact strength. It is understood that since the polyolefin of the present invention has a relatively wide molecular weight distribution, and since short chain branches are relatively more present in the high molecular weight component, a film produced therefrom is excellent in haze and impact resistance. Specifically, the film according to an embodiment of the present invention has a haze of 10% or less and a drop impact strength of 600 g or more. As an exemplary example, the film of the present invention has a haze of 8% or less, preferably 7% or less, and more preferably 6.5% or less. In addition, the film of the present invention has a drop impact strength of 650 g or more, preferably 700 g or more, and more preferably 800 g or more. There is no particular limitation to the method for producing a film according to the embodiment of the present invention, and any method known in the technical field of the present invention can be used. For example, the polyolefin according to an embodiment of the present invention may be molded by a conventional method such as blown film molding, extrusion molding, casting molding, or the like to prepare a film. Blown film molding among the above is the most preferred. EMBODIMENTS FOR CARRYING OUT THE INVENTION Hereinafter, the present invention is explained in detail with reference to the following examples and comparative examples. However, the following examples are intended to further illustrate the present invention. The scope of the present invention is not limited thereto only. Preparation Example 1 The transition metal compound of Formula A-1 purchased from sPCI was used without purification, and the transition metal compound of Formula B-1 was purchased from MCN and used without further purification. 2.7387 g of the compound of Formula A-1 and 3.3741 g of the compound of Formula B-1 were mixed with 991.69 g of a toluene solution of 10% by weight of methylaluminumoxane (MAO) (Al/Zr=150) in a glove box, which was stirred at room temperature for 1 hour. Meanwhile, 250 g of silica (XP2402) was charged to a reactor, and 500 ml of purified toluene was added thereto, followed by mixing thereof. Thereafter, the transition metal compounds solution was injected into the silica slurry, which was stirred in an oil bath at 75° C. for 3 hours. The supported catalyst was washed three times with toluene and dried at 60° C. under vacuum for 30 minutes to obtain 355 g of a hybrid supported catalyst in the form of a free-flowing powder. Preparation Example 2 356 g of a hybrid supported catalyst was obtained in the same manner as in Preparation Example 1, except that 1.8310 g of the compound of Formula A-1 and 5.0754 g of the compound of Formula B-1 were used. Preparation Example 3 359 g of a hybrid supported catalyst was obtained in the same manner as in Preparation Example 1, except that 8.5302 g of the compound of Formula B-1 alone was used. Example 1 An ethylene/1-hexene copolymer was prepared in the presence of the hybrid supported catalyst obtained in Preparation Example 1 in a fluidized-bed gas-phase reactor. The temperature in the reactor was maintained in the range of 80 to 90° C., and the degree of polymerization of the ethylene/1-hexene copolymer prepared was adjusted by adding hydrogen in addition to ethylene and 1-hexene. Subsequently, the ethylene/1-hexene copolymer was extruded in an extruder having a screw of 40 mm in diameter, a die of 75 mm in diameter, and a die gap of 2 mm at a screw speed of 80 rpm, and it was then subjected to blown film molding at a blow-up ratio of 2.0 to obtain a film having a thickness of 50 μm. Example 2 An ethylene/1-hexene copolymer was prepared in the same manner as in Example 1, except that the hybrid supported catalyst obtained in Preparation Example 2 was used. Subsequently, it was molded in the same manner as in Example 1 to obtain a film having a thickness of 50 μm. Comparative Example 1 An ethylene/1-hexene copolymer was prepared in the same manner as in Example 1, except that the supported catalyst obtained in Preparation Example 3 was used. Subsequently, it was molded in the same manner as in Example 1 to obtain a film having a thickness of 50 μm. Comparative Example 2 A linear low-density polyethylene (M1810HN) of Hanwha Chemical Corp. manufactured with a single metallocene catalyst was used. This resin was molded in the same manner as in Example 1 to obtain a film having a thickness of 50 μm. The reaction conditions such as the pressure of ethylene in the reactor and the molar ratio of the raw material gases added in Examples 1 and 2 and Comparative Example 1 are as shown in Table 1 below. TABLE 1EthyleneMolar ratio ofMolar ratio ofpressure1-hexene/ethylenehydrogen/ethyleneCatalytic activity(bar)(%)(%)(gPE/gCat · hr)Ex. 113.91.021.454,500Ex. 213.11.340.634,870C. Ex. 113.20.990.766,500 Test Example The physical properties of the resins and films prepared in the Examples and the Comparative Examples were measured according to the following methods and standards. The results are shown in Tables 2 and 3 below. (1) Melt Index It was measured at 190° C. under a load of 2.16 kg in accordance with ASTM D1238. (2) Melt Flow Ratio (MFR) It was measured at 190° C. under a load of 2.16 kg and 21.6 kg in accordance with ASTM D1238. Their ratio (MI21.6/MI2.16) was calculated. (3) Density It was measured in accordance with ASTM D638. (4) Molecular Weight and Molecular Weight Distribution They were measured using gel permeation chromatography-FTIR (GPC-FTIR). (5) BOCD Index It was measured using gel permeation chromatography-FTIR (GPC-FTIR). (6) Number of Long Chain Branches (LCB) The molecular weight distribution (MWD) value is fitted through the complex viscosity measured using MCR702 of Anton Parr, and the maximum peak value was taken. The maximum value of MWD through 3D-GPC was taken. The number of long chain branches was calculated from the ratio using Equations 1a and 1b above. (7) Complex Viscosity with Respect to Frequency It was measured using MCR702 of Anton Parr in a frequency range of 0.1 to 500 rad/s and a strain condition of 5% at 190° C. (8) Film Processing and Extrusion Load A blown film was prepared in a film processing machine having a die of 75 mmΦ and a die gap of 2 mm using a screw of 40 mmΦ. The screw speed was set to 80 rpm, and the blow-up ratio (BUR) was fixed to 2 to process a film having a thickness of 50 μm, and the extrusion load at that time was measured. (9) Drop Impact Strength (B-Type) It was measured in accordance with ASTM D1790. (10) Elmendorf Tear Strength It was measured in the machine direction (MD) and the transverse direction (TD) in accordance with ASTM D1922. (11) Tensile Strength It was measured in the machine direction (MD) and the transverse direction (TD) in accordance with ASTM D882. (12) Haze Haze of the blown film was measured in accordance with ASTM D1003. TABLE 2Properties ofresinUnitEx. 1Ex. 2C. Ex. 1C. Ex. 2MIg/10 min0.91.00.91.1MFR—22261916Densityg/cm30.9200.9200.9190.919Mng/mole13,7007,10036,70041,900Mwg/mole116,000100,300113,500109,700MWD—8.514.03.12.6BOCD index—0.290.500.140.16No. of LCDCount/1,000 C0.040.050.050CV index (c2)—−0.26447−0.28252−0.24333−0.20848Shear thinning—10.8812.69.106.51indexExtrusion loadA26.024.527.528.5 TABLE 3Properties of resinUnitEx. 1Ex. 2C. Ex. 1C. Ex. 2Drop impact strengthg800>1,000510650Elmendorf tearMDg320420490490strengthTDG820910900700TensileMDkg/cm2430450390510strengthTDkg/cm2500570460520Haze%4.96.110.212.2 As can be seen from Tables 2 and 3 andFIGS.1to3, the polyolefins prepared in the presence of the hybrid supported catalyst prepared in the Examples of the present invention had a wide molecular weight distribution, and short chain branches were relatively more present in the high molecular weight component. They also had long chain branches. By virtue of such structural characteristics, not only is the processability of the polyolefins excellent, but also such mechanical properties as drop impact strength and such optical properties as haze are excellent as compared with the films prepared from the polyolefins of Comparative Examples. INDUSTRIAL APPLICABILITY Accordingly, the hybrid supported catalyst according to the embodiment of the present invention can provide a polyolefin that has excellent processability, impact strength, and haze. The film made of this polyolefin can be advantageously used as a stretch film, an overlap film, a ramie, a silage wrap, an agricultural film, and the like. | 34,255 |
11859041 | DETAILED DESCRIPTION OF THE DISCLOSURE Among other things, this disclosure provides new methods for the design and development of ethylene polymerization catalysts, including Group 4 metallocene catalysts such as zirconocene catalysts, which are based on an improved ability to adjust or modulate co-monomer selectivity into the growing polymer chain. In an aspect, it has been unexpectedly discovered through computational analysis of catalysts that stabilizing non-covalent dispersion type interactions can be used to modulate co-monomer selectivity into the polyethylene chain. By designing catalysts that incorporate a greater number of such stabilizing non-covalent dispersion type interactions, α-olefin co-monomer incorporation can be enhanced. Definitions To define more clearly the terms used herein, the following definitions are provided, and unless otherwise indicated or the context requires otherwise, these definitions are applicable throughout this disclosure. If a term is used in this disclosure but is not specifically defined herein, the definition from the IUPAC Compendium of Chemical Terminology, 2ndEd (1997) can be applied, as long as that definition does not conflict with any other disclosure or definition applied herein, or render indefinite or non-enabled any claim to which that definition is applied. To the extent that any definition or usage provided by any document incorporated herein by reference conflicts with the definition or usage provided herein, the definition or usage provided herein controls. Regarding claim transitional terms or phrases, the transitional term “comprising”, which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. A “consisting essentially of” claim occupies a middle ground between closed claims that are written in a “consisting of” format and fully open claims that are drafted in a “comprising” format. Unless specified to the contrary, describing a compound or composition “consisting essentially of” is not to be construed as “comprising,” but is intended to describe the recited component that includes materials which do not significantly alter composition or method to which the term is applied. For example, a feedstock consisting essentially of a material A can include impurities typically present in a commercially produced or commercially available sample of the recited compound or composition. When a claim includes different features and/or feature classes (for example, a method step, feedstock features, and/or product features, among other possibilities), the transitional terms comprising, consisting essentially of, and consisting of, apply only to feature class to which is utilized and it is possible to have different transitional terms or phrases utilized with different features within a claim. For example, a method can comprise several recited steps (and other non-recited steps) but utilize a catalyst composition preparation consisting of specific steps but utilize a catalyst composition comprising recited components and other non-recited components. While compositions, processes, and computational methods are described in terms of “comprising” various components or steps, the compositions, processes, and computational methods can also “consist essentially of” or “consist of” the various components or steps. The terms “a,” “an,” and “the” are intended, unless specifically indicated otherwise, to include plural alternatives, e.g., at least one. For instance, the disclosure of “an organoaluminum compound” is meant to encompass one organoaluminum compound, or mixtures or combinations of more than one organoaluminum compound unless otherwise specified. For any particular compound disclosed herein, a general structure or name presented is also intended to encompass all structural isomers, conformational isomers, and stereoisomers that can arise from a particular set of substituents, unless indicated otherwise. Thus, a general reference to a compound includes all structural isomers unless explicitly indicated otherwise; e.g., a general reference to pentane includes n-pentane, 2-methyl-butane, and 2,2-dimethyl-propane, while a general reference to a butyl group includes an n-butyl group, a sec-butyl group, an iso-butyl group, and a tert-butyl group. Additionally, the reference to a general structure or name encompasses all enantiomers, diastereomers, and other optical isomers whether in enantiomeric or racemic forms, as well as mixtures of stereoisomers, as the context permits or requires. For any particular formula or name that is presented, any general formula or name presented also encompasses all conformational isomers, regioisomers, and stereoisomers that can arise from a particular set of substituents. Unless otherwise specified, any carbon-containing group for which the number of carbon atoms is not specified can have, according to proper chemical practice, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms, or any range or combination of ranges between these values. For example, unless otherwise specified or unless the context requires otherwise, any carbon-containing group can have from 1 to 30 carbon atoms, from 1 to 25 carbon atoms, from 1 to 20 carbon atoms, from 1 to 15 carbon atoms, from 1 to 10 carbon atoms, or from 1 to 5 carbon atoms, and the like. In an aspect, the context could require other ranges or limitations, for example, when the subject carbon-containing group is an aryl group or an alkenyl group, the lower limit of carbons in these subject groups is six carbon atoms and two carbon atoms, respectively. Moreover, other identifiers or qualifying terms may be utilized to indicate the presence or absence of a particular substituent, a particular regiochemistry and/or stereochemistry, or the presence of absence of a branched underlying structure or backbone, and the like. Various numerical ranges are disclosed herein. When Applicant discloses or claims a range of any type, Applicant's intent is to disclose or claim individually each possible number that such a range could reasonably encompass, including end points of the range as well as any sub-ranges and combinations of sub-ranges encompassed therein, unless otherwise specified. For example, by disclosing that a bond angle can be from 900 to 100°, Applicant's intent is to recite individually 90°, 91°, 92°, 93°, 94°, 95°, 96°, 99°, 98°, 99°, and 100°, including any sub-ranges and combinations of sub-ranges encompassed therein, and these methods of describing such ranges are interchangeable. Moreover, all numerical end points of ranges disclosed herein are approximate, unless excluded by proviso. As a representative example, if Applicant states that one or more steps in the processes disclosed herein can be conducted at a temperature in a range from 10° C. to 75° C., this range should be interpreted as encompassing temperatures in a range from “about” 10° C. to “about” 75° C. Values or ranges may be expressed herein as “about”, from “about” one particular value, and/or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In another aspect, use of the term “about” means ±15% of the stated value, ±10% of the stated value, 5% of the stated value, 3% of the stated value, ±2% of the stated value, or +1% of the stated value. Applicant reserves the right to proviso out or exclude any individual members of any such group of values or ranges, including any sub-ranges or combinations of sub-ranges within the group, that can be claimed according to a range or in any similar manner, if for any reason Applicant chooses to claim less than the full measure of the disclosure, for example, to account for a reference that Applicant may be unaware of at the time of the filing of the application. Further, Applicant reserves the right to proviso out or exclude any individual substituents, analogs, compounds, ligands, structures, or groups thereof, or any members of a claimed group, if for any reason Applicant chooses to claim less than the full measure of the disclosure, for example, to account for a reference or prior disclosure that Applicant may be unaware of at the time of the filing of the application. The term “substituted” when used to describe a group, for example, when referring to a substituted analog of a particular group, is intended to describe any non-hydrogen moiety that formally replaces a hydrogen in that group, and is intended to be non-limiting. A group or groups can also be referred to herein as “unsubstituted” or by equivalent terms such as “non-substituted,” which refers to the original group in which a non-hydrogen moiety does not replace a hydrogen within that group. Unless otherwise specified, “substituted” is intended to be non-limiting and include inorganic substituents or organic substituents as understood by one of ordinary skill in the art. Specific chemical groups may be specified according to the atom which is bonded to the metal or bonded to another chemical moiety as a substituent, such as an “oxygen-bonded group,” which is also called an “oxygen group.” For example, an oxygen-bonded group includes species such as hydrocarbyloxide (—OR where R is a hydrocarbyl group, also termed hydrocarboxy), alkoxide (—OR where R is an alkyl group), aryloxide (—OAr where Ar is an aryl group), or substituted analogs thereof, which function as ligands or substituents in the specified location. Therefore, an alkoxide group and an aryloxide group are each a subgenus of a hydrocarbyloxide (hydrocarbyloxy) group. A similar definition applies to chemical groups which may be specified according to the atom which is bonded to the metal or bonded to another chemical moiety as a substituent, in which the free valence is situated on a heteroatom (non-carbon atom), such as a “sulfur group,” “nitrogen group,” “phosphorus group,” “arsenic group,” “silicon group,” “germanium group,” “tin group,” “lead group,” “boron group,” “aluminum group,” and the like. A chemical “group” also may be described according to how that group is formally derived from a reference or “parent” compound, for example, by the number of hydrogen atoms formally removed from the parent compound to generate the group, even if that group is not literally synthesized in this manner. These groups can be utilized as substituents or coordinated or bonded to metal atoms. For example, an “alkyl group” formally can be derived by removing one hydrogen atom from an alkane, while an “alkanediyl group” (also referred to as a “alkylene group”) formally can be derived by removing two hydrogen atoms from an alkane. Moreover, a more general term can be used to encompass a variety of groups that formally are derived by removing any number (“one or more”) of hydrogen atoms from a parent compound, which in this example can be described as an “alkane group,” which encompasses an “alkyl group,” an “alkanediyl group,” and materials have three or more hydrogen atoms, as necessary for the situation, removed from the alkane. The disclosure that a substituent, ligand, or other chemical moiety can constitute a particular “group” implies that the known rules of chemical structure and bonding are followed when that group is employed as described. When describing a group as being “derived by,” “derived from,” “formed by,” or “formed from,” such terms are used in a formal sense and are not intended to reflect any specific synthetic method or procedure, unless specified otherwise or the context requires otherwise. The term “organyl group” is used herein in accordance with the definition set forth by IUPAC: an organic substituent group, regardless of functional type, having one free valence at a carbon atom. Similarly, an “organylene group” refers to an organic group, regardless of functional type, derived by removing two hydrogen atoms from an organic compound, either two hydrogen atoms from one carbon atom or one hydrogen atom from each of two different carbon atoms. An “organic group” refers to a generalized group formed by removing one or more hydrogen atoms from carbon atoms of an organic compound. Thus, an “organyl group,” an “organylene group,” and an “organic group” can contain organic functional group(s) and/or atom(s) other than carbon and hydrogen, that is, an organic group that can comprise functional groups and/or atoms in addition to carbon and hydrogen. For instance, non-limiting examples of atoms other than carbon and hydrogen include halogens, oxygen, nitrogen, phosphorus, and the like. Non-limiting examples of functional groups include ethers, aldehydes, ketones, esters, sulfides, amines, and phosphines, and so forth. In one aspect, the hydrogen atom(s) removed to form the “organyl group,” “organylene group,” or “organic group” can be attached to a carbon atom belonging to a functional group, for example, an acyl group (—C(O)R), a formyl group (—C(O)H), a carboxy group (—C(O)OH), a hydrocarboxycarbonyl group (—C(O)OR), a cyano group (—C≡N), a carbamoyl group (—C(O)NH2), a N-hydrocarbylcarbamoyl group (—C(O)NHR), or N,N′-dihydrocarbylcarbamoyl group (—C(O)NR2), among other possibilities. In another aspect, the hydrogen atom(s) removed to form the “organyl group,” “organylene group,” or “organic group” can be attached to a carbon atom not belonging to, and remote from, a functional group, for example, —CH2C(O)CH3, —CH2NR2, and the like. An “organyl group,” “organylene group,” or “organic group” can be aliphatic, inclusive of being cyclic or acyclic, or can be aromatic. “Organyl groups,” “organylene groups,” and “organic groups” also encompass heteroatom-containing rings, heteroatom-containing ring systems, heteroaromatic rings, and heteroaromatic ring systems. “Organyl groups,” “organylene groups,” and “organic groups” can be linear or branched unless otherwise specified. Finally, it is noted that the “organyl group,” “organylene group,” or “organic group” definitions include “hydrocarbyl group,” “hydrocarbylene group,” “hydrocarbon group,” respectively, and “alkyl group,” “alkylene group,” and “alkane group,” respectively, (among others known to those having ordinary skill in the art) as members. When bonded to a transition metal, an “organyl group,” “organylene group,” or “organic group” can be further described according to the usual 11 (eta-x) nomenclature, in which x is an integer corresponding to the number of atoms which are coordinated to the transition metal or are expected to be coordinated to the transition metal, for example, according to the 18-electron rule. The term “hydrocarbon” whenever used in this specification and claims refers to a compound containing only carbon and hydrogen. Other identifiers can be utilized to indicate the presence of particular groups in the hydrocarbon (e.g., halogenated hydrocarbon indicates that the presence of one or more halogen atoms replacing an equivalent number of hydrogen atoms in the hydrocarbon). The term “hydrocarbyl” group is used herein in accordance with the definition specified by IUPAC as follows: a univalent group formed by removing a hydrogen atom from a hydrocarbon (that is, a group containing only carbon and hydrogen). Non-limiting examples of hydrocarbyl groups include ethyl, phenyl, tolyl, propenyl, cyclopentyl, and the like. The term “hydrocarbylene” group is also used herein in accordance with the definition specified by IUPAC as follows: a “hydrocarbylene” group refers to a divalent group formed by removing two hydrogen atoms from a hydrocarbon or a substituted hydrocarbon, the free valencies of which are not engaged in forming a double bond. By way of example and comparison, examples of hydrocarbyl and hydrocarbylene groups include, respectively: aryl and arylene; alkyl and alkanediyl (or “alkylene”); cycloalkyl and cycloalkanediyl (or “cycloalkylene”); aralkyl and aralkanediyl (or “aralkylene”); and so forth. For example, an “arylene” group is used in accordance with IUPAC definition to refer to a bivalent group derived from arenes by removal of a hydrogen atom from two ring carbon atoms, which may also be termed an “arenediyl” group. Examples of hydrocarbylene groups include but are not limited to: 1,2-phenylene; 1,3-phenylene; 1,2-propandiyl; 1,3-propandiyl; 1,2-ethandiyl; 1,4-butandiyl; 2,3-butandiyl; and methylene (—CH2—). The term “heterohydrocarbyl” group is used herein to refer to a univalent group, which can be linear, branched or cyclic, formed by removing a single hydrogen atom from a heteroatom of a parent “heterohydrocarbon” molecule, the heterohydrocarbon molecule being one in which at least one carbon atom is replaced by a heteroatom. Therefore, a “heteroatom” refers to a non-carbon atom such as oxygen, sulfur, nitrogen, phosphorus, silicon, and the like. Examples of “heterohydrocarbyl” groups formed by removing a single hydrogen atom from a heteroatom of a heterohydrocarbon molecule include, for example: [1] a hydrocarbyloxide group, for example, an alkoxide (—OR) group such as tert-butoxide or aryloxide (—OAr) group such as a substituted or unsubstituted phenoxide formed by removing the hydrogen atom from the hydroxyl (OH) group of a parent alcohol or a phenol molecule; [2] a hydrocarbylsulfide group, for example, an alkylthiolate (—SR) group or arylthiolate (—SAr) group formed by removing the hydrogen atom from the hydrogensulfide (—SH) group of an alkylthiol or arylthiol; [3] a hydrocarbylamino group, for example, an alkylamino (—NHR) group or arylamino (—NHAr) group formed by removing a hydrogen atom from the amino (—NH2) group of an alkylamine or arylamine molecule; and [4] a trihydrocarbylsilyl group such as trialkylsilyl (—SiR3) or triarylsilyl (—SiAr3) group. An “aliphatic” compound is a class of acyclic or cyclic, saturated or unsaturated, carbon compounds, excluding aromatic compounds, e.g., an aliphatic compound is a non-aromatic organic compound. An “aliphatic group” is a generalized group formed by removing one or more hydrogen atoms (as necessary for the particular group) from a carbon atom of an aliphatic compound. Aliphatic compounds and therefore aliphatic groups can contain organic functional group(s) and/or atom(s) other than carbon and hydrogen. The term “olefin” is used herein in accordance with the definition specified by IUPAC: acyclic and cyclic hydrocarbons having one or more carbon-carbon double bonds apart from the formal ones in aromatic compounds. Thus, the term “olefin” includes aliphatic and aromatic, acyclic and cyclic, and/or linear and branched compounds having at least one carbon-carbon double bond that is not part of an aromatic ring or ring system unless specifically stated otherwise. The class “olefins” subsumes alkenes and cycloalkenes and the corresponding polyenes. Ethylene, propylene, 1-butene, 2-butene, 1-hexene and the like are non-limiting examples of olefins. The term “alpha olefin” as used in this specification and claims refers to an olefin that has a double bond between the first and second carbon atom of the longest contiguous chain of carbon atoms. The term “alpha olefin” includes linear and branched alpha olefins unless expressly stated otherwise. With respect to the olefin oligomerization reactions of this disclosure, the computational and reaction studies are conducted with ethylene, so use of the term “olefin” generally refers to ethylene, unless the context of the disclosure allows or requires otherwise. According to the context of the disclosure, and unless otherwise specified, the abbreviations which designate a carbon count, such as “C6” or “C6”, can be used to refer to all hydrocarbon compounds having the specified number of carbon atoms, such as six carbon atoms designated here. An “aromatic group” refers to a generalized group formed by removing one or more hydrogen atoms (as necessary for the particular group and at least one of which is an aromatic ring carbon atom) from an aromatic compound. Thus, an “aromatic group” as used herein refers to a group derived by removing one or more hydrogen atoms from an aromatic compound, that is, a compound containing a cyclically conjugated hydrocarbon that follows the Hückel (4n+2) rule and containing (4n+2) pi-electrons, where n is an integer from 1 to about 5. Aromatic compounds and hence “aromatic groups” may be monocyclic or polycyclic unless otherwise specified. Aromatic compounds include “arenes” (hydrocarbon aromatic compounds) and “heteroarenes,” also termed “hetarenes” (heteroaromatic compounds formally derived from arenes by replacement of one or more methine (—C═) carbon atoms by trivalent or divalent heteroatoms, in such a way as to maintain the continuous pi-electron system characteristic of aromatic systems and a number of out-of-plane pi-electrons corresponding to the Hückel rule (4n+2)). While arene compounds and heteroarene compounds are mutually exclusive members of the group of aromatic compounds, a compound that has both an arene group and a heteroarene group that compound generally is considered a heteroarene compound. Aromatic compounds, arenes, and heteroarenes may be mono- or polycyclic unless otherwise specified. Examples of arenes include, but are not limited to, benzene, naphthalene, and toluene, among others. Examples of heteroarenes include, but are not limited to furan, pyridine, and methylpyridine, among others. As disclosed herein, the term “substituted” may be used to describe an aromatic group wherein any non-hydrogen moiety formally replaces a hydrogen in that group, and is intended to be non-limiting. An arene is an aromatic hydrocarbon, with or without side chains (e.g., benzene, toluene, or xylene, among others). An “aryl group” is a group derived from the formal removal of a hydrogen atom from an aromatic hydrocarbon ring carbon atom from an arene compound. One example of an “aryl group” is ortho-tolyl (o-tolyl), the structure of which is shown here. The arene can contain a single aromatic hydrocarbon ring (e.g., benzene or toluene), contain fused aromatic rings (e.g., naphthalene or anthracene), and contain one or more isolated aromatic rings covalently linked via a bond (e.g., biphenyl) or non-aromatic hydrocarbon group(s) (e.g., diphenylmethane). A “heterocyclic compound” is a cyclic compound having at least two different elements as ring member atoms. For example, heterocyclic compounds may comprise rings containing carbon and nitrogen (for example, tetrahydropyrrole), carbon and oxygen (for example, tetrahydrofuran), or carbon and sulfur (for example, tetrahydrothiophene), among others. Heterocyclic compounds and heterocyclic groups may be either aliphatic or aromatic. An “aralkyl group” is an aryl-substituted alkyl group having a free valance at a non-aromatic carbon atom, for example, a benzyl group and a 2-phenylethyl group are examples of an “aralkyl” group. A “halide” has its usual meaning; therefore, examples of halides include fluoride, chloride, bromide, and iodide The term “co-catalyst” is used generally herein to refer to compounds such as organoaluminum compounds, organoboron compounds, organozinc compounds, organomagnesium compounds, organolithium compounds, and the like, that can constitute one component of a catalyst composition, when used, for example, with the Group 4 metallocene compounds such as zirconocenes of the disclosure. The term “co-catalyst” is used regardless of the actual function of the compound or any chemical mechanism by which the compound may operate. The terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, do not depend upon the actual product or composition resulting from the contact or reaction of the initial components of the claimed catalyst composition/mixture/system, the nature of the active catalytic site, or the fate of the co-catalyst, the transition metal catalyst compound(s), any olefin monomer used in the catalytic reaction, and the like. Therefore, the terms “catalyst composition,” “catalyst mixture.” “catalyst system,” and the like, encompass the initial starting components of the composition, as well as whatever product(s) may result from contacting these initial starting components, and this is inclusive of both heterogeneous and homogenous catalyst systems or compositions. The terms “catalyst composition,” “catalyst mixture,” “catalyst system,” and the like, are used interchangeably throughout this disclosure. An “organoaluminum compound,” is used to describe any compound that contains an aluminum-carbon bond. Thus, organoaluminum compounds include, but are not limited to, hydrocarbyl aluminum compounds such as trihydrocarbyl-, dihydrocarbyl-, or monohydrocarbylaluminum compounds; hydrocarbylaluminum halide compounds; hydrocarbylalumoxane compounds; and aluminate compounds which contain an aluminum-organyl bond such as tetrakis(p-tolyl)aluminate salts. An “organoboron” compound, an “organozinc compound,” an “organomagnesium compound,” and an “organolithium compound” are used in an analogous fashion to describe any compound that contains a direct metal-carbon bond between an organic group and the recited metal. References to gaseous, liquid, and/or solid materials refer to the physical state of the material at 25° C. and atmospheric pressure. Features within this disclosure that are provided as minimum values can be alternatively stated as “at least” or “greater than or equal to” any recited minimum value for the feature disclosed herein. Features within this disclosure that are provided as maximum values can be alternatively stated as “less than or equal to” for the feature disclosed herein. Within this disclosure, the normal rules of organic nomenclature will prevail. For instance, when referencing substituted compounds or groups, references to substitution patterns are taken to indicate that the indicated group(s) is (are) located at the indicated position and that all other non-indicated positions are hydrogen. For example, reference to a 4-substituted phenyl group indicates that there is a non-hydrogen substituent located at the 4 position and hydrogens located at the 2, 3, 5, and 6 positions. By way of another example, reference to a 3-substituted naphth-2-yl indicates that there is a non-hydrogen substituent located at the 3 position and hydrogens located at the 1, 4, 5, 6, 7, and 8 positions. References to compounds or groups having substitutions at positions in addition to the indicated position will be referenced using comprising or some other alternative language. For example, a reference to a phenyl group comprising a substituent at the 4 position refers to a group having a non-hydrogen atom at the 4 position and hydrogen or any non-hydrogen group at the 2, 3, 5, and 6 positions. Unless otherwise specified, the terms contacted, combined, and “in the presence of” refer to any addition sequence, order, or concentration for contacting or combining two or more components of the polymerization process. Combining or contacting of polymerization components, according to the various methods described herein, can occur in one or more contact zones under suitable contact conditions such as temperature, pressure, contact time, flow rates, etc. The contact zone can be disposed in a vessel (e.g., storage tank, tote, container, mixing vessel, reactor, etc.), a length of pipe (e.g., tee, inlet, injection port, or header for combining component feed lines into a common line), or any other suitable apparatus for bringing the components into contact. The processes can be carried out in a batch or continuous process as can be suitable for a given embodiment. Use of the term “optionally” with respect to any element of a claim is intended to mean that the subject element is required, or alternatively, is not required. Both alternatives are intended to be within the scope of the claim. Processes described herein can utilize steps, features, compounds and/or equipment which are independently described herein. The processes described herein may or may not utilize step identifiers (e.g., 1), 2), etc., a), b), etc., i), ii), etc., or first, second etc., among others), feature identifiers (e.g., 1), 2), etc., a), b), etc., i), ii), etc., or first, second etc., among others), and/or compound and/or composition identifiers (e.g., 1), 2), etc., a), b), etc., i), ii), etc., or first, second etc., among others). However, it should be noted that processes described herein can have multiple steps, features (e.g., reagent ratios, formation conditions, among other considerations), and/or multiple compounds and/or compositions using no descriptor or sometimes having the same general identifier. Consequently, it should be noted that the processes described herein can be modified to use an appropriate step or feature identifier (e.g., 1), 2), etc., a), b), etc., i), ii), etc., or first, second etc., among others), feature identifier (e.g., 1), 2), etc., a), b), etc., i), ii), etc., or first, second etc., among others), and/or compound identifier (e.g., first, second, etc.) regardless of step, feature, and/or compound identifier utilized in a particular aspect and/or embodiment described herein and that step or feature identifiers can be added and/or modified to indicate individual different steps/features/compounds utilized within the processes without detracting from the general disclosure. All publications and patents mentioned herein are incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which might be used in connection with the presently described invention. The publications discussed throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. Method Description In designing new catalysts, although there has been no single general strategy for computational catalyst design or optimization, one approach which has emerged uses ground-state ligand properties to develop structure-activity relationships. This approach has the advantage of being rapid and identifying chemical connections between seemingly unrelated ligands, however, this approach does not generally lead to the prediction and experimental testing of specific catalysts. Similarly, numerical or data science analysis approaches such as multivariate linear regression and machine learning methods, also generally do not identify chemical principles that enable further catalyst designs. Even quantum mechanical calculations which may screen and rank new catalysts are often limited by the computational resource costs required to calculate large structures and can be ineffective in cases where many conformations exist and there is no clear method to prioritize ligand screening. Therefore, cases in which specific organometallic catalyst predictions have been made followed by experimental realization are elusive. Non-hydrogen bonding non-covalent dispersion interactions are instantaneously induced attractive dipole-dipole interactions due to electronic fluctuation, and are typically modelled by electronic excitation (electron correlation) to generate the induced dipoles. In transition-metal catalysis, our notion that large ligands have the potential to act as stabilizing (especially those with 7-systems), rather than steric repulsive agents, through stabilizing non-covalent dispersion type interactions provided a design platform for new olefin polymerization catalysts with enhanced co-monomer selectivity. The inclusion of dispersion interaction energies in olefin migratory insertion transition states, was found to result in significantly tighter correlation with experiment with the metallocenes catalysts has not been recognized previously, and this aspect generally has been discouraged and taught away from in prior studies. For example, some studies showed that the inclusion of semiempirical dispersion resulted in an even greater deviation from experiment for gas phase ligand dissociation energies. See: Weymuth, T.; Couzijn, E. P. A.; Chen, P.; Reiher, M. New Benchmark Set of Transition-Metal Coordination Reactions for the Assessment of Density Functionals.J. Chem. Theory Comput.2014, 10 (8), 3092-3103; Husch, T.; Freitag, L.; Reiher, M. Calculation of Ligand Dissociation Energies in Large Transition-Metal Complexes.J. Chem. Theory Comput.2018, 14 (5), 2456-2468. Others suggested that including explicit dispersion results in overstabilization for BDA and COD ligand substitution by phosphines on Fe and Ru metal centers (BDA=benzylideneacetone, COD=cyclooctadiene). See Jacobsen, H.; Cavallo, L. On the Accuracy of DFT Methods in Reproducing Ligand Substitution Energies for Transition Metal Complexes in Solution: The Role of Dispersive Interactions.ChemPhysChem2012, 13 (2), 562-569. Still others showed that solvation rather than dispersion was the largest factor for deviation from experiment. See: Grimme, S. Comment on: “On the Accuracy of DFT Methods in Reproducing Ligand Substitution Energies for Transition Metal Complexes in Solution: The Role of Dispersive Interactions” by H. Jacobsen and L. Cavallo.ChemPhysChem2012, 13 (6), 1407-1409; Jacobsen, H.; Cavallo, L. Reply to the Comment by Grimme on: “On the Accuracy of DFT Methods in Reproducing Ligand Substitution Energies for Transition Metal Complexes in Solution: The Role of Dispersive Interactions.”ChemPhysChem2012, 13 (6), 1405-1406. Scheme 1 outlines and illustrates at (A) the use of metallocenes as ethylene/linear α-olefin (1-alkene) co-monomer polyethylene polymerization catalysts and the goal of developing new catalyst designs to enable modulation of branching to form short-chain branched polyethylene. Scheme 1 also outlines and illustrates at (B) the computational screening of new metallocene catalysts which provide one or more stabilizing non-covalent dispersion type interactions which can enhance co-monomer selectivity and incorporation into the polyethylene chain. Previous or conventional designs of metallocene olefin polymerization catalysts have centered around the metal center and the repulsive steric influences from the cycloalkadienyl (cyclopentadienyl-type) ligands. In contrast, the general catalyst design disclosed herein is based upon a transition state model which can computationally screen ligands in order to identify new catalyst candidates which can modulate co-monomer incorporation. In an aspect, it has been found for an experimentally validated test set of ethylene and propylene co-polymerization using zirconocene catalysts that determining non-covalent dispersion stabilization can provide quantitative accuracy in providing and validating co-monomer selectivity. In still a further aspect of the disclosure, computational screening may lead to the identification of Group 4 metallocene ligands, particularly zirconocene ligands, which either inhibit or enhance co-monomer such as 1-hexene incorporation during ethylene polymerization. This discovery provides a novel recognition, development, and use of non-covalent dispersion type interactions for catalyzed reactions. The methods of this disclosure employ accurate, experimentally calibrated density-functional theory (DFT) calculations to enable sound computational design of the new metallocene catalysts. In an aspect, after extensive testing it was found that using B3LYP in combination with Grimme's D3 dispersion (“D3”) and Becke-Johnson dampening (“BJ”) provided ethylene and propylene co-monomer insertion barrier heights that very reasonably matched experiment. This aspect is illustrated inFIG.2A, where the results of this B3LYP+D3BJ method are demonstrated.FIG.2Acorrelates the computed ΔΔG values against experimental ΔΔG values for a range of zirconocene catalysts such as disclosed herein; see Scheme 1. When the dispersion correction was disabled by using only B3LYP in the absence of Grimme's D3 dispersion and Becke-Johnson (BJ) dampening, the linear correlation with experiment disappeared as illustrated inFIG.2B. Scheme 2 below illustrates a general outline of an ethylene polymerization mechanism with metallocene catalyst, initially forming a metallocene-ethyl (or “metal-ethyl”) intermediate from the migratory insertion of an ethylene molecule into a metal-hydride bond. The mechanism branches from the resulting metal-ethyl intermediate to either (1) continue incorporation of ethylene by a migratory insertion of another ethylene molecule (lower pathway) or (2) incorporate an α-olefin (CH2═CHR) such as propylene, 1-butene, 1-hexene, or 1-octene, which results in an alkyl chain branch from the main, growing polyethylene backbone. Transition states for these two pathways are illustrated in Scheme 2. This methods of this disclosure allow modulating the selectivity of this process to favor one pathway or the other. The identification and influence of dispersion forces on the selectivity for the incorporation of propylene versus ethylene during polymerization were investigated, and while not intending to be theory bound, it was believed that larger linear α-olefins, such as 1-hexene, would be significantly more impacted by dispersion and other non-hydrogen bonding, non-covalent stabilizing interactions than would propylene. The metallocene cyclopentadienyl-type ligands seemed to provide an practical scaffold to design new ligands in which the resulting cyclopentadienyl-type ligand incorporate functional groups to enhance dispersion interactions. See, for example, Scheme 1(B). To investigate our belief that dispersion interactions act as a significant difference operator between ethylene and propylene migratory insertion transition states, we also used the non-covalent interaction (NCI) visualization technique described by Yang et al. (see Johnson, E. R.; Keinan, S.; Mori Sánchez, P.; Contreras García, J.; Cohen, A. J.; Yang, W.J. Am. Chem. Soc.2010, 132 (18), 6498-6506. The results of this visualization method are presented in the non-covalent interaction (NCI) graphics ofFIG.3AandFIG.3B, which compare the NCI plots for an ethylene migratory insertion transition state (FIG.3A) to a propylene migratory insertion transition state (FIG.3B). This NCI technique examined regions of the electron density where the reduced density gradient vanishes at low electron densities. Repulsive and attractive interactions are determined from the sign of the second eigenvalue of the electron density Hessian. NCIs are visualized in three dimensions, and dispersion interactions are revealed and visualized by green color. Therefore, cool colors (blue) represent a strong attractive forces such as a hydrogen bond that would involve a heteroatom, warm colors (red) represent strong repulsive forces, and the intermediate green color represents the NCI van der Waals type attractive forces.FIG.3AandFIG.3Bdisplay NCI plots for the ethylene and propylene insertion transition-state structures for catalyst 8, for insertion into a Zr—Pr (zirconium-propyl) bond. Consistent with the B3LYP+D3BJ calculations described herein, distinct dispersion interactions, shown in green, are observed between the methyl group of the incoming propylene and the aryl group of the ligand, which are not present in the ethylene transition state. With our identification of dispersion forces as a selectivity feature for controlling co-monomer incorporation, we hypothesized that this effect could be amplified for 1-hexene compared to propylene. In one aspect, new catalysts which provide ethylene/1-hexene selectivity modulation resulting from stabilizing dispersion-type interactions were computationally designed. Consistent with the computed results for ethylene/propylene selectivity, in all designs we assumed rapid equilibration of ethylene and 1-hexene coordination, which enabled direct comparison of transition-state energies as an estimate of selectivity. Generally, each pair-wise dispersion/non-covalent interaction was found to be worth approximately 1 kcal/mol, and because of the relatively small energy difference in transition states, several dispersion interactions may result in a significant impact on selectivity. In one aspect,FIG.1illustrates representative zirconocene catalyst frameworks or scaffolds which were used in the methods disclosed herein. These metallocene frameworks in their unsubstituted forms shown inFIG.1were compared to the analogous substituted metallocene frameworks and ΔΔG‡values for the transition states of ethylene versus α-olefin co-monomer were calculated. This method and the results are demonstrated in detail herein for catalyst scaffolds 6 and 8 ofFIG.1. Therefore, in an aspect, new catalysts based on catalyst scaffolds 6 and 8 illustrated below were computationally designed. These two ligand types were selected because they have substitution positions that can potentially interact to a significant extent with the n-butyl group of a coordinated 1-hexene. Therefore, 6 and 8 were examined and compared with their substituted analogs designated as 6-R and 8-R, shown below, where R represents a range of substituents. Catalyst scaffold 6 also provides an example of a so-called co-monomer “rejector” where the computed and experimental ΔΔG‡values for the transition states of ethylene versus co-monomer are relatively large. For the ethylene/1-hexene co-polymerization, the ΔΔG‡value is 2.41 kcal/mol, corresponding to 16.1 1-hexene/1000 total carbons, indicating that only a small amount of 1-hexene will be incorporated into the ethylene polymer. This ΔΔG‡ultimately results in a very small number or concentration of butyl branches in the poly(ethylene-co-1-hexene). In contrast, catalyst 8 has a much smaller ΔΔG‡value for ethylene/1-hexene co-polymerization of 1.58 kcal/mol, corresponding to 52.6 1-hexene/1000 total carbons, and this small energy difference between transition states suggests a larger amount of 1-hexene co-monomer will be incorporated into the ethylene polymer during catalysis. The functional groups attached to the 6 and 8 to provide 6-R and 8-R for computational analysis were selected for their ability to enhance dispersion or were selected as representative of groups that would likely not enhance dispersion interactions; seeFIG.4AandFIG.4B. Thus,FIG.4AandFIG.4Billustrate new ligand and catalyst designs according to aspects of this disclosure incorporating dispersion interactions.FIG.4Aillustrates substituents R to the indenyl-type ligand of catalyst scaffold 6, andFIG.4Billustrates substituents R to the 4-phenyl-indenyl type ligand of catalyst scaffold 8 which are used to examine dispersion type interactions with ethylene and an α-olefin co-monomer. Consistent with the hypothesis that non-covalent interactions may be important in olefin co-monomer selectivity, several functionalized forms for catalyst scaffolds 6 and 8 that enhanced dispersion (non-covalent interactions) were identified on the basis of an unexpected and surprising method of determining the extent of non-covalent dispersion-type interactions. Specifically, it was discovered that determining the number of CH—H, CH—X (X═F, Cl, Br, N, O), and CH-π interactions between the olefin substrate and the catalyst scaffold ligands that are present within a distance range of from 2.5 Å to 4.0 Å, and comparing the total number of these dispersion-type interactions in the ethylene versus 1-hexene transition states for migratory insertion into a Zr—Pr (zirconium-propyl) bond, specific substitutions in the functionalized forms for catalyst scaffolds 6 and 8 that enhanced dispersion and thereby enhance co-monomer incorporation could be identified. This “direct counting” method provides an estimate of dispersion stabilization, because it was unexpected found that each additional dispersion-type non-covalent interaction boosted the transition state stabilization which was observed in a corresponding boost in α-olefin co-monomer incorporation. To demonstrate that the direct counting method provides an estimate of dispersion stabilization, for each new catalyst ethylene and 1-hexene migratory insertion transition state,FIG.5plots the difference in total dispersion energy (determined with and without the D3(BJ) correction (|Δ Disp E‡|)) versus the counted non-covalent interactions (ΔNCI) that are present within a distance range of from 2.5 Å to 4.0 Å, as described herein. Thus,FIG.5demonstrates the relationship between the difference in the number of non-covalent interactions (ΔNCI) and the absolute difference in dispersion energy (|ΔDisp E|) comparing ethylene and 1-hexene transition states for insertion into a zirconium-propyl bond, for R-substituted indenyl-type ligands of catalyst scaffold 6 and R-substituted 4-phenyl-indenyl type ligand of catalyst scaffold 8. Dispersion energy differences are in kcal/mol. The circles (●) plot catalysts where ΔΔG‡values (comparing ethylene and 1-hexene transition states for insertion into a zirconium propyl bond) of the R-substituted scaffolds are smaller than the corresponding ΔΔG‡value without the R functional group, which would lead to enhanced 1-hexene selectivity and hence 1-hexene incorporation. The cross (x) symbols plot catalysts where ΔΔG‡values (comparing ethylene and 1-hexene transition states for insertion into a zirconium propyl bond) of the R-substituted scaffolds are larger than the corresponding ΔΔG‡value without the R functional group, which would lead to decreased 1-hexene selectivity, that is, 1-hexene rejection and enhanced ethylene selectivity. Table 1 also provides the ΔΔG‡, ΔNCI, and |Δ Disp E‡| values which are illustrated inFIG.5, along with the calculated 1-hexenes incorporation per 1000 total carbons (1-Hex/1000 TC) for the difference between 1-hexene and ethylene transition states for the selected substituent R. The order of results presented in Table 1 is with R═H (unsubstituted) first followed by increasing ΔΔG‡values. The ΔΔG‡and |Δ Disp E‡| values are in kcal/mol. As illustrated by the qualitative linear correlation shown inFIG.5, the greater difference in the number of interactions can be correlated with an increased difference in dispersion energy between the ethylene and 1-hexene transition states. This surprising result demonstrates that enhancement of non-covalent interactions can be rationally designed in these zirconocene-type catalysts as a method of enhancing α-olefin co-monomer incorporation. Consistent with our findings, three of the four catalysts with the largest dispersion energies provided a significant shift towards co-monomer incorporation. For example, the three catalysts based on catalyst scaffold 8 have between 22-25 kcal/mol more dispersion stabilization in their 1-hexene transition states as compared to their ethylene transition states. This energy difference resulted in an overall lower ΔΔG‡values (0.82 to 1.24 kcal/mol) with the exception of the CF3substituted version (1.77 kcal/mol), suggesting greater 1-hexene incorporation (85.4 to 155.4 1-hexene/1000 total carbons). This results demonstrates that significant shifts in selectivity may be possible through the sum of many weak dispersion interactions.FIG.5and Table 1 also reveal that the new catalysts based on the core of catalyst scaffold 8 provided more possible dispersion interactions (9-12 more interactions for the 1-hexene transition states) than new catalysts based on the core of catalyst scaffold 6 (1-6 more interactions for the 1-hexene transition states). Therefore some ligand scaffolds provide more advantageous frameworks for inserting new dispersion interactions. TABLE 1Computational results for ΔΔG‡, 1-hexenes incorporation per 1000 total carbons (1-Hex/1000 TC), ΔNCI, and |Δ Disp E‡|for the difference between 1-hexene and ethylenetransition states for the selected substituent R in catalyst scaffolds 6and 8. ΔΔG‡and |Δ Disp E‡| are reported in kcal/mol.RΔΔG‡1-Hex/1000 TCΔNCI|ΔDisp E‡|4-phenyl-H1.5852.6421.48indenylt-Butyl0.82155.41024.78CatalystCH30.99122.0922.39scaffold 8OCH31.2485.41122.91CF31.7143.71222.11IndenylH2.4116.1016.8CatalystOPh0.95129.1421.68scaffold 6F0.98123.7116.88SPh1.1893.0419.89Cl1.2979.5217.79OCH31.3771.0418.45Br1.4167.0218.24OCy1.6448.3619.58CH32.0128.5317.48Propellane2.0228.1219.7NCy22.1822.4621.28OCOCH32.3417.8418.57COCH32.7110.5518.52PH34.051.6618.25NMe24.450.9519.54PMe35.940.1316.26t-Butyl6.970.0618.51 The computational analysis described herein demonstrates another advantage in being able to analyze dispersion interactions in transition states in addition to any repulsive interactions introduced by the substituents that enhance dispersion. In an aspect, this disclosure demonstrates that dispersion interactions can be designed to enhance co-monomer incorporation; however, it may be possible that the introduction of groups which enhance dispersion interactions and selectivity for 1-hexene incorporation could induce repulsive type interactions, which may energetically exceed and overwhelm any dispersion effects. Indeed, the difficulty in making an a priori determination of the steric demand of a functional or ancillary group in a transition state has been described (Wagner, J. P.; Schreiner, P. R. London Dispersion in Molecular Chemistry—Reconsidering Steric Effects.Angew. Chemie—Int. Ed.2015, 54 (42), 12274-12296) and can make rational catalyst design difficult. Therefore, the computational analysis described herein demonstrates another advantage in being able to determine how many dispersion interactions are possible in transition states in addition to determining whether the dispersion is overall more favorable than repulsive interactions based on Gibbs energies. This aspect of analyzing dispersion interactions in transition states in addition to any repulsive interactions can be demonstrated by the date in Table 1. For example, catalyst scaffold 8 in Table 1 can be substituted with a t-butyl group which provides a high value of six (6) for calculated ΔNCI (non-covalent interactions) and a |ΔDisp E‡| of 18.51 kcal/mol, but also a large ΔΔG‡of 6.97 kcal/mol, leading to a calculated 0 (zero) 1-hexene co-monomers incorporated per 1000 total carbon atoms. In contrast, the unsubstituted analog of catalyst 8 has a calculated ΔNCI of 0 and a |ΔDisp E‡| of 16.80 kcal/mol, respectively, with a ΔΔG‡of 2.41 kcal/mol, leading to a calculated 16.1 1-hexene co-monomers incorporated per 1000 total carbon atoms. In addition to counting the number of non-covalent interactions, for representative examples of the new zirconocene catalysts, a qualitative examination of 3D pictures of the NCI plots was carried out, and quantitative absolutely localized molecular orbital energy decomposition calculations (ALMO-EDA) were performed. See: Horn, P. R.; Mao, Y.; Head-Gordon, M. Probing Non-Covalent Interactions with a Second Generation Energy Decomposition Analysis Using Absolutely Localized Molecular Orbitals.Phys. Chem. Chem. Phys.2016, 18 (33), 23067-23079. The ALMO-EDA method provides the ability to not only access the total dispersion energy in a transition state, but also the other key physical components, such as electrostatic interactions, Pauli repulsion, and orbital (charge transfer) interactions. Table 2 collects the ΔΔG‡(kcal/mol) for 1-hexene versus ethylene migratory insertion into the Zr—Pr (zirconium-propyl) bond, ΔNCI for 1-hexene versus ethylene migratory insertion into the Zr—Pr bond, and ALMO-EDA calculation results for 1-hexene transition states of the catalyst scaffolds 6 and 8 with selected substituents R. Specifically, Table 2 data is presented for catalyst scaffolds [μ-Me2Si(η5-C9H6)2Zr] (6), [μ-Me2Si(η5-4-FC9H5)2Zr] (6-F), {μ-Me2Si[η5-4-N(C6H11)2(C9H6)]2Zr} (6-NCy2, also 6-N(C6HH)2), and [μ-Me2Si(η5-4-(3-CH3C6H4)C9H6)2Zr](8-Me). Therefore, in Table 2 andFIG.6AthroughFIG.6D, L1 is unsubstituted indenyl, L2 is indenyl substituted with fluorine, L3 is indenyl substituted with dicyclohexylamine, and L4 is 4-phenyl indenyl substituted with methyl. From this analysis, these transition states were observed to clearly illustrate the interplay between repulsive steric type interactions and stabilizing dispersion interactions and the resulting effect on ΔΔG‡. The steric repulsion in Table 2 is the sum of the electrostatic and Pauli terms. FIG.6AthroughFIG.6Dillustrate graphics using the NCI visualization technique, which demonstrate the non-covalent interactions for 1-hexene migratory insertion transition states in a substituted zirconocene catalyst frameworks. The interactions are colored according to the key inFIG.3AandFIG.3B, thus, the cool colors (blue) represent a strong attractive forces such as a hydrogen bond that might involve a heteroatom, warm colors (red) represent strong repulsive forces, and the intermediate green colors represent the van der Waals attractive forces in effect. Quantitative data for theFIG.6AthroughFIG.6Dtransitions states are in Table 2. TABLE 2Absolutely localized molecular orbital energy decomposition analysis results, ΔΔG‡(1-hexene-ethylene) values, and difference in the number of non-covalent interactionsin the 1-hexene and ethylene transition states (ΔNCI). All energies are in kcal/mol.CatalystScaffoldLigandΔΔG‡Δ NCIElectrostaticPauliStericADispB6L1 = unsubstituted2.410−81.97140.3358.36−17.50indenyl6-FL2 = indenyl1.782−82.98141.7858.80−17.89substituted withfluorine6-NCy2L3 = indenyl3.576−90.39160.8070.41−26.51substituted withdicyclohexylamine8-MeL4 = 4-phenyl0.9911−82.17142.4660.29−25.12indenyl withmethyl groupsASteric repulsion is the sum of the electrostatic and Pauli repulsion terms.BDisp dispersion as used here is not the same as ΔDisp E‡in Table 1, but rather Disp energies are only for the 1-hexene transition state and the interaction between the 1-hexene transition-state fragment and the catalyst transition-state fragment. The energy values presented in Table 2 are for only the 1-hexene transition state and the interaction between the 1-hexene transition-state fragment and the catalyst transition-state fragment. Therefore, the dispersion energy (Disp) in Table 2 is not the same as the dispersion difference (ΔDisp E‡) presented in Table 1. The impact of steric repulsion can be quantified using the Pauli repulsion and electrostatic terms from the ALMO-EDA calculations, where the steric repulsion is calculated as the sum of Pauli and electrostatic terms. As the Table 2 data illustrate, the unsubstituted indenyl ligand (L1) has the lowest amount of destabilization due to steric repulsion as well as the least amount of stabilization from dispersion. In comparison, L2, which has a similar amount of destabilization from steric repulsion and stabilization from dispersion, has a smaller ΔΔG‡resulting in increased selectivity from 1-hexene (16.1 1-Hex/1000TC (L1) versus 123.7 1-Hex/1000TC (L2)). The L3 ligand has the greatest amount of stabilization from dispersion, however, it also has the greatest amount of destabilization due to steric repulsion. Because the stabilization from dispersion is offset by the destabilizing repulsion, the 1-hexene TS (transition state) is destabilized relative to the ethylene, indicated by the larger ΔΔG‡. On the other hand, L4 has an amount of destabilization due to steric repulsion comparable to L1 and L2, but ˜7 kcal/mol greater stabilization from dispersion. In this L4 example, because of the increased stabilization resulting from dispersion, 1-hexene TS is stabilized relative to the ethylene TS, resulting in a lower ΔΔG‡and greater incorporation of 1-hexene (122.0 1-Hex/1000TC). Therefore, in an aspect, this analysis of the Table 2 data highlights how the incorporation of dispersion in catalyst designs can be accomplished with functional group selections which minimize the impact of destabilizing steric repulsion while maximizing the stabilization gained from increased dispersion. Accordingly, in the B3LYP computational analysis disclosed herein which compared B3LYP+D3BJ and then disabled the dispersion correction by using only B3LYP, this dispersion deletion analysis and NCI visualization analysis revealed that despite a very subtle methyl group induced difference in for ethylene versus propylene transition states, transition-state dispersion interactions for ethylene versus propylene have a significant impact on α-olefin co-monomer selectivity with zirconocene catalysts. This discovery allowed the use of stabilizing non-covalent dispersion interactions as a design feature for ethylene/1-hexene copolymerization and a method for tuning selectivity for α-olefin co-monomer incorporation. The computational design of new catalysts derived from the catalyst scaffolds ofFIG.1such as catalysts 6 and 8 demonstrated that the amount of interactions and total stabilizing dispersion interactions can be greatly modulated by the ligand structure, which provides a platform to control co-monomer incorporation selectivity and develop catalysts with co-monomer insertion or rejection pathways. By providing the direct ability to design and control the co-monomer insertion rate according to the methods disclosed herein, the development of improved polyethylene resins with advantaged properties and improved performance can be achieved. Accordingly, this disclosure provides for a method for designing a Group 4 metallocene olefin polymerization catalyst, in which the method may comprise:(a) selecting a first metallocene catalyst framework comprising a Group 4 metal bonded to a hydrocarbyl ligand and to one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, and generating a first ground state model structure (GSA) derived from the first metallocene catalyst framework;(b) generating (1) a first transition state model structure (TSA1) derived from the migratory insertion of an ethylene molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework and (2) a second transition state model structure (TSA2) derived from the migratory insertion of an α-olefin co-monomer molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework;(c) determining, by at least one processor of a device, the relative energies of each of the first ground state model structure (GSA), the first transition state model structure (TSA1) and a dispersion energy (Disp EA1) associated with TSA1, and the second transition state model structure (TSA2) and a dispersion energy (Disp EA2) associated with TSA2, and determining values for ΔG‡A1(TSA1−GSA), ΔG‡A2(TSA2−GSA), ΔΔG‡A(TSA2−TSA1), and an absolute difference in dispersion energies |ΔDisp EA| calculated as |Δ(Disp EA2−Disp EA1|)| for migratory insertion of the ethylene molecule versus the α-olefin molecule in the first metallocene catalyst framework;(d) repeating steps (a)-(c) using a second metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a first test substituent, and generating a corresponding second ground state model structure (GSB), third transition state model structure (TSB1), and fourth transition state model structure (TSB2), and determining, by at least one processor of a device, the relative energies of each of a GSB, TSB1and a dispersion energy (Disp EB1) associated with TSB1, and TSB2and a dispersion energy (Disp EB2) associated with TSB2, and determining values for ΔG‡B1(TSB1−GSB), ΔG‡B2(TSB2−GSB), ΔΔG‡B(TSB2−TSB1), and an absolute difference in dispersion energies |ΔDisp EB| calculated as |Δ(Disp EB2−Disp EB1)| for migratory insertion of the ethylene molecule versus the α-olefin molecule in the second metallocene catalyst framework; and(e) identifying the first test substituent of the second metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B<ΔΔG‡A, when |ΔDisp EB|>|ΔDisp EA|, or a combination thereof, or (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B>ΔΔG‡A, when |ΔDisp EB|<|ΔDisp EA|, or a combination thereof. In this aspect, this method can further comprise the steps of:(f) repeating steps (a)-(c) using a third metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a second test substituent, and generating a corresponding third ground state model structure (GSC), fifth transition state model structure (TSC1), and sixth transition state model structure (TSC2), and determining, by at least one processor of a device, the relative energies of each of a GSC, TSC1and a dispersion energy (Disp EC1) associated with TSC1, and TSC2and a dispersion energy (Disp EC2) associated with TSC2, and determining values for ΔG‡C1(TSC1−GSC), ΔG‡C2(TSC2−GSC), ΔΔG‡C(TSC2−TSC1), and an absolute difference in dispersion energies |ΔDisp EC| calculated as |Δ(Disp EC2−Disp EC1)| for migratory insertion of the ethylene molecule versus the α-olefin molecule in the third metallocene catalyst framework; and(g) identifying the second test substituent of the third metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C<ΔΔG‡A, when |ΔDisp EC|>|ΔDisp EA|, or a combination thereof, (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C>ΔΔG‡A, when |ΔDisp EC|<|ΔDisp EA|, or a combination thereof, (3) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C<ΔΔG‡B, when |ΔDisp EC|>|ΔDisp EB|, or a combination thereof, or (4) enhancing ethylene incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C>ΔΔG‡B, when |ΔDisp EC|<|ΔDisp EB|, or a combination thereof. In this fashion, a series of substituents can be examined and ranked according to their ability to enhance α-olefin co-monomer incorporation into a polyethylene co-polymer relative to ethylene, and can serve to tune the catalyst for the desired co-monomer content. In accordance with a further aspect, this disclosure provides a method for designing a Group 4 metallocene olefin polymerization catalyst, the method comprising:(a) selecting a first metallocene catalyst framework comprising a Group 4 metal bonded to a hydrocarbyl ligand and to one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, and generating a first ground state model structure (GSA) derived from the first metallocene catalyst framework;(b) generating (1) a first transition state model structure (TSA1) derived from the migratory insertion of an ethylene molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework and (2) a second transition state model structure (TSA2) derived from the migratory insertion of an α-olefin co-monomer molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework;(c) determining, by at least one processor of a device, the relative energies of each of the first ground state model structure (GSA), the first transition state model structure (TSA1) including a dispersion energy (Disp EA1) associated with TSA1, and the second transition state model structure (TSA2) including a dispersion energy (Disp EA2) associated with TSA2, and determining values for ΔG‡A1(TSA1−GSA), ΔG‡A2(TSA2−GSA), and ΔΔG‡A(TSA2−TSA1) for migratory insertion of the ethylene molecule versus the α-olefin molecule in the first metallocene catalyst framework;(d) determining, by at least one processor of a device, the number of stabilizing, non-covalent (dispersion-type) interactions (NCI) within a distance of from 2.5 Å to 4.0 Å, inclusive, between (1) the ethylene molecule and the substituted or unsubstituted η5-cycloalkadienyl ligands in the first transition state model structure TSA1(NCIA1), and (2) the α-olefin molecule and the substituted or unsubstituted η5-cycloalkadienyl ligands in the second transition state model structure TSA2(NCIA2), and difference between the number of these NCI interactions (ΔNCIA);(e) repeating steps (a)-(d) using a second metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a first test substituent, and generating a corresponding second ground state model structure (GSB), third transition state model structure (TSB1), and fourth transition state model structure (TSB2), and determining, by at least one processor of a device, the relative energies of each of a GSB, TSB1including a dispersion energy (Disp EB1) associated with TSB1, TSB2including a dispersion energy (Disp EB2) associated with TSB2, and determining values for ΔG‡B1(TSB1−GSB), ΔG‡B2(TSB2−GSB), ΔΔG‡B(TSB2−TSB1), and the number of stabilizing, non-covalent (dispersion-type) interactions in TSB1(NCIB1) and TSB2(NCIB2), and difference between the numbers of these NCI interactions (ΔNCIB), for migratory insertion of the ethylene molecule versus the α-olefin molecule in the second metallocene catalyst framework; and(f) identifying the first test substituent of the second metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B<ΔΔG‡A, when ΔNCIB>ΔNCIA, or a combination thereof, or (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B>ΔΔG‡A, when ΔNCIB<ΔNCIA, or a combination thereof. In this aspect, this method can further comprise the steps of:(g) repeating steps (a)-(d) using a third metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a second test substituent, and generating a corresponding third ground state model structure (GSC), fifth transition state model structure (TSC1), and sixth transition state model structure (TSC2), and determining, by at least one processor of a device, the relative energies of each of a GSC, TSC1including a dispersion energy (Disp EC1) associated with TSC1, and TSC2including a dispersion energy (Disp EC2) associated with TSC2, and determining values for ΔG‡C1(TSC1−GSC), ΔG‡C2(TSC2−GSC), ΔΔG‡C(TSC2−TSC1), and the number of stabilizing, non-covalent (dispersion-type) interactions in TSC1(NCIC1) and TSC2(NCIC2), and difference between the numbers of these NCI interactions (ΔNCIC), for migratory insertion of the ethylene molecule versus the α-olefin molecule in the second metallocene catalyst framework; and(h) identifying the second test substituent of the third metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C<ΔΔG‡A, when ΔNCIC>ΔNCIA, or a combination thereof, (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C>ΔΔG‡A, when ΔNCIC<ΔNCIAor a combination thereof, (3) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C<ΔΔG‡B, when ΔNCIC>ΔNCIB, or a combination thereof, or (4) enhancing ethylene incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C>ΔΔG‡B, when ΔNCIC<ΔNCIB, or a combination thereof. When catalyst frameworks are being examined or ranked for their α-olefin co-monomer incorporation into a polyethylene co-polymer according to the number of dispersion-type interactions (NCI), the number of non-covalent dispersion-type interactions NCIA, NCIB, and NCICwithin a distance of from 2.5 Å to 4.0 Å can comprise the number of CH—H, CH—X (X═F, Cl, Br, N, O), and CH-π interactions between the ethylene molecule or the α-olefin molecule and the substituted or unsubstituted η5-cycloalkadienyl ligands and the first test substituent of the first metallocene catalyst framework or second metallocene catalyst framework within a distance range of 2.5 to 4.0 Å. Therefore, this disclosure provides for identifying the first test substituent of the second metallocene catalyst framework, the second test substituent of the third metallocene catalyst framework, or both the first test substituent and the second test substituent as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon (a) comparing the relative energies of ΔΔG‡A, ΔΔG‡B, and/or ΔΔG‡C, as appropriate, (b) comparing the relative energies of |ΔDisp EAI, |ΔDisp EB| and/or |ΔDisp EC, (c) based upon ΔNCIA, ΔNCIB, and/or ΔNCIC, or combinations of these parameters. For example, if examining the first test substituent of the second metallocene catalyst framework relative to first metallocene catalyst framework containing one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, comparisons are made between ΔΔG‡Aand ΔΔG‡B, |ΔDisp EA| and |ΔDisp EB|, ΔNCIAand ΔNCIB, or any combination thereof. If examining the second test substituent of the third metallocene catalyst framework relative to first metallocene catalyst framework containing one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, comparisons are made between ΔΔG‡Aand ΔΔG‡C, |ΔDisp EA| and |ΔDisp EC|, ΔNCIAand ΔNCIC, or any combination thereof. If examining the second test substituent of the third metallocene catalyst framework relative to the first test substituent of the second metallocene catalyst framework, comparisons are made between ΔΔG‡Band ΔΔG‡C, |ΔDisp EB| and |ΔDisp EC, ΔNCIBand ΔNCIC, or any combination thereof. As explained further herein, such as in the Examples section, in designing a Group 4 metallocene olefin polymerization catalyst according to this disclosure, the energies of any one or more of the ground state model structures (GSA, GSB, GSC) and any one or more of the transition state model structures (TSA1, TSA2, TSB1, TSB2, TSC1, TSC2) can be calculated as a B3LYP single point energy calculation with a D3BJ correction (B3LYP+D3BJ) using a density functional theory (DFT). In addition, any one or more of the dispersion energies (Disp EA, Disp EB, Disp EC) can be calculated as the difference between a B3LYP single point energy calculation with and without a D3BJ correction using a density functional theory (DFT). Further, any of the number of stabilizing, non-covalent (dispersion-type) interactions (NCIA1NCIA2, NCIB1, NCIB2, NCIC1, NCIC2) can be calculated using absolutely localized molecular orbital energy decomposition analysis (ALMO-EDA) of the respective transition state model structures (TSA1, TSA2, TSB1, TSB2, TSC1, and TSC2). Any test substituents can be used with the metallocene catalysts according to the methods of this disclosure, and these methods also allow comparing substituted metallocenes with unsubstituted metallocenes. In an aspect, the first test substituent, the second test substituent, or any additional test substituents can be selected from: a halide (F, Cl, or Br); a C1-C10heterohydrocarbyl group comprising a heteroatom selected from halide (F, Cl, or Br), N, O, P, or S; a C1-C10aliphatic group; or a C6-C10aromatic group. Any combinations of these substituents can be used in these tests, including multiple occurrences of the same substituent. For example, a mono-halide substituted metallocene can be compared with a di-halide substituted metallocene, and the like. Examples of test substituents can include but are not limited to F, Cl, Br, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C6-C10aryl, C1-C6alkoxide, —OC(O)R1, —CH2C(O)R1, —NR12, —PH3, PR13, or —SR1, wherein R1is independently selected from a C1-C6alkyl or a C6-C10aryl. For example, test substituents can comprise or can be selected from F, Cl, Br, —CH3, —CMe3, —C(CH2)3CH, —CF3, —OMe, —OC(O)Me, —CH2C(O)Me, —OC6H11, —OPh, —NMe2, —N(C6H11)2, PH3, PMe3, —SC6H11, or —SPh. The catalyst “framework” or “scaffold” used in developing the methods of this disclosure can comprise or can be selected from a metallocene catalyst framework based on titanium, zirconium, or hafnium, that is a Group 4 metallocene catalyst framework. The first or “baseline” metallocene catalyst framework for comparing other substituted metallocenes can comprise a Group 4 metal bonded to a hydrocarbyl ligand and to one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands. The second, third, or any subsequent metallocene catalyst frameworks comprise the same Group 4 metal, hydrocarbyl ligand, and the one or two η5-cycloalkadienyl ligands present in the first or “baseline” metallocene catalyst framework, in addition to one or more substituents present somewhere on the η5-cycloalkadienyl ligand(s) that are being tested and compared to the first metallocene. Examples of the substituted or unsubstituted η5-cycloalkadienyl ligands can include, but are not limited to, cyclopentadienyl, indenyl, and fluorenyl, which can be linked by a bridging group or unlinked. Further, the η5-cycloalkadienyl ligand(s) can be substituted or unsubstituted in the first baseline metallocene but are substituted in the second, third, or any subsequent metallocene catalyst frameworks. In the disclosed method, any substituent can be used in the second, third, or any subsequent metallocene catalyst frameworks for comparison with the first metallocene catalyst. For example, the substituted or unsubstituted η5-cycloalkadienyl ligands, absent the first test substituent and absent the second test substituent, can be selected independently from cyclopentadienyl, methylcyclopentadienyl, t-butylcyclopentadienyl, indenyl, 4-phenyl-indenyl, 2-methylindenyl, 3-t-butylindenyl, 2-methyl-4-phenylindenyl, fluorenyl, or 2-methylfluorenyl. In a further aspect of the disclosed method for designing a Group 4 metallocene olefin polymerization catalyst, the first metallocene catalyst framework can comprise two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands. Moreover, the first metallocene catalyst framework can comprise or can be selected from two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands which are bridged by a linking group of the formula (1) >ER1R2, wherein E is C or Si, R1is hydrogen or a C1-C12hydrocarbyl group, and R2is hydrogen, a C1-C12hydrocarbyl group, or a C3-C10alkenyl group having a terminal C═C double bond, or (2) CR12CR2—, wherein R1and R2are selected independently from hydrogen or a C1-C6hydrocarbyl group. In one aspect, the first metallocene catalyst framework can comprise two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands which are bridged by a linking group of the formula >CMe2, —CH2CH2, >SiMe2, or >CH[(CH2)2CH═CH2]. Examples of zirconocene catalyst frameworks or scaffolds which can be used in the methods disclosed herein are illustrated inFIG.1. Any hydrocarbyl ligand can be used for the computations of this disclosure, and examples of the hydrocarbyl ligand used for the calculations can be a C1-C6hydrocarbyl ligand, or alternatively a C1-C4hydrocarbyl ligand, or alternatively an ethyl (C2) ligand. In another aspect of this disclosure, any α-olefin co-monomer can be used in these computations. For example, the α-olefin co-monomer can comprise or can be selected from propylene, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, or styrene. According to another aspect, the first metallocene catalyst framework can comprise one substituted or unsubstituted η5-cycloalkadienyl ligand and can further comprises an anionic ligand in addition to the hydrocarbyl ligand which constitutes the growing polymer chain. In this aspect, the first metallocene catalyst framework can comprise one substituted or unsubstituted η5-cycloalkadienyl ligand and further comprise an anionic ligand selected from halide, hydride, a C1-C20hydrocarbyl group, a C1-C20heterohydrocarbyl group, tetrahydroborate, or OBRA2or OSO2RAwherein RAis independently a C1-C12hydrocarbyl group. For example, the first metallocene catalyst framework can comprise one substituted or unsubstituted η5-cycloalkadienyl ligand and further comprise an anionic ligand selected from F, Cl, Br, a hydride, a C1-C12hydrocarbyl group, a C1-C12hydrocarbyloxide group, a C1-C12hydrocarbylamino group, C1-C12dihydrocarbylamino, or a trihydrocarbylsilyl group wherein each hydrocarbyl is independently a C1-C12hydrocarbyl group. The second, third, or any subsequent metallocene catalyst frameworks can constitute the same ligands as the first metallocene, but are then further substituted for computational comparisons with the first metallocene. The method for designing a Group 4 metallocene olefin polymerization catalyst according to this disclosure can further comprise the step of synthesizing the Group 4 metallocene catalyst comprising the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises the first test substituent or the second test substituent or any subsequent test substituent. According the method for designing the Group 4 metallocene olefin polymerization catalyst may also further comprise the steps of: (a) providing a Group 4 metallocene catalyst comprising the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises the first test substituent or the second test substituent; and (b) contacting the Group 4 metallocene catalyst with ethylene and an α-olefin co-monomer molecule under polymerization conditions to form a polyethylene co-polymer. Regarding specific zirconocene catalyst systems that can be designed, in an aspect, this disclosure provides a catalyst system for polymerizing olefins, the catalyst system comprising:a zirconocene catalyst comprising two η5-cycloalkadienyl ligands independently selected from a substituted or an unsubstituted η5-cyclopentadienyl ligand or a substituted or an unsubstituted η5-indenyl ligand, whereinthe two η5-cycloalkadienyl ligands are optionally bridged by a linking group; andone of the η5-cycloalkadienyl ligands is substituted with at least one substituent which imparts enhanced dispersion-type interactions in a transition state for a migratory insertion of an α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of the zirconocene catalyst versus a migratory insertion of the α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of a zirconocene catalyst comprising the corresponding unsubstituted η5-cycloalkadienyl ligands. According to a further aspect, this disclosure provides a catalyst system for polymerizing olefins, the catalyst system comprising:a zirconocene catalyst comprising two η5-cycloalkadienyl ligands independently selected from a substituted or an unsubstituted η5-cyclopentadienyl ligand or a substituted or an unsubstituted η5-indenyl ligand, whereinthe two η5-cycloalkadienyl ligands are optionally bridged by a linking group; andone of the η5-cycloalkadienyl ligands is substituted with at least one substituent which imparts enhanced number of stabilizing, non-covalent (dispersion-type) interactions (NCI) within a distance of from 2.5 Å to 4.0 Å, inclusive, between the α-olefin molecule and the at least one substituent in a transition state for a migratory insertion of an α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of the zirconocene catalyst versus a migratory insertion of the α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of a zirconocene catalyst comprising the corresponding unsubstituted η5-cycloalkadienyl ligands. In an aspect, this catalyst systems for polymerizing olefins according to this disclosure can comprise zirconocene catalysts, which can comprise one of the following structures: wherein:R can be selected independently from F, Cl, Br, C1-C6alkyl, C6-C10aryl, C1-C6alkoxide, —OC(O)R1, —CH2C(O)R1, —NR12, —PH3, PR13, or —SR1, and wherein R1can be independently selected from a C1-C6alkyl or a C6-C10aryl, in the substituted η5-cycloalkadienyl ligands; andR is H in the corresponding unsubstituted η5-cycloalkadienyl ligands. In this aspect, R can be selected independently from F, Cl, Br, —CH3, —CMe3, —C(CH2)3CH, —CF3, —OMe, —OC(O)Me, —CH2C(O)Me, —OC6H11, —OPh, —NMe2, —N(C6H11)2, PH3, PMe3, —SC6H11, or —SPh, in the substituted η5-cycloalkadienyl ligands. This disclosure also provides for catalyst systems for polymerizing olefins, in which the catalyst system can comprise the Group 4 metallocene olefin polymerization catalyst comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, according to any of the aspects described herein, in which at least one of the η5-cycloalkadienyl ligands can comprise the first test substituent, the second test substituent, or any subsequent second test substituent, which can be identified as enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer, relative to an α-olefin co-monomer incorporation of the first metallocene catalyst framework. If desired, at least one of the η5-cycloalkadienyl ligands can comprise the first test substituent, the second test substituent, or any subsequent second test substituent, which can be identified as enhancing ethylene incorporation into a polyethylene co-polymer, relative to ethylene incorporation of the first metallocene catalyst framework. In addition to a metallocene, the catalyst system of this disclosure can include any additional components that are needed for polymerizing olefins. For example, the catalyst system may further comprise:(a) an activator comprising a solid oxide treated with an electron-withdrawing anion (activator-support), an organoboron compound, an organoborate compound, an ionizing ionic compound, an aluminoxane compound, or any combination thereof; and(b) optionally, a co-catalyst comprising an organoaluminum compound, an organoboron compound, an organozinc compound, an organomagnesium compound, an organolithium compound, or any combination thereof. In this aspect, the activator can comprises a solid oxide treated with an electron-withdrawing anion, and wherein:the solid oxide can comprise silica, alumina, titania, zirconia, magnesia, boria, calcia, zinc oxide, silica-alumina, silica-coated alumina, silica-titania, silica-zirconia, silica-magnesia, alumina-titania, alumina-zirconia, zinc-aluminate, alumina-boria, silica-boria, aluminum phosphate, aluminophosphate, aluminophosphate-silica, magnesium aluminate, titania-zirconia, boehmite, heteropolytungstates, mixed oxides thereof, or any combination thereof; andthe electron-withdrawing anion can comprise fluoride, chloride, bromide, iodide, sulfate, bisulfate, fluorosulfate, phosphate, fluorophosphate, triflate, mesylate, tosylate, thiosulfate, C1-C10alkyl sulfonate, C6-C14aryl sulfonate, trifluoroacetate, fluoroborate, fluorozirconate, fluorotitanate, or any combination thereof. Further to this aspect, the activator can comprises a solid oxide treated with an electron-withdrawing anion, and wherein:the solid oxide comprises alumina, silica-alumina, silica-coated alumina, or a mixture thereof, andthe electron-withdrawing anion comprises fluoride, sulfate, or phosphate. Examples of activators include, but are not limited to, fluorided alumina, fluorided silica, fluorided silica-alumina, or fluorided silica-coated alumina (mullite). According to a further aspect, the catalyst system for polymerizing olefins according to this disclosure can be present and can comprise alkyl aluminum compounds, such as trimethylaluminum, triethylaluminum, tri-n-propylaluminum, tri-n-butylaluminum, triisobutylaluminum, tri-n-hexylaluminum, tri-n-octylaluminum, diisobutylaluminum hydride, diethylaluminum ethoxide, diethylaluminum chloride, aluminoxanes, or any combination thereof. EXAMPLES General Methods. All structures were optimized using DFT (density-functional theory) and confirmed as transition states by frequency analysis in Gaussian 16 (Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; et al. Gaussian16, Rev. B.01. Gaussian Inc.: Wallingford, C T 2016). The Becke 3-parameter exchange functional (Becke, A. D. Density-Functional Thermochemistry. III. The Role of Exact Exchange.J. Chem. Phys.1993, 98 (7), 5648-5652) was combined with the correlation functional of Lee, Yang, and Parr (Gaussian version) (Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron Density.Phys. Rev. B1988, 37 (2), 785-789), the empirical dispersion correction of Grimme (D3) and the damping function of Becke and Johnson (Grimme, S.; Huenerbein, R.; Ehrlich, S. On the Importance of the Dispersion Energy for the Thermodynamic Stability of Molecules.ChemPhysChem2011, 12 (7), 1258-1261; Grimme, S.; Hansen, A.; Brandenburg, J. G.; Bannwarth, C. Dispersion-Corrected Mean-Field Electronic Structure Methods.Chem. Rev.2016, 116 (9), 5105-5154). The B3LYP+D3(BJ) functional was combined with the 6-31G** basis set for main group elements and LANL2DZ basis set and pseudo potential for Zr. The free energies reported were computed at the B3LYP+D3(BJ)/Def2TZVPP//B3LYP+D3BJ/6-31G**[LANL2DZ] level of theory (see below for further details). The dispersion energy was computed as the difference between B3LYP single point calculations with and without the D3BJ correction. Temperature corrections were applied according to the experimental conditions (323.15K) and no pressure corrections were applied. Standard rigid rotor and harmonic oscillator approximations were used. Additional analysis of the dispersion interactions was performed using the NCI method (Johnson, E. R.; Keinan, S.; Mori Sánchez, P.; Contreras García, J.; Cohen, A. J.; Yang, W. NCI: Revealing Non-Covalent Interactions.J. Am. Chem. Soc.2010, 132 (18), 6498-6506) as implemented in Multiwfn (Lu, T.; Chen, F. Multiwfn: A Multifunctional Wavefunction Analyze.J. Comput. Chem.2012, 33 (5), 580-592) and visualized in VMD (Humphrey, W.; Dalke, A.; Schulten, K. VMD—Visual Molecular Dynamics.J. Molec. Graph. 1996, 13, 33-38). Additional absolutely localized molecular orbital energy decomposition analysis (ALMO-EDA) (Horn, P. R.; Mao, Y.; Head-Gordon, M. Probing Non-Covalent Interactions with a Second Generation Energy Decomposition Analysis Using Absolutely Localized Molecular Orbitals.Phys. Chem. Chem. Phys.2016, 18 (33), 23067-23079) calculations were performed in QChem 5.2 (Shao, Y.; Gan, Z.; Epifanovsky, E.; Gilbert, A. T. B.; Wormit, M.; Kussmann, J.; Lange, A. W.; Behn, A.; Deng, J.; Feng, X.; et al.Advances in Molecular Quantum Chemistry Contained in the Q-Chem5.2 Program Package.Mol. Phys.2015, 113 (2), 184-215). ALMO-EDA is used to compute interactions between molecular fragments. The interaction is broken into electrostatic, Pauli repulsive, dispersion, charge-transfer, and polarization terms. The Pauli repulsive and electrostatic terms can be combined to quantify the steric repulsion of the molecular fragments. These calculations assume a bare cation as discussed by Linnolahti (Laine, A.; Coussens, B. B.; Hirvi, J. T.; Berthoud, A.; Friederichs, N.; Severn, J. R.; Linnolahti, M. Effect of Ligand Structure on Olefin Polymerization by a Metallocene/Borate Catalyst: A Computational Study.Organometallics2015, 34 (11), 2415-2421). The growing polymer chain was modeled as a propyl chain and olefin insertions were modelled as front side insertions (Margl, P.; Deng, L.; Ziegler, T. General Aspects of Ethylene Polymerization by D0 and D0fn Transition Metals.Top. Catal.1999, 7, 187-208) occurring in a 2,1-fashion. Additional Computational Details. All DFT calculations were performed in Gaussian 16 revision B.01. Geometry optimizations were performed at the B3LYP+D3BJ/6-31G** [LANL2DZ for Zr] (small) level of theory. Vibrational frequencies were computed to verify stationary points as first-order saddle points (transition states). The B3LYP+D3BJ functional was chosen because it most accurately reproduced the experimental co-polymerization ratios discussed in the main text. The electronic energies were further refined with single point calculations using the def2-TZVPP basis set (large). Final free energies reported are computed at the B3LYP+D3BJ/def2-TZVPP//B3LYP+D3BJ/6-31G** [LANL2DZ] level of theory. Free energies are the sum of Elarge+ΔEZPE(small)+ΔUvib(small)+ΔUrot(small)+ΔUtrans(small)+nRT−TΔSvib(small)−TΔSrot(small)−TΔStrans(small). E is the total SCF energy. ΔEZPE(small) is the zero-point energy correction. ΔUvib(small), ΔUrot(small), and ΔUtrans(small)are thermal energy vibrational, rotational, and translational corrects. R is the gas constant and T is the temperature. TΔSvib(small), TΔSrot(small), and TΔStrans(small)are temperature dependent vibrational, rotational, and translation entropy corrections. No pressure corrections were applied, and standard harmonic oscillator and rigid rotor approximations were applied. As discussed herein, the dispersion energy was computed by performing small basis set single point calculations with the D3BJ correction turned off. The dispersion energy was computed as the difference in the SCF energies with and without the correction applied. Absolutely localized molecular orbital energy decomposition analysis calculations (ALMO-EDA) calculations were performed in Q-Chem 5.2 at the B3LYP+D3BJ/6-31G**[LANL2DZ] level of theory using the geometries computed using Gaussian16 at the B3LYP+D3BJ/6-31G** [LANL2DZ] level of theory. In order to compute dispersion energy from ALMO-EDA a dispersion free density functional is specified, and these calculations used B3LYP. Molecular fragments used in the ALMO-EDA calculation were 1-hexene and the catalyst. ASPECTS OF THE DISCLOSURE These and other features of the invention can further include the various aspects, statements, embodiments, and features which are presented below.Aspect 1. A method for designing a Group 4 metallocene olefin polymerization catalyst, the method comprising:(a) selecting a first metallocene catalyst framework comprising a Group 4 metal bonded to a hydrocarbyl ligand and to one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, and generating a first ground state model structure (GSA) derived from the first metallocene catalyst framework;(b) generating (1) a first transition state model structure (TSA1) derived from the migratory insertion of an ethylene molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework and (2) a second transition state model structure (TSA2) derived from the migratory insertion of an α-olefin co-monomer molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework;(c) determining, by at least one processor of a device, the relative energies of each of the first ground state model structure (GSA), the first transition state model structure (TSA1) and a dispersion energy (Disp EA1) associated with TSA1, and the second transition state model structure (TSA2) and a dispersion energy (Disp EA2) associated with TSA2, and determining values for ΔG‡A1(TSA1−GSA), ΔG‡A2(TSA2−GSA), ΔΔG‡A(TSA2−TSA1), and an absolute difference in dispersion energies |ΔDisp EA| calculated as Δ(Disp EA2−Disp EA1)| for migratory insertion of the ethylene molecule versus the α-olefin molecule in the first metallocene catalyst framework;(d) repeating steps (a)-(c) using a second metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a first test substituent, and generating a corresponding second ground state model structure (GSB), third transition state model structure (TSB1), and fourth transition state model structure (TSB2), and determining, by at least one processor of a device, the relative energies of each of a GSB, TSB1and a dispersion energy (Disp EB1) associated with TSB1, and TSB2and a dispersion energy (Disp EB2) associated with TSB2, and determining values for ΔG‡B1(TSB1−GSB), ΔG‡B2(TSB2−GSB), ΔΔG‡B(TSB2−TSB1), and an absolute difference in dispersion energies |ΔDisp EB| calculated as |Δ(Disp EB2−Disp EB1)| for migratory insertion of the ethylene molecule versus the α-olefin molecule in the second metallocene catalyst framework; and(e) identifying the first test substituent of the second metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B<ΔΔG‡A, when |ΔDisp EB|>|ΔDisp EA|, or a combination thereof, or (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B>ΔΔG‡A, when |ΔDisp EB|<ΔDisp EA|, or a combination thereof.Aspect 2. The method for designing a Group 4 metallocene olefin polymerization catalyst according to Aspect 1, further comprising the steps of:(f) repeating steps (a)-(c) using a third metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a second test substituent, and generating a corresponding third ground state model structure (GSC), fifth transition state model structure (TSC1), and sixth transition state model structure (TSC2), and determining, by at least one processor of a device, the relative energies of each of a GSC, TSC1and a dispersion energy (Disp EC1) associated with TSC1, and TSC2and a dispersion energy (Disp EC2) associated with TSC2, and determining values for ΔG‡C1(TSC1−GSC), ΔG‡C2(TSC2−GSC), ΔΔG‡C(TSC2−TSC1), and an absolute difference in dispersion energies ΔDisp EC| calculated as |Δ(Disp EC2−Disp EC1)| for migratory insertion of the ethylene molecule versus the α-olefin molecule in the third metallocene catalyst framework; and(g) identifying the second test substituent of the third metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C<ΔΔG‡A, when |ΔDisp EC|>ΔDisp EA|, or a combination thereof, (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C>ΔΔG‡A, when |ΔDisp EC|<|ΔDisp EA|, or a combination thereof, (3) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C<ΔΔG‡B, when |ΔDisp EC|>ΔDisp EB|, or a combination thereof, or (4) enhancing ethylene incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C>ΔΔG‡B, when |ΔDisp EC|<|ΔDisp EB|, or a combination thereof.Aspect 3. A method for designing a Group 4 metallocene olefin polymerization catalyst, the method comprising:(a) selecting a first metallocene catalyst framework comprising a Group 4 metal bonded to a hydrocarbyl ligand and to one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, and generating a first ground state model structure (GSA) derived from the first metallocene catalyst framework;(b) generating (1) a first transition state model structure (TSA1) derived from the migratory insertion of an ethylene molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework and (2) a second transition state model structure (TSA2) derived from the migratory insertion of an α-olefin co-monomer molecule into the metal-hydrocarbyl ligand bond of the first metallocene catalyst framework;(c) determining, by at least one processor of a device, the relative energies of each of the first ground state model structure (GSA), the first transition state model structure (TSA) including a dispersion energy (Disp EA1) associated with TSA1, and the second transition state model structure (TSA2) including a dispersion energy (Disp EA2) associated with TSA2, and determining values for ΔG‡A1(TSA1−GSA), ΔG‡A2(TSA2−GSA), and ΔΔG‡A(TSA2−TSA1) for migratory insertion of the ethylene molecule versus the α-olefin molecule in the first metallocene catalyst framework;(d) determining, by at least one processor of a device, the number of stabilizing, non-covalent (dispersion-type) interactions (NCI) within a distance of from 2.5 Å to 4.0 Å, inclusive, between (1) the ethylene molecule and the substituted or unsubstituted η5-cycloalkadienyl ligands in the first transition state model structure TSA1(NCIA1), and (2) the α-olefin molecule and the substituted or unsubstituted η5-cycloalkadienyl ligands in the second transition state model structure TSA2(NCIA2), and difference between the number of these NCI interactions (ΔNCIA);(e) repeating steps (a)-(d) using a second metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a first test substituent, and generating a corresponding second ground state model structure (GSB), third transition state model structure (TSB1), and fourth transition state model structure (TSB2), and determining, by at least one processor of a device, the relative energies of each of a GSB, TSB1including a dispersion energy (Disp EB1) associated with TSB1, TSB2including a dispersion energy (Disp EB2) associated with TSB2, and determining values for ΔG‡B1(TSB1−GSB), ΔG‡B2(TSB2−GSB), ΔΔG‡B(TSB2−TSB1), and the number of stabilizing, non-covalent (dispersion-type) interactions in TSB1(NCIB1) and TSB2(NCIB2), and difference between the numbers of these NCI interactions (ΔNCIB), for migratory insertion of the ethylene molecule versus the α-olefin molecule in the second metallocene catalyst framework; and(f) identifying the first test substituent of the second metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B<ΔΔG‡A, when ΔNCIB>ΔNCIA, or a combination thereof, or (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡B>ΔΔG‡A, when ΔNCIB<ΔNCIA, or a combination thereof.Aspect 4. The method for designing a Group 4 metallocene olefin polymerization catalyst according to Aspect 3, further comprising the steps of:(g) repeating steps (a)-(d) using a third metallocene catalyst framework comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises a second test substituent, and generating a corresponding third ground state model structure (GSC), fifth transition state model structure (TSC1), and sixth transition state model structure (TSC2), and determining, by at least one processor of a device, the relative energies of each of a GSC, TSC1including a dispersion energy (Disp EC1) associated with TSC1, and TSC2including a dispersion energy (Disp EC2) associated with TSC2, and determining values for ΔG‡C1(TSC1−GSC), ΔG‡C2(TSC2−GSC), ΔΔG‡C(TSC2−TSC1), and the number of stabilizing, non-covalent (dispersion-type) interactions in TSC1(NCIC1) and TSC2(NCIC2), and difference between the numbers of these NCI interactions (ΔNCIC), for migratory insertion of the ethylene molecule versus the α-olefin molecule in the second metallocene catalyst framework; and(h) identifying the second test substituent of the third metallocene catalyst framework as (1) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C<ΔΔG‡A, when ΔNCIC>ΔNCIA, or a combination thereof, (2) enhancing ethylene incorporation into a polyethylene co-polymer relative to the first metallocene catalyst framework when ΔΔG‡C>ΔΔG‡A, when ΔNCIC<ΔNCIAor a combination thereof, (3) enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C<ΔΔG‡B, when ΔNCIC>ΔNCIB, or a combination thereof, or (4) enhancing ethylene incorporation into a polyethylene co-polymer relative to the second metallocene catalyst framework when ΔΔG‡C>ΔΔG‡B, when ΔNCIC<ΔNCIB, or a combination thereof.Aspect 5. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of Aspects 3-4, wherein the number of non-covalent (dispersion-type) interactions NCIA, NCIB, and NCICwithin a distance of from 2.5 Å to 4.0 Å comprises the number of CH—H, CH—X (X═F, Cl, Br, N, O), and CH-π interactions between the ethylene molecule or the α-olefin molecule and the substituted or unsubstituted η5-cycloalkadienyl ligands and the first test substituent of the first metallocene catalyst framework or second metallocene catalyst framework within a distance range of 2.5 to 4.0 Å.Aspect 6. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein:the first test substituent of the second metallocene catalyst framework is identified as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon the relative energies of ΔΔG‡Aand ΔΔG‡B; orthe second test substituent of the third metallocene catalyst framework is identified as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon the relative energies of two of ΔΔG‡A, ΔΔG‡B, and ΔΔG‡C.Aspect 7. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any of Aspects 1-2, wherein:the first test substituent of the second metallocene catalyst framework is identified as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon the relative energies of |ΔDisp EA| and |ΔDisp EB|; orthe second test substituent of the third metallocene catalyst framework is identified as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon the relative energies of two of |ΔDisp EA|, |ΔDisp EB| and |ΔDisp EC|.Aspect 8. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any of Aspects 3-5, wherein:the first test substituent of the second metallocene catalyst framework is identified as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon ΔNCIAand ΔNCIB; orthe second test substituent of the third metallocene catalyst framework is identified as enhancing α-olefin co-monomer incorporation or enhancing ethylene incorporation into the polyethylene co-polymer based upon ΔNCIA, ΔNCIB, and ΔNCIC.Aspect 9. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the energies of any one or more of the ground state model structures (GSA, GSB, GSC) and any one or more of the transition state model structures (TSA1, TSA2, TSB1, TSB2, TSC1, TSC2) is calculated as a B3LYP single point energy calculation with a D3BJ correction (B3LYP+D3BJ) using a density functional theory (DFT).Aspect 10. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of Aspects 1-2, 7, or 9, wherein the any one or more of the dispersion energies (Disp EA, Disp EB, Disp EC) is calculated as the difference between a B3LYP single point energy calculation with and without a D3BJ correction using a density functional theory (DFT).Aspect 11. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of Aspects 3-5 or 8-9, wherein the any of the number of stabilizing, non-covalent (dispersion-type) interactions (NCIA1, NCIA2, NCIB1, NCIB2, NCIC1, NCIC2) are calculated using absolutely localized molecular orbital energy decomposition analysis (ALMO-EDA) of the respective transition state model structures (TSA1, TSA2, TSB1, TSB2, TSC1, and TSC2).Aspect 12. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first test substituent or the second test substituent is selected from: a halide (F, Cl, or Br); a C1-C10heterohydrocarbyl group comprising a heteroatom selected from halide (F, Cl, or Br), N, O, P, or S; a C1-C10aliphatic group; or a C6-C10aromatic group.Aspect 13. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first test substituent or the second test substituent is selected from F, Cl, Br, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C6-C10aryl, C1-C6alkoxide, —OC(O)R1, —CH2C(O)R1, —NR12, —PH3, PR13, or —SR1, wherein R1is independently selected from a C1-C6alkyl or a C6-C10aryl.Aspect 14. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first test substituent or the second test substituent is selected from F, Cl, Br, —CH3, —CMe3, —C(CH2)3CH, —CF3, —OMe, —OC(O)Me, —CH2C(O)Me, —OC6H11, —OPh, —NMe2, —N(C6H11)2, PH3, PMe3, —SC6H11, or —SPh.Aspect 15. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises Ti, alternatively Zr, or alternatively Hf.Aspect 16. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the hydrocarbyl ligand is a C1-C6hydrocarbyl ligand, or alternatively a C1-C4hydrocarbyl ligand, or alternatively an ethyl (C2) ligand.Aspect 17. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the α-olefin co-monomer comprises or is selected from propylene, 1-butene, 1-pentene, 1-hexene, 1-heptene, 1-octene, or styrene.Aspect 18. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the substituted or unsubstituted η5-cycloalkadienyl ligands are selected independently from cyclopentadienyl, indenyl, or fluorenyl.Aspect 19. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the substituted or unsubstituted η5-cycloalkadienyl ligands, absent the first test substituent and absent the second test substituent, are selected independently from cyclopentadienyl, methylcyclopentadienyl, t-butyl-cyclopentadienyl, indenyl, 4-phenyl-indenyl, 2-methylindenyl, 3-t-butylindenyl, 2-methyl-4-phenylindenyl, fluorenyl, or 2-methylfluorenyl.Aspect 20. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands.Aspect 21. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands which are bridged by a linking group of the formula (1) >ER1R2, wherein E is C or Si, R1is hydrogen or a C1-C12hydrocarbyl group, and R2is hydrogen, a C1-C12hydrocarbyl group, or a C3-C10alkenyl group having a terminal C═C double bond, or (2) CR12CR2—, wherein R1and R2are selected independently from hydrogen or a C1-C6hydrocarbyl group.Aspect 22. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands which are bridged by a linking group of the formula >CMe2, —CH2CH2, >SiMe2, or >CH[(CH2)2CH═CH2].Aspect 23. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises one of the following structures: Aspect 24. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises one substituted or unsubstituted η5-cycloalkadienyl ligand and further comprises an anionic ligand.Aspect 25. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises one substituted or unsubstituted η5-cycloalkadienyl ligand and further comprises an anionic ligand selected from halide, hydride, a C1-C20hydrocarbyl group, a C1-C20heterohydrocarbyl group, tetrahydroborate, or OBRA2or OSO2RAwherein RAis independently a C1-C12hydrocarbyl group.Aspect 26. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, wherein the first metallocene catalyst framework comprises one substituted or unsubstituted η5-cycloalkadienyl ligand and further comprises an anionic ligand selected from F, Cl, Br, a hydride, a C1-C12hydrocarbyl group, a C1-C12hydrocarbyloxide group, a C1-C12hydrocarbylamino group, C1-C12dihydrocarbylamino, or a trihydrocarbylsilyl group wherein each hydrocarbyl is independently a C1-C12hydrocarbyl group.Aspect 27. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, further comprising the step of synthesizing the Group 4 metallocene catalyst comprising the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises the first test substituent or the second test substituent.Aspect 28. The method for designing a Group 4 metallocene olefin polymerization catalyst according to any one of the preceding Aspects, further comprising the steps of: providing a Group 4 metallocene catalyst comprising the one or two independently selected η5-cycloalkadienyl ligands, wherein at least one of the η5-cycloalkadienyl ligands comprises the first test substituent or the second test substituent; and contacting the Group 4 metallocene catalyst with ethylene and an α-olefin co-monomer molecule under polymerization conditions to form a polyethylene co-polymer.Aspect 29. A catalyst system for polymerizing olefins, the catalyst system comprising: a zirconocene catalyst comprising two η5-cycloalkadienyl ligands independently selected from a substituted or an unsubstituted η5-cyclopentadienyl ligand or a substituted or an unsubstituted η5-indenyl ligand, wherein the two η5-cycloalkadienyl ligands are optionally bridged by a linking group; and one of the η5-cycloalkadienyl ligands is substituted with at least one substituent which imparts enhanced dispersion-type interactions in a transition state for a migratory insertion of an α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of the zirconocene catalyst versus a migratory insertion of the α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of a zirconocene catalyst comprising the corresponding unsubstituted η5-cycloalkadienyl ligands.Aspect 30. A catalyst system for polymerizing olefins, the catalyst system comprising: a zirconocene catalyst comprising two η5-cycloalkadienyl ligands independently selected from a substituted or an unsubstituted η5-cyclopentadienyl ligand or a substituted or an unsubstituted η5-indenyl ligand, wherein the two η5-cycloalkadienyl ligands are optionally bridged by a linking group; and one of the η5-cycloalkadienyl ligands is substituted with at least one substituent which imparts enhanced number of stabilizing, non-covalent (dispersion-type) interactions (NCI) within a distance of from 2.5 Å to 4.0 Å, inclusive, between the α-olefin molecule and the at least one substituent in a transition state for a migratory insertion of an α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of the zirconocene catalyst versus a migratory insertion of the α-olefin co-monomer molecule into a metal-hydrocarbyl ligand bond of a zirconocene catalyst comprising the corresponding unsubstituted η5-cycloalkadienyl ligands.Aspect 31. The catalyst system for polymerizing olefins according to one of Aspects 29-30, wherein the zirconocene catalyst comprises one of the following structures: wherein:R is selected independently from F, Cl, Br, C1-C6alkyl, C6-C10aryl, C1-C6alkoxide, —OC(O)R1, —CH2C(O)R1, —NR12, —PH3, PR13, or —SR1, and wherein R1is independently selected from a C1-C6alkyl or a C6-C10aryl, in the substituted η5-cycloalkadienyl ligands; andR is H in the corresponding unsubstituted η5-cycloalkadienyl ligands.Aspect 32. The catalyst system for polymerizing olefins according to Aspect 31, wherein R is selected independently from F, Cl, Br, —CH3, —CMe3, —C(CH2)3CH, —CF3, —OMe, —OC(O)Me, —CH2C(O)Me, —OC6H11, —OPh, —NMe2, —N(C6H11)2, PH3, PMe3, —SC6H11, or —SPh, in the substituted η5-cycloalkadienyl ligands.Aspect 33. A catalyst system for polymerizing olefins, comprising:the Group 4 metallocene olefin polymerization catalyst comprising the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, according to any one of Aspects 1-28; andwherein at least one of the η5-cycloalkadienyl ligands comprises the first test substituent.Aspect 34. The catalyst system for polymerizing olefins according to Aspect 33, wherein the first test substituent is identified as enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer, relative to an α-olefin co-monomer incorporation of the first metallocene catalyst framework.Aspect 35. A catalyst system for polymerizing olefins, comprising:the Group 4 metallocene olefin polymerization catalyst comprises the Group 4 metal bonded to the hydrocarbyl ligand and to the one or two independently selected substituted or unsubstituted η5-cycloalkadienyl ligands, according to any of Aspects 2 and 4-28; andwherein at least one of the η5-cycloalkadienyl ligands comprises the second test substituent.Aspect 36. The catalyst system for polymerizing olefins according to Aspect 35, wherein the second test substituent is identified as enhancing α-olefin co-monomer incorporation into a polyethylene co-polymer, relative to an α-olefin co-monomer incorporation of the first metallocene catalyst framework.Aspect 37. A catalyst system for polymerizing olefins according to any one of Aspects 33-36, wherein the catalyst system further comprises:(a) an activator comprising a solid oxide treated with an electron-withdrawing anion (activator-support), an organoboron compound, an organoborate compound, an ionizing ionic compound, an aluminoxane compound, or any combination thereof; and(b) optionally, a co-catalyst comprising an organoaluminum compound, an organoboron compound, an organozinc compound, an organomagnesium compound, an organolithium compound, or any combination thereof.Aspect 38. The catalyst system for polymerizing olefins according to Aspect 37, wherein the activator comprises a solid oxide treated with an electron-withdrawing anion, and wherein: the solid oxide comprises silica, alumina, titania, zirconia, magnesia, boria, calcia, zinc oxide, silica-alumina, silica-coated alumina, silica-titania, silica-zirconia, silica-magnesia, alumina-titania, alumina-zirconia, zinc-aluminate, alumina-boria, silica-boria, aluminum phosphate, aluminophosphate, aluminophosphate-silica, magnesium aluminate, titania-zirconia, boehmite, heteropolytungstates, mixed oxides thereof, or any combination thereof; andthe electron-withdrawing anion comprises fluoride, chloride, bromide, iodide, sulfate, bisulfate, fluorosulfate, phosphate, fluorophosphate, triflate, mesylate, tosylate, thiosulfate, C1-C10alkyl sulfonate, C6-C14aryl sulfonate, trifluoroacetate, fluoroborate, fluorozirconate, fluorotitanate, or any combination thereof.Aspect 39. The catalyst system for polymerizing olefins according to Aspect 38, wherein:the solid oxide comprises alumina, silica-alumina, silica-coated alumina, or a mixture thereof, andthe electron-withdrawing anion comprises fluoride, sulfate, or phosphate.Aspect 40. The catalyst system for polymerizing olefins according to Aspect 37, wherein the activator comprises fluorided alumina, fluorided silica, fluorided silica-alumina, or fluorided silica-coated alumina (mullite).Aspect 41. The catalyst system for polymerizing olefins according to any one of Aspects 37-40, wherein the co-catalyst is present and comprises trimethylaluminum, triethylaluminum, tri-n-propylaluminum, tri-n-butylaluminum, triisobutylaluminum, tri-n-hexylaluminum, tri-n-octylaluminum, diisobutylaluminum hydride, diethylaluminum ethoxide, diethylaluminum chloride, or any combination thereof. 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11859042 | DETAILED DESCRIPTION OF THE INVENTION Before the present compositions and formulations of the invention are described, it is to be understood that this invention is not limited to particular compositions and formulations described, since such compositions and formulation may, of course, vary. It is also to be understood that the terminology used herein is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. The diversity of physical and mechanical properties exhibited by thermoplastic polyurethanes (hereinafter referred as TPU) arises from their ability to incorporate other chemical structures into these polymers. It is also well-known that TPUs tend to exhibit phase separation where the soft segment, also interchangeably referred as soft phase, units confer elastomeric behaviour while the microphase rich hard segment, also interchangeably referred as hard phase, provides physical cross linking. The hard segment and the soft segment do no generally mix. Thus, during cooling from above a particular temperature, spontaneous segregation of the soft segment and hard segment into separate soft and hard phases occurs. The prevalence of these segments and their fraction determines the properties exhibited by the final TPU. The thermoplastic polyurethane, as described hereinbelow, is a segmented thermoplastic polyurethane. For the purpose of the present invention, the soft segment is comprised of at least one polyol composition (P), as discussed hereinabove, while the hard segment is derived from the at least one polyisocyanate (PI) structure linked by the at least one low molecular weight diol (CE). The soft segment imparts amorphous properties to the TPU, while the hard segment imparts partially crystalline nature to the polyurethane. The soft segments primarily influence the elastic nature and low temperature performance while the hard segments particularly affect the modulus, hardness and upper-use temperature by their ability to remain associated. Thus, to obtain a TPU which minimizes or in fact prevents the occurrence of soft phase crystallization and further improves the mechanical performance of the resulting thermoplastic polyurethane for low temperature operation, the soft and hard segment fractions needs to be adjusted. According to the present invention, it was surprisingly found that the method for preparing a thermoplastic polyurethane, comprising the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P2) a second polyol (P2) which is different from the first polyol (P1),(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol, results in a thermoplastic polyurethane with a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode. Accordingly, a method for preparing a thermoplastic polyurethane according to the presently claimed invention also may comprise the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol,(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. According to a further embodiment, the present invention is also directed to a method for preparing a thermoplastic polyurethane, comprising the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P2) a second polyol (P2) which is different from the first polyol (P1),(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. In the thermoplastic polyurethane or TPU, as described hereinabove, the term “polyol” refers to polymer backbones containing nominally two or more hydroxyl groups, sometimes also referred as polyalcohol. According to step (A) of the present invention, at least one polyol composition (P) is provided comprising at least a poly-ε-caprolactone polyol as component (P1) and preferably also a second polyol (P2) which is different from the first polyol (P1). According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the weight ratio between the poly-ε-caprolactone polyol (P1) and the second polyol (P2) in the polyol composition (P) is in the range of ≥1:5 to ≤10:1. In step (A) of the above described method, the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03 and an OH value in the range of ≥10 mg KOH/g to ≤100 mg KOH/g determined according to DIN 53240-3:2016-03. Preferably, the number average molecular weight is in the range of ≥1500 g/mol to ≤9,000 g/mol determined according to DIN 55672-1: 2016-03. More preferably, it is in the range of ≥1500 g/mol to ≤8,000 g/mol determined according to DIN 55672-1: 2016-03. Most preferably, it is in the range of ≥1500 g/mol to ≤7,000 g/mol, or ≥1500 g/mol to ≤6,000 g/mol, or ≥1500 g/mol to ≤5,000 g/mol determined according to DIN 55672-1: 2016-03. In an embodiment, the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤4,000 g/mol determined according to DIN 55672-1: 2016-03. The at least one polyol composition (P), as described hereinabove, comprises a poly-ε-caprolactone polyol (P1). For the purpose of the present invention, the poly-ε-caprolactone polyol (P1), also interchangeably referred as polycaprolactone, preferably has a number average molecular weight in the range of ≥1500 g/mol to ≤2500 g/mol determined according to DIN 55672-1: 2016-03. Although, a person skilled in the art is well aware of the different poly-ε-caprolactone polyols available to him, the present invention TPU preferably employs a poly-ε-caprolactone polyol (P1) which is obtained or obtainable by reacting ε-caprolactone (P11) and a starter molecule (P12) having a number average molecular weight Mn in the range of ≥80 g/mol to ≤1500 g/mol as determined according to DIN 55672-1: 2016-03. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the polyol (P1) has a number average molecular weight in the range of ≥1500 g/mol to ≤2500 g/mol determined according to DIN 55672-1: 2016-03. The starter molecule (P12), as described hereinabove, is a difunctional starter having a number average molecular weight Mn in the range of ≥80 g/mol to ≤1500 g/mol as determined according to DIN 55672-1: 2016-03. Preferably, the molecular weight is in the range of ≥80 g/mol to ≤1500 g/mol, or ≥200 g/mol to ≤1500 g/mol as determined according to DIN 55672-1: 2016-03. More preferably, the molecular weight is in the range of ≥200 g/mol to ≤1400 g/mol, or ≥300 g/mol to ≤1400 g/mol, or ≥300 g/mol to ≤1400 g/mol, or ≥400 g/mol to ≤1400 g/mol, or ≥400 g/mol to ≤1300 g/mol, ≥500 g/mol to ≤1300 g/mol, or ≥600 g/mol to ≤1300 g/mol as determined according to DIN 55672-1: 2016-03. Most preferably, the molecular weight is in the range of ≥700 g/mol to ≤1300 g/mol, or ≥800 g/mol to ≤1300 g/mol, or ≥800 g/mol to ≤1200 g/mol, or ≥900 g/mol to ≤1200 g/mol as determined according to DIN 55672-1: 2016-03. In an embodiment, the starter molecule (P12) has a number average molecular weight Mn in the range of ≥900 g/mol to ≤1100 g/mol as determined according to DIN 55672-1: 2016-03. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the second polyol (P2) has a number average molecular weight Mn in the range of ≥1000 g/mol to ≤4000 g/mol determined according to DIN 55672-1: 2016-03. Suitable starter molecule (P12) for the purpose of the present invention include diols selected from the group consisting of neopentyl glycol, 1,4-butanediol, 1,6-hexanediol, polyethylene glycol, polypropylene glycol, α-hydro-ω-hydroxypoly(oxytetra-methylene) diol and α-hydro-ω-hydroxypoly(oxytri-methylene) diol. More preferably, the starter molecule (P12) is selected from the group consisting of 1,6-hexanediol, polyethylene glycol, polypropylene glycol, α-hydro-ω-hydroxypoly(oxytetra-methylene) diol and α-hydro-ω-hydroxypoly(oxytri-methylene) diol. Most preferably, the starter molecule (P12) is selected from the group consisting of polyethylene glycol, polypropylene glycol, α-hydro-ω-hydroxypoly(oxytetra-methylene) diol and α-hydro-ω-hydroxypoly(oxytri-methylene) diol. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the starter molecule (P12) in the poly-ε-caprolactone polyol (P1) in step (A) is selected from the group consisting of neopentyl glycol, 1,4-butanediol, 1,6-hexanediol, polyethylene glycol, polypropylene glycol, α-hydro-ω-hydroxypoly(oxytetra-methylene) diol and α-hydro-ω-hydroxypoly(oxytri-methylene) diol. In an embodiment, the starter molecule (P12) is α-hydro-ω-hydroxypoly(oxytetra-methylene) diol. The α-hydro-ω-hydroxy-poly(oxytetra-methylene) diol, also known as polytetramethylene glycol, is obtained by ring-opening polymerisation of tetrahydrofuran with the aid of highly acidic catalysts. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that in step (A) the poly-ε-caprolactone polyol (P1) is obtained by reacting ε-caprolactone (P11) and a starter molecule (P12) having a number average molecular weight in the range of ≥80 g/mol to ≤1500 g/mol as determined according to DIN 55672-1:2016-03. The poly-ε-caprolactone polyol (P1) in the at least one polyol composition (P) of step (A) preferably is a block copolymer synthesized from ε-caprolactone (P11) and the starter molecule (P12), as described hereinabove. More preferably, the poly-ε-caprolactone polyol (P1) is a A-B-A triblock copolymer, wherein A represents the ε-caprolactone (P11) while the starter molecule (P12) is represented by B. The presence of the at least one polyol composition (P) as the poly-ε-caprolactone polyol (P1) triblock copolymer, as described hereinabove, contributes in preventing the soft phase crystallization of the thermoplastic polyurethane obtained therefrom, with good mechanical and physical properties. In a particularly preferable embodiment, the poly-ε-caprolactone polyol (P1) comprises a (P11)-(P12)-(P11) triblock copolymer. In this manner, the resulting polyol comprises the starter molecule (P12) as a core which is extended by means of the ε-caprolactone (P11). The choice of the poly-ε-caprolactone polyol (P1), as described hereinabove, is independent of the method for obtaining the same. A person skilled in the art is well aware of these techniques. Commercially available poly-ε-caprolactone polyol (P1) such as, but not limited to, Capa™ from Perstorp can also be employed. The poly-ε-caprolactone polyol (P1) in step (A), as described hereinabove, preferably has a weight ratio between the ε-caprolactone (P11) and the starting molecule (P12) in the range of ≥1:10 to ≤10:1. Preferably, the weight ratio is in the range of ≥1:9 to ≤10:1, or ≥1:9 to ≤9:1, or ≥1:8 to ≤9:1. More preferably, the weight ratio is in the range of ≥1:8 to ≤8:1, or ≥1:7 to ≤8:1, or ≥1:7 to ≤7:1, or ≥1:6 to ≤7:1, or ≥1:6 to ≤6:1. Most preferably, the weight ratio is in the range of ≥1:5 to ≤6:1, or ≥1:5 to ≤5:1, or ≥1:4 to ≤5:1, or ≥1:4 to ≤4:1, or ≥1:3 to ≤4:1. In an embodiment, the weight ratio between the ε-caprolactone (P11) and the starting molecule (P12) in the poly-ε-caprolactone polyol (P1) in step (A) is in the range of ≥1:3 to ≤3:1. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the poly-ε-caprolactone polyol (P1) in step (A) has a weight ratio between the ε-caprolactone (P11) and the starting molecule (P12) in the range of ≥1:10 to ≤10:1. The at least one polyol composition (P) in step (A) of the method, as described hereinabove, preferably further comprises a second polyol (P2) which is different from the first polyol i.e. poly-ε-caprolactone polyol (P1). Although, the second polyol (P2) is different from the poly-ε-caprolactone polyol (P1), it is generally preferred to choose the second polyol (P2) which is similar or in fact same as the starter molecule (P12) in the poly-ε-caprolactone polyol (P1). This results in reduction or circumvention of the soft phase crystallization shown by the TPU comprising the second polyol only and further improves the physical and mechanical properties of the resulting TPU. Accordingly, in an embodiment the second polyol is optionally added to the at least one polyol composition (P) in step (A) along with the poly-ε-caprolactone polyol (P1). The second polyol is selected from the group consisting of a polyether polyol (P21), polyester polyol (P22) and polycarbonate polyol (P23). The terms “polyether polyol”, “polyester polyol” and “polycarbonate polyol” refer to the polyol derived from polyester, polyether and polycarbonate, respectively. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the second polyol (P2) is selected from the group consisting of a polyether polyol (P21), polyester polyol (P22) and polycarbonate polyol (P23). Therefore, in an embodiment of the present invention the method for preparing the thermoplastic polyurethane comprises the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P2) a second polyol, and(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. Suitable polyols are generally known to the person skilled in the art. The second polyol (P2), as described hereinabove, preferably has a number average molecular weight Mn in the range of ≥1000 g/mol to ≤4000 g/mol determined according to DIN 55672-1: 2016-03. Preferably, the molecular weight is in the range of ≥1000 g/mol to ≤3500 g/mol determined according to DIN 55672-1: 2016-03. More preferably, the molecular weight is in the range of ≥1000 g/mol to ≤3000 g/mol determined according to DIN 55672-1: 2016-03. Most preferably, the molecular weight is in the range of ≥1000 g/mol to ≤2500 g/mol determined according to DIN 55672-1: 2016-03. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that the second polyol (P2) has a number average molecular weight Mn in the range of ≥1000 g/mol to ≤4000 g/mol determined according to DIN 55672-1: 2016-03. Suitable polyether polyols, polyester polyols or polycarbonate polyols are known to the person skilled in the art. Typically, the polyether polyol (P21) is obtained by known methods, such as but not limited to, anionic polymerization of alkylene oxides with addition of at least one starter molecule which comprises from 2 to 8, preferably from 2 to 6, reactive hydrogen atoms in bound form in the presence of catalysts. As catalysts, it is possible to use alkali metal hydroxides such as, but not limited to, sodium or potassium hydroxide or alkali metal alkoxides, such as but not limited to, sodium methoxide, sodium or potassium ethoxide or potassium isopropoxide or, in the case of cationic polymerization, Lewis acids such as antimony pentachloride, boron trifluo-ride etherate or bleaching earth catalysts. Furthermore, double metal cyanide (or DMC) compounds can also be used as catalysts, as already described hereinabove. Other suitable polyether polyols (P21) include polyether diols and triols, such as polyoxypropyl-ene diols and triols and poly(oxyethylene-oxypropylene)diols and triols obtained by the simultaneous or sequential addition of ethylene and propylene oxides to di- or tri-functional initiators. Copolymers having oxyethylene contents in the range of ≥5 wt.-% to ≤90 wt.-%, based on the weight of the polyol component, of which the polyols may be block copolymers, random/block copolymers or random copolymers, can also be used. Preferably, the second polyol (P2) comprising the polyether polyol (P21) is derived from the group consisting of ethylene oxide, propylene oxide, butylene oxide, tetrahydrofuran (hereinafter referred to as THF) and a mixture thereof. By the term “derived”, as used herein, it refers to the building block of the polyether polyol. For the purpose of the present invention, suitable polyether polyols (P21) include polytetramethylene glycols obtained by the polymerization of cyclic ether, tetrahydrofuran. Polytetramethylene glycol or α-hydro-ω-hydroxypoly(oxytetra-methylene) diol having a number average molecular weight in the range of ≥1500 g/mol to ≤2500 g/mol as determined according to DIN 55672-1: 2016-03 are particularly preferable as polyether polyol (P21). Mixtures of two or more α-hydro-ω-hydroxypoly(oxytetra-methylene) diols having differing molecular weights are also employable in the context of the present invention. The choice of suitable polyether polyol (P21), as described hereinabove, is independent of the method for obtaining the same. Accordingly, a person skilled in the art is well aware of such polyether polyols (P21). However, commercially available polyether polyols (P21) such as, but not limited to, PolyTHF® from BASF can also be employed. Accordingly, in a further embodiment the method for preparing the thermoplastic polyurethane comprises the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P21) a polyether polyol, and(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. In another embodiment, the method for preparing the thermoplastic polyurethane comprises the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P21) α-hydro-ω-hydroxypoly(oxytetra-methylene) diol, and(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. Polyester polyol (P22) as suitable second polyol for the present invention comprise for example of at least one C4to C12dicarboxylic acid and at least one C2to C14diol. The at least one C4to C12dicarboxylic acid is selected from the group consisting of aliphatic dicarboxylic acid such as succinic acid, glutaric acid, adipic acid, suberic acid, azelaic acid and sebacic acid and aromatic dicarboxylic acid such as phthalic acid, isophthalic acid and terephthalic acid. The carboxylic acids can be utilized individually or in the form of mixtures, for example, a mixture succinic acid, glutaric acid and adipic acid. Preferably C2to C6diol such as ethylene glycol, diethylene glycol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,10-decanediol, 2,2-dimethyl-propane-1,3-diol, 1,3-propanediol, 2-methyl-1,3-propanediol and dipropylene glycol can be used individually or as mixtures. Alkoxylated diols obtained by alkoxylation of a diol with at least one C2to C4alkylene oxide can also be employed as suitable polyester polyol (P22) in the present invention. By the term “alkoxylated”, it is referred to the end capping of the at least one C2to C14diol by suitable alkylene oxides such as, but not limited to, at least one C2to C4alkylene oxide in an alkoxylation reaction. Preferably, the reaction between the at least one C2to C14diol with the at least one C2to C4alkylene oxide is carried out in the presence of at least one catalyst. Preferably the at least one catalyst is a base or a double metal cyanide catalyst (DMC catalyst). More preferably the at least one catalyst is selected from the group consisting of alkaline earth metal hydroxides such as calcium hydroxide, strontium hydroxide and barium hydroxide, alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, potassium hydroxide, rubidium hydroxide and caesium hydroxide and alkali metal alkoxylates such as potassium tert-butoxylate. Most preferably the at least one catalyst is potassium hydroxide or caesium hydroxide. In case the catalyst is a base, any inert solvents capable of dissolving alkoxylated C2to C14diol may be used as solvents during the reaction or as solvents required for working up the reaction mixture in cases where the reaction is carried out without solvents. The following solvents are mentioned as examples: toluene, methylene chloride, trichloroethylene, tetrahydrofuran, dioxane, methyl ethyl ketone, methylisobutyl ketone, ethyl acetate and isobutyl acetate. In case the catalyst is a base, the amount of catalysts used is preferably in the range ≥0.01 wt.-% to ≤3.0 wt.-%, more preferably in the range ≥0.05 wt.-% to ≤2.0 wt.-%, based on the total amount of the alkoxylated C2to C14diol. The reaction is preferably carried out at a temperature in the range of 70 to 200° C., more preferably from 100 to 160° C. The pressure is preferably in the range from 1 bar to 50 bar, more preferably in the range from 1 bar to 40 bar, even more preferably in the range from 1 bar to 30 bar or 2 bar to 30 bar. Particularly, the pressure is in the range from 3 bar to 30 bar. The alkoxylated C2to C14diol may also be obtained from DMC catalysts. The DMC catalysts are usually prepared as a solid and used as such. The catalyst is typically used as powder or in suspension. However, other ways known to those skilled in the art for using catalysts can likewise be employed. The DMC catalyst can be dispersed with an inert or non-inert suspension medium which can be, for example, the product to be produced or an intermediate by suitable measures, e.g. milling. The suspension produced in this way is used, if appropriate after removal of interfering amounts of water by methods known to those skilled in the art, e.g. stripping with or without use of inert gases such as nitrogen and/or noble gases. Suitable suspension media are, for example, toluene, xylene, tetrahydrofuran, acetone, 2-methylpentanone, cyclo-hexanone and also polyether alcohols and mixtures thereof. The catalyst is preferably used in a suspension in the polyester polyol as described, for example, in EP 0 090 444 A. Polycarbonate polyol (P23) as suitable second polyol (P2) in the at least one polyol composition (P) of step (A) may be obtained by, such as but not limited to, the reaction of phosgene or a carbonate monomer, usually dimethyl carbonate with a diol monomer or a mixture of diol monomers. Alternatively, suitable hydroxyl terminated polycarbonates include those prepared by reacting a glycol with a carbonate. U.S. Pat. No. 4,131,731 describes such hydroxyl terminated polycarbonates. The polycarbonates are linear and have terminal hydroxyl groups with essential exclusion of other terminal groups. The essential reactants are glycols and carbonates. Suitable glycols are selected from cycloaliphatic and aliphatic diols containing 4 to 40, and or even 4 to 12 carbon atoms, and from polyoxyalkylene glycols containing 2 to 20 alkoxy groups per molecule with each alkoxy group containing 2 to 4 carbon atoms. Suitable diols include aliphatic diols containing 4 to 12 carbon atoms such as 1,4-butanediol, 1,5-pentanediol, neopentyl glycol, 1,6-hexanediol, 1,6-2,2,4-trimethylhexanediol, 1,10-decanediol, hydrogenated dilinoleylglycol, hydrogenated dioleylglycol; and cycloaliphatic diols such as 1,3-cyclohexanediol, 1,4-dimethylolcyclohexane, 1,4-cyclohexanediol, 1,3-dimethylolcyclohexane, 1,4-endo methylene-2-hydroxy-5-hydroxymethyl cyclohexane, and polyalkylene glycol. The diols used in the reaction may be a single diol or a mixture of diols depending on the properties desired in the finished product. Polycarbonate intermediates which are hydroxyl terminated are generally those known to a person skilled in the art. Suitable carbonates are selected from alkylene carbonates composed of a 5 to 7-member ring. Suitable carbonates for use herein include ethylene carbonate, trimethylene carbonate, tetramethylene carbonate, 1,2-propylene carbonate, 1,2-butylene carbonate, 2,3-butylene carbonate, 1,2-ethylene carbonate, 1,3-pentylene carbonate, 1,4-pentylene carbonate, 2,3-pentylene carbonate and 2,4-pentylene carbonate. Also, suitable herein are dialkylcarbonates, cycloaliphatic carbonates and diarylcarbonates. The dialkylcarbonates can contain 2 to 5 carbon atoms in each alkyl group and specific examples thereof are diethylcarbonate and dipropylcarbonate. Cycloaliphatic carbonates, especially dicycloaliphatic carbonates, can contain 4 to 7 carbon atoms in each cyclic structure and there can be one or two of such structures. When one group is cycloaliphatic, the other can be either alkyl or aryl. On the other hand, if one group is aryl, the other can be alkyl or cycloaliphatic. Examples of suitable diarylcarbonates which can contain 6 to 20 carbon atoms in each aryl group, are diphenylcarbonate, ditolylcarbonate and dinaphthylcarbonate. The weight ratio of the components of composition (P) may vary in broad ranges. The polyol composition may also comprise further polyols or solvents. The weight ratio between the poly-ε-caprolactone polyol (P1) and the second polyol (P2) in the at least one polyol composition (P) of step (A), as described hereinabove, is in the range of ≥1:5 to ≤10:1. Preferably, the weight ratio is in the range of ≥1:5 to ≤9.5:1, or ≥1:5 to ≤9:1, or ≥1:5 to ≤8.5:1. More preferably, the ratio is in the range of ≥1:4 to ≤8:1, or 1:4 to ≤7.5:1, or ≥1:4 to ≤7:1, or ≥1:4 to ≤6.5:1, or ≥1:4 to ≤6:1, or ≥1:4 to ≤5.5:1. Most preferably, the ratio is in the range of ≥1:3 to ≤5:1, or ≥1:3 to ≤4.5:1, or ≥1:3 to ≤4:1, or ≥1:3 to ≤3.5:1. In an embodiment, weight ratio between the poly-ε-caprolactone polyol (P1) and the second polyol (P2) is in the range of ≥1:3 to ≤3:1. According to the present invention, according to step (B), the at least one polyol composition (P) of step (A) is reacted with the at least one polyisocyanate (PI) and the at least one low molecular weight diol (CE) in the step (B), as described hereinabove. Optionally, the step (B) proceeds in presence of the at least one catalyst (CA) and/or the at least one additive (AD). The ingredients for preparing the thermoplastic polyurethane are preferably reacted simultaneously. By the term “simultaneously”, it refers to the ingredients viz. the at least one polyol composition (P), the at least one polyisocyanate (PI), the at least one low molecular weight diol (CE), optionally the at least one catalyst (CA) and/or the at least one additive (AD) being reacted together at once. For instance, the at least one polyol composition (P), the at least one polyisocyanate (PI) and the at least one chain extender are reacted simultaneously in step (B) above. If required, the at least one catalyst (CA) and/or the at least one additive (AD) may also be optionally reacted simultaneously along with the others. This makes the process of the present invention a one-shot process for preparing thermoplastic polyurethane. This is another objective of the present invention to provide a one-shot method as an alternative to a two-shot method. As the name suggests, the one-shot process is a single step process and involves the simultaneous addition of the at least one polyol composition (P), the at least one polyisocyanate (PI) and the at least one low molecular weight diol, as described above in step (B). Accordingly, in a further embodiment of the present invention, the present invention is directed to a one-shot process for preparing the thermoplastic process comprises the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode, wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. According to the present invention, the polyol composition (P) is reacted with at least one polyisocyanate (PI). The term “polyisocyanate”, as used herein, refers to an isocyanate comprising at least two N═C═O groups, such as diisocyanates or triisocyanates, as well as dimers and trimers or biurets of the isocyanates discussed herein. Preferably, the NCO groups or functionality of the at least one polyisocyanate (PI) is in the range of ≥1.5 to ≤2.2. More preferably, it is in the range of ≥1.9 to ≤2.1. Most preferably, in the range of ≥1.9 to ≤2.1. The most chemically relevant attribute of isocyanate chemistry is its reactivity with molecules having active hydrogens. Such active hydrogens are typically found on molecules having alcohol and amine functionalities and water. Suitable conditions for step (B) are generally known to the person skilled in the art. The temperature in step (B) is preferably in the range of ≥70° C. to ≤120° C. Optionally, the ingredients in step (B) i.e. the at least one polyisocyanate (PI) the at least one low molecular weight diol (CE), the at least one polyol composition (P) and optionally the at least one catalyst (CA) and/or the at least one additive are mixed by means of suitable mixers and stirrers well known to the person skilled in the art. For the purpose of the present invention, the at least one polyisocyanate (PI) may be an aliphatic polyisocyanate (PI1), cycloaliphatic polyisocyanate (PI2), aromatic polyisocyanate (PI3) or mixtures thereof. In an embodiment, the at least one polyisocyanate (PI) is at least one diisocyanate of the abovementioned aliphatic, cycloaliphatic and aromatic polyisocyanates. Representative examples of these preferred diisocyanates may be found, for example, from U.S. Pat. Nos. 4,385,133, 4,522,975 and 5,167,899. Suitable cycloaliphatic polyisocyanates (PI2) include those in which two or more of the isocyanato groups are attached directly and/or indirectly to the cycloaliphatic ring. Aromatic polyisocyanates (PI3) include those in which two or more of the isocyanato groups are attached directly and/or indirectly to the aromatic ring. In an embodiment, the aliphatic polyisocyanates (PI1) and cycloaliphatic polyisocyanates (PI2) can comprise 6 to 100 carbon atoms linked in a straight chain or cyclized and having two isocyanate reactive end groups. Accordingly, the method for preparing the thermoplastic polyurethane comprising the at least one polyisocyanate (PI) as aliphatic polyisocyanate (PI1) is selected from the group consisting of tetramethylene 1,4-diisocyanate, pentamethylene 1,5-diisocyanate, hexamethylene 1,6-diisocyanate, decamethylene diisocyanate, 1,12-dodecane diisocyanate, 2,2,4-trimethyl-hexamethylene diisocyanate, 2,4,4-trimethyl-hexamethylene diisocyanate and 2-methyl-1,5-pentamethylene diisocyanate. The at least one polyisocyanate (PI) as cycloaliphatic polyisocyanate (PI2) is selected from the group consisting of cyclobutane-1,3-diisocyanate, 1,2-, 1,3- and 1,4-cyclohexane diisocyanates, 2,4- and 2,6-methylcyclohexane diisocyanate, 4,4′- and 2,4′-dicyclohexyldiisocyanates, 1,3,5-cyclohexane triisocyanates, isocyanatomethylcyclohexane isocyanates, isocyanatoethylcyclohexane isocyanates, bis(isocyanatomethyl)cyclohexane diisocyanates, 4,4′- and 2,4′-bis(isocyanato-methyl) dicyclohexane, isophorone diisocyanate and 4,4′-diisocyanatdicyclohexylmethane. The at least one polyisocyanate (PI) as aromatic polyisocyanate (PI3) is selected from the group consisting 2,4- and 2,6-hexahydrotoluenediisocyanate, 1,2-, 1,3-, and 1,4-phenylene diisocyanates, triphenyl methane-4,4′,4″-triisocyanate, naphthylene-1,5-diisocyanate, 2,4- and 2,6-toluene diisocyanate, 2,4′-, 4,4′- and 2,2-biphenyl diisocyanates, 2,2′-, 2,4′- and 4,4′-diphenylmethane diisocyanate, polyphenyl polymethylene polyisocyanates, 1,2-, 1,3- and 1,4-xylylene diisocyanates and m-tetramethylxylyene diisocyanate (TMXDI). Preferably, the at least one polyisocyanate (PI) is selected from the group consisting of 2,2′-, 2,4′- and 4,4′-diphenylmethane diisocyanate, 2,4- and 2,6-toluene diisocyanate, 1,2-, 1,3- and 1,4-cyclohexane diisocyanates, hexamethylene 1,6-diisocyanate, 2-methyl-1,5-pentamethylene diisocyanate, pentamethylene 1,5-diisocyanate, tetramethylene 1,4-diisocyanate, isophorone diisocyanate, p-phenyl diisocyanate, o-tolidine diisocyanate and 1,5-naphthalene diisocyanate and 4,4′-Diisocyanatdicyclohexylmethane. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that in step (B) the at least one polyisocyanate (PI) is selected from the group consisting of 2,2′-, 2,4′- and 4,4′-diphenylmethane diisocyanate, 2,4- and 2,6-toluene diisocyanate, 1,2-, 1,3- and 1,4-cyclohexane diisocyanates, hexamethylene 1,6-diisocyanate, 2-methyl-1,5-pentamethylene diisocyanate, pentamethylene 1,5-diisocyanate, tetramethylene 1,4-diisocyanate, isophorone diisocyanate, p-phenyl diisocyanate, o-tolidine diisocyanate and 1,5-naphthalene diisocyanate and 4,4′-diisocyanatdicyclohexylmethane. More preferably, the at least one polyisocyanate (PI) is selected from the group consisting of diphenylmethane 4,4′-diisocyanate, tolylene 2,6-diisocyanate, dicyclohexylmethane 2,2′-diisocyanate, hexamethylene 1,6-diisocyanate, tetramethylenexylene 2,4-diisocyanate and 1 isocyanato-3,3,5 trimethyl-5 isocyanatomethylcyclohexane. Most preferably, the at least one polyisocyanate (PI) is selected from the group consisting of diphenylmethane 4,4′-diisocyanate, tolylene 2,6-diisocyanate, dicyclohexylmethane 2,2′-diisocyanate and hexamethylene 1,6-diisocyanate. In a particularly preferred embodiment, the at least one polyisocyanate (PI) is a diphenylmethane 4,4′-diisocyanate (hereinafter referred as MDI). MDI is produced from aniline and formaldehyde feedstocks. Such methods are known to a person skilled in the art. The choice of MDI is not limited to any particular method for preparing the same. Accordingly, the person skilled in the art may obtain MDI by any suitable method. In fact, MDI may be commercially obtained such as, but not limited to, Lupranat® by BASF. It is important to employ the correct stoichiometry in carrying out polymerization reaction, thereby leading to the thermoplastic polyurethane having the desired properties. Typically, the total number of isocyanate groups will be greater than or equal to the sum of active hydrogen-containing groups in the polymer. By the term “active hydrogen-containing groups”, it is referred to the isocyanate-reactive groups or the hydroxyl groups of the at least one polyol composition (P) and the at least one low molecular weight diol (CE). This is expressed in terms of isocyanate index, which is usually greater than 1000. In other words, if the isocyanate index is greater than 1000, there is an excess of isocyanate groups. The isocyanate index in the context of the present invention is the stoichiometric ratio of isocyanate groups to the isocyanate-reactive groups, as described hereinabove. For the purpose of the present invention, the at least one polyol composition (P), the at least one polyisocyanate (PI) and the at least one low molecular weight diol (CE) are added in such amounts that the isocyanate index is in the range of ≥900 to ≤1500. More preferably, it is in the range of ≥900 to ≤1300. Most preferably, it is in the range of ≥900 to ≤1100. In an embodiment, the isocyanate index is in the range of ≥950 to ≤1050. Typically, the molar ratio between the at least one polyol composition (P) and the at least one polyisocyanate (PI) is in the range of ≥0.1:1 to ≤1:1 so that the resulting isocyanate index is in the range as prescribed hereinabove. Preferably, the weight ratio is in the range of ≥0.1:1 to ≤0.95:1, or ≥0.1:1 to ≤0.90:1, or ≥0.1:1 to ≤0.85:1, or ≥0.1:1 to ≤0.8:1, or ≥0.1:1 to ≤0.75:1. More preferably, the weight ratio is in the range of ≥0.1:1 to ≤0.7:1, or ≥0.1:1 to ≤0.65:1, or ≥0.1:1 to ≤0.6:1, or ≥0.1:1 to ≤0.55:1, or ≥0.1:1 to ≤0.5:1, or ≥0.1:1 to ≤0.45:1. Most preferably in the range of ≥0.15:1 to ≤0.45:1. In an embodiment, the molar ratio between the at least one polyol composition (P) and the at least one polyisocyanate (PI) is in the range of ≥0.15:1 to ≤0.4:1. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that in step (B) the molar ratio between the at least one polyol composition (P) and the at least one polyisocyanate (PI) is in the range of ≥0.1:1 to ≤1:1. In step (B) of the method described hereinabove, at least one low molecular weight diol (CE) is also present and generally functions as a chain extender thereby for example serving as a spacer between the neighbouring isocyanates. By the term “low molecular weight”, it refers to diols having a molecular weight in the range of ≥50 g/mol to ≤350 g/mol. The chain extender structure has a significant effect on the TPU properties because of its ability to drive phase separation, to complement or interfere with a regular hard segment structure and to promote inter-hard segment hydrogen bonding. The chain extenders are generally low molecular weight diol or diamine stringing together the isocyanate. These are preferably selected from the group of di- and/or tri-functional alcohols, di- to tetra-functional polyoxyalkylene polyols and of alkyl-substituted aromatic diamines, or of mixtures of two or more of the recited extenders. For the purpose of the present invention, the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol, as described hereinabove. Preferably, the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥60 g/mol to ≤350 g/mol. More preferably, the molecular weight is in the range of ≥60 g/mol to ≤330 g/mol, even more preferably in the range of ≥60 g/mol to ≤310 g/mol, or ≥60 g/mol to ≤310 g/mol, or ≥60 g/mol to ≤290 g/mol, or ≥60 g/mol to ≤290 g/mol, or ≥60 g/mol to ≤270 g/mol. Most preferably, the molecular weight is in the range of ≥70 g/mol to ≤270 g/mol, or ≥70 g/mol to ≤250 g/mol, or ≥70 g/mol to ≤250 g/mol, or ≥70 g/mol to ≤230 g/mol, or ≥70 g/mol to ≤230 g/mol, ≥70 g/mol to ≤210 g/mol. Even most preferably, the molecular weight is in the range of ≥70 g/mol to ≤190 g/mol, or ≥70 g/mol to ≤170 g/mol, or ≥70 g/mol to ≤150 g/mol. In a particularly preferable embodiment, the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥80 g/mol to ≤130 g/mol. The at least one low molecular weight diol (CE) as chain extenders are preferably C2to C12alkane diols, or C2to C6alkane diols. More preferably, ethanediol, 1,3-propanediol, 1,5-pentanediol, 1,6-hexanediol, 1,7-heptanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-decanediol and preferably 1,4-butanediol. Preferred chain extending and/or crosslinking agents further include dialkylene glycols having 4 to 8 carbon atoms, preferably diethylene glycol and dipropylene glycol and/or di-, tri- or tetrafunctional polyoxyalkylene polyols. The at least one low molecular weight diol (CE) may further include branched and/or unsaturated alkanediols having preferably not more than 12 carbon atoms, preferably 1,2-propanediol, 2 methylpropanediol-1,3,2,2-dimethylpropanediol-1,3, 2-butyl-2-ethylpropanediol-1,3, butene-2 diol-1,4 and butyne-2-diol-1,4, diesters of terephthalic acid with glycols of 2 to 4 carbon atoms, preferably terephthalic acid bis-ethylene glycol-1,4 or -butanediol-1,4, hydroxyalkylene ethers of hydroquinone or of resorcinol, preferably 1,4-di(β-hydroxyethyl)hydroquinone or 1,3 di(β-hydroxyethyl)resorcinol, alkanolamines having 2 to 12 carbon atoms, preferably ethanolamine, 2-aminopropanol and 3-amino-2,2-dimethylpropanol, N-alkyldialkanolamines, e.g., N-methyl- and N-ethyldiethanolamine. To obtain specific mechanical properties, the alkyl-substituted aromatic polyamines are preferably also used in admixture with the aforementioned low molecular weight polyhydric alcohols, preferably di- and/or tri-hydric alcohols or dialkylene glycols. In an embodiment, the at least one low molecular weight diol (CE) is selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, hydroquinone bis 2-hydroxyethyl ether and bis-2(hydroxy ethyl)-terephthalate. According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that in step (B) the at least one low molecular weight diol (CE) is selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, hydroquinone bis 2-hydroxyethyl ether and bis-2(hydroxy ethyl)-terephthalate. The weight ratio between the at least one polyol composition (P) and the at least one low molecular weight diol (CE) is in the range of ≥1:1 to ≤15:1. Preferably, the ratio is in the range of ≥1.4:1 to ≤15:1, or ≥1.4:1 to ≤14.8:1, or ≥1.8:1 to ≤14.6:1, or ≥1.8:1 to ≤14.6:1, or ≥2:1 to ≤14.4:1, or ≥2:1 to ≤14.2:1, or ≥2.4:1 to ≤14.2:1, or ≥2.4:1 to ≤14:1. More preferably, the ratio is in the range of ≥2.8:1 to ≤14:1, or ≥2.8:1 to ≤13.8:1, or ≥3:1 to ≤13.8:1, or ≥3:1 to ≤13.6:1, or ≥3.4:1 to ≤13.6:1, or ≥3.4:1 to ≤13.4:1, or ≥3.8:1 to ≤13.4:1, or ≥3.8:1 to ≤13.2:1, or ≥4:1 to ≤13.2:1, or ≥4:1 to ≤13:1. Most preferably, the ratio is in the range of ≥4.4:1 to ≤13:1, or ≥4.4:1 to ≤12.8:1, or ≥4.8:1 to ≤12.8:1, or ≥4.8:1 to ≤12.6:1, or ≥5:1 to ≤12.6:1, or ≥5:1 to ≤12.4:1, or ≥5:1 to ≤12.2:1. In an embodiment, weight ratio between the at least one polyol composition (P) and the at least one low molecular weight diol (CE) is in the range of ≥5:1 to ≤12:1. For the purpose of the present invention, the at least one catalyst (CA) may be optionally added in step (B) of the method, as described hereinabove. The at least one catalyst (CA) is preferably an organometallic compound, such as a tin(II) salt of an organic carboxylic acid, preferably tin(II) dioctoate, tin(II) dilaurate, dibutyltin diacetate or dibutyltin dilaurate, while other organometallic compounds are bismuth salts, preferably bismuth(III) neodecanoate, bismuth 2-ethylhexanoate and bismuth octanoate, or the catalyst is a tertiary amine such as tetramethylethylenediamine, N-methylmorpholine, diethylbenzylamine, triethylamine, dimethylcyclohexyl-amine, diazabicyclooctane, N,N′-dimethylpiperazine, N methyl,N′-(4-N-dimethylamino)butylpiperazine, N,N,N′,N″,N″-pentamethyldiethylenediamine. Similar substances can also be used as catalysts. Preferably, the at least one catalyst (CA) further includes amidines, preferably for example 2,3-dimethyl-3,4,5,6-tetra-hydropyrimidine, tris(dialkylaminoalkyl)-s-hexahydrotriazines, in particular tris(N,N-dimethyl-aminopropyl)-s-hexahydrotriazine, tetraalkylammonium hydroxides, preferably for example tetramethylammonium hydroxide. Preferred at least one catalyst (CA) further include N-methyl-N-dimethylaminoethylpiperazine and pentamethyldiethylenetriamine and also aromatic alkali metal carboxylates, alkali metal hydroxides, preferably for example sodium hydroxide, and alkali metal alkoxides, preferably for example sodium methoxide and potassium isopropoxide, and also alkali metal salts of long-chain fatty acids having 10 to 20 carbon atoms with or without OH side groups. The at least one catalyst (CA) is preferably used in amounts from 0.0001 parts by weight to 0.1 parts by weight per 100 parts by weight based on the at least one polyol composition (P). The person skilled in the art is well aware of such at least one catalyst (CA) and the amount to be added to obtain TPU. The choice and selection of the at least one catalyst (CA) does not limit the method of the present invention described hereinabove. The step (B) comprising the at least one additive (AD) is selected from the group consisting of antioxidant, hydrolysis stabilizer, light stabilizer, UV absorbers, blowing agents and other process stabilizers. The person skilled in the art is well aware of these additives and any further additives that may be added to obtain the thermoplastic polyurethane from the method as described hereinabove. For instance, commercially available additives such as, but not limited to, Citrofil® from Jungbunzlauer and Irganox® from BASF can be employed as the at least one additive (AD). According to a further embodiment, the present invention therefore is also directed to the method as disclosed above, characterized in that in step (B) the at least one additive (AD) is selected from the group consisting of antioxidant, hydrolysis stabilizer, light stabilizer, UV absorbers, blowing agents and other process stabilizers. Blowing agents are employable for example in the present invention. Commonly known chemically and/or physically acting compounds are yet additionally employable as blowing agents. Chemical blowing agents are compounds that react with isocyanate to form gaseous products. Physical blowing agents are compounds which are present in the TPU production ingredients in dissolved or emulsified form and vaporize under the conditions of TPU formation. Suitable blowing agents for the purposes of the present invention include, for example, low-boiling liquids which vaporize under the influence of the exothermic polyaddition reaction. Liquids that are inert with regard to the organic polyisocyanate and have boiling points below 100° C. are particularly suitable. Examples of liquids of this type, which are preferably used, are halogenated, preferably fluorinated, hydrocarbons, e.g., methylene chloride and dichloromonofluoromethane, per or partially fluorinated hydrocarbons, e.g., trifluoromethane, difluoromethane, difluoroethane, tetrafluoroethane and heptafluoropropane, hydrocarbons, e.g., n-butane, iso-butane, n-pentane, isopentane and also the technical-grade mixtures thereof, propane, propylene, hexane, heptane, cyclobutane, cyclopentane and cyclohexane, dialkyl ethers, preferably, for example, dimethyl ether, diethyl ether and furan, carboxylic acids, for example, farmic acid, carboxylic esters, preferably, for example, methyl and ethyl formates, ketones, preferably, for example, acetone, and/or fluorinated and/or perfluorinated, tertiary alkylamines, preferably, for example, perfluoro¬dimethylisopropylamine. Other blowing agents such as CO2and N2may also be employed for the purpose of the present invention. It is similarly possible to use mixtures of these low-boiling liquids with one another and/or with other substituted or unsubstituted hydrocarbons. The best amount of blowing agent depends on the target density and also on the amount of the preferably co-used water. Satisfactory results are generally obtained with amounts in the range of ≥1 wt.-% to ≤15 wt.-%, preferably ≥2 wt.-% to ≤11 wt.-%, based on the at least one polyol composition (P). A preferred embodiment employs a blowing agent comprising a mixture comprising one or more of these blowing agents and water, more preferably no physical blowing agents and yet more preferably water as sole blowing agent. The water content in a preferred embodiment is in the range of ≥0.1 wt.-% to ≤3 wt.-%, preferably in the range of ≥0.4 wt.-% to ≤2 wt.-% and more preferably in the range of ≥0.6 wt.-% to ≤1.5 wt.-%, based on the at least one polyol composition (P). Microbeads containing physical blowing agent may also be additionally admixed in the present invention. The microbeads are also employable in admixture with the aforementioned blowing agents. The microbeads typically consist of a shell of thermoplastic polymer and are filled in the core with a liquid, low-boiling substance based on alkanes. The production of such microbeads is described for example in U.S. Pat. No. 3,615,972. The microbeads are generally from 5 to 50 μm in diameter. Examples of suitable microbeads are available as Expancell® from Akzo Nobel. The microbeads are generally added in an amount in the range of ≥0.5 wt.-% to ≤5 wt.-%, based on the total weight of the at least one polyol composition (P). Alternatively, supercritical fluids may be used along with the blowing agents, as described hereinabove and mixed with molten thermoplastic polyurethane of the present invention. Said mixture can then be subjected to injection molding techniques in a mold to obtain low density foamed TPU. The supercritical fluids can be selected from the group consisting of supercritical CO2and/or N2. One such technique is described in US 2015/0038605 A1. Suitable techniques can also be employed in the present invention. Other blowing agents are selected based on the method and the precise conditions and include, but are not limited to organic liquids or inorganic gases, or a mixture thereof. Liquids that can be used comprise halogenated hydrocarbons, or saturated, aliphatic hydrocarbons, in particular those having from 3 to 8 carbon atoms. Suitable inorganic gases are nitrogen, air, ammonia, or carbon dioxide, as described hereinabove. Further details can be found in, for e.g. WO2005/023920, WO2007/082838, WO2010/136398, WO2013/153190, WO2014/198779, WO2015/055811, WO2017/030835, US2017/0036377, US2016/0271847, US2016/0108198, WO2014/150119, WO2014/150124 and WO2016/131671. Customary auxiliary substance materials and/or added substance materials are further employable. Auxiliary substance materials and/or added substance materials take the form of a single substance or of a mixture of two or more auxiliary substance materials and/or added substance materials. Examples include surface-active substances, fillers, flame retardants, nucleators, oxidation inhibitors, lubricating and demolding aids, dyes and pigments, optionally stabilizers, preferably against hydrolysis, light, heat or discoloration, organic and/or inorganic fillers, reinforcing agents and/or plasticizers. Stabilizers for the purposes of the present invention are additives to protect a plastic or a mixture of plastics from harmful environmental influences. Examples are primary and secondary antioxidants, hindered amine light stabilizers, UV absorbers, hydrolysis control agents, quench-ers and flame retardants. Examples of commercial stabilizers are given in Plastics Additive Handbook, 5th Edition, H. Zweifel, ed., Hanser Publishers, Munich, 2001 ([1]), p. 98 to 136. Useful surface-active substances include, for example, compounds to augment the homogenization of the starting materials and possibly also capable of acting as cell structure regulators. Examples include emulsifiers, e.g., the sodium salts of castor oil sulfates or of fatty acids, and also salts of fatty acids with amines, for example diethylamine oleate, diethanolamine stearate, diethanolamine ricinoleate, salts of sulfonic acids, e.g., alkali metal or ammonium salts of do-decylbenzene- or dinaphthylmethanedisulfonic acid and ricinoleic acid; foam stabilizers, such as siloxane-oxyalkylene interpolymers and other organosiloxanes, ethoxylated alkylphenols, ethoxylated fatty alcohols, paraffin oils, castor oil esters or, to be more precise, ricinoleic esters, Turkey red oil and peanut oil and cell regulators, such as paraffins, fatty alcohols and dimethylpolysiloxanes. To improve the emulsifying effect, the cell structure and/or its stabilization it is further possible to use oligomeric polyacrylates having polyoxyalkylene and fluoroalkane moieties as side groups. Surface-active substances are typically used in amounts from 0.01 part by weight to 5 parts by weight, based on 100 parts by weight of the at least one polyol composition (P). Fillers, especially reinforcing fillers, include the customary, familiar organic and inorganic fillers, reinforcing agents and weighting agents. Specific examples are inorganic fillers such as silicatic minerals, for example sheet-silicates such as antigorite, serpentine, hornblendes, amphibols, chrisotile, talc; metal oxides, such as kaolin, aluminum oxides, aluminum silicate, titanium oxides and iron oxides, metal salts such as chalk, barite and inorganic pigments, such as cadmium sulfide, zinc sulfide and also glass particles. Useful organic fillers include for example carbon black, melamine, expandable graphite, rosin, cyclopentadienyl resins, graft polyols and graft polymers. By way of reinforcing fillers, it is preferable to use fibers, for example carbon fibers or glass fibers, particularly when a high level of heat resistance or very high stiffness is demanded, in which case the fibers may be endowed with adhesion promoters and/or sizers. Organic and inorganic fillers may be used singly or as mixtures, and are typically added to the reaction mixture in an amount in the range of ≥0.5 wt.-% to ≤50 wt.-%, preferably ≥1 wt.-% to ≤30 wt.-% based on the weight of the at least one polyol composition (P) and the at least one polyisocyanate (PI). Suitable flame retardants include, for example, tricresyl phosphate, tris(2-chloroethyl) phosphate, tris(2-chloropropyl) phosphate, tris(1,3-dichloropropyl) phosphate, tris(2,3-dibromo¬propyl) phosphate and tetrakis(2-chloroethyl) ethylene diphosphate. Aside from the aforementioned halogen-substituted phosphates, it is also possible to use inorganic flame retardants such as red phosphorus, aluminum oxide hydrate, antimony trioxide, arsenic trioxide, ammonium polyphosphate and calcium sulfate or cyanuric acid derivatives, e.g., melamine, or mixtures of two or more flame retardants, e.g., ammonium phosphates and melamine, and also optionally starch and/or expandable graphite to confer flame retardancy on the TPU prepared according to the present invention. As nucleators there may be used, for example, talc, calcium fluoride, sodium phenyl-phosphinate, aluminum oxide and finely divided polytetrafluoroethylene in amounts up to 5 wt.-%, based on the total weight of the at least one polyol composition (P) and the at least one polyisocyanate (PI), as described hereinabove. Suitable oxidation retarders and heat stabilizers may be also added to the method of the present invention. These include, for example, halides of metals of group I of the periodic table, e.g., sodium halides, potassium halides, lithium halides, optionally combined with copper(I) halides, e.g., chlorides, bromides or iodides, sterically hindered phenols, hydroquinones, and also substituted compounds of these groups and mixtures thereof, which are preferably used in concentrations up to 1 wt.-% based on the weight of the at least one polyol composition (P) and of the at least one polyisocyanate (PI). Examples of hydrolysis control agents which may be added to in the method, as described hereinabove, are various substituted carbodiimides, such as preferably 2,2′,6,6′-tetraisopropyldiphenylcarbodiimide or carbodiimides based on 1,3-bis(1-methyl-1 isocyanatoethyl)benzene as described for example in the documents DE 19821668 A1, U.S. Pat. No. 6,184,410, DE 10004328 A1, U.S. Pat. No. 6,730,807, EP 0940389 B1 or U.S. Pat. No. 5,498,747, which are generally used in amounts up to 4.0 wt.-%, preferably in the range of ≥1.5 wt.-% to ≤2.5 wt.-% based on the weight of the at least one polyol composition (P) and of the at least one polyisocyanate (PI). Lubricating and demolding agents, generally likewise added in amounts up to 1 wt.-%, based on the weight of the at least one polyol composition (P) and of the at least one polyisocyanate (PI), are stearic acid, stearyl alcohol, stearic esters and amides and also the fatty acid esters of pentaerythritol. It is further possible to add organic dyes, such as nigrosine, pigments, e.g., titanium dioxide, cadmium sulfide, cadmium sulfide selenide, phthalocyanines, ultramarine blue or carbon black. Further particulars of the abovementioned auxiliary and added-substance materials are found in the trade literature, for example in Plastics Additive Handbook, 5th edition, H. Zweifel, ed, Hanser Publishers, Munich, 2001, p. 98-136. The at least one additive (AD) as described hereinabove, if present, may be in any suitable amount known to the person skilled in the art. For instance, the at least one additive (AD) may be in an amount in the range of ≥0.1 wt.-% to ≤60 wt.-% based on the total weight of the thermoplastic polyurethane. According to a further aspect, the present invention therefore is also directed to a thermoplastic polyurethane obtained or obtainable by the method of the present invention as disclosed above. In an aspect of the present invention, a thermoplastic polyurethane as obtained by the method, as described hereinabove, has a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode and a hard segment fraction in the range of ≥0.1 to ≤0.7. The hard segment fraction being defined by the formula: Hardsegmentfraction={∑x=1k[(mKV,CE/MKV,CE)*MIso+mKV,CE]}/mtotalwherein,mKV,CEis the mass of the at least one low molecular weight diol (CE) in g,MKV,CEis the molar mass of the at least one low molecular weight diol (CE) in g/mol,MIsois the molar mass of the at least one polyisocyanate (PI) in g/mol,mtotalis the total mass of all the starting materials in g,k is the number of the at least one low molecular weight diol (CE). By the term “number” in the definition of ‘k’ in the above formula, it is referred to the number of units of the said at least one low molecular weight diol (CE). For the purpose of the present invention, the term “mtotal” as used hereinabove in the formula for calculating the hard segment fraction and representing the total mass of all the starting materials in g, comprises the at least one polyol composition (P), the at least one polyisocyanate (PI), the at least one low molecular weight diol (CE), optionally the at least one additive (AD) and/or the at least one catalyst (CA), as described hereinabove. Preferably, the thermoplastic polyurethane has the hard segment fraction in the range of ≥0.15 to ≤0.70. More preferably the hard segment fraction is in the range of ≥0.20 to ≤0.70. Most preferably, the hard segment fraction is in the range of ≥0.20 to ≤0.60. In an embodiment, the hard segment fraction of the thermoplastic polyurethane obtained according to the method as described hereinabove is in the range of ≥0.20 to ≤0.50. Preferably, the Tgof the thermoplastic polyurethane is in the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode. More preferably, the Tgis in the range of ≥−60° C. to ≤5° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode. Most preferably, the Tgis in the range of ≥−60° C. to ≤0° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode. For the purpose of the present invention, the Tgof the thermoplastic polyurethane is determined by dynamic mechanical thermal analysis, as described hereinabove. Dynamic mechanical thermal analysis or dynamic mechanical analysis yields information about the mechanical properties of a specimen placed in minor, usually sinusoidal, oscillation of a function of time and temperature by subjecting it to a small, usually sinusoidal, oscillating force. In order to measure the Tgvalue of the TPU, storage modulus (G′) and loss modulus (G″) are first determined. The storage modulus (G′) represents the stiffness of the polymer material and is proportional to the energy stored during a loading cycle. The loss modulus (G″) is defined as being proportional to the energy dissipated during one loading cycle. It represents, for example, energy lost as heat, and is a measure of vibrational energy that has been converted during vibration and that cannot be recovered. Next, phase angle delta (δ) is measured which is the phase difference between dynamic stress and dynamic strain in the TPU subjected to a sinusoidal oscillation. Loss factor tan delta is the ratio of loss modulus (G′) to storage modulus (G″). It is a measure of the energy lost, expressed in terms of the recoverable energy, and represents mechanical damping or internal friction in the TPU. A high tan delta value is indicative of a material that has a high, non-elastic strain component, while a low value indicates one that is more elastic. Often, the Tgvalue is taken to be the temperature of the maximum loss modulus (G″max) or the maximum loss factor (max tan delta), as shown in the examples described hereinbelow. The thermoplastic polyurethane, as described hereinabove, preferably has a durometer Shore A hardness in the range of ≥20 to ≤100 determined according to ASTM D2240:2015. Preferably, it is in the range of ≥40 to ≤90, more preferably in the range of ≥65 to ≤90, most preferably in the range of ≥70 to ≤90 determined according to ASTM D2240:2015. Shore D hardness of the thermoplastic polyurethane preferably is in the range of ≥30 to ≤90 determined according to ASTM D2240:2015. Preferably, it is in the range of ≥50 to ≤90, more preferably in the range of ≥60 to ≤90, most preferably in the range of ≥65 to ≤85 determined according to ASTM D2240:2015. The density of the thermoplastic polyurethane, as obtained according to the method described hereinabove, may vary depending on the components present therein and the expansion of the TPU itself. Nevertheless, the thermoplastic polyurethane preferably has a DIN EN ISO 845:2009-10 density in the range of ≥800 kg/m3to ≤1500 kg/m3. Preferably, the density is in the range of ≥900 kg/m3to ≤1500 kg/m3. More preferably, the density is in the range of ≥1000 kg/m3to ≤1500 kg/m3. Most preferably, the density is in the range of ≥1000 kg/m3to ≤1400 kg/m3. In a particularly preferred embodiment, the density is in the range of ≥1000 kg/m3to ≤1300 kg/m3as determined according to DIN EN ISO 845:2009-10. According to a further aspect, the present invention therefore is also directed to a use of the thermoplastic polyurethane as disclosed above or the thermoplastic polyurethane obtained by the method as disclosed above in extruded article and injection molded article. The ability of the present invention thermoplastic polyurethane to withstand low temperatures, such as those in the vicinity of its Tg, as described hereinabove, along with improved mechanical properties of abrasion resistance, tensile strength, elongation at break, tear propagation at strength and compression set without any soft phase crystallization for example enables it to be used as part of a shoe or of a shoe sole, for example part of an insert sole or of a midsole, sealants, profiles and other similar applications. Thus, the present invention therefore is also directed to the use of the thermoplastic polyurethane as described above for the production of filling material for mattresses, parts of mattresses, mattresses as such, filling of tires, tires or part of tires, shoes, shoe-soles, shoe-midsoles gymnastic mats, protective clothing, cushioning elements for automotive, sound absorbers, anti-vibration devices, cushioning elements for bicycle saddles, toys, flooring or packaging materials. The thermoplastic polyurethane shoe soles of the present invention are preferably employed as a midsole, for example for footwear, sport shoes, sandals and boots. More particularly, the polyurethane shoe soles of the present invention are used as midsole for shoes. A shoe sole according to the present invention further also comprises shoe sole parts, for example heel parts or ball parts. Shoe soles of the present invention can also be used as insert soles or combi-soles. The present invention TPU, as described hereinabove, can be further used in, such as but not limited to, cable shielding, tubes, films, O-rings, sealings, conveyor belts, damping elements, laser or heat sintering techniques, stereo lithography, fused deposition modelling and slush molding. Additionally, it can also be used in making railway parts, pneumatic and non-pneumatic tires, bicycle seats, protection parts and tire parts. Another aspect of the present invention describes use of the thermoplastic polyurethane, as described hereinabove or as obtained according to the method also described hereinabove, in extruded article and injection molded article. The present invention therefore is also directed to the use of the thermoplastic polyurethane as described above in extruded articles or injection molded articles. By the term “extruded article”, it is referred to the articles obtained after extrusion of the thermoplastic polyurethane, as described hereinabove, in a suitable die or mould. Similarly, the term “injection molded article” refers to the articles obtained after injection molding of the thermoplastic polyurethane, as described hereinabove, in a suitable die or mould. Articles of any desired shape, size and dimension may be obtained using the present invention thermoplastic polyurethane, as described hereinabove, and with suitable techniques known to the person skilled in the art. Accordingly, the present invention is not limited by the choice of such article and/or the mould or die for obtaining the said article. As is known to those skilled in the art, injection molding is a cyclic process while extrusion is a steady-state process. Extruded products or articles are long and continuous and have a cross section that is usually constant with respect to the axis or direction of production. Injection molded products or articles, on the other hand, are discrete item with varying cross sections in each axis. The thermoplastic polyurethane of the present invention can be employed to obtain articles for a wide range of application, such as but not limited to, low temperature applications. By the term “low temperature”, it is referred to the temperature in the vicinity of the Tgof the thermoplastic polyurethane, as described hereinabove. Moreover, the low Tgof the thermoplastic polyurethane reduces the dynamic stiffening and heat build-up in applications where vibration plays a role. The absence of any soft phase crystallization further opens up a wide application area for the TPU. A person skilled in the art is well aware of the techniques involved in extrusion and injection molding. Accordingly, the use of the present invention thermoplastic polyurethane in extruded article and injection molded article is neither limited by the technique chosen by the skilled person and nor the mold or its type employed therefor. The present invention also relates to expanded thermoplastic polyurethane particles based on thermoplastic polyurethane as described herein as well as particle foams and methods to produce expanded thermoplastic polyurethane particles and particle foams based on expanded thermoplastic polyurethane particles. Thus, an aspect of the present invention relates to a method for producing expanded thermoplastic polyurethane particles, comprising:(a) melting the thermoplastic polyurethane, as described hereinabove, to obtain a melt,(b) mixing a blowing agent with the melt obtained in step (a), and(c) producing expanded thermoplastic polyurethane particles from the resulting melt. According to a further embodiment, the present invention therefore is also directed to a method for producing expanded thermoplastic polyurethane particles, comprising:(a) melting the thermoplastic polyurethane as disclosed above or the thermoplastic polyurethane obtained by the method as disclosed above to obtain a melt,(b) mixing a blowing agent with the melt obtained in step (a), and(c) producing expanded thermoplastic polyurethane particles from the resulting melt. Details of the methods to produce expanded thermoplastic polyurethane particles (or E-TPU particles) based on thermoplastic polyurethane, suitable blowing agents and further required auxiliaries are well-known to the person skilled in the art. For instance, the following procedures may be employed:a. impregnating particles of the thermoplastic polyurethane as described hereinabove (obtainable e.g. by extrusion) with average, minimal diameter from 0.2 mm to 10 mm determined by 3D evaluation of granules (e.g. by dynamic image analysis using a PartAn 3D, Microtrac) under pressure at a temperature in the range of ≥100° C. to ≤200° C. with blowing agents (e.g. in a supercritical fluid as blowing agent or in suspension with blowing agent) optionally further auxiliaries (e.g. suspension agents) followed by depressurizing; orb. by melting the thermoplastic polyurethane as described hereinabove, if appropriate with additives together with blowing agents (e.g. in the range of ≥0.1 wt.-% to ≤60 wt.-% based on the total weight of thermoplastic polyurethane) at elevated temperatures and under pressure in an extruder and pelletizing the melt without devices which inhibit uncontrolled foaming (e.g. by underwater granulation). Further details of these methods can be found in, for e.g. WO2005/023920, WO2007/082838, WO2010/136398, WO2013/153190, WO2014/198779, WO2015/055811 WO2017/030835, US2017/0036377, US2016/0271847, US2016/0108198, WO2014/150119, WO2014/150124 and WO2016/131671. Suitable blowing agents are selected based on the method and the precise conditions and include, but are not limited to organic liquids or inorganic gases, or a mixture thereof. Liquids that can be used comprise halogenated hydrocarbons, or saturated, aliphatic hydrocarbons, in particular those having from 3 to 8 carbon atoms. Suitable inorganic gases are nitrogen, air, ammonia, or carbon dioxide. Further details can be found in, for e.g. WO2005/023920, WO2007/082838, WO2010/136398, WO2013/153190, WO2014/198779, WO2015/055811 WO2017/030835, US2017/0036377, US2016/0271847, US2016/0108198, WO2014/150119, WO2014/150124 and WO2016/131671. A further aspect of the present invention relates to particle foams based on the expanded thermoplastic polyurethane particles as described hereinabove. Such particle foams are obtainable by fusing the expanded thermoplastic polyurethane particles, for e.g. by steam at a temperature in the range of ≥100° C. to ≤200° C., optionally at pressure in the range of ≥0.1 bar to ≤6 bar for temperature up to 150° C. For temperature in the range of ≥150° C. to ≤200° C., pressure up to 20 bar can be used, with the proviso that the temperature used for fusing is higher than the temperature of impregnation (e.g. in process variant “a.”, as described hereinabove) or by high energy radiation (e.g. microwave radiation for radiowave radiation). The resulting particle foam generally comprises fused expanded thermoplastic polyurethane particles with open cell to closed cell structure, preferably a closed cell structure with densities in the range of ≥50 kg/m3to ≤300 kg/m3, preferably 80 kg/m3to ≤150 kg/m3. In yet another aspect, the present invention relates to use of the expanded thermoplastic particles, as described hereinabove, or the particle foams, also described hereinabove, for the production of filling material for mattresses, parts of mattresses, mattresses as such, filling of tires, tires or part of tires, shoes, shoe-soles, shoe-midsoles gymnastic mats, protective clothing, cushioning elements for automotive, sound absorbers, anti-vibration devices e.g. for suspension fork absorbers, cushioning elements for bicycle saddles, toys, flooring, e.g. sport floorings or footpath surfacing or under- or interlayer of footpath or packaging materials The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. It will be appreciated that the terms “comprising”, “comprises” and “comprised of” as used herein comprise the terms “consisting of”, “consists” and “consists of”. Furthermore, the terms “first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms “first”, “second”, “third” or “(A)”, “(B)” and “(C)” or “(a)”, “(b)”, “(c)”, “(d)”, “i”, “ii” etc. relate to steps of a method or use or assay there is no time or time inter-val coherence between the steps, that is, the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below. Preferably, the terms “first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. and the like in the description and in the claims, are used for describing a sequential or chronological order in the context of the present invention. In the specification, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination. The present invention is illustrated in more detail by the following embodiments and combinations of embodiments which result from the corresponding dependency references and links. The present invention is further illustrated by the following embodiments and combinations of embodiments as indicated by the respective dependencies and back-references. In particular, it is noted that in each instance where a range of embodiments is mentioned, for example in the context of a term such as “The process of any one of embodiments 1 to 4”, every embodiment in this range is meant to be explicitly disclosed for the skilled person, i.e. the wording of this term is to be understood by the skilled person as being synonymous to “The process of any one of embodiments 1, 2, 3, and 4”.1. A method for preparing a thermoplastic polyurethane, comprising the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol,(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode,wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN55672-1:2016-03, andwherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol.2. The method according to embodiment 1, characterized in that in step (A) the poly-ε-caprolactone polyol (P1) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤4000 g/mol determined according to DIN 55672-1: 2016-03.3. The method according to embodiment 1 or 2, characterized in that in step (A) the poly-ε-caprolactone polyol (P1) is obtained by reacting ε-caprolactone (PI1) and a starter molecule (P12) having a number average molecular weight in the range of ≥80 g/mol to ≤1500 g/mol as determined according to DIN 55672-1: 2016-03.4. The method according to embodiment 3, characterized in that the starter molecule (P12) in the poly-ε-caprolactone polyol (P1) in step (A) is selected from the group consisting of neopentyl glycol, 1,4-butanediol, 1,6-hexanediol, polyethylene glycol, polypropylene glycol, α-hydro-ω-hydroxypoly(oxytetra-methylene) diol and α-hydro-ω-hydroxypoly(oxytri-methylene) diol.5. The method according to embodiment 3 or 4, characterized in that the starter molecule (P12) in the poly-ε-caprolactone polyol (P1) in step (A) is α-hydro-ω-hydroxypoly(oxytetra-methylene) diol.6. The method according to one or more of embodiments 1 to 5, characterized in that the poly-ε-caprolactone polyol (P1) in step (A) has a weight ratio between the ε-caprolactone (P11) and the starting molecule (P12) in the range of ≥1:10 to ≤10:1.7. The method according to one or more of embodiments 1 to 6, characterized in that in step (A) the at least one polyol composition (P) further comprises a second polyol (P2) which is different from poly-ε-caprolactone polyol (P1).8. The method according to embodiment 7, characterized in that the second polyol (P2) is selected from the group consisting of a polyether polyol (P21), polyester polyol (P22) and polycarbonate polyol (P23).9. The method according to embodiment 7 or 8, characterized in that the second polyol (P2) is a polyether polyol (P21).10. The method according to embodiment 9, characterized in that the polyether polyol (P21) is α-hydro-ω-hydroxypoly(oxytetra-methylene) diol.11. The method according to one or more of embodiments 7 to 10, characterized in that the second polyol (P2) has a number average molecular weight Mn in the range of ≥1000 g/mol to ≤4000 g/mol determined according to DIN 55672-1: 2016-03.12. The method according to one or more of embodiments 7 to 11, characterized in that the weight ratio between the poly-ε-caprolactone polyol (P1) and the second polyol (P2) is in the range of ≥1:5 to ≤10:1.13. The method according to one or more of embodiments 1 to 12, characterized in that in step (B) a temperature in the range of ≥70° C. to ≤120° C. is provided.14. The method according to one or more of embodiments 1 to 13, characterized in that in step (B) the at least one polyisocyanate (PI) is selected from the group consisting of 2,2′-, 2,4′- and 4,4′-diphenylmethane diisocyanate, 2,4- and 2,6-toluene diisocyanate, 1,2-, 1,3- and 1,4-cyclohexane diisocyanates, hexamethylene 1,6-diisocyanate, 2-methyl-1,5-pentamethylene diisocyanate, pentamethylene 1,5-diisocyanate, tetramethylene 1,4-diisocyanate, isophorone diisocyanate, p-phenyl diisocyanate, o-tolidine diisocyanate and 1,5-naphthalene diisocyanate and 4,4′-Diisocyanatdicyclohexylmethane.15. The method according to embodiment 14, characterized in that the at least one polyisocyanate (PI) is 4,4′-diphenylmethane diisocyanate.16. The method according to one or more of embodiments 1 to 15, characterized in that in step (B) the at least one low molecular weight diol (CE) is selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, hydroquinone bis 2-hydroxyethyl ether and bis-2(hydroxy ethyl)-terephthalate.17. The method according to one or more of embodiments 1 to 16, characterized in that in step (B) the at least one additive (AD) is selected from the group consisting of antioxidant, hydrolysis stabilizer, light stabilizer, UV absorbers, blowing agents and other process stabilizers.18. The method according to one or more of embodiments 1 to 17, characterized in that in step (A) the at least one polyol composition has an OH value in the range of ≥10 mg KOH/g to ≤100 mg KOH/g determined according to DIN 53240-3:2016-03.19. The method according to one or more of embodiments 1 to 18, characterized in that in step (B) the molar ratio between the at least one polyol composition (P) and the at least one polyisocyanate (PI) is in the range of ≥0.1:1 to ≤1:1.20. The method according to one or more of embodiments 1 to 19, characterized in that in step (B) the weight ratio between the at least one polyisocyanate (P) and the at least one low molecular weight diol (CE) is in the range of ≥1:1 to ≤15:1.21. The method according to one or more of embodiments 1 to 20, characterized in that in step (B) the at least one polyol composition (P), the at least one polyisocyanate (PI) and the at least one chain extender are reacted simultaneously.22. The method according to one or more of embodiments 1 to 21, characterized in that the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 to ≤250 g/mol.23. A thermoplastic polyurethane obtained by the method according to one or more of embodiments 1 to 22, characterized in that the thermoplastic polyurethane has a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode and a hard segment fraction in the range of ≥0.1 to ≤0.7, the hard segment fraction being defined by the formula: Hardsegmentfraction={∑x=1k[(mKV,CE/MKV,CE)*MIso+mKV,CE]}/mtotalwherein,mKV,CEis the mass of the at least one low molecular weight diol (CE) in g,MKV,CEis the molar mass of the at least one low molecular weight diol (CE) in g/mol,MIsois the molar mass of the at least one polyisocyanate (PI) in g/mol,mtotalis the total mass of all the starting materials in g,k is the number of the at least one low molecular weight diol (CE).24. Use of the thermoplastic polyurethane according to embodiment 23 or the thermoplastic polyurethane obtained by the method according to one or more of embodiments 1 to 22 in extruded article and injection molded article.25. A method for producing expanded thermoplastic polyurethane particles, comprising:(a) melting the thermoplastic polyurethane according to embodiment 23 or the thermoplastic polyurethane obtained by the method according to one or more of embodiments 1 to 22 to obtain a melt,(b) mixing a blowing agent with the melt obtained in step (a), and(c) producing expanded thermoplastic polyurethane particles from the resulting melt.26. Expanded thermoplastic polyurethane particles obtained by the method according to embodiment 25.27. A particle foam based on expanded thermoplastic polyurethane particles according to embodiment 26.28. Use of the expanded thermoplastic polyurethane particles according to embodiment 26 or the particle foam according to embodiment 27 for the production of filling material for mattresses, parts of mattresses, mattresses as such, filling of tires, tires or part of tires, shoes, shoe-soles, shoe-midsoles gymnastic mats, protective clothing, cushioning elements for automotive, sound absorbers, anti-vibration devices, cushioning elements for bicycle saddles, toys, flooring or packaging materials.29. A method for preparing a thermoplastic polyurethane, comprising the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P2) a second polyol (P2) which is different from the first polyol (P1),(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane,wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol.30. The method according to embodiment 29, wherein the thermoplastic polyurethane has a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode.31. A method for preparing a thermoplastic polyurethane, comprising the steps of:(A) providing at least one polyol composition (P) comprising(P1) a poly-ε-caprolactone polyol, and(P2) a second polyol (P2) which is different from the first polyol (P1),(B) reacting the at least one polyol composition (P) of step (A) with at least one polyisocyanate (PI) and at least one low molecular weight diol (CE) optionally in the presence of at least one catalyst (CA) and/or at least one additive (AD) to obtain a thermoplastic polyurethane having a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode,wherein the at least one polyol composition (P) has a number average molecular weight Mn in the range of ≥1500 g/mol to ≤10,000 g/mol determined according to DIN 55672-1: 2016-03, and wherein the at least one low molecular weight diol (CE) has a molecular weight in the range of ≥50 g/mol to ≤350 g/mol.32. The method according to any of embodiments 29 to 31, characterized in that the weight ratio between the poly-ε-caprolactone polyol (P1) and the second polyol (P2) in the polyol composition (P) is in the range of ≥1:5 to ≤10:1.33. The method according to any of embodiments 29 to 32, characterized in that the polyol (P1) has a number average molecular weight in the range of ≥1500 g/mol to ≤2500 g/mol determined according to DIN 55672-1: 2016-03.34. The method according to any of embodiments 29 to 33, characterized in that the second polyol (P2) has a number average molecular weight Mn in the range of ≥1000 g/mol to ≤4000 g/mol determined according to DIN 55672-1: 2016-03.35. The method according to any of embodiments 29 to 34, characterized in that in step (A) the poly-ε-caprolactone polyol (P1) is obtained by reacting ε-caprolactone (P11) and a starter molecule (P12) having a number average molecular weight in the range of ≥80 g/mol to ≤1500 g/mol as determined according to DIN 55672-1: 2016-03.36. The method according to any of embodiments 29 to 35, characterized in that the starter molecule (P12) in the poly-ε-caprolactone polyol (P1) in step (A) is selected from the group consisting of neopentyl glycol, 1,4-butanediol, 1,6-hexanediol, polyethylene glycol, polypropylene glycol, α-hydro-ω-hydroxypoly(oxytetra-methylene) diol and α-hydro-ω-hydroxypoly(oxytri-methylene) diol.37. The method according to one or more of embodiments 29 to 36, characterized in that the poly-ε-caprolactone polyol (P1) in step (A) has a weight ratio between the ε-caprolactone (P11) and the starting molecule (P12) in the range of ≥1:10 to ≤10:1.38. The method according to any one of embodiments 29 to 37, characterized in that the second polyol (P2) is selected from the group consisting of a polyether polyol (P21), polyester polyol (P22) and polycarbonate polyol (P23).39. The method according to one or more of embodiments 29 to 38, characterized in that in step (B) the at least one polyisocyanate (PI) is selected from the group consisting of 2,2′-, 2,4′- and 4,4′-diphenylmethane diisocyanate, 2,4- and 2,6-toluene diisocyanate, 1,2-, 1,3- and 1,4-cyclohexane diisocyanates, hexamethylene 1,6-diisocyanate, 2-methyl-1,5-pentamethylene diisocyanate, pentamethylene 1,5-diisocyanate, tetramethylene 1,4-diisocyanate, isophorone diisocyanate, p-phenyl diisocyanate, o-tolidine diisocyanate and 1,5-naphthalene diisocyanate and 4,4′-diisocyanatdicyclohexylmethane.40. The method according to one or more of embodiments 29 to 39, characterized in that in step (B) the at least one low molecular weight diol (CE) is selected from the group consisting of ethylene glycol, diethylene glycol, triethylene glycol, 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, hydroquinone bis 2-hydroxyethyl ether and bis-2(hydroxy ethyl)-terephthalate.41. The method according to one or more of embodiments 29 to 40, characterized in that in step (B) the at least one additive (AD) is selected from the group consisting of antioxidant, hydrolysis stabilizer, light stabilizer, UV absorbers, blowing agents and other process stabilizers.42. The method according to one or more of embodiments 29 to 41, characterized in that in step (B) the molar ratio between the at least one polyol composition (P) and the at least one polyisocyanate (PI) is in the range of ≥0.1:1 to ≤1:1.43. A thermoplastic polyurethane obtained or obtainable by the method according to one or more of embodiments 29 to 42.44. A thermoplastic polyurethane obtained by the method according to one or more of embodiments 29 to 42, characterized in that the thermoplastic polyurethane has a Tgin the range of ≥−60° C. to ≤10° C. determined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 at a heating rate of 2 K/min and 1 Hz torsion mode and a hard segment fraction in the range of ≥0.1 to ≤0.7, the hard segment fraction being defined by the formula: Hardsegmentfraction={∑x=1k[(mKV,CE/MKV,CE)*MIso+mKV,CE]}/mtotalwherein,mKV,CEis the mass of the at least one low molecular weight diol (CE) in g,MKV,CEis the molar mass of the at least one low molecular weight diol (CE) in g/mol,MIsois the molar mass of the at least one polyisocyanate (PI) in g/mol,mtotalis the total mass of all the starting materials in g,k is the number of the at least one low molecular weight diol (CE).45. Use of the thermoplastic polyurethane according to embodiment 43 or 44 or the thermoplastic polyurethane obtained by the method according to one or more of claims1to13in extruded article and injection molded article.46. A method for producing expanded thermoplastic polyurethane particles, comprising:(a) melting the thermoplastic polyurethane according to embodiment 43 or 44 or the thermoplastic polyurethane obtained by the method according to one or more of embodiments 29 to 42 to obtain a melt,(b) mixing a blowing agent with the melt obtained in step (a), and(c) producing expanded thermoplastic polyurethane particles from the resulting melt.47. Expanded thermoplastic polyurethane particles obtained by the method according to embodiment 46.48. A particle foam based on the expanded thermoplastic polyurethane particles according to embodiment 47.49. Use of the expanded thermoplastic polyurethane particles according to embodiment 47 or the particle foam according to embodiment 48 for the production of filling material for mattresses, parts of mattresses, mattresses as such, filling of tires, tires or part of tires, shoes, shoe-soles, shoe-midsoles gymnastic mats, protective clothing, cushioning elements for automotive, sound absorbers, anti-vibration devices, cushioning elements for bicycle saddles, toys, flooring or packaging materials. The invention is further illustrated by the following examples which do not limit the scope of the invention. Examples and Comparative Examples 1. Compounds Polyester Polyol OH valuePolyol(mg KOH/g)Type of polyolPolyol 156.4Polycaprolactonepolyol withPolytetrahydrofuranhaving Mn of 1000 asthe starter molecule,obtained fromPerstorpPolyol 2113.3Polytetrahydrofuranhaving Mn of 1000obtained from BASFPolyol 381.2Polytetrahydrofuranhaving Mn of 1400obtained from BASFPolyol 462.2Polytetrahydrofuranhaving Mn of 1800obtained from BASFPolyol 555.9Polytetrahydrofuranhaving Mn of 2000obtained from BASF Polyisocyanate-4,4′-MDI having an isocyanate content of 33.5 wt.-% obtained from BASF. Low molecular weight diolCE 11,6-HexanediolCE 21,4-Butanediolwere obtained from Sigma AldrichAdditivePhenolic antioxidantAD 1stabilizer obtained from BASF 2. Standard Methods Number average molecular weight (Mn)DIN 55672-1:2016 MarchOH valueDIN 53240-3:2016 MarchDensityDIN EN ISO 845:2009 OctoberShore hardnessASTM D2240:2015Tensile strengthDIN 53504:2017 MarchElongation at breakDIN 53504:2017 MarchTear strengthDIN ISO 34-1, B:2016 SeptemberAbrasion wearDIN ISO 4649:2014 MarchTgby DMADIN EN ISO 6721-1:2011 August All values given in the present application for the Tgdetermined by dynamic mechanical thermal analysis according to DIN EN ISO 6721-1:2011-08 were determined at a heating rate of 2 K/min and 1 Hz torsion mode. Deviant from the DIN norm, the temperature was adjusted step wise by 5 K and 35 s per step which corresponds to a continuous heating rate of 2 K/min. The measurements were conducted with a sample with a ratio of width:thickness of 1:6. 3. General TPU Synthesis In a 3.5 L metal container, polyol composition and low molecular weight diol were mixed with a mechanical stirrer and heated up to 80° C. In a separate vessel, polyisocyanate was heated to a temperature of 50° C. Once the temperature of the mixture reached 80° C., preheated polyisocyanate was added under constant stirring. Due to the exothermic reaction, the melt temperature increased rapidly. At a melt-temperature of 110° C., the mixture was poured into a teflon frame kept over a hot plate having a temperature of 125° C. for 10 minutes to obtain a TPU slab. Once the TPU slab turned solid, it was removed from the hot plate and subsequently annealed inside a hot oven at 80° C. for 15 h. The TPU was al-lowed to cool gradually, followed by milling in a miller and thereafter shredded to small granulates. The granulates were dried at 110° C. for 3 h and then injection molded to test plaques of size 2 mm×9 cm×12 cm. The test plaques were then used to determine the mechanical performance. Table 1a and 1b hereinbelow summarize both inventive examples (IE) and comparative examples (CE) obtained using the general synthesis, as described hereinabove. Table 2a and 2b hereinbelow summarize the properties of both the inventive examples (IE) and comparative examples (CE), as described in Table 1a and Table 1b respectively. TABLE 1aComparative and inventive TPU composition.TPUComp.Inv.Comp.Inv.Comp.Inv.ComponentsEx. 1Ex. 1Ex. 2Ex. 2Ex. 3Ex. 3Polyol 1—950g—676.02g—580.03gPolyol 2850g—————Polyol 3——————Polyol 4——————Polyol 5——1000g340.98g870g289.97gPolyisocyanate535.50g436.33g367.11g374.27g450.82g451.70gCE 1————161.68g161.79gCE 2114.41g114.48g87.31g88.85g——AD 115.16g15.16g——14.98g14.98g TABLE 1bInventive TPU composition.Compo-TPUnentsInv. Ex. 4Inv. Ex. 5Inv. Ex. 6Inv. Ex. 7Inv. Ex. 8Polyol 1870g566.70g566.70g290.06g664.72gPolyol 2—————Polyol 3—283.31g———Polyol 4——283.31g——Polyol 5———579.94g335.28gPolyisocyanate452.13g462.89g446.69g451.26g413.75gCE 1161.84g160.71g158.73g161.73g136.17gCE 2—————AD 114.98g14.88g14.70g14.98g15.66g TABLE 2aMechanical properties for TPU composition of Table 1a.TPUComp.Inv.Comp.Inv.Comp.Inv.PropertyEx. 1Ex. 1Ex. 2Ex. 2Ex. 3Ex. 3Hard segment0.290.290.230.230.340.34fractionDensity, kg/m3112011311075110410931117Shore A hardness878579798885Tensile strength,544330395149MPaElongation at510630720770550520break, (%)Tear strength,656552494857(kN/m)Abrasion wear,413557413844(mm3)Tgat max tan delta−25° C.−40° C.−55° C. &−45° C.−55° C. &−40° C.−15° C.−10° C.Tgat max G″−40° C.−50° C.−65° C.−55° C.−65° C.−55° C. TABLE 2bMechanical properties for TPU composition of Table 1b.TPUInv.Inv.Inv.Inv.Inv.Ex.Ex.Ex.Ex.Ex.Components45678Hard segment0.340.340.340.340.27fractionDensity, kg/m311301120111811061103Shore A hardness8584868879Tensile strength,4242414338MPaElongation at630620660660820break, (%)Tear strength,7073676451(kN/m)Abrasion wear,4346464939(mm3)Tgat max tan delta−35° C.−30° C.−35° C.−40° C.−40° C.Tgat max G″−45° C.−45° C.−50° C.−55° C.−55° C. The examples of the present invention do not show soft phase crystallization in the resulting TPU. In order for the TPU to showcase soft phase crystallization, the tan delta values when plotted against temperature show dual peaks at different temperatures. The temperatures at which the tan delta shows a peak or is maximum, the corresponding value on the temperature scale is the Tgvalue of the TPU. Accordingly, the soft phase crystallization can be observed in the comparative examples which show dual peaks of max tan delta values. On the contrary, the present invention TPU does not shows any such behaviour and has a single Tgat max tan delta value. As regards the comparative example 1, it is observed that the said example did not result in any soft phase crystallization. However, the resulting Tgvalue is sufficiently high in comparison with the Tgvalue of the inventive example 1. Moreover, the mechanical properties of the inventive example 1 are improved in comparison to the comparative example 1. The sufficiently low Tgvalues of the present invention TPU along with the improved mechanical properties renders them suitable for a wide variety of applications, as described hereinabove. 4. Synthesis of E-TPU by Underwater Granulation TPU extrudates may be obtained in a twin-screw extruder, such as but not limited to ZSK43 by Coperion GmbH. The polyol composition, polyisocyanate and low molecular weight diol in the presence of stabilizers are added in suitable amounts along with a catalyst, such as but not limited to tin(II) dioctoate, at a temperature in the range of 180° C. to 220° C. and reacted. Additionally, a further extruder may also be employed, such as but not limited to a ZSK92 twin-screw extruder from Coperion GmbH and the reaction can be further progressed at a temperature in the range of 200° C. to 240° C. Table 3 below provides possible amounts of the typical ingredients which may be used in the said extruder. Pelletization of the extrusion product or polymer melt, as obtained hereinabove, may be done using suitable methods, such as but not limited to, by forcing the polymer melt through a temperature-regulated pelletizing die at 200° C. into a water-flooded pelletizing chamber and cut-off with subsequent isolation and drying of the resulting pellets at a temperature of 70° C. for 4 h. TABLE 3Possible amounts of ingredients for extrusionTPUComponentsTPU 1TPU 2TPU 3Hard segment0.300.350.42Polyol 1613.36 g573.36 g513.36 gPolyol 5306.64 g286.64 g256.64 gPolyisocyanate416.77 g461.83 g522.79 gCE 1142.33 g167.16 g201.28 g For obtaining expanded beads or E-TPU, the TPUs obtained hereinabove are dried and mixed in an extruder, such as ZE75 twin-screw extruder by KraussMaffei Berstorff GmbH, followed by further addition of suitable additives, such as but not limited to talc as nucleating agent to obtain a mixture. Optionally, a TPU which has been separately obtained in an extrusion process by admixing the polyisocyanate, may also be added to the mixture. The mixture is melted at a temperature in the range of 160° C. to 220° C., while blowing agents such as but not limited to CO2and N2are, in the extruder, injected and mixed to form a homogeneous melt. The melt may be gear pumped at a temperature in the range of 160° C. to 200° C. into a pelletizing die and cut in an underwater pelletization (or UWP) cutting chamber into pellets. The pellets are then carried off by a temperature regulated and pressurized water stream, expanding in the process in a controlled manner to obtain expanded pellets. Once the expanded pellets have been separated out of the water by suitable means, such as but not limited to a centrifugal dryer, they are dried at 60° C. for 2 h. Table 4 below summarizes the suitable process parameters for obtaining the expanded pellets. TABLE 4Suitable process parameters for obtaining expanded pelletsWaterWaterE-TPUBulkpressuretemperaturebead massdensityCO2N2in UWPin UWPE-TPUTPU(mg)(kg/m3)(wt.-%)(wt.-%)(bar)(° C.)E-TPU 1TPU 125130-1501.3-1.50.19-0.2312-1530-40E-TPU 2TPU 132110-1301.3-1.50.19-0.2312-1530-40E-TPU 3TPU 225130-1601.3-1.50.19-0.2312-1535-45E-TPU 4TPU 325130-1601.3-1.50.19-0.2312-1535-45 LITERATURE CITED U.S. Pat. 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11859043 | DETAILED DESCRIPTION OF THE INVENTION It has been found that by employing an yttrium-containing catalyst, the resulting silylated polyurethane polymers unexpectedly exhibit reduced sensitivity to moisture and an increased storage stability compared to the same silylated polyurethane polymers prepared using non-yttrium containing catalysts. The silylated polyurethane can be made without using conventional tin or bismuth catalysts. The silylated polyurethane can be used to make coatings, adhesives, sealants and the other applications described herein. Other than in the working examples or where otherwise indicated, all numbers expressing amounts of materials, reaction conditions, time durations, quantified properties of materials, and so forth, stated in the specification and claims are to be understood as being modified in all instances by the term “about” whether or not the term “about” is used in the expression. It will be understood that any numerical range recited herein includes all sub-ranges within that range and any combination of the various endpoints of such ranges or sub-ranges, be it described in the examples or anywhere else in the specification. It will also be understood herein that any of the components of the invention herein as they are described by any specific genus or species detailed in the examples section of the specification, can be used in one embodiment to define an alternative respective definition of any endpoint of a range elsewhere described in the specification with regard to that component, and can thus, in one non-limiting embodiment, be used to supplant such a range endpoint, elsewhere described. It will be further understood that any compound, material or substance which is expressly or implicitly disclosed in the specification and/or recited in a claim as belonging to a group of structurally, compositionally and/or functionally related compounds, materials or substances includes individual representatives of the group and all combinations thereof. Reference is made to substances, components, or ingredients in existence at the time just before first contacted, formed in situ, blended, or mixed with one or more other substances, components, or ingredients in accordance with the present disclosure. A substance, component or ingredient identified as a reaction product, resulting mixture, or the like may gain an identity, property, or character through a chemical reaction or transformation during the course of contacting, in situ formation, blending, or mixing operation if conducted in accordance with this disclosure with the application of common sense and the ordinary skill of one in the relevant art (e.g., chemist). The transformation of chemical reactants or starting materials to chemical products or final materials is a continually evolving process, independent of the speed at which it occurs. Accordingly, as such a transformative process is in progress there may be a mix of starting and final materials, as well as intermediate species that may be, depending on their kinetic lifetime, easy or difficult to detect with current analytical techniques known to those of ordinary skill in the art. Reactants and components referred to by chemical name or formula in the specification or claims hereof, whether referred to in the singular or plural, may be identified as they exist prior to coming into contact with another substance referred to by chemical name or chemical type (e.g., another reactant or a solvent). Preliminary and/or transitional chemical changes, transformations, or reactions, if any, that take place in the resulting mixture, solution, or reaction medium may be identified as intermediate species, master batches, and the like, and may have utility distinct from the utility of the reaction product or final material. Other subsequent changes, transformations, or reactions may result from bringing the specified reactants and/or components together under the conditions called for pursuant to this disclosure. In these other subsequent changes, transformations, or reactions the reactants, ingredients, or the components to be brought together may identify or indicate the reaction product or final material. In accordance with the present invention, the silylated polyurethane composition comprises the reaction product of the components for forming a moisture-curable silylated polyurethane resin, which upon curing, provides a cured resin i.e., hydrolyzed and subsequently crosslinked, silylated polyurethane resin. In one embodiment herein the silylated polyurethane composition employed in the present invention in general is obtained by reacting the reaction-forming components therefore, as noted herein, with an yttrium-containing catalyst. The reaction of the components in the forming of the silylated polyurethane composition in the presence of the yttrium-containing catalyst may involve one or more of several different reaction routes for forming the silylated polyurethane. In an embodiment, the silylated polyurethane composition comprises (a) the reaction product of a polyurethane prepolymer and a silylating agent in the presence of an yttrium-containing catalyst, optionally in combination with one or more other polyurethane-forming catalysts, such as, but not limited to zinc, titanium, zirconium and the like. The nature of the silylating agent will depend on the termini of the polyurethane prepolymer, i.e., whether the prepolymer is hydroxyl-terminated or isocyanate-terminated. In the case of the prepolymer being hydroxyl-terminated, the silylating agent will be an isocyanate-containing silylating agent. In the case of the prepolymer being isocyanate-terminated, the silylating agent will have an active hydrogen moiety, such as mercaptan, primary and secondary amine, preferably the latter, etc. and can be a silyl-hydride containing silylating agent. Alternatively, in another embodiment the silylated polyurethane composition comprises (b) the reaction product of a polyol and an isocyanatosilane in the presence of an yttrium-containing catalyst, optionally in combination with one or more other polyurethane-forming catalysts, such as, but not limited to zinc, titanium, zirconium and the like. Still further, in yet another embodiment the silylated polyurethane composition comprises (c) the reaction product of a polyisocyanate, an active hydrogen-containing silane, such as those described herein, in the presence of an yttrium-containing catalyst, optionally in combination with one or more other polyurethane-forming catalysts, such as, but not limited to zinc, titanium, zirconium and the like. In preparing the polyurethane prepolymers herein, the isocyanate-terminated polyurethane prepolymers may be obtained by reacting one or more polyols, advantageously, diols, with one or more polyisocyanates, advantageously, diisocyanates, in such proportions that the resulting prepolymers will be terminated with isocyanate. In the case of reacting a diol with a diisocyanate, a molar excess of diisocyanate is employed. In an embodiment, the preparation of the isocyanate-terminated polyurethane prepolymer is conducted in the presence of an yttrium-containing catalyst as described herein. Included among the polyols that can be utilized for the preparation of the isocyanate-terminated polyurethane prepolymer are polyether polyols, polyester polyols such as the hydroxyl-terminated polycaprolactones, polyetherester polyols such as those obtained from the reaction of polyether polyol with ε-caprolactone, polyesterether polyols such as those obtained from the reaction of hydroxyl-terminated polycaprolactones with one or more alkylene oxides such as ethylene oxide and propylene oxide, hydroxyl-terminated polybutadienes, and the like. Specific suitable polyols include but are not limited to the polyether diols, in particular, the poly(oxyethylene) diols, the poly(oxypropylene) diols and the poly(oxyethylene-oxypropylene) diols, polyoxyalkylene triols, polytetramethylene glycols, polyacetals, polyhydroxy polyacrylates, polyhydroxy polyester amides and polyhydroxy polythioethers, polycaprolactone diols and triols, and the like. In one embodiment of the present invention, the polyols used in the production of the isocyanate-terminated polyurethane prepolymers are poly(oxyethylene) diols with molecular weights from about 500 to about 25,000. In another embodiment of the present invention, the polyols used in the production of the isocyanate-terminated polyurethane prepolymers are poly(oxypropylene) diols with molecular weights from about 1,000 to about 20,000. Mixtures of polyols of various structures, molecular weights and/or functionalities can also be used. As used herein, molecular weight is measured by Gel Permeation Chromatography (GPC) analysis. The polyether polyols can have a functionality up to about 8 but advantageously have a functionality of from about 2 to about 4 and more advantageously, a functionality of about 2 (i.e., diols). Especially suitable are the polyether polyols prepared in the presence of double-metal cyanide (DMC) catalysts, an alkaline metal hydroxide catalyst, or an alkaline metal alkoxide catalyst, such as those which are known by those skilled in the art. Polyether polyols produced in the presence of DMC catalysts tend to have high molecular weights and low levels of unsaturation, properties of which, without wishing to be bound by theory, it is believed are responsible for the improved performance of the inventive silylated polyurethane compositions. The polyether polyols preferably have a number average molecular weight of from about 1,000 to about 25,000, more preferably from about 2,000 to about 20,000, and even more preferably in the case of polyols made using DMC catalysts, from about 4,000 to about 18,000. The polyether polyols preferably have an end group unsaturation level of no greater than about 0.04 milliequivalents per gram of polyol. More preferably, the polyether polyol has an end group unsaturation of no greater than about 0.02 milliequivalents per gram of polyol. Examples of commercially available diols that are suitable for making the isocyanate-terminated polyurethane prepolymer include but are not limited to the Acclaim® polyols available from Covestro: Acclaim® Polyol 8200 N (number average molecular weight of ˜8,000), Acclaim® Polyol 4200 N (number average molecular weight of ˜4,000), Acclaim® Polyol 18200 N (number average molecular weight of ˜18,000), and Acclaim® Polyol 12200 N (number average molecular weight of ˜12,000). Any of numerous polyisocyanates, advantageously, diisocyanates, and mixtures thereof, can be used to provide the isocyanate-terminated polyurethane prepolymers. In one embodiment, the polyisocyanate can be diphenylmethane diisocyanate (“MDI”), polymeric diphenylmethane diisocyanate (“PMDI”), paraphenylene diisocyanate, naphthylene diisocyanate, liquid carbodiimide-modified MDI and derivatives thereof, isophorone diisocyanate (“IPDI”), dicyclohexylmethane-4,4′-diisocyanate, toluene diisocyanate (“TDI”), particularly the 2,6-TDI isomer, as well as various other aliphatic and aromatic polyisocyanates that are well-established in the art, and combinations thereof. Silylation reactants for reaction with the isocyanate-terminated polyurethane prepolymers of reaction (a) or the polyisocyanate of reaction (c) both as described herein must contain functionality that is reactive with isocyanate and at least one readily hydrolyzable and subsequently crosslinkable group, e.g., alkoxy. Particularly useful silylation reactants are active hydrogen-containing silanes, e.g., aminosilanes, especially those of the general formula: wherein R1is hydrogen, alkyl or cycloalkyl of up to 12 carbon atoms, optionally containing one or more heteroatom, or aryl of up to 8 carbon atoms, R2is a divalent alkylene group of up to 12 carbon atoms, optionally containing one or more heteroatoms, each R3is the same or different alkyl or aryl group of up to 8 carbon atoms, each R4is the same or different alkyl group of up to 6 carbon atoms and x is 0, 1 or 2. In one embodiment, R1is hydrogen or a methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, cyclohexyl or phenyl group, R2possesses 1 to 4 carbon atoms, each R4is the same or different methyl, ethyl, propyl or isopropyl group and x is 0. Specific aminosilanes for use herein include but are not limited to aminopropyltrimethoxysilane, aminopropyltriethoxysilane, aminobutyltriethoxysilane, N-(2-aminoethyl-3-aminopropyl)triethoxysilane, aminoundecyltrimethoxysilane, and aminopropylmethyldiethoxysilane, for example. Other suitable aminosilanes include, but are not limited to phenylaminopropyltrimethoxysilane, methylaminopropyltrimethoxysilane, n-butylaminopropyltrimethoxysilane, t-butyl aminopropyltrimethoxysilane, cyclohexylaminopropyltrimethoxysilane, dibutylmaleate aminopropyltrimethoxysilane, dibutylmaleate-substituted 4-amino-3,3-dimethylbutyl trimethoxy silane, N-methyl-3-amino-2-methylpropyltriemthoxysilane, N-ethyl-3-amino-2-methylpropyltrimethoxysilane, N-ethyl-3-amino-2-methylpropyldiethoxysilane, N-ethyl-3-amino-2-methylpropyltriethoxysilane, N-ethyl-3-amino-2-methylpropylmethyldimethoxysilane, N-butyl-3-amino-2-methylpropyltrimethoxysilane, 3-(N-methyl-3-amino-1-methyl-1-ethoxy)propyltrimethoxysilane, N-ethyl-4-amino-3,3-dimethylbutyldimethoxymethylsilane and N-ethyl-4-amino-3,3-dimethylbutyltrimethoxysilane. In one embodiment the isocyanate-terminated polyurethane prepolymer can be of the formula (I): wherein formula (I) is a polyisocyanate, or a larger polyisocyanate prepared from the reaction of a polyisocyanate and a polyol and/or a polyamine and P is an organic moiety of from 6 to 10,000 carbon atoms, which can optionally contain at least one heteroatom such as O, N or S and w is ≥1, preferably from 1 to 4. The silylated polyurethane polymer can be prepared by reaction of the above isocyanate-terminated polyurethane (I) with an aminosilane or mercaptosilane as is known by those of ordinary skill in the art. The silylated polyurethane polymer can preferably be of the general formula (IA): wherein P is a polymer derived from a polyisocyanate and/or a polyol, and preferably is an organic moiety of from 6 to 10,000 carbon atoms, which can optionally contain at least one heteroatom such as O, N or S, Z is S or N—R1, R1is hydrogen, alkyl or cycloalkyl of up to 12 carbon atoms, optionally containing one or more heteroatoms, such as an alkyl from 1 to 8 carbon atoms, or a cycloalkyl of from 5 to 8 carbon atoms, or aryl of up to 8 carbon atoms, or aryl of up to 8 carbon atoms, R2is a divalent alkylene group of up to 12 carbon atoms, optionally containing one or more heteroatoms, preferably from 1 to 12 carbon atoms, more preferably form 2 to 8 carbon atoms, each R3is the same or different alkyl or aryl group of up to 8 carbon atoms, such as an alkyl from 1 to 8 carbon atoms, or any aryl group of from 5 to 8 carbon atoms, each R4is the same or different alkyl group of up to 6 carbon atoms, preferably from 1 to 4 carbon atoms, w is greater than or equal to 1, a is 0, 1 or 2, and w is greater than or equal to 1, and w can be up to 4. In one embodiment herein the hydroxyl-terminated polyurethane prepolymer can be obtained in substantially the same manner employing substantially the same materials, i.e., polyols, polyisocyanates and yttrium-containing catalyst (optionally in combination with one or more other polyurethane-forming catalysts), described above for the preparation of isocyanate-terminated prepolymers, with the major difference being that the proportions of polyol and polyisocyanate will be such as to result in hydroxyl-termination in the resulting prepolymer. Thus, e.g., in the case of a diol and a diisocyanate, a molar excess of the former will be used thereby resulting in hydroxyl-terminated polyurethane prepolymer. In one embodiment the hydroxyl-terminated polyurethane prepolymer can be produced from a polyol of the formula (II): wherein P is an organic moiety of from 1 to 10,000 carbon atoms, such as a divalent alkyl, alkenyl, or aryl, which can optionally contain at least one heteroatom such as O, N or S. and w is ≥1, preferably from 1 to 4. The silylated polyurethane polymer can be prepared by reaction of the above polyol with an isocyanatosilane as is known by those of ordinary skill in the art. One embodiment of a silylated polyurethane polymer is of the general formula (IIA): wherein P is a polymer derived from a polyisocyanate and/or a polyol, R2is a divalent alkylene group of up to 12 carbon atoms, preferably from 1 to 12 carbon atoms, more preferably form 2 to 8 carbon atoms, optionally containing one or more heteroatoms, each R3is the same or different alkyl or aryl group of up to 8 carbon atoms, such as an alkyl from 1 to 8 carbon atoms, or any aryl group of from 5 to 8 carbon atoms, each R4is the same or different alkyl group of up to 6 carbon atoms, preferably from 1 to 4 carbon atoms, w is greater than or equal to 1. Useful silylation reactants for the hydroxyl-terminated polyurethane prepolymer in reaction (a), and for the polyol in reaction (b) are those containing isocyanate termination and readily hydrolyzable functionality, e.g., 1 to 3 alkoxy groups. Suitable silylating reactants are the isocyanatosilanes of the general formula (III): wherein R2is an alkylene group of up to 12 carbon atoms, optionally containing one or more heteroatoms, each R3is the same or different alkyl or aryl group of up to 8 carbon atoms, each R4is the same or different alkyl group of up to 6 carbon atoms and a is 0, 1 or 2. In one embodiment, R2possesses 1 to 4 carbon atoms, each R4is the same or different methyl, ethyl, propyl or isopropyl group and a is 0. Specific isocyanatosilanes that can be used herein to react with the foregoing hydroxyl-terminated polyurethane prepolymers or polyol to provide silylated polyurethane herein include but are not limited to isocyanatopropyltrimethoxysilane, isocyanatoisopropyltrimethoxysilane, isocyanato-n-butyltrimethoxysilane, isocyanato-t-butyltrimethoxysilane, isocyanatopropyltriethoxysilane, isocyanatoisopropyltriethoxysilane, isocyanato-n-butyltriethoxysilane, isocyanato-t-butyltriethoxysilane, isocyanatomethyltriethoxysilane, isocyanatomethyltrimethoxysilane, isocyanatomethylmethyldimethoxysilane, 3-isocyanatopropylmethyldimethoxysilane, isocyanatomethylmethyldiethoxysilane, 3-isocyanatopropylmethyldiethoxysilane and the like. In yet another embodiment, silylated polyurethane obtained from reacting isocyanatosilane directly with a polyol can be obtained from one or more polyols, advantageously, diols, reacting directly with isocyanatosilane without the initial formation of a polyurethane prepolymer. The materials, i.e., polyols and silanes (e.g., one possessing both hydrolyzable and isocyanato functionality), useful for this approach to produce silylated polyurethane are described above. As such, suitable polyols include, hydroxy-terminated polyols having a molecular weight from about 4,000 to about 20,000. However, mixtures of polyols of various structures, molecular weights and/or functionalities can also be used based on the desired application. Suitable isocyanatosilanes used to react with the foregoing polyols to provide silylated polyurethanes are described above. The silylated polyurethane polymer (i) is made using the same materials described above for the reactions (a), (b), and/or (c). The polyol (ii), hydroxyl-terminated polyurethane prepolymer (iii), isocyanato silylating agent (iv), polyisocyanate (v), isocyanate-terminated polyurethane prepolymer (vi), active hydrogen-containing silylating agent (vii), and yttrium-containing catalyst (viii) employed for the process of preparing silylated polyurethane polymer herein can be the same as those materials described herein for the contents of the silylated polyurethane-forming composition. The polyurethane prepolymer synthesis and subsequent silylation reaction (a), as well as the direct reaction of polyol and isocyanatosilane (b) or the direct reaction of polyisocyanate and an active-hydrogen containing silane (c) are conducted under anhydrous conditions and preferably under an inert atmosphere, such as a blanket of nitrogen, to prevent premature hydrolysis of the alkoxysilane groups, and in the presence of an yttrium-containing catalyst, and optionally also in the presence of one of the other alternate polyurethane-forming catalysts described above. Typical temperature range for each of the reaction steps, is 0° to 150° C., and more preferably between 60° and 90° C. Typically, the total reaction time for the synthesis of the silylated polyurethane is between 4 to 8 hours. The synthesis is monitored using a standard titration technique (ASTM 2572-87) or infrared analysis. Silylation of the urethane prepolymers is considered complete when no residual —NCO content can be detected by either technique. The yttrium-containing catalyst employed in one or more of the reactions (a)-(c) described herein can be selected from the group consisting of an yttrium salt, a hydrate of an yttrium salt, an yttrium complex, an yttrium alkoxide, an organic yttrium compound, an inorganic yttrium compound, and combinations thereof. Preferably, the yttrium-containing catalyst is an yttrium salt selected from the group consisting of yttrium halide, yttrium nitrate, yttrium sulfate, yttrium trifluoromethanesulfonate, yttrium acetate, yttrium trifluoroacetate, yttrium malonate, octylic acid (2-ethylhexanoic acid) salt of yttrium, yttrium naphthenate, versatic acid salt of yttrium, yttrium neodecanoate, and combinations thereof. In another embodiment, the yttrium-containing catalyst is an yttrium alkoxide selected from the group consisting of yttrium trimethoxide, yttrium triethoxide, yttrium triisopropoxide, yttrium isopropoxide oxide, yttrium tributoxide, yttrium triphenoxide, and combinations thereof. In one embodiment, the yttrium-containing catalyst has the general formula (IV): wherein, R1, R2and R3each denote hydrogen or a substituent having 1-12 carbon atoms, preferably from 1 to about 6 carbon atoms; n is 2 or 3; O denotes an oxygen atom; and, Y denotes an yttrium atom. Preferably, R1, R2and R3are each independently selected from hydrogen, methyl group, ethyl group, vinyl group, n-propyl group, isopropyl group, 1-propenyl group, allyl group, n-butyl group, s-butyl group, isobutyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, cyclohexyl group, methyl cyclohexyl group, ethyl cyclohexyl group, phenyl group, benzyl group, tolyl group, phenethyl group and trifluoromethyl group. The yttrium-containing catalyst can be an organic yttrium compound selected from the group consisting of yttrium tris(acetylacetonate), yttrium tris(hexanedionate), yttrium tris(heptanedionate), yttrium tris(dimethylheptanedionate, yttrium tris(tetramethylheptanedionate, yttrium tris(hexafluoroacetylacetonate), yttrium tris(trifluoroacetylacetonate), yttrium tris(acetoacetate), yttrium tris(ethylacetoacetate), cyclopentadienylyttrium dichloride, tris(methylcyclopentadienyl)yttrium, dicyclopentadienylyttrium chloride, tris(cyclopentadienyl)yttrium, tris(butylcyclopentadienyl) yttrium; tris[N,N-bis(trimethylsilyl)amide]yttrium, yttrium carbonate, yttrium hydrogen carbonate, and combinations thereof. The yttrium-containing catalyst can be an organobasic complex selected from the group consisting of pyridine complexes of yttrium salt, picoline complexes of yttrium salt, and combinations thereof; an alcohol complex of an yttrium salt; an inorganic yttrium catalyst selected from the group consisting of yttrium oxide, diyttrium trioxide, yttrium sulfide, diyttrium trisulfide, yttrium nitrate, yttrium nitrate hexahydrate, yttrium barium copper oxide, yttrium hydroxide, yttrium aluminum garnet, yttrium aluminum silicate, yttrium phosphide, yttrium sulfate, yttrium tripolyphosphite, yttrium iron garnet, yttrium dichromate, yttrium chromate, yttrium permanganate, yttrium peroxide, and combinations thereof and the like. The amounts of the various components described for reactions (a)-(c) described herein can vary greatly depending on various parameters, article or materials desired to be produced therewith and physical and processing parameters. In one embodiment, the reactants for forming the silylated polyurethane can be present in amounts of from 1 to about 99 weight percent, preferably from about 10 to about 75 weight percent, and most preferably from about 20-80 weight percent of the reaction components used to make the silylated polyurethane composition described herein. The amount of yttrium-containing catalyst can be from about 10 to about 5000 parts per million by weight, preferably from about 20 to about 200 parts per million by weight. The amount of the alternate catalyst(s) described above can also be used in like amounts. As noted above, the advantageous use of the yttrium-containing catalyst herein provides for a silylated polyurethane with reduced moisture sensitivity and increased storage stability compared to the same silylated polyurethane polymers prepared using non-yttrium containing catalysts. A reduced moisture sensitivity can be measured by exposing the silylated polyurethane to moisture and measuring the change in viscosity over time. In one embodiment the silylated polyurethane has approximately the same viscosity, e.g., within 5% of the original viscosity after formation, for a period of from about 12 hours to about 12 days, preferably from about 12 hours to about 15 days, more preferably from about 12 hours to about 30 days. Such viscosity measurements can be conducted in an atmosphere of approximately 50% relative humidity at 25° C. The silylated polyurethane material formed by conducting one or more of the reactions (a), (b) and (c), can be cured in the presence of moisture, e.g., atmospheric moisture or added water solutions added thereto to form a cured resin material having a tensile strength of from about 20 psi to about 1500 psi and preferably from about 50 psi to about 1000 psi determined by ASTM D 412; a modulus at 100% elongation of from about 10 psi to about 1200 psi, preferably from about 20 psi to about 800 psi, determined by ASTM D 412; an elongation at break of from about 15% to about 800%, preferably from about 40% to about 500%, determined by ASTM D 412; and, a hardness of from about 5 Shore A to about 100 Shore A, preferably from about 10 Shore A to about 90 Shore A, as determined by ASTM C 661. The silylated polyurethane composition described herein may also contain constituents that are also useful in crosslinkable materials, for example, silane moisture scavengers, plasticizers, adhesion promoter, organic solvents, catalyst and additives, all of which differ from the components described for the reactions (a), (b) and (c) for making the silylated polyurethane herein. The plasticizers optionally used in the compositions may be any useful plasticizers. Examples of plasticizers are high-boiling hydrocarbons, for example, liquid paraffins, dialkylbenzenes, dialkylnaphthalenes or mineral oils consisting of naphthenic and paraffinic units, polyglycols, in particular polyoxypropylene glycols, which can optionally be substituted, high-boiling esters such as phthalates, citric acid esters or diesters of dicarboxylic acids, liquid polyesters, polyacrylates or polymethacrylates and alkanesulfonic acid esters. If the compositions contain plasticizers, the amounts are preferably from about 1 to about 300 parts by weight, more preferably from 10 to 200 parts by weight, and in particular from about 20 to about 100 parts by weight, based in each case on 100 parts by weight of constituent. The compositions preferably contain plasticizers. In one embodiment, the silylated polyurethane composition can optionally comprise rheology additives, such as, for example, surface treated fumed silica having a particle size of less and 12 nm, preferably, less than 7 nm. Some such surface-treated fumed silicas are those such as Evonik Aerosil R 974, R 9200, R 8200, R 805, R 104, R812 and 812S, and R-106. Cabot CAB-O-SIL ULTRABOND. The amounts of such rheology additives can range from about 4% up to about 40%, and will depend on the required properties. The optional adhesion promoters may be any useful adhesion promoters, for example, organic compounds, silanes and organopolysiloxanes having functional groups such as those having epoxy, glycidoxypropyl, amino, amido, mercapto, carboxyl, anhydrido or methacryloyloxypropyl radicals, isocyanurate and tetraalkoxysilanes and siloxanes containing T or Q groups, which may optionally have alkoxy groups. If, however, another component, already has functional groups, an addition of adhesion promoter can be dispensed with. If the compositions contain adhesion promoters, the amounts are preferably from about 0.1 to about 50 parts by weight, more preferably from about 0.5 to about 20 parts by weight, and in particular from about 1 to about 10 parts by weight, based in each case on 100 parts by weight of moisture-curable silylated polyurethane. The compositions preferably contain adhesion promoters. All conventional organic solvents can be used as optionally used organic solvents. Examples of organic solvents are organic solvents having a water content of less than about 1% by weight, in particular of less than about 0.05% by weight, for example alcohols such as methanol, ethanol, isopropanol, and 1,2-propanediol; ketones such as acetone or cyclohexanone; methyl ethyl ketoxime; esters such as butyl acetate, ethyl oleate, diethyl adipate, propylene carbonate, triethyl phosphate, glyceryl triacetate or dimethyl phthalate; ethers such as dipropylene glycol monomethyl ether, tetrahydrofuran or butoxyethoxyethyl acetate; amides such as N,N-dimethylacetamide or N,N-dimethylformamide; sulfoxides such as dimethyl sulfoxide; pyrrolidones such as N-methyl-2 pyrrolidone or N-octyl-2-pyrrolidone; hydrocarbons such as hexane, cyclohexane, octane, or dodecane; halogenated hydrocarbons such as trichloroethane or difluorotetrachloroethane; and aromatics such as alkylnaphthenes or alkylbenzenes. If the compositions contain organic solvents (H), the amounts are preferably from about 0.1 to about 10 parts by weight, more preferably from about 0.2 to about 5 parts by weight, and in particular from about 0.5 to about 2 parts by weight, based in each case on 100 parts by weight of moisture-curable silylated polyurethane. The compositions preferably contain organic solvent. In another embodiment herein there is provided a sealant, an adhesive or coating comprising the silylated polyurethane formed by reacting the reactants present in the reactions (a), (b), or (c) described herein. The sealant, adhesive or coating can contain the silylated polyurethane in an amount of from about 1% to about 99% by weight, preferably from about 1 to about 95% and most preferably from about 1% to about 90% by weight. In one non-limiting embodiment herein the sealant, adhesive or coating is the same as the silylated polyurethane described herein. There is also provided herein a process of making a silylated polyurethane as described herein and above. The mixing can be conducted with conventional equipment as will be known by those skilled in the art. The addition of the components for one or more of the reactions (a), (b) and/or (c) and any optional components, can be conducted simultaneously, or with any permutation or combination of methods of addition of these components. The silylated polyurethane compositions of this invention, or sealant or adhesives containing the same, are useful in coating applications and in caulking and sealing applications on buildings, airplanes, bathroom fixtures, automotive equipment or wherever elastomeric polymers with improved elongation and flexibility are desired. Another desirable feature of these silylated polyurethane compositions is their ability to be applied to moist or wet surfaces and be cured into a cross-linked elastomer without deleterious effects, which cured product becomes tack-free within a relatively short period of time. Moreover, the cured compositions of this invention strongly adhere alone or with the aid of a primer to a wide variety of substrates such as glass, porcelain, wood, metals, polymeric materials and the like making them especially suited for any type of caulking, adhesive or laminating application. The compositions of the present invention provide a combination of the desirable properties of silylated polyurethane polymers such as tear resistance, extensibility, elastic recovery, and the like, while at the same time providing the desirable properties of improved elongation and flexibility and lower modulus of elasticity. Improved elongation and lower modulus of elasticity, e.g., can significantly reduce the stresses on polyurethane sealants at the interface of the substrate during expansions and contractions of joints. These properties help to minimize adhesive failure of the sealants. While the invention has been described with reference to a number of embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment disclosed herein. EXAMPLES The following nonrestrictive examples are further illustrative of the invention. As will be demonstrated herein below in Table 1, the silylated polyurethane polymers prepared using yttrium catalysts as detailed in the Examples 1-3 of the invention herein below, have a range of desirable properties depending on the reactants and reaction conditions employed, and these properties are similar to those achieved when using tin and bismuth catalysts such as those of the Comparative Examples 1 and 2 set out below. The properties of the various silylated polyurethane polymers are suitable for use in a range of adhesive and sealant applications. The silylated polyurethane polymers prepared in Examples 1-3 using yttrium compounds as catalysts show reduced sensitivity to moisture compared with the silylated polyurethane polymers prepared in Comparative Examples 1 and 2 using conventional tin and bismuth compounds. The silylated polyurethane polymers obtained in Examples 1-3 are formulated into sealant compositions and cured in Examples 5-7 and the properties of the cured sealants, as set out in Table 2 below demonstrate that the silylated polyurethane polymers of the invention prepared using yttrium catalysts can be formulated into sealants with desirable properties that are comparable to those produced from a silylated polyurethane polymer obtained by employing a tin catalyst (Comparative Example 3). Examples 5 and 6 show that silylated polyurethane polymers prepared with yttrium catalysts can be used to prepare sealant formulations that are completely tin-free by using a titanium curing catalyst. Examples 6 and 7 show that a silylated polyurethane polymer made using an yttrium catalyst can be formulated into sealants with very similar properties when using a titanium curing catalyst and a tin curing catalyst, respectively. Example 1 To a four-neck 1 L round bottom flask equipped with an overhead mechanical agitator, nitrogen line, stopper and thermocouple were charged 368.8 g of poly(propylene oxide) diol (HMBT-120, Zhejiang Huangma) and 1.80 g of Irganox 1135 (BASF). The mixture was dried by sparging with nitrogen while heating at 80° C. with a heating mantle for 120 minutes. The temperature was then set at 72° C. 0.02 g of yttrium tris(acetylacetonate) (Strem) was charged and mixed for 20 minutes. 3.79 g of isophorone diisocyanate (Desmodur I, Covestro) was charged and heated for 68 minutes until the isocyanate concentration of the mixture was 0.05% and the viscosity was 34,500 cP. 7.70 g of γ-isocyanatopropyltrimethoxysilane (Silquest A-Link 35, Momentive Performance Materials) was added and the reaction mixture was heated to 80° C. After a further 63 minutes the isocyanate concentration of the mixture was 0.12% and the viscosity was 60,000 cP at which point 7.92 g of a solution of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials) and methanol (6 wt. % methanol) was charged. The mixture was then allowed to cool slowly to room temperature. The viscosity of the finished silylated polyurethane polymer was 50,600 cP, and isocyanate was not detectable. Example 2 To a four-neck 1 L round bottom flask equipped with an overhead mechanical agitator, nitrogen line, stopper and thermocouple were charged 366.3 g of poly(propylene oxide) diol (HMBT-120, Zhejiang Huangma) and 1.80 g of Irganox 1135 (BASF). The mixture was dried by sparging with nitrogen while heating at 80° C. with a heating mantle for 140 minutes. The temperature was then set at 72° C. 0.067 g of yttrium triisopropoxide (Strem) was charged and mixed for 14 minutes. 3.75 g of isophorone diisocyanate (Desmodur I, Covestro) was charged and heated for 1402 minutes until the isocyanate concentration of the mixture was 0.06% and the viscosity was 63,300 cP. 7.46 g of γ-isocyanatopropyltrimethoxysilane (Silquest A-Link 35, Momentive Performance Materials) was added and the reaction mixture was heated to 80° C. After a further 308 minutes the isocyanate concentration of the mixture was 0.05% and the viscosity was 89,500 cP at which point 7.27 g of a solution of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials) and methanol (6 wt % methanol) was charged. The mixture was then allowed to cool slowly to room temperature. The viscosity of the finished silylated polyurethane polymer was 71,400 cP, and isocyanate was not detectable. Comparative Example 1 To a four-neck 1 L round bottom flask equipped with an overhead mechanical agitator, nitrogen line, stopper and thermocouple were charged 314.4 g of poly(propylene oxide) diol (HMBT-120, Zhejiang Huangma) and 1.60 g of Irganox 1135 (BASF). The mixture was dried by sparging with nitrogen while heating at 80° C. with a heating mantle for 120 minutes. The temperature was then set at 72° C. 0.233 mL of a 10% solution of dibutyltin dilaurate (Fomrez SUL-4, Momentive Performance Materials) in toluene was charged and mixed for 15 minutes. 3.21 g of isophorone diisocyanate (Desmodur I, Covestro) was charged and heated for 80 minutes until the isocyanate concentration of the mixture was 0.04% and the viscosity was 33,179 cP. 6.39 g of γ-isocyanatopropyltrimethoxysilane (Silquest A-Link 35, Momentive Performance Materials) was added and the reaction mixture was heated to 80° C. After a further 198 minutes the isocyanate concentration of the mixture was 0.03% and the viscosity was 61,040 cP at which point 6.56 g of a solution of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials) and methanol (6 wt. % methanol) was charged. The mixture was then allowed to cool slowly to room temperature. The viscosity of the finished silylated polyurethane polymer was 50,959 cP, and isocyanate was not detectable. Comparative Example 2 To a four-neck 1 L round bottom flask equipped with an overhead mechanical agitator, nitrogen line, stopper and thermocouple were charged 318.1 g of poly(propylene oxide) diol (HMBT-120, Zhejiang Huangma) and 1.60 g of Irganox 1135 (BASF). The mixture was dried by sparging with nitrogen while heating at 80° C. with a heating mantle for 85 minutes. The temperature was then set at 60° C. 0.024 g of an organobismuth compound (Coscat 83, Vertellus Performance Materials) was charged and mixed for 29 minutes. 3.25 g of isophorone diisocyanate (Desmodur I, Covestro) was charged and heated for 15 minutes until the isocyanate concentration of the mixture was 0.03% and the viscosity was 38,577 cP. 6.73 g of γ-isocyanatopropyltrimethoxysilane (Silquest A-Link 35, Momentive Performance Materials) was added. After a further 15 minutes the isocyanate concentration of the mixture was 0.04% and the viscosity was 82,392 cP at which point 6.91 g of a solution of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials) and methanol (6 wt. % methanol) was charged. The mixture was then allowed to cool slowly to room temperature. The viscosity of the finished silylated polyurethane polymer was 74,931 cP, and isocyanate was not detectable. Example 3 To a four-neck 1 L round bottom flask equipped with an overhead mechanical agitator, nitrogen line, stopper and thermocouple were charged 305.3 g of poly(propylene oxide) diol (Acclaim 18200N, Covestro) and 1.60 g of Irganox 1135 (BASF). The mixture was dried by sparging with nitrogen while heating at 80° C. with a heating mantle for 146 minutes. 0.019 g of yttrium tris(acetylacetonate) (Strem) was charged and mixed for 18 minutes. 7.19 g of γ-isocyanatopropyltrimethoxysilane (Silquest A-Link 35, Momentive Performance Materials) was added. After 151 minutes isocyanate was not detectable, and the viscosity was 38,497 cP at which point 6.47 g of a solution of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials) and methanol (6 wt. % methanol) was charged. The mixture was then allowed to cool slowly to room temperature. The viscosity of the finished silylated polyurethane polymer was 36,354 cP, and isocyanate was not detectable. Example 4 To a four-neck 1 L round bottom flask equipped with an overhead mechanical agitator, nitrogen line, stopper and thermocouple was charged 299.6 g of polybutadiene diol (Krasol LBH-P 3000, Total Cray Valley). The polyol was dried by sparging with nitrogen while heating at 80° C. with a heating mantle for 115 minutes. 0.043 g of yttrium tris(acetylacetonate) (Strem) was charged and mixed for 50 minutes. 6.22 g of isophorone diisocyanate (Desmodur I, Covestro) was charged and heated for 192 minutes until isocyanate was not detectable and the viscosity was 82,551 cP. 28.00 g of γ-isocyanatopropyltrimethoxysilane (Silquest A-Link 35, Momentive Performance Materials) was added. After a further 200 minutes isocyanate was not detectable and viscosity was 155,000 cP at which point 2.75 g of a solution of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials) and methanol (13 wt. % methanol) was charged. The mixture was then allowed to cool slowly to room temperature. The final product viscosity was 150,000 cP, and isocyanate was not detectable. Each silylated polyurethane polymer of Examples 1˜4 and Comparative Examples 1-2 was mixed with 0.5% dibutyltin dilaurate and 1% of a solution of potassium hydroxide/water/methanol (1:20:5 parts by weight), based on the weight of the silylated polyurethane polymer, and then cast into a film and cured in an oven at 50° C. for 1 day. Tensile properties of the cured sheets were tested according to ASTM D 412, and hardness was tested according to ASTM C 661. The test results are listed in the following Table 1: TABLE 1TensileModulusstrengthat 100%ElongationSilylatedat breakelongationat breakHardnessPolymerCatalyst(psi)(psi)(%)(Shore A)Example 1Y(acac)3694421515Example 2Y(OiPr)3976918624ComparativeDBTDL737717128Example 1ComparativeOrganobis-965824520Example 2muthExample 3Y(acac)3647615026Example 4Y(acac)398—4646 The moisture sensitivity of the silylated polyurethane polymers of Examples 1-3 and Comparative Examples 1-2 was evaluated by measuring viscosity after exposure to atmospheric moisture. A 15 g sample of each polymer was weighed into an aluminum pan of 5 cm diameter, and the sample was stored in a climate-controlled room at 50% relative humidity and 25° C. The viscosity of the aged samples was measured periodically. The samples prepared using yttrium compounds as catalysts show a much slower increase in viscosity, i.e. they display reduced sensitivity to moisture. The test results are shown in Chart 1. Example 5 The silylated polyurethane polymer of Example 1 was formulated into a sealant using the titanium-based catalyst Tytan S2. In a speed mixer were mixed: 22.95 g of the silylated polyurethane polymer of Example 1, 0.46 g of ultraviolet light absorber (BLS 1326, Mayzo), 18.35 g of diisodecyl phthalate (Jayflex DIDP, ExxonMobil), 33.00 g of precipitated calcium carbonate (Ultra-Pflex, Specialty Minerals), 22.00 g of ground calcium carbonate (Hi-Pflex, Specialty Minerals), 1.15 g of fumed silica (CAB-O-SIL TS-530, Cabot), 1.15 g of titanium dioxide (Ti-Pure, Chemours), 0.57 g of 3-aminopropyltrimethoxysilane (Silquest A-1110, Momentive Performance Materials), 0.34 g of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials), 0.07 of 1,8-Diazabicyclo[5.4.0]undec-7-ene (Sigma-Aldrich), and 0.07 g of titanium chelate complex (Tytan S2, Borica). The resulting sealant was cast into a film and cured in a climate-controlled room at 50% relative humidity and 25° C. for 7 days. Tensile properties of the cured sheets were tested according to ASTM D 412, and hardness was tested according to ASTM C 661. Example 6 The silylated polyurethane polymer of Example 2 was formulated into a sealant using the titanium-based catalyst Tytan S2. In a speed mixer were mixed: 22.95 g of the silylated polyurethane polymer of Example 2, 0.46 g of ultraviolet light absorber (BLS 1326, Mayzo), 18.35 g of diisodecyl phthalate (Jayflex DIDP, ExxonMobil), 33.00 g of precipitated calcium carbonate (Ultra-Pflex, Specialty Minerals), 22.00 g of ground calcium carbonate (Hi-Pflex, Specialty Minerals), 1.15 g of fumed silica (CAB-O-SIL TS-530, Cabot), 1.15 g of titanium dioxide (Ti-Pure, Chemours), 0.57 g of 3-aminopropyltrimethoxysilane (Silquest A-1110, Momentive Performance Materials), 0.34 g of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials), 0.07 of 1,8-Diazabicyclo[5.4.0]undec-7-ene (Sigma-Aldrich), and 0.07 g of titanium chelate complex (Tytan S2, Borica). The resulting sealant was cast into a film and cured in a climate-controlled room at 50% relative humidity and 25° C. for 7 days. Tensile properties of the cured sheets were tested according to ASTM D 412, and hardness was tested according to ASTM C 661. Example 7 The silylated polyurethane polymer of Example 2 was formulated into a sealant using the tin-based catalyst dibutyltin dilaurate. In a speed mixer were mixed: 22.95 g of the silylated polyurethane polymer of Example 2, 0.46 g of ultraviolet light absorber (BLS 1326, Mayzo), 18.35 g of diisodecyl phthalate (Jayflex DIDP, ExxonMobil), 33.00 g of precipitated calcium carbonate (Ultra-Pflex, Specialty Minerals), 22.00 g of ground calcium carbonate (Hi-Pflex, Specialty Minerals), 1.15 g of fumed silica (CAB-O-SIL TS-530, Cabot), 1.15 g of titanium dioxide (Ti-Pure, Chemours), 0.57 g of 3-aminopropyltrimethoxysilane (Silquest A-1110, Momentive Performance Materials), 0.34 g of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials), and 0.035 g of dibutyltin dilaurate (Fomrez SUL-4, Momentive Performance Materials). The resulting sealant was cast into a film and cured in a climate-controlled room at 50% relative humidity and 25° C. for 7 days. Tensile properties of the cured sheets were tested according to ASTM D 412, and hardness was tested according to ASTM C 661. Comparative Example 3 The silylated polyurethane polymer of Comparative Example 1 was formulated into a sealant using the tin-based catalyst dibutyltin dilaurate. In a speed mixer were mixed: 22.95 g of the silylated polyurethane polymer of Comparative Example 1, 0.46 g of ultraviolet light absorber (BLS 1326, Mayzo), 18.35 g of diisodecyl phthalate (Jayflex DIDP, ExxonMobil), 33.00 g of precipitated calcium carbonate (Ultra-Pflex, Specialty Minerals), 22.00 g of ground calcium carbonate (Hi-Pflex, Specialty Minerals), 1.15 g of fumed silica (CAB-O-SIL TS-530, Cabot), 1.15 g of titanium dioxide (Ti-Pure, Chemours), 0.57 g of 3-aminopropyltrimethoxysilane (Silquest A-1110, Momentive Performance Materials), 0.34 g of vinyltrimethoxysilane (Silquest A-171, Momentive Performance Materials), and 0.035 g of dibutyltin dilaurate (Fomrez SUL-4, Momentive Performance Materials). The resulting sealant was cast into a film and cured in a climate-controlled room at 50% relative humidity and 25° C. for 7 days. Tensile properties of the cured sheets were tested according to ASTM D 412, and hardness was tested according to ASTM C 661. The test results of the four sealants are listed in the following Table 2: TABLE 2TensileModulusstrengthat 100%ElongationSealantSilylatedat breakelongationat breakHardnessFormulationPolymer(psi)(psi)(%)(Shore A)Example 5Example 11808446319Example 6Example 22018769827Example 7Example 221210350430ComparativeComparative22712540539Example 3Example 1 While the invention has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out the process of the invention but that the invention will include all embodiments falling within the scope of the appended claims. | 50,083 |
11859044 | DESCRIPTION OF EMBODIMENTS Embodiments of the present disclosure will be described in detail below. Polyisocyanate Compound A polyisocyanate compound of the present disclosure is a polyisocyanate compound having an isocyanurate group represented by Formula (1) below. R1to R3in Formula (1) are identical or different and are a group represented by Formula (1a) below. In Formula (1a), L1and L2are identical or different and represent an alkylene group having from 1 to 10 carbons, m represents a number of 0 or greater, L3represents a divalent hydrocarbon group having from 4 to 18 carbons, and X represents an isocyanate group or a blocked isocyanate group blocked with a blocking agent. m is not simultaneously 0 for R1to R3, and the bond with the wavy line bonds to a nitrogen atom in Formula (1). Examples of the alkylene group having from 1 to 10 carbons in L1and L2above include linear or branched alkylene groups, such as a methylene group, a methylmethylene group, a dimethylmethylene group, an ethylene group, a propylene group, a trimethylene group, a butylene group, a 1-methyltrimethylene group, a 2-methyltrimethylene group, a 1,1′-dimethylethylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a 2-ethylhexylene group, a nonylene group, and a decylene group. L1above is among others preferably an alkylene group having from 1 to 3 carbons and more preferably an ethylene group. L2above is among others preferably an alkylene group having from 1 to 8 carbons and more preferably an alkylene group having from 4 to 6 carbons. In addition, the alkylene group is preferably a linear alkylene group. L3above is a divalent hydrocarbon group having from 4 to 18 carbons and preferably a divalent aliphatic hydrocarbon group having from 4 to 18 carbons, a divalent alicyclic hydrocarbon group having from 4 to 18 carbons, or a divalent aromatic hydrocarbon group having from 6 to 18 carbons. Examples of the divalent aliphatic hydrocarbon group having from 4 to 18 carbons include a tetramethylene group, a pentamethylene group, a hexamethylene group, a heptamethylene group, an octamethylene group, a nonamethylene group, a trimethylhexamethylene group, and a decamethylene group. Among these, a divalent aliphatic hydrocarbon group having from 6 to 12 carbons is more preferred, and a hexamethylene group is even more preferred. The divalent alicyclic hydrocarbon group having from 4 to 18 carbons is a group formed by removing two hydrogen atoms from an alicyclic hydrocarbon having from 4 to 18 carbons, and examples include a 1,3-cyclohexylene group, 1,4-cyclohexylene group, 1,3-cyclohexanedimethylene group, 1,4-cyclohexanedimethylene group, 4,4′-dicyclohexylmethane diisocyanate residue, and an isophorone residue. Among these, a divalent alicyclic hydrocarbon group having from 6 to 10 carbons is more preferred, and an isophorone residue is even more preferred. The divalent aromatic hydrocarbon group having from 6 to 18 carbons is a group formed by removing two hydrogen atoms from a divalent aromatic hydrocarbon group having from 6 to 18 carbons, and examples include a methylphenylene group, a methane diphenylene group, an ethane phenylene group, a naphthylene group, a dimethylphenylene group, and a phenylene group. m above is an average value of the degree of polymerization of the unit indicated by parentheses in Formula (1a) and is a number of 0 or greater, for example, of 0 to 7.0, preferably of 1.0 to 4.0, and more preferably of 1.0 to 3.0. The isocyanate group concentration of the polyisocyanate compound of the present disclosure is preferably from 6 to 14 wt. %, more preferably from 7 to 13 wt. %, even more preferably from 8 to 12 wt. %, and particularly preferably from 9 to 12 wt. %. The isocyanate group concentration can be calculated in accordance with HS K 1603-1A method by adding a tetrahydrofuran (THF) solution of dibutylamine (0.1 mol/L) to a sample diluted with THF to completely react isocyanate groups with an excess amount of dibutylamine (ureation), and then back-titrating the unreacted residual dibutylamine with a standard hydrochloric acid titration solution (0.1 mol/L). With the isocyanate group concentration of lower than 6 wt. %, the resulting polyurethane resin would tend to have lower chemical resistance, and with the isocyanate group concentration of higher than 14 wt. %, the resulting polyurethane resin would tend to have lower flexibility. Some or all of the isocyanate groups of the isocyanate compound of the present disclosure may be blocked isocyanate groups blocked with a blocking agent. The blocked isocyanate group is formed by reaction of the isocyanate group with a blocking agent. Exposing the blocked isocyanate group to heating during thermal curing dissociates the blocking agent from the blocked isocyanate group and regenerates the isocyanate group. Examples of the blocking agent include imidazole-based compounds, alcohol-based compounds, phenol-based compounds, active methylene-based compounds, oxime-based compounds, lactam-based compounds, amine-based compounds, pyrazole-based compounds, and bisulfites. Examples of the imidazole-based compound include imidazole, benzimidazole, 2-methylimidazole, 4-methylimidazole, and 2-ethylimidazole. Examples of the alcohol-based compound include methanol, ethanol, isopropanol, n-butanol, 2-ethoxyhexanol, 2-N,N-dimethylaminoethanol, 2-ethoxyethanol, cyclohexanol, butyl cellsolve, and ethyl cellsolve. Examples of the phenol-based compound include phenol, cresol, n-propylphenol, isopropylphenol, n-butylphenol, sec-butylphenol, tert-butylphenol, n-hexylphenol, nitrophenol, chlorophenol, cresol, and methyl 4-hydroxybenzoate. Examples of the active methylene-based compound include Meldrum's acid, acetylacetone, methyl acetoacetate, dimethyl malonate, ethyl cyanoacetate, and methyl isobutanoylacetate. Examples of the oxime-based compound include acetoxime, methyl ethyl ketoxime, methyl isobutyl ketoxime, and 2-heptanone oxime. Examples of the lactam-based compound include ε-caprolactam, δ-valerolactam, γ-butyrolactam, and β-propiolactam. Examples of the amine-based compound include dibutylamine, 4-phenylbutylamine, and 6-methyl-2-piperidine. Examples of the pyrazole-based compound include pyrazole, 3,5-dimethylpyrazole, and 3,5-diisopropylpyrazole. Examples of the bisulfate include sodium bisulfate. The blocking agent is preferably an alcohol-based compound or an active methylene-based compound from the viewpoints of low-temperature curability, chemical resistance, and flexibility. One blocking agent can be used alone or two or more in combination. The isocyanate group concentration when the polyisocyanate compound of the present disclosure has a blocked isocyanate group is a value obtained for a compound in which the blocked isocyanate group is replaced with an isocyanate group. The polyisocyanate compound of the present disclosure may contain a multimer (from a dimer to a hexamer) having a plurality of isocyanurate groups. The multimer is a compound in which two or more polyisocyanate compounds represented by Formula (1) above and a polyester polyol compound (1′) below are bonded by reaction of a terminal isocyanate group and a terminal hydroxyl group, where the isocyanurate groups are linked by a group represented by Formula (1b) below. In Formula (1b), L1, L2, L3, and m are identical to L1, L2, L3, and m in Formula (a) above, and two bonds with the wavy line each bonds to a nitrogen atom of the isocyanurate group. The polyisocyanate compound of the present disclosure can be produced, for example, by reacting a polyester polyol compound (1′) below having an isocyanurate group with at least one diisocyanate selected from aliphatic diisocyanates, alicyclic diisocyanates, and aromatic diisocyanates. The polyol and the diisocyanate are preferably reacted in an equivalent ratio of isocyanate groups of the diisocyanate to hydroxyl groups of the polyol compound (isocyanate groups/hydroxyl groups) ranging from 5 to 40, more preferably from 6 to 30, and even more preferably from 7 to 20. Reacted in an equivalent ratio (isocyanate groups/hydroxyl groups) within the above range, the polyester polyol compound (1′) below and the diisocyanate above do not excessively react and are more likely to react in a molar ratio of 1 to 3 (polyol compound/diisocyanate=1/3), and thus this makes it easier to obtain a polyisocyanate compound, which is a monomer having three isocyanate groups. The unreacted diisocyanate is removed by distillation, extraction, or the like, but the polyisocyanate compound of the present disclosure may contain 1.0 wt. % or lower of the unreacted diisocyanate. Polyol The polyol is a polyester polyol compound represented by Formula (1′) below. R1′to R3′in the formula are identical or different and are a group represented by Formula (1a′) below. L1, L2, and m in Formula (1a′) are identical to L1, L2, and m in Formula (1a) above. The number average molecular weight (Mn: in terms of standard polystyrene) of the polyester polyol compound (1′) is preferably from 570 to 2000, more preferably from 580 to 1500, even more preferably from 590 to 1200, particularly preferably from 590 to 1100, and most preferably from 590 to 900. In addition, the molecular weight dispersity (weight average molecular weight Mw/number average molecular weight Mn) of the polyester polyol compound (1′) is, for example, from 1.0 to 3.0. The hydroxyl value (KOH mg/g) of the polyester polyol (1′) is, for example, from 80 to 400 KOH mg/g, and in this range, from the viewpoints of being able to improve scratch resistance and chemical resistance of the resulting cured product, the hydroxyl value is preferably from 110 to 350 KOH mg/g, more preferably from 150 to 300 KOH mg/g, even more preferably from 160 to 290 KOH mg/g, and particularly preferably from 180 to 285 KOH mg/g. The hydroxyl value can be measured by the hydroxyl value measurement method described in JIS-K1557. The polyester polyol compound (1′) can be produced, for example, by ring-opening polymerization of a lactone using a hydroxyl group of a compound (1″) represented by Formula (1″) below as a starting point. L1in Formula (1″) is identical to L1in Formula (1a) and Formula (1a′). Examples of the lactone include α-acetolactone, β-propiolactone, γ-butyrolactone, δ-valerolactone, and ε-caprolactone. The number average molecular weight and the molecular weight dispersity (weight average molecular weight Mw/number average molecular weight Mn) of the polyester polyol compound (1′) can be measured with the following instrument and conditions.Measurement instrument: a high-speed GPC instrument “HLC-8220 GPC”, available from Tosoh CorporationMobile phase: tetrahydrofuran Diisocyanate The diisocyanate used in the present disclosure is a diisocyanate having from 4 to 18 carbons and preferably at least one selected from aliphatic diisocyanates having from 4 to 18 carbons, alicyclic diisocyanates having from 4 to 18 carbons, and aromatic diisocyanates having from 6 to 18 carbons. The number of carbons of the diisocyanate is the number not including two carbons involved in the isocyanate group. Examples of the aliphatic diisocyanate having from 4 to 18 carbons include 1,4-tetramethylene diisocyanate, 1,5-pentamethylene diisocyanate, 1,6-hexamethylene diisocyanate, 1,7-heptamethylene diisocyanate, 1,8-octamethylene diisocyanate, 1,9-nonamethylene diisocyanate, 2,2,4-trimethylhexamethylene diisocyanate, 2,4,4-trimethylhexamethylene diisocyanate, and 1,10-decamethylene diisocyanate. Examples of the alicyclic diisocyanate having from 4 to 18 carbons include 1,3-cyclohexane diisocyanate, 1,4-cyclohexane diisocyanate, 1,3-bis(isocyanatomethyl)cyclohexane, 1,4-bis(isocyanatomethyl)cyclohexane, 4,4′-dicyclohexylmethane diisocyanate, isophorone diisocyanate, and norbornene diisocyanate. In addition, examples of the aromatic diisocyanate having from 6 to 18 carbons include tolylene diisocyanate (TDI), diphenylmethane diisocyanate (MDI), 4,4′-dibenzyl diisocyanate, 1,5-naphthylene diisocyanate, xylylene diisocyanate, 1,3-phenylene diisocyanate, and 1,4-phenylene diisocyanate. Examples of the diphenylmethane diisocyanate include 2,2′-diphenylmethane diisocyanate, 2,4′-diphenylmethane diisocyanate, and 4,4′-diphenylmethane diisocyanate. Composition for Forming Polyurethane Resin The composition for forming a polyurethane resin of the present disclosure contains the polyisocyanate compound as a curing agent, and a polyacrylic polyol. From the viewpoint of being able to improve scratch resistance and chemical resistance of the resulting cured product, the polyisocyanate compound and the polyacrylic polyol are blended in an equivalent ratio of a total of isocyanate groups and blocked isocyanate groups contained in the polyisocyanate compound to hydroxyl groups contained in the polyacrylic polyol ((isocyanate groups+blocked isocyanate groups)/hydroxyl groups) of preferably from 0,5 to 2.0, more preferably from 0.5 to 1.5, even more preferably from 0.7 to 1.3, and particularly preferably from 0.9 to 1.2. In addition, the content of the polyacrylic polyol in the composition is, from the viewpoint of being able to improve scratch resistance and chemical resistance of the resulting cured product, preferably from 100 to 400 parts by weight, more preferably from 150 to 350 parts by weight, and even more preferably from 200 to 300 parts by weight per 100 parts by weight of the polyisocyanate compound. Polyacrylic polyol The polyacrylic polyol can be produced by copolymerizing a (meth)acrylic compound having a hydroxyl group and another (meth)acrylic compound other than the above (meth)acrylic compound having a hydroxyl group. Examples of the (meth)acrylic compound having a hydroxyl group include 2-hydroxyethyl (meth)acrylate, 3-hydroxypropyl (meth)acrylate, and 4-hydroxybutyl (meth)acrylate. Among these, 2-hydroxyethyl (meth)acrylate is preferably used. One of these can be used alone, or two or more in combination. Examples of another (meth)acrylic compound above include (meth)acrylic acid, methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, tert-butyl (meth)acrylate, neopentyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, stearyl (meth)acrylate, isostearyl (meth)acrylate, cetyl (meth)acrylate, lauryl (meth)acrylate, 2,2,2-trifluoroethyl (meth)acrylate, 2,2,3,3-tetrafluoropropyl (meth)acrylate, 1H,1H,5H-octafluoropentyl (meth)acrylate, 2-(perfluorooctyl)ethyl (meth)acrylate, cyclohexyl (meth)acrylate, dicyclopentanyl (meth)acrylate, dicyclopentenyloxyethyl (meth)acrylate, poly(ethylene glycol) mono(meth)acrylate, methoxyethyl (meth)acrylate, methoxybutyl (meth)acrylate, methoxy triethylene glycol (meth)acrylate, methoxy poly(ethylene glycol) (meth)acrylate, benzyl (meth)acrylate, 2-ethyl-2-methyl-[1,3]-dioxolan-4-yl-methyl (meth)acrylate, dimethylaminoethyl (meth)acrylate, and isobornyl (meth)acrylate. Among these, a (meth)acrylate having an alkyl group having from 1 to 10 carbons is preferred, and methyl (meth)acrylate or n-butyl (meth)acrylate is more preferred. One of these can be used alone, or two or more in combination. The hydroxyl value of the polyacrylic polyol is preferably from 30 to 100 KOH mg/g and more preferably from 40 to 80 KOH mg/g. With the hydroxyl value of the polyacrylic polyol of lower than 30 KOH mg/g, the resulting polyurethane resin would tend to have lower chemical resistance, and with the hydroxyl value of the polyacrylic polyol of higher than 100 KOH mg/g, the resulting polyurethane resin would tend to have lower flexibility. The composition for forming a polyurethane resin of the present disclosure may further contain as necessary an inorganic particle, an organic particle, an additive, a catalyst, or the like. Inorganic Particle Examples of the inorganic particle that the composition for forming a polyurethane resin of the present disclosure may further contain include silica, alumina, mica, synthetic mica, talc, calcium oxide, calcium carbonate, zirconium oxide, titanium oxide, barium titanate, kaolin, bentonite, diatomaceous earth, boron nitride, aluminum nitride, silicon carbide, zinc oxide, cerium oxide, cesium oxide, magnesium oxide, glass beads, glass fibers, graphite, carbon nanotubes, calcium hydroxide, magnesium hydroxide, and aluminum hydroxide. Among these, silica is preferred from the viewpoint of being able to improve chemical resistance and scratch resistance of the resulting cured product. Organic Particle Examples of the organic particle that the composition for forming a polyurethane resin of the present disclosure may further contain include polyethylene wax, polypropylene wax, acrylic beads, and urethane beads. Among these, a urethane bead is preferred from the viewpoint of improving the texture (soft feel properties) of the coating. Regardless of inorganic or organic, one type of these particles can be used alone, or two or more types in combination. The particle sizes of the inorganic particle and the organic particle are not particularly limited but are preferably from 0.01 nm to 1 μm from the viewpoint of good appearance. Additive Examples of the additive that the composition for forming a polyurethane resin of the present disclosure may further contain include surfactants, pigments, dyes, ultraviolet absorbers, light stabilizers, surface modifiers, antifoaming agents, wetting agents, dispersants, viscoelasticity modifiers, thixotropy-imparting agents, antiseptics, film-forming agents, plasticizers, penetrants, perfumes, bactericides, fungicides, antioxidants, antistatic agents, flame retardants, and matting agents. The contents of the inorganic particle, organic particle, and additive in the composition for forming a polyurethane resin of the present disclosure is not particularly limited but is preferably 10 wt. % or lower relative to a total weight (100 wt. %) of non-volatile content of the composition. Catalyst Examples of the catalyst that the composition for forming a polyurethane resin of the present disclosure may further contain include tin-based catalysts (such as stannous octoate and dibutyltin dilaurate); bismuth-based catalysts (such as bismuth neodecanoate and bismuth 2-ethylhexanoate); zirconia-based catalysts (such as zirconyl 2-ethylhexanoate and zirconyl naphthenate); other organometallic catalysts (such as phenylmercury propionate salt and lead octenoate); ammonium salt catalysts (such as tetraalkylammonium halides (such as tetramethylammonium chloride), tetraalkylammonium hydroxides (such as tetramethylammonium hydroxide salts), tetraalkylammonium organic salts (such as tetramethylammonium acetate salts, tetramethylammonium 2-ethylhexanoate salts, 2-hydroxypropyl trimethylammonium formate salts, and 2-hydroxypropyl trimethylammonium 2-ethylhexanoate salts)); and amine catalysts (such as triethylamine, triethylenediamine, diethanolamine, bis[2-(dimethylamino)ethyl]methylamine, dimethylamino morpholine, N-ethylmorpholine, and tetramethylguanidine). The blending amount of the catalyst is preferably from 0.001 to 0.5 parts by weight per 100 parts by weight of a total of the polyisocyanate compound and the polyacrylic polyol. Solvent A solvent is added to the composition for forming a polyurethane resin of the present disclosure, and the viscosity can be adjusted. Examples of the solvent include ester-based solvents, such as acetate esters (such as ethyl acetate and butyl acetate); ether-based solvents, such as dioxane and tetrahydrofuran; ketone-based solvents, such as acetone and methyl ethyl ketone, aromatic-based solvents, such as toluene and xylene; halogen-based solvents, such as dichloromethane and chloroform; alcohol-based solvents, such as methanol, ethanol, isopropanol, and butanol; nitrile-based solvents, such as acetonitrile and benzonitrile; and glycol-based solvents, such as alkylene glycol (such as ethylene glycol and propylene glycol), alkylene glycol monoalkyl ethers (such as ethylene glycol monomethyl ether and propylene glycol monomethyl ether), alkylene glycol dialkyl ethers (ethylene glycol dimethyl ether and propylene glycol dimethyl ether), and alkylene glycol monoalkyl ether monoalkyl esters (ethylene glycol monoethyl ether acetate and ethylene glycol monopropyl ether acetate). One of these can be used alone, or two or more in combination. The content of the solvent in the composition for forming a polyurethane resin of the present disclosure is adjusted to give a solid content concentration in the composition preferably of 70 to 99 wt. % and more preferably of 80 to 95 wt. %. In addition, the content of the solvent is preferably from 5 to 20 parts by weight, more preferably from 7 to 17 parts by weight, and even more preferably from 9 to 15 parts by weight per 100 parts by weight of a total of the polyisocyanate compound and the polyacrylic polyol. The composition for forming a polyurethane resin of the present disclosure can be produced by mixing the above components. The composition for forming a polyurethane resin of the present disclosure can be a two-component coating agent for which the polyisocyanate compound and the polyacrylic polyol are separately stored and mixed at the time of use. In addition, the composition for forming a polyurethane resin of the present disclosure can be a one-component coating agent for which the polyisocyanate compound having a blocked isocyanate group and the polyacrylic polyol are mixed in advance and stored. The composition for forming a polyurethane resin of the present disclosure has the above configuration, and thus heat treatment of the composition enables the polyisocyanate compound and the polyacrylic polyol to be urethane-bonded to form a cured product (i.e., a cured product composed of a polyurethane resin). The heat treatment conditions are, for example, at 50 to 100° C. for approximately 10 to 240 minutes. After completion of the heat treatment, the cured product may be further aged at room temperature (from 1 to 30° C.) for approximately 12 to 60 hours. The cured product thus obtained has excellent adhesion to a substrate (e.g., a plastic substrate, such as those of PET), scratch resistance, and flexibility. The cured product has high hardness; the pencil hardness (by a method in accordance with JIS K5600) is, for example, preferably 4B or harder, more preferably 3B or harder, and even more preferably B or harder. The cured product has excellent chemical resistance; for example, even if a sunscreen agent adheres to the cured product, the surface of the cured product does not swell or not exhibit a white cloudiness, and the cured product has excellent sunscreen resistance accordingly. The cured product has the above properties in combination. Thus, the composition for forming the cured product is suitable as a coating agent for plastic molded products, such as extrusion-molded products, injection-molded products, and compression-molded products; and as a material for molded products, such as films. Examples of the plastic molded article include enclosures for home appliances (such as refrigerators, washing machines, air conditioners, and televisions), enclosures for electronic devices (such as personal computers, mobile phones, and smartphones), a member constituting a musical instrument (such as pianos, electric organs, and electronic musical instruments), vehicle members, such as those for automobiles and railway vehicles (interior materials, such as those for instrument panels, door trims, headlining, and tonneau covers; and exterior materials, such as those for bumpers). In addition, examples of the plastic forming the plastic molded product include thermoplastic resins and thermosetting resins. Examples of the thermoplastic resin include styrene-based resins, such as polystyrene; polyolefin-based resins, such as polyethylene and polypropylene; polyesters, such as poly(ethylene terephthalate) (PET); vinyl chloride-based resins, such as vinyl chloride resins; polyamides, such as polyamide 46, polyamide 6, polyamide 66, polyamide 610, polyamide 612, polyamide 1010, polyamide 1012, polyamide 11, polyamide 12, and polyamide 1212; polyphenylene ethers, such as poly(2,6-dimethyl-1,4-phenylene ether); homopolymers or copolymers of acrylonitrile, such as PAN resins, AS resins, ABS resins, AAS resins, ACS resins, AES resins, and AXS resins; (meth)acrylic resins; polycarbonate; polyacetal; polyphenylene sulfide; polyether ether ketone; polyamide imide; polyimide; polyether imide; polysulfone; poly(ether sulfone); modified products and derivatives of a resin of these, and polymer blends and polymer alloys containing these resins. Examples of the thermosetting resin include phenolic resins, urea resins, melamine resins, unsaturated polyesters, furan resins, epoxy resins, polyurethane resins, allyl resins, and polyimide resins. Plastic Molded Product A plastic molded product provided with a coating composed of the cured product of the present disclosure on at least a portion of the surface has excellent properties, such as hardness, scratch resistance, and chemical resistance. The thickness of the coating is not particularly limited and is, for example, approximately from 10 to 150 μm. In addition, the plastic molded product formed of the composition for forming a polyurethane resin itself as a material has a surface composed of the cured product and thus likewise also has excellent properties, such as hardness, scratch resistance, and chemical resistance. Each configuration, a combination of the configurations, and the like in each embodiment are an example, and various additions, omissions, substitutions, and other changes are possible as appropriate without departing from the spirit of the present disclosure. The present disclosure is not limited by the embodiments and is limited only by the claims. EXAMPLES Hereinafter, the present disclosure will be described more specifically with reference to examples, but the present disclosure is not limited by these examples. Polyols, diisocyanates, and solvents used in examples are as follows. Polyol Polyol 1: a caprolactone adduct of tris(2-hydroxyethyl isocyanurate) (number average molecular weight 611, Mw/Mn=1.2, hydroxyl value 280 KOH mg/g)Polyol 2: a caprolactone adduct of tris(2-hydroxyethyl isocyanurate) (number average molecular weight 1068, Mw/Mn=1.3, hydroxyl value 169 KOH mg/g)303: PLACCEL 303 (poly(caprolactone triol), available from Daicel Corporation, number average molecular weight 400, Mw/Mn=1.2, hydroxyl value 544 KOH mg/g)305: PLACCEL 305 (poly(caprolactone triol), available from Daicel Corporation, number average molecular weight 640, Mw/Mn=1.4, hydroxyl value 308 KOH mg/g)309: PLACCEL 309 (poly(caprolactone triol), available from Daicel Corporation, number average molecular weight 987, Mw/Mn 1.5, hydroxyl value 188 KOH mg/g) Diisocyanate HDI: hexamethylene diisocyanate (available from Tosoh Corporation, molecular weight 168.2)IPDI: isophorone diisocyanate (available from Sumika Covestro Urethane Co., Ltd., molecular weight 222.3) Acrylic Polyol A-801: Acrydic A-801 (available from DIC Corporation, hydroxyl value 50 KOH mg/g) Solvent Butyl acetate: a reagent available from Tokyo Chemical Industry Co., Ltd. Synthesis Example A-1 To a five-neck flask equipped with a reflux condenser, a thermometer, a nitrogen gas inlet tube, and a stirring device were added 137 g of polyol 1 and 863 g of hexamethylene diisocyanate (HDI) under a nitrogen gas atmosphere, then the internal temperature was increased to 100° C. while the mixture was stirred, and the mixture was reacted for one hour. The resulting reaction solution was subjected to thin film distillation at 160° C. and 0.2 mm Hg, unreacted hexamethylene diisocyanate (HDI) was removed, and liquid polyisocyanate compound A-1 was obtained. The isocyanate group concentration of polyisocyanate compound A-1 was 11 wt. % as measured by the back titration method in accordance with the JIS K 1603-1 A method. Synthesis Examples A-2 and A-3, and Comparative Synthesis Examples B-1 to B-4 Polyol 1, polyol 2, 303, 305, or 309, and diisocyanate (HDI) or diisocyanate (IPDI) were blended according to the weight ratio shown in Table 1 to give a total of 1000 g, reacted under similar conditions to those in Synthesis Example A-1, and polyisocyanate compounds A-2, A-3, and B-1 to B-4 were obtained. Examples 1 to 3 and Comparative Examples 1 to 4 The polyisocyanate compound, acrylic polyol, and butyl acetate were mixed according to the weight ratio shown in Table 2, and a composition for forming a polyurethane resin was obtained. The resulting composition was applied by spraying to a poly(ethylene terephthalate) film (Cosmoshine A4100 #100, available from Toyobo Co., Ltd.) to give a coating thickness of 50 μm, cured and dried in an oven at 70° C. for 30 minutes, and a cured coating/PET film laminate was obtained. The cured coatings of the laminates obtained in the examples and comparative examples were evaluated for pencil hardness, scratch resistance, sunscreen resistance, elongation at break, and strength at break by the following methods. Pencil Hardness The pencil hardness of the cured coating side surface of the laminates obtained in the examples and comparative examples was evaluated by the method in accordance with JIS K5600, That is, the cured side surface of the laminate was rubbed with a pencil (pencil lead), and a laminate in which a scratch was observed on the surface was determined to be NG (poor). Specifically, the evaluation was performed using a pencil with a certain hardness, and when no scratch was made, another evaluation was performed with a pencil with a hardness one grade higher, and this operation was repeated. Once a scratch was observed, the laminate was re-evaluated with a hardness one grade lower, and when no scratch was observed, the laminate was evaluated again using a pencil with a hardness one grade higher. When reproducibility was confirmed twice or more, the hardness of the hardest pencil with which no scratch was made was determined as the pencil hardness of the cured coating.Pencil for evaluation: a “Pencil for Pencil Hardness Test” available from Mitsubishi Pencil Co., Ltd.Load: 750 gfScratch distance: 7 mm or longerScratch angle: 45°Measurement environment: 23° C., 50% RH In the test were used laminates moisture-controlled in a constant temperature and humidity chamber at 23° C. and 50% RH for 24 hours. Scratch Resistance For the scratch resistance of the cured coating side surface of the laminates obtained in the examples and comparative examples, a scratch test was performed by attaching a steel wool (B-204, Bonstar for commercial use #0000) to a rubbing tester (Standard Model, available from Nippon Rika Industries Corporation) and reciprocating the steel wool (10 reciprocations) on the coating with a load of 500 g applied. The initial gloss (60-degree gloss) (G0) before the scratch test on the cured coating side surface and the gloss (60-degree gloss) (G1) after two minutes of the scratch test were measured using a gloss meter (Gloss Meter VG7000, available from Nippon Denshoku Industries Co., Ltd.), and the scratch resistance was evaluated by calculating the retention rate of the gloss by the following equation. Retention rate of gloss after scratch test=(G1)/(G0)×100(%) Evaluation Criteria Excellent: The retention rate of the gloss was 95% or higherGood: The retention rate of the gloss was lower than 95% and 90% or higherSlightly poor: The retention rate of the gloss was lower than 90% and 80% or higherPoor: The retention rate of the gloss was lower than 80% Sunscreen Resistance (Drip Method) To the cured coating side surface of the laminates obtained in the examples and comparative examples was applied a sunscreen cream (“Ultra Sheer Dry-Touch SPF45” available from Neutrogena Corporation) to give 0.025 g/cm2, and the laminate was allowed to stand in an oven at 50° C. for one hour. The sunscreen cream was then wiped off, and the appearance of the cured coating was evaluated according to the following criteria. Less change in the appearance indicates better chemical resistance. Evaluation Criteria Excellent: The cured coating had almost no change in the appearanceGood: A trace of a chemical liquid remained on the cured coatingSlightly poor: The cured coating swelledPoor: The cured coating swelled and whitened Elongation at Break and Strength at Break A thermosetting polyurethane sheet 2 mm in thickness) with each composition (polyisocyanate composition+acrylic polyol) shown in Table 2 was prepared and formed into a type 3 dumbbell-shaped test piece (100 mm in length×25 mm in width×2 mm in thickness). Each dumbbell-shaped test piece was subjected to a tensile test using a Tensilon universal testing machine RTC-1350A (available from ORIENTEC Corporation) under an environment of 23° C. and 50% Rh in conditions of an inter-chuck distance of 60 mm and a tensile speed of 500 mm/min, and elongation (elongation at break) and maximum stress (strength at break) when the test piece was broken were measured. Higher elongation at break indicates superior flexibility. Evaluation Criteria for Elongation at Break Good: higher than 50%Slightly poor: from 10% to 50%Poor: lower than 10% Evaluation Criteria for Strength at Break Good: greater than 20 MPaSlightly poor: from 10 to 20 MPaPoor: less than 10 MPa The results are collectively shown in Table 2, The cured coatings shown in Examples 1 to 3 obtained using A-1 to A-3 as the polyisocyanate compound all had excellent hardness with a pencil hardness of 2B to H and exhibited excellent scratch resistance, excellent or good sunscreen resistance, good or slightly poor elongation at break, and good strength at break, and the cured coatings had excellent hardness, scratch resistance, chemical resistance, and flexibility accordingly. The cured coatings shown in Comparative Examples 1 to 4 obtained using B-1 to B-4 having no isocyanurate group as the polyisocyanate compound were insufficient for any of scratch resistance, chemical resistance, or flexibility as described below. The cured coating of Comparative Example 1 had slightly poor scratch resistance and poor flexibility with poor elongation at break. In addition, the cured coating of Comparative Example 2 had poor chemical resistance with slightly poor sunscreen resistance. Furthermore, the cured coating of Comparative Example 3 had good scratch resistance and good sunscreen resistance but had slightly poor elongation at break, indicating insufficient overall performance. Moreover, the cured coating of Comparative Example 4 had slightly poor scratch resistance and poor chemical resistance with poor sunscreen resistance. TABLE 1WeightSynthesis ExampleComparative Synthesis Exampleratio (wt. %)A-1A-2A-3B-1B-2B-3B-4Polyol113.714.10000020020.800003030007.6000305000012.613.0030900000019.1Diisocy-HDI86.3079.292.487.4080.9anateIPDI085.900087.00Isocyanate group11981511108concentration TABLE 2ExamplesComparative Examples1231234PolyisocyanateA-1Weight20.4000000compositionA-2ratio023.400000A-3(wt. %)0025.60000B-100016.0000B-2000020.400B-30000021.80B-400000025.6Acrylic polyolA-80159.656.654.464.059.658.254.4SolventButyl20.020.020.020.020.020.020.0acetateIsocyanate group/Equivalent1.01.01.01.01.01.01.0hydroxyl groupratioPencil hardnessBH2BB2B2B2BScratch resistance (glossExcellentExcellentExcellentSlightlyGoodGoodSlightlyretention ratio)poorpoorSunscreen resistanceExcellentExcellentGoodExcellentSlightlyGoodPoor(drip method)poorElongation at breakGoodSlightlyGoodPoorSlightlySlightlyGoodpoorpoorpoorStrength at breakGoodGoodGoodGoodGoodGoodGood To summarize the above, configurations of the present disclosure and their variations will be described in addition below.[1] A polyisocyanate compound represented by Formula (1).[2] The polyisocyanate compound according to [1], wherein L1of Formula (1) is an alkylene group having from 1 to 3 carbons (preferably an ethylene group).[3] The polyisocyanate compound according to [1] or [2], wherein L2of Formula (1) is an alkylene group having from 1 to 8 carbons (preferably an alkylene group having from 4 to 6 carbons and more preferably a pentylene group).[4] The polyisocyanate compound according to any one of [1] to [3], wherein L3of Formula (1) is a divalent aliphatic hydrocarbon group having from 4 to 18 carbons (preferably a divalent aliphatic hydrocarbon group having from 6 to 12 carbons and more preferably a hexamethylene group), a divalent alicyclic hydrocarbon group having from 4 to 18 carbons (preferably a divalent alicyclic hydrocarbon group having from 6 to 10 carbons and more preferably an isophorone residue), or a divalent aromatic hydrocarbon group having from 6 to 18 carbons.[5] The polyisocyanate compound according to any one of [1] to [4], wherein m of Formula (1) is from 0 to 7.0 (preferably from 1.0 to 4.0 and more preferably from 1.0 to 3.0).[6] The polyisocyanate compound according to any one of [1] to [5], wherein an isocyanate group concentration is from 6 to 14 wt. % (preferably from 7 to 13 wt. %, more preferably from 8 to 12 wt. %, and even more preferably from 9 to 12 wt. %), where an isocyanate group concentration of a compound having a blocked isocyanate group is a value obtained for a compound in which the blocked isocyanate group is replaced by an isocyanate group.[7] The polyisocyanate compound according to any one of [1] to [6], wherein some or all of the isocyanate groups are blocked isocyanate groups blocked with a blocking agent.[8] The polyisocyanate compound according to [7], wherein the blocking agent is one or more selected from imidazole-based compounds, alcohol-based compounds, phenol-based compounds, active methylene-based compounds, oxime-based compounds, lactam-based compounds, amine-based compounds, pyrazole-based compounds, and bisulfites.[9] The polyisocyanate compound according to any one of [1] to [8], containing a multimer (a dimer to a hexamer) having a plurality of isocyanurate groups linked by a group represented by Formula (1b).[10] A method of producing a polyisocyanate compound, the method of producing the polyisocyanate compound described in any one of [1] to [9], wherein a polyester polyol compound represented by Formula (1′) and at least one diisocyanate selected from aliphatic diisocyanates, alicyclic diisocyanates, and aromatic diisocyanates are reacted.[11] The method of producing a polyisocyanate compound according to [10], wherein the reaction is performed in an equivalent ratio of isocyanate groups of the diisocyanate to hydroxyl groups of the polyol compound (isocyanate groups/hydroxyl groups) ranging from 5 to 40 (preferably from 6 to 30 and more preferably from 7 to 20).[12] The method of producing a polyisocyanate compound according to [10] or [11], wherein a number average molecular weight of the polyester polyol compound is from 570 to 2000 (preferably from 580 to 1500, more preferably from 590 to 1200, even more preferably from 590 to 1100, and particularly preferably from 590 to 900).[13] The method of producing a polyisocyanate compound according to any one of [10] to [12], wherein a molecular weight dispersity (weight average molecular weight Mw/number average molecular weight Mn) of the polyester polyol compound is from 1.0 to 3.0.[14] The method of producing a polyisocyanate compound according to any one of [10] to [13], wherein a hydroxyl value (KOH mg/g) of the polyester polyol compound is from 80 to 400 KOH mg/g (preferably from 110 to 350 KOH mg/g, more preferably from 150 to 300 KOH mg/g, even more preferably from 160 to 290 KOH mg/g, and particularly preferably from 180 to 285 KOH mg/g).[15] A composition for forming a polyurethane resin, the composition containing the polyisocyanate compound described in any one of [1] to [9] and a polyacrylic polyol.[16] The composition for forming a polyurethane resin according to [15], wherein an equivalent ratio of a total of isocyanate groups and blocked isocyanate groups of the polyisocyanate compound to hydroxyl groups in the polyacrylic polyol ((isocyanate groups+blocked isocyanate groups)/hydroxyl groups) in the composition is from 0.5 to 2.0 (preferably from 0.5 to 1.5, more preferably from 0.7 to 1.3, and even more preferably from 0.9 to 1.2).[17] The composition for forming a polyurethane resin according to [15] or [16], wherein a content of the polyacrylic polyol in the composition is from 100 to 400 parts by weight (preferably from 150 to 350 parts by weight and more preferably from 200 to 300 parts by weight) per 100 parts by weight of the polyisocyanate compound.[18] The composition for forming a polyurethane resin according to any one of [15] to [17], wherein a hydroxyl value of the polyacrylic polyol is from 30 to 100 KOH mg/g (preferably from 40 to 80 KOH mg/g).[19] The composition for forming a polyurethane resin according to any one of [15] to [18], the composition further containing an inorganic particle and/or an organic particle.[20] The composition for forming a polyurethane resin according to [19], wherein a particle size of the inorganic particle and the organic particle is from 0.01 nm to 1 μm.[21] The composition for forming a polyurethane resin according to any one of [15] to [20], the composition further containing a solvent.[22] The composition for forming a polyurethane resin according to [21], wherein the solvent is an acetate ester (preferably butyl acetate).[23] The composition for forming a polyurethane resin according to [21] or [22], wherein a content of the solvent in the composition is from 5 to 20 parts by weight (preferably from 7 to 17 parts by weight and more preferably from 9 to 15 parts by weight) per 100 parts by weight of a total of the polyisocyanate compound and the polyacrylic polyol.[24] The composition according to any one of [15] to [23], wherein the composition is a coating agent.[25] A cured product of the composition described in any one of [15] to [24].[26] A plastic molded product, wherein a coating composed of the cured product described in [25] covers at least a portion of a surface of the plastic molded product. Industrial Applicability The polyisocyanate compound of the present disclosure can be used as a curing agent in a composition for forming a polyurethane resin, the composition to form a cured product with excellent hardness, scratch resistance, chemical resistance, and flexibility, and using the composition for forming a polyurethane resin of the present disclosure as a coating agent to cover a substrate forms a coating of a cured product, the coating having excellent flexibility and being less likely to peel off, and enables the substrate to have hardness, scratch resistance, and chemical resistance. Thus, the present disclosure has industrial applicability. | 43,092 |
11859045 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, multiple preparation examples are provided to illustrate raw materials used to produce the LCP film of the present application. Multiple examples are further provided to illustrate the implementation of the LCP film and the laminate of the present application, while multiple comparative examples are provided as comparison. A person having ordinary skill in the art can easily realize the advantages and effects of the present application from the following examples and comparative examples. The descriptions proposed herein are just preferable embodiments for the purpose of illustrations only, not intended to limit the scope of the present application. Various modifications and variations could be made in order to practice or apply the present application without departing from the spirit and scope of the present application. (LCP Resin) Preparation Example: LCP Resin A mixture of 6-hydroxy-2-naphthalene carboxylic acid (440 g), 4-hydroxybenzoic acid (1145 g), acetyl anhydride (1085 g), and sodium phosphite (1.3 g) was charged into a 3-liter autoclave and stirred for acetylation at 160° C. for about 2 hours under nitrogen atmosphere at normal pressure. Subsequently, the mixture was heated to 320° C. at a heating rate of 30° C. per hour, and then under this temperature condition, the pressure was reduced slowly from 760 torr to 3 torr or below, and the temperature was increased from 320° C. to 340° C. Afterwards, the stirring power and the pressure were increased, and steps of discharging polymers, drawing strands, and cutting strands into pellets were conducted to obtain an LCP resin having a melting point about 305° C. and a viscosity about 40 Pa·s @320° C. (LCP Film) Examples 1 to 12 and Comparative Examples 1 to 5: LCP Films The LCP resin obtained from Preparation Example was used as raw materials to prepare LCP films of Examples 1 to 12 (E1 to E12) and Comparative Examples 1 to 5 (C1 to C5) by the methods described below. First, the LCP resin was put into an extruder having a screw diameter of 27 millimeters (mm) (manufacturer: Leistritz, model: ZSE27) and heated to a temperature ranging from 300° C. to 330° C., and then extruded from a T-die having a temperature ranging from 300° C. to 330° C. and a width of 500 mm at a feeding speed ranging from 5.5 kilograms per hour (kg/hr) to 9.5 kg/hr. The LCP resin was then delivered to a space between two casting wheels, which were spaced from the T-die by about 5 mm to 40 mm and each had a temperature ranging from about 290° C. to 330° C. and a diameter ranging from about 35 centimeters (cm) to 45 cm, extruded with a force about 20 kilonewtons (kN) to 60 kN, and then transferred to a cooling wheel for cooling at room temperature, so as to obtain an LCP film having a thickness of 50 μm and a melting point about 250° C. to 360° C. The parameters of Examples 1 to 12 and Comparative Examples 1 to 5 are listed in Table 1 below. The processes of Examples 1 to 12 differ from those of Comparative Examples 1 to 5 in the distance from the T-die to the surfaces of the casting wheels, the feeding speed, and the temperature of the extruder. TABLE 1parameters of preparing LCP films of Examples 1to 12 and Comparative Examples 1 to 5Preparation parameterDistance fromthe T-die to thesurfacesFeedingTemperatureSampleof the castingspeedof theNo.wheels (mm)(kg/hr)extruder (° C.)E1106.5305E2107.5310E3108.5315E4205.5300E5206.5305E6207.5310E7208.5315E8208.5320E955.5310E1056.5315E1157.5320E12108.5325C1405.5305C2406.5310C3407.5315C4408.5320C5409.5330 The above-mentioned preparation method of LCP film is only used to exemplify implementation of the present application. A person having ordinary skill in the art may adopt conventional methods such as a laminate extension method, an inflation method, and a solvent casting method to prepare an LCP film. In one of the embodiments, after the LCP resin is extruded from the T-die, the LCP resin may be delivered with two high-temperature resistant films to a space between two casting wheels to form a three-layered laminate based on needs by a person having ordinary skill in the art. The two high-temperature resistant films are detached from the LCP resin at room temperature to obtain the LCP film of the present application. It should be understood that the diameters of the casting wheels are not particularly restricted. The high-temperature resistant films may be selected from, but not limited to, poly(tetrafluoroethene) (PTFE) film, polyimide (PI) film, and poly(ether sulfone) (PES) film. In addition, post treatments for the obtained LCP film may be conducted based on different needs by a person having ordinary skill in the art. The post treatments may be, but are not limited to, polishing, ultraviolet irradiation, heating, plasma, etc. For the plasma treatment, it may be applied with a plasma operated with a power of 1 kilowatt (kW) under nitrogen, oxygen, or air atmosphere at a reduced or normal pressure (1 atm) based on different needs by a person having ordinary skill in the art, but is not limited thereto. Test Example 1: Area Roughness of LCP Films In this test example, the LCP films of Examples 1 to 12 and Comparative Examples 1 to 5 were used as test samples. The area roughness, i.e., Sa and Sz, of either surface of each test sample was measured according to ISO 25178:2012. In order to measure Sa and Sz of each test sample, the surface morphology images of the test samples were taken by using a laser confocal scanning microscope (manufacturer: Olympus, model: LEXT OLS5000-SAF, objective lens: MPLAPON-50×LEXT) with an objective lens having a magnification power of 50×, 1.0× optical zoom, and a 405 nanometers (nm) wavelength of light source at a temperature of 24±3° C. and a relative humidity of 63±3%. Afterwards, Sa and Sz of the test samples were measured with a resolution of 1024 pixels×1024 pixels and a mode of auto tilt removal. According to the said method, the results of Sa and Sz of either surface of each LCP film of E1 to E12 and C1 to C5 are listed in Table 2 below. TABLE 2Sa and Sz of each LCP film of Examples 1to 12 and Comparative Examples 1 to 5Sa (μm)Sz (μm)E10.0984.856E20.0582.347E30.0431.602E40.2897.239E50.1955.609E60.1872.591E70.1791.806E80.1641.334E90.0514.386E100.0281.402E110.0360.921E120.0370.864C10.4415.826C20.4373.186C30.4262.394C40.3961.535C50.3200.893 As shown in Table 2, the Sa of either surface of each LCP film of E1 to E12 was less than 0.32 μm, but the Sa of either surface of each LCP film of C1 to C5 was more than or equal to 0.32 μm. Regarding the LCP films of E1 to E12, in addition to the Sa less than 0.32 μm, the Sz of either surface of each LCP film of E1 to E12 was more than or equal to 0.8 μm. That is, either surface of each LCP film of E1 to E12 had both characteristics of (1) Sa less than 0.32 μm and (2) Sz more than or equal to 0.8 μm. Test Example 2: Comparison Between Ra and Sa Properties of LCP Films To investigate the difference between line roughness (e.g., Ra) and surface roughness (e.g., Sa), the LCP films of E5, E6, and C5 were randomly selected as the test samples from the foresaid examples and comparative examples. According to JIS B 0601:1994, the line roughness of either surface of said LCP films were obtained. Additionally, according to ISO 25178:2012, the area roughness of either surface of said LCP films were obtained. In order to measure Ra of either surface of the LCP films of E5, E6, and C5, the surface morphology images of the test samples were taken by using a laser confocal scanning microscope (manufacturer: Olympus, model: LEXT OLS5000-SAF, objective lens: MPLAPON-50×LEXT) with an objective lens having a magnification power of 50×, 10× optical zoom, and a 405 nm wavelength of light source at a temperature of 24±3° C. and a relative humidity of 63±3%. Afterwards, Ra of the test samples were measured with a selection of an evaluation length of 4 mm and a cutoff value of 0.8 mm. Results are listed in Table 3 below. In the test example, the measurement method of area roughness was the same as Test Example 1. The results are the same as Table 2 above. To investigate the difference between Ra and Sa, in addition to Ra, the Sa of the LCP films of E5, E6, and C5 are listed together in Table 3 below. TABLE 3Ra and Sa of the LCP films of Examples5 to 6, and Comparative Example 5Ra (μm)Sa (μm)E50.0860.195E60.0890.187C50.0960.320 As shown in Table 3 above, the Ra and Sa of the three LCP films were ranked as follows. Ra: E5<E6<C5. Sa: E6<E5<C5. The relationship of increasing order in Ra of multiple LCP films was obviously different from the relationship of increasing order in Sa of multiple LCP films, which showed that Ra and Sa cannot be directly deduced from each other. Further, the difference of Ra or Sa was investigated by relative magnification deduced from maximum value and minimum value of these three LCP films. The Ra of the LCP film of C5 (maximum Ra among these three samples) was close to the Ra of the LCP film of E5 (minimum Ra among these three samples), but the Sa of the LCP film of C5 (maximum Sa among these three samples) was about 1.7 times more than the Sa of the LCP film of E6 (minimum Sa among these three samples). As shown by the results, although the Ra of the LCP films of E5 and C5 were close, the difference of Sa between E5 and C5 was still significant. The results proved that Ra and Sa cannot be directly deduced from each other. Text Example 3: Comparison Between Rz and Sz of LCP Films To investigate the difference between line roughness (e.g., Rz) and area roughness (e.g., Sz), the LCP films of E1 and E12 were randomly selected as the test samples from the foresaid examples. According to JIS B 0601:1994, the line roughness of either surface of said LCP films was obtained. Additionally, according to ISO 25178:2012, the area roughness of either surface of said LCP films was obtained. In order to measure Rz of either surface of the LCP films of E1 and E12, the surface morphology images of the test samples were taken by using a laser confocal scanning microscope (manufacturer: Olympus, model: LEXT OLS5000-SAF, objective lens: MPLAPON-50×LEXT) with an objective lens having a magnification power of 50×, 10× optical zoom, and a 405 nm wavelength of light source at a temperature of 24±3° C. and a relative humidity of 63±3%. Afterwards, Rz of the test samples were measured with a selection of an evaluation length of 4 mm and a cutoff value of 0.8 mm. Results are listed in Table 4 below. In this test example, the measurement method of area roughness was the same as said Test Example 1. The results are the same as Table 2 above. To investigate the difference between Rz and Sz, in addition to Rz, the Sz of the LCP films of E1 and E12 are listed together in Table 4 below. TABLE 4Rz and Sz of the LCP films of Examples 1 and 12Rz (μm)Sz (μm)E11.4064.856E121.8040.864 As shown in Table 4 above, the Rz of the LCP film of E1 was less than the Rz of the LCP film of E12, but the Sz of the LCP film of E1 was more than the Sz of the LCP film of E12, which showed that Rz and Sz cannot be directly deduced from each other. Moreover, either the difference of Ra or the difference of Sa between E1 and E12 was investigated by relative magnification. The Rz of the LCP film of E12 was only about 1.3 times more than the Rz of the LCP film of E1, but the Sz of the LCP film of E1 was about 5.6 times more than the Sz of the LCP film of E12. As shown by the results, although the Rz of the LCP films of E1 and E12 were close, the difference of Sz between E1 and E12 was still significant. The results proved that Rz and Sz cannot be directly deduced from each other. According to Test Examples 2 and 3, line roughness and area roughness were obviously different. A person having ordinary skill in the art cannot expect or deduce area roughness of the LCP film based on line roughness of the LCP film. Specifically, a person having ordinary skill in the art cannot expect or deduce Sa of LCP film based on Ra of the LCP film, Sz of LCP film based on Rz of the LCP film, and vice versa. (Laminate)) Examples 1A to 12A and Comparative Examples 1A to 5A: Laminates Laminates of Examples 1A to 12A (E1A to E12A) and Comparative Examples 1A to 5A (C1A to C5A) were respectively produced from the LCP films of Examples 1 to 12 and Comparative Examples 1 to 5 stacked to the same kind of commercially available copper foil. Specifically, the LCP film having a thickness about 50 μm and two identical copper foils each having a thickness about 12 μm were each first cut to a size of 20 cm×20 cm. The LCP film was then sandwiched between the two commercially available copper foils to form a laminated structure. The laminated structure was subjected to a pressure of 5 kilograms per square centimeter (kg/cm2) for 60 seconds at 180° C., followed by a pressure of 20 kg/cm2for 25 minutes (min) at 300° C., and then cooled to room temperature to obtain a laminate. The LCP films contained in each laminate are listed in Table 5 below. Herein, the lamination method for the laminates is not particularly restricted. A person having ordinary skill in the art may use conventional techniques such as a wire lamination or a surface lamination to conduct the lamination process. A laminator applicable to the present application may be, but is not limited to, an intermittent hot-press machine, a roll-to-roll wheeling machine, a double belt press machine, etc. According to different needs, a person having ordinary skill in the art can also align the LCP film with the copper foils to form a laminated structure, which may then be processed with surface lamination comprising a heating step and a pressing step. In another embodiment, a metal foil, such as a copper foil, on an LCP film may be formed through sputtering, electroplating, chemical plating, evaporation deposition, etc. based on different needs by a person having ordinary skill in the art. Or, a connection layer, such as a glue layer, a nickel layer, a cobalt layer, a chromium layer, or an alloy layer thereof, may be formed between an LCP film and a metal foil based on different needs by a person having ordinary skill in the art. Test Example 4: Insertion Loss of Laminate The laminates of Examples 1A to 12A and Comparative Examples 1A to 5A were each cut to a size of a length about 10 cm, a width about 140 μm, and a resistance about 50 Ohm (Q) as strip line specimens. The insertion loss of the strip line specimens were measured under 10 GHz by a microwave network analyzer (manufacturer: Agilent Technologies, Ltd., model: 8722ES) including a probe (manufacturer: Cascade Microtech, model: ACP40-250). The results of the laminates are listed in Table 5 below. TABLE 5Sa of the LCP films of Examples 1 to 12 and ComparativeExamples 1 to 5 and insertion loss of the laminates of Examples1A to 12A and Comparative Examples 1A to 5A (@ 10 GHz)LCP filmLaminateSampleSampleInsertion lossNo.Sa (μm)No.(dB/10 cm)1E10.098E1A−2.92E20.058E2A−2.93E30.043E3A−2.84E40.289E4A−3.05E50.195E5A−3.06E60.187E6A−3.07E70.179E7A−3.08E80.164E8A−2.99E90.051E9A−2.910E100.028E10A−2.811E110.036E11A−2.912E120.037E12A−2.813C10.441C1A−3.214C20.437C2A−3.115C30.426C3A−3.116C40.396C4A−3.117C50.320C5A−3.1 As shown in Table 5 above, since Sa of either surface of the LCP films of E1 to E12 were less than 0.32 μm, the insertion loss of the laminates (E1A to E12A) comprising said LCP films and commercially available copper foils could be controlled to be less than or equal to −3.0 dB/10 cm. That is, said insertion loss could be more than or equal to −2.8 dB/10 cm and less than or equal to −3.0 dB/10 cm. In contrast, since Sa of either surface of the LCP films of C1 to C5 were more than or equal to 0.32 μm, the insertion loss of the laminates of C1A to C5A were more than or equal to −3.1 dB/10 cm. Especially, Sa of either surface of the LCP film of C1 was 0.441 μm, which made the insertion loss of the laminate of C1A to be −3.2 dB/10 cm. Said laminate could not meet the need of high transmission quality of electronic products for the telecom industry. As shown by the results, controlling the Sa of either surface of the LCP film to be less than 0.32 μm could reduce the insertion loss of a laminate, which improved the performance of the laminate when applied to electronic products. Test Example 5: Peel Strength of Laminates The peel strength of the laminates was measured according to IPC-TM-650 No.: 2.4.9. The laminates of Examples 1A to 12A and Comparative Examples 1A to 5A were each cut to a size of a length about 228.6 mm and a width about 3.2 mm as etched specimens. Each etched specimen was placed at a temperature of 23±2° C. and a relative humidity of 50±5% for 24 hours to reach stabilization. Subsequently, each etched specimen was adhered to a clamp of a testing machine (manufacturer: Hung Ta Instrument Co., Ltd., model: HT-9102) with a double faced adhesive tape. Each etched specimen was then peeled from the clamp with a force at a peel speed of 50.8 mm/min, and the value of the force during the peeling process was continuously recorded. Herein, the force should be controlled within a range of 15% to 85% of the bearable force of the testing machine, the peeling distance from the clamp should be at least more than 57.2 mm, and the force for the initial distance of 6.4 mm was neglected and not recorded. The results are shown in Table 6. TABLE 6Sa and Sz of the LCP films of Examples 1 to 12 andComparative Examples 1 to 5 as well as the insertionloss and peel strength of the laminates of Examples1A to 12A and Comparative Examples 1A to 5 ALCP filmLaminateSampleSaSzSampleInsertion lossPeel strengthNo.(μm)(μm)No.(dB/10 cm)(kN/m)1E10.0984.856E1A−2.91.262E20.0582.347E2A−2.91.183E30.0431.602E3A−2.81.134E40.2897.239E4A−3.01.285E50.1955.609E5A−3.01.236E60.1872.591E6A−3.01.187E70.1791.806E7A−3.01.128E80.1641.334E8A−2.91.089E90.0514.386E9A−2.91.2310E100.0281.402E10A−2.81.1411E110.0360.921E11A−2.91.0612E120.0370.864E12A−2.80.9813C10.4415.826C1A−3.21.2414C20.4373.186C2A−3.11.1915C30.4262.394C3A−3.11.1816C40.3961.535C4A−3.11.1417C50.3200.893C5A−3.10.96 As shown in Table 6 above, the difference of the performance of the laminates of E1A to E11A between the performance of the laminates of E12A and C1A to C5A was obvious. Since either surface of the LCP films of E1 to E11 had both characteristics of (1) Sa less than 0.32 μm, and (2) Sz more than or equal to 0.9 μm, the laminates comprising copper foils and the LCP films of E1 to E11 had the advantages of low insertion loss (less than or equal to −3.0 dB/10 cm) and improved peel strength between an LCP film and a copper foil more than or equal to 1.0 kN/m. Accordingly, in addition to Sa of the LCP film, proper Sz of the LCP film could further optimize the lamination of the LCP film and copper foil, and thus the laminate exhibiting both high peel strength and low insertion loss could be obtained. In summary, by controlling the Sa of the first surface of the LCP film to be less than 0.32 μm, the insertion loss of the laminate comprising said LCP film can be reduced or suppressed. Therefore, the laminate of the present application is applicable to advanced high frequency products. | 19,350 |
11859046 | DETAILED DESCRIPTION OF THE INVENTION The polymer which is a subject of the invention is a thermoplastic polyester comprising:at least one 1,4:3,6-dianhydrohexitol unit (A);at least one alicyclic diol unit (B) other than the 1,4:3,6-dianhydrohexitol units (A);at least one terephthalic acid unit (C). The polyester according to the invention does not contain any aliphatic non-cyclic diol units, or comprises a small amount thereof. “Low molar amount of aliphatic non-cyclic diol units” is intended to mean, especially, a molar amount of aliphatic non-cyclic diol units of less than 5%. According to the invention, this molar amount represents the ratio of the sum of the aliphatic non-cyclic diol units, these units possibly being identical or different, relative to all the monomer units of the polyester. An aliphatic non-cyclic diol may be a linear or branched aliphatic non-cyclic diol. It may also be a saturated or unsaturated aliphatic non-cyclic diol. Aside from ethylene glycol, the saturated linear aliphatic non-cyclic diol may for example be 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, 1,8-octanediol and/or 1,10-decanediol. As examples of saturated branched aliphatic non-cyclic diol, mention may be made of 2-methyl-1,3-propanediol, 2,2,4-trimethyl-1,3-pentanediol, 2-ethyl-2-butyl-1,3-propanediol, propylene glycol and/or neopentyl glycol. As an example of an unsaturated aliphatic diol, mention may be made, for example, of cis-2-butene-1,4-diol. This molar amount of aliphatic non-cyclic diol units is advantageously less than 1%. Preferably, the polyester does not contain any aliphatic non-cyclic diol units. Despite the small amount of aliphatic non-cyclic diol, and hence ethylene glycol, used for producing the polyester, the polyester has a high reduced viscosity in solution. This high reduced viscosity in solution enables the polyester to be able to be used in numerous applications described below. This reduced viscosity in solution may be greater than 50 ml/g, this viscosity being able to be measured using an Ubbelohde capillary viscosimeter at 25° C. in an equi-mass mixture of phenol and ortho-dichlorobenzene after dissolving the polymer at 130° C. with stirring, the concentration of polymer introduced being 5 g/l. This test for measuring reduced viscosity in solution is, due to the choice of solvents and the concentration of the polymers used, perfectly suited to determining the viscosity of the viscous polymer of the present invention. According to the present invention, a polyester of reduced viscosity in solution of greater than 50 ml/g and extending up to 70 ml/g is considered to be a “high-viscosity polyester”. The Applicant has also succeeded in obtaining a polyester having an even higher viscosity, hereinafter referred to as “very high-viscosity polyester”. “Very high-viscosity polyester” is intended to mean, according to the invention, a polyester having a reduced viscosity in solution of greater than 70 ml/g, advantageously greater than 75 ml/g, preferably greater than 85 ml/g, most preferentially greater than 95 ml/g. In the case where the polyester according to the invention is a very high-viscosity polyester, it has excellent impact strength properties at room temperature but also good cold impact strength properties. Since this polyester can be used and mechanically stressed at low temperature, this enables it to be used in numerous applications, in various industries such as, for example, the automotive or household appliance industries. The monomer (A) is a 1,4:3,6-dianhydrohexitol. As explained previously, 1,4:3,6-dianhydrohexitols have the drawback of being secondary diols which are not very reactive in the production of polyesters. The 1,4:3,6-dianhydrohexitol (A) may be isosorbide, isomannide, isoidide, or a mixture thereof. Preferably, the 1,4:3,6-dianhydrohexitol (A) is isosorbide. Isosorbide, isomannide and isoidide may be obtained, respectively, by dehydration of sorbitol, of mannitol and of iditol. As regards isosorbide, it is sold by the Applicant under the brand name Polysorb® P. The alicyclic diol (B) is also referred to as aliphatic cyclic diol. It is a diol which may especially be chosen from 1,4-cyclohexanedimethanol, 1,2-cyclohexanedimethanol, 1,3-cyclohexanedimethanol or a mixture of these diols. The alicyclic diol (B) is very preferentially 1,4-cyclohexanedimethanol. The alicyclic diol (B) may be in the cis configuration, in the trans configuration, or may be a mixture of diols in the cis and trans configurations. The polyester of the invention may for example comprise:a molar amount of 1,4:3,6-dianhydrohexitol units (A) ranging from 1 to 54%;a molar amount of alicyclic diol units (B) other than the 1,4:3,6-dianhydrohexitol units (A) ranging from 1 to 54%;a molar amount of terephthalic acid units (C) ranging from 45 to 55%. The amounts of different units in the polyester may be determined by1H NMR or by chromatographic analysis of the mixture of monomers resulting from complete hydrolysis or methanolysis of the polyester, preferably by1H NMR. Those skilled in the art can easily find the analysis conditions for determining the amounts of each of the units of the polyester. For example, from an NMR spectrum of a poly(1,4-cyclohexanedimethylene-co-isosorbide terephthalate), the chemical shifts relating to the 1,4-cyclohexanedimethanol are between 0.9 and 2.4 ppm and 4.0 and 4.5 ppm, the chemical shifts relating to the terephthalate ring are between 7.8 and 8.4 ppm and the chemical shifts relating to the isosorbide are between 4.1 and 5.8 ppm. The integration of each signal makes it possible to determine the amount of each unit of the polyester. The polyester according to the invention may be semi-crystalline or amorphous. The semi-crystalline character of the polymer depends primarily on the amounts of each of the units in the polymer. Thus, when the polymer according to the invention comprises large amounts of 1,4:3,6-dianhydrohexitol units (A), the polymer is generally amorphous, whereas it is generally semi-crystalline in the opposite case. Preferably, the polyester according to the invention has a glass transition temperature ranging from 85 to 200° C. According to one advantageous embodiment, the polyester according to the invention comprises:a molar amount of 1,4:3,6-dianhydrohexitol units (A) ranging from 1 to 20%, advantageously from 5 to 15%;a molar amount of alicyclic diol units (B) other than the 1,4:3,6-dianhydrohexitol units (A) ranging from 25 to 54%, advantageously from 30 to 50%;a molar amount of terephthalic acid units (C) ranging from 45 to 55%. According to this advantageous embodiment, the polyester is generally semi-crystalline. The Applicant has succeeded in obtaining polyesters having semi-crystalline properties, even when the molar amount of 1,4:3,6-dianhydrohexitols reaches 20%. This polyester surprisingly exhibits excellent impact strength properties. Moreover, the crystallization rate of these novel polyesters is greater than that of PEITs and PEICTs, which makes it possible to transform them into articles having improved application properties. In particular, this semi-crystalline polyester has a particularly high thermomechanical strength, due to its high glass transition temperature and the presence of crystallinity which reinforces the mechanical properties at high temperature. Preferably, when the polyester according to the invention is semi-crystalline, it has a melting point ranging from 210 to 295° C., for example from 240 to 285° C. Preferably, when the polyester according to the invention is semi-crystalline, it has a glass transition temperature ranging from 85 to 140° C., for example from 90 to 115° C. The glass transition temperatures and melting points are measured by conventional methods, especially using differential scanning calorimetry (DSC) using a heating rate of 10° C./min. The experimental protocol is described in detail in the example section below. Advantageously, when the polyester according to the invention is semi-crystalline, it has a heat of fusion of greater than 10 J/g, preferably greater than 30 J/g, the measurement of this heat of fusion consisting in subjecting a sample of this polyester to a heat treatment at 170° C. for 10 hours, then in evaluating the heat of fusion by DSC by heating the sample at 10° C./min. According to another embodiment of the invention, the polyester comprises:a molar amount of 1,4:3,6-dianhydrohexitol units (A) ranging from 20 to 54%;a molar amount of alicyclic diol units (B) other than the 1,4:3,6-dianhydrohexitol units (A) ranging from 1 to 35%;a molar amount of terephthalic acid units (C) ranging from 45 to 55%. According to this other embodiment, the polymer is generally amorphous. Preferably, when the polyester according to the invention is amorphous, it has a glass transition temperature ranging from 120 to 200° C., for example from 140 to 190° C. The polyester according to the invention may have low coloration and especially have a lightness L* greater than 50. Advantageously, the lightness L* is greater than 55, preferably greater than 60, most preferentially greater than 65, for example greater than 70. The parameters L* may be determined using a spectrophotometer, via the CIE Lab model. The polyester according to the invention, especially that of very high viscosity, has a very good impact strength, in particular a very good cold impact strength. The polyester according to the invention, especially that of very high viscosity, advantageously has a non-notched Charpy impact strength greater than 100 kJ/m2(25° C., ISO 179-1/1eU: 2010). The polyester according to the invention, especially that of very high viscosity, advantageously has a notched Charpy impact strength greater than 5 kJ/m2, advantageously greater than 10 kJ/m2(−30° C., ISO 179-1/1eA: 2010). These very high impact strength properties were able to be obtained even when the polyester according to the invention is semi-crystalline. This goes against the teaching of application US2012/0177854, which teaches decreasing the crystallinity of the polyester with a view to improving the impact strength properties thereof. Another subject of the invention is a process for producing the polyester according to the invention. According to a first variant of the process of the invention, the Applicant has succeeded in obtaining a polyester, which may have a high reduced viscosity in solution, by a production process comprising:a step of introducing, into a reactor, monomers comprising at least one 1,4:3,6-dianhydrohexitol (A), at least one alicyclic diol (B) other than the 1,4:3,6-dianhydrohexitols (A) and at least one terephthalic acid (C), the molar ratio ((A)+(B))/(C) ranging from 1.05 to 1.5, said monomers not containing any aliphatic non-cyclic diols or comprising, relative to all of the monomers introduced, a molar amount of aliphatic non-cyclic diol units of less than 5%;a step of introducing, into the reactor, a catalytic system;a step of polymerizing said monomers to form the polyester, said step consisting of:a first stage of oligomerization, during which the reaction medium is stirred under an inert atmosphere at a temperature ranging from 265 to 280° C., advantageously from 270 to 280° C., for example 275° C.;a second stage of condensation of the oligomers, during which the oligomers formed are stirred under vacuum, at a temperature ranging from 278 to 300° C. so as to form the polyester, advantageously from 280 to 290° C., for example 285° C.;a step of recovering the polyester. Thus, contrary to that which was expected due to the use of high temperatures during the stages of oligomerization and condensation of the oligomers, it is entirely possible, using the first variant of the process according to the invention, to obtain high-viscosity polyesters with low coloration. Without being bound by any theory, the Applicant explains this low coloration by the fact that it is only when large amounts of aliphatic non-cyclic diol are used in combination with 1,4:3,6-dianhydrohexitol that the latter degrades in the reactor during polymerization. Quite unexpectedly, according to the process of the invention which uses small molar amounts of aliphatic non-cyclic diol (less than 5%), or even does not use this monomer, it is entirely possible to obtain polymers simultaneously exhibiting high viscosity and low coloration. The polymer obtained may thus at least have a reduced viscosity in solution of greater than 50 ml/g. This first stage of this variant of the process is carried out in an inert atmosphere, that is to say under an atmosphere of at least one inert gas. This inert gas may especially be dinitrogen. This first stage can be carried out under a gas stream. It can also be carried out under pressure, for example at a pressure of between 1.05 and 8 bar. Preferably, the pressure ranges from 3 to 8 bar, most preferentially from 5 to 7.5 bar, for example 6.6 bar. Under these preferred pressure conditions, the reaction of all the monomers with one another is promoted by limiting the loss of monomers during this stage. Prior to the first stage of oligomerization, a step of deoxygenation of the monomers is preferentially carried out. It can be carried out for example by generating a vacuum after having introduced the monomers into the reactor and then by introducing an inert gas such as nitrogen into the reactor. This vacuum-inert gas introduction cycle can be repeated several times, for example from 3 to 5 times. Preferably, this vacuum-nitrogen cycle is carried out at a temperature of between 60 and 80° C. so that the reagents, and especially the diols, are totally molten. This deoxygenation step has the advantage of improving the coloration properties of the polyester obtained at the end of the process. The second stage of condensation of the oligomers is carried out under vacuum. The pressure may decrease continuously during this second stage by using pressure decrease ramps, in steps, or else using a combination of pressure decrease ramps and steps. Preferably, at the end of this second stage, the pressure is less than 10 mbar, most preferentially less than 1 mbar. According to this first variant of the process, the first stage of the polymerization step preferably has a duration ranging from 20 minutes to 5 hours. Advantageously, the second stage has a duration ranging from 30 minutes to 6 hours, the beginning of this stage being the moment at which the reactor is placed under vacuum, that is to say at a pressure of less than 1 bar. The process of this first variant comprises a step of introducing a catalytic system into the reactor. This step may take place beforehand or during the polymerization step described above. Catalytic system is intended to mean a catalyst or a mixture of catalysts, optionally dispersed or fixed on an inert support. The catalyst is used in amounts suitable for obtaining a high-viscosity polymer in accordance with the invention. An esterification catalyst is advantageously used during the oligomerization stage. This esterification catalyst can be chosen from tin derivatives, titanium derivatives, zirconium derivatives, hafnium derivatives, zinc derivatives, manganese derivatives, calcium derivatives and strontium derivatives, organic catalysts such as para-toluenesulfonic acid (PTSA) or methanesulfonic acid (MSA), or a mixture of these catalysts. By way of example of such compounds, mention may be made of those given in application US 2011282020A1 in paragraphs [0026] to [0029], and on page 5 of application WO 2013/062408 A1. Preferably, a titanium derivative, a zinc derivative or a manganese derivative is used during the first stage of transesterification. By way of example of amounts by weight, use may be made of from 10 to 500 ppm of catalytic system during the oligomerization stage, relative to the amount of monomers introduced. At the end of transesterification, the catalyst from the first step can be optionally blocked by adding phosphorous acid or phosphoric acid, or else, as in the case of tin(IV), reduced with phosphites such as triphenyl phosphite or tris(nonylphenyl)phosphites or those cited in paragraph [0034] of application US 2011 282020A1. The second stage of condensation of the oligomers may optionally be carried out with the addition of a catalyst. This catalyst is advantageously chosen from tin derivatives, preferentially derivatives of tin, titanium, zirconium, germanium, antimony, bismuth, hafnium, magnesium, cerium, zinc, cobalt, iron, manganese, calcium, strontium, sodium, potassium, aluminum or lithium, or of a mixture of these catalysts. Examples of such compounds may for example be those given in patent EP 1 882 712 B1 in paragraphs [0090] to [0094]. Preferably, the catalyst is a tin, titanium, germanium, aluminum or antimony derivative. By way of example of amounts by weight, use may be made of from 10 to 500 ppm of catalytic system during the stage of condensation of the oligomers, relative to the amount of monomers introduced. Preferably, a catalytic system is used during the first stage and the second stage of polymerization. Said system advantageously consists of a catalyst based on tin or of a mixture of catalysts based on tin, titanium, germanium and aluminum. By way of example, use may be made of an amount by weight of 10 to 500 ppm of catalytic system relative to the amount of monomers introduced. According to the process of the first variant, an antioxidant is advantageously used during the step of polymerization of the monomers. These antioxidants make it possible to reduce the coloration of the polyester obtained. The antioxidants may be primary and/or secondary antioxidants. The primary antioxidant may be a sterically hindered phenol, such as the compounds Hostanox® 0 3, Hostanox® 0 10, Hostanox® 0 16, Ultranox® 210, Ultranox® 276, Dovernox® 10, Dovernox® 76, Dovernox® 3114, Irganox® 1010 or Irganox® 1076 or a phosphonate such as Irgamod® 195. The secondary antioxidant may be trivalent phosphorus compounds such as Ultranox® 626, Doverphos® S-9228, Hostanox® P-EPQ or Irgafos 168. It is also possible to introduce as polymerization additive into the reactor at least one compound that is capable of limiting spurious etherification reactions, such as sodium acetate, tetramethylammonium hydroxide or tetraethylammonium hydroxide. The process of the first variant comprises a step of recovering the polyester at the end of the polymerization step. The polyester can be recovered by extracting it from the reactor in the form of a molten polymer rod. This rod can be converted into granules using conventional granulation techniques. The polyester thus recovered has a reduced viscosity in solution of greater than 50 ml/g and generally less than 70 ml/g. According to a second variant of the process of the invention, the process for producing the polyester comprises a step of increasing the molar mass by post-polymerization of a polymer of lower reduced viscosity in solution, which comprises at least one 1,4:3,6-dianhydrohexitol unit (A), at least one alicyclic diol unit (B) other than the 1,4:3,6-dianhydrohexitol units (A) and at least one terephthalic acid unit (C), said polymer of lower reduced viscosity in solution not containing any aliphatic non-cyclic diol units or comprising a molar amount of aliphatic non-cyclic diol units, relative to all of the monomer units of the polymer, of less than 5%. According to this second advantageous variant of the invention, it is possible to obtain a polyester having a particularly high reduced viscosity in solution, for example greater than 70 ml/g. “Polymer of lower reduced viscosity in solution” is intended to mean a polyester having a reduced viscosity in solution which is lower than that of the polyester obtained at the end of the post-polymerization step. This polymer can be obtained according to the methods described in the documents US2012/0177854 and Yoon et al., using production processes using diols and diesters of terephthalic acid as monomers, or using the process of the first variant described above. The post-polymerization step may consist of a step of solid-state polycondensation (SSP) of the polymer of lower reduced viscosity in solution or of a step of reactive extrusion of the polymer of lower reduced viscosity in solution in the presence of at least one chain extender. According to a first particularly preferred embodiment of this second variant of the process, this post-polymerization step is carried out by SSP. SSP is generally carried out at a temperature between the glass transition temperature and the melting point of the polymer. Thus, in order to carry out the SSP, it is necessary that the polymer of lower reduced viscosity in solution is semi-crystalline. Preferably the latter has a heat of fusion of greater than 10 J/g, preferably greater than 30 J/g, the measurement of this heat of fusion consisting in subjecting a sample of this polymer of lower reduced viscosity in solution to a heat treatment at 170° C. for 10 hours, then in evaluating the heat of fusion by DSC by heating the sample at 10 K/min. Preferably, the polymer of lower reduced viscosity in solution comprises:a molar amount of 1,4:3,6-dianhydrohexitol units (A) ranging from 1 to 20%, advantageously from 5 to 15%;a molar amount of alicyclic diol units (B) other than the 1,4:3,6-dianhydrohexitol units (A) ranging from 25 to 54%, advantageously from 30 to 50%;a molar amount of terephthalic acid units (C) ranging from 45 to 55%. Advantageously, according to this embodiment of the process, the SSP step is carried out at a temperature ranging from 190 to 300° C., preferably from 200 to 280° C. The SSP step may be carried out in an inert atmosphere, for example under nitrogen or under argon or under vacuum. According to a second embodiment of the process of the invention, the post-polymerization step is carried out by reactive extrusion of the polymer of lower reduced viscosity in solution in the presence of at least one chain extender. The chain extender is a compound comprising two functions capable of reacting, in reactive extrusion, with alcohol, carboxylic acid and/or carboxylic acid ester functions of the polymer of lower reduced viscosity in solution. The chain extender may, for example, be chosen from compounds comprising two isocyanate, isocyanurate, lactam, lactone, carbonate, epoxy, oxazoline and imide functions, it being possible for said functions to be identical or different. The reactive extrusion may be carried out in an extruder of any type, especially a single-screw extruder, a co-rotating twin-screw extruder or a counter-rotating twin-screw extruder. However, it is preferred to carry out this reactive extrusion using a co-rotating extruder. The reactive extrusion step may be carried out by:introducing the polymer of lower reduced viscosity in solution into the extruder so as to melt said polymer;then introducing the chain extender into the molten polymer;then reacting the polymer with the chain extender in the extruder;then recovering the polyester obtained in the extrusion step. During extrusion, the temperature inside the extruder is regulated so as to be at a temperature above the glass transition temperature if the polymer is amorphous and above the melting point if the polymer is semi-crystalline. The temperature inside the extruder may range from 150 to 320° C. The invention also relates to the polyester able to be obtained by the process of the invention. The invention also relates to a composition comprising the polyester according to the invention, this composition possibly comprising at least one additive or at least one additional polymer or at least one mixture thereof. The polyester composition according to the invention may comprise the polymerization additives optionally used during the process. It may also comprise other additives and/or additional polymers that are generally added during a subsequent thermomechanical mixing step. By way of examples of additives, mention may be made of fillers or fibers of organic or mineral, nanometric or non-nanometric, functionalized or non-functionalized nature. They may be silicas, zeolites, glass fibers or beads, clays, mica, titanates, silicates, graphite, calcium carbonate, carbon nanotubes, wood fibers, carbon fibers, polymer fibers, proteins, cellulose-based fibers, lignocellulosic fibers and non-destructured granular starch. These fillers or fibers can make it possible to improve the hardness, the rigidity or the water- or gas-permeability. The composition may comprise from 0.1% to 75% by weight of fillers and/or fibers relative to the total weight of the composition, for example from 0.5% to 50%. The additive that is of use in the composition according to the invention may also comprise opacifiers, dyes and pigments. They may be chosen from cobalt acetate and the following compounds: HS-325 Sandoplast® Red BB (which is a compound bearing an azo function, also known under the name Solvent Red 195), HS-510 Sandoplast® Blue 2B which is an anthraquinone, Polysynthren® Blue R, and Clariant® RSB Violet. The composition may also comprise, as additive, a processing aid, for reducing the pressure in the processing tool. A demolding agent which makes it possible to reduce the adhesion to the materials for forming the polyester, such as the molds or the calendering rollers, may also be used. These aids may be chosen from fatty acid esters and fatty acid amides, metal salts, soaps, paraffins and hydrocarbon-based waxes. Particular examples of these agents are zinc stearate, calcium stearate, aluminum stearate, stearamides, erucamides, behenamides, beeswaxes or candelilla wax. The composition according to the invention may also comprise other additives, such as stabilizers, for example light stabilizers, UV stabilizers and heat stabilizers, fluidizers, flame retardants and antistatic agents. The composition may also comprise an additional polymer other than the polyester according to the invention. This polymer may be chosen from polyamides, polyesters other than the polyester according to the invention, polystyrene, styrene copolymers, styrene-acrylonitrile copolymers, styrene-acrylonitrile-butadiene copolymers, poly(methyl methacrylate)s, acrylic copolymers, poly(ether-imide)s, poly(phenylene oxide)s, such as poly(2,6-dimethylphenylene oxide), poly(phenylene sulfate)s, poly(ester-carbonate)s, polycarbonates, polysulfones, polysulfone ethers, polyether ketones, and mixtures of these polymers. The composition may also comprise, as additional polymer, a polymer which makes it possible to improve the impact properties of the polymer, especially functional polyolefins such as functionalized ethylene or propylene polymers and copolymers, core-shell copolymers or block copolymers. The composition according to the invention may also comprise polymers of natural origin, such as starch, cellulose, chitosans, alginates, proteins such as gluten, pea proteins, casein, collagen, gelatin or lignin, these polymers of natural origin possibly being physically or chemically modified. The starch may be used in destructured or plasticized form. In the latter case, the plasticizer may be water or a polyol, especially glycerol, polyglycerol, isosorbide, sorbitans, sorbitol, mannitol or else urea. The process described in document WO 2010/010 282 A1 may especially be used to prepare the composition. The composition according to the invention may be produced by conventional thermoplastics mixing methods. These conventional methods comprise at least one step of mixing the polymers in the molten or softened state and a step of recovering the composition. This process may be performed in paddle or rotor internal mixers, external mixers, or single-screw or twin-screw co-rotating or counter-rotating extruders. However, it is preferred to produce this mixture by extrusion, especially using a co-rotating extruder. The mixing of the constituents of the composition may take place under an inert atmosphere. In the case of an extruder, the various constituents of the composition may be introduced by means of feed hoppers located along the extruder. The invention also relates to a plastic, finished or semi-finished article comprising the polyester or the composition according to the invention. This article may be of any type and may be obtained using conventional transformation techniques. It may be, for example, fibers or threads that are of use in the textile industry or other industries. These fibers or threads may be woven so as to form fabrics, or else nonwovens. The article according to the invention may also be a film or a sheet. These films or sheets may be manufactured by the techniques of calendering, extrusion film cast, extrusion film blowing, followed or not by monoaxial or polyaxial stretching or orientation techniques. These sheets may be thermoformed or injected to be used, for example, for parts such as the viewing windows or covers for machines, the body of various electronic devices (telephones, computers, screens) or else as impact-resistant windows. The article may also be transformed also be processed by extrusion of profiled elements which may have applications in the building and construction sectors. The article according to the invention may also be a container for transporting gases, liquids and/or solids. The containers concerned may be baby bottles, flasks, bottles, for example sparkling or still water bottles, juice bottles, soda bottles, carboys, alcoholic drink bottles, small bottles, for example small medicine bottles, small bottles for cosmetic products, these small bottles possibly being aerosols, dishes, for example for ready meals, microwave dishes, or else lids. These containers may be of any size. They may be produced by extrusion blow molding, thermoforming or injection blow molding. These articles may also be optical articles, i.e. articles requiring good optical properties, such as lenses, disks, transparent or translucent panels, light-emitting diode (LED) components, optical fibers, films for LCD screens or else windows. These optical articles have the advantage of being able to be placed close to light sources and therefore to heat sources, while retaining excellent dimensional stability and good resistance to light. Among the applications of the article, mention may also be made of parts with a protective aim where impact strength is important, such as cell phone protectors, spherical packaging, but also, in the automotive sector, bumpers and dashboard elements. The articles may also be multilayer articles, at least one layer of which comprises the polymer or the composition according to the invention. These articles may be produced via a process comprising a coextrusion step in the case where the materials of the various layers are placed in contact in the molten state. By way of example, mention may be made of the techniques of tube coextrusion, profile coextrusion, coextrusion blow molding of a bottle, a small bottle or a tank, generally collated under the term “coextrusion blow molding of hollow bodies”, coextrusion blow molding, also known as film blowing, and cast coextrusion. They may also be produced according to a process comprising a step of applying a layer of molten polyester onto a layer based on organic polymer, metal or adhesive composition in the solid state. This step may be performed by pressing, by overmolding, stratification or lamination, extrusion-lamination, coating, extrusion-coating or spreading. The invention will now be illustrated in the examples below. It is specified that these examples do not in any way limit the present invention. EXAMPLES The properties of the polymers were studied via the following techniques: The reduced viscosity in solution is evaluated using an Ubbelohde capillary viscometer at 25° C. in an equi-mass mixture of phenol and ortho-dichlorobenzene after dissolving the polymer at 130° C. with magnetic stirring. For these measurements, the polymer concentration introduced is 5 g/l. The color of the polymer was measured on the granules (25 grams of granules in the measuring cell) using a Konica Minolta CM-2300d spectrophotometer. The mechanical properties of the polymers were evaluated according to the following standards: Bending test: ISO 178 Tensile test: ISO 527 Charpy impact test: ISO 179-1: 2010 (non-notched: ISO 179-1/1eU, notched: EN ISO 179-1/1eA) The impact strengths were determined as follows:in a first step, the test according to standard ISO 179-1 1eU is carried out at 25° C.;if, during this first test, the strength is greater than 155 kJ/m2, the ISO 179-1/1eA test is carried out at 25° C.;if, during this second test, the strength is greater than 155 kJ/m2, the ISO 179-1/1eA test is carried out at −30° C. HDT test, Method B, stress 0.45 MPa ISO 75 Vicat Method B50 ISO 306 DSC The thermal properties of the polyesters were measured by differential scanning calorimetry (DSC): The sample is first heated under a nitrogen atmosphere in an open crucible from 10° C. to 320° C. (10° C. min-1), cooled to 10° C. (10° C. min-1), then heated again to 320° C. under the same conditions as the first step. The glass transition temperatures were taken at the mid-point of the second heating. Any melting points are determined on the endothermic peak (onset) at the first heating. Similarly, the enthalpy of fusion (area under the curve) is determined at the first heating. For the illustrative examples presented below, the following reagents were used: Ethylene glycol (purity>99.8%) from Sigma-Aldrich 1,4-Cyclohexanedimethanol (99% purity, mixture of cis and trans isomers) Isosorbide (purity>99.5%) Polysorb® P from Roquette Freres Terephthalic acid (99+% purity) from Acros Germanium dioxide (>99.99%) from Sigma-Aldrich Irgamod 1010 from BASF AG Dibutyltin oxide (98% purity) from Sigma-Aldrich Carbonylbiscaprolactam (Allinco CBC) from DSM. Tritan TX2001: performance copolyester sold by Eastman® Preparation of the Polyesters Example 1 1680 g (11.6 mol) of 1,4-cyclohexanedimethanol, 233 g (1.6 mol) of isosorbide, 2000 g (12.0 mol) of terephthalic acid, 1.65 g of Irganox 1010 (antioxidant) and 1.39 g of dibutyltin oxide (catalyst) are added to a 7.5 I reactor. To extract the residual oxygen from the isosorbide crystals, four vacuum-nitrogen cycles are performed once the temperature of the reaction medium is between 60 and 80° C. The reaction mixture is then heated to 275° C. (4° C./min) under 6.6 bar of pressure and with constant stirring (150 rpm). The degree of esterification is estimated from the amount of distillate collected. The pressure is then reduced to 0.7 mbar over 90 minutes following a logarithmic ramp and the temperature is brought to 285° C. These vacuum and temperature conditions were maintained until an increase in torque of 15 Nm relative to the initial torque was obtained. Finally, a polymer rod is cast via the bottom valve of the reactor, cooled to 15° C. in a heat-regulated water bath and chopped in the form of granules of about 15 mg. The resin thus obtained has a reduced viscosity in solution of 69.9 ml/g-1.1H NMR analysis of the polyester shows that the final polyester contains 3.2 mol % of isosorbide relative to all the monomer units. With regard to the thermal properties (measured at the second heating), the polymer has a glass transition temperature of 91° C., a melting point of 276° C. with an enthalpy of fusion of 44.5 J/g. The mechanical properties of the polymer obtained are summarized in Table 1. The lightness L * is 53.2. Example 1a The polyester from Example 1 is used in a solid-state post-condensation step. First, the polymer is crystallized for 2 h in an oven under vacuum at 170° C. The crystallized polymer is then introduced into an oil bath rotavap fitted with a cannulated flask. The granules are then subjected to a temperature of 248° C. and a nitrogen flow of 3.3 l/min. After 23 h the polymer reaches a reduced viscosity in solution of 106.5 ml/g. Finally, after 54 h of post-condensation, the polymer will have a viscosity in solution of 121.3 ml/g. The mechanical properties of the polymer obtained are summarized in Table 1. Example 1 b According to another process according to the invention, the polymer of Ex. 1 was extruded in a DSM twin-screw microextruder in the presence of 1 w % carbonylbiscaprolactam (Allinco CBC). The extrusion was carried out on 12 g of polymer for 2 min at 300° C. The polymer has a viscosity in solution of 85.5 ml/g. Example 2 1432 g (9.9 mol) of 1,4-cyclohexanedimethanol, 484 g (3.3 mol) of isosorbide, 2000 g (12.0 mol) of terephthalic acid, 1.65 g of Irganox 1010 (antioxidant) and 1.39 g of dibutyltin oxide (catalyst) are added to a 7.5 I reactor. To extract the residual oxygen from the isosorbide crystals, four vacuum-nitrogen cycles are performed once the temperature of the reaction medium is between 60 and 80° C. The reaction mixture is then heated to 275° C. (4° C./min) under 6.6 bar of pressure and with constant stirring (150 rpm). The degree of esterification is estimated from the amount of distillate collected. The pressure is then reduced to 0.7 mbar over 90 minutes following a logarithmic ramp and the temperature is brought to 285° C. These vacuum and temperature conditions were maintained until an increase in torque of 12.1 Nm relative to the initial torque was obtained. Finally, a polymer rod is cast via the bottom valve of the reactor, cooled to 15° C. in a heat-regulated water bath and chopped in the form of granules of about 15 mg. The resin thus obtained has a reduced viscosity in solution of 80.1 ml/g-1.1H NMR analysis of the polyester shows that the final polyester contains 8.5 mol % of isosorbide relative to all the monomer units. With regard to the thermal properties, the polymer has a glass transition temperature of 96° C., a melting point of 253° C. with an enthalpy of fusion of 23.2 J/g. The lightness L * is 55.3. Example 2a The polyester from Example 2 is used in a solid-state post-condensation step. First, the polymer is crystallized for 2 h in an oven under vacuum at 170° C. The crystallized polymer is then introduced into an oil bath rotavap fitted with a cannulated flask. The granules are then subjected to a temperature of 230° C. and a nitrogen flow of 3.3 l/min. After 31 h of post-condensation, the polymer will have a viscosity in solution of 118.3 ml/g. The mechanical properties of the polymer obtained are summarized in Table 1. Example 2b According to another process according to the invention, the polymer of Ex. 2 was extruded in a DSM twin-screw microextruder in the presence of 1 w % carbonylbiscaprolactam (Allinco CBC). The extrusion was carried out on 12 g of polymer for 2 min at 300° C. The polymer has a viscosity in solution of 92.8 ml/g. Example 3 1194 g (8.3 mol) of 1,4-cyclohexanedimethanol, 726 g (5.0 mol) of isosorbide, 2000 g (12.0 mol) of terephthalic acid, 1.65 g of Irganox 1010 (antioxidant) and 1.39 g of dibutyltin oxide (catalyst) are added to a 7.5 I reactor. To extract the residual oxygen from the isosorbide crystals, four vacuum-nitrogen cycles are performed once the temperature of the reaction medium is between 60 and 80° C. The reaction mixture is then heated to 275° C. (4° C./min) under 6.6 bar of pressure and with constant stirring (150 rpm). The degree of esterification is estimated from the amount of distillate collected. The pressure is then reduced to 0.7 mbar over 90 minutes following a logarithmic ramp and the temperature is brought to 285° C. These vacuum and temperature conditions were maintained until an increase in torque of 11.1 Nm relative to the initial torque was obtained. Finally, a polymer rod is cast via the bottom valve of the reactor, cooled to 15° C. in a heat-regulated water bath and chopped in the form of granules of about 15 mg. The resin thus obtained has a reduced viscosity in solution of 66.2 ml/g-1.1H NMR analysis of the polyester shows that the final polyester contains 15.1 mol % of isosorbide relative to all the monomer units. With regard to the thermal properties (measured at the second heating), the polymer has a glass transition temperature of 109° C. The lightness L* is 51.5. Example 3a The polyester from Example 3 is used in a solid-state post-condensation step. First, the polymer is crystallized for 8 h 30 in an oven under vacuum at 170° C. The crystallized polymer is then introduced into an oil bath rotavap fitted with a cannulated flask. The granules are then subjected to a temperature of 210° C. and a nitrogen flow of 3.3 l/min. After 33 h of post-condensation, the polymer will have a viscosity in solution of 94.2 ml/g. The mechanical properties of the polymer obtained are summarized in Table 1. Example 3b According to another process according to the invention, the polymer of Ex. 2 was extruded in a DSM twin-screw microextruder in the presence of 1 w % carbonylbiscaprolactam (Allinco CBC). The extrusion was carried out on 12 g of polymer for 2 min at 300° C. The polymer has a viscosity in solution of 85.4 ml/g. Counter-example 1 This example was carried out according to the embodiment recommended by patent application US 2012/0177854 A1. 3038 g (21.0 mol) of 1,4-cyclohexanedimethanol, 440 g (3.0 mol) of isosorbide, 2000 g (12.0 mol) of terephthalic acid, and 0.38 g of germanium dioxide are added to a 7.5 I reactor. To extract the residual oxygen from the isosorbide crystals, four vacuum-nitrogen cycles are performed once the temperature of the reaction medium is between 60 and 80° C. The reaction mixture is then heated to 250° C. (4° C./min) under 6.6 bar of pressure and with constant stirring (150 rpm). The degree of esterification is estimated from the amount of distillate collected. The pressure is then reduced to 0.7 mbar over 90 minutes following a logarithmic ramp and the temperature is brought to 280° C. These vacuum and temperature conditions were maintained for 210 minutes without obtaining an increase in torque. The casting of the reactor did not make it possible to extrude a rod of polymer in order to carry out the granulation thereof. The resin thus obtained had a reduced viscosity in solution of 16.4 ml/g-1under the conditions as defined in the present invention, that is to say a much lower viscosity than that of the polymer according to the invention. This polymer exhibits insufficient properties to be able to evaluate its mechanical properties. Counter-example 2 859 g (13.8 mol) of ethylene glycol, 546 g (3.7 mol) of isosorbide, 2656 g (16.0 mol) of terephthalic acid, 1.65 g of Irganox 1010 (antioxidant) and 1.39 g of dibutyltin oxide (catalyst) are added to a 7.5 I reactor. To extract the residual oxygen from the isosorbide crystals, four vacuum-nitrogen cycles are performed once the temperature of the reaction medium is between 60 and 80° C. The reaction mixture is then heated to 275° C. (4° C./min) under 6.6 bar of pressure and with constant stirring (150 rpm). The degree of esterification is estimated from the amount of distillate collected. The pressure is then reduced to 0.7 mbar over 90 minutes following a logarithmic ramp and the temperature is brought to 285° C. These vacuum and temperature conditions were maintained until an increase in torque of 15.0 Nm relative to the initial torque was obtained. Finally, a polymer rod is cast via the bottom valve of the reactor, cooled to 15° C. in a heat-regulated water bath and chopped in the form of granules of about 15 mg. The resin thus obtained has a reduced viscosity in solution of 58.8 ml/g-1.1H NMR analysis of the polyester shows that the final polyester contains 8.7 mol % of isosorbide relative to all the monomer units. With regard to the thermal properties (measured at the second heating), the polymer has a glass transition temperature of 97° C. The lightness L* is 46.2. This sample does not exhibit sufficient crystallinity or a sufficient crystallization rate to allow a solid-state post-condensation step to be carried out (it has a zero heat of fusion after heat treatment for 10 hours at 170° C.). TABLE 1Mechanical propertiesImpact strength(KJ/m2)WithoutNotchedNotchedFlexuralTractionTensilenotch at2 mm at2 mm atModulusModulusstrengthDeformationShore D25° C.25° C.−30° C.(MPa)(MPa)(MPa)at break (%)VicatHDThardnessEx1No10NM1512711362471008476breaking(>155)Ex. 1aNoNo231459736432001008877breakingbreaking(>155)(>155)Ex. 2aNoNo221523760442031079581breakingbreaking(>155)(>155)Ex. 3aNoNo1916087984016411510381breakingbreaking(>155)(>155)C-Ex. 29.6NMNM24001110394968681TritanNo94NM1480.944.7147.4117.9103.879TX2001breaking(>155) Conclusions from Tests The comparative polyester 1, which is that described in the application US 2012/0177854 A1, has a very low viscosity, in comparison with the polyester according to the invention of example 1. These two examples show that, surprisingly, it is entirely possible to form viscous polymers using the process of the first variant of the invention. The polyesters according to the invention, produced under the same conditions as the comparative polyester of counter-example 2 (polyester further comprising a linear aliphatic diol) have lower coloration and also far superior impact strength properties. The polyesters according to the invention have a high viscosity or even a very high viscosity when a step of increasing the molar mass by SSP or reactive extrusion is carried out. The semi-crystalline polyesters, the molar mass of which was increased by SSP, have a higher viscosity than that of the polyesters the molar mass of which was increased by reactive extrusion, The very high-viscosity polyesters have excellent impact strength, at room temperature and in cold conditions. The polyesters according to the invention have excellent mechanical properties, similar to the Tritan™ type performance copolyesters sold by Eastman®. Their impact strength properties are even better. | 46,258 |
11859047 | DETAILED DESCRIPTION While the present invention is susceptible to various modifications and alternative forms, specific embodiments will be illustrated and described in detail as follows. It should be understood, however, that the description is not intended to limit the present invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention. Hereinafter, a polycarbonate resin composition according to specific embodiments of the present invention and a molded article thereof will be described in more detail. First, the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the present invention. The singular expression used herein can include the plural expression unless it is differently expressed contextually. Polycarbonate Resin Composition A polycarbonate resin composition according to one embodiment of the present invention can include:(a) a linear polycarbonate resin including a first repeating unit of the following Chemical Formula 1;(b) a branched polycarbonate resin including the first repeating unit of the following Chemical Formula 1;(c) 0.04 parts by weight to 0.15 parts by weight of a fluorinated sulfonate-based metal salt;(d) 1.5 parts by weight to 5.0 parts by weight of phenyl methyl silicone oil having a kinematic viscosity of 5 mm2/sec to 60 mm2/sec at 25° C.; and(e) 0.05 parts by weight to 0.2 parts by weight of a phenyl methyl silicone resin,wherein the parts by weight of the fluorinated sulfonate-based metal salt, the phenyl methyl silicone oil, and the phenyl methyl silicone resin are based on 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin, wherein in Chemical Formula 1:R1to R4are each independently hydrogen, C1-10alkyl, C1-10alkoxy, or halogen; andZ is C1-10alkylene unsubstituted or substituted with phenyl, C3-15cycloalkylene unsubstituted or substituted with C1-10alkyl, O, S, SO, SO2, or CO. The polycarbonate resin composition according to one embodiment of the present invention includes the linear polycarbonate resin and the branched polycarbonate resin at the same time. The branched polycarbonate resin includes the first repeating unit as a basic main chain, and has a structure in which a plurality of the first repeating units are connected to each other via the branched second repeating unit according to a branching agent included during polymerization, and thus an entanglement phenomenon is enhanced. Therefore, as compared with the use of the linear polycarbonate resin alone, the polycarbonate resin composition can have excellent melt strength, thereby exhibiting more excellent processability at the time of blow molding. Further, the polycarbonate resin composition according to the present invention includes the linear polycarbonate resin and the branched polycarbonate resin, together with a fluorinated sulfonate-based metal salt, phenyl methyl silicone oil, and a phenyl methyl silicone resin as flame retardants. Due to interactions therebetween, the polycarbonate resin composition can exhibit flame-retardancy of UL-94 V0 grade while exhibiting excellent fluidity, processability, weather resistance, and transparency. Accordingly, the polycarbonate resin composition according to one embodiment of the present invention can be, but is not limited to, used as a material for electrical and electronic parts, parts for lighting equipment, etc. which are required to have the above properties. Hereinafter, the polycarbonate resin composition according to one embodiment of the present invention will be described in more detail. (a) Linear Polycarbonate Resin The ‘linear polycarbonate resin’ according to the present invention refers to a resin including the polycarbonate-based first repeating unit of Chemical Formula 1, and is distinguished from the branched polycarbonate resin in that it does not include a branched repeating unit described below. Specifically, the repeating unit of Chemical Formula 1 is formed by reacting an aromatic diol compound with a carbonate precursor. In Chemical Formula 1, preferably, R1to R4are each independently hydrogen, methyl, chloro, or bromo. Further, preferably, Z is linear or branched C1-10alkylene unsubstituted or substituted with phenyl, and more preferably, methylene, ethane-1,1-diyl, propane-2,2-diyl, butane-2,2-diyl, 1-phenylethane-1,1-diyl, or diphenyl methylene. Further, Z is preferably cyclohexane-1,1-diyl, O, S, SO, SO2, or CO. The repeating unit of Chemical Formula 1 can be derived from an aromatic diol compound of the following Chemical Formula 1-1: wherein in Chemical Formula 1-1, R1to R4and Z are the same as defined in Chemical Formula 1. Non-limiting examples of the repeating unit of Chemical Formula 1 can be derived from one or more aromatic diol compounds selected from the group consisting of bis(4-hydroxyphenyl)methane, bis(4-hydroxyphenyl)ether, bis(4-hydroxyphenyl)sulfone, bis(4-hydroxyphenyl)sulfoxide, bis(4-hydroxyphenyl)sulfide, bis(4-hydroxyphenyl)ketone, 1,1-bis(4-hydroxyphenyl)ethane, bisphenol A, 2,2-bis(4-hydroxyphenyl)butane, 1,1-bis(4-hydroxyphenyl)cyclohexane, 2,2-bis(4-hydroxy-3,5-dibromophenyl)propane, 2,2-bis(4-hydroxy-3,5-dichlorophenyl)propane, 2,2-bis(4-hydroxy-3-bromophenyl)propane, 2,2-bis(4-hydroxy-3-chlorophenyl)propane, 2,2-bis(4-hydroxy-3-methylphenyl)propane, 2,2-bis(4-hydroxy-3,5-dimethylphenyl)propane, 1,1-bis(4-hydroxyphenyl)-1-phenylethane, bis(4-hydroxyphenyl)diphenylmethane, and a,ω-bis [3-(o-hydroxyphenyl)propyl]polydimethyl siloxane. The phrase ‘derived from aromatic diol compounds’ means that a hydroxy group of the aromatic diol compound of Chemical Formula 1-1 is reacted with the carbonate precursor to form the repeating unit of Chemical Formula 1. For non-limiting example, when bisphenol A, which is an aromatic diol compound, is polymerized with triphosgene, which is a carbonate precursor, the repeating unit of Chemical Formula 1 can have the following Chemical Formula 1-2: As the carbonate precursor, one or more selected from the group consisting of dimethyl carbonate, diethyl carbonate, dibutyl carbonate, dicyclohexyl carbonate, diphenyl carbonate, ditolyl carbonate, bis(chlorophenyl) carbonate, di-m-cresyl carbonate, dinaphthyl carbonate, bis(diphenyl) carbonate, phosgene, triphosgene, diphosgene, bromophosgene and bishaloformate can be used. Preferably, triphosgene or phosgene can be used. The linear polycarbonate resin can have a weight average molecular weight (Mw) of 1,000 g/mol to 100,000 g/mol, preferably 10,000 g/mol to 60,000 g/mol, and more preferably 15,000 g/mol to 50,000 g/mol. More preferably, the weight average molecular weight (Mw) is 17,000 g/mol or more, 18,000 g/mol or more, or 20,000 g/mol or more. Further, the weight average molecular weight is 43,000 g/mol or less, 42,000 g/mol or less, or 41,000 g/mol or less. In this regard, the weight average molecular weight refers to a converted value with respect to a standard polycarbonate (PC Standard), as measured by GPC (gel permeation chromatography). Further, the linear polycarbonate resin has a melt index (MI) of 2 g/10 min to 50 g/10 min, or 3 g/10 min to 40 g/10 min according to ASTM D1238 (as measured at 300° C. and a load of 1.2 kg for 10 minutes), which is preferred in terms of stable expression of physical properties of the resin composition. Further, the linear polycarbonate resin can be present in an amount of 50% by weight to 80% by weight, or 50% by weight to 70% by weight, or 55% by weight to 65% by weight, based on the total weight of the linear polycarbonate resin and the branched polycarbonate resin. If the amount of the linear polycarbonate resin is too small, there is a problem in that processability can be reduced. On the contrary, if the amount of the linear polycarbonate resin is too large, there is a problem in the effect of preventing dripping. Meanwhile, the above-described linear polycarbonate resin can be directly prepared according to a known method of polymerizing a general aromatic polycarbonate resin using the aromatic diol compound of Chemical Formula 1-1 and a carbonate precursor as starting materials. As the polymerization method, for example, an interfacial polymerization method can be used. In this case, the polymerization reaction can be carried out at an atmospheric pressure and a low temperature, and it is easy to control a molecular weight. The interfacial polymerization can be preferably conducted in the presence of an acid binder and an organic solvent. Furthermore, the interfacial polymerization can include, for example, the steps of conducting pre-polymerization, adding a coupling agent, and then conducting polymerization again. In this case, a polycarbonate having a high molecular weight can be obtained. The materials used in the interfacial polymerization are not particularly limited as long as they can be used in the polymerization of polycarbonates. The used amounts thereof can be adjusted as required. The acid binder can include, for example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, etc., or amine compounds such as pyridine, etc. The organic solvent is not particularly limited as long as it is a solvent that is usually used in the polymerization of polycarbonates. For example, halogenated hydrocarbons such as methylene chloride, chlorobenzene, etc., can be used. Further, during the interfacial polymerization, a reaction accelerator, for example, a tertiary amine compound, a quaternary ammonium compound, or a quaternary phosphonium compound, such as triethylamine, tetra-n-butylammonium bromide, tetra-n-butylphosphonium bromide, etc., can be further used for accelerating the reaction. In the interfacial polymerization, a reaction temperature can be preferably 0° C. to 40° C., and a reaction time can be preferably 10 minutes to 5 hours. Further, during the interfacial polymerization reaction, pH can be preferably maintained at 9 or more, or 11 or more. In addition, the interfacial polymerization reaction can be carried out by further including a molecular weight modifier. The molecular weight modifier can be added before the initiation of polymerization, during the initiation of polymerization, or after the initiation of polymerization. As the molecular weight modifier, mono-alkyl phenol can be used. For example, the mono-alkyl phenol is one or more selected from the group consisting of p-tert-butyl phenol, p-cumyl phenol, decyl phenol, dodecyl phenol, tetradecyl phenol, hexadecyl phenol, octadecyl phenol, eicosyl phenol, docosyl phenol, and triacontyl phenol. Preferably, the mono-alkyl phenol can be p-tert-butylphenol, and in this case, the effect of adjusting the molecular weight is great. The molecular weight modifier can be, for example, included in an amount of 0.01 part by weight or more, 0.1 part by weight or more, or 1 part by weight or more, and 10 parts by weight or less, 6 parts by weight or less, or 5 parts by weight, based on 100 parts by weight of the aromatic diol compound. Within the above range, a desired molecular weight can be obtained. (b) Branched Polycarbonate Resin The ‘branched polycarbonate resin’ according to the present invention refers to a branched polycarbonate resin including the polycarbonate-based first repeating unit of Chemical Formula 1. More specifically, it refers to a copolycarbonate resin further including a trivalent or tetravalent branched second repeating unit connecting a plurality of the first repeating units to each other, in addition to the polycarbonate-based first repeating unit of Chemical Formula 1. The description of the first repeating unit is the same as described above. Further, the trivalent or tetravalent second repeating unit refers to a repeating unit formed by being grafted as branches onto a main chain by a branching agent which is added during polymerization of the aromatic diol compound of Chemical Formula 1-1 and the carbonate precursor. Specifically, the trivalent or tetravalent second repeating unit can be derived from a branching agent which is a phenol derivative compound having three or four hydroxyl groups, for example, one or more branching agents selected from the group consisting of 1,1,1-tris(4-hydroxyphenyl)ethane, 1,3,5-tris-(2-hydroxyethyl)cyanuric acid, 4,6-dimethyl-2,4,6-tris-(4-hydroxyphenyl)-heptane-2,2,2-bis [4,4′-(dihydroxyphenyl)cyclohexyl] propane, 1,3,5-trihydroxybenzene, 1,2,3-trihydroxybenzene, 1,4-bis-(4′,4″-dihydroxytriphenyl methyl)-benzene, 2′, 3′,4′-trihydroxyacetophenone, 2,3,4-trihydroxybenzoic acid, 2,3,4,-trihydroxybenzophenone, 2,4,4′-trihydroxybenzophenone, 2′,4′,6′-trihydroxy-3-(4-hydroxyphenyl)propiophenone, pentahydroxyflavone, 3,4,5-trihydroxyphenylethylamine, 3,4-trihydroxyphenylethylalcohol, 2,4,5-trihydroxypyrimidine, tetrahydroxy-1,4-quinone hydrate, 2,2′,4,4′-tetrahydroxybenzophenone, and 1,2,5,8-tetrahydroxyanthraquinone. The phrase ‘derived from a branching agent’ means that three or four hydroxyl groups of the above-described branching agent are reacted with the carbonate precursor to be graft-polymerized with a plurality of the repeating units of Chemical Formula 1, thereby forming the trivalent or tetravalent second repeating unit. The second repeating unit can have the following Chemical Formula 2: wherein in Chemical Formula 2, R1to R4and Z are the same as defined in Chemical Formula 1, and Q is a trivalent or tetravalent phenol derivative compound derived from the branching agent. For a non-limiting example, when bisphenol A as the aromatic diol compound, triphosgene as the carbonate precursor, and 1,1,1,-tris(4′-hydroxyphenyl) ethane (THPE) as the branching agent are used to perform polymerization, the repeating unit of Chemical Formula 2 can have the following Chemical Formula 2-1: The branched polycarbonate resin can include 98 mol % to 99.999 mol % of the first repeating unit and 0.001 mol % to 2 mol % of the second repeating unit. Alternatively, the branched polycarbonate resin can include 98 mol % to 99.99 mol % of the first repeating unit and 0.01 mol % to 2 mol % of the second repeating unit. Alternatively, the branched polycarbonate resin can include 98 mol % to 99.9 mol % of the first repeating unit and 0.1 mol % to 2 mol % of the second repeating unit. If the amount of the second repeating unit is excessively small, it is difficult to sufficiently achieve improvement in the extensional viscosity property due to the branched structure. On the contrary, if the amount of the second repeating unit is excessively large, a large amount of gel can be formed to deteriorate physical properties. The branched polycarbonate resin can have a weight average molecular weight of 10,000 g/mol to 100,000 g/mol, preferably 20,000 g/mol to 50,000 g/mol. More preferably, the weight average molecular weight (Mw) can be 10,000 g/mol or more, 21,000 g/mol or more, 22,000 g/mol or more, 23,000 g/mol or more, 24,000 g/mol or more, 25,000 g/mol or more, 26,000 g/mol or more, 27,000 g/mol or more, or 28,000 g/mol or more. Further, the weight average molecular weight can be 100,000 g/mol or less, 50,000 g/mol or less, 45,000 g/mol or less, 42,000 g/mol or less, or 40,000 g/mol or less. In this regard, the weight average molecular weight refers to a converted value with respect to a standard polycarbonate (PC Standard), as measured by GPC (gel permeation chromatography). Further, the branched polycarbonate resin has a melt index (MI) of 1 g/10 min to 50 g/10 min, or 1.5 g/10 min to 40 g/10 min according to ASTM D1238 (as measured at 300° C. and a load of 1.2 kg for 10 minutes), which is preferred in terms of stable expression of physical properties of the resin composition. Further, the branched polycarbonate resin can be included in an amount of 20% by weight to 50% by weight, or 30% by weight to 50% by weight, or 35% by weight to 45% by weight, based on the total weight of the linear polycarbonate resin and the branched polycarbonate resin. If the amount of the branched polycarbonate resin is too small, there is a problem in the effect of preventing dripping. On the contrary, if the amount of the branched polycarbonate resin is too large, there is a problem in that processability can be reduced. Meanwhile, the above-described branched polycarbonate resin can be directly prepared according to a known method of polymerizing a general aromatic polycarbonate resin using the aromatic diol compound of Chemical Formula 1-1, the carbonate precursor, and the branching agent as starting materials. As the polymerization method, for example, an interfacial polymerization method can be used. The interfacial polymerization can be explained with reference to the above description. (c) Fluorinated Sulfonate-Based Metal Salt The flame retardant according to the present invention can be used as an alternative to Br- or Cl-based flame retardants having toxicity and environmental problems caused by generation of harmful gas, and can be used by adding to the polycarbonate for excellent flame retardancy. Polycarbonate has relatively excellent mechanical properties, electrical properties, and weather resistance, as compared with other kinds of resins, but its flame retardancy is poor. Thus, to apply polycarbonate in various fields requiring flame retardancy, it is necessary to improve flame retardancy. Therefore, in the present invention, in addition to the above-described polycarbonate resin, the fluorinated sulfonate-based metal salt, and phenyl methyl silicone oil and a phenyl methyl silicone resin described below are further included to improve flame retardancy. Further, very excellent transparency can be obtained due to low haze. Here, the ‘fluorinated sulfonate-based metal salt’ means a salt compound of a fluorinated sulfonic acid ion and a metal ion, which results in an increase in the char formation rate of the polycarbonate, thereby contributing to the improvement of the flame retardancy of the polycarbonate resin composition. For example, the fluorinated sulfonate-based metal salt can be one or more compounds selected from the group consisting of sodium trifluoromethyl sulfonate, sodium perfluoroethyl sulfonate, sodium perfluorobutyl sulfonate, sodium perfluoroheptyl sulfonate, sodium perfluorooctyl sulfonate, potassium perfluorobutyl sulfonate, potassium perfluorohexyl sulfonate, potassium perfluorooctyl sulfonate, calcium perfluoromethane sulfonate, rubidium perfluorobutyl sulfonate, rubidium perfluorohexyl sulfonate, cesium trifluoromethyl sulfonate, cesium perfluoroethyl sulfonate, cesium perfluorohexyl sulfonate, and cesium perfluorooctyl sulfonate. Preferably, the fluorinated sulfonate-based metal salt can be potassium perfluorobutyl sulfonate. Further, the fluorinated sulfonate-based metal salt can be included in an amount of 0.04 parts by weight or more, or 0.08 parts by weight or more and 0.15 parts by weight or less, or 0.12 parts by weight or less, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. If the amount of the fluorinated sulfonate-based metal salt is too small, there is a problem in that flame retardancy can be reduced. On the contrary, if the amount of the fluorinated sulfonate-based metal salt is too large, there is a problem in that transparency can be reduced. In this respect, therefore, the fluorinated sulfonate-based metal salt is preferably included within the above range. Meanwhile, the fluorinated sulfonate-based metal salt has excellent flame retardancy, but has a disadvantage of generating bubbles during injection molding of a composition including the same. For this reason, there has been an attempt to use sodium dodecylbenzene sulfonate as an organic sulfonate-based flame retardant replacing the fluorinated sulfonate-based metal salt. However, sodium dodecylbenzene sulfonate has a disadvantage of greatly reducing transparency or mechanical strength of the polycarbonate resin. Accordingly, the polycarbonate resin composition of the present invention can include no sodium dodecylbenzenesulfonate while including phenyl methyl silicone oil and the phenyl methyl silicone resin described below, in addition to the fluorinated sulfonate-based metal salt, thereby maintaining flame retardancy and preventing bubble generation by the fluorinated sulfonate-based metal salt, and achieving excellent flame retardancy and processability at the same time. (d) Phenyl Methyl Silicone Oil Meanwhile, the polycarbonate resin composition according to the present invention can further include phenyl methyl silicone oil having a kinematic viscosity of 5 mm2/sec to 60 mm2/sec at 25° C., in order to improve flame retardancy. Here, the ‘phenyl methyl silicone oil’ means a silicone polymer including a methyl group and a phenyl group as a side chain or terminal sub stituent of a siloxane repeating unit, and preferably, a silicone polymer including a methyl group as a terminal substituent and a phenyl group as a side chain substituent. Such a phenyl methyl silicone oil can contribute to improvement of heat resistance and flame retardancy of the polycarbonate resin composition by the repeating siloxane main chain, and can exhibit the effect of improving flame retardancy by essentially including the phenyl group. The phenyl methyl silicone oil can have the kinematic viscosity of 5 mm2/sec to 60 mm2/sec at 25° C. Specifically, the phenyl methyl silicone oil can have the kinematic viscosity (mm2/sec) of 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more at 25° C. Further, its kinematic viscosity (mm2/sec) can be 50 or less, 40 or less, 30 or less, 25 or less, or 20 or less at 25° C. If the kinematic viscosity of the phenyl methyl silicone oil is less than 5 mm2/sec, high volatility can generate bubbles. On the contrary, if the kinematic viscosity of the phenyl methyl silicone oil is more than 60 mm2/sec, transparency can be reduced. In this respect, therefore, phenyl methyl silicone satisfying the above-described range of kinematic viscosity is preferably used. Preferably, the phenyl methyl silicone oil can be a silicone polymer including a phenyl trimethicone repeating unit. Specifically, the phenyl methyl silicone oil can have the following Chemical Formula 3: wherein in Chemical Formula 3:R5and R6are each independently C1-10alkyl, C1-10alkenyl, or C6-10aryl;R7is phenyl;n is an integer from 0 to 10, andm is an integer from 1 to 10. More preferably, in Chemical Formula 3, at least one of R5and R6can be a phenyl group. For example, the phenyl methyl silicone oil can be a compound of the following Chemical Formula 3-1, in which all of R5to R7in Chemical Formula 3 are phenyl groups: wherein in Chemical Formula 3-1, n and m are the same as defined in Chemical Formula 3. Further, the phenyl methyl silicone oil can be included in an amount from 1.5 parts by weight or more, or 1.8 parts by weight or more to 5.0 parts by weight or less, or 4.5 parts by weight or less, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. If the amount of the phenyl methyl silicone oil is too small, there is a problem in that flame retardancy can be reduced. On the contrary, if the amount of the phenyl methyl silicone oil is too large, there is a problem in that transparency can be reduced. In this respect, therefore, the phenyl methyl silicone oil is preferably included within the above range. (e) Phenyl Methyl Silicone Resin Meanwhile, the polycarbonate resin composition according to the present invention can further include a phenyl methyl silicone resin in order to prevent dripping and to improve flame retardancy. Here, the ‘phenyl methyl silicone resin’ is a network structured polyorganosiloxane resin including both phenyl and methyl substituents and having solid-phase properties. The phenyl methyl silicone resin is distinguished from the aforementioned (d) phenyl methyl silicone oil in that it is in a solid phase. As the phenyl methyl silicone resin, KR-480 available from Shinetsu Co., Ltd., etc. can be used. As the phenyl methyl silicone resin having such a structure is used, the anti-dripping effect can be obtained. The silicone resin can be included in an amount from 0.05 parts by weight or more, or 0.08 parts by weight or more, to 0.2 parts by weight or less, or 0.1 part by weight or less, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. If the amount of the phenyl methyl silicone resin is too small, there is a problem in that the anti-dripping effect can be reduced. On the contrary, if the amount of the phenyl methyl silicone resin is too large, there is a problem in that transparency can be reduced. In this respect, therefore, the phenyl methyl silicone resin is preferably included within the above range. (f) Epoxy-Based Hydrolysis-Resistant Agent Meanwhile, the polycarbonate resin composition according to the present invention can further include an epoxy-based hydrolysis-resistant agent in order to improve hydrolysis resistance. As the epoxy-based hydrolysis-resistant agent, a compound having a structure in which an epoxy group is fused into an aliphatic ring, can be used, and examples of the hydrolysis-resistant agent having the epoxy-fused aliphatic ring can include 2021P(3,4-Epoxycyclohexylmethyl 3,4-epoxycyclohexanecarboxylate) available from Daicel, Corp. Further, the epoxy-based hydrolysis-resistant agent can be included in an amount from 0.05 parts by weight or more, or 0.06 parts by weight or more, or 0.08 parts by weight or more to 0.2 parts by weight or less, or 0.15 parts by weight or less, or 0.12 parts by weight or less, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. When the amount of the epoxy-based hydrolysis-resistant agent is within the above range, the effect of hydrolysis resistance can be sufficiently achieved without reduction in transparency and flame retardancy of the resin composition. In this respect, therefore, the epoxy-based hydrolysis-resistant agent is preferably included within the above range. (g) UV Absorber Meanwhile, the polycarbonate resin composition according to the present invention can further include a UV absorber in order to effectively block UV coming from the outside. The UV absorber applicable in the present invention can be any UV absorber without particular limitation, as long as it allows a molded film specimen of the polycarbonate resin composition according to the present invention to have light transmittance of 20% or less, preferably 10% or less at a wavelength of 380 nm under a thickness condition of 3 mm. Preferably, the UV absorber can be one or more selected from the group consisting of a benzotriazole compound, a benzophenone compound, an oxanilide compound, a benzoic acid ester compound, and a triazine compound. For example, the UV absorber can include benzotriazole compounds such as 2-(2′-hydroxyphenyl)-benzotriazole compounds including 2-(2′-hydroxy-5′-methylphenyl)benzotriazole, 2-(2′-hydroxy-5′-tert-octylphenyl)benzotriazole, 2-(3′,5′-di-tert-butyl-2′-hydroxyphenyl)benzotriazole, 2-(5′-tert-butyl-2′-hydroxyphenyl)benzotriazole, 2-(2-hydroxy-5-(1,1,3,3,tetramethylbutyl)phenyl)benzotriazole, 2-(3′,5′-di-tert-butyl-2′-hydroxyphenyl)-5-benzotriazole, 2-(3′-tert-butyl-2′-hydroxyphenyl-5′-methylphenyl)-5-benztrizol, 2-(3′-sec-butyl-5′-tert-butyl-2′-hydroxyphenyl)benzotriazole, 2-(2′-hydroxy-4′-octyloxyphenylphenyl)-5-benzotriazole or 2-(3′,5′-di-tert-butyl-2′-hydroxyphenyl)benzotriazole, etc.; benzophenone compounds such as 2-hydroxy benzophenone compounds having a 4-hydroxy, 4-methoxy, 4-octyloxy, 4-decyloxy, 4-dodecyloxy, 4-benzyloxy, 4,2′,4′-trihydroxy, or 2′-hydroxy-4,4′-dimethoxy functional group; benzoic acid ester compounds such as compounds having a substituted benzoic acid ester structure, including 4-tert-butyl-phenyl salicylate, phenyl salicylate, octylphenyl salicylate, dibenzoyl resorcinol, bis(4-tert-butyl-benzoyl)resorcinol, benzoyl resorcinol, 2,4-di-tert-butylphenyl-3,5′-di-tert-butyl-4-hydroxybenzoate, hexadecyl 3,5-di-tert-butyl-4-4hyroxybenzoate, octadecyl 3,5-di-tert-butyl-4-hydroxybenzoate or 2-methyl-4,6-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate, etc.; or triazine compounds having a 2,4,6-triphenyl-1,3,5-triazine skeleton, etc., but is not limited thereto. Further, the UV absorber can be included in an amount from 0.1 part by weight or more, or 0.2 parts by weight or more to 0.5 parts by weight or less, or 0.4 parts by weight or less, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. When the amount of the UV absorber is within the above range, the effect of weather resistance can be sufficiently achieved without reduction in transparency and flame retardancy of the resin composition. In this respect, therefore, the UV absorber is preferably included within the above range. (h) Additives In addition to the above-described components, the polycarbonate resin composition according to the present invention can further include, if necessary, additives such as an impact reinforcing agent; a rheology modifier; a flame retardant such as a phosphorus flame retardant, etc.; a surfactant; a nucleating agent; a coupling agent; a filler; a plasticizer; a lubricant; an antibacterial agent; a mold release agent; a heat stabilizer; an antioxidant; a UV stabilizer; a compatibilizer; a coloring agent; an antistatic agent; a pigment; a dye; a flame proofing agent, etc. The amount of the additives can vary depending on the physical properties to be provided for the composition. For example, the additives can be included in an amount of 0.01 part by weight to 10 parts by weight, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. However, to prevent reduction in heat resistance, impact strength, and chemical resistance of the polycarbonate resin composition, which is caused by adding the additives, the total amount of the additives can be preferably 20 parts by weight or less, or 15 parts by weight or less, or 10 parts by weight or less, based on the total 100 parts by weight of the linear polycarbonate resin and the branched polycarbonate resin. The polycarbonate resin composition of one embodiment of the present invention can exhibit excellent flame retardancy of V-0 grade, as measured for a specimen having a thickness of 1.5 mm in accordance with UL 94 standard. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit the flame retardancy of V-0 grade before and after a water exposure test (immersion protocol) and a UV exposure test (weather-O-meter protocol), as measured for a film specimen having a thickness of 1.5 mm in accordance with UL 746C, indicating excellent weather resistance. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit tensile impact from 310 kJ/m2or more, or 315 kJ/m2or more, or 320 kJ/m2or more, to 360 kJ/m2or less, or 350 kJ/m2or less, or 340 kJ/m2or less, as measured in accordance with ASTM D1822(¼ inch), indicating excellent impact strength. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit tensile strength from 60 MPa or more, or 62 MPa or more, or 64 MPa or more, to 80 MPa or less, or 75 MPa or less, or 70 MPa or less, as measured for a specimen having a thickness of 1.5 mm in accordance with ASTM D638 (1.5 mm), indicating excellent tensile strength. Further, when the tensile impact of the polycarbonate resin composition of one embodiment of the present invention is measured for a film specimen having a thickness of 1.5 mm in accordance with a water exposure test (immersion protocol) of UL 746C, the tensile impact value after the exposure test can be 50% or more of the value measured before the exposure test. Simultaneously, when the tensile impact and tensile strength of the polycarbonate resin composition of one embodiment of the present invention are measured for the film specimen having a thickness of 1.5 mm in accordance with a UV exposure test (weather-O-meter protocol) of UL 746C, each value after the exposure test can be 70% or more of the value measured before the exposure test. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit room-temperature impact strength from 790 J/m or more, or 800 J/m or more, or 810 J/m or more to 900 J/m or less, or 880 J/m or less, or 860 J/m or less, as measured at 23° C. in accordance with ASTM D256 (⅛ inch, Notched Izod), indicating excellent room-temperature impact strength. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit haze from 0.01% or more to 2.4% or less, or 2.0% or less, or 1.0% or less, or 0.8% or less, or 0.6% or less, or 0.5% or less, as measured for an injection-molded specimen having a thickness of 3 mm in accordance with ASTM D1003, indicating excellent weather resistance. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit a spiral length from 50 cm or more, or 51 cm or more, or 53 cm or more to 65 cm or less, or 60 cm or less, or 58 cm or less, as measured by injection-molding at 330° C. using a spiral mold having a thickness of 2.5 mm and a width of 10 mm, indicating excellent processability. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit a melt index (MI) from 7 g/10 min or more, or 8 g/10 min or more to 50 g/10 min or less, or 30 g/10 min or less, or 20 g/10 min or less, as measured in accordance with ASTM D1238 (at 300° C. under a load of 1.2 kg for 10 minutes), indicating excellent fluidity. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit the number of drips of 0, as measured under UL94 vertical test conditions, indicating excellent dripping flame retardancy. Further, the polycarbonate resin composition of one embodiment of the present invention can exhibit a total combustion time (sec) of 27 sec or more, or 28 sec or more, 29 sec or more, 30 sec or more, 31 sec or more, or 32 sec or more and 50 sec or less, or 45 sec or less, or 40 sec or less, as measured under UL94 vertical test conditions, indicating excellent flame retardancy. Resin Molded Article According to another embodiment of the present invention, provided is a molded article including the above-described polycarbonate resin composition. The molded article is an article obtained by performing molding such as extrusion, injection, or casting using the above-described polycarbonate resin composition as a raw material. The molding method and conditions can be appropriately selected and controlled according to the kind of the molded article. For non-limiting example, the molded article can be obtained by a method of mixing and extrusion-molding the polycarbonate resin composition to prepare a pellet, and drying the pellet, followed by injection. In particular, as the molded article is produced from the polycarbonate resin composition, it can exhibit excellent processability, flame retardancy, and transparency, thereby being appropriately used as a material for electrical and electronic parts, parts for lighting equipment, etc. Hereinafter, preferred examples will be provided for better understanding of the present invention. However, the following examples are provided only for illustrating the present invention, but the present invention is not limited thereby. EXAMPLES Materials Used The following materials were used in Examples and Comparative Examples. (a) Linear Copolycarbonate Resin(a-1) LUPOY PC1080-70 (weight average molecular weight: 19,600 g/mol) produced by LG Chem Ltd., which is a polycarbonate including a repeating unit of Chemical Formula 1-2.(a-2) LUPOY PC1300-30 (weight average molecular weight: 21,100 g/mol) produced by LG Chem Ltd., which is a polycarbonate including a repeating unit of Chemical Formula 1-2.(a-3) LUPOY PC1300-15 (weight average molecular weight: 27,800 g/mol) produced by LG Chem Ltd., which is a polycarbonate including a repeating unit of Chemical Formula 1-2. (b) Branched Polycarbonate Resin(b-1) LUPOY PC1600-03 produced by LG Chem Ltd., which is a polycarbonate having a weight average molecular weight of 37,600 g/mol and including repeating units of Chemical Formulae 1-2 and 2-1. (c) Organic Sulfonate-Based Metal Salt(c-1) FR-2025 produced by 3M, Com., which is a potassium perfluorobutyl sulfonate (KPFBS).(c-2) KSS-FR produced by Arichem, LLC., which is a potassium diphenylsulfone sulfonate (KSS).(c-3) SDBS (sodium dodecylbenzene sulfonate) produced by TCI Chemical, Co., Ltd. (d) Phenyl Methyl Silicone Oil(d-1) KR-56A produced by ShinEstu, Chemical Co., Ltd., Japan, which is a phenyl methyl silicone oil of the following Chemical Formula 3-1 having a kinematic viscosity of 15 mm2/sec at 25° C.: (d-2) KR-2710 produced by ShinEstu, Chemical Co., Ltd., Japan, which is a phenyl methyl silicone oil having a kinematic viscosity of 50 mm2/sec at 25° C.(d-3) KR-511 produced by ShinEstu, Chemical Co., Ltd., Japan, which is a phenyl methyl silicone oil having a kinematic viscosity of 100 mm2/sec at 25° C.(d-4) DC-550 produced by Dow Corning, Corp., which is a linear phenyl methyl silicone oil having a kinematic viscosity of 125 mm2/sec at 25° C. (e) Phenyl Methyl Silicone Resin(e-1) KR-480 produced by ShinEstu, Chemical Co., Ltd., Japan, which is a phenyl methyl silicone resin. (f) Epoxy-Based Hydrolysis-Resistant Agent(f-1) Celloxide 2021P produced by Daicel Corporation, which is (3′,4′-epoxycyclohexane)methyl 3,4-epoxycyclohexylcarboxylate. (g) UV Absorber(g-1) TINUVIN produced by 329 BASF, AG. (h) Additives(h-1) Alkanox 240 produced by Addivant, Corp., which is tris(2,4-di-tert-butylphenyl) phosphite.(h-2) FACI L348 produced by FACI, which is pentaerythrityl tetraethylhexanoate (PETS). Examples and Comparative Examples The respective components described in the following Tables 1 to 4 were mixed, and then pelletized at a speed of 80 kg per time using a biaxial extruder (L/D=36, Φ=45, barrel temperature: 240° C.), and injection-molded using an injection molding machine N-20C of JSW (Ltd.) at a cylinder temperature of 300° C. and a mold temperature of 80° C. to produce each specimen. Components and amounts used in Examples and Comparative Examples are shown in Tables 1 to 4 below, respectively. TABLE 1Example 1-1Example 1-2Example 1-3Example 1-4Example 1-5Linear PC1)aa-1(15)a-1(15)a-1(15)a-1(15)a-3(80)(wt %)a-2(45)a-2(45)a-2(45)a-2(45)Branched PC1)bb-1(40)b-1(40)b-1(40)b-1(40)b-1(20)(wt %)Organiccc-1(0.10)c-1(0.10)c-1(0.10)c-1(0.10)c-1(0.10)sulfonatemetal salt(phr)2)Phenyl methyldd-1(2.00)d-1(3.00)d-1(4.00)d-2(3.00)d-1(4.00)siliconeoil (phr)2)Phenyl methylee-1(0.10)e-1(0.10)e-1(0.10)e-1(0.10)e-1(0.10)silicone resin(phr)2)Additivehh-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)(phr)2)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)1)% by weight with respect to total weight of linear polycarbonate resin and branched carbonate resin2)parts by weight with respect to total 100 parts by weight of linear polycarbonate resin and branched carbonate resin TABLE 2ComparativeComparativeComparativeComparativeComparativeComparativeComparativeComparativeComparativeExample 1-1Example 1-2Example 1-3Example 1-4Example 1-5Example 1-6Example 1-7Example 1-8Example 1-9Linear PC1)aa-3 (100)a-3 (100)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)(wt %)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)Branched PC1)b——b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)(wt %)Organiccc-1(0.10)c-1 (0.10)c-1 (0.10)c-1 (0.10)c-2 (0.30)c-1 (0.10)c-1 (0.10)c-1 (0.10)c-3 (0.10)sulfonatemetal salt(phr)2)Phenyl methyld—d-1 (2.00)d-3 (2.00)d-4 (2.00)d-1 (3.00)d-1 (1.00)d-1 (2.00)d-1 (2.00)d-1 (3.00)silicone oil(phr)2)Phenyl methyle—e-1 (0.10)e-1 (0.10)e-1 (0.10)e-1 (0.10)e-1 (0.10)—e-1 (0.30)e-1 (0.10)silicone resin(phr)2)Additivehh-1 (0.05)h-1 (0.05)h-1 (0.05)h-1 (0.05)h-1 (0.05)h-1 (0.05)h-1 (0.05)h-1 (0.05)h-1 (0.05)(phr)2)h-2 (0.05)h-2 (0.05)h-2 (0.05)h-2 (0.05)h-2 (0.05)h-2 (0.05)h-2 (0.05)h-2 (0.05)h-2 (0.05)1)% by weight with respect to total weight of linear polycarbonate resin and branched carbonate resin2)parts by weight with respect to total 100 parts by weight of linear polycarbonate resin and branched carbonate resin TABLE 3Example 2-1Example 2-2Example 2-3Example 2-4Example 2-5Example 2-6Example 2-7Linear PC1)aa-1(15)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)(wt %)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)Branched PC1)bb-1(40)b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)(wt %)Organiccc-1(0.10)c-1(0.15)c-1(0.10)c-1(0.10)c-1(0.10)c-1(0.10)c-1(0.10)sulfonatemetal salt(phr)2)Phenyl methyldd-1(2.00)d-1(2.00)d-1(3.00)d-1(4.00)d-1(2.00)d-1(2.00)d-1(2.00)silicone oil(phr)2)Phenyl methylee-1(0.10)e-1(0.10)e-1(0.10)e-1(0.10)e-1(0.20)e-1(0.10)e-1(0.10)silicone resin(phr)2)Epoxy-basedff-1(0.10)f-1(0.10)f-1(0.10)f-1(0.10)f-1(0.10)f-1(0.20)f-1(0.10)hydrolysis-resistantagent (phr)2)UV absorbergg-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)g-1(0.50)(phr)2)Additivehh-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)(phr)2)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)1)% by weight with respect to total weight of linear polycarbonate resin and branched carbonate resin2)parts by weight with respect to total 100 parts by weight of linear polycarbonate resin and branched carbonate resin TABLE 4ComparativeComparativeComparativeComparativeComparativeComparativeComparativeExample 2-1Example 2-2Example 2-3Example 2-4Example 2-5Example 2-6Example 2-7Linear PC1)aa-3 (100)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)a-1(15)(wt %)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)a-2(45)Branched PC1)b—b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)b-1(40)(wt %)Organiccc-1(0.10)—c-1(0.10)c-1(0.10)c-1(0.10)c-1(0.10)c-3(0.10)sulfonatemetal salt(phr)2)Phenyl methyldd-1(2.00)d-1(2.00)—d-3(2.00)d-1(2.00)d-1(2.00)d-1(2.00)silicone oil(phr)2)Phenyl methylee-1(0.10)e-1(0.10)e-1(0.10)e-1(0.10)e-1(0.10)e-1(0.10)silicone resin(phr)2)Epoxy-basedff-1(0.10)f-1(0.10)f-1(0.10)f-1(0.10)f-1(0.20)—f-1(0.10)hydrolysis-resistantagent (phr)2)UV absorbergg-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)g-1(0.25)(phr)2)Additivehh-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)h-1(0.05)(phr)2)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)h-2(0.05)1)% by weight with respect to total weight of linear polycarbonate resin and branched carbonate resin2)parts by weight with respect to total 100 parts by weight of linear polycarbonate resin and branched carbonate resin Experimental Example The compositions of Examples and Comparative Examples and specimens produced therefrom were measured for physical properties by the following methods, respectively.1) Melt Index (MI): measured in accordance with ASTM D1238 (under conditions of 300° C. and 1.2 kg).2) Room temperature impact strength: measured in accordance with ASTM D256 (⅛ inch, Notched Izod) at 23° C.3) Haze: measured for a specimen having a thickness of 3 mm in accordance with ASTM D1003 using a haze meter.4) Spiral length: measured as an average length of a total of 10 injection molded-articles after injection molding at 330° C. using a spiral mold having a thickness of 2.5 mm and a width of 10 mm.5) Number of drips: the number of drips (in the case of dripping spark particles) was measured in accordance with UL 94 vertical test protocol.6) Total combustion time: measured as the sum of t1, t2, and t3 in accordance with UL94 vertical test criteria of the following Table 5.7) Flame retardancy (UL 94 Protocol): flame retardancy was tested in accordance with UL 94 standard. In detail, five flame retardant specimens having a thickness of 1.5 mm required for the flame retardancy test were prepared, and tested as follows. First, each specimen was left in contact with 20 mm high flame for 10 seconds, and then a combustion time (t1) of the specimen was measured, and a combustion aspect was recorded. Then, after the primary flame-contact, the combustion was terminated, and each specimen was left in contact with flame for another 10 seconds. Next, a combustion time (t2) and a glowing time (t3) of the specimen were measured, and a combustion aspect was recorded. The test was equally applied to five specimens, and the specimens were evaluated according to the criteria of Table 5 below. TABLE 5Flame retardancy ratingV-0V-1V-2Each combustion time10 sec or30 sec or30 sec or(t1 or t2 of each specimen)lesslesslessTotal combustion time of50 sec or250 sec or250 sec orfive specimens (sum of t1lesslesslessand t2 of five specimens)Combustion time and Glowing30 sec or60 sec or60 sec ortime after secondary flamelesslesslesscontact (sum of t2 and t3of each specimen)Whether particle causingNoneNoneDroppedflame9) Weather resistance 1 (UL 746C immersion protocol): weather resistance was evaluated by a water exposure test in accordance with UL 746C standard. In detail, five specimens having a thickness of 1.5 mm required for the test were prepared, and each specimen was measured for flame retardancy rating, tensile impact and tensile strength values. Next, each specimen was pre-immersed in distilled water at 70° C. for 7 days, and then immersed in distilled water at 23° C. for 30 minutes, and then the tensile impact value was measured. Each specimen was pre-immersed in distilled water at 70° C. for 7 days, and then immersed at 23° C. and relative humidity of 50% for 2 weeks, and then the flame retardancy was rated and compared with the value before exposure.10) Weather resistance 2 (UL 746C Weather-O-meter protocol): weather resistance was evaluated by a UV exposure test in accordance with UL 746C standard. In detail, five specimens having a thickness of 1.5 mm required for the test were prepared, and each specimen was measured for flame retardancy rating, tensile impact and tensile strength values. Next, a xenon-arc lamp was prepared according to ASTM G151, and each specimen was irradiated with UV for 102 minutes, and then exposed to UV and water spray for the remaining 18 minutes. This cycle of the total 120 minutes was repeatedly carried out until the total time reached 1000 hours. Each specimen was measured for flame retardancy rating, tensile impact and tensile strength values, and compared with the values before exposure. At this time, the test was carried out by irradiation at a wavelength of 340 nm with energy of 0.35 W/m2under operation conditions of a black-panel temperature of 63° C. (error: 3° C.). In the weather resistance tests 1 and 2, tensile impact was measured in accordance with ASTM D1822 (¼ inch), and tensile strength was measured in accordance with ASTM D638 (1.5 mm). The results of measuring the physical properties are shown in Tables 6 to 9 below, respectively. TABLE 6SectionExample 1-1Example 1-2Example 1-3Example 1-4Example 1-5Melt index (g/10 min)15.616.417.616.216.2Room temperature812821825798842impact strength (J/m)Haze (%)0.380.420.510.430.52Spiral length (cm)53.655.257.354.150.8Number of drips00000(number)Total combustion32.733.836.133.135.1time (second)UL94 ratingV-0V-0V-0V-0V-0 TABLE 7ComparativeComparativeComparativeComparativeComparativeComparativeComparativeComparativeComparativeSectionExample 1-1Example 1-2Example 1-3Example 1-4Example 1-5Example 1-6Example 1-7Example 1-8Example 1-9Melt index15.117.315.315.117.214.315.814.818.6(g/10 min)Room temperature845815866825788822792833615impact strength(J/m)Haze (%)0.230.332.1845.12.830.330.321.827.81Spiral length46.948.152.151.853.851.853.282.463.5(cm)Number of554243205drips (number)Total78.236.142.132.139.338.237.333.387.3combustiontime (second)UL94 ratingV-2V-2V-2V-2V-2V-2V-2V-0V-2 TABLE 8SectionExample 2-1Example 2-2Example 2-3Example 2-4Example 2-5Example 2-6Example 2-7InitialMelt index14.815.016.017.114.715.414.7physical(g/10 min)propertiesSpiral length52.051.854.857.052.453.751.6(cm)Haze (%)0.481.020.530.602.310.510.43Tensile impact324315330332319319326(kJ/m2)Tensile strength69646768656766(MPa)UL94Number of0000000protocoldrips (number)Total27.93829.232.230.137.539.5combustiontime (second)UL94 ratingV-0V-0V-0V-0V-0V-0V-0UL 746CTensile impact292300326329319300310immersion(kJ/m2)protocolTensile63606465636461strength (MPa)UL94 ratingV-0V-0V-0V-0V-0V-0V-0UL 746CTensile impact253236244252236230267Weather-(kJ/m2)O-meterTensile strength55495051484856protocol(MPa)UL94 ratingV-0V-0V-0V-0V-0V-0V-0 TABLE 9ComparativeComparativeComparativeComparativeComparativeComparativeComparativeSectionExample 2-1Example 2-2Example 2-3Example 2-4Example 2-5Example 2-6Example 2-7InitialMelt index15.214.013.214.715.114.316.8physical(g/10 min)propertiesSpiral length47.149.945.251.452.851.060.1(cm)Haze (%)0.330.420.312.320.440.483.41Tensile impact342339338348311319132(kJ/m2)Tensile strength70676665636442(MPa)UL94Number of5554205protocoldrips (number)Total57.360.749.547.735.730.879.6combustiontime (second)UL94 ratingV-2V-2V-2V-2V-2V-0V-2UL 746CTensile impact287immersion(kJ/m2)protocolTensile strength57(MPa)UL94 ratingV-2UL 746CTensile impact262Weather-(kJ/m2)O-meterTensile strength52protocol(MPa)UL94 ratingV-0 (In Table 9, the weather resistance was not tested for Comparative Examples which were rated as V-2 during the initial UL94 test) Referring to Tables 6 to 9, the flame retardant polycarbonate resin compositions of the present invention can exhibit all excellent properties in flame retardancy, melt index, impact strength, transparency, weather resistance, etc. | 50,053 |
11859048 | DETAILED DESCRIPTION Melt polymerization is an industrially used process to make polycarbonate by reacting a bisphenol and a carbonate compound in the molten form. The bisphenol and the carbonate compound are added to a monomer mixing tank along with a quaternary catalyst, where some oligomerization starts, as is evidenced by production of a phenol byproduct. From the monomer mixing tank, the melt is added to a series of oligomerization units that use Maxblend type, flat blade impellers from Sumitomo Heavy Industries Process Equipment Co., Ltd. and internal heat exchanger coils. These oligomerization units require large amounts of power per unit of solution volume to operate and also suffer from associated scale-up challenges, especially in terms of meeting heat transfer requirements. It was discovered that a reactor using a rotating impeller comprising a plurality of blades was capable of forming a polycarbonate oligomer using a reduced unit power as compared to a mixer using a flat blade impeller. For example, the present reactor can result in a more than 40% reduction in the unit power as compared to a mixer having the same capacity and using a flat blade impeller and an internal heat exchanger. The majority of the power reduction is achieved by using impellers with lower power numbers and a simplified internal configuration. It is believed that using the external heat exchanger will reduce the complexity of the reactor design as compared to a reactor with internal heating coils and will overcome the challenges associated with meeting the heat transfer requirements during scale-up of reactor design. The recirculation loop through the external heat exchanger can also help to reduce the mixing time of the reactor. FIG.1is an illustration of an embodiment of an improved reactor.FIG.1illustrates that reactor10can comprise cylindrical tank12comprising a top, a side, and a bottom, wherein the bottom is convex, extending away from the top. The reactor can be a vertical reactor such that axis100is perpendicular (within 10°, or 0 to 5°, or 0 to 1°) to at least one of a plane tangent to the bottom of the cylindrical tank or a top fluid plane as defined by a top surface of a resting liquid present in the cylindrical tank (also referred to herein as a level fluid line). Stirring shaft14is disposed within cylindrical tank12along axis100thereof so that it is rotatable from outside of cylindrical tank12. At least one impeller can extend from stirring shaft14in cylindrical tank12and can comprise a plurality of blades that are each independently at an angle α from the orthogonal of axis100. Externally located heat exchanger50can be in fluid communication with cylindrical tank12via recirculation stream52and a heated stream54. The temperature of the heated stream, Tf, can be greater than a temperature of the recirculation stream, Ti, for example, Tfcan be greater than or equal to Ti+10° C., or greater than or equal to Ti+20° C., or greater than or equal to Ti+50° C., optionally, less than or equal to Ti+100° C. The externally located heat exchanger can impart a heat to the recirculation stream to effect a rise in temperature. All of the recirculation stream withdrawn from the cylindrical tank, for example, 99 to 100 wt % based on the total weight of the recirculation stream withdrawn, can be reintroduced to the cylindrical tank as the heated stream. One or both of the recirculation stream and the heated stream can be in the liquid form. A mass flow rate of the recirculation stream entering the external heat exchanger can be equal to a mass flow rate of the heated stream exiting the external heat exchanger, for example, a mass flow rate of the heated stream can be within 5%, or within 1%, or within 0.1% of the mass flow rate of the recirculation stream. The heated stream upon entering the cylindrical tank can comprise, or can consist essentially of only the heated composition of the recirculation stream, i.e., it can be free of an additionally added component. An overhead stream can be withdrawn from the cylindrical tank. The overhead stream can be in fluid communication with a separation unit, for example, a scrubber, a distillation column, a pressure condenser, or an absorption unit. The overhead stream can be in the gas form. The overhead stream can comprise at least one of an unreacted monomer (such as bisphenol A or diphenyl carbonate) or a reaction by-product (such as phenol). At least a portion of the unreacted monomer can be returned to the cylindrical reactor via a return stream. At least one impeller, for example, 1 to 5, or 2 to 4, or 1 to 2 impellers can extend from the stirring shaft. For example,FIG.1andFIG.2illustrate a stirring shaft having two impellers, lower impeller16and upper impeller18, extending therefrom. Each impeller independently comprises a plurality of blades that are each independently at an angle α from the orthogonal of axis100, where the angle α is illustrated inFIG.2as the angle between line n that is along the face of blade b and line m is orthogonal to the axis100. The angle α of each blade independently can be 25 to 65 degrees, or 30 to 50 degrees. The angle α of each blade can be the same, or within 1 degree of each other. Each impeller independently can be a hydrofoil impeller. Each impeller independently can comprise 2 to 5 blades, or 2 to 4 blades, or 3 blades. For example, each impeller can be a three-blade impeller. When two or more impellers extend from the stirring shaft, for example, lower impeller16and upper impeller18, the respective impellers can be offset by an angle θ of 50 to 70 degrees, where the angle θ is the angle between a first blade on the lower impeller relative to a first blade on the second impeller that is encountered in a radial trajectory is illustrated inFIG.3. The impeller, for example, a hydrofoil impeller, can produce an axial flow pattern. As used herein, the axial flow pattern refers to the fluid flow flowing in the direction of the stirring shaft towards the bottom of the tank, flowing from the bottom of the tank towards the side of the tank, and flowing upwards along the side of the tank to form a complete circulation loop. A good axial flow pattern can ensure that there are no dead pockets or poorly mixed zones in the reactor and can provide a good volumetric renewal rate to ensure the produced phenol leaves the reactor.FIG.7andFIG.11illustrate examples of axial flow patterns achieved by the reactor. When two or more impellers extend from the stirring shaft, for example, lower impeller16and upper impeller18, the respective impellers can direct a fluid flow in the same direction, for example, toward the bottom of the cylindrical tank. This similar directionality can help to achieve an axial flow pattern of the mixing solution. The impeller can have a power number of less than or equal to 2, or less than or equal to 1, or 0.25 to 1. The power number is a dimensionless parameter used for estimating the power consumed by the agitating impeller in a cylindrical tank. The power number, NP, defined as Pu/(ρn3DI5), where Puthe power input per volume of the reaction mixture (also referred to herein as the unit power) in watts per meter cubed (W/m3), ρ is the density of the solution in kilograms per meter cubed (kg/m3), n is the impeller speed in revolutions per second (1/s), and DIis the impeller diameter in meters (m). When only one impeller is located on the stirring shaft, the power number can be 0.25 to 0.5. When two impellers are located on the stirring shaft, the respective power numbers can be 0.5 to 1. The impeller can have a flow number of less than or equal to 2, or less than or equal to 1, or 0.25 to 1. The flow number is a dimensionless parameter used for estimating the fluid movement resulting from the agitating impeller in a cylindrical tank. The flow number, Nq, is defined as Q/(nDI3), where Q is the volumetric flow rate of the discharge stream directly from the impeller in meters cubed per second (m3/s), and n and DIare defined as above. The flow number can be 0.1 to 1, or 0.4 to 0.7 for each impeller independently. A total volume of the cylindrical tank can be greater than or equal to 10 meters cubed (m3), or 20 to 100 m3, or 20 to 50 m3. The cylindrical tank can hold greater than or equal to 20,000 liters (L), or 20,000 to 50,000 L of liquid. The cylindrical tank can have an inner reactor diameter, DT, of 1 to 10 meters (m), or 2 to 5 m. The cylindrical tank can be defined by various lengths. For example,FIG.1illustrates that length C can be a length from a lowest point in the cylindrical tank12to a lowest point on the lower impeller16, length DILcan be two times the length of a blade of the lower impeller16, length S can be a length from level fluid line to a highest point of the upper impeller18, and length DIUcan be two times the length of the blades of the upper impeller18. These lengths are illustrated inFIG.1. It is noted that if there is only one impeller, then the one impeller is both the lower impeller and the upper impeller and both DILand DIUwould be equal to DI, for example, two times the length of the blades of the impeller. A ratio of C:DILcan be 0.05 to 0.5; or 0.1 to 0.2. A ratio of S:DIUcan be 0.1 to 2; 0.1 to 1. A reactant solution inlet can be located on the top of the cylindrical tank. A reactant solution inlet can be located on the side of the cylindrical tank. A reactant solution inlet can be located on the bottom of the cylindrical tank.FIG.4andFIG.5illustrate reactant solution inlet40located on the side of the cylindrical tank. The reactant solution inlet can allow for a polycarbonate precursor solution to be added to the reactor. One or both of additional catalyst and additional monomer can be added to the reactor through the same or through a different inlet. A reaction solution outlet can be located on the side of the cylindrical tank. A reaction solution outlet can be located on the bottom of the cylindrical tank. If located on the bottom of the cylindrical tank, the reaction solution outlet can be concentrically located on a central axis of the cylindrical tank. For example,FIG.1illustrates reaction solution outlet20concentrically located on axis100of cylindrical tank12. The reactant solution inlet can be in fluid communication with the externally located heat exchanger. For example, a heated stream leaving the externally located heat exchanger can be combined with a solution stream upstream of the reactant solution inlet and added as a combined stream. Conversely, the heated stream can be added to the cylindrical tank through a recirculation inlet that is different from the reactant solution inlet. The recirculation inlet can be located on the top of the cylindrical tank. The recirculation inlet can be located on the side of the cylindrical tank. The recirculation inlet can be located on the bottom of the cylindrical tank.FIG.1illustrates that recirculation inlet44can be in fluid communication with externally located heat exchanger50via heated stream54. The reaction solution outlet can be in fluid communication with the externally located heat exchanger. For example, a mixed solution stream exiting the reaction solution outlet can be split (for example, using a Y-junction or a T-junction) into at least two streams, where one of the streams is a recirculation stream that connects to the externally located heat exchanger. Conversely, the recirculation stream can exit the cylindrical tank via a recirculation outlet that is separate from the reaction solution outlet. The recirculation outlet can be located on the side of the cylindrical tank. The recirculation outlet can be located on the bottom of the cylindrical tank.FIG.1illustrates that recirculation outlet42can be in fluid communication with externally located heat exchanger50via recirculation stream52. The cylindrical tank can comprise a reactant solution inlet, a reaction solution outlet, a recirculation inlet, and a recirculation outlet. Both the recirculation inlet and the recirculation outlet can be located on the bottom of the cylindrical tank. The recirculation outlet can be located on the bottom of the cylindrical tank and the recirculation inlet can be located on the side of the cylindrical tank. The cylindrical tank can comprise a reactant solution inlet, a reaction solution outlet, and a recirculation inlet, where the reaction solution outlet is in fluid communication with both a second reactor via a mixed solution stream and the externally located heat exchanger via recirculation stream52. The reactor comprises a heat exchanger that is located external to the cylindrical tank and is referred to as the externally located heat exchanger. The externally located heat exchanger can comprise 1 or more externally located heat exchangers. When two or more externally located heat exchangers are present, the externally located heat exchangers can be configured in series and/or in parallel with each other. The cylindrical tank can be free of an internally located heat exchanger that is located inside the cylindrical tank, for example, internally located heating coils. Heating can be done through a reactor jacket. In other words, the heat management can be performed either through a reactor jacket and/or through an externally located heat exchanger. The reactor can comprise a heating jacket in physical contact with at least a portion of the outside wall of the cylindrical tank and also an externally located heat exchanger50, for example, as illustrated inFIG.1. A plurality of baffles can be located in the cylindrical tank. The plurality of baffles can comprise one or both of vertical baffles and circular baffles. The vertical baffles can be flat plate baffles vertically positioned in the tank such that the height (the longest side) of the flat plate baffles is parallel to the flat height of the side of the tank (or parallel to the impeller shaft axis) and the width (the shortest side) is perpendicular to a tangent of the round side of the cylindrical tank. The plurality of baffles can comprise 2 to 10, or 3 to 5 vertical baffles.FIG.4,FIG.6,FIG.7, andFIG.8are illustrations of a cylindrical reactor that comprises 4 vertical baffles62. The plurality of baffles can comprise 2 to 15, or 4 to 12, or 4 to 8 circular baffles.FIG.5,FIG.10, andFIG.11are illustrations of a cylindrical reactor that comprises 6 circular baffles64. An inner diameter of all of the circular baffles can be greater than or equal to DI. In other words, a rotation column defined by the rotation of the impeller, as illustrated inFIG.5as rotation column60, can be free of the circular baffles64. The presence of the circular baffles can result in a beneficial decrease in the mixing time. The reactor can be used to prepare a polycarbonate oligomer from a precursor solution. The precursor solution comprising the polycarbonate precursor can be formed in a monomer mixing unit. The monomer mixing unit can be maintained at atmospheric pressure and at a temperature of 100 to 250° C., or 150 to 200° C., or 165 to 185° C. The polycarbonate precursor can comprise a carbonate precursor, a bisphenol, a catalyst, and optionally a low molecular weight oligomer. The bisphenol and the carbonate precursor in the precursor solution can be present in a molar ratio of 0.5:1 to 1.5:1, or 0.9:1 to 1.1:1, or 0.99:1 to 1.01:1. The method of mixing a precursor solution in the reactor, can comprise adding the precursor solution comprising a polycarbonate precursor to the reactor through a reactant solution inlet; mixing and polymerizing the polycarbonate precursor at a reactor temperature and a reactor pressure to form a polycarbonate oligomer; and withdrawing a mixed solution comprising the polycarbonate oligomer having a weight average molecular weight that is greater than that of the polycarbonate precursor from a reaction solution outlet. The method can comprise removing a recirculation stream from the cylindrical reactor, flowing the recirculation stream through an externally located heat exchanger to form a heated stream, and reintroducing the heated stream to the cylindrical reactor. The reactor temperature can be 160 to 300 degrees Celsius (° C.), or 160 to 280° C., or 140 to 240° C., or 200 to 270° C., or 275 to 300° C. The reactor pressure can be 5 to 200 millibar absolute (mbar), or 30 to 200 mbar, or 2 to 25 mbar. The average residence time of the precursor solution in the reactor can be greater than or equal to a comparison average residence time of the precursor solution added to a same reactor but through a side feeder. The average residence time of the precursor solution in the reactor can be 0.1 to 15 hours. The mixing can result in the formation of an axial flow pattern, for example, as illustrated inFIG.7andFIG.11. The mixing can occur at a rotation speed of the stirring shaft14of 40 to 100 revolutions per minute (rpm). The mixing can achieve a normalized surface refresh rate of greater than or equal to 0.03 inverse seconds (s−1), or 0.04 to 0.4 s−1, or 0.06 to 0.1 s−1. As used herein, the normalized surface refresh rate is the volume of solution that passes across a plane located 200 millimeters (mm) below the liquid surface level per second per total volume of solution in the reactor (m3/s·m3or s−1). An example of plane, P, is illustrated inFIG.8. The mixing time can be less than or equal to 60 seconds (s), or 20 to 50 s. The reactor can comprise a plurality of circular baffles and the mixing time can be 20 to 40 s, or 20 to 30 s. The mixed solution can have a mixed solution viscosity that is greater than a precursor solution viscosity of the precursor solution. For example, the precursor solution can have a precursor solution viscosity that is less than or equal to 0.05 Pascal seconds (Pa·s) and the mixed solution can have a mixed solution viscosity of greater than or equal to 0.05 Pa·s, or greater than or equal to 0.5 Pa·s, or 0.05 to 0.5 Pa·s, or greater than or equal to 2.5 Pa·s, or 0.15 to 10 Pa·s, or 0.5 to 10 Pa·s. As used herein, the viscosity is determined using a parallel plate rheometer, AR-G2 from TA Instruments, using 25 mm diameter plates having a 0.5 mm gap between the plates. The measurements are made at a temperature of 250 to 300° C. and varying frequency from 100 and 1000 s−1. When the reactor is used in a melt polycarbonate polymerization plant, it can be used as an oligomerization reactor (also referred to as an oligomeriser). The oligomerization reactor can be in series with two or more oligomerizers. One or more of the oligomerisers can have an impeller mixer. For example, in a melt polymerization (also referred to herein as a melt transesterification reaction), the reactor can be a first reactor, the reactant solution inlet of the first reactor can be in fluid communication with a monomer mixing tank, the reaction solution outlet of the first reactor can be in fluid communication with a second reactor inlet of a second reactor, and a second reactor outlet of the second reactor can be in fluid communication with a polymerization reactor. The first reactor can have two impellers (for example, two hydrofoil impellers) located on the stirring shaft of the first reactor and the second reactor can have one impeller (for example, one hydrofoil impeller) located on the stirring shaft of the second reactor. The fluid flow in the first reactor can be turbulent flow and the first reactor can have a normalized surface refresh rate of 0.04 to 0.4 s−1, or 0.06 to 0.1 s−1. The fluid flow in the second reactor can be laminar flow and the second reactor can have a normalized surface refresh rate of 0.04 to 0.4 s−1, or greater than 0.04 to less than 0.4 s−1. The mixed solution can be added to a second reactor and the method can comprise adding the mixed solution to the second reactor, mixing and further polymerizing the mixed solution at a second temperature greater than the reactor temperature and a second pressure less than the reactor pressure, and withdrawing an oligomer solution comprising a high molecular weight polycarbonate oligomer having a weight average molecular weight that is greater than that of the polycarbonate oligomer. For example, the high molecular weight polycarbonate oligomer can have a weight average molecular weight of 1.5 to 15 kilodaltons, or 8 to 12 kilodaltons, 8 to 20 kilodaltons based on polycarbonate standards. The high molecular weight polycarbonate oligomer can have a viscosity of 1 to 10 Pa·s. The high molecular weight polycarbonate oligomer can then be polymerized in one or more polymerization vessels, for example, one or more wire wetting fall polymerization units, horizontal polymerizers, vertical polymerizers, reactive extruders, or a continuously stirred tanks. The second reactor can comprise a second cylindrical tank comprising a second top, a second side, and a second bottom, wherein the second bottom is convex, extending away from the second top; a second stirring shaft disposed within the second cylindrical tank along a second axis thereof so that it is rotatable from outside of the second cylindrical tank; a second impeller extending from the second stirring shaft in the second cylindrical tank and comprising a second plurality of blades that are each independently at a second angle α from a second orthogonal of the second axis; a second reactant solution inlet; a second reaction solution outlet; and a second externally located heat exchanger in fluid communication with the second cylindrical tank via a second recirculation stream and a second heated stream. It is noted that the term “second” is used for clarity to distinguish from the “first” reactor and that the term “downstream” could likewise be used. The first reactor temperature can be 160 to 300° C., or 160 to 275° C., or 160 to 250° C., or 200 to 270° C., or 230 to 270° C. The first reactor pressure can be 50 to 200 mbar, or 75 to 200 mbar. The mixed solution viscosity can be 0.05 to 1 Pa·s, or 0.05 to 0.5 Pa·s. The second reactor temperature can be 250 to 300° C., or 270 to 300° C. The second reactor pressure can be 5 to 50 mbar, or 10 to 40 mbar. The oligomer solution viscosity can be 0.5 to 10 Pa·s, or 1 to 5 Pa·s, or greater than or equal to 1 Pa·s. After polymerization, the polycarbonate can be extruded in an extruder where an optional quencher and an additive can be added to the molten polycarbonate. The extruder can be a twin-screw extruder and at least one of the components can be incorporated into the composition by feeding directly into the extruder at the throat and/or downstream of the throat through, for example, a side stuffer. The carbonate precursor can comprise a diaryl carbonate ester, for example, diphenyl carbonate or an activated diphenyl carbonate having electron-withdrawing substituents on each aryl, for example, at least one of bis(4-nitrophenyl)carbonate, bis(2-chlorophenyl)carbonate, bis(4-chlorophenyl)carbonate, bis(methyl salicyl)carbonate, bis(4-methylcarboxylphenyl) carbonate, bis(2-acetylphenyl) carboxylate, or bis(4-acetylphenyl) carboxylate. The diaryl carbonate ester can be free of an activated diphenyl carbonate having electron-withdrawing substituents on each aryl. For example, the diaryl carbonate ester can be free of bis(4-nitrophenyl)carbonate, bis(2-chlorophenyl)carbonate, bis(4-chlorophenyl)carbonate, bis(methyl salicyl)carbonate, bis(4-methylcarboxylphenyl) carbonate, bis(2-acetylphenyl) carboxylate, and bis(4-acetylphenyl) carboxylate. The diaryl carbonate ester can be free of bis(methyl salicyl)carbonate. As used herein, “can be free of” refers to none of the compounds being added in the melt polymerization, for example, less than or equal to 10 ppm, for example, 0 ppm of the compound being present. The carbonate precursor can comprise diphenyl carbonate. The bisphenol can comprise a dihydroxy compound of the formula HO—R1—OH, wherein the R1group can contain an aliphatic, an alicyclic, or an aromatic moiety. For example, the bisphenol can have the formula (2) HO-A1-Y1-A2-OH (2) wherein each of A1and A2is a monocyclic divalent aromatic group and Y1is a single bond or a bridging group having one or more atoms that separate A1from A2. One atom can separate A1from A2. The bisphenol can have the formula (3) wherein Raand Rbare each independently a halogen, C1-12alkoxy, or C1-12alkyl; and p and q are each independently integers of 0 to 4. It will be understood that Rais hydrogen when p is 0, and likewise Rbis hydrogen when q is 0. Also in formula (3), Xais a bridging group connecting the two hydroxy-substituted aromatic groups, where the bridging group and the hydroxy substituent of each C6arylene group are disposed ortho, meta, or para (specifically, para) to each other on the C6arylene group. The bridging group Xacan be single bond, —O—, —S—, —S(O)—, —S(O)2—, —C(O)—, or a C1-18organic bridging group. The C1-18organic bridging group can be cyclic or acyclic, aromatic or non-aromatic, and can further comprise heteroatoms, for example, halogens, oxygen, nitrogen, sulfur, silicon, or phosphorous. The C1-18organic bridging group can be disposed such that the C6arylene groups connected thereto are each connected to a common alkylidene carbon or to different carbons of the C1-18organic bridging group. p and q can each be 1, and Raand Rbare each a C1-3alkyl group, specifically, methyl, disposed meta to the hydroxy group on each arylene group. Xacan be a substituted or unsubstituted C3-18cycloalkylidene, a C1-25alkylidene of formula —C(Rc)(Rd)— wherein Rcand Rdare each independently hydrogen, C1-12alkyl, C1-12cycloalkyl, C7-12arylalkyl, C1-12heteroalkyl, or cyclic C7-12heteroarylalkyl, or a group of the formula —C(═Re)— wherein Reis a divalent C1-12hydrocarbon group. Groups of this type include methylene, cyclohexylmethylene, ethylidene, neopentylidene, and isopropylidene, as well as 2-[2.2.1]-bicycloheptylidene, cyclohexylidene, cyclopentylidene, cyclododecylidene, and adamantylidene. Xacan be a C1-18alkylene group, a C3-18cycloalkylene group, a fused C6-18cycloalkylene group, or a group of the formula —B1-G-B2— wherein B1and B2are the same or different C1-6alkylene group and G is a C3-12cycloalkylidene group or a C6-16arylene group. For example, Xacan be a substituted C3-18cycloalkylidene of formula (4) wherein Rr, Rp, Rq, and Rtare each independently hydrogen, halogen, oxygen, or C1-12hydrocarbon groups; Q is a direct bond, a carbon, or a divalent oxygen, sulfur, or —N(Z)— where Z is hydrogen, halogen, hydroxy, C1-12alkyl, C1-12alkoxy, or C1-12acyl; r is 0 to 2, t is 1 or 2, q is 0 or 1, and k is 0 to 3, with the proviso that at least two of Rr, Rp, Rq, and Rttaken together are a fused cycloaliphatic, aromatic, or heteroaromatic ring. It will be understood that where the fused ring is aromatic, the ring as shown in formula (4) will have an unsaturated carbon-carbon linkage where the ring is fused. When k is one and q is 0, the ring as shown in formula (4) contains 4 carbon atoms, when k is 2, the ring as shown in formula (4) contains 5 carbon atoms, and when k is 3, the ring contains 6 carbon atoms. Two adjacent groups (e.g., Rqand Rttaken together) can form an aromatic group or Rqand Rttaken together can form one aromatic group and Rrand Rptaken together form a second aromatic group. When Rqand Rttaken together form an aromatic group, Rpcan be a double-bonded oxygen atom, i.e., a ketone. Specific examples of bisphenol compounds of formula (3) include 1,1-bis(4-hydroxyphenyl) methane, 1,1-bis(4-hydroxyphenyl) ethane, 2,2-bis(4-hydroxyphenyl) propane (hereinafter “bisphenol A” or “BPA”), 2,2-bis(4-hydroxyphenyl) butane, 2,2-bis(4-hydroxyphenyl) octane, 1,1-bis(4-hydroxyphenyl) propane, 1,1-bis(4-hydroxyphenyl) n-butane, 2,2-bis(4-hydroxy-2-methylphenyl) propane, 1,1-bis(4-hydroxy-t-butylphenyl) propane, 3,3-bis(4-hydroxyphenyl) phthalimidine, 2-phenyl-3,3-bis(4-hydroxyphenyl) phthalimidine (PPPBP), and 1,1-bis(4-hydroxy-3-methylphenyl)cyclohexane (DMBPC). Combinations comprising at least one of the foregoing bisphenols can also be used. The bisphenol can comprise bisphenol A, in which each of A1and A2can be p-phenylene, and Y1can be isopropylidene in formula (3). The resultant “polycarbonate” as used herein is derived from the carbonate compound and the bisphenol and can have repeating structural carbonate units of formula (1) in which the R1groups contain aliphatic, alicyclic, and/or aromatic moieties (e.g., greater than or equal to 30 percent, specifically, greater than or equal to 60 percent, of the total number of R1groups can contain aromatic moieties and the balance thereof are aliphatic, alicyclic, or aromatic). Optionally, each R1can be a C6-30aromatic group that can contain at least one aromatic moiety. R1can be derived from the bisphenol. The precursor solution can comprise at least one of a quaternary compound or an alkali catalyst. The precursor solution can comprise a quaternary compound and an alkali catalyst can be added to the reactor as a separate catalyst stream. The quaternary catalyst comprises at least one of a quaternary ammonium compound or a quaternary phosphonium compound. The quaternary ammonium compound can be a compound of the structure (R4)4N+X−, wherein each R4is the same or different, and is a C1-20alkyl, a C4-20cycloalkyl, or a C4-20aryl; and X−is an organic or inorganic anion, for example, a hydroxide, halide, carboxylate, sulfonate, sulfate, formate, carbonate, or bicarbonate. Examples of organic quaternary ammonium compounds include tetramethyl ammonium hydroxide, tetrabutyl ammonium hydroxide, tetramethyl ammonium acetate, tetramethyl ammonium formate, and tetrabutyl ammonium acetate. The quaternary phosphonium compound can be a compound of the structure (R5)4P+X−, wherein each R5is the same or different, and is a C1-20alkyl, a C4-20cycloalkyl, or a C4-20aryl; and X−is an organic or inorganic anion, for example, a hydroxide, phenoxide, halide, carboxylate, for example, acetate or formate, sulfonate, sulfate, formate, carbonate, or bicarbonate. Where X−is a polyvalent anion, for example, carbonate or sulfate, it is understood that the positive and negative charges in the quaternary ammonium and phosphonium structures are properly balanced. For example, where R20to R23are each methyls and X−is carbonate, it is understood that X−represents 2(CO3−2). Examples of organic quaternary phosphonium compounds include tetramethyl phosphonium hydroxide, tetramethyl phosphonium acetate, tetramethyl phosphonium formate, tetrabutyl phosphonium hydroxide, tetraethyl phosphonium acetate, tetrapropyl phosphonium acetate, tetrabutyl phosphonium acetate (TBPA), tetrapentyl phosphonium acetate, tetrahexyl phosphonium acetate, tetraheptyl phosphonium acetate, tetraoctyl phosphonium acetate, tetradecyl phosphonium acetate, tetradodecyl phosphonium acetate, tetratolyl phosphonium acetate, tetramethyl phosphonium benzoate, tetraethyl phosphonium benzoate, tetrapropyl phosphonium benzoate, tetraphenyl phosphonium benzoate, tetraethyl phosphonium formate, tetrapropyl phosphonium formate, tetraphenyl phosphonium formate, tetramethyl phosphonium propionate, tetraethyl phosphonium propionate, tetrapropyl phosphonium propionate, tetramethyl phosphonium butyrate, tetraethyl phosphonium butyrate, tetrapropyl phosphonium butyrate, tetraphenyl phosphonium acetate (TPPA), and tetraphenyl phosphonium phenoxide (TPPP). The quaternary catalyst can comprise at least one of tetrabutyl phosphonium acetate, TPPP, or TPPA. The amount of the quaternary catalyst can be added based upon the total number of moles of bisphenol employed in the polymerization reaction. When referring to the ratio of catalyst, for example, phosphonium salt, to all bisphenols employed in the polymerization reaction, it is convenient to refer to moles of phosphonium salt per mole of the bisphenol(s), meaning the number of moles of phosphonium salt divided by the sum of the moles of each individual bisphenol present in the reaction mixture. The amount of the optional quaternary catalyst (e.g., organic ammonium or phosphonium salts) can each independently be employed in an amount of 1×10−2to 1×10−5, or 1×10−3to 1×104moles per total mole of the bisphenol(s) in the monomer mixture. The alkali catalyst comprises a source of one or both of alkali ions and alkaline earth ions. The sources of these ions can include alkaline earth hydroxides, for example, magnesium hydroxide and calcium hydroxide. Sources of alkali metal ions can include the alkali metal hydroxides, for example, at least one of lithium hydroxide, sodium hydroxide, or potassium hydroxide. Examples of alkaline earth metal hydroxides are calcium hydroxide and magnesium hydroxide. The alkali catalyst can comprise sodium hydroxide. Other possible sources of alkaline earth and alkali metal ions include salts of carboxylic acids (for example, sodium acetate) or derivatives of ethylene diamine tetraacetic acid (EDTA) (for example, EDTA tetrasodium salt, and EDTA magnesium disodium salt). For example, the alkali catalyst can comprise at least one of an alkali metal salt(s) of a carboxylic acid or an alkaline earth metal salt(s) of a carboxylic acid. In another example, the alkali catalyst comprises Na2Mg EDTA or a salt thereof. The alkali catalyst can also, or alternatively, comprise salt(s) of a non-volatile inorganic acid. For example, the alkali catalyst can comprise salt(s) of a non-volatile inorganic acid, for example, at least one of NaH2PO3, NaH2PO4, Na2HPO3, KH2PO4, CsH2PO4, or Cs2HPO4. Alternatively, or in addition, the alkali catalyst can comprise mixed alkali metal salt(s) of phosphoric acid, for example, at least one of NaKHPO4, CsNaHPO4, or CsKHPO4. The alkali catalyst can comprise KNaHPO4, wherein a molar ratio of Na to K is 0.5 to 2. The alkali catalyst typically can be used in an amount sufficient to provide 1×10−2to 1×10−8moles, or 1×10−4to 1×10−7moles of metal hydroxide per mole of the bisphenol(s). Quenching of the transesterification catalysts and any reactive catalyst residues with an acidic compound after polymerization can be completed can also be useful in some melt polymerization processes. Among the many quenchers that can be used are alkyl sulfonic esters of the formula R8SO3R9wherein R8is hydrogen, C1-12alkyl, C6-18aryl, or C7-19alkylaryl, and R9is C1-12alkyl, C6-18aryl, or C7-19alkylaryl. Examples of quenchers include benzenesulfonate, p-toluenesulfonate, methylbenzene sulfonate, ethylbenzene sulfonate, n-butyl benzenesulfonate, octyl benzenesulfonate and phenyl benzenesulfonate, methyl p-toluenesulfonate, ethyl p-toluenesulfonate, n-butyl p-toluene sulfonate, octyl p-toluenesulfonate, and phenyl p-toluenesulfonate. In particular, the quencher can comprise an alkyl tosylate, for example, n-butyl tosylate. The following examples are provided to illustrate the impeller reactor. The examples are merely illustrative and are not intended to limit devices made in accordance with the disclosure to the materials, conditions, or process parameters set forth therein. EXAMPLES Example 1 Flow Evaluation of an Impeller Reactor A computational fluid dynamics evaluation of fluid flow in a reactor having an upper impeller and a lower impeller was performed using ANSYS CFD software. In the evaluation, a reactor as illustrated inFIG.1having 4 vertical baffles and the heating loop was modelled, with the parameters listed in Table 1. TABLE 1Design ParameterValueLiquid volume (L)22,360Liquid Level, fluid height/DT0.61DI/DTratio0.55Impeller speed (RPM)60Number of impellers2Type of impellerHydrofoilBaffles typeVerticalNumber of baffles4Baffle width (m)0.316Baffle clearance (m)0.063C/DILRatio0.15S/DIURatio0.5Mass flow (kg/h)29,596Recirculation flow (kg/h)592,310Temperature (° C.)258Density (kg/m3)1040Viscosity (cP)150 The resulting flow pattern is shown inFIG.6that illustrates the resulting velocity contours as well as inFIG.7andFIG.8that illustrate the resulting stream lines. These figures illustrate the presence of a single axial flow loop and that good mixing is achieved in the impeller mixer.FIG.8further illustrates Plane, P, located 200 mm below the surface of the solution. The software determined that there was a normalized surface refresh rate through this surface of 0.072 s−1. It is noted that the normalized surface refresh rate of 0.072 s−1is greater than the target volumetric surface refresh rate of 0.07 s−1. Examples 2-3 Residence Time Evaluation Residence time evaluations were performed, where, after a steady state was achieved, a tracer was injected upstream of the reactor and the concentration of the tracer in a line downstream of the reactor was determined with time. In Example 2, the simulation was performed in accordance with the reactor of Example 1. In Example 3, the simulation was performed based on mixing in a Maxblend flat blade mixer from Sumitomo. The performance metrics of Examples 2 and 3 relative to the performance objectives are shown in Table 2. The concentration profiles were then compared to that of a theoretical continuously stirred tank (CSTR). The results are illustrated inFIG.9. TABLE 2ExamplePerformance23ObjectiveReactor Volume (L)22,36011,18022,360Normalized Surface Refresh0.0720.069≥0.07Rate at 0.2 m below the liquidlevel (s−1)Unit Power (kW/m3)0.741.36≤1.36Mixing Time (s)3223≤29Average residence time (min)474646 Surprisingly, merely by utilizing an impeller mixer, the peak observed in Example 2 is nearly eliminated as compared to the flat blade mixer of Example 3 and the reactor of Example 2 achieves almost ideal mixing.FIG.9further clearly shows a delayed peak formation of Example 2 as compared to Example 3. This result indicates that using the impeller mixer, a longer residence time in the reactor is achieved for some fluid elements in the beginning part of residence time distribution (RTD) profile. In other words, the residence time distribution profile was improved and is closer to ideal residence time distribution behavior. Therefore, when the impeller mixer is used in an oligomerization reaction, a longer residence time can be achieved for some of the early leaving fluid elements, which can ultimately result in an increased reaction time for the reactants in the reactor and potentially allowing for the increased higher molecular weight with a reduced polydispersity. It is further noted that the specific unit power of Example 2 was 40% less than the unit power of Example 3. Here, it is noted that not only is the mixing performance improved as shown by the improvement in the residence time distribution, but the mixing performance is improved using a reduced mixing power. Examples 4-8 Effect of Baffles The impeller reactors of Examples 4-7 were modelled in accordance with Example 1 except the type and number of baffles and the mixing speed was varied. The reactor of Example 8 was a Maxblend Sumitomo reactor of Example 3. The results are shown in Table 3. Surface refresh rate and mixing time are important metrics for mixing performance as they indicate the mixing effectiveness inside the reactor. In comparing Examples 4 and 5 and Examples 6 and 7, it is shown that at the same mixing speed, although the surface refresh rate is reduced, the mixing times are beneficially reduced when circular baffles are used as opposed to vertical baffles. Further, as compared to Example 8, there is a significant reduction in unit power. TABLE 3Example45678BafflesCircularVerticalCircularVerticalVerticalwithinternalheating coilsMixing Speed (rpm)100100606053Normalized Surface0.0380.0790.0320.0510.069Refresh Rate (s−1)Unit Power (kW/m3)0.951.240.220.281.36Mixing Time (s)2436384823 Set forth below are non-limiting embodiments of the present disclosure. Aspect 1: A reactor for carrying out a melt transesterification reaction at a reactor temperature of 160 to 300° C. and a reactor pressure of 5 to 200 mbar, comprising: a cylindrical tank comprising a top, a side, and a bottom, wherein the bottom is convex, extending away from the top; a stirring shaft disposed within the cylindrical tank along an axis thereof so that it is rotatable from outside of the cylindrical tank; an impeller extending from the stirring shaft in the cylindrical tank and comprising a plurality of blades that are each independently at an angle α from an orthogonal of the axis; a reactant solution inlet; a reaction solution outlet; and an externally located heat exchanger in fluid communication with the cylindrical tank via a recirculation stream and a heated stream. The reactor can be a vertical reactor such that axis is perpendicular (within 10°, or 0 to 5°, or 0 to 1°) to at least one of a plane tangent to the bottom of the cylindrical tank or a top fluid plane as defined by a top surface of a resting liquid present in the cylindrical tank. Aspect 2: The reactor of Aspect 1, wherein a lower impeller and an upper impeller extend from the stirring shaft, wherein the lower impeller and the upper impeller are both three blade impellers. Aspect 3: The reactor of Aspect 2, wherein the lower impeller and the upper impeller are offset by an angle θ of 50 to 70 degrees. Aspect 4: The reactor of any one or more of Aspects 2 to 3, wherein the angle α of the blades of the lower impeller and the angle α of the upper impeller are each independently 25 to 65 degrees. Aspect 5: The reactor of any one or more of Aspects 2 to 4, wherein the lower impeller and the upper impeller direct a fluid flow in a direction of the stirring shaft towards the bottom of the cylindrical tank, from the bottom of the cylindrical tank towards the side of the cylindrical tank, and upward along the side of the cylindrical tank to form a circulation loop. Aspect 6: The reactor of any one or more of the preceding aspects, wherein the externally located heat exchanger is in fluid communication with the cylindrical tank via the recirculation stream that connects a recirculation outlet with the externally located heat exchanger and the heated stream that connects a recirculation inlet with the externally located heat exchanger. In an aspect, the externally located heat exchanger is only in fluid communication with the cylindrical tank via the recirculation stream that connects a recirculation outlet with the externally located heat exchanger and the heated stream that connects a recirculation inlet with the externally located heat exchanger Aspect 7: The reactor of any one or more of the preceding aspects, wherein the impeller has a power number of less than or equal to 2. Aspect 8: The reactor of any one or more of the preceding aspects, wherein the cylindrical tank is free of internally located heating coils. Aspect 9: The reactor of any one or more of the preceding aspects, wherein a ratio of C:DIis 0.05 to 0.5; or 0.1 to 0.2 and a ratio of S:DIis 0.1 to 2; 0.1 to 1, wherein C is a length from a lowest point in the cylindrical tank to a lowest point on the impeller; DIis two times the length of a blade of the impeller; and S is a length from a level fluid line to a highest point of the impeller. Aspect 10: A method of melt polymerizing a polycarbonate, comprising adding a precursor solution comprising a polycarbonate precursor to the reactor of any one or more of the preceding aspects through the reactant solution inlet; mixing and polymerizing the polycarbonate precursor at the reactor temperature of 160 to 300° C., preferably, 230 to 280° C., and the reactor pressure of 5 to 200 mbar to form a polycarbonate oligomer; and withdrawing a mixed solution comprising the polycarbonate oligomer having a weight average molecular weight that is greater than that of the polycarbonate precursor from the reaction solution outlet. Aspect 11: The method of Aspect 10, wherein the mixed solution has a mixed solution viscosity of greater than or equal to 0.05 Pa·s. Aspect 12: The method of any one or more of Aspects 10 to 11, wherein the polycarbonate precursor comprises bisphenol A and diphenyl carbonate. Aspect 13: The method of any one or more of Aspects 10 to 12, further comprising directing the mixed solution into a second reactor optionally of any one or more of Aspects 1 to 9; mixing the mixed solution at a second temperature greater than the reactor temperature and a second pressure less than the reactor pressure; and withdrawing an oligomer solution from the second reactor comprising a high molecular weight polycarbonate oligomer having a weight average molecular weight that is greater than that of the polycarbonate oligomer. Aspect 14: The method of any one or more of Aspects 10 to 13, wherein the mixing occurs at a rotation speed of the stirring shaft of 40 to 100 revolutions per minute. Aspect 15: A method of polymerizing a polycarbonate, comprising: adding a carbonate precursor, a bisphenol, and a quaternary catalyst to a monomer mixing tank to form a precursor solution; adding the precursor solution to a first oligomeriser and mixing and polymerizing the polycarbonate precursor in the first oligomeriser at a first reactor temperature of 200 to 270° C., preferably, 245 to 265° C., and a first reactor pressure of 50 to 200 mbar to form a polycarbonate oligomer having a first viscosity of 0.05 to 0.5 Pa·s; withdrawing a mixed solution from the first oligomeriser comprising the polycarbonate oligomer having a weight average molecular weight that is greater than that of the polycarbonate precursor from the reaction solution outlet; directing the mixed solution into a second oligomeriser and mixing the mixed solution at a second temperature of 275 to 300° C. and a second pressure of 2 to 25 mbar; withdrawing an oligomer solution comprising a high molecular weight polycarbonate oligomer having a weight average molecular weight of 8 to 20 kilodaltons based on polystyrene standards and a viscosity of greater than or equal to 1 Pa·s; and directing the high molecular weight polycarbonate oligomer to a series of polymerization vessels; wherein at least of the first oligomeriser and the second oligomeriser are described by the reactor of any one or more of Aspects 1 to 9. Aspect 16: The method of Aspect 15, wherein the first oligomeriser and the second oligomeriser are described by the reactor of any one or more of Aspects 1 to 9, wherein the first oligomeriser has two impellers located on the stirring shaft and the second oligomeriser has one impeller located on the stirring shaft. Aspect 17: Use of the reactor of any one or more of Aspects 1 to 9 in preparing a polycarbonate oligomer. Aspect 18: The method of any one or more of the preceding method aspects, wherein the impeller has a power number of less than or equal to 2. Aspect 19: The method of any one or more of the preceding method aspects, further comprising directing a recirculation stream from the cylindrical tank to the externally located heat exchanger; heating the recirculation stream in the externally located heat exchanger to form the heated stream; and directing the heated stream back into the cylindrical tank. Aspect 20: The method of Aspect 19, wherein an increased temperature of the heated stream, Tf, is greater than an initial temperature of the recirculation stream, Ti, for example, Tfis greater than or equal to Ti+10° C., or greater than or equal to Ti+20° C., or greater than or equal to Ti+50° C., optionally, less than or equal to Ti+100° C. Aspect 21: The method of any one or more of Aspects 19 to 20, wherein all of the recirculation stream withdrawn from the cylindrical tank, for example, 99 to 100 wt % based on the total weight of the recirculation stream withdrawn, is reintroduced to the cylindrical tank as the heated stream. Aspect 22: The method of any one or more of Aspects 19 to 21, wherein one or both of the recirculation stream and the heated stream are in the liquid form. Aspect 23: The method of any one or more of Aspects 19 to 22, wherein a mass flow rate of the recirculation stream entering the external heat exchanger is equal to a mass flow rate of the heated stream exiting the external heat exchanger. For example, a mass flow rate of the heated stream can be within 5%, or within 1%, or within 0.1% of the mass flow rate of the recirculation stream. Aspect 24: The method of any one or more of Aspects 19 to 23, wherein the heated stream entering the cylindrical tank comprises, or consists essentially of only the heated composition of the recirculation stream. In an aspect, the heated stream can be free of an additionally added component. Aspect 25: The reactor of any one or more of the preceding aspects further comprising a controller configured to control at least one of a flow rate, a pressure, or a temperature in the reactor and a method one or more of the preceding aspects can comprise monitoring at least one of a flow rate, a pressure, or a temperature in the reactor and adjusting one or more of said variables via a controller. Aspect 25: The reactor of any one or more of the preceding aspects, wherein the heated stream is only in fluid communication with the externally located heat exchanger and the cylindrical tank. Aspect 26: The reactor of any one or more of the preceding aspects, wherein the recirculation stream are only in fluid communication with the cylindrical tank and the externally located heat exchanger. Aspect 27: The reactor of any one or more of the preceding aspects, wherein the externally located heat exchanger is configured to impart a heat to the recirculation stream. The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any appropriate materials, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), steps, or components, that are otherwise not necessary to the achievement of the function or objectives of the compositions, methods, and articles. The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term “or” means “and/or” unless clearly indicated otherwise by context. Reference throughout the specification to “an embodiment”, “another embodiment”, “some embodiments”, “an aspect”, and so forth, means that a particular element (e.g., feature, structure, step, or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not. The term “combination” is inclusive of blends, mixtures, alloys, reaction products, and the like. The suffix “(s)” as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the colorant(s) includes one or more colorants). Unless specifically stated, the terms “first,” “second,” and the like, “primary,” “secondary,” and the like, as used herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. Also, “at least one of” means that the list is inclusive of each element individually, as well as combinations of two or more elements of the list, and combinations of at least one element of the list with like elements not named. In general, the compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any ingredients, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated, conducted, or manufactured so as to be devoid, or substantially free, of any ingredients, steps, or components not necessary to the achievement of the function or objectives of the present claims. The endpoints of all ranges directed to the same component or property are inclusive of the endpoints, are independently combinable, and include all intermediate points and ranges. For example, ranges of “up to 25 wt %, or 5 to 20 wt %” is inclusive of the endpoints and all intermediate values of the ranges of “5 to 25 wt %,” for example, 10 to 23 wt %, etc. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference. While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to embrace all such alternatives, modifications variations, improvements, and substantial equivalents. | 53,586 |
11859049 | DETAILED DESCRIPTION OF THE EMBODIMENTS As used herein, terms such as first, second, and the like can be used to describe various components, and the terms are used only to discriminate one component from another component. Further, the terms used herein are used only to describe exemplary embodiments, and are not intended to limit the present disclosure. A singular expression includes a plural expression unless they have definitely opposite meanings in the context. It should be understood that the terms “comprise”, “include”, and “have” as used herein are intended to designate the presence of stated features, numbers, steps, constitutional elements, or combinations thereof, but it should be understood that they do not preclude a possibility of existence or addition of one or more other features, numbers, steps, constitutional elements, or combinations thereof. Although various modifications can be made to the present disclosure and the present disclosure can have various forms, hereinafter, specific embodiments will be illustrated and explained in detail. However, it should be understood that the present disclosure is not limited to specific disclosed forms, and the present disclosure includes all changes, equivalents and substitutions included in the spirit and technical scope of the invention. Hereinafter, a method for preparing a polyalkylene carbonate according to a preferred embodiment of the present disclosure will be described. The present disclosure relates to a method for preparing a polyalkylene carbonate in which unreacted epoxide compounds generated in the preparation process of polyalkylene carbonate resins are removed before the high-temperature process, thereby preventing side reactions caused by unreacted epoxide compounds and reduce steam energy during solvent removal. Specifically, according to one embodiment of the invention, there is provided a method for preparing a polyalkylene carbonate comprising the steps of: polymerizing a monomer containing an epoxide compound and carbon dioxide in a solvent in the presence of an organic zinc catalyst to provide a first mixture containing a polyalkylene carbonate, an unreacted epoxide compound, an unreacted carbon dioxide, a residual catalyst, a by-product and a solvent; removing the unreacted carbon dioxide and residual catalyst from the first mixture; removing the unreacted epoxide compound from the first mixture from which the unreacted carbon dioxide and residual catalyst have been removed by using a stripper to provide a second mixture containing a polyalkylene carbonate, a by-product, and a solvent; heat-exchanging the second mixture; heating the heat-exchanged second mixture; and removing the solvent and by-product from the heated second mixture. According to the present disclosure, by using a stripper using a stripping agent having a low boiling point, an unreacted epoxide compound is removed at a lower temperature than the prior art, and therefore, side reactions due to the residual epoxide compound can be prevented. At this time, since carbon dioxide is too light during the polyalkylene carbonate polymerization process, carbon dioxide remaining after the polymerization process can be easily vaporized and removed. Further, according to the present disclosure, after the unreacted epoxide compound is removed before proceeding to the high temperature process in the preparation of a polyalkylene carbonate resin, the step of increasing the temperature of the polymeric solution containing the polyalkylene carbonate to the maximum level by using a heat exchanger is performed. Through this step, the present disclosure can reduce the amount of steam required for a heater when removing the solvent from the polymer solution. Therefore, the method of the present disclosure can reduce the amount of steam energy used for removing the solvent, and thus contribute to reducing the overall process cost. In addition, the present disclosure can reduce the content of a by-product (polyalkylene glycol) due to a side reaction product of an unreacted epoxide compound, thereby minimizing deterioration in physical properties of a polymer product. More specifically, the method for preparing the polyalkylene carbonate according to the present disclosure will be described step by step. First, the present disclosure polymerizes a monomer containing an epoxide compound and carbon dioxide in a solvent in the presence of an organic zinc catalyst, thereby providing a first mixture containing a polyalkylene carbonate, an unreacted epoxide compound, an unreacted carbon dioxide, a residual catalyst, a by-product and a solvent. In this case, the by-product can be removed together with the solvent in the step of removing the solvent. The by-product can be an alkylene carbonate generated during the preparation of a polyalkylene carbonate. For example, it can include an alkylene carbonate having 2 to 5 carbon atoms. More specifically, the by-product is ethylene carbonate. The step of providing the first mixture can be carried out through polymerization of a monomer containing carbon dioxide and an epoxide compound under a catalyst and a solvent, according to a method well known in the art. Further, the method can include a step of removing the unreacted carbon dioxide and the residual catalyst from the first mixture before being charged into a stripper for providing the second mixture. At this time, the residual catalyst can be removed from the second mixture according to a well-known method. Further, the unreacted carbon dioxide can be easily removed using a vaporization method using a compression means (compression system). Further, the method can further include a step of purifying raw materials before the polymerization step of the monomer. This step is a step of purifying and preparing an epoxide compound and carbon dioxide for use in the reaction. The polymerization step can be performed at 50 to 100° C. under 20 to 40 bar for 2 to 20 hours. Through such a step, a polymerization solution of polyalkylene carbonate containing a polyalkylene carbonate, an unreacted epoxide compound, an unreacted carbon dioxide, a residual catalyst, a by-product and a solvent is provided. In addition, the step of removing the unreacted carbon dioxide and the residual catalyst in advance can be further performed before removing the unreacted carbon dioxide. The epoxide compound used in the polymerization of the polyalkylene carbonate can be one or more selected from the group consisting of an alkylene oxide having 2 to 20 carbon atoms which is unsubstituted or substituted with a halogen or an alkyl group having 1 to 5 carbon atoms; a cycloalkylene oxide having 4 to 20 carbon atoms which is unsubstituted or substituted with a halogen or an alkyl group having 1 to 5 carbon atoms; and a styrene oxide having 8 to 20 carbon atoms which is unsubstituted or substituted with a halogen or an alkyl group having 1 to 5 carbon atoms. More preferably, the epoxide compound can include an alkylene oxide having 2 to 20 carbon atoms which is unsubstituted or substituted with a halogen or an alkyl group having 1 to 5 carbon atoms. Further, specific examples of the epoxide compound include ethylene oxide, propylene oxide, butene oxide, pentene oxide, hexene oxide, octene oxide, decene oxide, dodecene oxide, tetradecene oxide, hexadecene oxide, octadecene oxide, butadiene monoxide, 1,2-epoxy-7-octene, epifluorohydrin, epichlorohydrin, epibromohydrin, isopropyl glycidyl ether, butyl glycidyl ether, t-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, allyl glycidyl ether, cyclopentene oxide, cyclohexene oxide, cyclooctene oxide, cyclododecene oxide, alpha-pinene oxide, 2,3-epoxy norbornene, limonene oxide, dieldrin, 2,3-epoxypropylbenzene, styrene oxide, phenyl propylene oxide, stilbene oxide, chlorostilbene oxide, dichloro stilbene oxide, 1,2-epoxy-3-phenoxypropane, benzyloxymethyloxirane, glycidyl-methylphenyl ether, chlorophenyl-2,3-epoxypropyl ether, epoxypropyl methoxyphenyl ether, biphenyl glycidyl ether, glycidyl naphthyl ether, and the like. Preferably, the epoxide compound is ethylene oxide. The carbon dioxide can be charged continuously or discontinuously during the reaction, but is preferably continuously charged. In this case, it is preferable to use a continuous type or a semi-batch type as the polymerization reactor. If the carbon dioxide is not continuously charged, the production of by-products such as polyethylene glycol can be increased apart from the carbonate copolymerization reaction intended in the present disclosure. In addition, when carbon dioxide is continuously charged in the polymerization, the reaction pressure can be 5 to 50 bar or 10 to 40 bar. The catalyst used in the present disclosure can include a metal complex compound such as zinc, aluminum, or cobalt, and preferably, a zinc-based catalyst is used. The type of the zinc-based catalyst is not limited, and it can include zinc complex compounds that are well known in the art. As the solvent, methylene chloride, ethylene dichloride, dioxolane or the like can be used, and more preferably, methylene chloride can be used. Further, the present disclosure performs a step of removing the unreacted epoxide compound from the first mixture using a stripper to provide a second mixture containing a polyalkylene carbonate, a by-product, and a solvent. The present disclosure performs a step of first removing the unreacted epoxide compound from the first mixture before proceeding to the high temperature step in the preparation of a polyalkylene carbonate, and then removing the solvent from the polymer solution through a heat exchange step described later, so that side reactions due to unreacted epoxides can be prevented, steam energy consumption can be reduced, and production costs can be reduced. Since the unreacted epoxide monomer is converted to a polyalkylene glycol due to self-polymerization in the subsequent high-temperature step so that the physical properties of the final product can be reduced, it is desirable to minimize and reduce its content in order to improve the physical properties of the product. Thus, the method according to the present disclosure can provide the effect of excellently maintaining the product properties because the content of polyalkylene glycol in the final product is small. Specifically, according to the present disclosure, a first mixture (mixture solution) that has undergone the step of removing residual unreacted carbon dioxide and a catalyst after polymerization is charged into a stripper. At this time, to prevent side reactions due to unreacted epoxide contained in the first mixture, the first mixture is charged into the stripper under a temperature condition of 90° C. or less, preferably 80 to 90° C., and residual epoxide compounds in the first mixture are first removed. When the first mixture is charged into a stripper, the problem of self-polymerization of the unreacted epoxide occurs when the temperature condition is 90° C. or higher. This tendency is rapidly accelerated as the temperature rises. Further, even if the temperature is too low, the efficiency of removing unreacted epoxide from the stripper is reduced. This is because as the temperature is lowered, the epoxide removal efficiency is rapidly reduced. Therefore, when the first mixture is charged into a stripper, it is preferable to increase the charging temperature to the maximum level under the conditions of suppressing the self-polymerization of the unreacted epoxide, but if the range is too high or too low, it causes the above-described problems. Accordingly, in the present disclosure, it is preferable to remove the unreacted epoxide by charging the first mixture into the stripper within the above-described range. Thus, in the present disclosure, by optimizing the temperature of the first mixture charged into the stripper as described above, the side reaction due to the unreacted epoxide can be reduced and the content of the by-product (polyalkylene glycol) due to the side reaction product of the unreacted epoxide compound can be reduced compared to the prior art, thereby minimizing the deterioration of the physical properties of the polymer product. Further, the step of removing the unreacted epoxide compound using the stripper can include charging carbon dioxide as a stripping agent. Preferably, the carbon dioxide charged as the stripping agent is unreacted carbon dioxide, and is preferably charged in an amount of 75 to 85% by weight based on the total content of the unreacted carbon dioxide (gas) which is in an uncondensed state. Specifically, most of the unreacted carbon dioxide is obtained as a gas in an uncondensed state even through a compression means (compression system). Of these, 15 to 25% by weight is purged to prevent concentration, and the remaining 75 to 85% by weight can be utilized as a stripping agent of a stripper. When the first mixture passes through the stripper, a part of the solvent can be removed together with the unreacted epoxide compound in the first mixture. Therefore, a second mixture containing a polyalkylene carbonate, a by-product, and a solvent can be provided through the above step. Next, the present disclosure performs a step of heat-exchanging a second mixture containing the polyalkylene carbonate from which the unreacted epoxide compound has been removed, a by-product, and a solvent. The step of heat-exchanging the second mixture means a step of heat-exchanging a high-temperature vapor stream and a low-temperature liquid stream in the process (heat integration). By performing such a step, the low-temperature liquid stream can be heated to some extent by utilizing the sensible heat and latent heat of the high-temperature vapor stream, so that the amount of steam energy used can be reduced. Preferably, the heat-exchanging step can include raising the temperature of the second mixture containing the polyalkylene carbonate, by-product and solvent that have passed through the stripper using at least one heat exchanger. Specifically, the second mixture that has passed through the stripper is obtained at a temperature of 40° C. or less, or about 30 to 40° C. This second mixture can be heated through a heat exchanger and a heater. The heat exchanger can be installed to connect with a condenser and a means for removing the solvent from the second mixture containing a polyalkylene carbonate, a by-product and a solvent. More specifically, the heat exchanger can be connected through a line connected to the upper part of the means for removing the solvent, and through this line, heat from the means for removing the solvent can be transferred to the heat exchanger. Therefore, the temperature of the second mixture of 40° C. or less charged into the heat exchanger can be raised to the maximum level due to the heat of the means for removing the solvent connected to the heat exchanger. For example, the temperature of the second mixture through the heat exchanger can be about 80° C. or higher or 85 to 95° C. Through this step, the stream temperature of the second mixture is increased to the maximum level, so that the amount of steam required for the heater when removing the solvent contained in the second mixture can be reduced. Following the above step, it is possible to perform a step of removing the unreacted epoxide compound, and removing the solvent from the polymerization solution (i.e., the heat-exchanged second mixture) whose temperature has risen to the maximum level during heat exchange. That is, after the second mixture is heated through a heat exchanger and a heater in the above step, the solvent is removed by vaporization from the heated second mixture, and by-products can also be removed in this process. More preferably, since the second mixture that has undergone the heat exchange step contains a by-product together with polyalkylene carbonate and the solvent, the by-product can be removed together when removing the solvent. Specifically, a step of heating a second mixture containing the heat-exchanged polyalkylene carbonate, by-product and solvent; and a step of removing the solvent and by-products from the second mixture containing the heated polyalkylene carbonate, solvent and by-product are performed. The step of heating the second mixture can be performed through a heater connected to a heat exchanger. The second mixture charged into the heater can be heated through steam connected to the heater. The second mixture heated by the heater can be charged into a means for removing one or more solvents to perform a step of removing the solvent contained in the second mixture, and in this step, by-products can also be removed. Preferably, the step of removing the solvent and by-product can include a step of removing the solvent and by-products at least two or more times from the second mixture containing the polyalkylene carbonate and the solvent by utilizing a means for removing one or more solvents. The step for removing the solvent and by-products can use a combination of one or more devices selected from the group consisting of a flash vessel, a simple flash drum, a falling film evaporator, a thin film evaporator, an extruder DV, and a kneader or a film extruder. According to an embodiment of the present disclosure, when two kinds of solvents are contained in the second mixture, the step of removing the solvent and by-products can include a step in which the heated second mixture is charged into a means for removing the first solvent to firstly remove the first solvent, and then a second mixture from which the first solvent has been firstly removed is charged into the means for removing the second solvent to secondly remove the remaining solvent and remove by-products. For example, the second mixture having the temperature range raised to the maximum level in the heat exchanger can be charged into a heater and then charged into a means for removing the solvent at a temperature of about 110° C. or higher or in a temperature range of 115 to 125° C. Further, the second mixture charged at a temperature of about 110° C. or higher or in a temperature range of 115 to 125° C. is charged into a means for removing the first solvent, and then the solvent is firstly removed. The removed solvent can have a temperature in the range of about 95° C. to 105° C. Such a solvent can be charged into a condenser, liquefied and then recovered as a solvent at about 50° C. Further, in the second mixture that has passed through the means for removing the first solvent, some are charged into a means for removing the second solvent in a temperature range of about 95 to 105° C., and then the polymer can be recovered after the remaining solvent is removed. At this time, the removed solvent can exhibit a temperature of about 160° C. or higher, and can be recovered through a heat exchanger and a condenser. Further, the by-product can be removed together with the solvent in a means for removing at least one second solvent. According to the present disclosure, by performing these steps, the solvent contained in the second mixture can be removed through two steps, and recover after being liquefied through a condenser. The recovered liquid solvent can be reused for the polymerization of polyalkylene carbonate. In addition, since by-products are also removed in the above step, a product having excellent physical properties can be recovered. Therefore, in the present disclosure, steam energy can be reduced in the step of removing the solvent according to the above-described method, so that the solvent contained in the second mixture can be more economically removed than before. On the other hand,FIG.1briefly shows the structure for removing an unreacted epoxide compound and a solvent in the method for preparing a polyalkylene carbonate according to an embodiment of the present disclosure. Referring toFIG.1a stripper, a heat exchanger, a heater, a flash vessel and an extruder DV are connected and installed, and the solvent removed from the flash vessel and the extruder DV is connected to a condenser, so that a liquid solvent can be recovered. That is, as described above, the second mixture solution, which has undergone the steps of removing the residual carbon dioxide and removing the catalyst after polymerization, is charged into the stripper at a temperature of 90° C. or less in order to prevent side reactions due to the epoxide compound, thereby firstly removing the residual epoxide compound in the mixture. Subsequently, before the second mixture is charged into the heater, it is heat-exchanged with the high-temperature steam discharged from the second solvent removing means (for example, the upper part of extruder DV) via the heat exchanger to thereby increase the temperature of the second mixture to the maximum level. Through such a step, the amount of steam required by the heater can be reduced. Next, the solvent is partially removed in the first solvent removal means (flash vessel) through a heater, and the remaining polymer solution can be charged into the second solvent removing means (extruder DV) to remove the remaining solvent. InFIG.1, the second solvent removal means is shown as a single extruder DV (Extruder DV) for convenience, but this can include an extruder DV consisting of two or more. Therefore, the extruder DV, which is the second solvent removal means, can be composed of two or more extruder DVs. Most preferably, the second solvent removal means can use an extruder DV consisting of two. In the first extruder DV among the second solvent removal means, most of the residual solvent is removed from the second mixture from which the organic solvent is first removed, and in the second extruder DV, it is possible to perform a step of removing the remaining solvents and especially by-products together. In the present disclosure, through the above method, the residual monomer can be removed from the polymerization mixture of polyalkylene carbonate, and then the solvent can be recovered in a liquid form. In addition, the recovered solvent can be reused in polymerization reaction. Hereinafter, preferred examples of the present disclosure will be described in detail. However, these examples are for illustrative purposes only, and the scope of the present disclosure will not be construed as being limited by these examples. Example 1 In accordance with the process diagram ofFIG.1, residual carbon dioxide, residual EO, catalyst, by-product and solvent (MC) were removed from the first mixture containing residual carbon dioxide, residual EO, catalyst, solvent, and polymer (PEC) obtained after a typical PEC polymerization process. At this time, the first mixture ofFIG.1includes those provided by the following method. A polymerization reaction was carried out using a diethyl-zinc catalyst, a solvent, ethylene oxide (EO) and carbon dioxide, and a solvent (methylene chloride) to prepare PEC. Then, residual unreacted carbon dioxide and residual catalyst were removed by a conventional method. Thus, the first mixture was a mixture that has undergone a step of removing residual carbon dioxide and a catalyst after PEC polymerization, and a mixture in a stream state in which the residual EO content was 1,200 kg/hr, the solvent (dioxolane) content was 19,400 kg/hr, and the PEC polymer content was 2,400 kg/hr was used. That is, the first mixture containing residual EO, a solvent, and a polymer (PEC) was charged into the stripper at a temperature of 90° C. At this time, unreacted carbon dioxide was used as a stripping agent and supplied to a stripper under the condition of 4,000 kg/hr (80% by weight of unreacted carbon dioxide gas in the uncondensed state was charged). Through the above process, 1,140 kg/hr of EO and 4,100 kg/hr of some solvent (MC) were removed. By these processes, a second mixture was obtained. Here, the first and second mixtures contain ethylene carbonate (EC) as a by-product. After passing through the stripper, the temperature of the second mixture was about 30 to 40° C. In order to heat the second mixture passed through the stripper, a heat exchanger was used to heat-exchange with the vapor at 160° C. discharged to the upper part of the extruder DV. Through the heat exchange process, the temperature of the mixture was raised to 85 to 95° C. Then, the temperature of the mixture was raised to 115 to 125° C. via a heater, and steam was used as a temperature raising means. Thereafter, a second mixture passed through the heat exchanger was charged into a flash vessel, and the solvent (MC) was removed by 5,500 kg/hr. The temperature of the second mixture passed through the flash vessel was 95 to 105·, and this was charged into an extruder DV and operated at 160° C. to completely remove the remaining solvent. The solvent vapor removed here was utilized for heat exchange as mentioned above. In addition, the solvent removed in the form of vapor from the flash vessel and the extruder DV was recovered as a liquid solvent at 50° C. using a condenser. The recovered solvent can be reused in the polymerization reaction. Here, the extruder DV is an extruder DV consisting of two, and in the first extruder DV, most of the residual solvent (MC) was removed from the second mixture in which the solvent was removed through a flash vessel. In the second extruder DV, the remaining solvent (MC) and ethylene carbonate (EC) as a by-product were removed together. Example 2 The process was performed in the same manner as in Example 1, except that when using unreacted carbon dioxide as a stripper, 75% by weight of the unreacted carbon dioxide gas in the uncondensed state was charged into the stripper (supplied under the condition of 3400 kg/hr). Example 3 The process was performed in the same manner as in Example 1, except that when using unreacted carbon dioxide as a stripper, 85% by weight of the unreacted carbon dioxide gas in the uncondensed state was charged into the stripper (supplied under the condition of 4800 kg/hr). Example 4 The process was performed in the same manner as in Example 1, except that the first mixture containing residual EO, solvent, and polymer (PEC) was charged into a stripper at a temperature of 80° C. Comparative Example 1 The mixture containing residual EO, solvent, and polymer (PEC) was charged directly to a heater without passing through a stripper or heat exchanger, and the temperature was raised to 115 to 125° C. Then, the mixture was charged into a flash vessel to remove 9,600 kg/hr of the solvent. At this time, the amount of solvent remaining in the mixture passed through the flash vessel was the same as in Example 1. And, the temperature of the mixture was 95 to 105·, and this was charged into the extruder DV and operated under the condition of 160° C. to completely remove the remaining solvent. The solvent removed in the form of vapor from the flash vessel and the extruder DV was recovered as a liquid solvent (50° C.) using a condenser. Comparative Example 2 The mixture containing residual EO, solvent, and polymer (PEC) was heat-exchanged with steam (160° C.) discharged to the upper part of the extruder DV using a heat exchanger without passing through a stripper. At this time, the temperature of the mixture was raised to 85 to 95·. Thereafter, the liquid solvent was recovered in the same manner as in Comparative Example 1 from the process of charging the mixture into a heater. Comparative Example 3 A mixture containing a residual EO, a solvent, and a polymer (PEC) was charged into a stripper at a temperature of 90° C. At this time, CO2was used as a stripping agent and supplied at 4,000 kg/hr to the stripper. Through the above process, 1,140 kg/hr of EO and 4,100 kg/hr of solvent (MC) were removed. After passing through a stripper, the temperature of the mixture was about 30 to 40·. The mixture passed through the stripper was immediately charged into a heater, and the temperature of the mixture was raised to 115 to 125° C. Subsequent process was performed in the same manner as in Example 1, except that steam discharged from the extruder DV was not utilized for heat exchange (because the heat exchanger was not configured), and thereby, a liquid solvent was recovered. Reference Example 1 The process was performed in the same manner as in Example 1, except that when using unreacted carbon dioxide as a stripper, 70% by weight of the unreacted gas in the uncondensed state was charged into the stripper (supplied under the condition of 2850 kg/hr). Reference Example 2 The process was performed in the same manner as in Example 1, except that the first mixture containing a residual EO, a solvent, and a polymer (PEC) was charged into a stripper at a temperature of 100° C. Experimental Example 1 With respect to Examples 1 to 3, Comparative Examples 1 to 3, and Reference Example 1, the amount of steam energy used by the heater and the content of the EO side reaction product contained in the mixture are shown in Table 1 below. (device configuration, use amount of steam energy, PEG content in polymer products (EO side reaction products)) TABLE 1Use amountof steamenergy inPEG contentheaterin productsDevice configurationkg/hrwt %Example1Stripper + Heat exchanger + Heater +1,1001.2Flash Vessel + Extruder DV +Condenser(Stripping agent: 80 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged)Example 2Stripper + Heat exchanger + Heater +1,0802.0Flash Vessel + Extruder DV +Condenser(Stripping agent: 75 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged)Example 3Stripper + Heat exchanger + Heater +1,1201.0Flash Vessel + Extruder DV +Condenser(Stripping agent: 85 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged)ComparativeHeater + Flash Vessel + Extruder DV +2,25016.7Example 1CondenserComparativeHeat exchanger + Heater + Flash1,00016.7Example 2Vessel + Extruder DV + CondenserComparativeStripper + Heater + Flash Vessel +2,1001.2Example 3Extruder DV + CondenserReferenceStripper + Heat exchanger + Heater +1,0503.2Example 1Flash Vessel + Extruder DV +Condenser(Stripping agent: 70 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged) As shown in Table 1, in the case of Example 1, the conversion of unreacted EO to PEG (side reaction) was minimized by configuring the stripper and the heat exchanger before the heater, and at the same time, steam energy in the heater was minimized. In the case of Example 2, if the ratio of utilizing a stripping agent in the unreacted carbon dioxide gas in an uncondensed state was slightly reduced, the amount of the stripping agent charged into the stripper was reduced, so the EO removal efficiency slightly decreased. As a result, EO that was not removed had a slightly higher PEG content in the product as it proceeds to subsequent the high-temperature process, but the range of PEG content (within 2 wt %) of a normal product normally required can be satisfied. Further, in Example 2, the amount of heater steam energy used can be reduced. In the case of Example 3, the charging amount of the stripping agent was slightly increased, so that the EO removal efficiency was slightly higher. However, it was confirmed that also in the case of Example 3, the PEG content in the product can be reduced while reducing the amount of heater steam energy used, as compared with Comparative Examples 1 to 4, thereby exhibiting a remarkable effect. At this time, if the amount vaporized to the upper part of the stripper increases (in addition to EO, some solvents are also contained and vaporized), the polymer solution solids content (TSC) is increased and the temperature is lowered, resulting in the increase of the viscosity. This should be careful as it can cause problems such as fouling in stripper operation. Therefore, even if the charging amount of the stripping agent is increased too much, it becomes a problem, and thus, the charging amount of the stripping agent must be adjusted within the scope of the present disclosure. In contrast, in the case of Comparative Example 1, it can be seen that as the stripper and the heat exchanger are not configured, the PEG content in the product is increased and the amount of steam energy used in the heater is higher. Further, in the case of Comparative Example 2, it can be seen that the PEG content in the product is high as the stripper is not configured. In the case of Comparative Example 3, it can be seen that the amount of steam energy used in the heater is high as heat integration using a heat exchanger is excluded. In the case of Reference Example 1, since the charging amount of the stripping agent was smaller than that of Example 2, the PEG content of the product was finally increased to a level of 3%. Experimental Example 2 Comparison of the effect according to the temperature when the first mixture is charged into the stripper With respect to Examples 1 and 4 and Reference Example 2, the amount of steam energy used in the heater and the amount of EO side reaction products contained in the mixture are shown in Table 2 below. (device configuration, use amount of steam energy, PEG content in polymer products (EO side reaction products)) TABLE 2Use amountof steamenergy inPEG contentheaterin productsDevice configurationkg/hrwt %ExampleStripper + Heat exchanger + Heater +1,1001.21Flash Vessel + Extruder DV +Condenser(Stripping agent: 80 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged, charging temperatureof first mixture: 90° C.)ExampleStripper + Heat exchanger + Heater +1,1001.74Flash Vessel + Extruder DV +Condenser(Stripping agent: 80 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged, charging temperatureof first mixture: 80° C.)ReferenceStripper + Heat exchanger + Heater +1,1005.0ExampleFlash Vessel + Extruder DV +2Condenser(Stripping agent: 80 wt % of unreactedcarbon dioxide gas in an uncondensedstate was charged, charging temperatureof first mixture: 100° C.) According to Table 2, in the case of Reference Example 2 where the temperature at the time of charging the first mixture as a stripper is 90° C. or higher, the self-polymerization of the unreacted epoxide contained in the first mixture is accelerated, and the PEG content is increased before being charged into the stripper, so that the PEG content in the final product was high. Therefore, the polyalkylene carbonate according to Reference Example 2 can be deteriorated in physical properties. On the other hand, when the temperature at the time of charging the first mixture into the stripper is set to 90° C. or less, preferably 80 to 90° C. as in Examples 1 and 4, the amount of steam energy used in the heater and the PEG content in the product can be reduced by minimizing the self-polymerization of unreacted epoxides. In addition, in the present disclosure, by optimizing the charging temperature of the first mixture charged into the stripper, it is possible to suppress the self-polymerization of unreacted epoxides than before. Thus, by reducing the amount of PEG produced (EO side reaction product) in the subsequent high-temperature process, the deterioration of the physical properties of the final polymer product can be minimized. | 35,398 |
11859050 | DESCRIPTION OF EMBODIMENTS Hereinafter, the present invention is described in more detail. <Silanol-Group-Terminated Polyoxyalkylene Compound> A silanol-group-terminated polyoxyalkylene compound of the present invention is a novel polyoxyalkylene compound having at least one, preferably two or more silanol group-containing reactive silicon groups represented by the following structural formula (1) at a molecular chain terminal (particularly at both molecular chain terminals) in one molecule thereof as a partial structure and having a main chain of a polyoxyalkylene polymer: wherein R1and R2may be the same or different, and each represents an unsubstituted or substituted monovalent hydrocarbon group having 1 to 20 carbon atoms, a hydrogen atom, or a triorganosiloxy group represented by (R3)3Si—O— (R3represents a monovalent hydrocarbon group having 1 to 20 carbon atoms, and three R3s may be the same or different), n represents an integer of 2 or more, m represents an integer of 1 or more, and the broken line represents a linking bond. If the number of the silanol group-containing reactive silicon groups represented by the structural formula (1) contained in one molecule is less than 1 on average, curability of a composition containing the reactive silicon groups as a main component is insufficient. If the number of the reactive silicon groups is too large, a network structure is too dense, and therefore there is a possibility that an obtained cured product does not exhibit favorable mechanical properties. Therefore, the number of the silanol group-containing reactive silicon groups contained in one molecule is 1 or more, preferably 1.1 to 5, more preferably 2 to 4, and still more preferably 2 (for example, one at each molecular chain terminal). In the formula (1), R1and R2may be the same or different from each other, and each represents an unsubstituted or substituted monovalent hydrocarbon group having 1 to 20 carbon atoms, a hydrogen atom, or a triorganosiloxy group represented by (R3)3Si—O— (R3represents a monovalent hydrocarbon group having 1 to 20 carbon atoms, and three R3s may be the same or different from one another). The unsubstituted or substituted monovalent hydrocarbon group of each of R1and R2has the carbon number of 1 to 20, preferably of 1 to 10, more preferably of about 1 to 8. R1and R2may be the same or different from each other, and each preferably represents an alkyl group having 1 to 20 to carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms. The carbon number of each of these groups is more preferably 1 to 10, and particularly preferably 1 to 8. The monovalent hydrocarbon group of R3has the carbon number of 1 to 20, preferably of 1 to 10, more preferably of about 1 to 8. R3may be the same or different from each other, and each preferably represents an alkyl group having 1 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 20 carbon atoms. The carbon number of each of these groups is more preferably 1 to 10, and particularly preferably 1 to 8. Specific examples of R1and R2include: an alkyl group such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, a neopentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, or an eicosyl group; a cycloalkyl group such as a cyclopentyl group or a cyclohexyl group; an alkenyl group such as a vinyl group, an allyl group, a propenyl group, an isopropenyl group, a butenyl group, a pentenyl group, or a hexenyl group; an aryl group such as a phenyl group, a tolyl group, a xylyl group, or an α-, β-naphthyl group; an aralkyl group such as a benzyl group, a 2-phenylethyl group, or a 3-phenylpropyl group; a group obtained by replacing a part or all of hydrogen atoms in these groups with a halogen atom such as F, Cl, or Br, a cyano group, or the like, for example, a 3-chloropropyl group, a 3,3,3-trifluoropropyl group, or a 2-cyanoethyl group; a hydrogen atom; and a triorganosiloxy group such as a trimethylsiloxy group, a triethylsiloxy group, or a triphenylsiloxy group. Among these groups, R1is preferably a methyl group, an ethyl group, or a phenyl group, and particularly preferably a methyl group or a phenyl group from viewpoints of easy availability, productivity, and cost. R2is preferably a hydrogen atom. As R3, groups similar to the groups exemplified for the monovalent hydrocarbon group of R1and R2can be exemplified. R3is preferably a methyl group, an ethyl group, or a phenyl group. In the formula (1), n represents an integer of 2 or more, m represents an integer of 1 or more, n preferably represents an integer of 2 to 8, m preferably represents an integer of 1 to 8, n more preferably represents an integer of 2 to 4, and m more preferably represents an integer of 2 to 4. A main chain skeleton of the silanol-group-terminated polyoxyalkylene compound preferably has a repeating unit (for example, an oxyalkylene group) represented by the following formula (3): —R4—O— (3) wherein R4represents a divalent hydrocarbon group. The R4is not particularly limited as long as being a divalent hydrocarbon group (particularly an aliphatic divalent hydrocarbon group), but a linear or branched alkylene group having 1 to 14 carbon atoms is preferable. The R4is more preferably a linear or branched alkylene group having 2 to 4 carbon atoms. The repeating unit represented by the formula (3) is not particularly limited, and examples thereof include an oxyalkylene group such as —CH2O—, —CH2CH2O—, —CH2CH2CH2O—, —CH2CH(CH3)O—, —CH2CH(CH2CH3)O—, —CH2C(CH3)2O—, or CH2CH2CH2CH2O—. The main chain skeleton of the polyoxyalkylene compound may be formed of one kind or two or more kinds selected from the repeating units represented by the formula (3). Particularly, in a case where the polyoxyalkylene compound is used for a sealant or the like, a polymer mainly containing propylene oxide (—CH2CH(CH3)O—) is preferable. The silanol-group-terminated polyoxyalkylene compound of the present invention has viscosity at 25° C. preferably of 10 to 100,000 mPa·s, more preferably of 50 to 50,000 mPa·s, particularly preferably of 100 to 10,000 mPa·s. If the viscosity of the silanol-group-terminated polyoxyalkylene compound is 10 mPa·s or more, it is easy to obtain a coating film having excellent physical/mechanical strength. The viscosity of the silanol-group-terminated polyoxyalkylene compound of 100,000 mPa·s or less is preferable because a composition does not have too high viscosity and have good workability at the time of use. Here, the viscosity is a numerical value according to a rotational viscometer (for example, a BL type, a BH type, a BS type, a cone plate type, or a rheometer, the same below). Note that the silanol-group-terminated polyoxyalkylene compound may be linear or branched, but is preferably linear. The silanol-group-terminated polyoxyalkylene compound has a molecular weight usually of 200 to 50,000, preferably of 800 to 40,000, more preferably of 1,000 to 30,000, still more preferably of 2,000 to 20,000, particularly preferably of 3,000 to 15,000, most preferably of about 4,000 to 10,000. Here, the molecular weight or degree of polymerization (repeating number of an oxyalkylene unit) can be determined, for example, by regarding the molecular weight or degree of polymerization as a number average molecular weight (or number average degree of polymerization) or the like in terms of polystyrene in gel permeation chromatography (GPC) analysis using tetrahydrofuran (THF) or the like as a developing solvent. Incidentally, in the present invention, the “linear” of the silanol-group-terminated polyoxyalkylene compound means that divalent oxyalkylene groups which are repeating units constituting the polyoxyalkylene structure are linearly connected to each other, and each of the oxyalkylene groups itself may be linear or branched (for example, a propyleneoxy group such as —CH2CH(CH3)O—. Examples of such a silanol-group-terminated polyoxyalkylene compound include a compound represented by the following structural formula (2): wherein R1, n, and m are the same as defined above, and Z represents a polyoxyalkylene polymer as a main chain. In the formula (2), Z has the above-described repeating unit represented by the formula (3), and examples thereof include a compound represented by the following formula (4): wherein R4is the same as defined above, preferably a linear or branched alkylene group having 1 to 14 carbon atoms, more preferably a linear or branched alkylene group having 2 to 4 carbon atoms, p represents an integer of 2 or more, preferably an integer of 10 to 700, more preferably an integer of 20 to 500, still more preferably an integer of 50 to 200, and the broken line represents a linking bond. Examples of the silanol-group-terminated polyoxyalkylene compound represented by the formula (2) include the following compounds: wherein p1 is the same as p, and m1 is the same as m. The above silanol-group-terminated polyoxyalkylene compound may be used singly or in combination of two or more kinds thereof. <Process for Producing Silanol-Group-Terminated Polyoxyalkylene Compound> The novel silanol-group-terminated polyoxyalkylene compound according to an embodiment of the present invention can be easily produced, for example, by causing a hydrosilylation addition reaction between an organosilane or an organopolysiloxane compound (silicon compound) having a hydrogen atom bonded to a silicon atom (Si—H group) at one molecular chain terminal and having a hydroxy group bonded to a silicon atom (silanol group) at the other terminal, represented by the following formula (5), and a polyoxyalkylene polymer having both molecular chain terminals blocked with alkenyl groups, represented by the following formula (6): wherein R1, Z, and m are the same as defined above, r represents an integer of 0 or more, preferably an integer of 0 to 8, and more preferably an integer of 0 to 2. Specific examples of the silicon compound represented by the formula (5) include compounds represented by the following structural formulae (here, Ph represents a phenyl group), but are not limited thereto. Any silicon compound having a Si—H group at one molecular chain terminal and having a Si—OH group at the other molecular chain terminal can be used. Specific examples of the polyoxyalkylene polymer having both molecular chain terminals blocked with alkenyl groups, represented by the formula (6), include compounds represented by the following structural formulae (here, p is the same as defined above), but are not limited thereto. Any polyoxyalkylene polymer having both terminals blocked with alkenyl groups can be used. The polyoxyalkylene polymer represented by the formula (6) only needs to have a molecular weight (particularly a number average molecular weight) usually of 150 to 49,000, preferably of 700 to 39,000, more preferably of 900 to 29,000, still more preferably of 1,500 to 19,000, particularly preferably of 2,500 to 14,000, most preferably of about 3,500 to 9,500. If the molecular weight of the polyoxyalkylene polymer represented by formula (6) is too small, physical properties of a cured product after curing is insufficient. If the molecular weight is too large, not only the viscosity is extremely high to deteriorate workability, but also curability of the cured product may be lowered. A reaction ratio between the silicon compound represented by the formula (5) and the polyoxyalkylene polymer represented by the formula (6) is preferably 0.8 to 1.5 (mol/mol), and particularly preferably about 0.9 to 1.1 (mol/mol) in terms of a molar ratio of a Si—H group in the silicon compound represented by formula (5) with respect to an alkenyl group in the polyoxyalkylene polymer represented by formula (6). If the molar ratio is too small, a cured product after curing is not be completely cured, and rubber properties are not sufficiently obtained in some cases. If the molar ratio is too large, rubber strength after curing is lowered to make it difficult to obtain rubber elasticity, and this may be disadvantageous also in cost. Examples of an addition reaction catalyst used for adding the silicon compound include a platinum group metal-based catalyst such as a platinum-based catalyst, a palladium-based catalyst, a rhodium-based catalyst, or a ruthenium-based catalyst, and a platinum-based catalyst is particularly preferable. Examples of the platinum-based catalyst include platinum black, a catalyst in which solid platinum is carried on a carrier such as alumina or silica, chloroplatinic acid, alcohol-modified chloroplatinic acid, a complex of chloroplatinic acid and an olefin, and a complex of platinum and vinyl siloxane. The use amount of the platinum group metal-based catalyst only needs to be a so-called catalytic amount. For example, with respect to the total weight of the silicon compound represented by the formula (5) and the polyoxyalkylene polymer represented by the formula (6), the platinum group metal-based catalyst is used in an amount preferably of 0.1 to 1,000 ppm, particularly preferably of 0.5 to 100 ppm in terms of the weight of a platinum group metal. This reaction is desirably performed at a temperature of 50 to 120° C., particularly at a temperature of 60 to 100° C., for 0.5 to 12 hours, particularly for 1 to 6 hours, and can be performed without using a solvent. However, an appropriate solvent such as toluene or xylene may be used as necessary as long as not adversely affecting the addition reaction or the like. This reaction is represented by the following formula [1], for example, in a case where polypropylene having both molecular chain terminals blocked with allyl groups is used as an alkenyl group-blocked polyoxyalkylene polymer. wherein R1is the same as defined above, and p2 and m2 each represents an integer of 1 or more. The polyoxyalkylene compound of the present invention can be used as a main component (base polymer) of a room-temperature-curable composition using a crosslinking agent component such as an oxime type, an amide type, an aminoxy type, an acetic acid type, or an alcohol type. The composition has excellent curability, and can be preferably used as a sealing material, a one-liquid type adhesive, a pressure-sensitive adhesive, a paint, a coating material, a filling material, a casting material, a covering material, or the like. <Room-Temperature-Curable Composition> The room-temperature-curable composition of the present invention comprises the polyoxyalkylene compound as a main component, and preferably comprises the following components: (a) the polyoxyalkylene compound; (b) the following component (b-1) and/or component (b-2): (b-1) a hydrolyzable organosilicon compound having two alkoxysilyl-vinylene groups on the same silicon atom, represented by the following general formula (7), and/or a partial hydrolytic condensate thereof: wherein R5independently represents an unsubstituted or substituted monovalent hydrocarbon group having 1 to 20 carbon atoms, R6represents an unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, or an unsubstituted or substituted cycloalkyl group having 3 to 20 carbon atoms, and a represents an integer of 1 to 3; (b-2) a hydrolyzable organosilane free of an amino group, having one methyl group, one vinyl group, or one phenyl group, and having at least two hydrolyzable groups in one molecule thereof, being other than the component (b-1), and/or a partial hydrolytic condensate thereof; and (c) a curing catalyst. Component (a): Silanol-Group-Terminated Polyoxyalkylene Compound The component (a) is a main component (base polymer) of the room-temperature-curable composition of the present invention. The above-described polyoxyalkylene compound can be used as the component (a). Among the polyoxyalkylene compounds, the component (a) is preferably a silanol-group-terminated polyoxyalkylene compound free of an aliphatic unsaturated bond. That is, in the formula (1), R1and R2may be the same or different from each other, and each represents an alkyl group having 1 to 20 carbon atoms, in which an alkyl group having 3 or more carbon atoms may be a cyclic cycloalkyl group, an aryl group having 6 to 20 carbon atoms, an aralkyl group having 7 to 20 carbon atoms, a hydrogen atom, or a triorganosiloxy group represented by (R3)3Si—O— (R3is the same as defined above). Among these groups, R1is preferably a methyl group, an ethyl group, or a phenyl group, and particularly preferably a methyl group or a phenyl group from viewpoints of easy availability, productivity, and cost, R2is preferably a hydrogen atom, and R3is preferably a group free of an aliphatic unsaturated bond, and more preferably a methyl group, an ethyl to group, or a phenyl group. Examples of such a silanol-group-terminated polyoxyalkylene compound include a compound free of an aliphatic unsaturated bond among the above-described polyoxyalkylene compounds. Component (b): Hydrolyzable Organosilane and/or Partial Hydrolytic Condensate Thereof The component (b) according to the present invention acts as a curing agent (crosslinking agent) component in the room-temperature-curable composition of the present invention, and includes the following component (b-1) and/or component (b-2). Unless the room-temperature-curable composition of the present invention includes at least one of the component (b-1) and the component (b-2), an excellent cured product cannot be obtained. The component (b-1) is a hydrolyzable organosilicon compound (hydrolyzable organosilane) having two alkoxysilyl-vinylene groups (alkoxysilyl-ethenylene groups) on the same silicon atom represented by the following general formula (7) and/or a partial hydrolytic condensate thereof. Incidentally, in the present invention, the partial hydrolytic condensate refers to an organosiloxane oligomer having at least 2, preferably 3 or more residual hydrolyzable groups in a molecule thereof, produced by partially hydrolyzing and condensing the hydrolyzable organosilane. wherein R5independently represents an unsubstituted or substituted monovalent hydrocarbon group having 1 to 20 carbon atoms, R6represents an unsubstituted or substituted alkyl group having 1 to 20 carbon atoms, or an unsubstituted or substituted cycloalkyl group having 3 to 20 carbon atoms, and a represents an integer of 1 to 3. Here, in the formula (7), the unsubstituted or substituted monovalent hydrocarbon group of R5has 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably about 1 to 8 carbon atoms. R5may be the same or different from each other. Examples thereof include an alkyl group such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, a neopentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, or an eicosyl group; a cycloalkyl group such as a cyclopentyl group or a cyclohexyl group; an aryl group such as a phenyl group, a tolyl group, a xylyl group, or an α-, β-naphthyl group; an aralkyl group such as a benzyl group, a 2-phenylethyl group, or a 3-phenylpropyl group; and a group obtained by replacing a part or all of hydrogen atoms in these groups with a halogen atom such as F, Cl, or Br, a cyano group, or the like, for example, a 3-chloropropyl group, a 3,3,3-trifluoropropyl group, or a 2-cyanoethyl group. Among these groups, a methyl group, an ethyl group, and a phenyl group are preferable, and a methyl group and a phenyl group are particularly preferable from viewpoints of easy availability, productivity, and cost. The unsubstituted alkyl group of R6has 1 to 20 carbon atoms, preferably 1 to 6 carbon atoms, and more preferably about 1 to 4 carbon atoms, and examples thereof include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, a neopentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, and an eicosyl group. The unsubstituted cycloalkyl group has 3 to 20 carbon atoms, preferably 4 to 8 carbon atoms, and more preferably about 5 or 6 carbon atoms, and examples thereof include a cyclopentyl group and a cyclohexyl group. A part or all of hydrogen atoms of the alkyl group or the cycloalkyl group may be replaced with a halogen atom such as F, Cl, or Br, a cyano group, or the like, and examples thereof include a 3-chloropropyl group, a 3,3,3-trifluoropropyl group, and a 2-cyanoethyl group. Among these groups, R6is preferably a methyl group or an ethyl group, and particularly preferably a methyl group from a viewpoint of hydrolyzability or the like. The hydrolyzable organosilicon compound represented by the general formula (7) as the component (b-1) is mainly used as a curing agent. In general formula (7), a independently represent an integer of 1 to 3 for each silicon atom, but preferably represent 2 or 3 from a viewpoint of curability. Particularly, a compound having three alkoxy groups such as methoxy groups (that is, a compound having six alkoxy groups in total in a molecule) as two alkoxysilyl-vinylene groups on the same silicon atom in a molecule has two trifunctional alkoxyalkoxysilane moieties in one molecule, and therefore is useful as a curing agent (crosslinking agent) of a dealcoholated type modified silicone (room-temperature-curable composition). A synthesis example of the component (b-1) is described below. <Production of Hydrolyzable Organosilicon Compound Having Two Alkoxysilyl-Vinylene Groups on the Same Silicon Atom> A hydrolyzable organosilicon compound having two alkoxysilyl-vinylene groups (alkoxysilyl-ethenylene groups) on the same silicon atom as the component (b-1) can be easily produced, for example, through an addition reaction caused by a hydrosilylation reaction between an organosilane having two ethynyl groups on the same silicon atom and two alkoxyhydrosilanes. This reaction formula is represented, for example, by the following formula [2]. wherein R5, R6, and a are the same as defined in the general formula (7). Examples of an addition reaction catalyst used for adding an alkoxyhydrosilane include a platinum group metal-based catalyst such as a platinum-based catalyst, a palladium-based catalyst, a rhodium-based catalyst, or a ruthenium-based catalyst, and a platinum-based catalyst is particularly preferable. Examples of the platinum-based catalyst include platinum black, a catalyst in which solid platinum is carried on a carrier such as alumina or silica, chloroplatinic acid, alcohol-modified chloroplatinic acid, a complex of chloroplatinic acid and an olefin, and a complex of platinum and vinyl siloxane. The use amount of the platinum only needs to be a so-called catalytic amount. For example, with respect to the total weight of silanes (the total weight of an organosilane having two ethynyl groups on the same silicon atom and an alkoxyhydrosilane), platinum can be used in an amount of 0.1 to 1,000 ppm, particularly of 0.5 to 100 ppm in terms of the weight of a platinum group metal. This reaction is desirably performed generally at a temperature of 50 to 120° C., particularly at a temperature of 60 to 100° C., for 0.5 to 12 hours, particularly for 1 to 6 hours, and can be performed without using a solvent. However, an appropriate solvent such as toluene or xylene can be used as necessary as long as not adversely affecting the addition reaction or the like. Through the addition reaction of an alkoxyhydrosilane to an acetylene group (ethynyl group), for example, geometric isomers (that is, a mixture of an E-isomer and a Z-isomer) are generated as illustrated by the following reaction formula [3]. In this case, the E-isomer (trans-isomer) is generated in higher selectivity and is an active species having higher reactivity. However, for the alkoxysilyl-vinylene group-containing hydrolyzable organosilane as the component (b-1) of the present invention, a mixture of the geometric isomers can be used as it is without separating the geometric isomers because even coexistence of a small amount of the Z-isomer (cis-isomer) does not adversely affect properties of the alkoxysilyl-vinylene group-containing hydrolyzable organosilane. wherein a broken line represents a linking bond. Specific examples of the hydrolyzable organosilicon compound having two alkoxysilyl-vinylene groups on the same silicon atom represented by the formula (7) include compounds represented by the following structural formulae. The component (b-1) can be used singly or in combination of two or more kinds thereof. The component (b-2) is a hydrolyzable organosilane free of an amino group, having one methyl group, one vinyl group, or one phenyl group, and having at least two, preferably three hydrolyzable groups in one molecule thereof, being other than the component (b-1), and/or a partial hydrolytic condensate thereof, and is used as a crosslinking agent. Here, examples of the hydrolyzable group include: an alkoxy group having 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 4 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, or a tert-butoxy group; an alkoxyalkoxy group having 2 to 40 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 4 carbon atoms, such as a methoxymethoxy group or a methoxyethoxy group; an alkenyloxy group having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 5 carbon atoms, such as a vinyloxy group, an allyloxy group, a propenoxy group, or an isopropenoxy group; a ketoxime group having 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms, more preferably 3 to 6 carbon atoms, such as a dimethylketoxime group, a diethylketoxime group, or a methylethylketoxime group; and an acyloxy group having 2 to 20 carbon atoms, preferably 2 to 10 carbon atoms, more preferably 2 to 5 carbon atoms, such as an acetoxy group. Specific examples of the component (b-2) include methyltrimethoxysilane, methyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, vinyltriisopropoxysilane, methyltris(methoxyethoxy) silane, vinyltris(methoxyethoxy) silane, methyltripropenoxysilane, vinyltriisopropenoxysilane, phenyltriisopropenoxysilane, methyltriacetoxysilane, vinyltriacetoxysilane, and partial hydrolytic condensates thereof, but the component (b-2) is not limited thereto. These compounds can be used singly or in combination of two or more kinds thereof. A hydrolyzable organosilicon compound and/or a partial hydrolytic condensate thereof as the component (b) (that is, the total amount of the components (b-1) and (b-2)) is used in an amount of 0.1 to 30 parts by weight, preferably of 0.5 to 20 parts by weight, more preferably of 3 to 15 parts by weight per 100 parts by weight of the polyoxyalkylene polymer as the component (a). If the use amount is less than 0.1 parts by weight, sufficient crosslinking cannot be obtained, and it is difficult to obtain a composition having desired rubber elasticity. If the use amount exceeds 30 parts by weight, mechanical properties of rubber properties are lowered, and a problem of further causing an economic disadvantage occurs. Incidentally, as the component (b) which is a curing agent (crosslinking agent) in the room-temperature-curable composition of the present invention, the component (b-1) and the component (b-2) may be used singly or in combination thereof. In a case where the component (b-1) and the component (b-2) are used in combination, a use weight ratio of the component (b-1): the component (b-2) can be 99:1 to 1:99, preferably 90:10 to 10:90, more preferably 70:30 to 30:70, and still more preferably about 60:40 to 40:60. Component (c): Curing Catalyst (Nonmetallic Organic Catalyst and/or Metallic Catalyst) The component (c) is a curing catalyst (nonmetallic organic catalyst and/or metallic catalyst) and acts to promote curing of the room-temperature-curable composition of the present invention. As the nonmetallic organic catalyst of the curing catalyst, a known curing promoter for a condensation-curable organopolysiloxane composition can be used, and the nonmetallic organic catalyst is not particularly limited. Examples of the nonmetallic organic catalyst include: a phosphazene-containing compound such as N,N,N′,N′,N″,N″-hexamethyl-N′″-(trimethylsilylmethyl)-phosphorimidic triamide; an aminoalkyl group-substituted alkoxysilane such as 3-aminopropyltriethoxysilane or N-β (aminoethyl) γ-aminopropyltrimethoxysilane; an amine compound such as hexylamine or dodecylamine phosphate and salts thereof; a quaternary ammonium salt such as benzyltriethylammonium acetate; a dialkylhydroxylamine such as dimethylhydroxylamine or diethylhydroxylamine; a guanidyl group-containing silane such as tetramethylguanidylpropyl trimethoxysilane, tetramethylguanidylpropylmethyl dimethoxysilane, or tetramethylguanidylpropyl tris(trimethylsiloxy) silane; and a siloxane. However, the nonmetallic organic catalyst is not limited thereto. The nonmetallic organic catalyst may be used singly or in combination of two or more kinds thereof. As the metallic catalyst of the curing catalyst, a known curing promoter for a condensation-curable organopolysiloxane can be used, and the metallic catalyst is not particularly limited. Examples of the metallic catalyst include: an alkyl tin ester compound such as dibutyl tin diacetate, dibutyl tin dilaurate, dibutyl tin dioctoate, dioctyl tin dineodecanoate, or di-n-butyl-dimethoxy tin; a titanate or a titanium chelate compound such as tetraisopropoxy titanium, tetra-n-butoxy titanium, tetrakis(2-ethylhexoxy) titanium, dipropoxybis(acetylacetonato) titanium, or titanium isopropoxy octylene glycol; zinc naphthenate; zinc stearate; zinc-2-ethyl octoate; iron-2-ethylhexoate; cobalt-2-ethylhexoate; manganese-2-ethylhexoate; cobalt naphthenate; an alcoholate aluminum compound such as aluminum isopropylate or aluminum secondary butyrate; an aluminum chelate compound such as aluminum alkyl acetate-diisopropylate or aluminum bisethyl acetoacetate-monoacetylacetonate; an organometallic compound such as bismuth(III) neodecanoate, bismuth(III) 2-ethylhexanoate, bismuth(III) citrate, or bismuth octylate; and a lower fatty acid salt of an alkali metal such as potassium acetate, sodium acetate, or lithium oxalate. However, the metallic catalyst is not limited thereto. The metallic catalyst may be used singly or in combination of two or more kinds thereof. The use amount of the curing catalyst only needs to be a small catalyst amount. The blending amount of the component (c) is 0.01 to 20 parts by weight, particularly preferably 0.05 to 10 parts by weight, and more preferably 0.05 to 5 parts by weight per 100 parts by weight of the component (a). If the use amount is less than 0.01 parts by weight, favorable curability cannot be obtained, and therefore a curing rate is small disadvantageously. On the other hand, if the use amount exceeds 20 parts by weight, the curability of the composition is too large, and therefore an allowable range of working time after application of the composition may be shortened, or mechanical properties of an obtained rubber may be lowered. Component (d): Filler The component (d) is a filler (an inorganic filler and/or an organic resin filler), is an optional component that can be blended in the room-temperature-curable composition of the present invention as necessary, and is used in order to impart sufficient mechanical strength to a cured product formed from this composition. As the filler, a known filler can be used. Examples thereof include: finely powdered silica; fumed silica; precipitated silica; silica obtained by subjecting surfaces of the silica to a hydrophobic treatment with an organosilicon compound; a glass bead; a glass balloon; a transparent resin bead; silica aerogel; diatomaceous earth; a metal oxide such as iron oxide, zinc oxide, titanium oxide, or a fumed metal oxide; wet silica; products obtained by subjecting surfaces of these materials to a silane treatment; quartz powder, carbon black; talc; a reinforcing agent such as zeolite or bentonite; asbestos; a glass fiber; a carbon fiber; a metal carbonate such as calcium carbonate, magnesium carbonate, or zinc carbonate; glass wool; finely powdered mica; fused silica powder; and synthetic resin powder such as polystyrene, polyvinyl chloride, or polypropylene. Among these fillers, an inorganic filler such as silica, calcium carbonate, or zeolite is preferable, and fumed silica and calcium carbonate having surfaces hydrophobized are particularly preferable. The blending amount of the component (d) is preferably 0 to 1,000 parts by weight, and more preferably 0 to 300 parts by weight per 100 parts by weight of the component (a). If the component (d) is used in an amount larger than 1,000 parts by weight, the viscosity of a composition increases to deteriorate workability, and in addition, the rubber strength after curing is lowered, and it may be difficult to obtain rubber elasticity. Incidentally, in a case where the component (d) is blended, the blending amount of the component (d) is usually 3 parts by weight or more, and particularly preferably 5 parts by weight or more. By blending the component (d), the mechanical strength of an obtained cured product can be sufficiently high. Component (e): Adhesion Promoter The component (e) is an adhesion promoter, is an optional component that can be blended in the room-temperature-curable composition of the present invention as necessary, and is used in order to impart sufficient adhesiveness to a cured product formed from this composition. As the adhesion promoter (a silane coupling agent such as a functional group-containing hydrolyzable silane), a known adhesion promoter is preferably used. Examples of the adhesion promoter include a vinylsilane coupling agent, a (meth)acrylic silane coupling agent, an epoxy silane coupling agent, an aminosilane coupling agent, and a mercaptosilane coupling agent, and specific examples thereof include vinyltris(β-methoxyethoxy) silane, γ-methacryloxypropyltrimethoxysilane, β-(3,4-epoxycyclohexyl) ethyltrimethoxysilane, γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropylmethyldiethoxysilane, N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, 3-2-(aminoethylamino) propyltrimethoxysilane, γ-mercaptopropyl trimethoxysilane, isocyanate silane, and compounds obtained by partially hydrolyzing and condensing these compounds. Incidentally, among the adhesion promoters, a vinylsilane coupling agent such as vinyltris(β-methoxyethoxy) silane can function also as the component (b-2). Among these adhesion promoters, an aminosilane such as to γ-aminopropyltriethoxysilane or 3-2-(aminoethylamino) propyltrimethoxysilane; an epoxysilane such as γ-glycidoxypropyltrimethoxysilane or β-(3,4-epoxycyclohexyl) ethyltrimethoxysilane; and isocyanate silane are particularly preferable. The component (e) is blended in an amount preferably of 0 to 30 parts by weight, particularly preferably of 0.1 to 20 parts by weight per 100 parts by weight of the component (a). In a case where adhesion is performed using a filler or an adherend without using an adhesion promoter, the adhesion promoter does not have to be used. Component (f): Organopolysiloxane In addition to the components (a) to (e), the room-temperature-curable composition of the present invention may further contain a linear diorganopolysiloxane (f) represented by the following general formula (8) (so-called non-functional silicone oil): wherein R7independently represents an unsubstituted or substituted monovalent hydrocarbon group free of an aliphatic unsaturated bond having 1 to 20 carbon atoms, and q represents such a numerical value that the organopolysiloxane has viscosity of 1.5 to 1,000,000 mPa·s at 23° C. In the formula (8), the unsubstituted or substituted monovalent hydrocarbon group of R7free of an aliphatic unsaturated bond has 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably about 1 to 8 carbon atoms. R7may be the same or different from each other. Examples thereof include an alkyl group such as a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a tert-butyl group, a pentyl group, a neopentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, a hexadecyl group, a heptadecyl group, an octadecyl group, a nonadecyl group, or an eicosyl group; a cycloalkyl group such as a cyclopentyl group or a cyclohexyl group; an aryl group such as a phenyl group, a tolyl group, a xylyl group, or an α-, β-naphthyl group; an aralkyl group such as a benzyl group, a 2-phenylethyl group, or a 3-phenylpropyl group; and a group obtained by replacing a part or all of hydrogen atoms in these groups with a halogen atom such as F, Cl, or Br, a cyano group, or the like, for example, a 3-chloropropyl group, a 3,3,3-trifluoropropyl group, or a 2-cyanoethyl group. Among these groups, a methyl group, an ethyl group, and a phenyl group are preferable, and a methyl group and a phenyl group are more preferable from viewpoints of easy availability, productivity, and cost. Particularly, any one of R7is preferably a methyl group, and a dimethylpolysiloxane having both molecular chain terminals blocked with trimethylsiloxy groups is preferable. q represents such a numerical value that the viscosity of the diorganopolysiloxane is 1.5 to 1,000,000 mPa·s at 23° C., and preferably 30 to 100,000 mPa·s. Particularly, r represents an integer of 2 or more, preferably an integer of 20 to 2,000. In a case where the component (f) is blended, the blending amount of the component (f) is preferably 0.1 to 100 parts by weight, and more preferably 10 to 80 parts by weight per 100 parts by weight of the component (a). The amount of the component (f) within the above range is preferable because the viscosity can be adjusted to a viscosity easily handled for construction without impairing mechanical properties and flame retardancy of the room-temperature-curable composition of the present invention. --Other Components-- In addition to the components (a) to (f), the room-temperature-curable composition of the present invention may contain various other additives as necessary. For example, the room-temperature-curable composition may contain a known additive such as a pigment including carbon black, iron oxide, and titanium oxide, a dye, an anti-aging agent, an antioxidant, an antistatic agent, an adhesion imparting agent, an antiseptic agent, a flame retardancy imparting agent such as zinc carbonate, an antifungal agent, or an antibacterial agent. These other additives only need to be added in amounts not hindering the effect of the present invention. An organic solvent may be used as necessary for the room-temperature-curable composition of the present invention. Examples of the organic solvent include: an aliphatic hydrocarbon compound such as n-hexane, n-heptane, isooctane, or isododecane; an aromatic hydrocarbon compound such as toluene or xylene; a linear siloxane such as hexamethyldisiloxane, octamethyltrisiloxane, decamethyltetrasiloxane, dodecamethylpentasiloxane, or 2-(trimethylsiloxy)-1,1,1,2,3,3,3-heptamethyltrisiloxane; and a cyclic siloxane such as octamethylcyclopentasiloxane or decamethylcyclopentasiloxane. The amount of the organic solvent only needs to be appropriately adjusted within a range not hindering the effect of the present invention. The room-temperature-curable composition of the present invention can be obtained by uniformly mixing the components and, in addition, predetermined amounts of the various additives in a dry atmosphere. Specifically, the room-temperature-curable composition is preferably prepared by uniformly mixing the components (a) to (c) and, as necessary, the components (d) to (f) and various other additives while bubbles are removed under a state where moisture is blocked or under reduced pressure. A mixing apparatus is not particularly limited, but a universal mixing stirrer (manufactured by DALTON Corporation) connected to a vacuum pump, a planetary mixer (manufactured by INOUE MFG. INC.), or the like is preferably used. The room-temperature-curable composition is cured by being left at room temperature (23° C.±10° C.). As a method for molding the room-temperature-curable composition, a condition for curing the room-temperature-curable composition, and the like, known methods and conditions can be adopted depending on the kind of the composition. The room-temperature-curable composition of the present invention is easily cured at room temperature (23° C.±10° C.) by being stored, in the absence of moisture, that is, in a sealed container with moisture blocked and by being exposed to moisture in air at the time of use. When the room-temperature-curable composition of the present invention is cured, the room-temperature-curable composition becomes a cured product having excellent flame retardancy, favorable adhesiveness without a primer to glass and coated aluminum, and excellent deformation followability. An obtained cured product has favorable rubber elasticity. Therefore, the cured product is useful as a sealing material used for a building sealing material. A method of using the room-temperature-curable composition of the present invention as a sealing material is not particularly limited as long as being in accordance with a conventionally known method of using a sealing material. Examples of an article to be bonded and/or sealed with a cured product of the room-temperature-curable composition of the present invention include articles made of glasses, various metals, and the like. The room-temperature-curable composition of the present invention thus obtained is rapidly cured at room temperature (23° C.±10° C.) due to moisture in air to form a rubber elastic body cured product having excellent heat resistance, weather resistance, and adhesion to various substrates. The room-temperature-curable composition of the present invention particularly has excellent storage stability and curability. For example, even after storage for six months, the room-temperature-curable composition is rapidly cured by being exposed to air to provide a cured product having excellent physical properties as described above. Furthermore, the room-temperature-curable composition does not release a toxic or corrosive gas during curing and does not generate rust on a surface to which this composition has been applied. This composition can be cured and molded to obtain various molded articles. EXAMPLES Hereinafter, the present invention is described specifically with reference to Examples and Comparative Examples, but the present invention is not limited to the following Examples. Incidentally, in the following specific examples, “part” means “part by weight”, the room temperature is 23° C.±10° C., and the viscosity indicates a value measured by a rotational viscometer at 25° C., and the molecular weight and the degree of polymerization (the repeating number of polyoxyalkylene units) are a number average molecular weight and a number average degree of polymerization in terms of polystyrene in GPC analysis using THF as a developing solvent. <Synthesis of Silanol-Group-Terminated Polyoxyalkylene Compound> Example 1 Into a 500 mL four-neck separable flask equipped with a stirrer, a reflux condenser, a thermometer, and a dropping funnel, 500 g of an allyl group-terminated polypropylene glycol corresponding to a molecular weight of 7,400 (0.160 mol in terms of functional group of terminal allyl group) and 1.0 g of a platinum catalyst (a solution of a vinylsiloxane complex of chloroplatinic acid, platinum concentration: 1% by weight) were put, and the temperature was raised to 90° C. while the resulting mixture was heated and stirred. Subsequently, under stirring, 50 g of 1-hydroxy-octamethyltetrasiloxane (that is, 1-hydroxy-7-hydrogen-1,1,3,3,5,5,7,7-octamethyltetrasiloxane) (the functional group amount of terminal Si—H: 0.167 mol) was added dropwise thereto. At this time, heat was generated, the reaction temperature became 90 to 95° C., and this reaction system was held for six hours. After completion of the reaction, a small excess of 1-hydroxy-octamethyltetrasiloxane was removed under reduced pressure. The temperature was lowered to room temperature. Thereafter, filtration was performed to obtain 520 g of a silanol-group-terminated polypropylene glycol (polymer A) (viscosity: 4.0 Pa·s, molecular weight: 8,000, yield: 95%). This reaction formula is represented by the following formula [4]. wherein p′ represents 127. Example 2 Into a 500 mL four-neck separable flask equipped with a stirrer, a reflux condenser, a thermometer, and a dropping funnel, 500 g of an allyl group-terminated polypropylene glycol corresponding to a molecular weight of 8,400 (0.112 mol in terms of functional to group of terminal allyl group) and 1.0 g of a platinum catalyst (a solution of a vinylsiloxane complex of chloroplatinic acid, platinum concentration: 1% by weight) were put, and the temperature was raised to 90° C. while the resulting mixture was heated and stirred. Subsequently, under stirring, 35 g of 1-hydroxy-octamethyltetrasiloxane (the functional group amount of terminal Si—H: 0.117 mol) was added dropwise thereto. At this time, heat was generated, the reaction temperature became 90 to 95° C., and this reaction system was held for six hours. After completion of the reaction, a small excess of 1-hydroxy-octamethyltetrasiloxane was removed under reduced pressure. The temperature was lowered to room temperature. Thereafter, filtration was performed to obtain 505 g of a silanol-group-terminated polypropylene glycol (polymer B) (viscosity: 10.0 Pa·s, molecular weight: 9,000, yield: 95%). Example 3 Into a 500 mL four-neck separable flask equipped with a stirrer, a reflux condenser, a thermometer, and a dropping funnel, 250 g of an allyl group-terminated polypropylene glycol corresponding to a molecular weight of 4,300 (0.107 mol in terms of functional group of terminal allyl group) and 1.0 g of a platinum catalyst (a solution of a vinylsiloxane complex of chloroplatinic acid, platinum concentration: 1% by weight) were put, and the temperature was raised to 90° C. while the resulting mixture was heated and stirred. Subsequently, under stirring, 33 g of 1-hydroxy-octamethyltetrasiloxane (the to functional group amount of terminal Si—H: 0.110 mol) was added dropwise thereto. At this time, heat was generated, the reaction temperature became 90 to 95° C., and this reaction system was held for six hours. After completion of the reaction, a small excess of 1-hydroxy-octamethyltetrasiloxane was removed under reduced pressure. The temperature was lowered to room temperature. Thereafter, filtration was performed to obtain 268 g of a silanol-group-terminated polypropylene glycol (polymer C) (viscosity: 1.3 Pa·s, molecular weight: 4,900, yield: 95%). Example 4 Into a 500 mL four-neck separable flask equipped with a stirrer, a reflux condenser, a thermometer, and a dropping funnel, 250 g of an allyl group-terminated polypropylene glycol corresponding to a molecular weight of 7,190 (0.063 mol in terms of functional group of terminal allyl group) and 1.0 g of a platinum catalyst (a solution of a vinylsiloxane complex of chloroplatinic acid, platinum concentration: 1% by weight) were put, and the temperature was raised to 90° C. while the resulting mixture was heated and stirred. Subsequently, under stirring, 19 g of 1-hydroxy-octamethyltetrasiloxane (the functional group amount of terminal Si—H: 0.066 mol) was added dropwise thereto. At this time, heat was generated, the reaction temperature became 90 to 95° C., and this reaction system was held for six hours. After completion of the reaction, a small excess of 1-hydroxy-octamethyltetrasiloxane was removed under reduced pressure. The temperature was lowered to room temperature. Thereafter, filtration was performed to obtain 255 g of a silanol-group-terminated polypropylene glycol (polymer D) (viscosity: 9.2 Pa·s, molecular weight: 7,800, yield: 95%). Example 5 Into a 500 mL four-neck separable flask equipped with a stirrer, a reflux condenser, a thermometer, and a dropping funnel, 250 g of an allyl group-terminated polypropylene glycol corresponding to a molecular weight of 7,500 (0.048 mol in terms of functional group of terminal allyl group) and 1.0 g of a platinum catalyst (a solution of a vinylsiloxane complex of chloroplatinic acid, platinum concentration: 1% by weight) were put, and the temperature was raised to 90° C. while the resulting mixture was heated and stirred. Subsequently, under stirring, 15 g of 1-hydroxy-octamethyltetrasiloxane (the functional group amount of terminal Si—H: 0.050 mol) was added dropwise thereto. At this time, heat was generated, the reaction temperature became 90 to 95° C., and this reaction system was held for six hours. After completion of the reaction, a small excess of 1-hydroxy-octamethyltetrasiloxane was removed under reduced pressure. The temperature was lowered to room temperature. Thereafter, filtration was performed to obtain 251 g of a silanol-group-terminated polypropylene glycol (polymer E) (viscosity: 26.5 Pa·s, molecular weight: 8,100, yield: 95%). Example 6 100 parts of the silanol-group-terminated polypropylene glycol (polymer A) having viscosity of 4.0 Pa·s, synthesized according to the method of Example 1, 13.2 parts of bis(trimethoxysilyl-ethylene) dimethylsilane (the following structural formula), and 0.5 parts of dioctyltin dineodecanoate were mixed under a moisture blocking condition until the resulting mixture became homogeneous to prepare a composition. Example 7 A composition was prepared in a similar manner to Example 6 except that the same amount of tetramethylguanidylpropyl trimethoxysilane was used in place of dioctyltin dineodecanoate. Example 8 A composition was prepared in a similar manner to Example 7 except that the same amount of di-n-butyl-dimethoxy tin was used in place of tetramethylguanidylpropyl to trimethoxysilane. Example 9 A composition was prepared in a similar manner to Example 8 except that 100 parts of the silanol-group-terminated polypropylene glycol (polymer B) having viscosity of 10.0 Pa·s, synthesized according to the method of Example 2, was used in place of the silanol-group-terminated polypropylene glycol (polymer A) having viscosity of 4.0 Pa·s, synthesized according to the method of Example 1. Example 10 A composition was prepared in a similar manner to Example 9 except that 6.6 parts of vinyltrimethoxysilane was used in place of bis(trimethoxysilyl-ethylene) dimethylsilane. Example 11 A composition was prepared in a similar manner to Example 9 except that 13.9 parts of vinyltriisopropoxysilane was used in place of bis(trimethoxysilyl-ethylene) dimethylsilane and that the same amount of dioctyltin dineodecanoate was used in place of di-n-butyl-dimethoxy tin. Examples 12 to 14 A composition was prepared in a similar manner to Examples 9 to 11 except that 100 parts of the silanol-group-terminated polypropylene glycol (polymer C) having viscosity of 1.3 Pa·s, synthesized according to the method of Example 3, was used in place of the silanol-group-terminated polypropylene glycol (polymer B) having viscosity of 10.0 Pa·s, synthesized according to the method of Example 2. Examples 15 to 17 A composition was prepared in a similar manner to Examples 9 to 11 except that 100 parts of the silanol-group-terminated polypropylene glycol (polymer D) having viscosity of 9.2 Pa·s, synthesized according to the method of Example 4, was used in place of the silanol-group-terminated polypropylene glycol (polymer B) having viscosity of 10.0 Pa·s, synthesized according to the method of Example 2. Examples 18 to 20 A composition was prepared in a similar manner to Examples 9 to 11 except that 100 parts of the silanol-group-terminated polypropylene glycol (polymer E) having viscosity of 26.5 Pa·s, synthesized according to the method of Example 5, was used in place of the silanol-group-terminated polypropylene glycol (polymer B) having viscosity of 10.0 Pa·s, synthesized according to the method of Example 2. Comparative Example 1 A composition was prepared in a similar manner to Example 6 except that the same amount of MS polymer S303H (polyoxypropylene polymer having both molecular chain terminals blocked with dimethoxysilyl groups) manufactured by Kaneka Corporation was used in place of polymer A. Subsequently, each of the compositions immediately after preparation in Examples 6 to 20 and Comparative Example 1 was extruded into a sheet shape having a thickness of 2 mm and exposed to air at 23° C. and 50% RH. Subsequently, the physical properties (initial physical properties) of a cured product obtained by leaving the sheet for seven days in the same atmosphere were measured in accordance with JIS K-6249. Note that the hardness was measured using a durometer A hardness meter of JIS K-6249. The results are illustrated in Tables 1 and 2. TABLE 1Example678910111213Hardness3130322110105522Elongation (%)1005090501801702060Tensile0.390.340.420.320.170.190.630.51strength (MPa) TABLE 2ComparativeExampleExample141516171819201Hardness213410152711103Elongation (%)8040150185100200225205Tensile strength (MPa)0.310.360.180.240.490.230.190.21 It was confirmed that each of the cured products obtained in Examples 6 to 20 satisfied equivalent rubber properties (elongation and tensile strength) to a conventional modified silicone rubber cured product based on MS polymer S303H having a terminal blocked with a dialkoxysilyl group, manufactured by Kaneka Corporation, illustrated in Comparative Example 1. As for the hardness, it was confirmed that a rubber cured product having higher hardness than the conventional modified silicone rubber cured product could be obtained. Therefore, by using the novel polyoxyalkylene compound having a silanol group-containing reactive silicon group at a molecular chain terminal according to the present invention as a base polymer of a room-temperature-curable composition (modified silicone RTV composition), it is theoretically possible to use not only an alkoxy type group as a hydrolyzable group of a crosslinking agent but also various curing agents (for example, an organosilicon compound containing various hydrolyzable groups such as oxime, amide, aminoxy, acetic acid (acetoxy group), and alcohol (alkoxy group)) as a crosslinking component. This makes it possible to obtain various curing reaction (condensation reaction) types of room-temperature-curable compositions (modified silicone RTV compositions). | 56,034 |
11859051 | DETAILED DESCRIPTION The present disclosure relates to compositions, synthesis methods, and application methods of polyamides having an optical absorber in the backbone of the polyamide. More specifically, the polyamide syntheses described herein use amine and/or carboxyl functionalized optical absorbers as comonomers with the polyamide monomers. The result is a polyamide having optical absorbers in the backbone of the polyamide, also referred to herein as an in-backbone optical absorber polyamide or IBOA-polyamide. Because the optical absorbers are in the backbone of the polyamide, objects produced by additive manufacturing methods that include IBOA-polyamide-containing particles should maintain an even color and/or fluorescence over time because the optical absorbers cannot migrate within or leach from the object. The present disclosure also relates to particles comprising a polyamide having an optical absorber in the backbone of the polyamide (also referred to herein as an in-backbone optical absorber polyamide or IBOA-polyamide) and related methods. More specifically, the present disclosure includes methods of making highly spherical polymer particles comprising the one or more IBOA-polyamides and optionally one or more other thermoplastic polymers. Said polymer particles may be useful, among other things, as starting material for additive manufacturing. The polymer particles described herein are produced by melt emulsification methods where one or more IBOA-polyamides and optionally one or more additional thermoplastic polymers are dispersed as a melt in a carrier fluid that is immiscible with the IBOA-polyamide and additional thermoplastic polymers, if used. A sufficient amount of shear is applied to the mixture to cause the polymer melt to form droplets in the carrier fluid. Because the optical absorber is in the backbone of the polyamide, objects produced by additive manufacturing methods that include these particles should maintain an even color over time because the optical absorber cannot migrate within or leach from the object. Definitions and Test Methods As used herein, the term “immiscible” refers to a mixture of components that, when combined, form two or more phases that have less than 5 wt % solubility in each other at ambient pressure and at room temperature or the melting point of the component if it is solid at room temperature. For example, polyethylene oxide having 10,000 g/mol molecular weight is a solid at room temperature and has a melting point of 65° C. Therefore, said polyethylene oxide is immiscible with a material that is liquid at room temperature if said material and said polyethylene oxide have less than 5 wt % solubility in each other at 65° C. As used herein, the term “optical absorber” refers to a molecule or portion thereof that absorbs ultraviolet or visible light. As used herein, the term “chromophore” refers to an optical absorber where the light absorption imparts color. As used herein, the term “fluorophore” refers to an optical absorber that re-emits an absorbed photon at a different wavelength. As used herein, the term “polyamide monomer(s)” refers to a monomer(s) that form a polyamide. As used herein, the term “polyacid” when referring to a compound refers to a compound having two or more carboxylic acid moieties. Herein, anhydride moieties are considered carboxylic acid moieties because the anhydrides open to carboxylic acids during synthesis. As used herein, the term “polyamine” when referring to a compound refers to a compound having two or more amine moieties. As used herein, the term “amino acid” when referring to a compound refers to a compound having one or more carboxylic acid moieties and one or more amine moieties. Again, anhydride moieties are considered carboxylic acid moieties because the anhydrides open to carboxylic acids during synthesis. When referring to a polymer in terms of the -mer units (e.g., polyamide monomers and/or chromophores in the backbone of a polyamide), it would be understood by one skilled in the art that the -mer units are in the polymerized form in the polymer. As used herein, the term “thermoplastic polymer” refers to a plastic polymer material that softens and hardens reversibly on heating and cooling. Thermoplastic polymers encompass thermoplastic elastomers. As used herein, the term “elastomer” refers to a copolymer comprising a crystalline “hard” section and an amorphous “soft” section. In the case of a polyurethane, the crystalline section may include a portion of the polyurethane comprising the urethane functionality and optional chain extender group, and the soft section may include the polyol, for instance. As used herein, the term “polyurethane” refers to a polymeric reaction product between a diisocyanate, a polyol, and an optional chain extender. As used herein, the term “oxide” refers to both metal oxides and non-metal oxides. For purposes of the present disclosure, silicon is considered to be a metal. As used herein, the terms “associated,” “association,” and grammatical variations thereof between emulsion stabilizers and a surface refers to chemical bonding and/or physical adherence of the emulsion stabilizers to the surface. Without being limited by theory, it is believed that the associations described herein between polymers and emulsion stabilizers are primarily physical adherence via hydrogen bonding and/or other mechanisms. However, chemical bonding may be occurring to some degree. As used herein, the term “embed” relative to nanoparticles and a surface of a polymer particle refers to the nanoparticle being at least partially extended into the surface such that polymer is in contact with the nanoparticle to a greater degree than would occur if the nanoparticle were simply laid on the surface of the polymer particle. Herein, D10, D50, D90, and diameter span are primarily used herein to describe particle sizes. As used herein, the term “D10” refers to a diameter at which 10% of the sample (on a volume basis unless otherwise specified) is comprised of particles having a diameter less than said diameter value. As used herein, the term “D50” refers to a diameter at which 50% of the sample (on a volume basis unless otherwise specified) is comprised of particles having a diameter less than said diameter value. As used herein, the term “D90” refers to a diameter at which 90% of the sample (on a volume basis unless otherwise specified) is comprised of particles having a diameter less than said diameter value. As used herein, the terms “diameter span” and “span” and “span size” when referring to diameter provides an indication of the breadth of the particle size distribution and is calculated as (D90−D10)/D50 (again each D-value is based on volume, unless otherwise specified). Particle size can be determined by light scattering techniques using a Malvern MASTERSIZER™ 3000 or analysis of optical digital micrographs. Unless otherwise specified, light scattering techniques are used for analyzing particle size. For light scattering techniques, the control samples were glass beads with a diameter within the range of 15 μm to 150 μm under the tradename Quality Audit Standards QAS4002™ obtained from Malvern Analytical Ltd. Samples were analyzed as dry powders, unless otherwise indicated. The particles analyzed were dispersed in air and analyzed using the AERO S dry powder dispersion module with the MASTERSIZER™ 3000. The particle sizes were derived using instrument software from a plot of volume density as a function of size. Particle size measurement and diameter span can also be determined by optical digital microscopy. The optical images are obtained using a Keyence VHX-2000 digital microscope using version 2.3.5.1 software for particle size analysis (system version 1.93). As used herein, when referring to sieving, pore/screen sizes are described per U.S.A. Standard Sieve (ASTM E11-17). As used herein, the terms “circularity” and “sphericity” relative to the particles refer to how close the particle is to a perfect sphere. To determine circularity, optical microscopy images are taken of the particles. The perimeter (P) and area (A) of the particle in the plane of the microscopy image is calculated (e.g., using a SYSMEX FPIA 3000 particle shape and particle size analyzer, available from Malvern Instruments). The circularity of the particle is CEA/P, where CEAis the circumference of a circle having the area equivalent to the area (A) of the actual particle. As used herein, the term “shear” refers to stirring or a similar process that induces mechanical agitation in a fluid. As used herein, the term “aspect ratio” refers to length divided by width, wherein the length is greater than the width. The melting point of a polymer, unless otherwise specified, is determined by ASTM E794-06(2018) with 10° C./min ramping and cooling rates. The softening temperature or softening point of a polymer, unless otherwise specified, is determined by ASTM D6090-17. The softening temperature can be measured by using a cup and ball apparatus available from Mettler-Toledo using a 0.50 gram sample with a heating rate of 1° C./min. Angle of repose is a measure of the flowability of a powder. Angle of repose measurements were determined using a Hosokawa Micron Powder Characteristics Tester PT-R using ASTM D6393-14 “Standard Test Method for Bulk Solids Characterized by Carr Indices.” Hausner ratio (Hr) is a measure of the flowability of a powder and is calculated by Hr=ρtap/ρbulk, where ρbulkis the bulk density per ASTM D6393-14 and ρtapis the tapped density per ASTM D6393-14. As used herein, the viscosity of carrier fluids is the kinematic viscosity at 25° C., unless otherwise specified, measured per ASTM D445-19. For commercially procured carrier fluids (e.g., PDMS oil), the kinematic viscosity data cited herein was provided by the manufacturer, whether measured according to the foregoing ASTM or another standard measurement technique. Polyamides with in-Backbone Optical Absorbers Polyamides may be synthesized by condensation polymerization (also referred to herein as polycondensation) or by ring-opening polymerization. Herein, these polymerizations are performed in the presence of one or more diamino optical absorbers, one or more diacid optical absorbers, one or more amino acid optical absorbers, or any combination thereof. This results in the optical absorbers being incorporated into the backbone of the polyamide. The resultant IBOA-polyamides can then be used in a variety of applications. Several example polycondensation and ring-opening polymerization reactions are presented below followed by nonlimiting examples of polyamide monomers and optical absorbers suitable for use in one or more of said reactions. The following examples are nonlimiting to the scope of polyamide monomers and optical absorbers. The nonlimiting polycondensation examples presented in Schemes 1-4 use amino acid polyamide monomers with one or more polyamine optical absorbers, one or more polyacid optical absorbers, one or more amino acid optical absorbers, or any combination thereof to yield an IBOA-polyamide. Scheme 1 illustrates a polycondensation reaction between an amino acid polyamide monomer and an amino acid optical absorber (OA1). Scheme 2 illustrates a polycondensation reaction between an amino acid polyamide monomer and a polyamine optical absorber (OA2). In this example, there will be one optical absorber per polymer chain. Scheme 3 illustrates a polycondensation reaction between an amino acid polyamide monomer and a polyacid optical absorber (OA3). In this example, there will be one optical absorber per polymer chain. Scheme 4 illustrates a polycondensation reaction between an amino acid polyamide monomer, a polyamine optical absorber (OA2), and a polyacid optical absorber (OA3). Again, Schemes 1-4 are nonlimiting examples. One skilled in the art will recognize other polycondensation reactions that utilize amino acid polyamide monomers with amino acid optical absorbers, polyamine optical absorbers, and/or polyacid optical absorbers. For example, more than one amino acid polyamide monomer may be used. In another example, other combinations of the three types of optical absorbers may be used (e.g., OA1 with OA2, OA1 with OA3, and OA1, OA2, and OA3 together). In yet another example, two or more amino acid optical absorbers, two or more polyamine optical absorbers, and/or two or more polyacid optical absorbers may be used. Further, combinations of these variations may be implemented. Further, Schemes 1-4 illustrate random polymerization. However, one skilled in the art will recognize that block polymers can be produced (e.g., by adding different optical absorbers at different times or having a portion of the polymerization occur with no optical absorber present). Other polymerization techniques can be used like grafting where the optical absorber is in the backbone of the polyamide. The nonlimiting polycondensation examples presented in Schemes 5-7 use polyamine polyamide monomers and polyacid polyamide monomers with one or more polyamide optical absorbers, one or more polyacid optical absorbers, one or more amino acid optical absorbers, or any combination thereof to yield an IBOA-polyamide. Scheme 5 illustrates a polycondensation reaction between a polyamine polyamide monomer, a polyacid polyamide monomer, and an amino acid optical absorber (OA1). Scheme 6 illustrates a polycondensation reaction between a polyamine polyamide monomer, a polyacid polyamide monomer, and a polyamine optical absorber (OA2). Scheme 7 illustrates a polycondensation reaction between a polyamine polyamide monomer, a polyacid polyamide monomer, and a polyacid optical absorber (OA3). Again, Schemes 5-7 are nonlimiting examples. One skilled in the art will recognize other polycondensation reactions that utilize polyamine polyamide monomers and polyacid polyamide monomers with amino acid optical absorbers, polyamine optical absorbers, and/or polyacid optical absorbers. For example, more than one polyamine polyamide monomers and/or more than one polyacid polyamide monomers may be used. In another example, combinations of the three types of optical absorbers may be used (e.g., OA1 with OA2, OA1 with OA3, OA2 with OA3, and OA1, OA2, and OA3 together). In yet another example, two or more amino acid optical absorbers, two or more polyamine optical absorbers, and/or two or more polyacid optical absorbers may be used. Further, combinations of these variations may be implemented. Further, Schemes 5-7 illustrate random polymerization. However, one skilled in the art will recognize that block polymers can be produced (e.g., by adding different optical absorbers at different times or having a portion of the polymerization occur with no optical absorber present). Other polymerization techniques can be used like grafting where the optical absorber is in the backbone of the polyamide. The nonlimiting ring-opening polymerization reaction examples presented in Schemes 8-11 use amino acid polyamide monomers with one or more polyamide optical absorbers, one or more polyacid optical absorbers, one or more amino acid optical absorbers, or any combination thereof to yield an IBOA-polyamide. Scheme 8 illustrates a ring-opening polymerization reaction between a cyclic polyamide monomer and an amino acid optical absorber (OA1). Scheme 9 illustrates a ring-opening polymerization reaction between a cyclic polyamide monomer and a polyamine optical absorber (OA2). In this example, there will be one optical absorber per polymer chain. Scheme 10 illustrates a ring-opening polymerization reaction between a cyclic acid polyamide monomer and a polyacid optical absorber (OA3). In this example, there will be one optical absorber per polymer chain. Scheme 11 illustrates a ring-opening polymerization reaction between a cyclic acid polyamide monomer, a polyamine optical absorber (OA2), and a polyacid optical absorber (OA3). Again, Schemes 8-11 are nonlimiting examples. One skilled in the art will recognize other ring-opening polymerization reactions that utilize cyclic polyamide monomers with amino acid optical absorbers, polyamine optical absorbers, and/or polyacid optical absorbers. For example, more than one cyclic polyamide monomer may be used. In another example, other combinations of the three types of optical absorbers may be used (e.g., OA1 with OA2, OA1 with OA3, and OA1, OA2, and OA3 together). In yet another example, two or more amino acid optical absorbers, two or more polyamine optical absorbers, and/or two or more polyacid optical absorbers may be used. Further, combinations of these variations may be implemented. Examples of amino acid polyamide monomers suitable for use in polycondensations include, but are not limited to, H2N—(CH2)n—COOH where n is 1-20; branched aliphatic amino acids (e.g., C4-C20); cyclic-aliphatic amino acids (e.g., C4-C20); aromatic amino acids (e.g., 3-aminobenzoic acid, 4-aminobenzoic acid); and the like; and any combination thereof. Examples of polyacid polyamide monomers suitable for use in polycondensations include, but are not limited to, HOOC—(CH2)n—COOH where n is 1-20 (e.g., adipic acid, terephthalic acid, isophthalic acid, pimelic acid, suberic acid, decanedioic acid, dodecanedioic acid); isophthalic acid; terephthalic acid; pent-2-enedioic acid; dodec-2-enedioic acid; succinic acid; glutaric acid; adipic acid; pimelic acid; suberic acid; azeleic acid; sebacic acid; undecanedioic acid; dodecanedioic acid; 1,3-cyclohexanedicarboxylic acid; and the like; and any combination thereof. Examples of polyamine polyamide monomers suitable for use in polycondensations include, but are not limited to, H2N—(CH2)n—NH2where n is 1-20; 1,5-diamino-2-methylpentane; 1,2-diaminopropane; trimethylhexamethylenediamine; 2-methyloctane-1,8-diamine; n-methyl 1,6-hexamethylene diamine where n is 2 or 3; n-methyl 1,7-heptamethylene diamine where n is 2-4; n-methyl 1,8-octamethylene diamine where n is 2-4; n-methyl 1,12-dodecamethylene diamine where n is 2-6; 1,3-bis(aminomethyl)benzene; ortho-phenylene-bis(methylamine); 1,4-bis(aminomethyl)benzene; 1,4-cyclohexanediamine; 4-methyl cyclohexane-1,3-diamine; 4-methylcyclohexane-1,3-diamine; diphenylethylenediamine; diphenylethylenediamine; 1,3-bis(aminomethyl)cyclohexane; 4,4′-methylenebis(cyclohexylamine); 4,4′-biphenyldiamine; 1,8-diaminonaphthalene; and the like; and any combination thereof. Examples of cyclic polyamide monomers suitable for use in ring-opening polymerizations include, but are not limited to, azeridinone, 2-azetidinone, 2-pyrrolidinone, 2-piperidinone, ε-caprolactam, 2-azacyclooctanone, 2-azacyclononanone, 2-azacyclodecanone, 2-azacycloundecanone, 2-azacyclododecanone, laurolactam, and the like, and any combination thereof. Generally, optical absorbers may belong to the following classes of optical absorbers: naphthalimides, fluoresceins, rhodamines, coumarins, azo-dyes, oxadiazoles, perylenes, calceins, and other aromatic dyes. However, other optical absorbers may be suitable for use in the methods and compositions described herein. As stated above, anhydride moieties are considered carboxylic acid moieties because the anhydrides open to carboxylic acids during synthesis. Examples of amino acid optical absorbers suitable for use in polycondensations include, but are not limited to, 4-amino-1,8-naphthalimide; 7-amino-4-methyl-3-coumarinylacetic acid; and the like; and any combination thereof. Preferably, amino acid optical absorbers used in the polycondensation reactions described herein have one amine moiety and one carboxylic acid moiety. Examples of polyacid optical absorbers suitable for use in polycondensations include, but are not limited to, calcein (also known as flourexon); 4-methylumbelliferone-8-methyliminodiacetic acid (also known as calein blue); 6-carboxyfluorescein (also known as 6-FAM); 3,9-perylenedicarboxylic acid; N,N-bis(4-tert-butylphenyl)-N′,N′-bis(4-carboxyphthalimido)-1,4-phenylenediamine; perylene-3,4,9,10-tetracarboxylic dianhydride; alizarin-3-methyliminodiacetic acid; and the like; and any combination thereof. Preferably, polyacid optical absorbers used in the polycondensation reactions described herein have two carboxylic acid moieties. Examples of polyamine optical absorbers suitable for use in polycondensations include, but are not limited to, N,N-di(4-aminophenyl)-1-aminopyrene; N,N-bis(4-aminophenyl)-N′-4-methoxyphenyl-N′-4-(1,2,2-triphenylethenyl)phenyl-1,4-phenylenediamine; 4,5-diamino-rhodamine B (also known as DAR-1); rhodamine 123; 2,7-dimethyl acridine-3,6-diamine; 2-nitro-1,4-phenylenediamine; 4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-di en-1-ylidene)methyl]aniline hydrochloride (also known as Pararosaniline and Basic Red 9); 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2-sulfonate sodium (also known as Acid Blue 43); and the like; and any combination thereof. Preferably, polyamine optical absorbers used in the polycondensation reactions described herein have two amine moieties. Examples of polyamides that can be synthesized with optical absorbers in the backbone include, but are not limited to, polycaproamide (nylon 6, polyamide 6, or PA6), polyhexamethylene succinamide (nylon 4,6, polyamide 4,6, or PA4,6), polyhexamethylene adipamide (nylon 6,6, polyamide 6,6, or PA6,6), polypentamethylene adipamide (nylon 5,6, polyamide 5,6, or PA5,6), polyhexamethylene sebacamide (nylon 6,10, polyamide 6,10, or PA6,10), polyundecamide (nylon 11, polyamide 11, or PA11), polydodecamide (nylon 12, polyamide 12, or PA12), and polyhexamethylene terephthalamide (nylon 6T, polyamide 6T, or PA6T), nylon 10,10 (polyamide 10,10 or PA10,10), nylon 10,12 (polyamide 10,12 or PA10,12), nylon 10,14 (polyamide 10,14 or PA10,14), nylon 10,18 (polyamide 10,18 or PA10,18), nylon 6,18 (polyamide 6,18 or PA6,18), nylon 6,12 (polyamide 6,12 or PA6,12), nylon 6,14 (polyamide 6,14 or PA6,14), nylon 12,12 (polyamide 12,12 or PA12,12), semi-aromatic polyamide, aromatic polyamides (aramides), and the like, and any combination thereof. Copolyamides may also be used. Examples of copolyamides include, but are not limited to, PA 11/10,10, PA 6/11, PA 6,6/6, PA 11/12, PA 10,10/10,12, PA 10,10/10,14, PA 11/10,36, PA 11/6,36, PA 10,10/10,36, PA 6T/6,6, and the like, and any combination thereof. Examples of polyamide elastomers include, but are not limited to, polyesteramide, polyetheresteramide, polycarbonate-esteramide, and polyether-block-amide elastomers. Herein, a polyamide followed by a single number is a polyamide having that number of backbone carbons between each nitrogen. A polyamide followed by a first number comma second number is a polyamide having the first number of backbone carbons between the nitrogens for the section having no pendent ═O and the second number of backbone carbons being between the two nitrogens for the section having the pendent ═O. By way of nonlimiting example, nylon 6,10 is [NH—(CH2)6—NH—CO—(CH2)8—CO]n. A polyamide followed by number(s) backslash number(s) are a copolymer of the polyamides indicated by the numbers before and after the backslash. Polycondensation reactions (e.g., Schemes 1-7 and variations thereof) may be performed in the presence of an activator and/or metal salts. Examples of activators include, but are not limited to, triphenyl phosphine; and the like; and any combination thereof. Examples of metal salts include, but are not limited to, calcium chloride; cesium fluoride; and the like; and any combination thereof. Polycondensation reactions (e.g., Schemes 1-7 and variations thereof) may be performed at about 50° C. to about 200° C. (or about 50° C. to about 100° C., or about 75° C. to about 150° C., or about 125° C. to about 200° C.). Polycondensation reactions (e.g., Schemes 1-7 and variations thereof) may be performed for about 5 minutes to about 24 hours (or about 5 minutes about 6 hours, or about 2 hours to about 12 hours, or about 6 hours to about 24 hours). Polycondensation reactions (e.g., Schemes 1-7 and variations thereof) may be performed in a solvent that includes, but is not limited to, N-methyl pyrrolidone (NMP), pyridine, dichloromethane, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and the like, and any combination thereof. Polycondensation reactions (e.g., Schemes 1-7 and variations thereof) may be performed with a molar ratio of polyamide monomers (cumulatively) to optical absorbers (cumulatively) of about 500:1 to about 10:1 (or about 500:1 to about 100:1, or about 250:1 to about 50:1, or about 100:1 to about 10:1). Ring-opening polymerization reactions (e.g., Schemes 8-11 and variations thereof) may be performed in the presence of an activator and/or metal salts. Examples of activators include, but are not limited to, triphenyl phosphine; and the like; and any combination thereof. Examples of metal salts include, but are not limited to, calcium chloride; cesium fluoride; and the like; and any combination thereof. Ring-opening polymerization reactions (e.g., Schemes 8-11 and variations thereof) may be performed at about 50° C. to about 200° C. (or about 50° C. to about 100° C., or about 75° C. to about 150° C., or about 125° C. to about 200° C.). Ring-opening polymerization reactions (e.g., Schemes 8-11 and variations thereof) may be performed for about 5 minutes to about 24 hours (or about 5 minutes about 6 hours, or about 2 hours to about 12 hours, or about 6 hours to about 24 hours). Ring-opening polymerization reactions (e.g., Schemes 8-11 and variations thereof) may be performed in a solvent that includes, but is not limited to, N-mehtyl pyrrolidone (NMP), pyridine, dichloromethane, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, acetonitrile, tetrahydrofuran, and the like, and any combination thereof. Ring-opening polymerization reactions (e.g., Schemes 8-11 and variations thereof) may be performed with a molar ratio of polyamide monomers (cumulatively) to optical absorbers (cumulatively) of about 500:1 to about 10:1 (or about 500:1 to about 100:1, or about 250:1 to about 50:1, or about 100:1 to about 10:1). The resultant IBOA-polyamide from any suitable synthesis route may have a molar equivalent of non-optical absorber polyamide units to optical absorber units of about 500:1 to about 10:1 (or about 500:1 to about 100:1, or about 250:1 to about 50:1, or about 100:1 to about 10:1). Applications of IBOA-Polyamides The IBOA-polyamides described herein may be used to produce a variety of objects (or articles). The IBOA-polyamides described herein may be used alone or in combination with other thermoplastic polymers (e.g., polyamides without an optical absorber and/or other thermoplastic polymers). Examples of thermoplastic polymers that may be used in conjunction with one or more IBOA-polyamides of the present disclosure include, but are not limited to, polyamides, polyurethanes, polyethylenes, polypropylenes, polyacetals, polycarbonates, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), polyhexamethylene terephthalate, polystyrenes, polyvinyl chlorides, polytetrafluoroethenes, polyesters (e.g., polylactic acid), polyethers, polyether sulfones, polyetherether ketones, polyacrylates, polymethacrylates, polyimides, acrylonitrile butadiene styrene (ABS), polyphenylene sulfides, vinyl polymers, polyarylene ethers, polyarylene sulfides, polysulfones, polyether ketones, polyamide-imides, polyetherimides, polyetheresters, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), grafted or ungrafted thermoplastic polyolefins, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylates, functionalized or nonfunctionalized (meth)acrylic acid polymers, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymers, ethylene/vinyl monomer/carbonyl terpolymers, ethylene/alkyl (meth)acrylate/carbonyl terpolymers, methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, chlorinated or chlorosulphonated polyethylenes, polyvinylidene fluoride (PVDF), phenolic resins, poly(ethylene/vinyl acetate)s, polybutadienes, polyisoprenes, styrenic block copolymers, polyacrylonitriles, silicones, and the like, and any combination thereof. Copolymers comprising one or more of the foregoing may also be used in the methods and systems described herein. If needed, compatibilizers may be used when combining the IBOA-polyamides described herein with other thermoplastic polymers. Compatibilizers may improve the blending efficiency and/or efficacy of the polymers. Examples of polymer compatibilizers include, but are not limited to, PROPOLDER™ MPP2020 20 (polypropylene, available from Polygroup Inc.), PROPOLDER™ MPP2040 40 (polypropylene, available from Polygroup Inc.), NOVACOM™ HFS2100 (maleic anhydride functionalized high density polyethylene polymer, available from Polygroup Inc.), KEN-REACT™ CAPS™ L™ 12/L (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ L™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ LICA™ 12 (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPS™ KPR™ 12/LV (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ KPR™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ titanates & zirconates (organometallic coupling agent, available from Kenrich Petrochemicals), VISTAMAXX™ (ethylene-propylene copolymers, available from ExxonMobil), SANTOPRENE™ (thermoplastic vulcanizate of ethylene-propylene-diene rubber and polypropylene, available from ExxonMobil), VISTALON™ (ethylene-propylene-diene rubber, available from ExxonMobil), EXACT™ (plastomers, available from ExxonMobil) EXXELOR™ (polymer resin, available from ExxonMobil), FUSABOND™ M603 (random ethylene copolymer, available from Dow), FUSABOND™ E226 (anhydride modified polyethylene, available from Dow), BYNEL™ 41E710 (coextrudable adhesive resin, available from Dow), SURLYN™ 1650 (ionomer resin, available from Dow), FUSABOND™ P353 (a chemically modified polypropylene copolymer, available from Dow), ELVALOY™ PTW (ethylene terpolymer, available from Dow), ELVALOY™ 3427AC (a copolymer of ethylene and butyl acrylate, available from Dow), LOTADER™ AX8840 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3210 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3410 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3430 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4700 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ AX8900 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4720 (ethylene acrylate-based terpolymer, available from Arkema), BAXXODUR™ EC 301 (amine for epoxy, available from BASF), BAXXODUR™ EC 311 (amine for epoxy, available from BASF), BAXXODUR™ EC 303 (amine for epoxy, available from BASF), BAXXODUR™ EC 280 (amine for epoxy, available from BASF), BAXXODUR™ EC 201 (amine for epoxy, available from BASF), BAXXODUR™ EC 130 (amine for epoxy, available from BASF), BAXXODUR™ EC 110 (amine for epoxy, available from BASF), styrenics, polypropylene, polyamides, polycarbonate, EASTMAN™ G-3003 (a maleic anhydride grafted polypropylene, available from Eastman), RETAIN™ (polymer modifier, available from Dow), AMPLIFY TY™ (maleic anhydride grafted polymer, available from Dow), INTUNE™ (olefin block copolymer, available from Dow), and the like, and any combination thereof. Methods for producing objects include, but are not limited to, melt extrusion, injection molding, compression molding, melt spinning, melt emulsification, spray drying (e.g., to form particles), cryogenic milling (or cryogenic grinding), freeze drying polymer dispersions, precipitation of polymer dispersions, and the like, and any hybrid thereof. Examples of articles that may be produced by such methods where the IBOA-polyamide may be all or a portion of said articles include, but are not limited to, particles, films, packaging, toys, household goods, automotive parts, aerospace/aircraft-related parts, containers (e.g., for food, beverages, cosmetics, personal care compositions, medicine, and the like), shoe soles, furniture parts, decorative home goods, plastic gears, screws, nuts, bolts, cable ties, jewelry, art, sculpture, medical items, prosthetics, orthopedic implants, production of artifacts that aid learning in education, 3D anatomy models to aid in surgeries, robotics, biomedical devices (orthotics), home appliances, dentistry, electronics, sporting goods, and the like. Further, particles may be useful in applications that include, but are not limited to, paints, powder coatings, ink jet materials, electrophotographic toners, 3D printing, and the like. In addition, the IBOA-polyamides described herein may have a specific chemical fingerprint that is useful in identifying objects, tracking objects, authenticating objects, and/or determining the health of objects. Further, the placement of where the IBOA-polyamides are located in the objects has another layer of fingerprinting the objects for identifying objects, tracking objects, authenticating objects, and/or determining the health of objects. Methods of identifying objects, tracking objects, authenticating objects, and/or determining the health of objects may include (a) exposing the object comprising IBOA-polyamides to electromagnetic radiation (e.g., for fluorophores preferably at a wavelength of 302 nm or less or 700 nm or greater); (b) sensing one or more spectra related to the electromagnetic radiation absorbed and/or reemitted (e.g., for fluorophores preferably the photoluminescence emitted between 302 nm to 700 nm); and (c) comparing the spectra to the known spectra for the optical absorbers used in said object or portion thereof. Optionally, the location of where the spectra area is located on the object may be compared to the known location where the spectra area should be. The comparison(s) can be used for identifying and/or authenticating the object. For tracking, the comparison(s) may be done and/or the detected spectra and/or spectra area may be logged into a database along with the physical location of the object. Further, the health of objects that wear and/or crack can be ascertained. For example, a core portion of the article may comprise optical absorbers and an outer portion may cover the core portion and not comprise the optical absorbers (or comprise different optical absorbers). Then, when comparing spectra, the appearance of spectral features for the optical absorbers in the core may indicate that the object is at or near the end of life. By way of nonlimiting example, 3-D printing processes of the present disclosure may comprise: depositing particles comprising one or more IBOA-polyamides of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) upon a surface in a specified shape, and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body (object), such that the consolidated body has a void percentage of about 1% or less after being consolidated. For example, heating and consolidation of the thermoplastic polymer particles may take place in a 3-D printing apparatus employing a laser, such that heating and consolidation take place by selective laser sintering. By way of nonlimiting example, 3-D printing processes of the present disclosure may comprise: extruding a filament comprising one or more IBOA-polyamides of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) through an orifice, wherein the filament becomes a polymer melt upon extrusion; depositing the polymer melt as a first layer on a platform; cooling the layer; depositing an additional layer of the polymer melt on the first layer; cooling the additional layer; repeating depositing and cooling for at least one additional layer to produce a 3-D shape. Yet another nonlimiting example is a method comprising: extruding a polymer melt comprising one or more IBOA-polyamides of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) through an orifice to produce a film, a fiber (or a filament), particles, pellets, or the like. Thermoplastic Polymer Particles and Methods of Making The FIGURE is a flow chart of a nonlimiting example method100of the present disclosure. Thermoplastic polymer102(comprising one or more IBOA-polyamides and optionally one or more other thermoplastic polymers), carrier fluid104, and optionally emulsion stabilizer106are combined108to produce a mixture110. The components102,104, and106can be added in any order and include mixing and/or heating during the process of combining108the components102,104, and106. The mixture110is then processed112by applying sufficiently high shear to the mixture110at a temperature greater than the melting point or softening temperature of the thermoplastic polymer102to form a melt emulsion114. Because the temperature is above the melting point or softening temperature of the thermoplastic polymer102, the thermoplastic polymer102becomes a polymer melt. The shear rate should be sufficient enough to disperse the polymer melt in the carrier fluid104as droplets (i.e., the polymer emulsion114). Without being limited by theory, it is believed that, all other factors being the same, increasing shear should decrease the size of the droplets of the polymer melt in the carrier fluid104. However, at some point there may be diminishing returns on increasing shear and decreasing droplet size or may be disruptions to the droplet contents that decrease the quality of particles produced therefrom. The melt emulsion114inside and/or outside the mixing vessel is then cooled116to solidify the polymer droplets into thermoplastic polymer particles (also referred to as solidified thermoplastic polymer particles). The cooled mixture118can then be treated120to isolate the thermoplastic polymer particles122from other components124(e.g., the carrier fluid104, excess emulsion stabilizer106, and the like) and wash or otherwise purify the thermoplastic polymer particles122. The thermoplastic polymer particles122comprise the thermoplastic polymer102and, when included, at least a portion of the emulsion stabilizer106coating the outer surface of the thermoplastic polymer particles122. Emulsion stabilizers106, or a portion thereof, may be deposited as a uniform coating on the thermoplastic polymer particles122. In some instances, which may be dependent upon non-limiting factors such as the temperature (including cooling rate), the type of thermoplastic polymer102, and the types and sizes of emulsion stabilizers106, the nanoparticles of emulsion stabilizers106may become at least partially embedded within the outer surface of thermoplastic polymer particles122in the course of becoming associated therewith. Even without embedment taking place, at least the nanoparticles within emulsion stabilizers106may remain robustly associated with thermoplastic polymer particles122to facilitate their further use. In contrast, dry blending already formed thermoplastic polymer particulates (e.g., formed by cryogenic grinding or precipitation processes) with a flow aid like silica nanoparticles does not result in a robust, uniform coating of the flow aid upon the thermoplastic polymer particulates. Advantageously, carrier fluids and washing solvents of the systems and methods described herein (e.g., method100) can be recycled and reused. One skilled in the art will recognize any necessary cleaning of used carrier fluid and solvent necessary in the recycling process. The thermoplastic polymer102and carrier fluid104should be chosen such that at the various processing temperatures (e.g., from room temperature to process temperature) the thermoplastic polymer102and carrier fluid104are immiscible. An additional factor that may be considered is the differences in (e.g., a difference or a ratio of) viscosity at process temperature between the molten polyamide102and the carrier fluid104. The differences in viscosity may affect droplet breakup and particle size distribution. Without being limited by theory, it is believed that when the viscosities of the molten polyamide102and the carrier fluid104are too similar, the circularity of the product as a whole may be reduced where the particles are more ovular and more elongated structures are observed. The thermoplastic polymers102comprises one or more IBOA-polyamides and optionally one or more other thermoplastic polymers. Examples of other thermoplastic polymers include, but are not limited to, polyamides, polyurethanes, polyethylenes, polypropylenes, polyacetals, polycarbonates, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), polyhexamethylene terephthalate, polystyrenes, polyvinyl chlorides, polytetrafluoroethenes, polyesters (e.g., polylactic acid), polyethers, polyether sulfones, polyetherether ketones, polyacrylates, polymethacrylates, polyimides, acrylonitrile butadiene styrene (ABS), polyphenylene sulfides, vinyl polymers, polyarylene ethers, polyarylene sulfides, polysulfones, polyether ketones, polyamide-imides, polyetherimides, polyetheresters, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), grafted or ungrafted thermoplastic polyolefins, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylates, functionalized or nonfunctionalized (meth)acrylic acid polymers, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymers, ethylene/vinyl monomer/carbonyl terpolymers, ethylene/alkyl (meth)acrylate/carbonyl terpolymers, methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, chlorinated or chlorosulphonated polyethylenes, polyvinylidene fluoride (PVDF), phenolic resins, poly(ethylene/vinyl acetate)s, polybutadienes, polyisoprenes, styrenic block copolymers, polyacrylonitriles, silicones, and the like, and any combination thereof. Copolymers comprising one or more of the foregoing may also be used in the methods and systems of the present disclosure. The other thermoplastic polymers in the compositions and methods of the present disclosure may be elastomeric or non-elastomeric. Some of the foregoing examples of other thermoplastic polymers may be elastomeric or non-elastomeric depending on the exact composition of the polymer. For example, polyethylene that is a copolymer of ethylene and propylene may be elastomeric or not depending on the amount of propylene in the polymer. Thermoplastic elastomers generally fall within one of six classes: styrenic block copolymers, thermoplastic polyolefin elastomers, thermoplastic vulcanizates (also referred to as elastomeric alloys), thermoplastic polyurethanes, thermoplastic copolyesters, and thermoplastic polyamides (typically block copolymers comprising polyamide). Examples of thermoplastic elastomers can be found in the “Handbook of Thermoplastic Elastomers,” 2nd ed., B. M. Walker and C. P. Rader, eds., Van Nostrand Reinhold, New York, 1988. Examples of thermoplastic elastomers include, but are not limited to, elastomeric polyamides, polyurethanes, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), methyl methacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, polybutadienes, polyisoprenes, styrenic block copolymers, and polyacrylonitriles), silicones, and the like. Elastomeric styrenic block copolymers may include at least one block selected from the group of: isoprene, isobutylene, butylene, ethylene/butylene, ethylene-propylene, and ethylene-ethylene/propylene. More specific elastomeric styrenic block copolymer examples include, but are not limited to, poly(styrene-ethylene/butylene), poly(styrene-ethylene/butylene-styrene), poly(styrene-ethylene/propylene), styrene-ethylene/propylene-styrene), poly(styrene-ethylene/propylene-styrene-ethylene-propylene), poly(styrene-butadiene-styrene), poly(styrene-butylene-butadiene-styrene), and the like, and any combination thereof. Examples of polyamides include, but are not limited to, polycaproamide, polyhexamethylene succinamide, polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecamide, polydodecamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Copolyamides may also be used. Examples of copolyamides include, but are not limited to, PA 11/10.10, PA 6/11, PA 6.6/6, PA 11/12, PA 10.10/10.12, PA 10.10/10.14, PA 11/10.36, PA 11/6.36, PA 10.10/10.36, and the like, and any combination thereof. Examples of polyamide elastomers include, but are not limited to, polyesteramide, polyetheresteramide, polycarbonate-esteramide, and polyether-block-amide elastomers. Examples of polyurethanes include, but are not limited to, polyether polyurethanes, polyester polyurethanes, mixed polyether and polyester polyurethanes, and the like, and any combination thereof. Examples of thermoplastic polyurethanes include, but are not limited to, poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone], ELASTOLLAN® 1190A (a polyether polyurethane elastomer, available from BASF), ELASTOLLAN® 1190A10 (a polyether polyurethane elastomer, available from BASF), and the like, and any combination thereof. Compatibilizers may optionally be used to improve the blending efficiency and efficacy IBOA-polyamides with one or more thermoplastic polymers. Examples of polymer compatibilizers include, but are not limited to, PROPOLDER™ MPP2020 20 (polypropylene, available from Polygroup Inc.), PROPOLDER™ MPP2040 40 (polypropylene, available from Polygroup Inc.), NOVACOM™ HFS2100 (maleic anhydride functionalized high density polyethylene polymer, available from Polygroup Inc.), KEN-REACT™ CAPS™ L™ 12/L (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ L™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ LICA™ 12 (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPS™ KPR™ 12/LV (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ KPR™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ titanates & zirconates (organometallic coupling agent, available from Kenrich Petrochemicals), VISTAMAXX™ (ethylene-propylene copolymers, available from ExxonMobil), SANTOPRENE™ (thermoplastic vulcanizate of ethylene-propylene-diene rubber and polypropylene, available from ExxonMobil), VISTALON™ (ethylene-propylene-diene rubber, available from ExxonMobil), EXACT™ (plastomers, available from ExxonMobil) EXXELOR™ (polymer resin, available from ExxonMobil), FUSABOND™ M603 (random ethylene copolymer, available from Dow), FUSABOND™ E226 (anhydride modified polyethylene, available from Dow), BYNEL™ 41E710 (coextrudable adhesive resin, available from Dow), SURLYN™ 1650 (ionomer resin, available from Dow), FUSABOND™ P353 (a chemically modified polypropylene copolymer, available from Dow), ELVALOY™ PTW (ethylene terpolymer, available from Dow), ELVALOY™ 3427AC (a copolymer of ethylene and butyl acrylate, available from Dow), LOTADER™ AX8840 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3210 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3410 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3430 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4700 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ AX8900 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4720 (ethylene acrylate-based terpolymer, available from Arkema), BAXXODUR™ EC 301 (amine for epoxy, available from BASF), BAXXODUR™ EC 311 (amine for epoxy, available from BASF), BAXXODUR™ EC 303 (amine for epoxy, available from BASF), BAXXODUR™ EC 280 (amine for epoxy, available from BASF), BAXXODUR™ EC 201 (amine for epoxy, available from BASF), BAXXODUR™ EC 130 (amine for epoxy, available from BASF), BAXXODUR™ EC 110 (amine for epoxy, available from BASF), styrenics, polypropylene, polyamides, polycarbonate, EASTMAN™ G-3003 (a maleic anhydride grafted polypropylene, available from Eastman), RETAIN™ (polymer modifier available from Dow), AMPLIFY TY™ (maleic anhydride grafted polymer, available from Dow), INTUNE™ (olefin block copolymer, available from Dow), and the like, and any combination thereof. The thermoplastic polymers102(comprising one or more IBOA-polyamides and optionally one or more other thermoplastic polymers) may have a melting point or softening temperature of about 50° C. to about 450° C. (or about 50° C. to about 125° C., or about 100° C. to about 175° C., or about 150° C. to about 280° C., or about 200° C. to about 350° C., or about 300° C. to about 450° C.). The thermoplastic polymers102may have a glass transition temperature (ASTM E1356-08(2014) with 10° C./min ramping and cooling rates) of about −50° C. to about 400° C. (or about −50° C. to about 0° C., or about −25° C. to about 50° C., or about 0° C. to about 150° C., or about 100° C. to about 250° C., or about 150° C. to about 300° C., or about 200° C. to about 400° C.). The thermoplastic polymers102may optionally comprise an additive. Typically, the additive would be present before addition of the thermoplastic polymers102to the mixture110. Therefore, in the thermoplastic polymer melt droplets and resultant thermoplastic polymer particles, the additive is dispersed throughout the thermoplastic polymer. Accordingly, for clarity, this additive is referred to herein as an “internal additive.” The internal additive may be blended with the thermoplastic polymer just prior to making the mixture110or well in advance. When describing component amounts in the compositions described herein (e.g., the mixture110and thermoplastic polymer particles122), a weight percent based on the thermoplastic polymer102not inclusive of the internal additive. For example, a composition comprising 1 wt % of emulsion stabilizer by weight of 100 g of a thermoplastic polymer102comprising 10 wt % internal additive and 90 wt % thermoplastic polymer is a composition comprising 0.9 g of emulsion stabilizer, 90 g of thermoplastic polymer, and 10 g of internal additive. The internal additive may be present in the thermoplastic polymer102at about 0.1 wt % to about 60 wt % (or about 0.1 wt % to about 5 wt %, or about 1 wt % to about 10 wt %, or about 5 wt % to about 20 wt %, or about 10 wt % to about 30 wt %, or about 25 wt % to about 50 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer102. For example, the thermoplastic polymer102may comprise about 70 wt % to about 85 wt % of a thermoplastic polymer and about 15 wt % to about 30 wt % of an internal additive like glass fiber or carbon fiber. Examples of internal additives include, but are not limited to, fillers, strengtheners, pigments, pH regulators, and the like, and combinations thereof. Examples of fillers include, but are not limited to, glass fibers, glass particles, mineral fibers, carbon fiber, oxide particles (e.g., titanium dioxide and zirconium dioxide), metal particles (e.g., aluminum powder), and the like, and any combination thereof. Examples of pigments include, but are not limited to, organic pigments, inorganic pigments, carbon black, and the like, and any combination thereof. The thermoplastic polymer102may be present in the mixture110at about 5 wt % to about 60 wt % (or about 5 wt % to about 25 wt %, or about 10 wt % to about 30 wt %, or about 20 wt % to about 45 wt %, or about 25 wt % to about 50 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer102and carrier fluid104combined. Suitable carrier fluids104have a viscosity at 25° C. of about 1,000 cSt to about 150,000 cSt (or about 1,000 cSt to about 60,000 cSt, or about 40,000 cSt to about 100,000 cSt, or about 75,000 cSt to about 150,000 cSt). Examples of carrier fluids104include, but are not limited to, silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, alkyl-terminal polyethylene glycols (e.g., C1-C4 terminal alkyl groups like tetraethylene glycol dimethyl ether (TDG)), paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and the like, and any combination thereof. Examples of silicone oils include, but are not limited to, polydimethylsiloxane, methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and the like, and any combination thereof. When the carrier fluid104comprises two or more of the foregoing, the carrier fluid104may have one or more phases. For example, polysiloxanes modified with fatty acids and polysiloxanes modified with fatty alcohols (preferably with similar chain lengths for the fatty acids and fatty alcohols) may form a single-phase carrier fluid104. In another example, a carrier fluid104comprising a silicone oil and an alkyl-terminal polyethylene glycol may form a two-phase carrier fluid104. The carrier fluid104may be present in the mixture110at about 40 wt % to about 95 wt % (or about 75 wt % to about 95 wt %, or about 70 wt % to about 90 wt %, or about 55 wt % to about 80 wt %, or about 50 wt % to about 75 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer102and carrier fluid104combined. In some instances, the carrier fluid104may have a density of about 0.6 g/cm3to about 1.5 g/cm3, and the thermoplastic polymer102has a density of about 0.7 g/cm3to about 1.7 g/cm3, wherein the thermoplastic polymer has a density similar, lower, or higher than the density of the carrier fluid. The emulsion stabilizers used in the methods and compositions of the present disclosure may comprise nanoparticles (e.g. oxide nanoparticles, carbon black, polymer nanoparticles, and combinations thereof), surfactants, and the like, and any combination thereof. Oxide nanoparticles may be metal oxide nanoparticles, non-metal oxide nanoparticles, or mixtures thereof. Examples of oxide nanoparticles include, but are not limited to, silica, titania, zirconia, alumina, iron oxide, copper oxide, tin oxide, boron oxide, cerium oxide, thallium oxide, tungsten oxide, and the like, and any combination thereof. Mixed metal oxides and/or non-metal oxides, like aluminosilicates, borosilicates, and aluminoborosilicates, are also inclusive in the term metal oxide. The oxide nanoparticles may by hydrophilic or hydrophobic, which may be native to the particle or a result of surface treatment of the particle. For example, a silica nanoparticle having a hydrophobic surface treatment, like dimethyl silyl, trimethyl silyl, and the like, may be used in methods and compositions of the present disclosure. Additionally, silica with functional surface treatments like methacrylate functionalities may be used in methods and compositions of the present disclosure. Unfunctionalized oxide nanoparticles may also be suitable for use as well. Commercially available examples of silica nanoparticles include, but are not limited to, AEROSIL® particles available from Evonik (e.g., AEROSIL® R812S (about 7 nm average diameter silica nanoparticles having a hydrophobically modified surface and a BET surface area of 260±30 m2/g), AEROSIL® RX50 (about 40 nm average diameter silica nanoparticles having a hydrophobically modified surface and a BET surface area of 35±10 m2/g), AEROSIL® 380 (silica nanoparticles having a hydrophilically modified surface and a BET surface area of 380±30 m2/g)), and the like, and any combination thereof. Carbon black is another type of nanoparticle that may be present as an emulsion stabilizer in the compositions and methods disclosed herein. Various grades of carbon black will be familiar to one having ordinary skill in the art, any of which may be used herein. Other nanoparticles capable of absorbing infrared radiation may be used similarly. Polymer nanoparticles are another type of nanoparticle that may be present as an emulsion stabilizer in the disclosure herein. Suitable polymer nanoparticles may include one or more polymers that are thermosetting and/or crosslinked, such that they do not melt when processed by melt emulsification according to the disclosure herein. High molecular weight thermoplastic polymers having high melting or decomposition points may similarly comprise suitable polymer nanoparticle emulsion stabilizers. The nanoparticles may have an average diameter (D50 based on volume) of about 1 nm to about 500 nm (or about 10 nm to about 150 nm, or about 25 nm to about 100 nm, or about 100 nm to about 250 nm, or about 250 nm to about 500 nm). The nanoparticles may have a BET surface area of about 10 m2/g to about 500 m2/g (or about 10 m2/g to about 150 m2/g, or about 25 m2/g to about 100 m2/g, or about 100 m2/g to about 250 m2/g, or about 250 m2/g to about 500 m2/g). Nanoparticles may be included in the mixture110at a concentration of about 0.01 wt % to about 10 wt % (or about 0.01 wt % to about 1 wt %, or about 0.1 wt % to about 3 wt %, or about 1 wt % to about 5 wt %, or about 5 wt % to about 10 wt %) based on the weight of the thermoplastic polymer102. Surfactants may be anionic, cationic, nonionic, or zwitterionic. Examples of surfactants include, but are not limited to, sodium dodecyl sulfate, sorbitan oleates, poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propylmethylsiloxane], docusate sodium (sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate), and the like, and any combination thereof. Commercially available examples of surfactants include, but are not limited to, CALFAX® DB-45 (sodium dodecyl diphenyl oxide disulfonate, available from Pilot Chemicals), SPAN® 80 (sorbitan maleate non-ionic surfactant), MERPOL® surfactants (available from Stepan Company), TERGITOL™ TMN-6 (a water-soluble, nonionic surfactant, available from DOW), TRITON™ X-100 (octyl phenol ethoxylate, available from SigmaAldrich), IGEPAL® CA-520 (polyoxyethylene (5) isooctylphenyl ether, available from SigmaAldrich), BRIJ® S10 (polyethylene glycol octadecyl ether, available from SigmaAldrich), and the like, and any combination thereof. Surfactants may be included in the mixture110at a concentration of about 0.01 wt % to about 10 wt % (or about 0.01 wt % to about 1 wt %, or about 0.5 wt % to about 2 wt %, or about 1 wt % to about 3 wt %, or about 2 wt % to about 5 wt %, or about 5 wt % to about 10 wt %) based on the weight of the polyamide102. Alternatively, the mixture110may comprise no (or be absent of) surfactant. A weight ratio of nanoparticles to surfactant may be about 1:10 to about 10:1 (or about 1:10 to about 1:1, or about 1:5 to about 5:1, or about 1:1 to about 10:1). As described above, the components102,104, and106can be added in any order and include mixing and/or heating during the process of combining108the components102,104, and106. For example, the emulsion stabilizer106may first be dispersed in the carrier fluid104, optionally with heating said dispersion, before adding the thermoplastic polymer102. In another nonlimiting example, the thermoplastic polymer102may be heated to produce a polymer melt to which the carrier fluid104and emulsion stabilizer106are added together or in either order. In yet another nonlimiting example, the thermoplastic polymer102and carrier fluid104can be mixed at a temperature greater than the melting point or softening temperature of the thermoplastic polymer102and at a shear rate sufficient enough to disperse the thermoplastic polymer melt in the carrier fluid104. Then, the emulsion stabilizer106can be added to form the mixture110and maintained at suitable process conditions for a set period of time. Combining108the components102,104, and106in any combination can occur in a mixing apparatus used for the processing112and/or another suitable vessel. By way of nonlimiting example, the thermoplastic polymer102may be heated to a temperature greater than the melting point or softening temperature of the thermoplastic polymer102in the mixing apparatus used for the processing112, and the emulsion stabilizer106may be dispersed in the carrier fluid104in another vessel. Then, said dispersion may be added to the melt of the thermoplastic polymer102in the mixing apparatus used for the processing112. The mixing apparatuses used for the processing112to produce the melt emulsion114should be capable of maintaining the melt emulsion114at a temperature greater than the melting point or softening temperature of the thermoplastic polymer102and applying a shear rate sufficient to disperse the polymer melt in the carrier fluid104as droplets. Examples of mixing apparatuses used for the processing112to produce the melt emulsion114include, but are not limited to, extruders (e.g., continuous extruders, batch extruders, and the like), stirred reactors, blenders, reactors with inline homogenizer systems, and the like, and apparatuses derived therefrom. Processing112and forming the melt emulsion114at suitable process conditions (e.g., temperature, shear rate, and the like) for a set period of time. The temperature of processing112and forming the melt emulsion114should be a temperature greater than the melting point or softening temperature of the thermoplastic polymer102and less than the decomposition temperature of any components102,104, and106in the mixture110. For example, the temperature of processing112and forming the melt emulsion114may be about 1° C. to about 50° C. (or about 1° C. to about 25° C., or about 5° C. to about 30° C., or about 20° C. to about 50° C.) greater than the melting point or softening temperature of the thermoplastic polymer102provided the temperature of processing112and forming the melt emulsion114is less than the decomposition temperature of any of components102,104, and106in the mixture110. The shear rate of processing112and forming the melt emulsion114should be sufficiently high to disperse the polymer melt in the carrier fluid104as droplets. Said droplets should comprise droplets having a diameter of about 1000 μm or less (or about 1 μm to about 1000 μm, or about 1 μm to about 50 μm, or about 10 μm to about 100 μm, or about 10 μm to about 250 μm, or about 50 μm to about 500 μm, or about 250 μm to about 750 μm, or about 500 μm to about 1000 μm). The time for maintaining said temperature and shear rate for processing112and forming the melt emulsion114may be 10 seconds to 18 hours or longer (or 10 seconds to 30 minutes, or 5 minutes to 1 hour, or 15 minutes to 2 hours, or 1 hour to 6 hours, or 3 hours to 18 hours). Without being limited by theory, it is believed that a steady state of droplet sizes will be reached at which point processing112can be stopped. That time may depend on, among other things, the temperature, shear rate, thermoplastic polymer102composition, the carrier fluid104composition, and the emulsion stabilizer106composition. The melt emulsion114may then be cooled116. Cooling116can be slow (e.g., allowing the melt emulsion to cool under ambient conditions) to fast (e.g., quenching). For example, the rate of cooling may range from about 10° C./hour to about 100° C./second to almost instantaneous with quenching (for example in dry ice) (or about 10° C./hour to about 60° C./hour, or about 0.5° C./minute to about 20° C./minute, or about 1° C./minute to about 5° C./minute, or about 10° C./minute to about 60° C./minute, or about 0.5° C./second to about 10° C./second, or about 10° C./second to about 100° C./second). During cooling, little to no shear may be applied to the melt emulsion114. In some instances, the shear applied during heating may be applied during cooling. The cooled mixture118resulting from cooling116the melt emulsion114comprises solidified thermoplastic polymer particles122(or simply thermoplastic polymer particles) and other components124(e.g., the carrier fluid104, excess emulsion stabilizer106, and the like). The thermoplastic polymer particles may be dispersed in the carrier fluid or settled in the carrier fluid. The cooled mixture118may then be treated120to the separate thermoplastic polymer particles122(or simply thermoplastic polymer particles122) from the other components124. Suitable treatments include, but are not limited to, washing, filtering, centrifuging, decanting, and the like, and any combination thereof. Solvents used for washing the thermoplastic polymer particles122should generally be (a) miscible with the carrier fluid104and (b) nonreactive (e.g., non-swelling and non-dissolving) with the thermoplastic polymer102. The choice of solvent will depend on, among other things, the composition of the carrier fluid and the composition of the thermoplastic polymer102. Examples of solvents include, but are not limited to, hydrocarbon solvents (e.g., pentane, hexane, heptane, octane, cyclohexane, cyclopentane, decane, dodecane, tridecane, and tetradecane), aromatic hydrocarbon solvents (e.g., benzene, toluene, xylene, 2-methyl naphthalene, and cresol), ether solvents (e.g., diethyl ether, tetrahydrofuran, diisopropyl ether, and dioxane), ketone solvents (e.g., acetone and methyl ethyl ketone), alcohol solvents (e.g., methanol, ethanol, isopropanol, and n-propanol), ester solvents (e.g., ethyl acetate, methyl acetate, butyl acetate, butyl propionate, and butyl butyrate), halogenated solvents (e.g., chloroform, bromoform, 1,2-dichloromethane, 1,2-dichloroethane, carbon tetrachloride, chlorobenzene, and hexafluoroisopropanol), water, and the like, and any combination thereof. Solvent may be removed from the thermoplastic polymer particles122by drying using an appropriate method such as air drying, heat drying, reduced pressure drying, freeze drying, or a hybrid thereof. The heating may be performed preferably at a temperature lower than the glass transition point of the thermoplastic polymer (e.g., about 50° C. to about 150° C.). The thermoplastic polymer particles122after separation from the other components124may optionally be further classified to produce purified thermoplastic polymer particles128. For example, to narrow the particle size distribution (or reduce the diameter span), the thermoplastic polymer particles122can be passed through a sieve having a pore size of about 10 μm to about 250 μm (or about 10 μm to about 100 μm, or about 50 μm to about 200 μm, or about 150 μm to about 250 μm). In another example of a purification technique, the thermoplastic polymer particles122may be washed with water to remove surfactant while maintaining substantially all of the nanoparticles associated with the surface of the thermoplastic polymer particles122. In yet another example of a purification technique, the thermoplastic polymer particles122may be blended with additives to achieve a desired final product. For clarity, because such additives are blended with the thermoplastic particles122or other particles resultant from the methods described herein after the particles are solidified, such additives are referred to herein as “external additives.” Examples of external additives include flow aids, other polymer particles, fillers, and the like, and any combination thereof. In some instances, a surfactant used in making the thermoplastic polymer particles122may be unwanted in downstream applications. Accordingly, yet another example of a purification technique may include at least substantial removal of the surfactant from the thermoplastic polymer particles122(e.g., by washing and/or pyrolysis). The thermoplastic polymer particles122and/or purified thermoplastic polymer particles128(referred to as particles122/128) may be characterized by composition, physical structure, and the like. As described above, the emulsion stabilizers are at the interface between the polymer melt and the carrier fluid. As a result, when the mixture is cooled, the emulsion stabilizers remain at, or in the vicinity of, said interface. Therefore, the structure of the particles122/128, in general, includes emulsion stabilizers (a) dispersed on an outer surface of the particles122/128and/or (b) embedded in an outer portion (e.g., outer 1 vol %) of the particles122/128. Further, where voids form inside the polymer melt droplets, emulsion stabilizers106should generally be at (and/or embedded in) the interface between the interior of the void and the thermoplastic polymer. The voids generally do not contain the thermoplastic polymer. Rather, the voids may contain, for example, carrier fluid, air, or be void. The particles122/128may comprise carrier fluid at about 5 wt % or less (or about 0.001 wt % to about 5 wt %, or about 0.001 wt % to about 0.1 wt %, or about 0.01 wt % to about 0.5 wt %, or about 0.1 wt % to about 2 wt %, or about 1 wt % to about 5 wt %) of the particles122/128. The thermoplastic polymer102may be present in the particles122/128at about 90 wt % to about 99.5 wt % (or about 90 wt % to about 95 wt %, or about 92 wt % to about 97 wt %, or about 95 wt % to about 99.5 wt %) of the particles122/128. When included, the emulsion stabilizers106may be present in the particles122/128at about 10 wt % or less (or about 0.01 wt % to about 10 wt %, or about 0.01 wt % to about 1 wt %, or about 0.5 wt % to about 5 wt %, or about 3 wt % to about 7 wt %, or about 5 wt % to about 10 wt %) of the particles122/128. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers106may be present in the particles128at less than 0.01 wt % (or 0 wt % to about 0.01 wt %, or 0 wt % to 0.001 wt %). Upon forming thermoplastic particulates according to the disclosure herein, at least a portion of the nanoparticles, such as silica nanoparticles, may be disposed as a coating upon the outer surface of the thermoplastic particulates. At least a portion of the surfactant, if used, may be associated with the outer surface as well. The coating may be disposed substantially uniformly upon the outer surface. As used herein with respect to a coating, the term “substantially uniform” refers to even coating thickness in surface locations covered by the coating composition (e.g., nanoparticles and/or surfactant), particularly the entirety of the outer surface. The emulsion stabilizers106may form a coating that covers at least 5% (or about 5% to about 100%, or about 5% to about 25%, or about 20% to about 50%, or about 40% to about 70%, or about 50% to about 80%, or about 60% to about 90%, or about 70% to about 100%) of the surface area of the particles122/128. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers106may be present in the particles128at less than 25% (or 0% to about 25%, or about 0.1% to about 5%, or about 0.1% to about 1%, or about 1% to about 5%, or about 1% to about 10%, or about 5% to about 15%, or about 10% to about 25%) of the surface area of the particles128. The coverage of the emulsion stabilizers106on an outer surface of the particles122/128may be determined using image analysis of the scanning electron microscope images (SEM micrographs). The emulsion stabilizers106may form a coating that covers at least 5% (or about 5% to about 100%, or about 5% to about 25%, or about 20% to about 50%, or about 40% to about 70%, or about 50% to about 80%, or about 60% to about 90%, or about 70% to about 100%) of the surface area of the particles122/128. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers106may be present in the particles128at less than 25% (or 0% to about 25%, or about 0.1% to about 5%, or about 0.1% to about 1%, or about 1% to about 5%, or about 1% to about 10%, or about 5% to about 15%, or about 10% to about 25%) of the surface area of the particles128. The coverage of the emulsion stabilizers106on an outer surface of the particles122/128may be determined using image analysis of the SEM micrographs The particles122/128may have a D10 of about 0.1 μm to about 125 μm (or about 0.1 nm to about 5 μm, about 1 μm to about 10 μm, about 5 μm to about 30 μm, or about 1 μm to about 25 μm, or about 25 μm to about 75 μm, or about 50 μm to about 85 μm, or about 75 μm to about 125 μm), a D50 of about 0.5 μm to about 200 μm (or about 0.5 μm to about 10 μm, or about 5 μm to about 50 μm, or about 30 μm to about 100 μm, or about 30 μm to about 70 μm, or about 25 μm to about 50 μm, or about 50 μm to about 100 μm, or about 75 μm to about 150 μm, or about 100 μm to about 200 μm), and a D90 of about 3 μm to about 300 μm (or about 3 μm to about 15 μm, or about 10 μm to about 50 μm, or about 25 μm to about 75 μm, or about 70 μm to about 200 μm, or about 60 μm to about 150 μm, or about 150 μm to about 300 μm), wherein D10<D50<D90. The particles122/128may also have a diameter span of about 0.4 to about 3 (or about 0.6 to about 2, or about 0.4 to about 1.5, or about 1 to about 3). Without limitation, diameter span values of 1.0 or greater are considered broad, and diameter span values of 0.75 or less are considered narrow. For example, the particles122/128may have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 100 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. The particles122/128may also have a diameter span of about 0.4 to about 3 (or about 0.6 to about 2, or about 0.4 to about 1.5, or about 1 to about 3). In a first nonlimiting example, the particles122/128may have a D10 of about 0.5 μm to about 5 μm, a D50 of about 0.5 μm to about 10 μm, and a D90 of about 3 μm to about 15 μm, wherein D10<D50<D90. In a second nonlimiting example, the particles122/128may have a D10 of about 1 μm to about 50 μm, a D50 of about 25 μm to about 100 μm, and a D90 of about 60 μm to about 300 μm, wherein D10<D50<D90. In a third nonlimiting example, the particles122/128may have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 1.0 to about 2.5. In a fourth nonlimiting example, the particles122/128may have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 0.6 to about 1.5. In a fifth nonlimiting example, the particles122/128may have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 0.2 to about 1.2. The particles122/128may have a circularity of about 0.7 or greater (or about 0.90 to about 1.0, or about 0.93 to about 0.99, or about 0.95 to about 0.99, or about 0.97 to about 0.99, or about 0.98 to 1.0). The particles122/128may have an angle of repose of about 25° to about 45° (or about 25° to about 35°, or about 30° to about 40°, or about 35° to about 45°). The particles122/128may have a Hausner ratio of about 1.0 to about 1.5 (or about 1.0 to about 1.2, or about 1.1 to about 1.3, or about 1.2 to about 1.35, or about 1.3 to about 1.5). The particles122/128may have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3(or about 0.3 g/cm3to about 0.6 g/cm3, or about 0.4 g/cm3to about 0.7 g/cm3, or about 0.5 g/cm3to about 0.6 g/cm3, or about 0.5 g/cm3to about 0.8 g/cm3). Depending on the temperature and shear rate of processing112and the composition and relative concentrations of the components102,104, and106, different shapes of the structures that compose the particles122/128have been observed. Typically, the particles122/128comprise substantially spherical particles (having a circularity of about 0.97 or greater). However, other structures including disc and elongated structures have been observed in the particles122/128. Therefore, the particles122/128may comprise one or more of: (a) substantially spherical particles having a circularity of 0.97 or greater, (b) disc structures having an aspect ratio of about 2 to about 10, and (c) elongated structures having an aspect ratio of 10 or greater. Each of the (a), (b), and (c) structures have emulsion stabilizers dispersed on an outer surface of the (a), (b), and (c) structures and/or embedded in an outer portion of the (a), (b), and (c) structures. At least some of the (a), (b), and (c) structures may be agglomerated. For example, the (c) elongated structures may be laying on the surface of the (a) substantially spherical particles. The particles122/128may have a sintering window that is within 10° C., preferably within 5° C., of the sintering window of the thermoplastic polymer102(comprising one or more IBOA-polyamides and optionally one or more other thermoplastic polymers). Applications of IBOA-Polyamide Particles The IBOA-polyamide particles described herein may be used to produce a variety of objects (or articles). The IBOA-polyamides described herein may be used alone or in combination with other particles comprising other thermoplastic polymers (e.g., polyamides without an optical absorber and/or other thermoplastic polymers). Examples of thermoplastic polymers that may be used in such other particles include, but are not limited to, polyamides, polyurethanes, polyethylenes, polypropylenes, polyacetals, polycarbonates, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), polyhexamethylene terephthalate, polystyrenes, polyvinyl chlorides, polytetrafluoroethenes, polyesters (e.g., polylactic acid), polyethers, polyether sulfones, polyetherether ketones, polyacrylates, polymethacrylates, polyimides, acrylonitrile butadiene styrene (ABS), polyphenylene sulfides, vinyl polymers, polyarylene ethers, polyarylene sulfides, polysulfones, polyether ketones, polyamide-imides, polyetherimides, polyetheresters, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), grafted or ungrafted thermoplastic polyolefins, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylates, functionalized or nonfunctionalized (meth)acrylic acid polymers, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymers, ethylene/vinyl monomer/carbonyl terpolymers, ethylene/alkyl (meth)acrylate/carbonyl terpolymers, methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, chlorinated or chlorosulphonated polyethylenes, polyvinylidene fluoride (PVDF), phenolic resins, poly(ethylene/vinyl acetate)s, polybutadienes, polyisoprenes, styrenic block copolymers, polyacrylonitriles, silicones, and the like, and any combination thereof. Copolymers comprising one or more of the foregoing may also be used in the methods and systems described herein. The IBOA-polyamide particles may be useful in applications that include, but are not limited to, paints, powder coatings, ink jet materials, electrophotographic toners, 3-D printing, and the like. By way of nonlimiting example, 3-D printing processes of the present disclosure may comprise: depositing IBOA-polyamide particles of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) optionally in combination with other particles comprising one or more thermoplastic polymers and/or one or more compatibilizers upon a surface in a specified shape, and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body (or object or article), such that the consolidated body has a void percentage of about 1% or less after being consolidated. For example, heating and consolidation of the thermoplastic polymer particles may take place in a 3-D printing apparatus employing a laser, such that heating and consolidation take place by selective laser sintering. Examples of articles that may be produced by such methods where the IBOA-polyamide may be all or a portion of said articles include, but are not limited to, particles, films, packaging, toys, household goods, automotive parts, aerospace/aircraft-related parts, containers (e.g., for food, beverages, cosmetics, personal care compositions, medicine, and the like), shoe soles, furniture parts, decorative home goods, plastic gears, screws, nuts, bolts, cable ties, jewelry, art, sculpture, medical items, prosthetics, orthopedic implants, production of artifacts that aid learning in education, 3-D anatomy models to aid in surgeries, robotics, biomedical devices (orthotics), home appliances, dentistry, electronics, sporting goods, and the like. The IBOA-polyamides described herein may have a specific chemical fingerprint that is useful in identifying objects, tracking objects, authenticating objects, and/or determining the health of objects. Further, the placement of where the IBOA-polyamides are located in the objects is another layer of fingerprinting the objects for identifying objects, tracking objects, authenticating objects, and/or determining the health of objects. Methods of identifying objects, tracking objects, authenticating objects, and/or determining the health of objects may include (a) exposing the object comprising IBOA-polyamides to electromagnetic radiation (e.g., for fluorophores preferably at a wavelength of 302 nm or less or 700 nm or greater); (b) sensing one or more spectra related to the electromagnetic radiation absorbed and/or reemitted (e.g., for fluorophores preferably the photoluminescence emitted between 302 nm to 700 nm); and (c) comparing the spectra to the known spectra for the optical absorbers used in said object or portion thereof. Optionally, the location of where the spectra area is located on the object may be compared to the known location where the spectra area should be. The comparison(s) can be used for identifying and/or authenticating the object. For tracking, the comparison(s) may be done and/or the detected spectra and/or spectra area may be logged into a database along with the physical location of the object. Further, the health of objects that wear and/or crack can be ascertained. For example, a core portion of the article may comprise optical absorbers and an outer portion may cover the core portion and not comprise the optical absorbers (or comprise different optical absorbers). Then, when comparing spectra, the appearance of spectral features for the optical absorbers in the core may indicate that the object is at or near the end of life. Nonlimiting Examples A first nonlimiting embodiment of the present disclosure is a method comprising: polymerizing polyamide monomers in the presence of an optical absorber selected from the group consisting of a polyamine optical absorber, a polyacid optical absorber, an amino acid optical absorber, and any combination thereof to yield a polyamide having the optical absorber in the backbone of the polyamide. The first nonlimiting embodiment may further include one or more of: Element 1: wherein polymerizing is a ring opening polymerization; Element 2: Element 1 and wherein the polyamide monomers are selected from the group consisting of: 2-azetidinone, 2-pyrrolidinone, 2-piperidinone, ε-caprolactam, 2-azacyclooctanone, 2-azacyclononanone, 2-azacyclodecanone, 2-azacycloundecanone, 2-aza[H]cyclododecanone, laurolactam, and any combination thereof; Element 3: wherein polymerizing is a polycondensation reaction and the polyamide monomers comprise a polyacid polyamide monomer and a polyamine polyamide monomer; Element 4: Element 3 and wherein the polyacid polyamide monomer is selected from the group consisting of: HOOC—(CH2)n—COOH where n is 1-20; isophthalic acid; terephthalic acid; pent-2-enedioic acid; dodec-2-enedioic acid; succinic acid; glutaric acid; adipic acid; pimelic acid; suberic acid; azeleic acid; sebacic acid; undecanedioic acid; dodecanedioic acid; 1,3-cyclohexanedicarboxylic acid; and any combination thereof; Element 5: Element 3 and wherein the polyamine polyamide monomer is selected from the group consisting of: H2N—(CH2)n—NH2where n is 1-20; 1,5-diamino-2-methylpentane; 1,2-diaminopropane; trimethylhexamethylenediamine; 2-methyloctane-1,8-diamine; n-methyl 1,6-hexamethylene diamine where n is 2 or 3; n-methyl 1,7-heptamethylene diamine where n is 2-4; n-methyl 1,8-octamethylene diamine where n is 2-4; n-methyl 1,12-dodecamethylene diamine where n is 2-6; 1,3-bis(aminomethyl)benzene; ortho-phenylene-bis(methylamine); 1,4-bis(aminomethyl)benzene; 1,4-cyclohexanediamine; 4-methyl cyclohexane-1,3-diamine; 4-methylcyclohexane-1,3-diamine; diphenylethylenediamine; 1,3-bis(aminomethyl)cyclohexane; 4,4′-methylenebis(cyclohexylamine); 4,4′-biphenyl diamine; 1,8-diaminonaphthalene; and any combination thereof; Element 6: wherein polymerizing is a polycondensation reaction and the monomers comprise an amino acid polyamide monomer; Element 7: Element 6 and wherein the amino acid polyamide monomer are selected from the group consisting of: H2N—(CH2)n—COOH where n is 1-20; branched aliphatic amino acids; cyclic-aliphatic amino acids; aromatic amino acids; and the like; and any combination thereof; Element 8: wherein the polyamide is selected from the group consisting of: polycaproamide, polyhexamethylene succinamide, polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene seb acami de, polyundecamide, polydodecamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof; Element 9: wherein the optical absorber comprises the polyamine optical absorber selected from the group consisting of: N,N-di(4-aminophenyl)=1-aminopyrene; N,N-bis(4-aminophenyl)-N′-4-methoxyphenyl-N′-4-(1,2,2-triphenylethenyl)phenyl-1,4-phenylenediamine; 4,5-diamino-rhodamine B; rhodamine 123; 2,7-dimethylacridine-3,6-diamine; 2-nitro-1,4-phenylenediamine; 4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-dien-1-ylidene)methyl]aniline hydrochloride; 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2-sulfonate sodium; and any combination thereof; Element 10: wherein the optical absorber comprises the polyacid optical absorber selected from the group consisting of: calcein; 4-methylumbelliferone-8-methyliminodiacetic acid; 6-carboxyfluorescein; 3,9-perylenedicarboxylic acid; N,N-bis(4-tert-butylphenyl)-N′,N′-bis(4-carboxyphthalimido)-1,4-phenylenediamine; perylene-3,4,9,10-tetracarboxylic dianhydride; alizarin-3-methyliminodiacetic acid; and any combination thereof; Element 11: wherein the optical absorber comprises the amino acid optical absorber selected from the group consisting of: 4-amino-1,8-naphthalimide; 7-amino-4-methyl-3-coumarinylacetic acid; and any combination thereof; and Element 12: wherein the molar ratio in the polyamide having the optical absorber in the backbone of the polyamide of the polyamide monomers (cumulatively) to optical absorbers (cumulatively) of about 500:1 to about 10:1. Examples of combinations include, but are not limited to, Element 1 (optionally in combination with Element 2) in combination with one or more of Elements 9-11; Element 3 (optionally in combination with Element 4 and/or Element 5) in combination with one or more of Elements 9-11; Element 6 (optionally in combination with Element 7) in combination with one or more of Elements 9-11; and Element 12 in combination with one or more of Elements 1-11. A second nonlimiting embodiment of the present disclosure is an article comprising: the polyamide of the first nonlimiting embodiment (optionally including one or more of Elements 1-12). A third nonlimiting embodiment of the present disclosure is a composition comprising: a polyamide having the optical absorber in the backbone of the polyamide. The third nonlimiting embodiment may include one or more of: Element 8; Element 9; Element 10; Element 11; and Element 12. A fourth nonlimiting embodiment of the present disclosure is an article comprising: the polyamide of the third nonlimiting embodiment. A fifth nonlimiting embodiment of the present disclosure is a method comprising: a polymer melt comprising the polyamide of the third nonlimiting embodiment and optionally one or more other thermoplastic polymers and/or one or more compatibilizers through an orifice to produce a film, a fiber (or a filament), particles, pellets, or the like. A sixth nonlimiting embodiment of the present disclosure is a method comprising: extruding a filament comprising the polyamide of the third nonlimiting embodiment and optionally one or more other thermoplastic polymers and/or one or more compatibilizers through an orifice, wherein the filament becomes a polymer melt upon extrusion; depositing the polymer melt as a first layer on a platform; cooling the layer; depositing an additional layer of the polymer melt on the first layer; cooling the additional layer; repeating depositing and cooling for at least one additional layer to produce a 3-D shape. A seventh nonlimiting embodiment of the present disclosure is a method comprising: mixing a mixture comprising a polyamide having an optical absorber in a backbone of the polyamide (1130A-polyamide), a carrier fluid that is immiscible with the IBOA-polyamide, and optionally an emulsion stabilizer at a temperature greater than a melting point or softening temperature of the IBOA-polyamide and at a shear rate sufficiently high to disperse the 1130A-polyamide in the carrier fluid; and cooling the mixture to below the melting point or softening temperature of the IBOA-polyamide to form solidified particles comprising the IBOA-polyamide and the emulsion stabilizer, when present, associated with an outer surface of the solidified particles. The seventh nonlimiting embodiment may further include one or more of: Element 13: wherein the emulsion stabilizer is included in the mixture, and wherein the emulsion stabilizer associated with an outer surface of the solidified particles; Element 14: wherein the mixture further comprises a thermoplastic polymer that is not the MOA-polyamide; Element 15: wherein the mixture further comprises a second polyamide but without an optical absorber in a backbone of the second polyamide therefrom; Element 16: wherein the optical absorber is from a family selected from the group consisting of: rhodamines, fluoresceins, coumarins, naphthalimides, benzoxanthenes, acridines, cyanines, oxazins, phenanthridine, pyrrole ketones, benzaldehydes, polymethines, triarylmethanes, anthraquinones, pyrazolones, quinophthalones, carbonyl dyes, diazo dyes, perinones, diketopyrrolopyrrole (DPP), dioxazine dyes, phthalocyanines, indanthrenes, benzanthrone, violanthrones, azo dyes, phthalocyanine dyes, quinacridone dyes, anthraquinone dyes, indigo dyes, thioindigo dyes, perinonc dyes, perylene dyes, isoindolene dyes, aromatic amino acids, flavins, derivatives of pyridoxyl, derivatives of chlorophyll, and any combination thereof; Element 17: wherein the polyamide is selected from the group consisting of: polycaproamide, polyhexamethylene succinamide, polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacami de, polyundecamide, polydodecamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof; Element 18: wherein the optical absorber is a polyamine optical absorber selected from the group consisting of: N,N-di(4-aminophenyl)-1-aminopyrene; N,N-bis(4-aminophenyl)-N′-4-methoxyphenyl-N′-4-(1,2,2-triphenyl ethenyl)phenyl-1,4-phenylenediamine; 4,5 diamino-rhodamine B; rhodamine 123; 2,7-dimethylacridine-3,6-diamine; 2-nitro-1,4-phenylenediamine; 4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-di en-1-ylidene)methyl]aniline hydrochloride; 4,8-diamino-1,5-dihydroxy-9,10-dioxoanthracene-2-sulfonate sodium; and any combination thereof; Element 19: wherein the optical absorber is a polyacid optical absorber selected from the group consisting of: calcein; 4-methylumbelliferone-8-methyliminodiacetic acid; 6-carboxyfluorescein; 3,9-perylenedicarboxylic acid; N,N-bis(4-tert-butylphenyl)-N′,N′-bis(4-carboxyphthalimido)-1,4-phenylenediamine; perylene-3,4,9,10-tetracarboxylic dianhydride; alizarin-3-methyliminodiacetic acid; and any combination thereof; Element 20: wherein the optical absorber is an amino acid optical absorber selected from the group consisting of: 4-amino-1,8-naphthalimide; 7-amino-4-methyl-3-coumarinylacetic acid; and any combination thereof; Element 21: wherein the emulsion stabilizer is included in the mixture, wherein at least some of the solidified particles have a void comprising the emulsion stabilizer at a void/polymer interface; Element 22: Element 21 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface; Element 23: Element 21 and wherein the void contains the carrier fluid; Element 24: wherein the emulsion stabilizer is included in the mixture, wherein the solidified particles further comprises elongated structures on the surface of the solidified particles, wherein the elongated structures comprises the IBOA-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures; Element 25: wherein the emulsion stabilizer is included in the mixture, wherein the emulsion stabilizer forms a coating that covers less than 5% of the surface of the solidified particles; Element 26: wherein the emulsion stabilizer is included in the mixture, wherein the emulsion stabilizer forms a coating that covers at least 5% of the surface of the solidified particles; Element 27: wherein the emulsion stabilizer is included in the mixture, wherein the emulsion stabilizer forms a coating that covers at least 25% of the surface of the solidified particles; Element 28: wherein the emulsion stabilizer is included in the mixture, wherein the emulsion stabilizer forms a coating that covers at least 50% of the surface of the solidified particles; Element 29: wherein the emulsion stabilizer is included in the mixture, wherein the IBOA-polyamide is present in the mixture at 5 wt % to 60 wt % of the mixture; Element 30: wherein the emulsion stabilizer is present in the mixture at 0.05 wt % to 5 wt % by weight of the IBOA-polyamide; Element 31: wherein the emulsion stabilizer is included in the mixture, wherein the emulsion stabilizer comprises nanoparticles have an average diameter of 1 nm to 500 nm; Element 32: wherein the carrier fluid is selected from the group consisting of: silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and any combination thereof; Element 33: Element 32 and wherein the silicone oil is selected from the group consisting of: polydimethylsiloxane, methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and any combination thereof; Element 34: wherein the carrier fluid has a viscosity at 25° C. of 1,000 cSt to 150,000 cSt; Element 35: wherein the carrier fluid has a density of 0.6 g/cm3to 1.5 g/cm3; Element 36: wherein mixing occurs in an extruder; Element 37: wherein mixing occurs in a stirred reactor; Element 38: wherein the emulsion stabilizer is included in the mixture, wherein the emulsion stabilizer comprises a surfactant; Element 39: wherein the particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90; Element 30: wherein the particles have a diameter span of about 0.2 to about 10; Element 41: wherein the particles have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90; Element 42: wherein the particles have a diameter span of about 1.0 to about 2.5; Element 43: wherein the particles have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90; Element 44: wherein the particles have a diameter span of about 0.6 to about 1.5; Element 45: wherein the particles have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90; Element 46: wherein the particles have a diameter span of about 0.2 to about 1.2; Element 47: wherein the solidified particles have a circularity of about 0.90 to about 1.0; Element 48: wherein the solidified particles have a Hausner ratio of about 1.0 to about 1.5; Element 49: wherein the emulsion stabilizer is included in the mixture, wherein emulsion stabilizer comprises nanoparticles that comprise oxide nanoparticles; Element 50: wherein the emulsion stabilizer is included in the mixture, wherein emulsion stabilizer comprises nanoparticles that comprise carbon black; and Element 51: wherein the emulsion stabilizer is included in the mixture, wherein emulsion stabilizer comprises nanoparticles that comprise polymer nanoparticles. Examples of combinations include, but are not limited to, Element 13 in combination with one or more of Elements 14-51; Element 14 in combination with one or more of Elements 15-51; Element 15 in combination with one or more of Elements 16-51; Element 16 in combination with one or more of Elements 17-51; Element 17 in combination with one or more of Elements 18-51; two or more of Elements 18-20 in combination; one or more of Elements 18-20 in combination with one or more of Elements 21-51; two or more of Elements 21-24 in combination; one or more of Elements 21-24 in combination with one or more of Elements 25-51; one of Elements 15-28 in combination with one or more of Elements 30, 31, 38, 49, 50, and 51; two or more of Elements 30, 31, 38, 49, 50, and 51 in combination; Element 29 and Element 30 in combination; two or more of Elements 32-35 in combination; one or more of Elements 32-35 in combination with one or more of Elements 36-51; Elements 39 and 40 in combination; Elements 41 and 42 in combination; Elements 43 and 44 in combination; Elements 45 and 46 in combination; two or more of Elements 38-46 in combination with one or more of Elements 30, 31, 38, 49, 50, and 51; and Element 47 and/or Element 48 in combination with one or more of Elements 13-46. A eighth nonlimiting example embodiment of the present disclosure is a composition comprising: particles comprising a polyamide having an optical absorber in a backbone of the polyamide (IBOA-polyamide) and optionally an emulsion stabilizer, wherein the particles have a circularity of about 0.90 to about 1.0. The eighth nonlimiting example embodiment may include one or more of: Element 39; Element 40; Element 41; Element 42; Element 43; Element 44; Element 45; Element 46; Element 48; Element 52: wherein the particles further comprise a thermoplastic polymer that is not the IBOA-polyamide; Element 53: Element 42 and wherein the particles further comprise a second polyamide but without an optical absorber in a backbone of the second polyamide therefrom; Element 54: wherein the particles further comprise an emulsion stabilizer associated with an outer surface of the particles; Element 55: wherein at least some of the particles have a void comprising the emulsion stabilizer at a void/polymer interface; Element 56: Element 55 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface; Element 57: Element 55 and wherein the void contains the carrier fluid; Element 58: wherein the particles further comprises elongated structures on the surface of the particles, wherein the elongated structures comprises the IBOA-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures; Element 59: wherein the emulsion stabilizer forms a coating that covers less than 5% of the surface of the particles; Element 60: wherein the emulsion stabilizer forms a coating that covers at least 5% of the surface of the particles; Element 61: wherein the emulsion stabilizer forms a coating that covers at least 25% of the surface of the particles; Element 62: wherein the emulsion stabilizer forms a coating that covers at least 50% of the surface of the particles; and Element 63: wherein the emulsion stabilizer comprises nanoparticles having an average diameter of 1 nm to 500 nm. A ninth nonlimiting example embodiment is a method comprising: depositing IBOA-polyamide particles of the eighth nonlimiting example optionally in combination with other thermoplastic polymer particles upon a surface in a specified shape; and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body. Clauses Clause 1. A method comprising: polymerizing polyamide monomers in the presence of an optical absorber selected from the group consisting of a polyamine optical absorber, a polyacid optical absorber, an amino acid optical absorber, and any combination thereof to yield a polyamide having the optical absorber in the backbone of the polyamide. Clause 2. The method of Clause 1, wherein polymerizing is a ring opening polymerization. Clause 3. The method of Clause 2, wherein the polyamide monomers are selected from the group consisting of: 2-azetidinone, 2-pyrrolidinone, 2-piperidinone, ε-caprolactam, 2-azacyclooctanone, 2-azacyclononanone, 2-azacyclodecanone, 2-azacycloundecanone, 2-azacyclododecanone, laurolactam, and any combination thereof. Clause 4. The method of Clause 1, wherein polymerizing is a polycondensation reaction and the polyamide monomers comprise a polyacid polyamide monomer and a polyamine polyamide monomer. Clause 5. The method of Clause 4, wherein the polyacid polyamide monomer is selected from the group consisting of: HOOC—(CH2)n—COOH where n is 1-20; isophthalic acid; terephthalic acid; pent-2-enedioic acid; dodec-2-enedioic acid; succinic acid; glutaric acid; adipic acid; pimelic acid; suberic acid; azeleic acid; sebacic acid; undecanedioic acid; dodecanedioic acid; 1,3-cyclohexanedicarboxylic acid; and any combination thereof. Clause 6. The method of Clause 4 or Clause 5, wherein the polyamine polyamide monomer is selected from the group consisting of: H2N—(CH2)n—NH2where n is 1-20; 1,5-diamino-2-methylpentane; 1,2-diaminopropane; trimethylhexamethylenediamine; 2-methyl octane-1,8-diamine; n-methyl 1,6-hexamethylene diamine where n is 2 or 3; n-methyl 1,7-heptamethylene diamine where n is 2-4; n-methyl 1,8-octamethylene diamine where n is 2-4; n-methyl 1,12-dodecamethylene diamine where n is 2-6; 1,3-bis(aminomethyl)benzene; ortho-phenylene-bis(methylamine); 1,4-bis(aminomethyl)benzene; 1,4-cyclohexanediamine; 4-methyl cycl ° hexane-1,3-diamine; 4-methyl cyclohexane-1,3-di amine; diphenyl ethylenediamine; 1,3-bis(aminomethyl)cyclohexane; 4,4′-methylenebis(cyclohexylamine); 4,4′-biphenyldiamine; 1,8-diaminonaphthalene; and any combination thereof. Clause 7. The method of Clause 1, wherein polymerizing is a polycondensation reaction and the monomers comprise an amino acid polyamide monomer. Clause 8. The method of Clause 7, wherein the amino acid polyamide monomer is are selected from the group consisting of: H2N—(CH2)n—COOH where n is 1-20; branched aliphatic amino acids; cyclic-aliphatic amino acids; aromatic amino acids; and the like; and any combination thereof. Clause 9. The method of Clause 1, wherein the polyamide is selected from the group consisting of: polycaproamide, polyhexamethylene succinamide, polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethyl ene sebacamide, polyundecamide, polydodecamide polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 10. The method of Clause 1, wherein the optical absorber comprises the polyamine optical absorber selected from the group consisting of: N,N-di(4-aminophenyl)-1-aminopyrene; N,N-bis(4-aminophenyl)-N′-4-methoxyphenyl-N′-4-(1,2,2-triphenylethenyl)phenyl-1,4-phenylenediamine; 1,5 diamino-rhodamine B; rhodamine 123; 2,7-dimethylacridine-3,6-diamine; 2-nitro-1,4-phenylenediamine; 4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-dien-1-ylidene)methyl]aniline hydrochloride; 4,8-di amino-1,5-dihydroxy-9,10-dioxoanthracene-2-sulfonate sodium; and any combination thereof. Clause 11. The method of Clause 1, wherein the optical absorber comprises the polyacid optical absorber selected from the group consisting of: calcein; 4-methylumbelliferone-8-methyliminodiacetic acid; 6-carboxyfluorescein; 3,9-perylenedicarboxylic acid; N,N-bis(4-tert-butylphenyl)-N′,N′-bis(4-carboxyphthalimido)-1,4-phenylenediamine; perylene-3,4,9,10-tetracarboxylic dianhydride; alizarin-3-methyliminodiacetic acid; and any combination thereof. Clause 12. The method of Clause 1, wherein the optical absorber comprises the amino acid optical absorber selected from the group consisting of: 4-amino-1,8-naphthalimide; 7-amino-4-methyl-3-coumarinylacetic acid; and any combination thereof. Clause 13. The method of Clause 1, wherein the molar ratio in the polyamide having the optical absorber in the backbone of the polyamide of the polyamide monomers (cumulatively) to optical absorbers (cumulatively) of about 500:1 to about 10:1. Clause 14. An article comprising: the polyamide of Clause 1. Clause 15. A composition comprising: a polyamide having the optical absorber in the backbone of the polyamide. Clause 16. The composition of Clause 15, wherein the polyamide is selected from the group consisting of: polycaproamide, polyhexamethylene succinamide, polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethyl ene sebacamide, polyundecamide, polydodecamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 17. The composition of Clause 15, wherein the optical absorber comprises the polyamine optical absorber selected from the group consisting of: N,N-di(4-aminophenyl)-1-aminopyrene; N,N-bis(4-aminophenyl)-N′-4-methoxyphenyl-N′-4-(1,2,2-triphenylethenyl)phenyl-1,4-phenylenediamine; 4,5-diamino-rhodamine B; rhodamine 123; 2,7-dimethylacridine-3,6-diamine; 2-nitro-1,4-phenylenediamine; 4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-dien-1-ylidene)methyl]aniline hydrochloride; 4,8-di amino-1,5-dihydroxy-9,10-dioxoanthracene-2-sulfonate sodium; and any combination thereof. Again, one skilled in the art will recognize that these are -mer units in this context of the polyamide having been synthesized. Therefore, the optical absorber -mer units described are in the polyamide in the polymerized form of said optical absorber -mer units. Clause 18. The composition of Clause 15, wherein the optical absorber is a polyacid optical absorber selected from the group consisting of: calcein; 4-methylumbelliferone-8-methyliminodiacetic acid; 6-carboxyfluorescein; 3,9-perylenedicarboxylic acid; N,N-bis(4-tert-butylphenyl)-N′,N′-bis(4-carboxyphthalimido)-1,4-phenylenediamine; perylene-3,4,9,10-tetracarboxylic dianhydride; alizarin-3-methyliminodiacetic acid; and any combination thereof. Clause 19. The composition of Clause 15, wherein the optical absorber is an amino acid optical absorber selected from the group consisting of: 4-amino-1,8-naphthalimide; 7-amino-4-methyl-3-coumarinylacetic acid; and any combination thereof. Clause 20. The composition of Clause 15, wherein the molar ratio in the polyamide having the optical absorber in the backbone of the polyamide of polyamide monomers (cumulatively) to optical absorbers (cumulatively) of about 500:1 to about 10:1. Clause 21. A method comprising: depositing particles upon a surface in a specified shape, wherein the particles comprise the polyamide of Clause 15 and optionally one or more other thermoplastic polymers and/or one or more compatibilizers; and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body. Clause 22. An article comprising: the polyamide of Clause 15. Clause 23. A method comprising: extruding a filament comprising the polyamide of Clause 15 and optionally one or more other thermoplastic polymers and/or one or more compatibilizers through an orifice, wherein the filament becomes a polymer melt upon extrusion; depositing the polymer melt as a first layer on a platform; cooling the layer; depositing an additional layer of the polymer melt on the first layer; cooling the additional layer; repeating depositing and cooling for at least one additional layer to produce a 3-D shape. Clause 24. A method comprising: a polymer melt comprising the polyamide of Clause 15 and optionally one or more other thermoplastic polymers and/or one or more compatibilizers through an orifice to produce a film, a fiber (or a filament), particles, pellets, or the like. Clause 25. A method comprising: mixing a mixture comprising a polyamide having an optical absorber in a backbone of the polyamide (IBOA-polyamide), a carrier fluid that is immiscible with the IBOA-polyamide, and optionally an emulsion stabilizer at a temperature greater than a melting point or softening temperature of the IBOA-polyamide and at a shear rate sufficiently high to disperse the IBOA-polyamide in the carrier fluid; and cooling the mixture to below the melting point or softening temperature of the IBOA-polyamide to form solidified particles comprising the IBOA-polyamide and the emulsion stabilizer, when present, associated with an outer surface of the solidified particles. Clause 26. The method of Clause 25, wherein the emulsion stabilizer is included in the mixture, and wherein the emulsion stabilizer associated with an outer surface of the solidified particles. Clause 27. The method of Clause 26, wherein the emulsion stabilizer comprises nanoparticles, and wherein the nanoparticles are embedded in an outer surface of the solidified particles. Clause 28. The method of Clause 25, wherein the mixture further comprises a thermoplastic polymer that is not the IBOA-polyamide. Clause 29. The method of Clause 25, wherein the mixture further comprises a second polyamide but without an optical absorber in a backbone of the second polyamide therefrom. Clause 30. The method of Clause 25, wherein the optical absorber is from a family selected from the group consisting of: rhodamines, fluoresceins, coumarins, naphthalimides, benzoxanthenes, acridines, cyanines, oxazins, phenanthridine, pyrrole ketones, benzaldehydes, polymethines, triarylmethanes, anthraquinones, pyrazolones, quinophthalones, carbonyl dyes, diazo dyes, perinones, diketopyrrolopyrrole (DPP), dioxazine dyes, phthalocyanines, indanthrenes, benzanthrone, violanthrones, azo dyes, phthalocyanine dyes, quinacridone dyes, anthraquinone dyes, indigo dyes, thioindigo dyes, perinone dyes, perylene dyes, isoindolene dyes, aromatic amino acids, flavins, derivatives of pyridoxyl, derivatives of chlorophyll, and any combination thereof. Clause 31. The method of Clause 25, wherein the polyamide is selected from the group consisting of: polycaproamide, polyhexamethylene succinamide, polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecamide, polydodecamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 32. The method of Clause 25, wherein the optical absorber is a polyamine optical absorber selected from the group consisting of: N,N-di(4-aminophenyl)-1-aminopyrene; N,N-bis(4-aminophenyl)-N′-4-methoxyphenyl-N′-4-(1,2,2-triphenyl ethenyl)phenyl-1,4-phenylenediamine, 4,5-diamino-rhodamine B; rhodamine 123; 2,7-dimethylacridine-3,6-diamine; 2-nitro-1,4-phenylenediamine; 4-[(4-aminophenyl)-(4-iminocyclohexa-2,5-dien-1-ylidene)methyl]aniline hydrochloride; 4,8-di amino-1,5-dihydroxy-9,10-dioxoanthracene-2-sulfonate sodium; and any combination thereof. Again, one skilled in the art will recognize that these are -mer units in this context of the polyamide having been synthesized. Therefore, the optical absorber -mer units described are in the polyamide in the polymerized form of said optical absorber -mer units. Clause 33. The method of Clause 25, wherein the optical absorber is a polyacid optical absorber selected from the group consisting of: calcein; 4-methylumbelliferone-8-methyliminodiacetic acid; 6-carboxyfluorescein; 3,9-perylenedicarboxylic acid; N,N-bis(4-tert-butylphenyl)-N′,N′-bis(4-carboxyphthalimido)-1,4-phenylenediamine; perylene-3,4,9,10-tetracarboxylic dianhydride; alizarin-3-methyliminodiacetic acid; and any combination thereof. Clause 34. The method of Clause 25, wherein the optical absorber is an amino acid optical absorber selected from the group consisting of: 4-amino-1,8-naphthalimide; 7-amino-4-methyl-3-coumarinylacetic acid; and any combination thereof. Clause 35. The method of Clause 25, wherein at least some of the solidified particles have a void comprising the emulsion stabilizer at a void/polymer interface. Clause 36. The method of Clause 35, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface. Clause 37. The method of Clause 35, wherein the void contains the carrier fluid. Clause 38. The method of Clause 25, wherein the solidified particles further comprises elongated structures on the surface of the solidified particles, wherein the elongated structures comprises the IBOA-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures. Clause 39. The method of Clause 25, wherein the emulsion stabilizer forms a coating that covers less than 5% of the surface of the solidified particles. Clause 40. The method of Clause 25, wherein the emulsion stabilizer forms a coating that covers at least 5% of the surface of the solidified particles. Clause 41. The method of Clause 25, wherein the emulsion stabilizer forms a coating that covers at least 25% of the surface of the solidified particles. Clause 42. The method of Clause 25, wherein the emulsion stabilizer forms a coating that covers at least 50% of the surface of the solidified particles. Clause 43. The method of Clause 25, wherein the IBOA-polyamide is present in the mixture at 5 wt % to 60 wt % of the mixture. Clause 44. The method of Clause 25, wherein the emulsion stabilizer is present in the mixture at 0.05 wt % to 5 wt % by weight of the IBOA-polyamide. Clause 45. The method of Clause 25, wherein the emulsion stabilizer comprises nanoparticles having an average diameter of 1 nm to 500 nm. Clause 46. The method of Clause 25, wherein the carrier fluid is selected from the group consisting of: silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and any combination thereof. Clause 47. The method of Clause 46, wherein the silicone oil is selected from the group consisting of: polydimethylsiloxane, methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and any combination thereof. Clause 48. The method of Clause 25, wherein the carrier fluid has a viscosity at 25° C. of 1,000 cSt to 150,000 cSt. Clause 49. The method of Clause 25, wherein the carrier fluid has a density of 0.6 g/cm3 to 1.5 g/cm3. Clause 50. The method of Clause 25, wherein mixing occurs in an extruder. Clause 51. The method of Clause 25, wherein mixing occurs in a stirred reactor. Clause 52. The method of Clause 25, wherein the mixture further comprises a surfactant. Clause 53. The method of Clause 25, wherein the particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 54. The method of Clause 25, wherein the particles have a diameter span of about 0.2 to about 10. Clause 55. The method of Clause 25, wherein the particles have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Clause 56. The method of Clause 55, wherein the particles have a diameter span of about 1.0 to about 2.5. Clause 57. The method of Clause 25, wherein the particles have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Clause 58. The method of Clause 57, wherein the particles have a diameter span of about 0.6 to about 1.5. Clause 59. The method of Clause 25, wherein the particles have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Clause 60. The method of Clause 59, wherein the particles have a diameter span of about 0.2 to about 1.2. Clause 61. The method of Clause 25, wherein the solidified particles have a circularity of about 0.90 to about 1.0. Clause 62. The method of Clause 25, wherein the solidified particles have a Hausner ratio of about 1.0 to about 1.5. Clause 63. The method of Clause 25, wherein emulsion stabilizer comprises nanoparticles that comprise oxide nanoparticles. Clause 64. The method of Clause 25, wherein emulsion stabilizer comprises nanoparticles that comprise carbon black. Clause 65. The method of Clause 25, wherein emulsion stabilizer comprises nanoparticles that comprise polymer nanoparticles. Clause 66. A composition comprising: particles comprising a polyamide having an optical absorber in a backbone of the polyamide (IBOA-polyamide) and having a circularity of about 0.90 to about 1.0. Clause 67. The composition of Clause 66, wherein the particles further comprise a thermoplastic polymer that is not the IBOA-polyamide. Clause 68. The composition of Clause 66, wherein the particles further comprise a second polyamide but without an optical absorber in a backbone of the second polyamide therefrom. Clause 69. The composition of Clause 66, wherein the particles further comprise an emulsion stabilizer associated with an outer surface of the particles. Clause 70. The composition of Clause 69, wherein the emulsion stabilizer comprise nanoparticles, and wherein at least some of the nanoparticles are embedded in a surface of the particles. Clause 71. The composition of Clause 69, wherein at least some of the particles have a void comprising the emulsion stabilizer at a void/polymer interface. Clause 72. The composition of Clause 69, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface. Clause 73. The composition of Clause 69, wherein the void contains the carrier fluid. Clause 74. The composition of Clause 66, wherein the solidified particles further comprises elongated structures on the surface of the solidified particles, wherein the elongated structures comprises the IBOA-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures. Clause 75. The composition of Clause 66, wherein the emulsion stabilizer forms a coating that covers less than 5% of the surface of the solidified particles. Clause 76. The composition of Clause 66, wherein the emulsion stabilizer forms a coating that covers at least 5% of the surface of the solidified particles. Clause 77. The composition of Clause 66, wherein the emulsion stabilizer forms a coating that covers at least 25% of the surface of the solidified particles. Clause 78. The composition of Clause 66, wherein the emulsion stabilizer forms a coating that covers at least 50% of the surface of the solidified particles. Clause 79. The composition of Clause 66, wherein the emulsion stabilizer comprises nanoparticles having an average diameter of 1 nm to 500 nm. Clause 80. The composition of Clause 66, wherein the particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 81. The composition of Clause 66, wherein the particles have a diameter span of about 0.2 to about 10. Clause 82. The composition of Clause 66, wherein the particles have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Clause 83. The composition of Clause 82, wherein the particles have a diameter span of about 1.0 to about 2.5. Clause 84. The composition of Clause 66, wherein the particles have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Clause 85. The composition of Clause 84, wherein the particles have a diameter span of about 0.6 to about 1.5. Clause 86. The composition of Clause 66, wherein the particles have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Clause 87. The composition of Clause 86, wherein the particles have a diameter span of about 0.2 to about 1.2. Clause 88. The composition of Clause 66, wherein the solidified particles have a Hausner ratio of about 1.0 to about 1.5. Clause 89. A method comprising: depositing the composition of Clause 66 optionally in combination with other thermoplastic polymer particles upon a surface in a specified shape; and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the present specification and associated claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the incarnations of the present inventions. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claim, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. One or more illustrative incarnations incorporating one or more invention elements are presented herein. Not all features of a physical implementation are described or shown in this application for the sake of clarity. It is understood that in the development of a physical embodiment incorporating one or more elements of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints, which vary by implementation and from time to time. While a developer's efforts might be time-consuming, such efforts would be, nevertheless, a routine undertaking for those of ordinary skill in the art and having benefit of this disclosure. While compositions and methods are described herein in terms of “comprising” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. To facilitate a better understanding of the embodiments of the present invention, the following examples of preferred or representative embodiments are given. In no way should the following examples be read to limit, or to define, the scope of the invention. EXAMPLES Prophetic Example 1—Preparation of Modified-Alizarin About 1.5 mmol DMAP was added to a stirred solution of about 5.5 mmol bromoacetate in DMSO. The mixture was stirred at room temperature for 5 minutes before adding 6.0 mmol DCC. After 10 minutes, 5.5 mmol alizarin was added and stirred for 4 hours. The organic layer was separated, mixed with ethyl acetate, washed with water, and dried over Na2SO4. After evaporation of the solvent, the crude residue was purified by column chromatography using cyclohexane-EtOAc (10:1) as an eluent. Prophetic Example 2—Preparation of Alizarin-Modified Nylon Nylon 6, nylon 6,6, nylon 6,10, and nylon 12 were modified with the modified-alizarin prepared in Example 1. 150 mL DMSO and 5.5 mmol nylon polymer were mixed. To the mixture, 5.5 mmol potassium t-butoxide was added. The mixture was blanketed with argon and heated to a temperature of 150° C. The suspension was allowed to mix at 150° C. for about 1 hour or until most of nylon was dissolved. Next, 5.5 mmol modified alizarin was added to the flask, and the reaction was allowed to proceed overnight. The next day the reaction mixture was cooled to room temperature and precipitated into 800 mL of deionized water. The mixture comprising alizarin-modified nylon, unmodified nylon, and unreacted modified-alizarin was then isolated by filtration and repeatedly washed with water to remove the DMSO solvent. Next, the solid was rinsed with methanol to remove the water then stirred in hexanes to remove the unreacted modified-alizarin. The resulted nylon mixture (modified and unmodified) was then isolated by filtration and allowed to dry in a vacuum oven at 60° C. overnight. These examples illustrate that optical absorbers can be modified and then reacted with polyamides to produce optical absorber-modified polyamides. Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular examples and configurations disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative examples disclosed above may be altered, combined, or modified and all such variations are considered within the scope and spirit of the present invention. The invention illustratively disclosed herein suitably may be practiced in the absence of any element that is not specifically disclosed herein and/or any optional element disclosed herein. While compositions and methods are described in terms of “comprising,” “containing,” or “including” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. All numbers and ranges disclosed above may vary by some amount. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In particular, every range of values (of the form, “from about a to about b,” or, equivalently, “from approximately a to b,” or, equivalently, “from approximately a-b”) disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the element that it introduces. | 131,991 |
11859052 | DETAILED DESCRIPTION The present disclosure relates to compositions, synthesis methods, and application methods of polyamides having a pigment pendent from the backbone of the polyamide, also referred to herein as an polyamide having pendent pigments or PP-polyamide. Because the polyamides are functionalized with the pigment particles, objects that incorporate the PP-polyamide will have a more evenly dispersed pigment. The present disclosure also relates to particles comprising polyamides having a pigment pendent from the backbone of the polyamide (also referred to herein as a pigment-pendent polyamide or PP-polyamide) and related methods. More specifically, the present disclosure includes methods of making highly spherical polymer particles comprising the one or more PP-polyamides and optionally one or more other thermoplastic polymers. Said polymer particles may be useful, among other things, as starting material for additive manufacturing. The polymer particles described herein may be, for example, produced by melt emulsification methods where one or more PP-polyamides and optionally one or more additional thermoplastic polymers are dispersed as a melt in a carrier fluid that is immiscible with the PP-polyamide and additional thermoplastic polymers, if used. A sufficient amount of shear is applied to the mixture to cause the polymer melt to form droplets in the carrier fluid. Because the pigment is pendent from the backbone of the polyamide, objects produced by additive manufacturing methods that include these particles should maintain an even color over time because the pigment cannot migrate within the object. Definitions and Test Methods As used herein, the term “pigment” refers to a particle that absorbs and/or refracts ultraviolet or visible light. As used herein, a “surface treated pigment” refers to a pigment having oxide particles chemically bonded and/or physically bonded to the surface of the pigment particle. As used herein, the term “immiscible” refers to a mixture of components that, when combined, form two or more phases that have less than 5 wt % solubility in each other at ambient pressure and at room temperature or the melting point of the component if it is solid at room temperature. For example, polyethylene oxide having 10,000 g/mol molecular weight is a solid at room temperature and has a melting point of 65° C. Therefore, said polyethylene oxide is immiscible with a material that is liquid at room temperature if said material and said polyethylene oxide have less than 5 wt % solubility in each other at 65° C. As used herein, the term “thermoplastic polymer” refers to a plastic polymer material that softens and hardens reversibly on heating and cooling. Thermoplastic polymers encompass thermoplastic elastomers. As used herein, the term “elastomer” refers to a copolymer comprising a crystalline “hard” section and an amorphous “soft” section. In the case of a polyurethane, the crystalline section may include a portion of the polyurethane comprising the urethane functionality and optional chain extender group, and the soft section may include the polyol, for instance. As used herein, the term “polyurethane” refers to a polymeric reaction product between a diisocyanate, a polyol, and an optional chain extender. As used herein, the term “oxide” refers to both metal oxides and non-metal oxides. For purposes of the present disclosure, silicon is considered to be a metal. As used herein, the terms “associated,” “association,” and grammatical variations thereof between emulsion stabilizers and a surface refers to chemical bonding and/or physical adherence of the emulsion stabilizers to the surface. Without being limited by theory, it is believed that the associations described herein between polymers and emulsion stabilizers are primarily physical adherence via hydrogen bonding and/or other mechanisms. However, chemical bonding may be occurring to some degree. As used herein, the term “embed” relative to nanoparticles and a surface of a polymer particle refers to the nanoparticle being at least partially extended into the surface such that polymer is in contact with the nanoparticle to a greater degree than would occur if the nanoparticle were simply laid on the surface of the polymer particle. Herein, D10, D50, D90, and diameter span are primarily used herein to describe particle sizes. As used herein, the term “D10” refers to a diameter at which 10% of the sample (on a volume basis unless otherwise specified) is comprised of particles having a diameter less than said diameter value. As used herein, the term “D50” refers to a diameter at which 50% of the sample (on a volume basis unless otherwise specified) is comprised of particles having a diameter less than said diameter value. As used herein, the term “D90” refers to a diameter at which 90% of the sample (on a volume basis unless otherwise specified) is comprised of particles having a diameter less than said diameter value. As used herein, the terms “diameter span” and “span” and “span size” when referring to diameter provides an indication of the breadth of the particle size distribution and is calculated as (D90−D10)/D50 (again each D-value is based on volume, unless otherwise specified). Particle size can be determined by light scattering techniques using a Malvern MASTERSIZER™ 3000 or analysis of optical digital micrographs. Unless otherwise specified, light scattering techniques are used for analyzing particle size. For light scattering techniques, the control samples were glass beads with a diameter within the range of 15 μm to 150 μm under the tradename Quality Audit Standards QAS4002™ obtained from Malvern Analytical Ltd. Samples were analyzed as dry powders, unless otherwise indicated. The particles analyzed were dispersed in air and analyzed using the AERO S dry powder dispersion module with the MASTERSIZER™ 3000. The particle sizes were derived using instruments software from a plot of volume density as a function of size. Particle size measurement and diameter span can also be determined by optical digital microscopy. The optical images are obtained using a Keyence VHX-2000 digital microscope using version 2.3.5.1 software for particle size analysis (system version 1.93). As used herein, when referring to sieving, pore/screen sizes are described per U.S.A. Standard Sieve (ASTM E11-17). As used herein, the terms “circularity” and “sphericity” relative to the particles refer to how close the particle is to a perfect sphere. To determine circularity, optical microscopy images are taken of the particles. The perimeter (P) and area (A) of the particle in the plane of the microscopy image is calculated (e.g., using a SYSMEX FPIA 3000 particle shape and particle size analyzer, available from Malvern Instruments). The circularity of the particle is CEA/P, where CEAis the circumference of a circle having the area equivalent to the area (A) of the actual particle. As used herein, the term “sintering window” refers to the difference between the melting temperature (Tm) onset and the crystallization temperature (Tc) onset, or (Tm−Tc) onset. Tm, Tm (onset), Tc, and Tc (onset) are determined by differential scanning calorimetry per ASTM E794-06(2018) with a 10° C./min ramp rate and a 10° C./min cool rate. As used herein, the term “shear” refers to stirring or a similar process that induces mechanical agitation in a fluid. As used herein, the term “aspect ratio” refers to length divided by width, wherein the length is greater than the width. The melting point of a polymer, unless otherwise specified, is determined by ASTM E794-06(2018) with 10° C./min ramping and cooling rates. The softening temperature or softening point of a polymer, unless otherwise specified, is determined by ASTM D6090-17. The softening temperature can be measured by using a cup and ball apparatus available from Mettler-Toledo using a 0.50 gram sample with a heating rate of 1° C./min. Angle of repose is a measure of the flowability of a powder. Angle of repose measurements were determined using a Hosokawa Micron Powder Characteristics Tester PT-R using ASTM D6393-14 “Standard Test Method for Bulk Solids” Characterized by Carr Indices.” Hausner ratio (Hr) is a measure of the flowability of a powder and is calculated by Hr=ρtap/ρbulk, where ρbulkis the bulk density per ASTM D6393-14 and ρtapis the tapped density per ASTM D6393-14. As used herein, viscosity of carrier fluids are the kinematic viscosity at 25° C., unless otherwise specified, measured per ASTM D445-19. For commercially procured carrier fluids (e.g., PDMS oil), the kinematic viscosity data cited herein was provided by the manufacturer, whether measured according to the foregoing ASTM or another standard measurement technique. Pigment-Pendent Polyamides Generally, the compositions, synthesis methods, and application methods of the present disclosure use a linker that chemically bonds to the oxide particles of the surface treated pigment to a nitrogen of the polyamide. The result is a PP-polyamide. Because surface treated pigments have several oxide particles on the surface, the surface treated pigment particles can act as crosslinkers for the polyamides. Examples of polyamides include, but are not limited to, polycaproamide (nylon 6, polyamide 6, or PA6), poly(hexamethylene succinamide) (nylon 4,6, polyamide 4,6, or PA4,6), polyhexamethylene adipamide (nylon 6,6, polyamide 6,6, or PA6,6), polypentamethylene adipamide (nylon 5,6, polyamide 5,6, or PA5,6), polyhexamethylene sebacamide (nylon 6,10, polyamide 6,10, or PA6,10), polyundecaamide (nylon 11, polyamide 11, or PA11), polydodecaamide (nylon 12, polyamide 12, or PA12), polyhexamethylene terephthalamide (nylon 6T, polyamide 6T, or PA6T), nylon 10,10 (polyamide 10,10 or PA10,10), nylon 10,12 (polyamide 10,12 or PA10,12), nylon 10,14 (polyamide 10,14 or PA10,14), nylon 10,18 (polyamide 10,18 or PA10,18), nylon 6,18 (polyamide 6,18 or PA6,18), nylon 6,12 (polyamide 6,12 or PA6,12), nylon 6,14 (polyamide 6,14 or PA6,14), nylon 12,12 (polyamide 12,12 or PA12,12), semi-aromatic polyamide, aromatic polyamides (aramides), and the like, and any combination thereof. Copolyamides may also be used. Examples of copolyamides include, but are not limited to, PA 11/10,10, PA 6/11, PA 6,6/6, PA 11/12, PA 10,10/10,12, PA 10,10/10,14, PA 11/10,36, PA 11/6,36, PA 10,10/10,36, PA 6T/6,6, and the like, and any combination thereof. Examples of polyamide elastomers include, but are not limited to, polyesteramide, polyetheresteramide, polycarbonate-esteramide, and polyether-block-amide elastomers. Herein, a polyamide followed by a single number is a polyamide having that number of backbone carbons between each nitrogen. A polyamide followed by a first number comma second number is a polyamide having the first number of backbone carbons between the nitrogens for the section having no pendent ═O and the second number of backbone carbons being between the two nitrogens for the section having the pendent ═O. By way of nonlimiting example, nylon 6,10 is [NH—(CH2)6—NH—CO−(CH2)8—CO]n. A polyamide followed by number(s) backslash number(s) are a copolymer of the polyamides indicated by the numbers before and after the backslash. Pigments may impart a color, a metallic color, and/or a pearlescent color such as gold, silver aluminum, bronze, gold bronze, stainless steel, zinc, iron, tin and copper pigments to the polyamide. Examples of pigments include, but are not limited to, synthetic mica (e.g., fluorphlogopite), natural mica (e.g., muscovite), talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments (e.g., BiOCl, colored glass fibers, α-Fe2O3, and α-FeOOH), CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments (e.g., Al flakes and bronzes), optically variable pigments (OVPs), liquid crystal polymer pigments (LCPs), holographic pigments, and the like, and any combination thereof. Examples of metal oxides that may be coating the surface of a pigment include, but are not limited to, titanium dioxide, titanium suboxides, titanium oxynitrides, Al2O3, Fe2O3, Fe3O4, SnO2, Cr2O3, ZnO, CuO, NiO, zirconium oxide, iron titanium oxides (iron titanates), other metal oxides, and the like, and any combination thereof. Preferred metal oxides include TiO2and/or Fe2O3. Surface treated pigments may be any combination of the foregoing pigments with one or more metal oxides, alone or in a mixture in a uniform layer or in successive layers. For example, the surface treated pearlescent pigments may be pigments based on platelet-shaped, transparent or semitransparent substrates like sheet silicates, which are coated with colored or colorless metal oxides like titanium oxides and iron oxides, alone or in a mixture in a uniform layer or in successive layers. Particularly preferred surface treated pigments contain TiO2-coated mica, Fe2O3-coated mica, TiO2/Fe2O3-coated mica, and any combination thereof. Examples of commercially available surface treated pigments include, but are not limited to, REFLEX™ pigments (synthetic mica-based pearlescent pigments, available from CQV), IRIODIN™ (mica-based, metal oxide-coated pearlescent pigments, available from Merck) (e.g., IRIODIN™ 300 “Gold Pearl” and IRIODIN™ 100 “Silver Pearl”), SUNGEM™ (glass platelet-based pigments, available from Sun Chemical), SUNMICA™ (mica-based pigments, available from Sun Chemical), SYNCRYSTAL™ (metal oxide coated synthetic fluorphlogopite flakes, available from Eckart), and the like, and any combination thereof. Other metallic color pearlescent pigments from Merck include TIMIRON® Bronze MP60 with a D50 volume average particle size (50% of the pigments have a volume size of less than the stated size) of 22-37 microns, TIMIRON® Copper MP-65 D50 size of 22-37 microns, COLORONA® Oriental Beige D50 size of 3-10 microns, COLORONA® Aborigine Amber D50 size of 18-25 microns, COLORONA® Passion Orange with D50 size of 18-25 microns, COLORONA® Bronze Fine of D50 size of 7-14, COLORONA® Bronze with D50 size of 18-25 microns, COLORONA® Bronze Sparkle of D50 size of 28-42 microns, COLORONA® Copper Fine with D50 size of 7-14 microns, COLORONA® Copper with D50 size of 18-25, COLORONA® Copper Sparkle with D50 size of 25-39 microns, COLORONA® Red Brown with D50 size of 18-25 microns, COLORONA® Russet with D50 size of 18-25 microns, COLORONA® Tibetan Ochre with D50 size of 18-25 microns, COLORONA® Sienna Fine with D50 size of 7-14 microns, COLORONA® Sienna with D50 size of 18-25 microns, COLORONA® Bordeaux with D50 size of 18-25 microns, COLORONA® Glitter Bordeaux, COLORONA® Chameleon with D50 size of 18-25 microns. Also suitable are Merck mica based pigments with metal oxide particle coatings such as the Merck silvery white pigments including TIMIRON® Super Silk MP-1005 with D50 size of 3-10 microns, TIMIRON® Super Sheen MP-1001 with D50 size of 7-14 microns, TIMIRON® Super Silver Fine with D50 size of 9-13 microns, TIMIRON® Pearl Sheen MP-30 with D50 size of 15-21 microns, TIMIRON® Satin MP-11171 with D50 size of 11-20 microns, TIMIRON® Ultra Luster MP-111 with D50 size of 18-25 microns, TIMIRON® Star Luster MP-111 with D50 size of 18-25 microns, TIMIRON® Pearl Flake MP-10 with D50 size of 22-37 microns, TIMIRON® Super Silver with D50 size of 17-26 microns, TIMIRON® Sparkle MP-47 with D50 size of 28-38 microns, TIMIRON® Arctic Silver with D50 size of 19-25 microns, XIRONA® Silver with D50 size of 15-22 microns, RONASTAR® Silver with D50 size of 25-45 microns, and the like, and any combination thereof. For very bright colors, examples from Merck include COLORONA® Carmine Red with D50 size of 10-60 microns giving a Red lustrous effect, COLORONA® Magenta with D50 size of 18-25 microns, giving a pink-violet lustrous effect, COLORONA® Light Blue with D50 size of 18-25 microns, to give a light blue lustrous effect, COLORONA® Dark Blue with D50 size of 18-25 microns to give a dark blue lustrous effect, COLORONA® Majestic Green with 18-25 microns to give a green lustrous color, COLORONA® Brilliant Green of D5 19-26 microns to give a Green-golden lustrous color, COLORONA® Egyptian Emerald of D50 18-25 microns to give a dark green lustrous effect, COLORONA® Patagonian Purple of 18-25 microns size to give a purple lustrous effect, and the like, and any combination thereof. Mica based special effect pigments having a D50 from about 18 microns to about 50 microns from Eckart may also be used, such as DORADO® PX 4001, DORADO® PX 4261, DORADO® PX 4271, DORADO® PX 4310, DORADO® PX 4331, DORADO® PX 4542, PHOENIX® XT, PHOENIX® XT 2001, PHOENIX® XT 3001, PHOENIX® XT 4001, PHOENIX® XT 5001, PHOENIX® PX 1000, PHOENIX® PX 1001, PHOENIX® PX 1221, PHOENIX® PX 1231, PHOENIX® PX 1241, PHOENIX® PX 1251, PHOENIX® PX 1261, PHOENIX® PX 1271, PHOENIX® PX 1310, PHOENIX® PX 1320, PHOENIX® PX 1502, PHOENIX® PX 1522, PHOENIX® PX 1542, PHOENIX® PX 2000, PHOENIX® PX 2000 L, PHOENIX® PX 2001, PHOENIX® PX 2011, PHOENIX® PX 2021, PHOENIX® PX 2221, PHOENIX® PX 2231, PHOENIX® PX 2241, PHOENIX® PX 2251, PHOENIX® PX 2261, PHOENIX® PX 2271, PHOENIX® PX 3001, PHOENIX® PX 4000, PHOENIX® PX 4001, PHOENIX® PX 4221, PHOENIX® PX 4231, PHOENIX® PX 4241, PHOENIX® PX 4251, PHOENIX® PX 4261, PHOENIX® PX 4271, PHOENIX® PX 4310, PHOENIX® PX 4320, PHOENIX® PX 4502, PHOENIX® PX 4522, PHOENIX® PX 4542, PHOENIX® PX 5000, PHOENIX® PX 5001, PHOENIX® PX 5310, PHOENIX® PX 5331, and the like, and any combination thereof. Special effect pigments such as Silberline aluminum flake pigments may be used, such as 16 micron DF-1667, 55 micron DF-2750, 27 micron DF-3500, 35 micron DF-3622, 15 micron DF-554, 20 micron DF-L-520AR, 20 micron LED-1708AR, 13 micron LED-2314AR, 55 micron SILBERCOTE™ PC 0452Z, 47 micron SILBERCOTE™ PC 1291X, 36 micron SILBERCOTE™, 36 micron SILBERCOTE™ PC 3331X, 31 micron SILBERCOTE™ PC 4352Z, 33 micron SILBERCOTE™ PC 4852X, 20 micron SILBERCOTE™ PC 6222X, 27 micron SILBERCOTE™ PC 6352Z, 25 micron SILBERCOTE™ PC 6802X, 14 micron SILBERCOTE™ PC 8152Z, 14 micron SILBERCOTE™ PC 8153X, 16 micron SILBERCOTE™ PC 8602X, 20 micron SILVET®/SILVEX® 890 Series, 16 micron SILVET®/SILVEX® 950 Series, and the like, and any combination thereof. Surface treated pigments may have an average diameter (or D50) of about 1 micron to about 500 microns (or about 1 micron to about 25 microns, or about 5 microns to about 50 microns, or about 25 microns to about 200 microns, or about 100 microns to about 300 microns, or about 250 microns to about 500 microns). Without being limited by theory, it is believed that larger pigment particles impart greater coloring, metallic, and/or pearlescent effects to the polyamide. Scheme 1 below is a first nonlimiting example synthesis of a PP-polyamide. More specifically, a difunctional linkage reagent containing for example an alkoxysilane end group and a glycidyl functional group. The alkoxysilane group (illustrated with Si(—OCH3)3groups where one or more of such groups may be hydrolyzed to —OH groups) is coupled to the surface of the oxide particles (MOx particles) on the surface of the pigment. While the silane is shown as coupling only to the metal oxide particles, the silane may also couple to the surface of the pigment depending on the composition of the pigment. Then, the epoxy of the glycidyl moiety reacts with the nitrogen of the amide in the polyamide (illustrated using nylon 6) to yield a PP-polyamide. While the epoxy is illustrated as a terminal epoxy, the difunctional linkage reagent may have one or more glycidyl functional groups (or epoxy groups) that are terminal, pendent, or include both terminal and pendent of such groups. As illustrated, the pigment is chemically linked to four polyamide chains, which illustrates that the pigment may also be a crosslinker. Examples of silanes having a glycidyl moiety include, but are not limited to, (3-glycidoxypropyl)trimethoxysilane, (3-glycidoxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane and 1,3-bis(3-dlycidyloxypropyl)tetramethylsiloxane, 2-(3,4 epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropyl methyldimethoxysilane, 3-glycidoxypropyl methyldiethoxysilane, 5,6-epoxyhexyltriethoxysilane, and the like, and any combination thereof. The silane coupling reaction can be performed by known methods. For example, as outlined in reference by X. Guillory et al. calledGlycidyl Alkoxysilanc Reactivities Towards Simple Nucleophiles in Organic Media for Improved Molecular Structure Definition in Hybrid Materialsin Royal Society of Chemistry Advances 2016, 6, 74087-74099. The outcome of the reactions are highly dependent on the reaction conditions; selection of solvent whether it be aqueous, organic or ionic liquid media, temperature concentration, catalyst and acidic or basic conditions. Also from the cited U.S. Pat. No. 7,998,649, the metallic oxide coated mica pigment that is a silica or titania particle surface is reacted with (3-glycidoxypropyl)trimethoxysilane or (3-glycidoxypropyl)triethoxysilane or diethoxy(3-glycidyloxypropyl)methylsilane or 1,3-bis(3-dlycidyloxypropyl)tetramethylsiloxane in a molar ratio of 0.85 to 0.15 with the appropriate amount of ethanol or methanol, water, and ammonia to prepare the epoxide containing component. The solution is then stirred for a period of time of about 2 to about 10 hours at room temperature or slightly elevated up to 40° C. In another example, the silane coupling reaction can be performed by hydrolytic deposition of silanes (B. Arkles, CHEMTECH, 7, 766, 1977) where the silane oligomers hydrogen bond with OH groups of the substrate. During drying or curing, a covalent linkage is formed with the substrate with concomitant loss of water. Methods for enhancing reactivity include transesterification catalysts and agents which increase the acidity of hydroxyl groups on the substrate by hydrogen bonding. Transesterification catalysts include tin compounds such as dibutyldiacetoxytin and titanates such as titanium isopropoxide. Incorporation of transesterification catalysts at about 2 wt % to about 3 wt % of the silane effectively promotes reaction and deposition in many instances, others include tetrabutyl titanate or the reaction can be promoted by addition of catalytic amounts of amines such as benzyldimethylamine The reaction conditions may include a coupling agent may be present at about 0.1 wt % to about 5 wt % relative to the pigment, reaction times may be about 4 hours to about 12 hours, temperatures may be about 50° C. to about 120° C., and ethanol solvent may be preferred over methanol, where water in the solvent is at levels of about 1 wt % to about 5 wt % to promote hydrolysis of the silane during attachment to the surface. In yet another example, the silane coupling reaction can be performed by anhydrous liquid phase deposition where toluene, tetrahydrofuran, and/or hydrocarbon solutions are prepared containing about 1 wt % to about 10 wt % silane. The mixture is refluxed for about 12 hours to about 24 hours with the substrate (pigment in this instance) to be treated. The treated pigment is washed with the solvent. The solvent is then removed by air or explosion proof oven drying. No further cure is necessary. The epoxide group on the metallic pigment reactions with the polyamide may be performed under an atmosphere (nitrogen or argon) at temperatures of about 70° C. to about 200° C. (or about 70° C. to about 150° C., or about 125° C. to about 200° C.) in the presence of an organic solvent such as tetrahydrofuan, dimethylformamide or toluene or the like. The mixture is then stirred for about 24 hours at an elevated temperature. After cooling the mixture to room temperature, the grafted polymer is filtered and washed to remove organic impurities and unreacted starting reagents. Scheme 2 below is a second nonlimiting example synthesis of a PP-polyamide. More specifically, silica particles having a surface functionality with at least one carboxylic acid (which may be terminal as shown, pendent, or both terminal and pendent) are grafted onto the metal oxide particle (but some may graft to the pigment depending on the pigment compositions). Then, the carboxylic acids from the functionalized silica particles are converted to acid chlorides, which react with the nitrogen of the amide in the polyamide (illustrated using nylon 6) to yield a PP-polyamide. As illustrated, the pigment is chemically linked to four polyamide chains, which illustrates that the surface coated pigment (surface of the pigment and/or surface of the metal oxide particles) may also be a crosslinker. Examples of functionalized silica particles include, but are not limited to, 3-aminopropyl-(3-oxobutanoic) acid functionalized silica, 3-propylsulphonic acid-functionalized silica gel, propylcarboxylic acid functionalized silica, triaminetetraacetic acid-functionalized silica gel, propionyl chloride-functionalized silica gel, 3-carboxypropyl functionalized silica gel, aminomethylphosphonic acid (AMPA)-functionalized silica gel, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized silica gel, and the like, and any combination thereof. The grafting reaction of the functionalized silica to the pigment can be performed by known methods. Similar to the glycidyl functionalized coated pigments of Scheme 1, silanes that contain reactive alkoxy (OR) functional groups can react with the surface hydroxyl groups of the metal oxide particles and/or the pigments. When the silane solution is applied onto the surface hydroxyl groups of the metal oxide particles and/or the pigments, the free silanol groups first form hydrogen bonding with the hydroxyl (—OH) groups on the metal oxide particles and/or the pigments at ambient temperature. Then, a SiO2linkage is formed between the silanol and the —OH groups on the surface of the SiO2linkage by condensation. The conversion of the carboxylic acid to the more reactive acid chloride can be performed by known methods. For example, oxalyl chloride (COCl)2and/or thionyl chloride SOCl2may be used as chlorinating agents in conjunction with a catalyst. Solvents for said reactions may include, but are not limited to, dimethylformamide, dichloromethane, and the like, and any combination thereof. The acid chloride/polyamide reaction with the polyamide may be performed by known methods. For example, the pigment having the acid chloride functionality may be melt mixed with polyamide at about 125° C. to about 250° C. (or about 125° C. to about 200° C., or about 150° C. to about 225° C., or about 200° C. to about 250° C.) for about 15 minutes to about 1 hour or longer (or about 15 minutes to about 30 minutes, or about 20 minutes to about 40 minutes, or about 30 minutes to about 1 hour). Scheme 1 and Scheme 2 include nonlimiting examples of synthetic routes to producing a surface treated pigment having pendent epoxy or carboxylic acid moieties. Other reaction schemes will be apparent to those skilled in the art. The surface treated pigment having pendent epoxy or carboxylic acid moieties are reacted with the polyamide to yield the PP-polyamides described herein. Whether by Scheme 1, Scheme 2, or another coupling reaction scheme, the PP-polyamides described herein may have a weight ratio of pigment to polyamide of about 1:10 to about 1:1000 (or about 1:10 to about 1:200, or about 1:100 to about 1:500, or about 1:250 to about 1:1000). Applications of PP-Polyamides The PP-polyamides described herein may be used to produce a variety of objects (or articles). The PP-polyamides described herein may be used alone or in combination with other thermoplastic polymers (e.g., polyamides without an optical absorber and/or other thermoplastic polymers). Examples of thermoplastic polymers that may be used in conjunction with one or more PP-polyamides of the present disclosure include, but are not limited to, polyamides (e.g., polyamides not coupled to a pigment), polyurethanes, polyethylenes, polypropylenes, polyacetals, polycarbonates, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), polyhexamethylene terephthalate, polystyrenes, polyvinyl chlorides, polytetrafluoroethenes, polyesters (e.g., polylactic acid), polyethers, polyether sulfones, polyetherether ketones, polyacrylates, polymethacrylates, polyimides, acrylonitrile butadiene styrene (ABS), polyphenylene sulfides, vinyl polymers, polyarylene ethers, polyarylene sulfides, polysulfones, polyether ketones, polyamide-imides, polyetherimides, polyetheresters, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), grafted or ungrafted thermoplastic polyolefins, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylates, functionalized or nonfunctionalized (meth)acrylic acid polymers, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymers, ethylene/vinyl monomer/carbonyl terpolymers, ethylene/alkyl (meth)acrylate/carbonyl terpolymers, methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, chlorinated or chlorosulphonated polyethylenes, polyvinylidene fluoride (PVDF), phenolic resins, poly(ethylene/vinyl acetate)s, polybutadienes, polyisoprenes, styrenic block copolymers, polyacrylonitriles, silicones, and the like, and any combination thereof. Copolymers comprising one or more of the foregoing may also be used in the methods and systems described herein. If needed, compatibilizers may be used when combining the PP-polyamides described herein with other thermoplastic polymers. Compatibilizers may improve the blending efficiency and/or efficacy of the polymers. Examples of polymer compatibilizers include, but are not limited to, PROPOLDER™ MPP2020 20 (polypropylene, available from Polygroup Inc.), PROPOLDER™ MPP2040 40 (polypropylene, available from Polygroup Inc.), NOVACOM™ HFS2100 (maleic anhydride functionalized high density polyethylene polymer, available from Polygroup Inc.), KEN-REACT™ CAPS™ L™ 12/L (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ L™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ LICA™ 12 (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPS™ KPR™ 12/LV (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ CAPOW™ KPR™ 12/H (organometallic coupling agent, available from Kenrich Petrochemicals), KEN-REACT™ titanates & zirconates (organometallic coupling agent, available from Kenrich Petrochemicals), VISTAMAXX™ (ethylene-propylene copolymers, available from ExxonMobil), SANTOPRENE™ (thermoplastic vulcanizate of ethylene-propylene-diene rubber and polypropylene, available from ExxonMobil), VISTALON™ (ethylene-propylene-diene rubber, available from ExxonMobil), EXACT™ (plastomers, available from ExxonMobil) EXXELOR™ (polymer resin, available from ExxonMobil), FUSABOND™ M603 (random ethylene copolymer, available from Dow), FUSABOND™ E226 (anhydride modified polyethylene, available from Dow), BYNEL™ 41E710 (coextrudable adhesive resin, available from Dow), SURLYN™ 1650 (ionomer resin, available from Dow), FUSABOND™ P353 (a chemically modified polypropylene copolymer, available from Dow), ELVALOY™ PTW (ethylene terpolymer, available from Dow), ELVALOY™ 3427AC (a copolymer of ethylene and butyl acrylate, available from Dow), LOTADER™ AX8840 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3210 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3410 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 3430 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4700 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ AX8900 (ethylene acrylate-based terpolymer, available from Arkema), LOTADER™ 4720 (ethylene acrylate-based terpolymer, available from Arkema), BAXXODUR™ EC 301 (amine for epoxy, available from BASF), BAXXODUR™ EC 311 (amine for epoxy, available from BASF), BAXXODUR™ EC 303 (amine for epoxy, available from BASF), BAXXODUR™ EC 280 (amine for epoxy, available from BASF), BAXXODUR™ EC 201 (amine for epoxy, available from BASF), BAXXODUR™ EC 130 (amine for epoxy, available from BASF), BAXXODUR™ EC 110 (amine for epoxy, available from BASF), styrenics, polypropylene, polyamides, polycarbonate, EASTMAN™ G-3003 (a maleic anhydride grafted polypropylene, available from Eastman), RETAIN™ (polymer modifier available from Dow), AMPLIFY TY™ (maleic anhydride grafted polymer, available from Dow), INTUNE™ (olefin block copolymer, available from Dow), and the like and any combination thereof. Methods for producing objects include, but are not limited to, melt extrusion, injection molding, compression molding, melt spinning, melt emulsification, spray drying (e.g., to form particles), cryogenic milling (or cryogenic grinding), freeze drying polymer dispersions, precipitation of polymer dispersions, and the like, and any hybrid thereof. Examples of articles that may be produced by such methods where the PP-polyimide may be all or a portion of said articles include, but are not limited to, particles, films, packaging, toys, household goods, automotive parts, aerospace/aircraft-related parts, containers (e.g., for food, beverages, cosmetics, personal care compositions, medicine, and the like), shoe soles, furniture parts, decorative home goods, plastic gears, screws, nuts, bolts, cable ties, jewelry, art, sculpture, medical items, prosthetics, orthopedic implants, production of artifacts that aid learning in education, 3D anatomy models to aid in surgeries, robotics, biomedical devices (orthotics), home appliances, dentistry, electronics, sporting goods, and the like. Further, particles may be useful in applications that include, but are not limited to, paints, powder coatings, ink jet materials, electrophotographic toners, 3D printing, and the like. In addition, the PP-polyamides described herein may have a specific chemical fingerprint that is useful in identifying objects, tracking objects, authenticating objects, and/or determining the health of objects. Further, the placement of where the PP-polyamides are located in the objects is another layer of fingerprinting the objects for identifying objects, tracking objects, authenticating objects, and/or determining the health of objects. Methods of identifying objects, tracking objects, authenticating objects, and/or determining the health of objects may include (a) exposing the object comprising PP-polyamides to electromagnetic radiation; (b) sensing one or more spectra related to the electromagnetic radiation absorbed and/or reemitted; and (c) comparing the spectra to the known spectra for the optical absorbers used in said object or portion thereof. Optionally, the location of where the spectra area is located on the object may be compared to the known location where the spectra area should be. The comparison(s) can be used for identifying and/or authenticating the object. For tracking, the comparison(s) may be done and/or the detected spectra and/or spectra area may be logged into a database along with the physical location of the object. Further, the health of objects that wear and/or crack can be ascertained. For example, a core portion of the article may comprise optical absorbers and an outer portion may cover the core portion and not comprise the optical absorbers (or comprise different optical absorbers). Then, when comparing spectra, the appearance of spectral features for the optical absorbers in the core may indicate that the object is at or near the end of life. By way of nonlimiting example, 3-D printing processes of the present disclosure may comprise: depositing particles comprising one or more PP-polyamides of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) upon a surface in a specified shape, and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body (object), such that the consolidated body has a void percentage of about 1% or less after being consolidated. For example, heating and consolidation of the thermoplastic polymer particles may take place in a 3-D printing apparatus employing a laser, such that heating and consolidation take place by selective laser sintering. By way of nonlimiting example, 3-D printing processes of the present disclosure may comprise: extruding a filament comprising one or more PP-polyamides of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) through an orifice, wherein the filament becomes a polymer melt upon extrusion; depositing the polymer melt as a first layer on a platform; cooling the layer; depositing an additional layer of the polymer melt on the first layer; cooling the additional layer; repeating depositing and cooling for at least one additional layer to produce a 3-D shape. Yet another nonlimiting example is a method comprising: extruding a polymer melt comprising one or more PP-polyamides of the present disclosure (and optionally one or more other thermoplastic polymers and/or one or more compatibilizers) through an orifice to produce a film, a fiber (or a filament), particles, pellets, or the like. Thermoplastic Polymer Particles and Methods of Making TheFIG.1sa flow chart of a nonlimiting example method100of the present disclosure. Thermoplastic polymer102(comprising one or more PP-polyamides and optionally one or more other thermoplastic polymers), carrier fluid104, and optionally emulsion stabilizer106are combined108to produce a mixture110. The components102,104, and106can be added in any order and include mixing and/or heating during the process of combining108the components102,104, and106. The mixture110is then processed112by applying sufficiently high shear to the mixture110at a temperature greater than the melting point or softening temperature of the thermoplastic polymer102to form a melt emulsion114. Because the temperature is above the melting point or softening temperature of the thermoplastic polymer102, the thermoplastic polymer102becomes a polymer melt. The shear rate should be sufficient enough to disperse the polymer melt in the carrier fluid104as droplets (i.e., the polymer emulsion114). Without being limited by theory, it is believed that, all other factors being the same, increasing shear should decrease the size of the droplets of the polymer melt in the carrier fluid104. However, at some point there may be diminishing returns on increasing shear and decreasing droplet size or may be disruptions to the droplet contents that decrease the quality of particles produced therefrom. The melt emulsion114inside and/or outside the mixing vessel is then cooled116to solidify the polymer droplets into thermoplastic polymer particles (also referred to as solidified thermoplastic polymer particles). The cooled mixture118can then be treated120to isolate the thermoplastic polymer particles122from other components124(e.g., the carrier fluid104, excess emulsion stabilizer106, and the like) and wash or otherwise purify the thermoplastic polymer particles122. The thermoplastic polymer particles122comprise the thermoplastic polymer102and, when included, at least a portion of the emulsion stabilizer106coating the outer surface of the thermoplastic polymer particles122. Emulsion stabilizers106, or a portion thereof, may be deposited as a uniform coating on the thermoplastic polymer particles122. In some instances, which may be dependent upon non-limiting factors such as the temperature (including cooling rate), the type of thermoplastic polymer102, and the types and sizes of emulsion stabilizers106, the nanoparticles of emulsion stabilizers106may become at least partially embedded within the outer surface of the thermoplastic polymer particles122in the course of becoming associated therewith. Even without embedment taking place, at least the nanoparticles within emulsion stabilizers106may remain robustly associated with thermoplastic polymer particles122to facilitate their further use. In contrast, dry blending already formed thermoplastic polymer particulates (e.g., formed by cryogenic grinding or precipitation processes) with a flow aid like silica nanoparticles does not result in a robust, uniform coating of the flow aid upon the thermoplastic polymer particulates. Advantageously, carrier fluids and washing solvents of the systems and methods described herein (e.g., method100) can be recycled and reused. One skilled in the art will recognize any necessary cleaning of used carrier fluid and solvent necessary in the recycling process. The thermoplastic polymer102and carrier fluid104should be chosen such that at the various processing temperatures (e.g., from room temperature to process temperature) the thermoplastic polymer102and carrier fluid104are immiscible. An additional factor that may be considered is the differences in (e.g., a difference or a ratio of) viscosity at process temperature between the molten polyamide102and the carrier fluid104. The differences in viscosity may affect droplet breakup and particle size distribution. Without being limited by theory, it is believed that when the viscosities of the molten polyamide102and the carrier fluid104are too similar, the circularity of the product as a whole may be reduced where the particles are more ovular and more elongated structures are observed. The thermoplastic polymers102comprises one or more PP-polyamides and optionally one or more other thermoplastic polymers. Examples of other thermoplastic polymers include, but are not limited to, polyamides, polyurethanes, polyethylenes, polypropylenes, polyacetals, polycarbonates, polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polytrimethylene terephthalate (PTT), polyhexamethylene terephthalate, polystyrenes, polyvinyl chlorides, polytetrafluoroethenes, polyesters (e.g., polylactic acid), polyethers, polyether sulfones, polyetherether ketones, polyacrylates, polymethacrylates, polyimides, acrylonitrile butadiene styrene (ABS), polyphenylene sulfides, vinyl polymers, polyarylene ethers, polyarylene sulfides, polysulfones, polyether ketones, polyamide-imides, polyetherimides, polyetheresters, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), grafted or ungrafted thermoplastic polyolefins, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylates, functionalized or nonfunctionalized (meth)acrylic acid polymers, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymers, ethylene/vinyl monomer/carbonyl terpolymers, ethylene/alkyl (meth)acrylate/carbonyl terpolymers, methylmethacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, chlorinated or chlorosulphonated polyethylenes, polyvinylidene fluoride (PVDF), phenolic resins, poly(ethylene/vinyl acetate)s, polybutadienes, polyisoprenes, styrenic block copolymers, polyacrylonitriles, silicones, and the like, and any combination thereof. Copolymers comprising one or more of the foregoing may also be used in the methods and systems of the present disclosure. The other thermoplastic polymers in the compositions and methods of the present disclosure may be elastomeric or non-elastomeric. Some of the foregoing examples of other thermoplastic polymers may be elastomeric or non-elastomeric depending on the exact composition of the polymer. For example, polyethylene that is a copolymer of ethylene and propylene may be elastomeric or not depending on the amount of propylene in the polymer. Thermoplastic elastomers generally fall within one of six classes: styrenic block copolymers, thermoplastic polyolefin elastomers, thermoplastic vulcanizates (also referred to as elastomeric alloys), thermoplastic polyurethanes, thermoplastic copolyesters, and thermoplastic polyamides (typically block copolymers comprising polyamide). Examples of thermoplastic elastomers can be found in theHandbook of Thermoplastic Elastomers,2nd ed., B. M. Walker and C. P. Rader, eds., Van Nostrand Reinhold, New York, 1988. Examples of thermoplastic elastomers include, but are not limited to, elastomeric polyamides, polyurethanes, copolymers comprising a polyether block and a polyamide block (PEBA or polyether block amide), methyl methacrylate-butadiene-styrene (MBS)-type core-shell polymers, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) (SBM) block terpolymers, polybutadienes, polyisoprenes, styrenic block copolymers, and polyacrylonitriles), silicones, and the like. Elastomeric styrenic block copolymers may include at least one block selected from the group of: isoprene, isobutylene, butylene, ethylene/butylene, ethylene-propylene, and ethylene-ethylene/propylene. More specific elastomeric styrenic block copolymer examples include, but are not limited to, poly(styrene-ethylene/butylene), poly(styrene-ethylene/butylene-styrene), poly(styrene-ethylene/propylene), styrene-ethylene/propylene-styrene), poly(styrene-ethylene/propylene-styrene-ethylene-propylene), poly(styrene-butadiene-styrene), poly(styrene-butylene-butadiene-styrene), and the like, and any combination thereof. Examples of polyamides include, but are not limited to, those described above. Examples of polyamide elastomers include, but are not limited to, polyesteramide, polyetheresteramide, polycarbonate-esteramide, and polyether-block-amide elastomers. Examples of polyurethanes include, but are not limited to, polyether polyurethanes, polyester polyurethanes, mixed polyether and polyester polyurethanes, and the like, and any combination thereof. Examples of thermoplastic polyurethanes include, but are not limited to, poly[4,4′-methylenebis(phenylisocyanate)-alt-1,4-butanediol/di(propylene glycol)/polycaprolactone], ELASTOLLAN® 1190A (a polyether polyurethane elastomer, available from BASF), ELASTOLLAN® 1190A10 (a polyether polyurethane elastomer, available from BASF), and the like, and any combination thereof. Compatibilizers may optionally be used to improve the blending efficiency and efficacy of PP-polyamides with one or more thermoplastic polymers. Examples of polymer compatibilizers include, but are not limited to, the compatibilizers described above. The thermoplastic polymers102(comprising one or more PP-polyamides and optionally one or more other thermoplastic polymers) may have a melting point or softening temperature of about 50° C. to about 450° C. (or about 50° C. to about 125° C., or about 100° C. to about 175° C., or about 150° C. to about 280° C., or about 200° C. to about 350° C., or about 300° C. to about 450° C.). The thermoplastic polymers102may have a glass transition temperature (ASTM E1356-08(2014) with 10° C./min ramping and cooling rates) of about −50° C. to about 400° C. (or about −50° C. to about 0° C., or about −25° C. to about 50° C., or about 0° C. to about 150° C., or about 100° C. to about 250° C., or about 150° C. to about 300° C., or about 200° C. to about 400° C.). The thermoplastic polymers102may optionally comprise an additive. Typically, the additive would be present before addition of the thermoplastic polymers102to the mixture110. Therefore, in the thermoplastic polymer melt droplets and resultant thermoplastic polymer particles, the additive is dispersed throughout the thermoplastic polymer. Accordingly, for clarity, this additive is referred to herein as an “internal additive.” The internal additive may be blended with the thermoplastic polymer just prior to making the mixture110or well in advance. When describing component amounts in the compositions described herein (e.g., the mixture110and the thermoplastic polymer particles122), a weight percent based on the thermoplastic polymer102not inclusive of the internal additive. For example, a composition comprising 1 wt % of emulsion stabilizer by weight of 100 g of a thermoplastic polymer102comprising 10 wt % internal additive and 90 wt % thermoplastic polymer is a composition comprising 0.9 g of emulsion stabilizer, 90 g of thermoplastic polymer, and 10 g of internal additive. The internal additive may be present in the thermoplastic polymer102at about 0.1 wt % to about 60 wt % (or about 0.1 wt % to about 5 wt %, or about 1 wt % to about 10 wt %, or about 5 wt % to about 20 wt %, or about 10 wt % to about 30 wt %, or about 25 wt % to about 50 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer102. For example, the thermoplastic polymer102may comprise about 70 wt % to about 85 wt % of a thermoplastic polymer and about 15 wt % to about 30 wt % of an internal additive like glass fiber or carbon fiber. Examples of internal additives include, but are not limited to, fillers, strengtheners, pigments, pH regulators, and the like, and combinations thereof. Examples of fillers include, but are not limited to, glass fibers, glass particles, mineral fibers, carbon fiber, oxide particles (e.g., titanium dioxide and zirconium dioxide), metal particles (e.g., aluminum powder), and the like, and any combination thereof. Examples of pigments include, but are not limited to, organic pigments, inorganic pigments, carbon black, and the like, and any combination thereof. The thermoplastic polymer102may be present in the mixture110at about 5 wt % to about 60 wt % (or about 5 wt % to about 25 wt %, or about 10 wt % to about 30 wt %, or about 20 wt % to about 45 wt %, or about 25 wt % to about 50 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer102and carrier fluid104combined. Suitable carrier fluids104have a viscosity at 25° C. of about 1000 cSt to about 150,000 cSt (or about 1000 cSt to about 60,000 cSt, or about 40,000 cSt to about 100,000 cSt, or about 75,000 cSt to about 150,000 cSt). Examples of carrier fluids104include, but are not limited to, silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, alkyl-terminal polyethylene glycols (e.g., C1-C4 terminal alkyl groups like tetraethylene glycol dimethyl ether (TDG)), paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and the like, and any combination thereof. Examples of silicone oils include, but are not limited to, polydimethylsiloxane, methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and the like, and any combination thereof. The carrier fluid104may have one or more phases. For example, polysiloxanes modified with fatty acids and polysiloxanes modified with fatty alcohols (preferably with similar chain lengths for the fatty acids and fatty alcohols) may form a single-phase carrier fluid104. In another example, a carrier fluid104comprising a silicone oil and an alkyl-terminal polyethylene glycol may form a two-phase carrier fluid104. The carrier fluid104may be present in the mixture110at about 40 wt % to about 95 wt % (or about 75 wt % to about 95 wt %, or about 70 wt % to about 90 wt %, or about 55 wt % to about 80 wt %, or about 50 wt % to about 75 wt %, or about 40 wt % to about 60 wt %) of the thermoplastic polymer102and carrier fluid104combined. In some instances, the carrier fluid104may have a density of about 0.6 g/cm3to about 1.5 g/cm3, and the thermoplastic polymer102has a density of about 0.7 g/cm3to about 1.7 g/cm3, wherein the thermoplastic polymer has a density similar, lower, or higher than the density of the carrier fluid. The emulsion stabilizers used in the methods and compositions of the present disclosure may comprise nanoparticles (e.g. oxide nanoparticles, carbon black, polymer nanoparticles, and combinations thereof), surfactants, and the like, and any combination thereof. Oxide nanoparticles may be metal oxide nanoparticles, non-metal oxide nanoparticles, or mixtures thereof. Examples of oxide nanoparticles include, but are not limited to, silica, titania, zirconia, alumina, iron oxide, copper oxide, tin oxide, boron oxide, cerium oxide, thallium oxide, tungsten oxide, and the like, and any combination thereof. Mixed metal oxides and/or non-metal oxides, like aluminosilicates, borosilicates, and aluminoborosilicates, are also inclusive in the term metal oxide. The oxide nanoparticles may by hydrophilic or hydrophobic, which may be native to the particle or a result of surface treatment of the particle. For example, a silica nanoparticle having a hydrophobic surface treatment, like dimethyl silyl, trimethyl silyl, and the like, may be used in methods and compositions of the present disclosure. Additionally, silica with functional surface treatments like methacrylate functionalities may be used in methods and compositions of the present disclosure. Unfunctionalized oxide nanoparticles may also be suitable for use as well. Commercially available examples of silica nanoparticles include, but are not limited to, AEROSIL® particles available from Evonik (e.g., AEROSIL® R812S (about 7 nm average diameter silica nanoparticles having a hydrophobically modified surface and a BET surface area of 260±30 m2/g), AEROSIL® RX50 (about 40 nm average diameter silica nanoparticles having a hydrophobically modified surface and a BET surface area of 35±10 m2/g), AEROSIL® 380 (silica nanoparticles having a hydrophilically modified surface and a BET surface area of 380±30 m2/g)), and the like, and any combination thereof. Carbon black is another type of nanoparticle that may be present as an emulsion stabilizer in the compositions and methods disclosed herein. Various grades of carbon black will be familiar to one having ordinary skill in the art, any of which may be used herein. Other nanoparticles capable of absorbing infrared radiation may be used similarly. Polymer nanoparticles are another type of nanoparticle that may be present as an emulsion stabilizer in the disclosure herein. Suitable polymer nanoparticles may include one or more polymers that are thermosetting and/or crosslinked, such that they do not melt when processed by melt emulsification according to the disclosure herein. High molecular weight thermoplastic polymers having high melting or decomposition points may similarly comprise suitable polymer nanoparticle emulsion stabilizers. The nanoparticles may have an average diameter (D50 based on volume) of about 1 nm to about 500 nm (or about 10 nm to about 150 nm, or about 25 nm to about 100 nm, or about 100 nm to about 250 nm, or about 250 nm to about 500 nm). The nanoparticles may have a BET surface area of about 10 m2/g to about 500 m2/g (or about 10 m2/g to about 150 m2/g, or about 25 m2/g to about 100 m2/g, or about 100 m2/g to about 250 m2/g, or about 250 m2/g to about 500 m2/g). Nanoparticles may be included in the mixture110at a concentration of about 0.01 wt % to about 10 wt % (or about 0.01 wt % to about 1 wt %, or about 0.1 wt % to about 3 wt %, or about 1 wt % to about 5 wt %, or about 5 wt % to about 10 wt %) based on the weight of the thermoplastic polymer102. Surfactants may be anionic, cationic, nonionic, or zwitterionic. Examples of surfactants include, but are not limited to, sodium dodecyl sulfate, sorbitan oleates, poly[dimethylsiloxane-co-[3-(2-(2-hydroxyethoxy)ethoxy)propylmethylsiloxane], docusate sodium (sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate), and the like, and any combination thereof. Commercially available examples of surfactants include, but are not limited to, CALFAX® DB-45 (sodium dodecyl diphenyl oxide disulfonate, available from Pilot Chemicals), SPAN® 80 (sorbitan maleate non-ionic surfactant), MERPOL® surfactants (available from Stepan Company), TERGITOL™ TMN-6 (a water-soluble, nonionic surfactant, available from DOW), TRITON™ X-100 (octyl phenol ethoxylate, available from SigmaAldrich), IGEPAL® CA-520 (polyoxyethylene (5) isooctylphenyl ether, available from SigmaAldrich), BRIJ® S10 (polyethylene glycol octadecyl ether, available from SigmaAldrich), and the like, and any combination thereof. Surfactants may be included in the mixture110at a concentration of about 0.01 wt % to about 10 wt % (or about 0.01 wt % to about 1 wt %, or about 0.5 wt % to about 2 wt %, or about 1 wt % to about 3 wt %, or about 2 wt % to about 5 wt %, or about 5 wt % to about 10 wt %) based on the weight of the polyamide102. Alternatively, the mixture110may comprise no (or be absent of) surfactant. A weight ratio of nanoparticles to surfactant may be about 1:10 to about 10:1 (or about 1:10 to about 1:1, or about 1:5 to about 5:1, or about 1:1 to about 10:1). As described above, the components102,104, and106can be added in any order and include mixing and/or heating during the process of combining108the components102,104, and106. For example, the emulsion stabilizer106may first be dispersed in the carrier fluid104, optionally with heating said dispersion, before adding the thermoplastic polymer102. In another nonlimiting example, the thermoplastic polymer102may be heated to produce a polymer melt to which the carrier fluid104and emulsion stabilizer106are added together or in either order. In yet another nonlimiting example, the thermoplastic polymer102and carrier fluid104can be mixed at a temperature greater than the melting point or softening temperature of the thermoplastic polymer102and at a shear rate sufficient enough to disperse the thermoplastic polymer melt in the carrier fluid104. Then, the emulsion stabilizer106can be added to form the mixture110and maintained at suitable process conditions for a set period of time. Combining108the components102,104, and106in any combination can occur in a mixing apparatus used for the processing112and/or another suitable vessel. By way of nonlimiting example, the thermoplastic polymer102may be heated to a temperature greater than the melting point or softening temperature of the thermoplastic polymer102in the mixing apparatus used for the processing112, and the emulsion stabilizer106may be dispersed in the carrier fluid104in another vessel. Then, said dispersion may be added to the melt of the thermoplastic polymer102in the mixing apparatus used for the processing112. The mixing apparatuses used for the processing112to produce the melt emulsion114should be capable of maintaining the melt emulsion114at a temperature greater than the melting point or softening temperature of the thermoplastic polymer102and applying a shear rate sufficient to disperse the polymer melt in the carrier fluid104as droplets. Examples of mixing apparatuses used for the processing112to produce the melt emulsion114include, but are not limited to, extruders (e.g., continuous extruders, batch extruders, and the like), stirred reactors, blenders, reactors with inline homogenizer systems, and the like, and apparatuses derived therefrom. Processing112and forming the melt emulsion114at suitable process conditions (e.g., temperature, shear rate, and the like) for a set period of time. The temperature of processing112and forming the melt emulsion114should be a temperature greater than the melting point or softening temperature of the thermoplastic polymer102and less than the decomposition temperature of any components102,104, and106in the mixture110. For example, the temperature of processing112and forming the melt emulsion114may be about 1° C. to about 50° C. (or about 1° C. to about 25° C., or about 5° C. to about 30° C., or about 20° C. to about 50° C.) greater than the melting point or softening temperature of the thermoplastic polymer102provided the temperature of processing112and forming the melt emulsion114is less than the decomposition temperature of any components102,104, and106in the mixture110. The shear rate of processing112and forming the melt emulsion114should be sufficiently high to disperse the polymer melt in the carrier fluid104as droplets. Said droplets should comprise droplets having a diameter of about 1000 μm or less (or about 1 μm to about 1000 μm, or about 1 μm to about 50 μm, or about 10 μm to about 100 μm, or about 10 μm to about 250 μm, or about 50 μm to about 500 μm, or about 250 μm to about 750 μm, or about 500 μm to about 1000 μm). The time for maintaining said temperature and shear rate for processing112and forming the melt emulsion114may be 10 seconds to 18 hours or longer (or 10 seconds to 30 minutes, or 5 minutes to 1 hour, or 15 minutes to 2 hours, or 1 hour to 6 hours, or 3 hours to 18 hours). Without being limited by theory, it is believed that a steady state of droplet sizes will be reached at which point processing112can be stopped. That time may depend on, among other things, the temperature, shear rate, thermoplastic polymer102composition, the carrier fluid104composition, and the emulsion stabilizer106composition. The melt emulsion114may then be cooled116. Cooling116can be slow (e.g., allowing the melt emulsion to cool under ambient conditions) to fast (e.g., quenching). For example, the rate of cooling may range from about 10° C./hour to about 100° C./second to almost instantaneous with quenching (for example in dry ice) (or about 10° C./hour to about 60° C./hour, or about 0.5° C./minute to about 20° C./minute, or about 1° C./minute to about 5° C./minute, or about 10° C./minute to about 60° C./minute, or about 0.5° C./second to about 10° C./second, or about 10° C./second to about 100° C./second). During cooling, little to no shear may be applied to the melt emulsion114. In some instances, the shear applied during heating may be applied during cooling. The cooled mixture118resulting from cooling116the melt emulsion114comprises solidified thermoplastic polymer particles122(or simply thermoplastic polymer particles) and other components124(e.g., the carrier fluid104, excess emulsion stabilizer106, and the like). The thermoplastic polymer particles may be dispersed in the carrier fluid or settled in the carrier fluid. The cooled mixture118may then be treated120to the separate thermoplastic polymer particles122(or simply thermoplastic polymer particles122) from the other components124. Suitable treatments include, but are not limited to, washing, filtering, centrifuging, decanting, and the like, and any combination thereof. Solvents used for washing the thermoplastic polymer particles122should generally be (a) miscible with the carrier fluid104and (b) nonreactive (e.g., non-swelling and non-dissolving) with the thermoplastic polymer102. The choice of solvent will depend on, among other things, the composition of the carrier fluid and the composition of the thermoplastic polymer102. Examples of solvents include, but are not limited to, hydrocarbon solvents (e.g., pentane, hexane, heptane, octane, cyclohexane, cyclopentane, decane, dodecane, tridecane, and tetradecane), aromatic hydrocarbon solvents (e.g., benzene, toluene, xylene, 2-methyl naphthalene, and cresol), ether solvents (e.g., diethyl ether, tetrahydrofuran, diisopropyl ether, and dioxane), ketone solvents (e.g., acetone and methyl ethyl ketone), alcohol solvents (e.g., methanol, ethanol, isopropanol, and n-propanol), ester solvents (e.g., ethyl acetate, methyl acetate, butyl acetate, butyl propionate, and butyl butyrate), halogenated solvents (e.g., chloroform, bromoform, 1,2-dichloromethane, 1,2-dichloroethane, carbon tetrachloride, chlorobenzene, and hexafluoroisopropanol), water, and the like, and any combination thereof. Solvent may be removed from the thermoplastic polymer particles122by drying using an appropriate method such as air-drying, heat-drying, reduced pressure drying, freeze drying, or a hybrid thereof. The heating may be performed preferably at a temperature lower than the glass transition point of the thermoplastic polymer (e.g., about 50° C. to about 150° C.). The thermoplastic polymer particles122after separation from the other components124may optionally be further classified to produce purified thermoplastic polymer particles128. For example, to narrow the particle size distribution (or reduce the diameter span), the thermoplastic polymer particles122can be passed through a sieve having a pore size of about 10 μm to about 250 μm (or about 10 μm to about 100 μm, or about 50 μm to about 200 μm, or about 150 μm to about 250 μm). In another example purification technique, the thermoplastic polymer particles122may be washed with water to remove surfactant while maintaining substantially all of the nanoparticles associated with the surface of the thermoplastic polymer particles122. In yet another example purification technique, the thermoplastic polymer particles122may be blended with additives to achieve a desired final product. For clarity, because such additives are blended with the thermoplastic particles122or other particles resultant from the methods described herein after the particles are solidified, such additives are referred to herein as “external additives.” Examples of external additives include flow aids, other polymer particles, fillers, and the like, and any combination thereof. In some instances, a surfactant used in making the thermoplastic polymer particles122may be unwanted in downstream applications. Accordingly, yet another example purification technique may include at least substantial removal of the surfactant from the thermoplastic polymer particles122(e.g., by washing and/or pyrolysis). The thermoplastic polymer particles122and/or purified thermoplastic polymer particles128(referred to as particles122/128) may be characterized by composition, physical structure, and the like. As described above, the emulsion stabilizers are at the interface between the polymer melt and the carrier fluid. As a result, when the mixture is cooled, the emulsion stabilizers remain at, or in the vicinity of, said interface. Therefore, the structure of the particles122/128, in general, includes emulsion stabilizers (a) dispersed on an outer surface of the particles122/128and/or (b) embedded in an outer portion (e.g., outer 1 vol %) of the particles122/128. Further, where voids form inside the polymer melt droplets, emulsion stabilizers106should generally be at (and/or embedded in) the interface between the interior of the void and the thermoplastic polymer. The voids generally do not contain the thermoplastic polymer. Rather, the voids may contain, for example, carrier fluid, air, or be void. The particles122/128may comprise carrier fluid at about 5 wt % or less (or about 0.001 wt % to about 5 wt %, or about 0.001 wt % to about 0.1 wt %, or about 0.01 wt % to about 0.5 wt %, or about 0.1 wt % to about 2 wt %, or about 1 wt % to about 5 wt %) of the particles122/128. The thermoplastic polymer102may be present in the particles122/128at about 90 wt % to about 99.5 wt % (or about 90 wt % to about 95 wt %, or about 92 wt % to about 97 wt %, or about 95 wt % to about 99.5 wt %) of the particles122/128. When included, the emulsion stabilizers106may be present in the particles122/128at about 10 wt % or less (or about 0.01 wt % to about 10 wt %, or about 0.01 wt % to about 1 wt %, or about 0.5 wt % to about 5 wt %, or about 3 wt % to about 7 wt %, or about 5 wt % to about 10 wt %) of the particles122/128. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers106may be present in the particles128at less than 0.01 wt % (or 0 wt % to about 0.01 wt %, or 0 wt % to 0.001 wt %). Upon forming thermoplastic particulates according to the disclosure herein, at least a portion of the nanoparticles, such as silica nanoparticles, may be disposed as a coating upon the outer surface of the thermoplastic particulates. At least a portion of the surfactant, if used, may be associated with the outer surface as well. The coating may be disposed substantially uniformly upon the outer surface. As used herein with respect to a coating, the term “substantially uniform” refers to even coating thickness in surface locations covered by the coating composition (e.g., nanoparticles and/or surfactant), particularly the entirety of the outer surface. The emulsion stabilizers106may form a coating that covers at least 5% (or about 5% to about 100%, or about 5% to about 25%, or about 20% to about 50%, or about 40% to about 70%, or about 50% to about 80%, or about 60% to about 90%, or about 70% to about 100%) of the surface area of the particles122/128. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers106may be present in the particles128at less than 25% (or 0% to about 25%, or about 0.1% to about 5%, or about 0.1% to about 1%, or about 1% to about 5%, or about 1% to about 10%, or about 5% to about 15%, or about 10% to about 25%) of the surface area of the particles128. The coverage of the emulsion stabilizers106on an outer surface of the particles122/128may be determined using image analysis of the scanning electron microscope images (SEM micrographs). The emulsion stabilizers106may form a coating that covers at least 5% (or about 5% to about 100%, or about 5% to about 25%, or about 20% to about 50%, or about 40% to about 70%, or about 50% to about 80%, or about 60% to about 90%, or about 70% to about 100%) of the surface area of the particles122/128. When purified to at least substantially remove surfactant or another emulsion stabilizer, the emulsion stabilizers106may be present in the particles128at less than 25% (or 0% to about 25%, or about 0.1% to about 5%, or about 0.1% to about 1%, or about 1% to about 5%, or about 1% to about 10%, or about 5% to about 15%, or about 10% to about 25%) of the surface area of the particles128. The coverage of the emulsion stabilizers106on an outer surface of the particles122/128may be determined using image analysis of the SEM micrographs. The particles122/128may have a D10 of about 0.1 μm to about 125 μm (or about 0.1 μm to about 5 μm, about 1 μm to about 10 μm, about 5 μm to about 30 μm, or about 1 μm to about 25 μm, or about 25 μm to about 75 μm, or about 50 μm to about 85 μm, or about 75 μm to about 125 μm), a D50 of about 0.5 μm to about 200 μm (or about 0.5 μm to about 10 μm, or about 5 μm to about 50 μm, or about 30 μm to about 100 μm, or about 30 μm to about 70 μm, or about 25 μm to about 50 μm, or about 50 μm to about 100 μm, or about 75 μm to about 150 μm, or about 100 μm to about 200 μm), and a D90 of about 3 μm to about 300 μm (or about 3 μm to about 15 μm, or about 10 μm to about 50 μm, or about 25 μm to about 75 μm, or about 70 μm to about 200 μm, or about 60 μm to about 150 μm, or about 150 μm to about 300 μm), wherein D10<D50<D90. The particles122/128may also have a diameter span of about 0.2 to about 10 (or about 0.2 to about 0.5, or about 0.4 to about 0.8, or about 0.5 to about 1.0, or about 1 to about 3, or about 2 to about 5, or about 5 to about 10). Without limitation, diameter span values of 1.0 or greater are considered broad, and diameter spans values of 0.75 or less are considered narrow. Without limitation, diameter span values of 1.0 or greater are considered broad, and diameter spans values of 0.75 or less are considered narrow. In a first nonlimiting example, the particles122/128may have a D10 of about 0.1 μm to about 10 μm, a D50 of about 0.5 μm to about 25 μm, and a D90 of about 3 μm to about 50 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 0.2 to about 2. In a second nonlimiting example, the particles122/128may have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 1.0 to about 2.5. In a third nonlimiting example, the particles122/128may have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 0.6 to about 1.5. In a fourth nonlimiting example, the particles122/128may have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Said particles122/128may have a diameter span of about 0.2 to about 1.2. In a fifth nonlimiting example, the particles122/128may have a D10 of about 1 μm to about 50 μm (or about 5 μm to about 30 μm, or about 1 μm to about 25 μm, or about 25 μm to about 50 μm), a D50 of about 25 μm to about 100 μm (or about 30 μm to about 100 μm, or about 30 μm to about 70 μm, or about 25 μm to about 50 μm, or about 50 μm to about 100 μm), and a D90 of about 60 μm to about 300 μm (or about 70 μm to about 200 μm, or about 60 μm to about 150 μm, or about 150 μm to about 300 μm), wherein D10<D50<D90. The particles122/128may also have a diameter span of about 0.4 to about 3 (or about 0.6 to about 2, or about 0.4 to about 1.5, or about 1 to about 3). The particles122/128may have a circularity of about 0.9 or greater (or about 0.90 to about 1.0, or about 0.93 to about 0.99, or about 0.95 to about 0.99, or about 0.97 to about 0.99, or about 0.98 to 1.0). The particles122/128may have an angle of repose of about 25° to about 45° (or about 25° to about 35°, or about 30° to about 40°, or about 35° to about 45°). The particles122/128may have a Hausner ratio of about 1.0 to about 1.5 (or about 1.0 to about 1.2, or about 1.1 to about 1.3, or about 1.2 to about 1.35, or about 1.3 to about 1.5). The particles122/128may have a bulk density of about 0.3 g/cm3to about 0.8 g/cm3(or about 0.3 g/cm3to about 0.6 g/cm3, or about 0.4 g/cm3to about 0.7 g/cm3, or about 0.5 g/cm3to about 0.6 g/cm3, or about 0.5 g/cm3to about 0.8 g/cm3). Depending on the temperature and shear rate of processing112and the composition and relative concentrations of the components102,104, and106, different shapes of the structures that compose the particles122/128have been observed. Typically, the particles122/128comprise substantially spherical particles (having a circularity of about 0.97 or greater). However, other structures including disc and elongated structures have been observed in the particles122/128. Therefore, the particles122/128may comprise one or more of: (a) substantially spherical particles having a circularity of 0.97 or greater, (b) disc structures having an aspect ratio of about 2 to about 10, and (c) elongated structures having an aspect ratio of 10 or greater. Each of the (a), (b), and (c) structures have emulsion stabilizers dispersed on an outer surface of the (a), (b), and (c) structures and/or embedded in an outer portion of the (a), (b), and (c) structures. At least some of the (a), (b), and (c) structures may be agglomerated. For example, the (c) elongated structures may be laying on the surface of the (a) substantially spherical particles. The particles122/128may have a sintering window that is within 10° C., preferably within 5° C., of the sintering window of the thermoplastic polymer102(comprising one or more PP-polyamides and optionally one or more other thermoplastic polymers). 3-Dimensional Printing The particles comprising PP-polyamides described herein may be useful in a variety of applications including 3-D printing. 3-D printing processes of the present disclosure may comprise: depositing PP-polyamide particles of the present disclosure (e.g., particles comprising one or more PP-polyamides and optionally one or more other thermoplastic polymers) upon a surface in a specified shape, and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body (object), such that the consolidated body has a void percentage of about 1% or less after being consolidated. For example, heating and consolidation of the thermoplastic polymer particles may take place in a 3-D printing apparatus employing a laser, such that heating and consolidation take place by selective laser sintering. Examples of objects that may be 3-D printed using the thermoplastic polymer particles of the present disclosure include, but are not limited to, containers (e.g., for food, beverages, cosmetics, personal care compositions, medicine, and the like), shoe soles, toys, furniture parts and decorative home goods, plastic gears, screws, nuts, bolts, cable ties, automotive parts, medical items, prosthetics, orthopedic implants, aerospace/aircraft-related parts, production of artifacts that aid learning in education, 3-D anatomy models to aid in surgeries, robotics, biomedical devices (orthotics), home appliances, dentistry, electronics, sporting goods, and the like. Other applications for particles comprising one or more PP-polyamides of the present disclosure may include, but are not limited to, use as a filler in paints and powder coatings, inkjet materials and electrophotographic toners, and the like. Example Embodiments A first nonlimiting example embodiment is a method comprising: functionalizing metal oxide particles that are bound to a pigment particle with a compound having an epoxy (terminal, pendent, or include both terminal and pendent of such group) to produce a surface treated pigment having a pendent epoxy; and reacting the pendent epoxy with a polyamide to yield a pigment-pendent polyamide (PP-polyamide). The first nonlimiting example embodiment may further include one or more of: Element 1: wherein the compound having the epoxide is selected from the group consisting of: (3-glycidoxypropyl)trimethoxysilane, (3-glycidoxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane and 1,3-bis(3-dlycidyloxypropyl)tetramethylsiloxane, 2-(3,4 epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropyl methyldimethoxysilane, 3-glycidoxypropyl methyldiethoxysilane, 5,6-epoxyhexyltriethoxysilane, and any combination thereof; Element 2: wherein the metal oxide particles comprise one or more selected from the group consisting of: titanium dioxide, a titanium suboxide, a titanium oxynitride, Al2O3, Fe2O3, Fe3O4, SnO2, Cr2O3, ZnO, CuO, NiO, zirconium oxide, and an iron titanium oxide; Element 3: wherein the pigment particle comprises one or more selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, and holographic pigments; Element 4: wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof; Element 5: wherein the reacting of the pendent epoxy with a polyamide is at about 70° C. to about 200° C.; and Element 6: wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000. Examples of combinations include, but are not limited to, Element 1 in combination with one or more of Elements 2-6; Element 2 in combination with one or more of Elements 3-6; Element 3 in combination with one or more of Elements 4-6; and two or more of Elements 4-6 in combination. A second nonlimiting example embodiment is the PP-polyamide produced according to the method of the first nonlimiting example embodiment (optionally including one or more of Elements 1-6). A third nonlimiting example embodiment is a method comprising: functionalizing metal oxide particles that are bound to a pigment particle with a silica particle having a carboxylic acid (terminal, pendent, or include both terminal and pendent of such group) surface treatment to produce a surface treated pigment having a pendent carboxylic acid; converting the pendent carboxylic acid to a pendent acid chloride; and reacting the pendent acid chloride with a polyamide to yield a pigment-pendent polyamide (PP-polyamide). The third nonlimiting example embodiment can further include one or more of: Element 7: wherein the metal oxide particles comprise one or more selected from the group consisting of: titanium dioxide, a titanium suboxide, a titanium oxynitride, Al2O3, Fe2O3, Fe3O4, SnO2, Cr2O3, ZnO, CuO, NiO, zirconium oxide, and an iron titanium oxide; Element 8: wherein the pigment particle comprises one or more selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, and holographic pigments; Element 9: wherein the silica particle having the carboxylic acid surface treatment comprises one or more selected from the group consisting of: 3-aminopropyl-(3-oxobutanoic) acid functionalized silica, 3-propylsulphonic acid-functionalized silica gel, propylcarboxylic acid functionalized silica, triaminetetraacetic acid-functionalized silica gel, propionyl chloride-functionalized silica gel, 3-carboxypropyl functionalized silica gel, aminomethylphosphonic acid (AMPA)-functionalized silica gel, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized silica gel; Element 10: wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof; Element 11: wherein the reacting of the pendent acid chloride with the polyamide comprises melt mixing the pigment particle having a functionality with the pendent acid chloride with the polyamide for 15 minutes to about 1 hour at about 125° C. to about 250° C.; and Element 12: wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000. Examples of combinations include, but are not limited to, Element 7 in combination with one or more of Elements 8-12; Element 8 in combination with one or more of Elements 9-12; Element 9 in combination with one or more of Elements 10-12; and two or more of Elements 10-12 in combination. A fourth nonlimiting example embodiment is the PP-polyamide produced according to the method of the third nonlimiting example embodiment (optionally including one or more of Elements 7-12). A fifth nonlimiting example embodiment is a composition comprising: a polyamide having a pigment pendent from a backbone of the polyamide, wherein the pigment comprises metal oxide particles on the surface of a pigment particle. The third nonlimiting example embodiment may further include one or more of: Element 13: wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof; Element 14: wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000; and Element 15: wherein the pigment particle comprises one or more selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, and holographic pigments. A sixth nonlimiting example embodiment is an object comprising the composition of the fifth nonlimiting example embodiment (optionally including one or more of Elements 13-15), which may be produced by the first or third nonlimiting example embodiments. A seventh nonlimiting example embodiment is a method comprising: depositing particles upon a surface in a specified shape, and once deposited, wherein the particles comprise the composition of the fifth nonlimiting example embodiment (optionally including one or more of Elements 13-15); and heating at least a portion of the particles to promote consolidation thereof and form a consolidated body. Further, the particles of the seventh nonlimiting example embodiment may further comprise a thermoplastic polymer selected from the group consisting of: polyurethane, polyethylene, polypropylene, polyacetal, polycarbonate, polybutylene terephthalate, polyethylene terephthalate, polyethylene naphthalate, polytrimethylene terephthalate, polyhexamethylene terephthalate, polystyrene, polyvinyl chloride, polytetrafluoroethene, polyester, polyether, polyether sulfone, polyetherether ketone, polyacrylate, polymethacrylate, polyimide, acrylonitrile butadiene styrene, polyphenylene sulfide, vinyl polymer, polyarylene ether, polyarylene sulfide, polysulfone, polyether ketone, polyamide-imide, polyetherimide, polyetherester, copolymers comprising a polyether block and a polyamide block, grafted or ungrafted thermoplastic polyolefin, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, functionalized or nonfunctionalized (meth)acrylic acid polymer, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, ethylene/vinyl monomer/carbonyl terpolymer, ethylene/alkyl (meth)acrylate/carbonyl terpolymer, methylmethacrylate-butadiene-styrene type core-shell polymer, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) block terpolymer, chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride, phenolic resin, poly(ethylene/vinyl acetate), polybutadiene, polyisoprene, styrenic block copolymer, polyacrylonitrile, silicone, and any combination thereof. A eighth nonlimiting example embodiment is a method comprising: mixing a mixture comprising a polyamide having a pigment pendent from a backbone of the polyamide (PP-polyamide), a carrier fluid that is immiscible with the PP-polyamide, and optionally an emulsion stabilizer at a temperature greater than a melting point or softening temperature of the PP-polyamide and at a shear rate sufficiently high to disperse the PP-polyamide in the carrier fluid; and cooling the mixture to below the melting point or softening temperature of the PP-polyamide to form solidified particles comprising the PP-polyamide and, when present, the emulsion stabilizer associated with an outer surface of the solidified particles. A ninth nonlimiting example embodiment is a composition comprising: solidified particles comprising a polyamide having a pigment pendent from a backbone of the polyamide (PP-polyamide) and having a circularity of about 0.90 to about 1.0. The eighth and ninth example embodiments may include one or more of: Element 16: wherein the emulsion stabilizer is included in the mixture (or wherein the solidified particles further comprise an emulsion stabilizer), and wherein the emulsion stabilizer associated with an outer surface of the solidified particles; Element 17: Element 16 and wherein at least some of the solidified particles have a void comprising the emulsion stabilizer at a void/polymer interface; Element 18: Element 17 and wherein the void contains the carrier fluid; Element 19: Element 17 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface; Element 20: Element 16 and wherein the solidified particles further comprises elongated structures on the surface of the solidified particles, wherein the elongated structures comprises the PP-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures; Element 21: Element 16 and wherein the emulsion stabilizer forms a coating that covers less than 5% of the surface of the solidified particles; Element 22: Element 16 and wherein the emulsion stabilizer forms a coating that covers at least 5% of the surface of the solidified particles; Element 23: Element 16 and wherein the emulsion stabilizer forms a coating that covers at least 25% of the surface of the solidified particles; Element 24: Element 16 and wherein the emulsion stabilizer forms a coating that covers at least 50% of the surface of the solidified particles; Element 25: Element 16 and wherein the emulsion stabilizer is present in the mixture (or the solidified particles) at 0.05 wt % to 5 wt % by weight of the PP-polyamide; Element 26: Element 16 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles have an average diameter of 1 nm to 500 nm; Element 27: Element 16 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles comprise oxide nanoparticles; Element 28: Element 16 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles comprise carbon black; Element 29: Element 16 and wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles comprise polymer nanoparticles; Element 30: wherein the mixture further comprises a thermoplastic polymer that is not the PP-polyamide (or wherein the solidified particles further comprise a thermoplastic polymer that is not the PP-polyamide); Element 31: wherein the mixture further comprises the polyamide of the PP-polyamide but without a pigment pendent therefrom (or wherein the solidified particles further comprise the polyamide of the PP-polyamide but without a pigment pendent therefrom); Element 32: wherein the pigment is selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, holographic pigments, and any combination thereof; Element 33: wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof; Element 34: wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000; Element 35: wherein the solidified particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90; Element 36: wherein the solidified particles have a diameter span of about 0.2 to about 10; Element 37: wherein the solidified particles have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90; Element 38: Element 37 and wherein the solidified particles have a diameter span of about 1.0 to about 2.5; Element 39: wherein the solidified particles have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90; Element 40: Element 39 and wherein the solidified particles have a diameter span of about 0.6 to about 1.5; Element 41: wherein the solidified particles have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90; Element 42: Element 41 and wherein the solidified particles have a diameter span of about 0.2 to about 1.2; Element 43: wherein the solidified particles have a circularity of about 0.90 to about 1.0; and Element 44: wherein the solidified particles have a Hausner ratio of about 1.0 to about 1.5. Examples of combinations include, but are not limited to, Element 16 in combination with one or more of Elements 17-19; Element 16 in combination with one of Elements 20-24 optionally in further combination with one or more of Elements 17-19; Element 16 (optionally in combination with one of Elements 20-24 and/or optionally in combination with one or more of Elements 17-19) in combination with one or more of Elements 25-29; Element 16 (optionally in combination with one or more of Elements 17-29) in combination with one or more of Elements 30-44; two or more of Elements 30-34 in combination; one or more of Elements 30-34 in combination with one or more of Elements 35-44; and Element 43 and/or 44 in combination with one or more of Elements 35-42. Further, the eighth nonlimiting example embodiment may further include one or more of: Element 45: wherein the PP-polyamide is present in the mixture at 5 wt % to 60 wt % of the mixture; Element 46: wherein the carrier fluid is selected from the group consisting of: silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and any combination thereof; Element 47: wherein the silicone oil is selected from the group consisting of: polydimethylsiloxane, methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and any combination thereof; Element 48: wherein the carrier fluid has a viscosity at 25° C. of 1000 cSt to 150,000 cSt; and Element 49: wherein the carrier fluid has a density of 0.6 g/cm3to 1.5 g/cm3. Examples of combinations include, but are not limited to, one or more of Elements 16-44 in combination with one or more of Elements 45-49; and two or more of Elements 45-49. Clauses Clause 1. A method comprising: functionalizing metal oxide particles that are bound to a pigment particle with a compound having an epoxy (terminal, pendent, or include both terminal and pendent of such group) to produce a surface treated pigment having a pendent epoxy; and reacting the pendent epoxy with a polyamide to yield a pigment-pendent polyamide (PP-polyamide). Clause 2. The method of Clause 1, wherein the compound having the epoxide is selected from the group consisting of: (3-glycidoxypropyl)trimethoxysilane, (3-glycidoxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane and 1,3-bis(3-dlycidyloxypropyl)tetramethylsiloxane, 2-(3,4 epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropyl methyldimethoxysilane, 3-glycidoxypropyl methyldiethoxysilane, 5,6-epoxyhexyltriethoxysilane, and any combination thereof. Clause 3. The method of Clause 1, wherein the metal oxide particles comprise one or more selected from the group consisting of: titanium dioxide, a titanium suboxide, a titanium oxynitride, Al2O3, Fe2O3, Fe3O4, SnO2, Cr2O3, ZnO, CuO, NiO, zirconium oxide, and an iron titanium oxide. Clause 4. The method of Clause 1, wherein the pigment particle comprises one or more selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, and holographic pigments. Clause 5. The method of Clause 1, wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 6. The method of Clause 1, wherein the reacting of the pendent epoxy with a polyamide is at about 70° C. to about 200° C. Clause 7. The method of Clause 1, wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000. Clause 8. A method comprising: functionalizing metal oxide particles that are bound to a pigment particle with a silica particle having a carboxylic acid (terminal, pendent, or include both terminal and pendent of such group) surface treatment to produce a surface treated pigment having a pendent carboxylic acid; converting the pendent carboxylic acid to a pendent acid chloride; and reacting the pendent acid chloride with a polyamide to yield a pigment-pendent polyamide (PP-polyamide). Clause 9. The method of Clause 8, wherein the metal oxide particles comprise one or more selected from the group consisting of: titanium dioxide, a titanium suboxide, a titanium oxynitride, Al2O3, Fe2O3, Fe3O4, SnO2, Cr2O3, ZnO, CuO, NiO, zirconium oxide, and an iron titanium oxide. Clause 10. The method of Clause 8, wherein the pigment particle comprises one or more selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, and holographic pigments. Clause 11. The method of Clause 8, wherein the silica particle having the carboxylic acid surface treatment comprises one or more selected from the group consisting of: 3-aminopropyl-(3-oxobutanoic) acid functionalized silica, 3-propylsulphonic acid-functionalized silica gel, propylcarboxylic acid functionalized silica, triaminetetraacetic acid-functionalized silica gel, propionyl chloride-functionalized silica gel, 3-carboxypropyl functionalized silica gel, aminomethylphosphonic acid (AMPA)-functionalized silica gel, and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-functionalized silica gel. Clause 12. The method of Clause 8, wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 13. The method of Clause 8, wherein the reacting of the pendent acid chloride with the polyamide comprises melt mixing the pigment particle having a functionality with the pendent acid chloride with the polyamide for 15 minutes to about 1 hour at about 125° C. to about 250° C. Clause 14. The method of Clause 8, wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000. Clause 15. A composition comprising: a polyamide having a pigment pendent from a backbone of the polyamide, wherein the pigment comprises metal oxide particles on the surface of a pigment particle. Clause 16. The composition of Clause 15, wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 17. The composition of Clause 15, wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000. Clause 18. The composition of Clause 15, wherein the pigment particle comprises one or more selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, and holographic pigments. Clause 19. An article comprising: the composition of Clause 15. Clause 20. A method comprising: depositing particles upon a surface in a specified shape, and once deposited, wherein the particles comprise the polyamide of one of claims15-18; and heating at least a portion of the particles to promote consolidation thereof and form a consolidated body. Clause 21. The method of Clause 20, wherein the particles further comprise a thermoplastic polymer selected from the group consisting of: polyurethane, polyethylene, polypropylene, polyacetal, polycarbonate, polybutylene terephthalate, polyethylene terephthalate, polyethylene naphthalate, polytrimethylene terephthalate, polyhexamethylene terephthalate, polystyrene, polyvinyl chloride, polytetrafluoroethene, polyester, polyether, polyether sulfone, polyetherether ketone, polyacrylate, polymethacrylate, polyimide, acrylonitrile butadiene styrene, polyphenylene sulfide, vinyl polymer, polyarylene ether, polyarylene sulfide, polysulfone, polyether ketone, polyamide-imide, polyetherimide, polyetherester, copolymers comprising a polyether block and a polyamide block, grafted or ungrafted thermoplastic polyolefin, functionalized or nonfunctionalized ethylene/vinyl monomer polymer, functionalized or nonfunctionalized ethylene/alkyl (meth)acrylate, functionalized or nonfunctionalized (meth)acrylic acid polymer, functionalized or nonfunctionalized ethylene/vinyl monomer/alkyl (meth)acrylate terpolymer, ethylene/vinyl monomer/carbonyl terpolymer, ethylene/alkyl (meth)acrylate/carbonyl terpolymer, methylmethacrylate-butadiene-styrene type core-shell polymer, polystyrene-block-polybutadiene-block-poly(methyl methacrylate) block terpolymer, chlorinated or chlorosulphonated polyethylene, polyvinylidene fluoride, phenolic resin, poly(ethylene/vinyl acetate), polybutadiene, polyisoprene, styrenic block copolymer, polyacrylonitrile, silicone, and any combination thereof. Clause 22. A method comprising: mixing a mixture comprising a polyamide having a pigment pendent from a backbone of the polyamide (PP-polyamide), a carrier fluid that is immiscible with the PP-polyamide, and optionally an emulsion stabilizer at a temperature greater than a melting point or softening temperature of the PP-polyamide and at a shear rate sufficiently high to disperse the PP-polyamide in the carrier fluid; and cooling the mixture to below the melting point or softening temperature of the PP-polyamide to form solidified particles comprising the PP-polyamide and, when present, the emulsion stabilizer associated with an outer surface of the solidified particles. Clause 23. The method of Clause 22, wherein the emulsion stabilizer is included in the mixture, and wherein the emulsion stabilizer associated with an outer surface of the solidified particles. Clause 24. The method of Clause 23, wherein emulsion stabilizer comprises nanoparticles, and wherein the nanoparticles are embedded in the outer surface of the solidified particles. Clause 25. The method of Clause 22, wherein the mixture further comprises a thermoplastic polymer that is not the PP-polyamide. Clause 26. The method of Clause 22, wherein the mixture further comprises the polyamide of the PP-polyamide but without a pigment pendent therefrom. Clause 27. The method of Clause 22, wherein the pigment is selected from the group consisting of: synthetic mica, natural mica, talc, sericite, kaolin, glass, SiO2flakes, Al2O3flakes, glass flakes, acicular pigments, CaSO4, iron oxides, chromium oxides, carbon black, metal effect pigments, optically variable pigments, liquid crystal polymer pigments, holographic pigments, and any combination thereof. Clause 28. The method of Clause 22, wherein the polyamide is selected from the group consisting of: polycaproamide, poly(hexamethylene succinamide), polyhexamethylene adipamide, polypentamethylene adipamide, polyhexamethylene sebacamide, polyundecaamide, polydodecaamide, polyhexamethylene terephthalamide, nylon 10,10, nylon 10,12, nylon 10,14, nylon 10,18, nylon 6,18, nylon 6,12, nylon 6,14, nylon 12,12, a semi-aromatic polyamide, an aromatic polyamide, any copolymer thereof, and any combination thereof. Clause 29. The method of Clause 22, wherein a weight ratio of the pigment to the polyamide is about 1:10 to about 1:1000. Clause 30. The method of Clause 22, wherein at least some of the solidified particles have a void comprising the emulsion stabilizer at a void/polymer interface. Clause 31. The method of Clause 30, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface. Clause 32. The method of Clause 30, wherein the void contains the carrier fluid. Clause 33. The method of Clause 22, wherein the solidified particles further comprises elongated structures on the surface of the solidified particles, wherein the elongated structures comprises the PP-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures. Clause 34. The method of Clause 22, wherein the emulsion stabilizer is included and forms a coating that covers less than 5% of the surface of the solidified particles. Clause 35. The method of Clause 22, wherein the emulsion stabilizer is included and forms a coating that covers at least 5% of the surface of the solidified particles. Clause 36. The method of Clause 22, wherein the emulsion stabilizer is included and forms a coating that covers at least 25% of the surface of the solidified particles. Clause 37. The method of Clause 22, wherein the emulsion stabilizer is included and forms a coating that covers at least 50% of the surface of the solidified particles. Clause 38. The method of Clause 22, wherein the PP-polyamide is present in the mixture at 5 wt % to 60 wt % of the mixture. Clause 39. The method of Clause 22, wherein the emulsion stabilizer is included and is present in the mixture at 0.05 wt % to 5 wt % by weight of the PP-polyamide. Clause 40. The method of Clause 22, wherein the emulsion stabilizer is included and comprises nanoparticles and the nanoparticles have an average diameter of 1 nm to 500 nm. Clause 41. The method of Clause 22, wherein the carrier fluid is selected from the group consisting of: silicone oil, fluorinated silicone oils, perfluorinated silicone oils, polyethylene glycols, paraffins, liquid petroleum jelly, vison oils, turtle oils, soya bean oils, perhydrosqualene, sweet almond oils, calophyllum oils, palm oils, parleam oils, grapeseed oils, sesame oils, maize oils, rapeseed oils, sunflower oils, cottonseed oils, apricot oils, castor oils, avocado oils, jojoba oils, olive oils, cereal germ oils, esters of lanolic acid, esters of oleic acid, esters of lauric acid, esters of stearic acid, fatty esters, higher fatty acids, fatty alcohols, polysiloxanes modified with fatty acids, polysiloxanes modified with fatty alcohols, polysiloxanes modified with polyoxy alkylenes, and any combination thereof. Clause 42. The method of Clause 41, wherein the silicone oil is selected from the group consisting of: polydimethylsiloxane, methylphenylpolysiloxane, an alkyl modified polydimethylsiloxane, an alkyl modified methylphenylpolysiloxane, an amino modified polydimethylsiloxane, an amino modified methylphenylpolysiloxane, a fluorine modified polydimethylsiloxane, a fluorine modified methylphenylpolysiloxane, a polyether modified polydimethylsiloxane, a polyether modified methylphenylpolysiloxane, and any combination thereof. Clause 43. The method of Clause 22, wherein the carrier fluid has a viscosity at 25° C. of 1000 cSt to 150,000 cSt. Clause 44. The method of Clause 22, wherein the carrier fluid has a density of 0.6 g/cm3to 1.5 g/cm3. Clause 45. The method of Clause 22, wherein mixing occurs in an extruder. Clause 46. The method of Clause 22, wherein mixing occurs in a stirred reactor. Clause 47. The method of Clause 22, wherein the mixture further comprises a surfactant. Clause 448. The method of Clause 22, wherein the solidified particles have a D10 of about 0.1 μm to about 125 μm, a D50 of about 0.5 μm to about 200 μm, and a D90 of about 3 μm to about 300 μm, wherein D10<D50<D90. Clause 49. The method of Clause 22, wherein the solidified particles have a diameter span of about 0.2 to about 10. Clause 50. The method of Clause 22, wherein the solidified particles have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Clause 51. The method of Clause 50, wherein the solidified particles have a diameter span of about 1.0 to about 2.5. Clause 52. The method of Clause 22, wherein the solidified particles have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Clause 53. The method of Clause 52, wherein the solidified particles have a diameter span of about 0.6 to about 1.5. Clause 54. The method of Clause 22, wherein the solidified particles have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Clause 55. The method of Clause 54, wherein the solidified particles have a diameter span of about 0.2 to about 1.2. Clause 56. The method of Clause 22, wherein the solidified particles have a circularity of about 0.90 to about 1.0. Clause 57. The method of Clause 22, wherein the solidified particles have a Hausner ratio of about 1.0 to about 1.5. Clause 58. The method of Clause 22, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles comprise oxide nanoparticles. Clause 59. The method of Clause 22, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles comprise carbon black. Clause 60. The method of Clause 22, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles comprise polymer nanoparticles. Clause 61. A composition comprising: particles comprising a polyamide having a pigment pendent from a backbone of the polyamide (PP-polyamide) and having a circularity of about 0.90 to about 1.0. Clause 62. The composition of Clause 61, wherein the particles further comprise a thermoplastic polymer that is not the PP-polyamide. Clause 63. The composition of Clause 61, wherein the particles further comprise the polyamide of the PP-polyamide but without a pigment pendent therefrom. Clause 64. The composition of Clause 61, wherein the particles further comprise an emulsion stabilizer associated with an outer surface of the particles. Clause 65. The composition of Clause 61, wherein at least some of the particles have a void comprising the emulsion stabilizer at a void/polymer interface. Clause 66. The composition of Clause 65, wherein the emulsion stabilizer comprises nanoparticles and the nanoparticles are embedded in the void/polymer interface. Clause 67. The composition of Clause 65, wherein the void contains the carrier fluid. Clause 68. The composition of Clause 61, wherein the particles further comprises elongated structures on the surface of the particles, wherein the elongated structures comprises the PP-polyamide with the emulsion stabilizer associated with an outer surface of the elongated structures. Clause 69. The composition of Clause 61, wherein the emulsion stabilizer forms a coating that covers less than 5% of the surface of the solidified particles. Clause 70. The composition of Clause 61, wherein the emulsion stabilizer forms a coating that covers at least 5% of the surface of the solidified particles. Clause 71. The composition of Clause 61, wherein the emulsion stabilizer forms a coating that covers at least 25% of the surface of the solidified particles. Clause 72. The composition of Clause 61, wherein the emulsion stabilizer forms a coating that covers at least 50% of the surface of the solidified particles. Clause 73. The composition of Clause 61, wherein the emulsion stabilizer comprises nanoparticles having an average diameter of 1 nm to 500 nm. Clause 74. The composition of Clause 61, wherein the solidified particles have a D10 of about 0.5 μm to about 125 μm, a D50 of about 1 μm to about 200 μm, and a D90 of about 70 μm to about 300 μm, wherein D10<D50<D90. Clause 75. The composition of Clause 61, wherein the solidified particles have a diameter span of about 0.2 to about 10. Clause 76. The composition of Clause 61, wherein the solidified particles have a D10 of about 5 μm to about 30 μm, a D50 of about 30 μm to about 70 μm, and a D90 of about 70 μm to about 120 μm, wherein D10<D50<D90. Clause 77. The composition of Clause 76, wherein the solidified particles have a diameter span of about 1.0 to about 2.5. Clause 78. The composition of Clause 61, wherein the solidified particles have a D10 of about 25 μm to about 60 μm, a D50 of about 60 μm to about 110 μm, and a D90 of about 110 μm to about 175 μm, wherein D10<D50<D90. Clause 79. The composition of Clause 78, wherein the solidified particles have a diameter span of about 0.6 to about 1.5. Clause 80. The composition of Clause 61, wherein the solidified particles have a D10 of about 75 μm to about 125 μm, a D50 of about 100 μm to about 200 μm, and a D90 of about 125 μm to about 300 μm, wherein D10<D50<D90. Clause 81. The composition of Clause 80, wherein the solidified particles have a diameter span of about 0.2 to about 1.2. Clause 82. The composition of Clause 61, wherein the solidified particles have a Hausner ratio of about 1.0 to about 1.5. Clause 83. A method comprising: depositing the composition of Clause 61 optionally in combination with other thermoplastic polymer particles upon a surface in a specified shape; and once deposited, heating at least a portion of the particles to promote consolidation thereof and form a consolidated body. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the present specification and associated claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the incarnations of the present inventions. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claim, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. One or more illustrative incarnations incorporating one or more invention elements are presented herein. Not all features of a physical implementation are described or shown in this application for the sake of clarity. It is understood that in the development of a physical embodiment incorporating one or more elements of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints, which vary by implementation and from time to time. While a developer's efforts might be time-consuming, such efforts would be, nevertheless, a routine undertaking for those of ordinary skill in the art and having benefit of this disclosure. While compositions and methods are described herein in terms of “comprising” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. To facilitate a better understanding of the embodiments of the present invention, the following examples of preferred or representative embodiments are given. In no way should the following examples be read to limit, or to define, the scope of the invention. EXAMPLES Prophetic Example 1: IRIODIN® 100 Silver Pearl Pigment Reacted with (3-glycidyloxypropyl)trimethoxysilane and Crosslinked by Polyamide Resin in Solution Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of IRIODIN® 100 Silver Pearl pigment (available from E. Merck KGaA, Darmstadt) suspended in 900 mL of deionized water and heated to 40° C. with vigorous stirring. The suspension is adjusted to pH of 3.3 using 2.5% hydrochloric acid and the temperature is raised to 75° C. Subsequently, 3.0 g of (3-glycidyloxypropyl)trimethoxysilane (available from Millipore Sigma) is added over the course of 10 minutes and the pH is kept constant using the 2.5% hydrochloric acid solution. At the end of the addition, stirring is continued at 75° C. for 2 hours during which the silane hydrolyzes and the resulting silanols associate with the inorganic pigment surface. Subsequently, the system is adjusted to a pH of 8.0 while maintaining the reaction temperature of 75° C. using 2.5% sodium hydroxide solution very slowly over the course of 1 hour during which time the condensation reaction occurs and the resulting siloxane bonds to the pigment surface leaving the unreacted epoxy end group free for subsequent functionalization. Stirring is continued at 75° C. for an additional 1 hour to complete the reaction and the pH falls to 7.0. The product is filtered off using vacuum filtration, washed with deionized water and dried at 140° C. for approximately 16 hours. Subsequently, 100 g of polyamide resin, such as nylon 6,6, is dissolved in N-methyl-2-pyrrolidone (NMP) with vigorous agitation. To this mixture is added 10 g of the epoxide functionalized IRIODIN® 100 Silver Pearl pigment from above and the reaction mixture with continuous agitation is increased to 150° C. for 2 hours to facilitate the curing reaction of the amino functional groups of the polyamide resin with the pendent glycidyloxypropyl (epoxide) group coating the surface of IRIODIN® 100 Silver Pearl pigment. After the polyamide resin has cured and coated the suspended IRIODIN® 100 Silver Pearl pigment the solvent is removed by filtering the particles using vacuum filtration and the material is thoroughly dried in a vacuum oven for 24 hours. Then a portion of this mixture is mixed with non-pigment-pendant polyamide in the Haake reaction with PDMS to form the particles. Using 2.5 g of IRIODIN® 100 Silver Pearl pigment-pendent crosslinked by polyamide resin onto the pigment surface and 27.5 g of nylon 6,6 is melt mixed with 150 g of polydimethylsiloxane (PDMS) of 60,000 specific viscosity by hot melt emulsification in a Haake mixer fitted with a 300 ml mixing vessel. The mixer is heated to 230° C. and mixed at 200 rpm for 20 minutes. Then, the mixture is discharged from the Haake onto a cold surface to provide rapid quench cooling. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamide particles from the carrier fluid. The PP-polyamide particles are washed three times with 1000 mL of ethyl acetate. The PP-polyamide particles are then allowed to air dry overnight in an aluminum pan in a fume hood. Optionally, the dried PP-polyamide particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 50 μm with a span of about 0.85. Prophetic Example 2: BLONDIEE® Metallic Super Gold Pigment Reacted with (3-glycidyloxypropyl)trimethoxysilane and In-Situ Crosslinking with Polyamide Resin Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of BLONDIEE® Metallic Super Gold pigment, product code N-2002S (available from Creation of Quality Value Company Ltd.) suspended in 900 mL of deionized water and heated to 40° C. with vigorous stirring. The suspension is adjusted to pH of 3.3 using 2.5% hydrochloric acid and the temperature is raised to 75° C. Subsequently, 3.0 g of (3-glycidyloxypropyl)trimethoxysilane (available from Millipore Sigma) is added over the course of 10 minutes and the pH is kept constant using the 2.5% hydrochloric acid solution. At the end of the addition, stirring is continued at 75° C. for 2 hours during which the silane hydrolyzes and the resulting silanols associate with the inorganic pigment surface. Subsequently, the system is adjusted to a pH of 8.0 while maintaining the reaction temperature of 75° C. using 2.5% sodium hydroxide solution very slowly over the course of 1 hour during which time the condensation reaction occurs and the resulting siloxane bonds to the pigment surface leaving the unreacted epoxy end group free for subsequent functionalization. Stirring is continued at 75° C. for an additional 1 hour to complete the reaction and the pH falls to 7.0. The product is filtered off using vacuum filtration, washed with deionized water and dried at 140° C. for approximately 16 hours. Subsequently, 50 g of polyamide resin such as nylon 6,6 is melt mixed with 10 g of the above epoxide surface functionalized mica pigment Blondiee® Metallic Super Gold melt mixed in the Haake mixer at 150° C. to 200° C. for 20 to 30 minutes to facilitate the crosslinking reaction of the epoxide with the amino group of the polyamide resin. The resulting pigment-pendent polyamide resin concentrate is discharged from the Haake mixer, cooled and grounded into a fine powder for subsequent incorporation into pigmented polyamide micron particles. Using 1.5 g of BLONDIEE® Metallic Super Gold pigment-pendent crosslinked polyamide resin onto the pigment surface and 28.5 g of nylon 6,6 is melt mixed with 150 g of polydimethylsiloxane (PDMS) of 30,000 specific viscosity by hot melt emulsification in a Haake mixer fitted with a 300 ml mixing vessel. The mixer is heated to 230° C. and mixed at 200 rpm for 20 minutes. Then, the mixture is discharged from the Haake onto a cold surface to provide rapid quench cooling. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 1000 mL of ethyl acetate. The particles are then allowed to air dry overnight in an aluminum pan in a fume hood. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 65 μm with a span of about 1.20. Prophetic Example 3: REFLEX® 100 Sparkle Violet Pigment Reacted with (3-glycidyloxypropyl)trimethoxysilane and Crosslinked by Polyamide Resin in Solution Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of REFLEX® 100 Sparkle Violet R-706E pigment (available from Creation of Quality Value Company Ltd.) suspended in 900 mL of deionized water and heated to 40° C. with vigorous stirring. The suspension is adjusted to pH of 3.3 using 2.5% hydrochloric acid and the temperature is raised to 75° C. Subsequently, 3.0 g of (3-glycidyloxypropyl)trimethoxysilane (available from Millipore Sigma) is added over the course of 10 minutes and the pH is kept constant using the 2.5% hydrochloric acid solution. At the end of the addition, stirring is continued at 75° C. for 2 hours during which the silane hydrolyzes and the resulting silanols associate with the inorganic pigment surface. Subsequently, the system is adjusted to a pH of 8.0 while maintaining the reaction temperature of 75° C. using 2.5% sodium hydroxide solution very slowly over the course of 1 hour during which time the condensation reaction occurs and the resulting siloxane bonds to the pigment surface leaving the unreacted epoxy end group free for subsequent functionalization. Stirring is continued at 75° C. for an additional 1 hour to complete the reaction and the pH falls to 7.0. The product is filtered off using vacuum filtration, washed with deionized water and dried at 140° C. for approximately 16 hours. Subsequently, 100 g of polyamide resin such as nylon 6,6 is dissolved in N-methyl-2-pyrrolidone (NMP) with vigorous agitation. To this mixture is added 10 g of the epoxide functionalized REFLEX® 100 Sparkle Violet R-706E pigment from above and the reaction mixture with continuous agitation is increased to 150° C. for 2 hours to facilitate the curing reaction of the amino functional groups of the polyamide resin with the pendent glycidyloxypropyl (epoxide) group coating the surface of REFLEX® 100 Sparkle Violet R-706E pigment. After the polyamide resin has cured and coated the suspended REFLEX® 100 Sparkle Violet R-706E pigment the solvent is removed by filtering the particles using vacuum filtration and the material is thoroughly dried in a vacuum oven for 24 hours. Then a portion of this mixture is mixed with non-pigment-pendant polyamide in the Haake reaction with PDMS to form the particles. Using 50 g of REFLEX® 100 Sparkle Violet R-706E pigment-pendent crosslinked by polyamide resin onto the pigment surface and 550 g of nylon 6,6 is melt mixed with 2000 g of polydimethylsiloxane (PDMS) of 10,000 specific viscosity by hot melt emulsification in a 25 mm twin-screw extruder (Werner & Pfleiderer ZSK-25). The polymer pellets are added to the extruder first, brought to the temperature of 230° C. and rpm of 900, and then preheated carrier fluid having AEROSIL® R812S silica nanoparticles (1.1-wt. % relative to PP-polyamide) dispersed therein is added to the molten polymer in the extruder. Then the mixture is discharged into a container and allowed to cool to room temperature over several hours. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 2000 mL of ethyl acetate. The particles are then allowed to dry overnight in vacuum oven at ambient temperature. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 75 μm with a span of about 1.30. Prophetic Example 4: Reflex® Glitter Blue R-781E Pigment Reacted with (3-glycidyloxypropyl)trimethoxysilane and In-Situ Crosslinking with Polyamide Resin Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of Reflex® Glitter Blue pigment, product code R-781E (available from Creation of Quality Value Company Ltd.) suspended in 900 mL of deionized water and heated to 40° C. with vigorous stirring. The suspension is adjusted to pH of 3.3 using 2.5% hydrochloric acid and the temperature is raised to 75° C. Subsequently, 3.0 g of (3-glycidyloxypropyl)trimethoxysilane (available from Millipore Sigma) is added over the course of 10 minutes and the pH is kept constant using the 2.5% hydrochloric acid solution. At the end of the addition, stirring is continued at 75° C. for 2 hours during which the silane hydrolyzes and the resulting silanols associate with the inorganic pigment surface. Subsequently, the system is adjusted to a pH of 8.0 while maintaining the reaction temperature of 75° C. using 2.5% sodium hydroxide solution very slowly over the course of 1 hour during which time the condensation reaction occurs and the resulting siloxane bonds to the pigment surface leaving the unreacted epoxy end group free for subsequent functionalization. Stirring is continued at 75° C. for an additional 1 hour to complete the reaction and the pH falls to 7.0. The product is filtered off using vacuum filtration, washed with deionized water and dried at 140° C. for approximately 16 hours. Subsequently, 50 g of polyamide resin such as nylon 6,6 is melt mixed with 10 g of the above epoxide surface functionalized mica pigment Reflex® Glitter Blue R-871E melt mixed in the Haake mixer at 150° C. to 200° C. for 20 to 30 minutes to facilitate the crosslinking reaction of the epoxide with the amino group of the polyamide resin. The resulting pigment-pendent polyamide resin concentrate is discharged from the Haake mixer, cooled and grounded into a fine powder for subsequent incorporation into pigmented polyamide micron particles. Using 30 g of REFLEX® Glitter Blue R-871E pigment-pendent crosslinked polyamide resin onto the pigment surface and 570 g of nylon 6,6 is melt mixed with 2000 g of polydimethylsiloxane (PDMS) of 10,000 specific viscosity by hot melt emulsification in a 25 mm twin-screw extruder (Werner & Pfleiderer ZSK-25). The polymer pellets are added to the extruder first, brought to the temperature of 230° C. and rpm of 900, and then preheated carrier fluid having AEROSIL® R812S silica nanoparticles (1.1 wt % relative to PP-polyamide) dispersed therein is added to the molten polymer in the extruder. Then the mixture is discharged into a container and allowed to cool to room temperature over several hours. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 2000 mL of ethyl acetate. The particles are then allowed to dry overnight in vacuum oven at ambient temperature. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 65 μm with a span of about 1.10. Prophetic Example 5: IRIODIN® 100 Silver Pearl Pigment Reacted with 3-Aminopropyl(3-oxobutaonoic)acid Functionalized Silica Nanoparticles and In-situ Crosslinked by Polyamide Resin Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of IRIODIN® 100 Silver Pearl pigment (available from E. Merck KGaA, Darmstadt, Germany) suspended in 900 mL of deionized water and 100 mL of 3-aminopropyl(3-oxobutaonoic)acid functionalized silica nanoparticles as a colloidal dispersion at 2.5 wt % loading in dimethylformamide (DMF) (available from Millipore Sigma). The mixture was heated to 40° C. with vigorous stirring for 4 hours to facilitate the adsorption of the silica nanoparticles onto the surface of the pigment. The suspension is cooled to room temperature and filtered to remove the water and DMF solvent. The functionalized pigment particles with free carboxylic acid functional groups are dried in a vacuum oven at 40° C. for 24 hours to produce a functionalized pigment powder. A clear viscous stock solution (1.5 molar) of thionyl chloride (5.46 mL, 0.075 mol) and benzotriazole (8.93 g, 0.075 mol) in 50 mL of dry methylene chloride was prepared at room temperature with mixing. A portion of this solution (1.25 mmol) is added slowly to convert the pendent carboxylic acid functional groups on the surface of the mica pigment to acid chloride to enable curing with the amino polyamide group in the polyamide resin. The dried mica pigment IRIODIN® 100 Silver Pearl functionalized with surface carboxylic acid (approximately 110 g) is suspended in 500 mL of dry methylene chloride with constant agitation. To this mixture is added 20 mL of the thionyl chloride-benzotriazole mixture slowly over 30 minutes at room temperature. As the reaction proceeds benzotriazole hydrochloride salt starts to precipitate out of the solution indicating the conversion of the carboxylic acid is converted to the acid chloride. The reaction mixture is mixed for an additional 30 minutes and then the mixture is filtered to remove the solvent and thoroughly washed with water then dried in a vacuum oven for 24 hours. Subsequently, 50 g of polyamide resin such as nylon 6,6 is melt mixed with 10 g of the above acid chloride surface functionalized mica pigment IRIODIN® 100 Silver Pearl melt mixed in the Haake mixer at 150° C. to 200° C. for 20 to 30 minutes to facilitate the crosslinking reaction of the acid chloride with the amino group of the polyamide resin. The resulting pigment-pendent polyamide resin concentrate is discharged from the Haake mixer, cooled and grounded into a fine powder for subsequent incorporation into pigmented polyamide micron particles. Using 1.5 g of IRIODIN® 100 Silver Pearl pigment-pendent crosslinked polyamide resin onto the pigment surface and 28.5 g of nylon 6,6 is melt mixed with 150 g of polydimethylsiloxane (PDMS) of 30,000 specific viscosity containing AEROSIL® R812S silica nanoparticles (0.75 wt % relative to PP-polyamide) dispersed therein by hot melt emulsification in a Haake mixer fitted with a 300 ml mixing vessel. The mixer is heated to 230° C. and mixed at 200 rpm for 15 minutes. Then, the mixture is discharged from the Haake onto a cold surface to provide rapid quench cooling. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 1000 mL of ethyl acetate. The particles are then allowed to air dry overnight in an aluminum pan in a fume hood. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 55 μm with a span of about 1.10. Prophetic Example 6: BLONDIEE® Metallic Super Gold Pigment Reacted with 3-Aminopropyl(3-oxobutaonoic)acid Functionalized Silica Nanoparticles and In-Situ Crosslinked by Polyamide Resin Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of BLONDIEE® Metallic Super Gold (available from Creation of Quality Value Company Ltd.) suspended in 900 mL of deionized water and 100 mL of 3-aminopropyl(3-oxobutaonoic)acid functionalized silica nanoparticles as a colloidal dispersion at 2.5 wt % loading in DMF (available from Millipore Sigma). The mixture was heated to 40° C. with vigorous stirring for 4 hours to facilitate the adsorption of the silica nanoparticles onto the surface of the pigment. The suspension is cooled to room temperature and filtered to remove the water and DMF solvent. The functionalized pigment particles with free carboxylic acid functional groups are dried in a vacuum oven at 40° C. for 24 hours to produce a functionalized pigment powder. A clear viscous stock solution (1.5 molar) of thionyl chloride (5.46 mL, 0.075 mol) and benzotriazole (8.93 g, 0.075 mol) in 50 mL of dry methylene chloride was prepared at room temperature with mixing. A portion of this solution (1.25 mmol) is added slowly to convert the pendent carboxylic acid functional groups on the surface of the mica pigment to acid chloride to enable curing with the amino polyamide group in the polyamide resin. The dried mica pigment BLONDIEE® Metallic Super Gold functionalized with surface carboxylic acid (approximately 110 g) is suspended in 500 mL of dry methylene chloride with constant agitation. To this mixture is added 20 mL of the thionyl chloride-benzotriazole mixture slowly over 30 minutes at room temperature. As the reaction proceeds benzotriazole hydrochloride salt starts to precipitate out of the solution indicating the conversion of the carboxylic acid is converted to the acid chloride. The reaction mixture is mixed for an additional 30 minutes and then the mixture is filtered to remove the solvent and thoroughly washed with water then dried in a vacuum oven for 24 hours. Subsequently, 50 g of polyamide resin such as nylon 6,6 is melt mixed with 10 g of the above acid chloride surface functionalized mica pigment Blondiee® Metallic Super Gold melt mixed in the Haake mixer at 150° C. to 200° C. for 20 to 30 minutes to facilitate the crosslinking reaction of the acid chloride with the amino group of the polyamide resin. The resulting pigment-pendent polyamide resin concentrate is discharged from the Haake mixer, cooled and grounded into a fine powder for subsequent incorporation into pigmented polyamide micron particles. Using 1.5 g of BLONDIEE® Metallic Super Gold pigment-pendent crosslinked polyamide resin onto the pigment surface and 28.5 g of nylon 6,6 is melt mixed with 150 g of polydimethylsiloxane (PDMS) of 30,000 specific viscosity containing AEROSIL® R812S silica nanoparticles (1.00 wt % relative to PP-polyamide) dispersed therein by hot melt emulsification in a Haake mixer fitted with a 300 ml mixing vessel. The mixer is heated to 230° C. and mixed at 200 rpm for 15 minutes. Then, the mixture is discharged from the Haake onto a cold surface to provide rapid quench cooling. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 1000 mL of ethyl acetate. The particles are then allowed to air dry overnight in an aluminum pan in a fume hood. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 50 μm with a span of about 0.95. Prophetic Example 7: REFLEX® 100 Sparkle Violet Pigment Reacted with 3-Aminopropyl(3-oxobutaonoic)acid Functionalized Silica Nanoparticles and In-Situ Crosslinked by Polyamide Resin Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of REFLEX® 100 Sparkle Violet R-706E (available from Creation of Quality Value Company Ltd.) suspended in 900 mL of deionized water and 100 mL of 3-aminopropyl(3-oxobutaonoic)acid functionalized silica nanoparticles as a colloidal dispersion at 2.5 wt % loading in DMF (available from Millipore Sigma). The mixture was heated to 40° C. with vigorous stirring for 4 hours to facilitate the adsorption of the silica nanoparticles onto the surface of the pigment. The suspension is cooled to room temperature and filtered to remove the water and DMF solvent. The functionalized pigment particles with free carboxylic acid functional groups are dried in a vacuum oven at 40° C. for 24 hours to produce a functionalized pigment powder. A clear viscous stock solution (1.5 molar) of thionyl chloride (5.46 mL, 0.075 mol) and benzotriazole (8.93 g, 0.075 mol) in 50 mL of dry methylene chloride was prepared at room temperature with mixing. A portion of this solution (1.25 mmol) is added slowly to convert the pendent carboxylic acid functional groups on the surface of the mica pigment to acid chloride to enable curing with the amino polyamide group in the polyamide resin. The dried mica pigment REFLEX® 100 Sparkle Violet R-706E functionalized with surface carboxylic acid (approximately 110 g) is suspended in 500 mL of dry methylene chloride with constant agitation. To this mixture is added 20 mL of the thionyl chloride-benzotriazole mixture slowly over 30 minutes at room temperature. As the reaction proceeds benzotriazole hydrochloride salt starts to precipitate out of the solution indicating the conversion of the carboxylic acid is converted to the acid chloride. The reaction mixture is mixed for an additional 30 minutes and then the mixture is filtered to remove the solvent and thoroughly washed with water then dried in a vacuum oven for 24 hours. Subsequently, 50 g of polyamide resin such as nylon 6,6 is melt mixed with 10 g of the above acid chloride surface functionalized mica pigment Reflex® 100 Sparkle Violet R-706E melt mixed in the Haake mixer at 150° C. to 200° C. for 20 to 30 minutes to facilitate the crosslinking reaction of the acid chloride with the amino group of the polyamide resin. The resulting pigment-pendent polyamide resin concentrate is discharged from the Haake mixer, cooled and grounded into a fine powder for subsequent incorporation into pigmented polyamide micron particles. Using 30 g of REFLEX® 100 Sparkle Violet R-706E pigment-pendent crosslinked polyamide resin onto the pigment surface and 570 g of nylon 6,6 is melt mixed with 2000 g of polydimethylsiloxane (PDMS) of 20,000 specific viscosity by hot melt emulsification in a 25 mm twin-screw extruder (Werner & Pfleiderer ZSK-25). The polymer pellets are added to the extruder first, brought to the temperature of 230° C. and rpm of 1100, and then preheated carrier fluid having AEROSIL® R812S silica nanoparticles (1.1 wt % relative to PP-polyamide) dispersed therein is added to the molten polymer in the extruder Then the mixture is discharged into a container and allowed to cool to room temperature over several hours. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 2000 mL of ethyl acetate. The particles are then allowed to dry overnight in vacuum oven at ambient temperature. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 55 μm with a span of about 1.30. Prophetic Example 8: REFLEX® Glitter Blue R-781E Pigment Reacted with 3-Aminopropyl(3-oxobutaonoic)acid Functionalized Silica Nanoparticles and In-Situ Crosslinked by Polyamide Resin Into a 2 liter glass reactor equipped with an overhead mechanical stirrer and a heating mantle is added 100 g of REFLEX® Glitter Blue pigment, product code R-781E (available from Creation of Quality Value Company Ltd.) suspended in 900 mL of deionized water and 100 mL of 3-aminopropyl(3-oxobutaonoic)acid functionalized silica nanoparticles as a colloidal dispersion at 2.5 wt % loading in DMF (available from Millipore Sigma). The mixture was heated to 40° C. with vigorous stirring for 4 hours to facilitate the adsorption of the silica nanoparticles onto the surface of the pigment. The suspension is cooled to room temperature and filtered to remove the water and DMF solvent. The functionalized pigment particles with free carboxylic acid functional groups are dried in a vacuum oven at 40° C. for 24 hours to produce a functionalized pigment powder. A clear viscous stock solution (1.5 molar) of thionyl chloride (5.46 mL, 0.075 mol) and benzotriazole (8.93 g, 0.075 mol) in 50 mL of dry methylene chloride was prepared at room temperature with mixing. A portion of this solution (1.25 mmol) is added slowly to convert the pendent carboxylic acid functional groups on the surface of the mica pigment to acid chloride to enable curing with the amino polyamide group in the polyamide resin. The dried mica pigment REFLEX® Glitter Blue functionalized with surface carboxylic acid (approximately 110 g) is suspended in 500 mL of dry methylene chloride with constant agitation. To this mixture is added 20 mL of the thionyl chloride-benzotriazole mixture slowly over 30 minutes at room temperature. As the reaction proceeds benzotriazole hydrochloride salt starts to precipitate out of the solution indicating the conversion of the carboxylic acid is converted to the acid chloride. The reaction mixture is mixed for an additional 30 minutes and then the mixture is filtered to remove the solvent and thoroughly washed with water then dried in a vacuum oven for 24 hours. Subsequently, 50 g of polyamide resin such as nylon 6,6 is melt mixed with 10 g of the above acid chloride surface functionalized mica pigment Reflex® Glitter Blue melt mixed in the Haake mixer at 150° C. to 200° C. for 20 to 30 minutes to facilitate the crosslinking reaction of the acid chloride with the amino group of the polyamide resin. The resulting pigment-pendent polyamide resin concentrate is discharged from the Haake mixer, cooled and grounded into a fine powder for subsequent incorporation into pigmented polyamide micron particles. Using 1.5 g of Reflex® Glitter Blue pigment-pendent crosslinked polyamide resin onto the pigment surface and 28.5 g of Nylon 6,6 is melt mixed with 150 g of polydimethylsiloxane (PDMS) of 60,000 specific viscosity containing AEROSIL® R812S silica nanoparticles (0.75 wt % relative to PP-polyamide) dispersed therein by hot melt emulsification in a Haake mixer fitted with a 300 ml mixing vessel. The mixer is heated to 230° C. and mixed at 200 rpm for 20 minutes. Then, the mixture is discharged from the Haake onto a cold surface to provide rapid quench cooling. The resultant mixture is then filtered through a 90 mm WHATMAN® #1 paper filter (available from SigmaAldrich) to separate the PP-polyamides particles from the carrier fluid. The particles are washed three times with 1000 mL of ethyl acetate. The particles are then allowed to air dry overnight in an aluminum pan in a fume hood. Optionally, the dried particles can be screened through a 150-μm sieve. The PP-polyamide particles are then characterized for size with a Malvern MASTERSIZER™ 3000 and morphology with SEM micrographs. The D50 (μm) is predicted to be around 75 μm with a span of about 0.85. Prophetic Example 9: General Epoxide and Polyamide Reaction Conditions The epoxide group on the metallic pigment reactions with the polyamide may be performed under an atmosphere (nitrogen or argon) at temperatures of about 70° C. to about 200° C. (or about 70° C. to about 150° C., about 125° C. to about 200° C.) in the presence of an organic solvent such as tetrahydrofuan, dimethylformamide, toluene, and the like, and any combination thereof. The mixture is then stirred for about 24 hours at an elevated temperature. After cooling the mixture to room temperature, the grafted polymer is filtered and washed to remove organic impurities and unreacted starting reagents. Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular examples and configurations disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative examples disclosed above may be altered, combined, or modified and all such variations are considered within the scope and spirit of the present invention. The invention illustratively disclosed herein suitably may be practiced in the absence of any element that is not specifically disclosed herein and/or any optional element disclosed herein. While compositions and methods are described in terms of “comprising,” “containing,” or “including” various components or steps, the compositions and methods can also “consist essentially of” or “consist of” the various components and steps. All numbers and ranges disclosed above may vary by some amount. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range is specifically disclosed. In particular, every range of values (of the form, “from about a to about b,” or, equivalently, “from approximately a to b,” or, equivalently, “from approximately a-b”) disclosed herein is to be understood to set forth every number and range encompassed within the broader range of values. Also, the terms in the claims have their plain, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. Moreover, the indefinite articles “a” or “an,” as used in the claims, are defined herein to mean one or more than one of the element that it introduces. | 150,810 |
11859053 | DETAILED DESCRIPTION The invention provides aprocess for producing SiOC-bonded, linear polydimethylsiloxane-polyoxyalkylene block copolymers comprising repeating (AB) units, comprising the steps of:(a) pretreating acidified, preferably superacid-acidified, preferably trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes with a base with optional subsequent distillative purification of the α,ω-diacetoxypolydimethylsiloxanes previously pretreated with base(b) reacting the α,ω-diacetoxypolydimethylsiloxanes resulting from (a) with polyether diols in the presence of a solid, liquid or gaseous base, optionally using inert solvents. The invention further provides SiOC-bonded, linear polydimethylsiloxane-polyoxyalkylene block copolymers comprising repeating (AB) units produced by the process according to the invention and to the use thereof as surface-active additives for production of polyurethane ether foams. The process according to the invention will initially be precisely elucidated. The process according to the invention provides for pretreating acidified, preferably superacid-acidified, preferably trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes with a base with optional subsequent distillative purification of the α,ω-diacetoxypolydimethylsiloxanes previously pretreated with base. The acidified, preferably superacid-acidified, especially trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes to be employed are obtainable for example by reacting cyclic siloxanes, in particular comprising D4and/or D5, with acetic anhydride using acid, preferably superacid, in particular trifluoromethanesulfonic acid, as catalyst. The acid, preferably superacid, especially trifluoromethanesulfonic acid is preferably employed in amounts of 0.1 to 0.3 percent by mass based on the reaction matrix consisting of acetic anhydride and cyclic siloxanes. The employed acids are preferably superacids. Superacids are well known to those skilled in the art; these are generally acids which are stronger than concentrated 100% sulfuric acid (H2SO4: pKa=−3.0). Acid strength is generally quantified using the Hammett acidity function. Particular preference is given to superacids having a pKa of less than −3.0, preferably fluorinated and/or perfluorinated sulfonic acids, fluorosulfonic acid HSO3F, fluoroantimonic acid HSbF6, perfluorobutanesulfonic acid C4F9SO3H and very particularly preferably trifluoromethanesulfonic acid CF3SO3H. The reaction is preferably performed at temperatures of 140° C. to 160° C. and preferably over a period of 4 to 8 hours. Particularly suitable trifluoromethanesulfonic acid-acidified, equilibrated α,ω-diacetoxysiloxanes and the production thereof are described for example in EP18189075.7 and EP18189074.0. The pretreatment of the acidified, preferably superacid-acidified, preferably trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes may in principle be carried out with any base. Preferred simple bases to be employed according to the invention are for example alkali metal and/or alkaline earth metal carbonates and/or hydrogencarbonates and/or gaseous ammonia and/or amines. Taking account of the known tendency to condensation of acetoxysiloxanes, very particular preference is given to those bases which on account of their chemical composition do not introduce any water into the reaction system. Thus anhydrous carbonates are preferred over hydrogencarbonates and bases free from water of hydration are preferred over bases containing water of hydration. However, the use of gaseous ammonia as the base is very particularly preferred according to the invention. This corresponds to a very particularly preferred embodiment. In a particularly preferred embodiment the pretreatment of the acidified end-equilibrated α,ω-diacetoxypolydimethylsiloxane in step (a) is performed with base, in particular ammonia, in the temperature range from 0° C. to 50° C., preferably between 15° C. to 35° C. The minimum molar amount of the employed base, in particular ammonia, is preferably chosen such that it corresponds to 1/20 to ⅛ of the molar amount of Si-bonded acetoxy groups in the α,ω-diacetoxypolydimethylsiloxane. This base treatment further ensures that the acid, especially trifluoromethanesulfonic acid, present in the system is neutralized. The pretreatment of the acidified, preferably superacid-acidified, especially trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes with a base may be followed by a purification. For example any solids may be removed, for example by filtration. A distillative purification of the previously base-treated α,ω-diacetoxypolydimethylsiloxane may in particular be carried out if desired. Especially the inventive pretreatment of the acidified, preferably superacid-acidified, especially trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes with a base distinguishes the present subject-matter from the subject-matter of the unpublished European patent application having filing number 18189072.4. This inventive pretreatment especially provides a further contribution to the desired achievement of a high molecular weight SiOC-bonded A(BA)n-polyethersiloxane structure. The process according to the invention comprises reacting the α,ω-diacetoxypolydimethylsiloxanes resulting from (a) with polyether diols in the presence of a solid, liquid or gaseous base, optionally using inert solvents. Preferred bases to be employed according to the invention in step (b) correspond to the abovementioned bases, cf. step (a). The use of gaseous ammonia as the base is likewise very particularly preferred according to the invention. In a preferred embodiment of the invention the amount of the solid, liquid or gaseous base introduced into the reaction system in step (b) is chosen such that it is at least stoichiometric based on the Si-bonded acetoxy groups present in the acetoxysiloxane treated according to the invention, particularly preferably superstoichiometric based on said groups. In a preferred embodiment of the invention the reaction is performed in step (b) at temperatures between 20° C. to 120° C., preferably between 20° C. and 70° C., over the duration of 1 to 10, preferably at least over the duration of 1 to 3, hours. As is already known from the unpublished European patent application having filing number 18189072.4, the quality of the employed acidified, preferably superacid-acidified, especially trifluoromethanesulfonic acid-acidified α,ω-diacetoxypolydimethylsiloxane is also of decisive importance for the achievement of a high molecular weight SiOC-bonded A(BA)n-polyethersiloxane structure. According to the unpublished European patent application having filing number 18189072.4 it is desirable to achieve a perfect equilibration result for the employed acidified, preferably superacid-acidified, especially trifluoromethanesulfonic acid-acidified α,ω-diacetoxypolydimethylsiloxane for the construction of high molecular weight SiOC-bonded A(BA)n-polyethersiloxane structures. The term “end-equilibrated” is accordingly to be understood as meaning that the equilibrium established at a temperature of 23° C. and a pressure of 1013.25 hPa has been reached. Employed as an indicator for reaching the abovementioned equilibrium is the total cycles content determined by gas chromatography and defined as the sum of the D4, D5, D6contents based on the siloxane matrix and ascertained after derivatization of the α,ω-diacetoxypolydimethylsiloxanes to the corresponding α,ω-diisopropoxypolydimethylsiloxanes. The derivatization to afford the α,ω-diisopropoxypolydimethylsiloxanes is intentionally chosen in order to prevent a thermally induced retrocleavage reaction of the α,ω-diacetoxypolydimethylsiloxanes which may take place under the conditions of analysis by gas chromatography (regarding the retrocleavage reaction see inter alia J. Pola et al., Collect. Czech. Chem. Commun. 1974, 39(5), 1169-1176 and also W. Simmler, Houben-Weyl, Methods of Organic Chemistry, Vol. VI/2, 4thEdition, 0-Metal Derivates of Organic Hydroxy Compounds p. 162 ff). According to the invention the total cycles content present therein defined as the sum of the content fractions of the cyclic siloxanes comprising D4, D5and D6based on the siloxane matrix shall preferably be less than 13 percent by weight, particularly preferably less than 12 percent by weight, based on the siloxane matrix consisting of α,ω-diisopropoxypolydimethylsiloxanes. Equilibrated α,ω-diacetoxypolydimethylsiloxanes of this quality, i.e. end-equilibrated α,ω-diacetoxypolydimethylsiloxanes, may be produced very advantageously, i.e. even after a very short reaction time, by reaction of siloxane cycles (particularly comprising D4and/or D5) with acetic anhydride in the presence of trifluoromethanesulfonic acid and acetic acid. It is preferable when acetic acid is added in amounts of 0.4 to 3.5 percent by weight, preferably 0.5 to 3 percent by weight, more preferably 0.8 to 1.8 percent by weight, particularly preferably in amounts of 1.0 to 1.5 percent by weight, based on the reaction matrix consisting of acetic anhydride and cyclic siloxanes. The provision of trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes employable according to the invention is described for example in Example 1 of unpublished European patent application having filing number 18189072.4. In the context of the present invention the inventors have found that a pretreatment of the acidified, preferably superacid-acidified, preferably trifluoromethanesulfonic acid-acidified, end-equilibrated α,ω-diacetoxypolydimethylsiloxanes with a base before the subsequent conversion with polyether diols results in particularly good product mixtures, especially in respect of the achievable degree of polymerization. Since the degree of polymerization of the linear polydimethylsiloxane-polyoxyalkylene block copolymers is quality-determining, in particular for its effectiveness as a surfactant in polyurethane ether foams, reaction monitoring plays an important role. What has proven useful in this regard according to the invention is the method of withdrawing over the course of the reaction time samples of the reaction matrix which are then analyzed for example using29Si-NMR and/or13C-NMR spectroscopy. The reduction in the integral of the signals characteristic of the presence of acetoxydimethylsiloxy groups —OSi(CH3)2OCOCH3accompanies the intended molar mass increase of the copolymer having an A(BA)n structure and is a reliable indicator of the reaction conversion achieved. Obtained in this way in a manner unforeseeable to those skilled in the art are structures which as stabilizers in the production of polyurethane foams (PU foams), in particular flexible PU foams, exhibit markedly better properties. The present invention accordingly provides a process for producing SiOC-bonded, linear polydimethylsiloxane-polyoxyalkylene block copolymers comprising repeating (AB) units by reaction of polyether diols with inventively pretreated α,ω-diacetoxypolydimethylsiloxanes, wherein the reaction is undertaken by adding a solid, liquid or gaseous base, optionally using inert solvents. Inert solvents employed in a preferred embodiment of the invention are alkanes, cycloalkanes, alkylaromatics, end-capped polyethers and/or emollient esters, such as the esters derived from lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, ricinoleic acid and behenic acid combined with cetyl, stearyl, isostearyl, oleyl, octyldodecyl, myristyl and behenyl alcohol and/or glycerol, preferably myristyl myristate. In a preferred embodiment of the invention the siloxane blocks (A) of the block copolymers that result according to the invention are linear siloxane polymers or chains comprising repeating siloxane units which may be represented by the formula (—R2SiO—), wherein R=methyl. In a preferred embodiment of the invention the polyoxyalkylene block (B) of the linear block copolymers that result according to the invention is an oxyalkylene polymer containing the repeating oxyalkylene units, here in particular the oxyethylene and propenyloxy units. In a preferred embodiment the weight-average molecular weight of each siloxane block (A) is between 650 to 6500 g/mol, preferably 800 to 1500 g/mol, particularly preferably 1000 to 1200 g/mol. In a preferred embodiment the weight-average molecular weight of each polyoxyalkylene block of the copolymers produced according to the invention is between 600 and 10,000 g/mol, preferably 1000 to 5000 g/mol. The size of the individual oxyalkylene units or siloxane blocks is not necessarily uniform but may be varied as desired within the specified limits. In a preferred embodiment of the invention the individual polyoxyalkylene units are addition products of at least one oxyalkylene monomer selected from the group of ethylene oxide, propylene oxide, butylene oxide, tetrahydrofuran, preferably mixed products composed of at least two monomer units, in particular of ethylene oxide and propylene oxide. In a preferred embodiment the polyoxyalkylene blocks consist essentially of oxyethylene units or oxypropylene units, preference being given to mixed oxyethylene and oxypropylene units having an oxyethylene proportion of about 30 to 70 percent by weight and an oxypropylene proportion of 70 to 30 percent by weight based on the total content of oxyalkylene units in the block. In a preferred embodiment the total siloxane block proportion (A) in the copolymer is between 20 and 50 percent by weight, preferably 25% to 40% by weight, and the proportion of the polyoxyalkylene blocks is between 80% and 50% by weight. In a preferred embodiment the block copolymer has an average weight-average molecular weight Mw of at least 10,000 g/mol to about 160,000 g/mol, preferably 15 000 g/mol to about 100,000 g/mol, in particular 20,000 g/mol to about 36,000 g/mol. The determination of the average molecular weights is based on the known methods of GPC analysis using a polystyrene standard. In a preferred embodiment the molar ratio of α,ω-diacetoxysiloxanes to polyether diols is in the range from 0.90 to 1.10, preferably in the range 0.95 to 1.05, particularly preferably in the range 0.99 to 1.01. It is readily apparent to those skilled in the art that the achievable degree of polymerization depends on achieving a virtually perfect stoichiometry of the reactants. In a preferred embodiment the process according to the invention is implemented by reacting inventively pretreated acetoxy-Si-containing polyorganosiloxanes of general formula (II) wherein:R represents methyl radicals,b represents 8 to 80, preferably 10 to 50, particularly preferably 10 to 25, with at least one alcohol selected from the group of polyether diols of general formula (III) HO—(CnH(2n-m)R1mO—)x—H (III)whereinR1represents methyln represents 2 to 4,m represents 0 or 1x represents a value of 1 to 200, preferably 10 to 100, in particular 35 to 60, wherein the oxyalkylene segments —(CnH(2n-m)R1mO—) within an oxyalkylene ether radical may differ from one another and the sequence of the individual segments—(CnH(2n-m)R1mO—) is freely choosable and comprises in particular block copolymers, random polymers and combinations thereof. According to the invention, preference is given to polyether diols in which ethylene oxide (EO) and propylene oxide (PO) are present as copolymers. Particular preference is given to EO/PO copolymers having a block-type construction and containing an EO proportion of about 30% to 70% by weight based on the total content of oxyalkylene units. In order to guarantee increased storage stability, the linear SiOC-linked polyethersiloxanes produced by the process according to the invention can additionally also be admixed with small amounts of organic amines, such as for example N-methylmorpholine, triisopropanolamine or triethanolamine. This corresponds to a preferred embodiment of the invention. A particularly sensitive and informative assessment of the compounds according to the invention is possible using a performance test in which the obtained copolymer is introduced as a foam stabilizer into polyurethane formulations for producing in particular ether foams or open-celled rigid foams. Structural deficits in the foam stabilizer manifest during foaming as technical inadequacies, for example shrinkage or collapse. Production of the SiOC-bonded, linear polydimethylsiloxane-polyoxyalkylene block copolymers claimed by the process according to the invention may be carried out with or without use of a suitable solvent as desired. If high molecular weight and thus high-viscosity SiOC-bonded copolymers are sought the production thereof for ease of handling during and after synthesis may advantageously be carried out by reaction of the respective polyether diol with the respective α,ω-acetoxysiloxane in a suitable solvent. Suitable solvents are alkanes, cycloalkanes, alkylaromatics, end-capped polyethers but also emollient esters such as myristyl myristate or the like, high-boiling solvents having boiling points>120° C. being preferred in particular. The inventors have found that rapid and complete conversion of the pretreated α,ω-diacetoxypolydimethylsiloxanes with polyether diols while avoiding discoloration of the reaction product is especially performed in the presence of ammonia. The use of ammonia both in step (a) (pretreatment) and in step (b) (reaction with polyether diols) corresponds to a very particularly preferred embodiment of the invention. In a preferred embodiment of the invention the reaction in step (b) (reaction with polyether diols) is performed at temperatures between preferably 20° C. and 70° C. over a duration of preferably 1 to 3 hours. In another embodiment preferred according to the invention base(s) are initially charged with stirring into the polyetherol or, respectively, polyetherol mixture provided for bonding even before the inventively pretreated α,ω-diacetoxypolydimethylsiloxane is added. In a preferred embodiment of the invention this reaction is performed preferably at temperatures between 50° C. to 90° C. and preferably over a duration of 2 to 6 hours. It has surprisingly further been found that the polyethersiloxanes produced according to the invention using pretreated α,ω-diacetoxypolydimethylsiloxane have extremely good storage stability. As a criterion for assessing the storage stability of the SiOC-bonded polyethersiloxanes produced in accordance with the inventive teaching, viscosity is monitored as a function of time at a constant storage temperature by sampling since any possible degradation and/or growth processes sensitively manifest therein. In a preferred embodiment the reaction temperature for producing the copolymers according to the invention should be at 20° C. to 120° C., preferably at 20° C. to 70° C. EXAMPLES The examples which follow serve merely to elucidate this invention to those skilled in the art and do not constitute any limitation of the claimed process whatsoever. The inventive determination of the water contents is in principle performed by the Karl Fischer method based on DIN 51777, DGF E-III 10 and DGF C-III 13a.29Si-NMR spectroscopy was used for reaction monitoring in all examples. In the context of the present invention the29Si-NMR samples are analysed at a measurement frequency of 79.49 MHz in a Bruker Avance III spectrometer equipped with a 287430 sample head with gap width of 10 mm, dissolved at 22° C. in CDCl3and against a tetramethylsilane (TMS) external standard [δ(29Si)=0.0 ppm]. The gas chromatograms are recorded on an Agilent Technologies GC 7890B GC instrument fitted with an HP-1 column having dimensions of 30 m×0.32 mm ID×0.25 μm dF (Agilent Technologies No. 19091Z-413E) using hydrogen as a carrier gas and employing the following parameters:Detector: FID; 310° C.Injector: split; 290° C.Mode: constant flow, 2 ml/minTemperature programme: 60° C. at 8° C./min-150° C. at 40° C./min-300° C. 10 min. Employed as an indicator for reaching the equilibrium is the total cycles content determined by gas chromatography and defined as the sum of the D4, D5, D6contents based on the siloxane matrix and ascertained after derivatization of the α,ω-diacetoxypolydimethylsiloxanes to the corresponding α,ω-diisopropoxypolydimethylsiloxanes. The derivatization to afford the α,ω-diisopropoxypolydimethylsiloxanes is intentionally chosen in order to prevent a thermally induced retrocleavage reaction of the α,ω-diacetoxypolydimethylsiloxanes which may take place under the conditions of analysis by gas chromatography (regarding the retrocleavage reaction see inter alia J. Pola et al., Collect. Czech. Chem. Commun. 1974, 39(5), 1169-1176 and also W. Simmler, Houben-Weyl, Methods of Organic Chemistry, Vol. VI/2, 4th Edition, 0-Metal Derivates of Organic Hydroxy Compounds p. 162 ff.). The employed polyether diols have water contents of about 0.2% by mass and are used after pre-drying thereof. Employed toluene and alkylbenzene (C10-C13) each have a water content of 0.03% by mass and are used without pre-drying. The OH number of the polyether diols is determined according to DGF C-V 17 a (53) or according to Ph. Eur. 2.5.3 Method A, wherein the hydroxyl groups of the sample to be analysed are firstly acetylated with acetic anhydride in the presence of pyridine and then within the scope of a differential titration (blank sample, accounting for acetic anhydride excess) the liberated acetic acid is titrated as the consumption of KOH in mg per gram of polyether diol. Example 1 Production of an End-Equilibrated, Acetoxy-Terminated, Linear Polydimethylsiloxane In a 1000 ml four-necked flask fitted with a KPG stirrer, an internal thermometer and a reflux cooler 77.3 g (0.757 mol) of acetic anhydride together with 732.8 g (1.98 mol) of decamethylcyclopentasiloxane (D5) and 24.3 g of acetic acid (3.0 percent by weight based on the total mass of the reactants) are initially charged with stirring and admixed with 1.62 g (0.88 ml) of trifluoromethanesulfonic acid (0.2 percent by mass based on the total batch) and swiftly heated to 150° C. The initially slightly cloudy reaction mixture is left at this temperature for 4 hours with continued stirring. After cooling of the batch a colorless, clear, mobile liquid is isolated, whose29Si-NMR spectrum demonstrates the presence of Si-acetoxy groups in a yield of about 93% based on employed acetic anhydride corresponding to an α,ω-diacetoxypolydimethylsiloxane having an average total chain length of about 14. Conversion of the α,ω-Diacetoxypolydimethylsiloxane into the Corresponding α,ω-Diisopropoxypolydimethylsiloxane for Analytical Characterization Immediately after the synthesis in a 250 ml four-necked round-bottomed flask fitted with a KPG stirrer, an internal thermometer and a reflux cooler 50.0 g of this trifluoromethanesulfonic acid-acidified, equilibrated α,ω-diacetoxypolydimethylsiloxane are mixed together with 11.3 g of a molecular sieve-dried isopropanol by stirring at 22° C. Gaseous ammonia (NH3) is then introduced to the reaction mixture until alkaline reaction (moist universal indicator paper) and the mixture is then stirred at this temperature for a further 45 minutes. The precipitated salts are removed using a fluted filter. A colorless, clear liquid is isolated, whose accompanying29Si-NMR spectrum demonstrates the quantitative conversion of the α,ω-diacetoxypolydimethylsiloxane into an α,ω-diisopropoxypolydimethylsiloxane. An aliquot of this α,ω-diisopropoxypolydimethylsiloxane is withdrawn and analysed by gas chromatography. The gas chromatogram shows the following contents (reported in percent by mass): Sum ofIsopropanolD4D5D6(D4-D6)content4.09%2.62%0.86%7.57%4.60% Example 2 (Inventive) a) Pretreatment of the Trifluoromethanesulfonic Acid-Acidified, End-Equilibrated α,ω-Diacetoxypolydimethylsiloxane In a 250 ml four-necked flask fitted with a KPG stirrer, a contact thermometer and a gas introduction tube 100 g of the trifluoromethanesulfonic acid-acidified, end-equilibrated, acetoxy-terminated, linear polydimethylsiloxane produced in example 1 are subjected to a moderate ammonia stream at 22° C. for 30 minutes with stirring and a salt precipitation is observed. Once gas introduction is complete and the stirrer is switched off a sample of the clear supernatant is withdrawn and characterized using29Si-NMR analysis. The integral intensity over the signal layers characteristic for short-chain α,ω-diacetoxysiloxanes have markedly reduced compared to the29Si-NMR spectrum of the non-pretreated, end-equilibrated, acetoxy-terminated, linear polydimethylsiloxane and altogether represent only about 27% of the Si-bonded acetoxy groups originally appearing in this shift range in the starting spectrum. Calculating the average chain length based on the integral intensities of the new spectrum results in a length of about N=15. A pleated filter is used to separate the mixture from the precipitated salts and the α,ω-diacetoxypolydimethylsiloxane is isolated. b) Reacting the α,ω-Diacetoxypolydimethylsiloxane Resulting from a) with Polyether Diol in the Presence of Ammonia as the Base In a 250 ml four-necked flask fitted with a KPG stirrer, a contact thermometer and a water separator 56.1 g (0.02 mol) of a polyether diol constructed from ethylene oxide and propylene oxide units and having an average molar mass of about 2800 g/mol and a propylene oxide proportion of 40 percent by mass are admixed with 91.3 g of toluene and subjected to azeotropic drying at 120° C. After cooling 35.2 g (0.029 mol) of the α,ω-diacetoxypolydimethylsiloxane from step a) are added and then a moderate stream of dry ammonia is introduced at 22° C. over 3 hours with stirring. The resulting salts are subsequently separated using a filter press. The obtained clear filtrate is concentrated to about 75% of its original volume in a rotary evaporator at a bottom temperature of 150° C. and an applied auxiliary vacuum of <1 mbar and then mixed with 91.3 g of a butanol-started polyetherol consisting solely of propyleneoxy units having an average molar mass of 700 g/mol before distillation under the previously chosen conditions is continued until volatiles no longer pass over. Cooling affords a clear, colorless, high viscosity material whose29Si-NMR spectrum verifies quantitative conversion. | 26,717 |
11859054 | DETAILED DESCRIPTION OF THE INVENTION A method of preparing alkoxy-functional organosilicon compounds is disclosed. The alkoxy-functional organosilicon compounds prepared may be utilized in diverse end use applications. For example, the alkoxy-functional organosilicon compounds may be utilized as a starting component and/or precursor when preparing silicone-organic hybrid materials, e.g. via copolymerization, grafting, etc. The alkoxy-functional organosilicon compounds may also be utilized in a composition or formulation, as provided herein. The method comprises reacting (A) an initial organosilicon compound having at least one alkoxysilyl group and (B) an alcohol component comprising an organic alcohol in the presence of (C) a catalyst comprising (C1) an ammonium carboxylate compound or (C2) a titanate compound. In general, reacting the initial organosilicon compound (A) and the alcohol component (B) comprises combining the initial organosilicon compound (A) and the alcohol component (B) in the presence of the catalyst (C). Said differently, there is generally no proactive step required for the reaction beyond combining the initial organosilicon compound (A) and the alcohol component (B) in the presence of the catalyst (C). As will be appreciated by those of skill in the art, the reaction may be generally defined or otherwise characterized as a transalkoxylation reaction or, more simply, a “transalkoxylation” or “alkoxylation”, e.g. a selective alkoxylation reaction, a catalytic alkoxylation reaction, an alkoxyltic conversion reaction, etc. The initial organosilicon compound (A) is an organosilicon compound having at least one alkoxysilyl group, and is otherwise not particularly limited. In general, the alkoxysilyl group of the initial organosilicon compound (A) has the formula: where each R1and each R2is an independently selected hydrocarbyl group; and subscript a is 1, 2, or 3. Each R1and each R2is independently selected from hydrocarbyl groups. However, as will be appreciated from the description herein, each R1and each R2may comprise a combination of such hydrocarbyl groups, e.g. as substituents of one another. As such, suitable hydrocarbyl groups may be substituted or unsubstituted. With regard to such hydrocarbyl groups, the term “substituted” describes hydrocarbon moieties where either one or more hydrogen atoms is replaced with atoms other than hydrogen (e.g. a halogen atom, such as chlorine, fluorine, bromine, etc.), a carbon atom within a chain of the hydrocarbon is replaced with an atom other than carbon (i.e., R1and/or R2may include one or more heteroatoms (oxygen, sulfur, nitrogen, etc.) within a carbon chain), or both. As such, it will be appreciated that R1and/or R2may include hydrocarbon moieties that have substituents in and/or on (i.e., appended to and/or integral with) carbon chains/backbones thereof, such that R1and/or R2may comprise or be an ether, an ester, etc. Hydrocarbyl groups suitable for R1and/or R2may independently be linear, branched, cyclic, or combinations thereof. Cyclic hydrocarbyl groups encompass aryl groups as well as saturated or non-conjugated cyclic groups. Cyclic hydrocarbyl groups may independently be monocyclic or polycyclic. Linear and branched hydrocarbyl groups may independently be saturated or unsaturated. One example of a combination of a linear and cyclic hydrocarbyl group is an aralkyl group. General examples of hydrocarbyl groups include alkyl groups, aryl groups, alkenyl groups, halocarbon groups, and the like, as well as derivatives, modifications, and combinations thereof. Examples of suitable alkyl groups include methyl, ethyl, propyl (e.g. isopropyl and/or n-propyl), butyl (e.g. isobutyl, n-butyl, tert-butyl, and/or sec-butyl), pentyl (e.g., isopentyl, neopentyl, and/or tert-pentyl), hexyl, as well as branched saturated hydrocarbon groups having from 6 to 18 carbon atoms. Examples of suitable aryl groups include phenyl, tolyl, xylyl, naphthyl, benzyl, and dimethyl phenyl. Examples of suitable alkenyl groups include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, heptenyl, hexenyl, and cyclohexenyl groups. Examples of suitable monovalent halogenated hydrocarbon groups (i.e., halocarbon groups) include halogenated alkyl groups, aryl groups, and combinations thereof. Examples of halogenated alkyl groups include the alkyl groups described above where one or more hydrogen atoms is replaced with a halogen atom such as F or Cl. Specific examples of halogenated alkyl groups include fluoromethyl, 2-fluoropropyl, 3,3,3-trifluoropropyl, 4,4,4-trifluorobutyl, 4,4,4,3,3-pentafluorobutyl, 5,5,5,4,4,3,3-heptafluoropentyl, 6,6,6,5,5,4,4,3,3-nonafluorohexyl, and 8,8,8,7,7-pentafluorooctyl, 2,2-difluorocyclopropyl, 2,3-difluorocyclobutyl, 3,4-difluorocyclohexyl, and 3,4-difluoro-5-methylcycloheptyl, chloromethyl, chloropropyl, 2-dichlorocyclopropyl, and 2,3-dichlorocyclopentyl groups, as well as derivatives thereof. Examples of halogenated aryl groups include the aryl groups described above where one or more hydrogen atoms is replaced with a halogen atom, such as F or Cl. Specific examples of halogenated aryl groups include chlorobenzyl and fluorobenzyl groups. Typically, each R1and each R2is an independently selected substituted or unsubstituted hydrocarbyl group. Each R1may be the same as or different from any other R1in the initial organosilicon compound (A). In certain embodiments, each R1is the same. In other embodiments, at least one R1is different from at least one other R1of the initial organosilicon compound (A). In some embodiments, each R1is an independently selected hydrocarbyl group having from 1 to 18, alternatively from 1 to 12, alternatively from 1 to 6, alternatively from 1 to 4 carbon atoms. Typically, each R1is independently selected from alkyl groups, such as methyl groups, ethyl groups, etc. In certain embodiments, each R1is methyl. Each R2may be the same as or different from any other R2in the initial organosilicon compound (A). In certain embodiments, each R2is the same. In other embodiments, at least one R2is different from at least one other R2of the initial organosilicon compound (A). In some embodiments, each R2is typically an independently selected substituted or unsubstituted hydrocarbyl group having from 1 to 20, alternatively from 1 to 18, alternatively from 1 to 16 carbon atoms. Subscript a is 1, 2, or 3, such that the initial organosilicon compound (A) comprises a monoalkoxysilyl group, dialkoxysilyl group, or trialkoxysilyl group, respectively. In particular embodiments, subscript a is 3, such that the initial organosilicon compound (A) comprises at least one trialkoxysilyl group. As introduced above, the initial organosilicon compound (A) is not particularly limited aside from the alkoxysilyl group. In some embodiments, however, the initial organosilicon compound (A) has the general formula: where R1, R2, and subscript a are as defined above, D1is a divalent linking group, and R3is an independently selected hydrocarbyl group. In general, D1is a divalent linking group, and may be linear or branched and substituted or unsubstituted. When branched, D1may optionally be bonded (e.g. cross-linked) to a siloxane segment or a silane moiety (i.e., other than the alkoxysilyl group in the general initial organosilicon compound formula above). Typically, D1is selected from divalent substituted or unsubstituted hydrocarbon groups, which may optionally be modified or substituted, e.g. with ether, ester, siloxy, and/or silyl groups. For example, in some embodiments, D1comprises a hydrocarbon moiety having the formula —(CH2)c—, where subscript c is from 1 to 16. In these or other embodiments, D1may comprise a substituted hydrocarbon, i.e., a hydrocarbon group comprising a backbone having at least one heteroatom (e.g. O, N, S, etc.). For example, in some embodiments, D1is a hydrocarbon having a backbone comprising an ether moiety. R3is an independently selected hydrocarbyl group, such as any of those described above. Typically, R3is an independently selected substituted hydrocarbyl group. For example, in particular embodiments, R3comprises at least one functional group, and may thus be referred to as a functional moiety (i.e., the functional moiety R3). In some such embodiments, the functional group is selected from epoxide groups, ester groups, anhydride groups, and acryloxy groups. As such, in these embodiments, R3comprises an epoxide group, an ester group, an anhydride group, and/or an acryloxy group. In certain embodiments, R3comprises, alternatively is, an epoxide group, an ester group, an anhydride group, or an acryloxy group. For example, in certain embodiments, R3comprises, alternatively is, an epoxyethyl group of the following formula: In some embodiments, R3comprises, alternatively is, an epoxycyclohexyl group of the following formula: In particular embodiments, R3may comprise, alternatively may be, a 3-glycidoxypropyl group, a 4-glycidoxybutyl group, or similar glycidoxyalkyl groups; a 2-(3,4-epoxycyclohexyl)ethyl group, a 3-(3,4-epoxycyclohexyl)propyl group, or similar epoxycyclohexylalkyl groups; and a 4-oxiranylbutyl group, an 8-oxiranyloctyl group. In certain embodiments, R3comprises, alternatively is, an alkyl glycidyl ether group, such as a methyl glycidyl ether group, ethyl glycidyl ether group, propyl glycidyl ether group, etc. In some embodiments, R3comprises, alternatively is, an epoxycyclohexylalkyl group, such as an epoxycyclohexylmethyl group, an epoxycyclohexylethyl group, an epoxycyclohexylpropyl group, etc. In some embodiments, R3comprises, alternatively is, an acryloxy group of the formula: where R12is independently selected from hydrocarbyl groups and H. Examples of hydrocarbyl groups suitable for R12include any of those described above. In certain embodiments, R12is H, such that R3comprises an acryloxy group that may be defined as an acrylate group. In other embodiments, R12is selected from substituted or unsubstituted hydrocarbyl groups. In some such embodiments, R12is an alkyl group, such that R3comprises, alternatively is, an alkylacrylate group. In specific embodiments, R12is methyl, such that R3comprises, alternatively is, a methacrylate group. In some embodiments, R3comprises, alternatively is, an ester group. For example, in some such embodiments, R3comprises, alternatively is, an ester group of the formula: where R13is an independently selected hydrocarbyl group. Examples of hydrocarbyl groups suitable for R13include any of those described above. In some embodiments, R13is selected from substituted or unsubstituted hydrocarbyl groups. In some such embodiments, R13is an alkyl group, such that R3comprises, alternatively is, an alkyl ester group. In specific embodiments, R13is methyl, ethyl, or propyl, such that R3comprises, alternatively is, a methyl ester group, an ethyl ester group, or a propyl ester group, respectively. In certain embodiments, R3comprises, alternatively is, an anhydride group. The anhydride group may comprise saturated or unsaturated substituents, and may be linear or cyclic. In certain embodiments, the method comprises utilizing more than one initial organosilicon compound (A), such as 2, 3, 4, or more initial organosilicon compounds (A). In such embodiments, each initial organosilicon compound (A) is independently selected, and may be the same as or different from any other initial organosilicon compound (A). The initial organosilicon compound (A) may be utilized in any form, such as neat (i.e., absent solvents, carrier vehicles, diluents, etc.), or disposed in a carrier vehicle, such as a solvent or dispersant. The carrier vehicle, if present, may comprise an organic solvent (e.g. aromatic hydrocarbons such as benzene, toluene, xylene, etc.; aliphatic hydrocarbons such as heptane, hexane, octane, etc.; halogenated hydrocarbons such as dichloromethane, 1,1,1-trichloroethane, chloroform; etc.; ethers such as diethyl ether, tetrahydrofuran, etc.), a silicone fluid, or combinations thereof. When utilized, the carrier vehicle will be selected based on the particular components of the reaction, such as the particular initial organosilicon compound (A) selected. For example, in certain embodiments, the method is carried out in the presence of a carrier vehicle or solvent comprising a polar component, such as an ether, acetonitrile, dimethylformamide, dimethylsulfoxide, and the like, or combinations thereof. In some embodiments, the carrier vehicle may comprise a halogenated hydrocarbon, such as those described above. In such embodiments, the carrier vehicle in general, and/or the halogenated hydrocarbon in particular, is typically purified and/or processes to reduce, alternatively to remove, any hydrochloric acid (HCl) therefrom. It will be appreciated that the initial organosilicon compound (A) may be combined with the carrier vehicle, if utilized, prior to, during, or after being combined with components (B) and (C). In certain embodiments, the initial organosilicon compound (A) is free from, alternatively substantially free from carrier vehicles. In some such embodiments, the initial organosilicon compound (A) is free from, alternatively substantially free from, water and carrier vehicles/volatiles reactive with the initial organosilicon compound (A), the alcohol component (B) (e.g. the organic alcohol thereof), and/or the catalyst (C). In some embodiments, the method is carried out in the absence of carrier vehicles/volatiles that are reactive with the initial organosilicon compound (A), the alcohol component (B), and/or the catalyst (C). For example, in certain embodiments, the method may comprise stripping a mixture of the initial organosilicon compound (A) of volatiles and/or solvents prior to combining the same with components (B) and/or (C). Techniques for stripping the initial organosilicon compound (A) are known in the art, and may include heating, drying, applying reduced pressure/vacuum, azeotroping with solvents, utilizing molecular sieves, etc., and combinations thereof. The initial organosilicon compound (A) may be utilized in any amount, which will be selected by one of skill in the art, e.g. dependent upon the particular catalyst (C) selected, the reaction parameters employed, the scale of the reaction (e.g. total amounts of component (A) to be reacted and/or alkoxy-functional organosilicon compound to be prepared), etc. The alcohol component (B) comprises an organic alcohol, and is otherwise not particularly limited. As will be appreciated by one of skill in the art, the organic alcohol is also not particularly limited, but will be selected in view of the particular initial organosilicon compound (A) utilized, including the boiling point and/or other properties of the conjugate acids of the individual alkoxides of the alkoxysilyl group to be transalkoxylated with the organic alcohol of the alcohol component (B). Typically, the organic alcohol of the alcohol component (B) has the formula R4OH, where R4is an independently selected hydrocarbyl group. Examples of hydrocarbyl groups suitable for R4include any of those described above. For example, in certain embodiments, R4is selected from substituted and unsubstituted hydrocarbyl groups. In some such embodiments, R4is a substituted or unsubstituted hydrocarbyl group having at least 3, alternatively at least 4, alternatively at least 5, alternatively at least 6, alternatively greater than 6 carbon atoms. In particular embodiments, R4is an independently selected hydrocarbyl group having from 3 to 30, alternatively from 3 to 28, alternatively from 3 to 26, alternatively from 3 to 24, alternatively from 3 to 22, alternatively from 4 to 22, alternatively from 5 to 22, alternatively from 6 to 22, alternatively from 6 to 20 carbon atoms. In general, as will be appreciated by those of skill in the art, R4is different from R1of the alkoxysilyl group of the initial organosilicon compound (A) to facilitate the transalkoxylation reaction. The difference between R4and R1may be selected, e.g. to increase the ease of purifying the alkoxy-functional organosilicon compound to be prepared (i.e., with regard to removal of alcohol of formula R1OH produced during the reaction, via distillation, evaporation, etc.), as described below. For example, in certain embodiments, R4is selected to have at least 1, alternatively at least 2, alternatively at least 3, alternatively at least 4 more carbon atoms than R1of the alkoxysilyl group of the initial organosilicon compound (A). In these or other embodiments, R4and R1are cooperatively selected such that the organic alcohol has a higher boiling point and/or lower vapor pressure than the alcohol of formula R1OH produced during the transalkoxylation reaction. Examples of suitable organic alcohols include 2,2-dimethyl-3-(3-methylphenyl)-1-propanol, 2,2-dimethyl-3-phenyl-1-propanol, 3-(2-bornyloxy)2-methyl-1-propanol, 2-tert-butylcyclohexanol, 4-tert-butylcyclohexanol, dihydroterpineol, 2,4-dimethyl-4-cyclohexen-1-yl methanol, 2,4-dimethylcyclohexyl methanol, 2,6-dimethyl-2-heptanol, 2,6-dimethyl-4-heptanol, 2,6-dimethyl-2,7-octadien-6-ol (linalool), cis-3,7-dimethyl-2,6-octadien-1-ol (nerol), trans-3,7-dimethyl-2,6-octadien-1-ol (geraniol), 1-octanol, 2-octanol, 3,7-dimethyl-1,7-octanediol, 3,7-dimethyl-1-octanol (tetrahydrogeraniol), 2,6-dimethyl-2-octanol (tetrahydromyrcenol), 3,7-dimethyl-3-octanol (tetrahydrolinalool), 2,6-dimethyl-7-octen-2-ol (dihydromyrcenol), 3,7-dimethyl-6-octen-1-ol (citronellol), 3,7-dimethyl-1,6-nonadien-3-ol,1-decanol, 9-decen-1-ol, 2-ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, cis-3-hexen-1-ol, 4-(4-hydroxy-3-methoxyphenyl)-2-butanone, 3-(hydroxymethyl)-2-nonanone, 3a,4,5,6,7,7a-hexahydro-2,4-dimethyl-4,7-methano[H]inden-5-ol, 1-hydroxy-2-(1-methyl-1-hydroxyethyl)-5-methylcyclohexane, 4-hydroxy-3-methoxybenzaldehyde (vanillin), 3-ethoxy-4-hydroxybenzaldehyde (ethylvanillin), 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde, isoborneol, 3-isocamphylcyclohexanol, 2-isopropenyl-5-methylcyclohexanol (isopulegol), 1-isopropyl-4-methylcyclohex-3-enol (terpinenol), 4-isopropylcyclohexanol, 1-(4-isopropylcyclohexyl) ethanol, 4-isopropylcyclohexylmethanol, 2-isopropyl-5-methylcyclohexanol (menthol), 2-isopropyl-5-methylphenol (thymol), 5-isopropyl-2-methylphenol (carvacrol), 2-(4-methyl-3-cyclohexenyl)-2-propanol (terpineol), 2-(4-methylcyclohexyl)-2-propanol (dihydroterpineol), benzyl alcohol, 4-methoxybenzyl alcohol, 2-methoxy-4-methylphenol, 3-methoxy-5-methylphenol, 2-ethoxy-4-methoxymethylphenol, 4-allyl-2-methoxyphenol (eugenol), 2-methoxy-4-propenylphenol (isoeugenol), 1-methoxy-4-propenylbenzene (anethol), 4-methyl-3-decen-5-ol, 2-methyl-6-methylene-7-octen-2-ol (myrcenol), 2-methyl-2-butanol (2M2B, tert-amyl alcohol, TAA), 3-methyl-4-phenyl-2-butanol,3-methyl-1-butanol (isoamyl alcohol, isopentyl alcohol) 2-(2-methylphenyl) ethanol, 2-methyl-4-phenyl-1-pentanol, 3-methyl-5-phenyl-1-pentanol, 2-methyl-1-phenyl-2-propanol, (1-methyl-2-(1,2,2-trimethylbicyclo[3.1.0]hex-3-ylmethyl) cyclopropyl) methanol, 3-methyl-4-(2,2,6-trimethylcyclohexen-1-yl)-2-butanol, 2-methyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol, (3-methyl-1-(2,2,3-trimethyl-3-cyclopentenyl)-3-cyclohexen-1-yl) methanol, 3-methyl-5-(2,2,3-trimethyl-3-cyclopenten-1-yl)-4-penten-2-ol, 2-methyl-2-vinyl-5-(1-hydroxy-1-methylethyl) tetrahydrofuran, (2E,6Z)-nona-2,6-dien-1-ol, 1-nonanol, 3,5,5-trimethyl-1-hexanol (isononanol),nopol, 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol, 3,4,5,6,6-pentamethyl-2-heptanol, 2-phenylethanol, 2-phenylpropanol, 3-phenylpropanol (hydrocinnamic alcohol), 3-phenyl-2-propen-1-ol (cinnamic alcohol), 4-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl) cyclohexan-1-ol, 3,5,5-trimethylcyclohexanol, 2,4,6-trimethyl-4-cyclohexen-1-ylmethanol, 5-(2,2,3-trimethyl-3-cyclopentenyl)-3-methylpentan-2-ol, 3,7,11-trimethyl-2,6,10-dodecatrien-1-ol (farnesol), 3,7,11-trimethyl-1,6,10-dodecatrien-3-ol (nerolidol), 1-undecanol, 10-undecen-1-ol, vetiverol, and the like, as well as derivatives, modifications, and combinations thereof. In particular embodiments, the organic alcohol is selected from geraniol, 2E,6Z)-nona-2,6-dien-1-ol, isoamyl alcohol, benzyl alcohol, 2-octanol, and 2-methyl-2-butanol. In certain embodiments, the organic alcohol of the alcohol component (B) may comprise, alternatively may be, a fragrance alcohol or a nonfragrance alcohol. Typically, the distinction as to whether a particular organic alcohol is considered a fragrance alcohol or a nonfragrance alcohol is based on whether the particular organic alcohol exhibits an odiferous effect detectable by a human nose. However, because the organic alcohol can be considered either a fragrance alcohol and/or a nonfragrance alcohol, such distinction is only relevant, if at all, to the selection of the organic alcohol of the alcohol component (B) by one of skill in the art based on end use applications. In some embodiments, the alcohol component (B) is substantially free from, alternatively free from, a fragrance alcohol. In these or other embodiments, the organic alcohol of the alcohol component (B) is substantially free from, alternatively free from pro-fragrance and/or fragrance precursor groups. In certain embodiments, the alcohol component (B) comprises more than one organic alcohol, such as 2, 3, 4, or more organic alcohols. In such embodiments, each organic alcohol is independently selected, and may be the same as or different from any other organic alcohol, e.g. in terms of number of carbon atoms, structure (e.g. stereochemistry, etc.), boiling point, vaporization point, vapor pressure, etc. The organic alcohol of the alcohol component (B) may be utilized in any form, such as neat (i.e., absent solvents, carrier vehicles, diluents, etc.), or disposed in a carrier vehicle, such as a solvent or dispersant. As such, the alcohol component (B) itself may comprise the organic alcohol and other components, such as the carrier vehicle, or may consist essentially of, alternatively consist of, the organic alcohol. The carrier vehicle, if present, may comprise an organic solvent (e.g. aromatic hydrocarbons such as benzene, toluene, xylene, etc.; aliphatic hydrocarbons such as heptane, hexane, octane, etc.; halogenated hydrocarbons such as dichloromethane, 1,1,1-trichloroethane, chloroform; etc.; ethers such as diethyl ether, tetrahydrofuran, etc.), a silicone fluid, or combinations thereof. It will be appreciated that the alcohol component (B) may be combined with such a carrier vehicle, if utilized, prior to, during, or after being combined with components (A) and (C). In certain embodiments, the alcohol component (B) itself is utilized as a carrier vehicle for the reaction, e.g. when the organic alcohol is liquid under the reaction conditions employed. In certain embodiments, the alcohol component (B) is free from, alternatively substantially free from carrier vehicles. In some such embodiments, the alcohol component (B) is free from, alternatively substantially free from, water and carrier vehicles/volatiles reactive with the alcohol component (B) (e.g. the organic alcohol thereof), the initial organosilicon compound (A), and/or the catalyst (C). For example, in certain embodiments, the method may comprise stripping the alcohol component (B) of volatiles (i.e., aside from the organic alcohol, if volatile) and/or solvents (e.g. water, reactive solvents, etc.) prior to combining the same with components (A) and/or (C). Techniques for stripping the alcohol component (B) are known in the art, and may include heating, drying, applying reduced pressure/vacuum, azeotroping with solvents, utilizing molecular sieves, etc., and combinations thereof. The alcohol component (B) may be utilized in any amount, which will be selected by one of skill in the art, e.g. dependent upon the particular initial organosilicon compound (A) selected, the particular catalyst (C) selected, the reaction parameters employed, the scale of the reaction (e.g. total amounts of component (A) to be converted and/or alkoxy-functional organosilicon compound to be prepared), etc. The relative amounts of the initial organosilicon compound (A) and the alcohol component (B) utilized may vary, e.g. based upon the particular initial organosilicon compound (A) selected, the particular organic alcohol of component (B) selected, the reaction parameters employed, etc. As understood by those of skill in the art, the transalkoxylation of the initial organosilicon compound (A) with the organic alcohol of the alcohol component (B) occurs at a theoretical maximum based on the number of alkoxysilyl functionalities present within the initial organosilicon compound (A). In particular, with reference to the general alkoxysilyl group above, each alkoxy group designated by subscript a can be transalkoxylated, such that one molar equivalent of the organic alcohol of the alcohol component (B) is needed for every alkoxysilyl group of the initial organosilicon compound (A). In this fashion, when the initial organosilicon compound (A) comprises a single alkoxysilyl group where subscript a is 3, the transalkoxylation of the initial organosilicon compound (A) with the organic alcohol of the alcohol component (B) occurs at a theoretical maximum molar ratio of 1:3 (A):(B), where (B) is the organic alcohol of the alcohol component (B). Likewise, when the initial organosilicon compound (A) comprises two alkoxysilyl groups where each subscript a is 2, the transalkoxylation of the initial organosilicon compound (A) with the organic alcohol of the alcohol component (B) occurs at a theoretical maximum molar ratio of 1:4 (A):(B), where (B) is the organic alcohol of the alcohol component (B). Typically, however, an excess (e.g. molar and/or stoichiometric) of one of the components is utilized to fully consume the initial organosilicon compound (A) or the organic alcohol of the alcohol component (B), e.g. to simplify purification of the reaction product formed. For example, in certain embodiments, the alcohol component (B) is utilized in relative excess (e.g. where the organic alcohol is present in a molar excess of the number of silicon-bonded alkoxy groups of the initial organosilicon compound (A)) to maximize a conversion rate of the initial organosilicon compound (A) to the alkoxy-functional organosilicon compound. In some such embodiments, the alcohol component (B) may also be utilized, or otherwise function, as a carrier vehicle in the reaction. It will be appreciated that the initial organosilicon compound (A) may be used in excess of the organic alcohol of the alcohol component (B), such as when maximum consumption of the organic alcohol is desired. In general, the initial organosilicon compound (A) and the organic alcohol of the alcohol component (B) are typically reacted in a molar ratio of from 10:1 to 1:10 (A):(B). In certain embodiments, the initial organosilicon compound (A) and the organic alcohol of the alcohol component (B) are reacted in a molar ratio of from 1:1 to 1:9, such as from 1:2 to 1:9, alternatively of from 1:2 to 1:8, alternatively of from 1:2 to 1:7, alternatively of from 1:3 to 1:7, alternatively of from 1:3.1 to 1:6.1, (A):(B). It will be appreciated that ratios outside of these ranges may be utilized as well. For example, in certain embodiments, the organic alcohol of the alcohol component (B) is utilized in a gross excess (e.g. in an amount of ≥10, alternatively ≥15, alternatively ≥20, times the molar amount of the initial organosilicon compound (A)), such as when the organic alcohol of the alcohol component (B) is utilized as a carrier (i.e., a solvent, diluent, etc.) during the reaction. However, one of skill in the art will readily select the particular ratio of the initial organosilicon compound (A) and the organic alcohol of the alcohol component (B) to be reacted in view of the description above, including with respect to the theoretical maximum ratio of (A):(B) for a particular transalkoxylation according to the method, the presence of any carrier vehicle, the particular initial organosilicon compound (A) utilized (e.g. the nature of the alcohol R1OH to be formed therefrom), etc. The catalyst (C) comprises (C1) an ammonium carboxylate compound or (C2) a titanate compound. The particular catalyst (C) utilized is selected in view of the particular initial organosilicon compound (A) to be transalkoxylated. In particular, the selection of the functional group of R3(e.g. where the initial organosilicon compound comprises the functional moiety R3) will control which particular catalyst (C) may be utilized. In general, the use of the ammonium carboxylate compound (C1) is not limited, and may be utilized with any of the initial organosilicon compounds (A) described above. As such, in certain embodiments, the catalyst (C) comprises, alternatively is, the ammonium carboxylate compound (C1). In particular embodiments, the catalyst (C) comprises, alternatively is, the titanate compound (C2). Ammonium Carboxylate Compound (C1) In certain embodiments, the catalyst (C) comprises the ammonium carboxylate compound (C1). The ammonium carboxylate compound (C1) is not particularly limited, and generally comprises the reaction product of an amine compound and a carboxylic acid. One of skill in the art will appreciate that the reaction of the amine compound and the carboxylic acid is generally an acid-base reaction, where the amine compound (i.e., a base) is protonated by the carboxylic acid to give an ammonium cation and a carboxylate anion, which are collectively referred to as the ammonium carboxylate compound, regardless of whether such ions are closely or transiently coordinated. In general, suitable amine compounds include amino-functional organic compounds (e.g. amine-substituted hydrocarbon compounds). In particular, the amine compound typically comprises a moiety having the general formula: where each R5is an independently selected substituted or unsubstituted hydrocarbyl group having from 1 to 18 carbon atoms, and subscript b is 0, 1, or 2. Examples of hydrocarbyl groups suitable for R5include any of those described above. For example, in certain embodiments, each R5is a substituted or unsubstituted hydrocarbyl group having from 1 to 16, alternatively from 1 to 14, alternatively from 1 to 12, alternatively from 1 to 10, alternatively from 1 to 9, alternatively from 1 to 8, alternatively from 1 to 7 carbon atoms. In some such embodiments, each R5is a linear, branched, and/or cyclic alkyl group. In some embodiments, subscript b is 0, such that the amine compound is a primary amine. In other embodiments, subscript b is 1, such that the amine compound is a secondary amine. In additional embodiments, subscript b is 2, such that the amine compound is a tertiary amine. In some embodiments, the amine compound is an organic amine having the general formula: where each R5and subscript b are as defined above and R14is an independently selected substituted or unsubstituted hydrocarbyl group having from 1 to 22 carbon atoms. Examples of hydrocarbyl groups suitable for R14include any of those described above, such that R14may be the same as or different from any R5, if present, of the amine compound. For example, in certain embodiments, R14is a substituted or unsubstituted hydrocarbyl group having from 1 to 20, alternatively from 2 to 20, alternatively from 2 to 18 carbon atoms. In particular embodiments, R14is a linear, branched, and/or cyclic alkyl group. In particular embodiments, the amine compound is an organic amine having the general formula above where subscript b is 0 or 1, such that the amine compound may be defined as a primary or secondary organic amine, respectively. In some such embodiments, subscript b, each R5, and R14are selected such that the amine compound comprises a total of from 3 to 20, alternatively from 4 to 20, alternatively from 5 to 20, alternatively from 5 to 18 carbon atoms. It is to be appreciated that the amine compound may be a cyclic amine, such as a secondary or tertiary amine with at least two nitrogen-bonded substituents being joined to one another in a ring structure (i.e., the amine compound may be a heterocyclic amine, such as a pyrrole, pyrrolidine, imidazole, thiazole, pyridine, piperidine, morpholine, etc.). Typically, the amine compound is selected from volatile organic amines. For example, in certain embodiments the organic amine has a vaporization point of less than 300, alternatively less than 250, alternatively less than 240, alternatively less than 230, alternatively less than 220, alternatively less than 210, alternatively less than 200° C., at atmospheric pressure. It is to be understood that the term vaporization point, as used herein, refers to a temperature at which a compound in a solid or liquid phase is converted to a vapor/gaseous phase (e.g. via evaporation, sublimation, etc.). In this sense, the vaporization point may correspond to a boiling point of such a compound (e.g. where the compound is a liquid). In particular embodiments, the amine compound has a vaporization point of from 50 to 250, alternatively from 60 to 250, alternatively from 60 to 235, alternatively from 70 to 235, alternatively from 70 to 220° C., at atmospheric pressure. Examples of particular amine compounds suitable for use in preparing the ammonium carboxylate compound (C1) include: alkylamines, such as aliphatic primary alkylamines including methylamine, ethylamine, propyl amines (e.g. n-propylamine, isopropylamine, etc.), butyl amines (e.g. n-butylamine, sec-butylamine, isobutylamine, t-butylamine, etc.), pentyl amines (e.g. pentylamine, 2-aminopentane, 3-aminopentane, 1-amino-2-methylbutane, 2-amino-2-methylbutane, 3-amino-2-methylbutane, 4-amino-2-methylbutane, etc.), hexylamines (e.g. hexylamine, 5-amino-2-methylpentane, etc.), heptylamines, octylamines, nonylamines, decylamines, undecylamines, dodecylamines, tridecylamines, tetradecylamines, pentadecylamines, hexadecylamines, heptadecylamines, octadecylamines, and the like; aliphatic secondary alkylamines, such as dimethylamine, diethylamine, dipropylamine, diisopropylamine, dibutylamine, diisobutylamine, di-sec-butylamine, di-tert-butylamine, dipentylamine, dihexylamine, diheptylamine, dioctylamine, dinonylamine, didecylamine, methylethylamine, methypropylamine, methylisopropylamine, methylbutylamine, methylisobutylamine, methyl-sec-butylamine, methyl-tert-butylamine, methylamylamine, methylisoamylamine, ethylpropylamine, ethylisopropylamine, ethylbutylamine, ethylisobutylamine, ethyl-sec-butylamine, ethyl-tert-butylamine, ethylisoamylamine, propylbutylamine, propylisobutylamine, and the like; as well as derivatives, modifications, and combinations thereof. For example, mixed secondary alkylamines (e.g. N-ethylisopropylamine, etc.), such as any of those comprising a combination of the alkyl groups listed in the above examples, may also be utilized. In particular embodiments, the amine compound comprises, alternatively is, octadecylamine and/or diethylamine. In certain embodiments, the ammonium carboxylate compound (C1) comprises (i.e., is formed from) more than one amine compound, such as 2, 3, 4, or more amine compounds. In such embodiments, each amine compound is independently selected, and may be the same as or different from any amine compound of the ammonium carboxylate compound (C1). In certain embodiments, the ammonium carboxylate compound (C1) comprises (i.e., is formed from) more than one amine compound, such as 2, 3, 4, or more amine compounds. In such embodiments, each amine compound is independently selected, and may be the same as or different from any amine compound of the ammonium carboxylate compound (C1). Likewise, the catalyst (C) may comprise more than one ammonium carboxylate compound (C1), such as such as 2, 3, 4, or more ammonium carboxylate compounds (C1). In such embodiments, the amine compound of each ammonium carboxylate compound (C1) is independently selected, and may independently complex, coordinate, ion pair, or otherwise associate with any carboxylic acid of the catalyst (C) (i.e., when the amine compound is protonated to a corresponding ammonium cation and the carboxylic acid is deprotonated to a corresponding carboxylate anion). In general, suitable carboxylic acid compounds for use in preparing the ammonium carboxylate compound (C1) have the general formula: where R6is an independently selected substituted or unsubstituted hydrocarbyl group having from 1 to 18 carbon atoms. Examples of hydrocarbyl groups suitable for R6include any of those described above. For example, in certain embodiments, R6is a substituted or unsubstituted hydrocarbyl group having from 1 to 16, alternatively from 1 to 14, alternatively from 1 to 12 carbon atoms. In particular embodiments, R6is a linear, branched, and/or cyclic alkyl group. In some embodiments, R5is propyl or methyl. Typically, the carboxylic acid is selected from volatile carboxylic acids. For example, in certain embodiments the carboxylic acid comprising a vaporization point of less than 300, alternatively less than 255, alternatively less than 240, alternatively less than 230, alternatively less than 220, alternatively less than 220, alternatively less than 200, alternatively less than 190, alternatively less than 180° C., at atmospheric pressure. In particular embodiments, the carboxylic acid has a boiling point of from 100 to 250, alternatively from 100 to 225, alternatively from 100 to 200, alternatively from 100 to 175, alternatively from 100 to 150° C., at atmospheric pressure. Examples of particular carboxylic acids suitable for use in preparing the ammonium carboxylate compound (C1) include ethanoic acids (e.g. acetic acid), propanoic acids (e.g. propionic acid), butanoic acids (e.g. butyric acid), pentanoic acids (e.g. valeric acid), hexanoic acids (e.g. caproic acid), heptanoic acids (e.g. enanthic acid), octanoic acids (e.g. caprylic acid), nonanoic acids (e.g. pelargonic acid), decanoic acids (e.g. capric acid), and the like, as well as derivatives, modifications, and combinations thereof. In certain embodiments, the carboxylic acid is acetic acid and/or propionic acid, such that the ammonium carboxylate compound (C1) comprises acetate and/or propionate. While linear carboxylic acids are exemplified above, it will be appreciated that cyclic and/or branched carboxylic acids may also be utilized. In certain embodiments, the ammonium carboxylate compound (C1) comprises (i.e., is formed from) more than one carboxylic acid, such as 2, 3, 4, or more carboxylic acids. In such embodiments, each carboxylic acid is independently selected, and may be the same as or different from any carboxylic acid of the ammonium carboxylate compound (C1). Likewise, the catalyst (C) may comprise more than one ammonium carboxylate compound (C1), such as such as 2, 3, 4, or more ammonium carboxylate compounds (C1). In such embodiments, the carboxylic acid of each ammonium carboxylate compound (C1) is independently selected, and may independently complex, coordinate, ion pair, or otherwise associate with any amine compound of the catalyst (C) (i.e., when the amine compound is protonated to a corresponding ammonium cation and the carboxylic acid is deprotonated to a corresponding carboxylate anion). In certain embodiments, the catalyst (C) comprises the titanate compound (C2). Typically, the titanate compound (C2) is an organotitanium compound, such as a titanium (IV) complex comprising alkoxide and/or enolate-type ligands (i.e., a titanium chelate catalyst). The titanate compound (C2) may comprise any combination of alkoxide and enolate-type ligands, which are described in further detail below, limited only by the particular ligands selected and the ability of the same to coordinate with titanium to give the titanate compound (C2). For example, the titanate compound (C2) may comprise one ligand that is the same as one, two, or each other ligand, e.g. in terms of ligand type, structure, etc. Likewise, the titanate compound (C2) may one or more ligand that is different from every other ligand. In certain embodiments, the titanate compound (C2) comprises at least one alkoxide ligand. In such embodiments, the titanate compound (C2) may comprise one, two, three, or four such alkoxide ligands, such that the titanate compound (C2) is a titanium mono-, di-, tri-, or tetraalkoxide, respectively. It is to be appreciated that the titanate compound (C2) may comprise a bidentate alkoxide ligand (e.g. a dialkoxide ligand) as well as the monoalkoxides described in detail herein. In some embodiments, the titanate compound (C2) comprises at least one enolate-type ligand. Suitable enolate-type ligands are exemplified by enolates formed from parent beta-dicarbonyl compounds, such from beta-diketones (i.e., diones), beta-ketoesters, and/or beta-diesters, which may be referred to as a beta-dicarbonyl enolates. In such embodiments, the titanate compound (C2) may comprise two, three, or four such enolate-type ligands, such that the titanate compound (C2) is a titanium bis-, tris-, or tetrakis(beta-carbonyl enolate). In particular embodiments, the titanate compound (C2) comprises at least one alkoxide ligand and at least one beta-carbonyl enolate. It will be appreciated that the titanate compound (C2) may comprise multiple titanium complexes (e.g. due to ligand exchange, preparation, etc.), and thus may comprise, alternatively may be, a titanium tetrakis(beta-carbonyl enolate), titanium alkoxide tris(beta-carbonyl enolate), titanium dialkoxide bis(beta-carbonyl enolate), titanium trialkoxide (beta-carbonyl enolate), a titanium tetraalkoxide, or combinations thereof. In particular embodiments, the titanate compound (C2) has the general formula TiX4, where each X is independently of formula Y—CH—Y or —OR7, wherein each Y is independently of formula R7C(O)— or R7OC(O)— and each R7is an independently selected hydrocarbyl group. As will be understood by those of skill in the art in view of the embodiments described herein, each X of formula —OR7may be defined as an alkoxide ligand and each X of formula Y—CH—Y may be defined as a beta-dicarbonyl enolate ligand. With regard to beta-dicarbonyl enolate ligands in particular, as introduced above, each Y is independently of formula R7C(O)— or R7OC(O)—. Accordingly, in certain embodiments, the titanate compound (C2) may comprise a ligand X selected from beta-diketo enolates of formula R7C(O)—CH—C(O)R7, beta-ketoester enolates of formula R7OC(O)—CH—C(O)R7, and beta-diester enolates of formula R7OC(O)—CH—C(O)R7. In general, for any of ligands X described above, each R7is an independently selected substituted or unsubstituted hydrocarbyl group. Hydrocarbyl groups suitable for R7are exemplified by, and may include, any of those described above, as well as methyl, ethyl, trifluoromethyl, 4-methoxyphenyl, 4-chlorophenyl, tert-butyl, 2-pyridyl, heptafluoropropyl, isobutyl, 2-mesetylenyl, phenyl, benzyl, 2-thienyl, and 2-napthyl groups. Typically, each R7is independently selected from substituted and unsubstituted hydrocarbyl groups having from 1 to 18, alternatively from 1 to 10, alternatively from 1 to 8, alternatively from 1 to 6 carbon atoms. For example, in certain embodiments, each R7is independently selected from substituted, unsubstituted, linear, and/or branched methyl, propyl, butyl, pentyl, and hexyl groups. Examples of titanium chelate catalysts suitable for use in or as the titanate compound (C2) include titanium methoxides, titanium ethoxides, titanium propoxides (e.g. titanium propoxide, titanium isopropoxide, etc.), titanium butoxides (e.g. titanium butoxide, titanium isobutoxide, titanium tertbutoxide etc.), titanium methylacetoacetates, titanium isopropylacetoatates, titanium butylacetoacetates, titanium acetylacetonates, and the like, as well as derivatives, modifications, and combinations thereof (e.g. titanium diisopropoxide bis(ethyl acetoacetate), titanium diisopropoxide bis(acetylacetonate), titanium dibutoxide bis(methyl acetoacetate), etc.). Methods of preparing catalysts (C1) and (C2) are known in the art, with the catalysts themselves and/or compounds used to prepare the same being commercially available from various suppliers. As such, the catalyst (C) may be prepared as part of the method, or otherwise obtained (i.e., as a prepared compound). Preparing the catalyst (C) may be performed prior to the reaction of components (A) and (B), or in situ (i.e., during the reaction of components (A) and (B), e.g. via combining components of the catalyst (C) with components (A) and/or (B)). For example, in some embodiments, the method comprises combining the amine compound and the carboxylic acid compound with the initial organosilicon compound (A) and/or the alcohol component (B), thereby forming the ammonium carboxylate compound (C1) (i.e., the catalyst (C)), in situ. The catalyst (C) may be utilized in any form, such as neat (i.e., absent solvents, carrier vehicles, diluents, etc.), or disposed in a carrier vehicle, such as a solvent or dispersant (e.g. such as any of those listed above with respect to the initial organosilicon compound (A) and/or the alcohol component (B)). In some embodiments, the catalyst (C) is utilized in a form absent water and/or carrier vehicles/volatiles reactive with the initial organosilicon compound (A), the alcohol component (B), and/or the catalyst (C) itself (i.e., until combined with components (A) and (B). For example, in certain embodiments, the method may comprise stripping the catalyst (C) of volatiles and/or solvents (e.g. water, organic solvents, etc.). Techniques for stripping the catalyst (C) are known in the art, and may include heating, drying, applying reduced pressure/vacuum, azeotroping with solvents, utilizing molecular sieves, etc., and combinations thereof. The catalyst (C) may be utilized in any amount, which will be selected by one of skill in the art, e.g. dependent upon the particular catalyst (C) selected (i.e., whether (C1) or (C2), the particular species utilized, etc.), the reaction parameters employed, the scale of the reaction (e.g. total amounts of components (A) and (B)), etc. The molar ratio of the catalyst (C) to components (A) and/or (B) utilized in the reaction may influence the rate and/or amount transalkoxylation to prepare the alkoxy-functional organosilicon compound. Thus, the amount of the catalyst (C) as compared to components (A) and/or (B), as well as the molar ratios therebetween, may vary. Typically, these relative amounts and the molar ratio are selected to maximize the transalkoxylation of component (A) to the alkoxy-functional organosilicon compound, while minimizing the loading of the catalyst (C) (e.g. for increased economic efficiency of the reaction, increased ease of purification of the reaction product formed, etc.). In certain embodiments, the catalyst (C) is utilized in the reaction in an amount of from 0.001 to 30 mol % based on the total amount of component (A) utilized. For example, the catalyst (C) may be used in an amount of from 0.005 to 25, alternatively of from 0.005 to 20, alternatively of from 0.01 to 20, mol % based on the total amount of component (A) utilized. Typically, components (A) and (B) are reacted in a vessel or reactor to prepare the alkoxy-functional organosilicon compound. When the reaction is carried out at an elevated or reduced temperature as described below, the vessel or reactor may be heated or cooled in any suitable manner, e.g. via a jacket, mantle, exchanger, bath, coils, etc. Components (A), (B), and (C) may be fed together or separately to the vessel, or may be disposed in the vessel in any order of addition, and in any combination. For example, in certain embodiments, components (B) and (C) are added to a vessel containing component (A). In such embodiments, components (B) and (C) may be first combined prior to the addition, or may be added to the vessel sequentially (e.g. (C) then (B)). In other embodiments, component (C) is added to a vessel containing components (A) and (B). In general, reference to the “reaction mixture” herein refers generally to a mixture comprising components (A), (B), and (C) (e.g. as obtained by combining such components, as described above). The method may further comprise agitating the reaction mixture. The agitating may enhance mixing and contacting together components (A), (B), and (C) when combined, e.g. in the reaction mixture thereof. Such contacting independently may use other conditions, with (e.g. concurrently or sequentially) or without (i.e., independent from, alternatively in place of) the agitating. The other conditions may be tailored to enhance the contacting, and thus reaction (i.e., transalkoxylation), of the initial organosilicon compound (A) with the alcohol component (B) to form the alkoxy-functional organosilicon compound. Other conditions may be result-effective conditions for enhancing reaction yield or minimizing amount of a particular reaction by-product included within the reaction product along with the alkoxy-functional organosilicon compound. In certain embodiments, the reaction of components (A) and (B) is carried out in the presence of a carrier vehicle or solvent, such as one or more of those described above. For example, portions of carrier vehicle or solvent may be added to or otherwise combined with the initial organosilicon compound (A), the alcohol component (B), and/or the catalyst (C) discretely, collectively with mixtures of components (A), (B), and/or (C), or with the reaction mixture as a whole. The total amount of carrier vehicle/solvent present in the reaction mixture will be selected by one of skill in the art, e.g. based on the particular component (A), (B), and/or (C) selected, the reaction parameters employed, etc.). In certain embodiments, the reaction of components (A) and (B) is carried out in the absence of any carrier vehicle or solvent. For example, no carrier vehicle or solvent may be combined discretely with the initial organosilicon compound (A), the alcohol component (B), and/or the catalyst (C). In these or other embodiments, none of components (A), (B), and (C), are disposed in any carrier vehicle or solvent, such that no carrier vehicle or solvent is present in the reaction mixture during the transesterification (i.e., the reaction mixture is free from, alternatively substantially free from, solvents). The above notwithstanding, in certain embodiments, one of components (A), (B), and/or (C) may be a carrier, e.g. when utilized as a fluid in an amount sufficient to carry, dissolve, or disperse any other component of the reaction mixture. In specific embodiments, the alcohol component (B) is utilized as a carrier. Additionally, it will be appreciated that the transesterification of the initial organosilicon compound (A) with the alcohol component (B) results in the production of the alcohol of formula R1—OH (hereinafter the “alcohol byproduct”), where R1is as defined above with respect to the initial organosilicon compound (A). The alcohol byproduct may itself be utilized as a carrier (i.e., once produced). In certain embodiments, the alcohol byproduct is removed from the reaction mixture once produced. As understood in the art, transesterifications are reversible reactions, such that removing the alcohol byproduct from the reaction mixture influences the reaction in terms of selectivity in favor, and/or overall yields, of the alkoxy-functional organosilicon compound (e.g. by selectively driving the equilibrium of the reaction). Typically, the alcohol byproduct is volatile, or at least more volatile than components (A), (B), and/or (C) in the reaction mixture. The removal of the alcohol byproduct may include distillation, heating, applying reduced pressure/vacuum, azeotroping with solvents, utilizing molecular sieves, etc., and combinations thereof. In certain embodiments, the alcohol byproduct is distilled from the reaction mixture during the reaction, such that the reaction is carried out under distillation conditions. The distillation conditions typically include (i) an elevated temperature; (ii) a reduced pressure; or (iii) both an elevated temperature and reduced pressure. By elevated or reduced, it is meant as compared to room temperature and atmospheric pressure. As understood in the art, the number of trays utilized in any distillation may be optimized, and may influence the rate and/or recovery of the alcohol byproduct with respect to the distillate produced. The distillation may be continuous or batched, and may include use of a solvent (e.g. hexane, toluene, etc.), such that the distillation may be an azeotropic distillation. The distillate comprising the azeotropic solvent utilized may be reused and/or recycled after removing the alcohol byproduct therefrom (e.g. via solvent phase extraction). In some embodiments, the reaction is carried out at the elevated temperature. The elevated temperature will be selected and controlled depending on the particular initial organosilicon compound (A) selected, the particular the alcohol component (B) selected, the particular alcohol byproduct being produced (e.g. as a factor of substituent(s) R1O— of component (A)), the reaction vessel selected (e.g. whether open to ambient pressure, sealed, under reduced pressure, etc.) and combinations thereof. Accordingly, the elevated temperature will be readily selected by one of skill in the art in view of the reaction conditions and parameters selected and the description herein. The elevated temperature is typically from greater than ambient temperature to 140° C., such as from 30 to 130, alternatively from 40 to 130, alternatively from 40 to 120, alternatively from 50 to 120, alternatively from 50 to 110, alternatively from 50 to 100, alternatively from 60 to 100° C. In certain embodiments, the reaction is carried out at reduced pressure. The reduced pressure will be selected and controlled depending on the particular initial organosilicon compound (A) selected, the particular the alcohol component (B) selected, the particular alcohol byproduct being produced (e.g. as a factor of substituent(s) R1O— of component (A)), and combinations thereof. Accordingly, the reduced pressure will be readily selected by one of skill in the art in view of the reaction conditions and parameters selected and the description herein. The reduced pressure is typically operated as a vacuum although any reduced pressure between vacuum and atmospheric pressure (i.e., 101.325 kPa) may be utilized. For example, the reduced pressure may be from greater than 0 to 30, alternatively from greater than 0 to 20, alternatively from greater than 0 to 15, alternatively from greater than 0 to 10, alternatively from greater than 0 to 8, alternatively from greater than 0 to 6, alternatively from greater than 0 to 5, alternatively from greater than 0 to 4, alternatively from greater than 0 to 3, alternatively from greater than 0 to 2, kPa (e.g. as measured by mmHg). It is to be appreciated that the elevated temperature and/or reduced pressure may also differ from the ranges set forth above, especially when both elevated temperature and reduced pressure are utilized. For example, in certain embodiments, the reduced pressure is utilized in order to maintain reaction progression while utilizing a lower reaction temperature, which may lead to a decrease in the formation of undesirable byproducts (e.g. polymerization byproducts when the R3of the initial organosilicon compound (A) comprises, alternatively is, an acryloxy group). Likewise, it is also to be appreciated that reaction parameters may be modified during the reaction of components (A) and (B). For example, temperature, pressure, and other parameters may be independently selected or modified during the reaction. Any of these parameters may independently be an ambient parameter (e.g. room temperature and/or atmospheric pressure) and/or a non-ambient parameter (e.g. reduced or elevated temperature and/or reduced or elevated pressure). Any parameter, may also be dynamically modified, modified in real time, i.e., during the method, or may be static (e.g. for the duration of the reaction, or for any portion thereof.) The time during which the reaction of components (A) and (B) to prepare the alkoxy-functional organosilicon compound is carried out is a function of scale, reaction parameters and conditions, selection of particular components, etc. On a relatively large scale (e.g. greater than 1, alternatively 5, alternatively 10, alternatively 50, alternatively 100 kg), the reaction may be carried out for hours, such as from 1 to 48, alternatively from 2 to 36, alternatively from 4 to 24, alternatively of 6, 12, 18, 24, 36, or 48 hours, as will be readily determined by one of skill in the art (e.g. by monitoring conversion of the initial organosilicon compound (A), production of the alkoxy-functional organosilicon compound, etc., such as via chromatographic and/or spectroscopic methods). In certain embodiments, the time during which the reaction is carried out is from greater than 0 to 48 hours, alternatively from 1 to 36 hours, alternatively from 1 to 24 hours, alternatively from 1 to 12 hours, alternatively from 2 to 12 hours, alternatively from 2 to 8 hours, after components (A) and (B) are combined in the presence of component (C). Generally, the reaction of components (A) and (B) prepares a reaction product comprising the alkoxy-functional organosilicon compound. In particular, over the course of the reaction, the reaction mixture comprising components (A), (B), and (C) comprises increasing amounts of the alkoxy-functional organosilicon compound and decreasing amounts of components (A) and (B). Once the reaction is complete (e.g. one of components (A) and (B) is consumed, no additional alkoxy-functional organosilicon compound is being prepared, etc.), the reaction mixture may be referred to as a reaction product comprising the alkoxy-functional organosilicon compound. In this fashion, the reaction product typically includes any remaining amounts of components (A), (B), and (C), as well as degradation and/or reaction products thereof (e.g. materials which were not previously removed via any distillation, stripping, etc.). If the reaction is carried out in any carrier vehicle or solvent, the reaction product may also include such carrier vehicle or solvent. However, because the method is typically carried out neat (i.e., in the absence of added solvents) and performed under distillation and/or other heated conditions, this is typically not the case. In certain embodiments, the method further comprises isolating and/or purifying the alkoxy-functional organosilicon compound from the reaction product. As used herein, isolating the alkoxy-functional organosilicon compound is typically defined as increasing the relative concentration of the alkoxy-functional organosilicon compound as compared to other compounds in combination therewith (e.g. in the reaction product or a purified version thereof). As such, as is understood in the art, isolating/purifying may comprise removing the other compounds from such a combination (i.e., decreasing the amount of impurities combined with the alkoxy-functional organosilicon compound, e.g. in the reaction product) and/or removing the alkoxy-functional organosilicon compound itself from the combination. Any suitable technique and/or protocol for isolation may be utilized. Examples of suitable isolation techniques include distilling, stripping/evaporating, extracting, filtering, washing, partitioning, phase separating, chromatography, and the like. As will be understood by those of skill in the art, any of these techniques may be used in combination (i.e., sequentially) with any another technique to isolate the alkoxy-functional organosilicon compound. It is to be appreciated that isolating may include, and thus may be referred to as, purifying the alkoxy-functional organosilicon compound. However, purifying the alkoxy-functional organosilicon compound may comprise alternative and/or additional techniques as compared to those utilized in isolating the alkoxy-functional organosilicon compound. Regardless of the particular technique(s) selected, isolation and/or purification of alkoxy-functional organosilicon compound may be performed in sequence (i.e., in line) with the reaction itself, and thus may be automated. In other instances, purification may be a stand-alone procedure to which the reaction product comprising the alkoxy-functional organosilicon compound is subjected. In particular embodiments, isolating the alkoxy-functional organosilicon compound comprises distilling and/or stripping volatiles from the reaction product. For example, in certain embodiments, such as where component (B) is used in excess of component (A), remaining amounts of component (B) are distilled and/or stripped from the reaction mixture comprising the alkoxy-functional organosilicon compound. In these or other embodiments, isolating the alkoxy-functional organosilicon compound comprises filtering the reaction product to remove remaining amounts of the catalyst (C) and/or solids formed therefrom. In both or either case (e.g. after removing components (B) and/or (C) via stripping/distillation and/or filtration), the reaction product may be referred to as a purified reaction product comprising the alkoxy-functional organosilicon compound. In particular embodiments, the method further comprises purifying the alkoxy-functional organosilicon compound. Any suitable technique for purification may be utilized. In certain embodiments, purifying the alkoxy-functional organosilicon compound comprises distillation, to either remove the alkoxy-functional organosilicon compound (e.g. as a distillate) or to strip other compounds/components therefrom (i.e., leaving the alkoxy-functional organosilicon compound in the pot as a high-boiling component of the reaction mixture or purified reaction mixture. As will be appreciated by those of skill in the art, distilling the reaction product or purified reaction product to purify and/or isolate the alkoxy-functional organosilicon compound is typically carried out at an elevated temperature and a reduced pressure. The elevated temperature and reduced pressure are independently selected, e.g. based on the particular components of the reaction, the particular alkoxy-functional organosilicon compound prepared, other isolation/purification techniques utilized, etc. For example, any of the elevated temperatures and reduced pressures described herein may be utilized in purifying the alkoxy-functional organosilicon compound. As will be appreciated by those of skill in the art in view of the description above, the particular alkoxy-functional organosilicon compound prepared in accordance with the method is a function of the particular initial organosilicon compound (A) and the alcohol component (B) utilized. According, in some embodiments, reacting components (A) and (B) in the presence of the catalyst (C) prepares an alkoxy-functional organosilicon compound having the general formula (I): where each of R2, R3, D1, and subscript a is defined above with respect to the initial organosilicon compound (A), and R4is defined above with respect to the alcohol component (B). For example, in certain embodiments each R2is an independently selected substituted or unsubstituted hydrocarbyl group; R3comprises an epoxide group, an ester group, an anhydride group, or an acryloxy group; each R4is an independently selected substituted or unsubstituted hydrocarbyl group; D1is a divalent linking group; and subscript a is 1, 2, or 3. As will be understood by one of skill in the art in view of the description herein, the particular initial organosilicon compound (A) utilized in the method forms all of the alkoxy-functional organosilicon compound of general formula (I) with the exclusion of the alkoxy groups of formula (R4O—), which are formed by the alcohol component (B) utilized. As such, where formulas, structures, moieties, groups, or other such motifs are shared between the alkoxy-functional organosilicon compound of formula (I) and components (A) and/or (B), the description above with respect to such shared motifs may equally describe the alkoxy-functional organosilicon compound prepared. However, the alkoxy-functional organosilicon compound prepared according to the method is not limited to the general formula (I) shown above, as described below. In certain embodiments, reacting the initial organosilicon compound (A) and the alcohol component (B) in the presence of the catalyst (C) produces an intermediate alkoxy-functional organosilicon compound (i.e., via an “initial reaction”), and the method further comprises reacting the intermediate alkoxy-functional organosilicon compound with (D) an amino-functional organosiloxane compound, thereby preparing the alkoxy-functional organosilicon compound (i.e., via a “second reaction”). Said differently, in such embodiments, the alkoxy-functional organosilicon compound having the general formula (I) described above may be alternatively defined as an intermediate alkoxy-functional organosilicon compound, which is subsequently reacted with the amino-functional organosiloxane compound (D) to prepare the alkoxy-functional organosilicon compound. It is to be understood that, in such embodiments, the description above regarding reacting components (A) and (B), the reaction product prepared thereby, and isolating and/or purifying the alkoxy-functional organosilicon compound therefrom, may thus describe preparing, isolating, and/or purifying the intermediate alkoxy-functional organosilicon compound prepared according to the initial reaction. However, the intermediate alkoxy-functional organosilicon compound may be utilized at any time after preparation, such that no isolating and/or purifying procedures need be utilized. For example, the initial reaction and the second reaction may performed concurrently (e.g. in a one-pot reaction, etc.). Amino-Functional Organosiloxane Compound (D) The amino-functional organosiloxane compound (D) is not particularly limited, and may be any organosiloxane compound comprising an amine functional group (i.e., an amino group) suitable for bonding the organosiloxane compound to the functional group of R3described above with respect to the intermediate alkoxy-functional organosilicon compound (e.g. via a substitution reaction, displacement reaction, alkylation reaction, etc.), as will be understood by one of skill in the art in view of the description herein. In general, the amino-functional organosiloxane compound (D) has the following general formula: where each R8is an independently selected substituted or unsubstituted hydrocarbyl group; each R9is independently R8or an amino group of formula -D2-NH2, where D2is an independently selected divalent linking group, with the proviso that at least one R9is the amino group; subscript m is from 0 to 1000; and subscript n is from 1 to 100. In general, hydrocarbyl groups suitable for R8include those described above, such as those described above with particular reference to R1and/or R2of the initial organosilicon compound (A). In specific embodiments, each R8is independently an alkyl group having from 1 to 10, alternatively from 1 to 8, alternatively from 1 to 6, alternatively from 1 to 4, alternatively from 1 to 3, alternatively from 1 to 2, alternatively 1, carbon atom(s). For example, in some such embodiments, each R8is methyl. Each R9is independently selected from R8and amino groups of formula -D2-NH2. In general, each D2is independently a divalent linking group, and may be linear or branched and substituted or unsubstituted. Typically, D2is selected from divalent substituted or unsubstituted hydrocarbon groups, which may optionally be modified or substituted, e.g. with ether, ester, amino, and/or silyl groups. For example, in some embodiments, D2comprises a hydrocarbon moiety having the formula —(CH2)c—, where subscript c is ≥1, such as from 1 to 18, alternatively from 1 to 16, alternatively from 1 to 12, alternatively from 1 to 10, alternatively from 1 to 8, alternatively from 1 to 6, alternatively from 2 to 6. In these or other embodiments, D2may comprise a substituted hydrocarbon, i.e., a hydrocarbon group comprising a backbone substituted with (e.g. in and/or on the carbon chain) at least one heteroatom (e.g. O, N, S, etc.). For example, in some embodiments, D2is a hydrocarbon comprising one or more N atoms in, or bonded to one of the carbon atoms of, the backbone. In some such embodiments, D2may comprise, alternatively may be, an aminoalkyl moiety, such the amino group is a diamino group (i.e., at least one R9of the amino-functional organosiloxane compound (D) is a diamine). As will be appreciated by those of skill in the art, subscripts m and n represent the number of siloxy units in, and thus the degree of polymerization (DP) of, the amino-functional organosiloxane compound (D). It will be appreciated that the siloxy units indicated by subscripts m and n may be in any order (e.g. randomized and/or block from, etc.) in the amino-functional organosiloxane compound (D). In general, the amino-functional organosiloxane compound (D) has a DP of from 1 to 1100. In particular embodiments, the amino-functional organosiloxane compound (D) has a DP greater than 400, alternatively greater than 500, alternatively greater than 600, alternatively greater than 700, alternatively greater than 800. In some embodiments, the amino-functional organosiloxane compound (D) has a DP less than 400, alternatively less than 350, alternatively less than 300, alternatively less than 250, alternatively less than 200, alternatively less than 150, alternatively less than 100. Subscript m is from (and including) 0 to 1000. In some embodiments, subscript m is from 1 to 1000, alternatively from 100 to 1000, alternatively from 200 to 1000, alternatively from 300 to 1000, alternatively from 400 to 1000, alternatively from 500 to 1000. In certain embodiments, subscript m is from 0 to 100, alternatively from 0 to 80, alternatively from 0 to 60, alternatively from 0 to 40, alternatively from 0 to 20, alternatively from 1 to 20. In particular embodiments, subscript m is 0. Subscript n is from (and including) 1 to 100. In some embodiments, subscript n is from 1 to 90, alternatively from 1 to 80, alternatively from 1 to 70, alternatively from 1 to 60, alternatively from 1 to 50, alternatively from 1 to 40, alternatively from 1 to 30, alternatively from 1 to 20, alternatively from 2 to 20, alternatively from 3 to 20, alternatively from 4 to 20, alternatively from 5 to 20. In general, the amino-functional organosiloxane compound (D) comprises at least one of the amino groups. However, in some embodiments, the amino-functional organosiloxane compound (D) comprises at least two, alternatively at least three, alternatively at least four, alternatively at least five of the amino groups. As will be understood by those of skill in the art, each R9is independently selected in each moiety indicated by subscript n, such that the amino-functional organosiloxane compound (D) may comprise a total of from 1 to n+2 number of the amino groups. For example, in certain embodiments, the amino-functional organosiloxane compound (D) comprises from 1 to 102 of the amino groups, such as from 1 to 100, alternatively from 1 to 90, alternatively from 1 to 80, alternatively from 1 to 70, alternatively from 1 to 60 of the amino groups. In these or other embodiments, the amino-functional organosiloxane compound (D) comprises from 1 to 20 of the amino groups, such as from 1 to 18, alternatively from 1 to 16, alternatively from 1 to 14, alternatively from 1 to 12, alternatively from 1 to 10 of the amino groups. The amino-functional organosiloxane compound (D) may be utilized in any form, such as neat (i.e., absent solvents, carrier vehicles, diluents, etc.), or disposed in a carrier vehicle, such as a solvent or dispersant (e.g. such as any of those listed above). In some embodiments, the amino-functional organosiloxane compound (D is utilized in the absence of water and carrier vehicles/volatiles reactive with the intermediate alkoxy-functional organosilicon compound, the amino-functional organosiloxane compound (D) itself, and/or any other components utilized in the second reaction. For example, in certain embodiments, the method may comprise stripping the amino-functional organosiloxane compound (D) of volatiles and/or solvents (e.g. water, organic solvents, etc.) prior to reacting the same with the intermediate alkoxy-functional organosilicon compound. Techniques for stripping the amino-functional organosiloxane compound (D) are known in the art, and may include heating, drying, applying reduced pressure/vacuum, azeotroping with solvents, utilizing molecular sieves, etc., and combinations thereof. In certain embodiments, the method comprises utilizing more than one amino-functional organosiloxane compound (D), such as 2, 3, 4, or more amino-functional organosiloxane compounds (D). In such embodiments, each amino-functional organosiloxane compound (D) is independently selected, and may be the same as or different from any other amino-functional organosiloxane compound (D). The amino-functional organosiloxane compound (D) may be utilized in any amount, which will be selected by one of skill in the art, e.g. dependent upon the particular intermediate alkoxy-functional organosilicon compound and/or amino-functional organosiloxane compound (D) utilized, the reaction parameters employed, the scale of the reaction (e.g. total amounts of the intermediate alkoxy-functional organosilicon compound and/or component (D) to be reacted and/or the alkoxy-functional organosilicon compound to be prepared), etc. As will be understood by those of skill in the art in view of the description herein, the second reaction typically occurs at a maximum ratio of 1:1 [AG]:R3, where [AG] represents the amino group of formula -D2-NH2and R3is the functional group of the intermediate alkoxy-functional organosilicon compound as described above. As such, the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are typically reacted in a stoichiometric ratio of from 1.5:1 to 1:1.5, alternatively from 1.4:1 to 1:1.4, alternatively from 1.3:1 to 1:1.3, alternatively from 1.2:1 to 1:1.2, alternatively from 1.1:1 to 1:1.1, alternatively from 1.1:1 to 1:1 [AG]:R3. In general, the amino-functional organosiloxane compound (D) is typically utilized at a molar amount equal to (e.g. where the component (D) comprises but 1 of the amino groups) or less than (e.g. where the component (D) comprises >1 of the amino groups) the amount of the intermediate alkoxy-functional organosilicon compound. However, an excess of one of the components is typically utilized to fully consume the intermediate alkoxy-functional organosilicon compound or the amino-functional organosiloxane compound (D), e.g. to simplify purification of the reaction product formed, etc. For example, in certain embodiments, component (D) is utilized in relative excess (e.g. in a stoichiometric ratio of 1:>1 [AG]:R3) to maximize a conversion rate of the intermediate alkoxy-functional organosilicon compound to the alkoxy-functional organosilicon compound. In certain embodiments, the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are reacted in the presence of (E) a catalyst (herein referred to as the “second catalyst” (E) for clarity). The second catalyst (E) is not limited, and may be any catalyst suitable for facilitating the bonding of the intermediate alkoxy-functional organosilicon compound to the organosiloxane of the amino-functional organosiloxane compound (D) (i.e., via the reaction amino groups of component (D) with the functional group R3of the intermediate alkoxy-functional organosilicon compound, e.g. via substitution reaction, displacement, alkylation, etc.), as will be understood by one of skill in the art in view of the description herein. In certain embodiments, the second catalyst (E) is a base, such as carbonate base (e.g. Na2CO3, CaCO3, MgCO3, etc.), a hydroxide base (e.g. Mg(OH)2, etc.), a metal oxide base (e.g. ZnO, MgO, etc.), an amine base (e.g. pyridine, etc.), and combinations thereof. In certain embodiments, the method comprises utilizing more than one second catalyst (E), such as 2 different second catalysts (E). In such embodiments, each second catalyst (E) is independently selected, and may be the same as or different from any other second catalyst (E) being utilized. In particular embodiments, the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are reacted in the absence of any discreet second catalyst (E), e.g. such as when the amino-functional organosiloxane compound (D) is utilized in excess of the intermediate alkoxy-functional organosilicon compound and itself acts as a base (i.e., a basic catalyst) in the second reaction. Methods of preparing compounds suitable for use in, or as, second catalyst (E) are well known in the art, and many of the compounds listed herein are commercially available from various suppliers. As such, the second catalyst (E) may be prepared as part of the method, or otherwise obtained (i.e., as a prepared compound). The second catalyst (E) may be utilized in any form, such as neat (i.e., absent solvents, carrier vehicles, diluents, etc.), or disposed in a carrier vehicle, such as a solvent or dispersant (e.g. such as any of those listed above). In some embodiments, the second catalyst (E) is utilized in the absence of water and carrier vehicles/volatiles reactive with any of the components of the second reaction. For example, in certain embodiments, the method may comprise stripping the second catalyst (E) of volatiles and/or solvents (e.g. water, organic solvents, etc.). Techniques for stripping the second catalyst (E) are known in the art, and may include heating, drying, applying reduced pressure/vacuum, azeotroping with solvents, utilizing molecular sieves, etc., and combinations thereof. The second catalyst (E) may be utilized in any amount, which will be selected by one of skill in the art, e.g. dependent upon the particular second catalyst (E) selected, the reaction parameters employed, the scale of the second reaction, etc. The molar ratio of the second catalyst (E) to the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) utilized in the second reaction may influence the rate and/or amount of the reaction of these components to prepare the alkoxy-functional organosilicon compound therewith. Thus, the amount of the second catalyst (E) as compared to the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D), as well as the molar ratios therebetween, may vary. Typically, these relative amounts and the molar ratio are selected to maximize coupling of the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) to prepare the alkoxy-functional organosilicon compound (e.g. for increased economic efficiency of the reaction, increased ease of purification of the reaction product formed, etc.). In certain embodiments, the second catalyst (E) is utilized in a molar ratio of from 0.1 to 2 (D):(E). For example, in certain embodiments, the amino-functional organosiloxane compound (D) and the second catalyst (E) are utilized in a molar ratio of from 1:1 to 1:10, such as from 1:1.1 to 1:10, alternatively of from 1:1.2 to 1:10, alternatively of from 1:1.2 to 1:8, alternatively of from 1:1.2 to 1:6, alternatively of from 1:1.2 to 1:4, alternatively of from 1:1.1 to 1:2, (D):(E). It will be appreciated that ratios outside of these ranges may be utilized as well. For example, in certain embodiments, the second catalyst (E) is utilized in a gross excess (e.g. in an amount of ≥10, alternatively ≥15, alternatively ≥20, times the molar amount of the amino-functional organosiloxane compound (D)), such as when the second catalyst (E) is utilized as a carrier (i.e., a solvent, diluent, etc.) during the reaction. Typically, the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are reacted in a vessel or reactor. As such, in some embodiments, the second reaction is carried out in a vessel that is the same, or same type of, vessel/reactor utilized to prepare the intermediate alkoxy-functional organosilicon compound. In other embodiments, the second reaction is carried out in a vessel that is different from the vessel/reactor utilized to prepare the intermediate alkoxy-functional organosilicon compound. In some embodiments, the second reaction is carried out the same vessel utilized for the initial reaction, such that the method may be defined or otherwise described as a continuous, in-line, or one-pot reaction. As with the initial reaction, the second reaction may be carried out at an elevated or reduced temperature as described below, and thus the vessel or reactor may be heated or cooled in any suitable manner, e.g. via a jacket, mantle, exchanger, bath, coils, etc. The intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D), and optionally the second catalyst (E), may be fed together or separately to the vessel, or may be disposed in the vessel in any order of addition, and in any combination. For example, in certain embodiments, component (D) is added to a vessel containing the intermediate alkoxy-functional organosilicon compound. In some embodiments, the intermediate alkoxy-functional organosilicon compound is added to a vessel containing component (D). In either of such embodiments, the second catalyst (E) may optionally be added to the vessel in isolation or combined with another component of the second reaction. In general, reference to the “second reaction mixture” herein refers generally to a mixture comprising the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D), and optionally the second catalyst (E) (e.g. as obtained by combining such components, as described above). The method may further comprise agitating the second reaction mixture. The agitating may enhance mixing and contacting together the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D), and optionally the second catalyst (E), when combined, e.g. in the reaction mixture thereof. Such contacting independently may use other conditions, with (e.g. concurrently or sequentially) or without (i.e., independent from, alternatively in place of) the agitating. The other conditions may be tailored to enhance the contacting, and thus the second reaction of the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) to form the alkoxy-functional organosilicon compound. Other conditions may be result-effective conditions for enhancing reaction yield or minimizing amount of a particular reaction by-product included within the second reaction product along with the alkoxy-functional organosilicon compound. In certain embodiments, the second reaction is carried out in the presence of a carrier vehicle or solvent, such as one or more of those described above. For example, portions of carrier vehicle or solvent may be added to or otherwise combined with the intermediate alkoxy-functional organosilicon compound and/or the amino-functional organosiloxane compound (D) discretely, collectively with mixtures of the components utilized, or with the second reaction mixture as a whole. The total amount of carrier vehicle/solvent present in the second reaction mixture will be selected by one of skill in the art, e.g. based on the particular components selected, the reaction parameters employed, etc.). In some embodiments, the second reaction is carried out at an elevated temperature. The elevated temperature will be selected and controlled depending on the particular intermediate alkoxy-functional organosilicon compound prepared and utilized, the particular amino-functional organosiloxane compound (D) selected, the particular carrier and/or solvent being utilized, if any, etc. Accordingly, the elevated temperature will be readily selected by one of skill in the art in view of the second reaction conditions and parameters selected and the description herein. The elevated temperature is typically from greater than ambient temperature to 200° C., such as from 30 to 180, alternatively from 60 to 180, alternatively from 90 to 180, alternatively from 120 to 180, ° C. It is to be appreciated that the elevated temperature may also differ from the ranges set forth above, or be more specific subsets thereof. For example, in particular embodiments, the second reaction is carried out at an elevated temperature of from 110 to 120, alternatively from 120 to 130, alternatively from 130 to 140, alternatively from 140 to 150, alternatively from 150 to 160° C. It is also to be appreciated that reaction parameters may be modified during the second reaction as well. For example, temperature, pressure, and other parameters may be independently selected or modified during the second reaction. Any of these parameters may independently be an ambient parameter (e.g. room temperature and/or atmospheric pressure) and/or a non-ambient parameter (e.g. reduced or elevated temperature and/or reduced or elevated pressure). Any parameter, may also be dynamically modified, modified in real time, i.e., during the method, or may be static (e.g. for the duration of the reaction, or for any portion thereof). In certain embodiments, the second reaction is carried out at ambient temperature (e.g. when the second reaction is run over a long time scale, as described below). The time during which the second reaction to prepare the alkoxy-functional organosilicon compound is carried out is a function of scale, reaction parameters and conditions, selection of particular components, etc. In certain embodiments, the time during which the second reaction is carried out is from greater than 0 to 48 hours, alternatively from 1 to 36 hours, alternatively from 6 to 36 hours, alternatively from 12 to 24 hours after the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are combined (e.g. in the presence of component (E), when utilized). In particular embodiments, the time during which the second reaction is carried out is from 0.5 to 24 hours, alternatively from 0.5 to 12 hours, alternatively from 1 to 12 hours, alternatively from 2 to 8 hours after the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are combined (e.g. in the presence of component (E), when utilized). It is to be appreciated that times outside these ranges may also be utilized, as will be understood by those of skill in the art. For example, in certain embodiments, the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) are reacted over time scales around or longer than 48 hours (i.e., 2 days), such as from 2 to 60, alternatively from 2 to 30, alternatively from 4 to 21, alternatively from 7 to 21, alternatively from 7 to 14, days. Generally, reacting the intermediate alkoxy-functional organosilicon compound and the amino-functional organosiloxane compound (D) prepares a second reaction product comprising the alkoxy-functional organosilicon compound. In particular, over the course of the second reaction, the second reaction mixture comprising the intermediate alkoxy-functional organosilicon compound and component (D) comprises increasing amounts of the alkoxy-functional organosilicon compound and decreasing amounts of the intermediate alkoxy-functional organosilicon compound and component (D). Once the second reaction is complete (e.g. one of the intermediate alkoxy-functional organosilicon compound and component (D) is consumed, no additional alkoxy-functional organosilicon compound is being prepared, etc.), the second reaction mixture may be referred to as a reaction product comprising the alkoxy-functional organosilicon compound (i.e., the “second reaction product”). In this fashion, the second reaction product typically includes any remaining amounts of the intermediate alkoxy-functional organosilicon compound and component (D), and any remaining amounts of components (A), (B), and/or (C) if carried over, as well as degradation and/or reaction products thereof (e.g. materials which were not previously removed via any distillation, stripping, etc.). If the second reaction is carried out in any carrier vehicle or solvent, the second reaction product may also include such carrier vehicle or solvent. In certain embodiments, the method further comprises isolating and/or purifying the alkoxy-functional organosilicon compound from the second reaction product. Any suitable technique and/or protocol for isolation may be utilized, including any of those listed above. For example, in certain embodiments, such as where a carrier vehicle is utilized, volatiles are distilled and/or stripped from the second reaction mixture comprising the alkoxy-functional organosilicon compound. Once purified and/or isolated from the second reaction product, the alkoxy-functional organosilicon compound may be referred to as the isolated alkoxy-functional organosilicon compound. As will be appreciated by those of skill in the art in view of the description above, the particular alkoxy-functional organosilicon compound prepared in accordance with the method is a function of the particular initial organosilicon compound (A) and the alcohol component (B) reacted, as well as whether the alkoxy-functional organosilicon compound thereby prepared is utilized as the intermediate alkoxy-functional organosilicon compound in the second reaction and, if so, the particular amino-functional organosiloxane compound (D) utilized. As such, in addition to the alkoxy-functional organosilicon compounds having the general formula (I) described above, in particular embodiments the method prepares alkoxy-functional organosilicon compounds having the following general formula (II): where each R8is an independently selected substituted or unsubstituted hydrocarbyl group; subscript m is from 0 to 1000; subscript n is from 1 to 100; and each R10is independently R8, an amino group of formula -D2-NH2, or an amino group of formula -D2-N(H)Y, with the proviso that at least one R10is an amino group of formula -D2-N(H)Y; wherein each D2is an independently selected divalent linking group, and each Y comprises an independently selected alkoxysilyl moiety having the formula: where each R2is an independently selected substituted or unsubstituted hydrocarbyl group; each R4is an independently selected substituted or unsubstituted hydrocarbyl group; D1is a divalent linking group; and subscript a is 1, 2, or 3. As will be understood by one of skill in the art in view of the description herein, the amino-functional organosiloxane compound (D) utilized in the method (e.g. in the second reaction) forms the organosiloxane backbone of the alkoxy-functional organosilicon compound of general formula (II), and the intermediate alkoxy-functional organosilicon compound utilized forms the alkoxysilyl moieties Y. As such, where formulas, structures, moieties, groups, or other such motifs are shared between the alkoxy-functional organosilicon compound of formula (II) and the amino-functional organosiloxane compound (D) and the intermediate alkoxy-functional organosilicon compound, the description above with respect to such shared motifs may equally describe the alkoxy-functional organosilicon compound prepared. A composition comprising the alkoxy-functional organosilicon compound is also provided. The composition generally comprises the alkoxy-functional organosilicon compound and at least one other component, such as a non-reactive component (e.g. a carrier vehicle, solvent, etc.), a reactive component (e.g. a compound reactive with, or capable of being made reactive with, the alkoxy-functional organosilicon compound), or combinations thereof. In some embodiments, the composition comprises the alkoxy-functional organosilicon compound of general formula (I) above. In these or other embodiments, the composition comprises the alkoxy-functional organosilicon compound of general formula (II) above. In certain embodiments, the composition comprises less than 0.1% cyclic polydiorganosiloxanes based on the total amount of components therein (e.g. wt. %, based on the total weight of the composition). In general, the composition comprising the alkoxy-functional organosilicon compound may comprise, or be, the reaction product (or second reaction product) prepared according to the embodiments described above. In some embodiments however, the method includes purifying and/or isolating the alkoxy-functional organosilicon compound from the reaction product, and the composition thus comprises the isolated and/or purified alkoxy-functional organosilicon compound. In particular embodiments, the method comprises preparing the reaction product comprising the intermediate alkoxy-functional organosilicon compound (i.e., of general formula (I)) with the catalyst (C1), removing the catalyst (C1) from the reaction product, and reacting the thus purified intermediate alkoxy-functional organosilicon compound to prepare the second reaction product comprising the alkoxy-functional organosilicon compound of general formula (II). In such embodiments, the second reaction product comprising the alkoxy-functional organosilicon compound comprises a cyclic polydiorganosiloxane content of less than 1, alternatively less than 0.8, alternatively less than 0.6, alternatively less than 0.4, alternatively less than 0.2, alternatively less than 0.1%, based on the total amount of components therein (e.g. wt. %, based on the total weight of the composition). In some such embodiments, the method further comprises preparing a composition comprising the alkoxy-functional organosilicon compound via combining the second reaction product comprising the alkoxy-functional organosilicon compound and at least one other component. In these embodiments, the composition comprises less than 0.1, alternatively less than 0.05%, alternatively less than 0.01%, % cyclic polydiorganosiloxanes based on the total amount of components therein (e.g. wt. %, based on the total weight of the composition). The following examples, illustrating embodiments of this disclosure, are intended to illustrate and not to limit the invention. The brief summary immediately below provides information as to certain abbreviations, shorthand notations, and components utilized in the Examples. The various components utilized in the Examples are set forth in Table 1 below. TABLE 1Compounds Utilized in Examples 1-20ComponentDescriptionStructure/FormulaInitial Organosilicon Compound (A1)(3-glycidoxypropyl)trimethoxysilaneAlcohol Component (B1)GeraniolAlcohol Component (B2)(2E,6Z)-nona-2,6-dien-1-olAlcohol Component (B3)Isoamyl alcoholAlcohol Component (B4)Benzyl alcoholAlcohol Component (B5)2-octanolAlcohol Component (B6)2-methyl-2-butanolAmine Compound (C1-a1)OctadecylamineAmine Compound (C1-a2)DiethylamineCarboxylic Acid (C1-b1)Propionic acidCarboxylic Acid (C1-b2)Glacial acetic acidTitanate Compound (C2-1)Titanium(IV) butoxideTi(OnBu)4Titanate Compound (C2-2)Titanium(IV) tert-butoxideTi(OtBu)4Amino-functional Organosiloxane Compound DAminopropyl-functional polydimethylsiloxane (viscosity = 520 mPa-s (25° C.); N content = 0.68 wt.%) In each of the Examples below, viscosity is determined by loading a sample into a cup, letting the cup stand for one minute to reach room temperature, and then measuring the viscosity of the sample on a Brookfield DV-Ill viscometer at 25° C., with rpm adjusted to reach a stress of from 45-55%. Example 1: Preparation of Alkoxy-Functional Organosilicon Compound with an Ammonium Carboxylate Catalyst A glass vial equipped with a magnetic stir bar is charged with Initial Organosilicon Compound (A1) (2.5 g. 10 mmol, 1 eq.) and Alcohol Component (B1) (4.9 g, 30 mmol, 3 eq.). Amine Compound (C1-a1) (150 mg, 0.56 mmol, 2 wt. %) and Carboxylic Acid (C1-b1) (42 μL, 0.56 mmol) are then added to the vial to form a reaction mixture, which is then heated to and held at 80° C. for 16 h. A sample of the reaction mixture is then analyzed via1H NMR to show ˜40% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1), with intact epoxide functionality. The reaction mixture is then placed on a rotary evaporator under full vacuum, and heated to and held at 80° C. for 1 h, then 100° C. for 1 h. Another sample of the reaction mixture is then analyzed via1H NMR to show that all methanol has been evaporated, but no further conversion transformation had occurred (˜44% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1)). The reaction mixture is then placed in a heating block at 80° C. and left for 7 h exposed to ambient air give a reaction product comprising an alkoxy-functional organosilicon compound (˜70% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1) with intact epoxide functionality, via1H NMR). Example 2: Preparation of Alkoxy-Functional Organosilicon Compound with an Ammonium Carboxylate Catalyst A glass vial equipped with a magnetic stir bar is charged with Initial Organosilicon Compound (A1) (2.5 g. 10 mmol, 1 eq.) and Alcohol Component (B1) (3.2 g, 20 mmol, 2 eq.). Amine Compound (C1-a1) (75 mg, 0.28 mmol, 1 wt. %) and Carboxylic Acid (C1-b1) (21 μL, 0.28 mmol) are then added to the vial to form a reaction mixture. The vial is sealed, and the reaction mixture is heated to and held at 80° C. for 1 h. A sample of the reaction mixture is then analyzed via1H NMR to show ˜26% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1). The reaction mixture is then held at 80° C. for one additional hour to give a reaction product comprising an alkoxy-functional organosilicon compound (˜28% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1) with intact epoxide functionality, via1H NMR). Example 3: Preparation of Alkoxy-Functional Organosilicon Compound with an Ammonium Carboxylate Catalyst A glass vial equipped with a magnetic stir bar and nitrogen inlet is charged with Initial Organosilicon Compound (A1) (2.5 g. 10 mmol, 1 eq.) and Alcohol Component (B1) (4.9 g, 30 mmol, 3 eq.). Amine Compound (C1-a2)(40 mg, 0.56 mmol) and Carboxylic Acid (C1-b1) (41 μL, 0.56 mmol) are then added to the vial to form a reaction mixture. The reaction mixture is placed under nitrogen and heated to and held at 100° C. for 2 h. A sample of the reaction mixture is then analyzed via1H NMR to show ˜36% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1). The reaction mixture is then held at 100° C. for another 18 h, and another sample of the reaction mixture is analyzed via1H NMR to show ˜63% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1). The reaction mixture is then heated to and held at 120° C. for 16 h to give an orange reaction mixture, which is then stripped under vacuum (rotary evaporator) at 130° C. for 2 h to give a reaction product comprising an alkoxy-functional organosilicon compound (˜75% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1) with intact epoxide functionality, via1H NMR). Example 4: Preparation of Alkoxy-Functional Organosilicon Compound with a Titanate Catalyst A glass vial equipped with a magnetic stir bar and nitrogen inlet is charged with Initial Organosilicon Compound (A1) (2.5 g. 10 mmol, 1 eq.) and Alcohol Component (B1) (4.8 g, 30 mmol, 3 eq.). Titanate Compound (C2-1) (70 mg) is then added to the vial to form a reaction mixture, which is then heated to and held at 60° C. on a rotary evaporator for 4 h to give a reaction product comprising an alkoxy-functional organosilicon compound (˜97% Alcohol Component (B1) bound to the Initial Organosilicon Compound (A1) with intact epoxide functionality, via1H NMR). Examples 5-16: Preparation of Alkoxy-Functional Organosilicon Compounds with Titanate Catalysts For each of Examples 5-16, a glass vial equipped with a magnetic stir bar and nitrogen inlet is charged with Initial Organosilicon Compound (A1) (1.5 g, 6.3 mmol, 1 eq.) and an Alcohol Component (B). A Titanate Compound (C2) (25 mg, 7.3 mmol, 1.1 mol %) is then added to the vial to form a reaction mixture, which is then heated to and held at 80° C. on a rotary evaporator for 4 h to give a reaction product comprising an alkoxy-functional organosilicon compound, which is then analyzed via1H NMR to confirm the epoxide functionality of the initial organosilicon compound remains intact during the conversation to the alkoxy-functional organosilicon compound. TABLE 3Examples 5-10Example:5678910Alcohol Component (B):B1B2B3B4B5B6Equivalents (B):336333Titanate Compound (C2):C2-1C2-1C2-1C2-1C2-1C2-1Conv. (%) (1H NMR):9698889678100Epoxide Intact (1HYesYesYesYesYesYesNMR): TABLE 4Examples 11-16Example:111213141516Alcohol Component (B):B1B2B3B4B5B6Equivalents (B):336333Titanate Compound (C2):C2-2C2-2C2-2C2-2C2-2C2-2Conv. (%) (1H NMR):9899869584100Epoxide Intact* (1HYesYesYesYesYesYesNMR): Example 17: Preparation of an Alkoxy-Functional Organosilicon Compound A mixing cup is charged with Amino-functional Organosiloxane Compound (D) (5 g), water (deionized; 0.5 g), and an emulsification component (polyoxyethylene (12) tridecanol; 100% active; 0.4 g) and placed in a high-speed mixer (SpeedMixer; DAC 150). The cup is then spun (2×30 second cycles; maximum speed), and charged with water (incremental additions; 4.1 g) to give an emulsion comprising the Amino-functional Organosiloxane Compound (D) (amino-functional organosiloxane content=50%; pH 9.5) having a particle size of Dv(50)=0.39 μm and Dv(90)=0.885 μm (via laser diffraction particle size analyzer; Malvern Mastersizer 3000). The emulsion comprising the Amino-functional Organosiloxane Compound (D) (10 g; 4.9 mmol N; 1 equiv) is then combined with the Alkoxy-functional Organosilicon Compound prepared in Example 8, the resulting combination mixed (dental mixer), and the mixture oven-dried (45° C.; 8 days) to give a composition comprising an akoxy-functional organosilicon compound (emulsion; particle size: Dv(50)=0.246 μm and Dv(90)=0.602 μm), which is allowed to cool to room temperature and then transferred to a capped glass bottle containing CaCl2(1.5 g; pellets). The bottle is then vigorously stirred. The composition is then diluted four times via charging the bottle with pentane (25 mL) and methanol (20 mL), and stirred with venting (via cracking open the cap) between each dilution to give the diluted composition as an opaque mixture. The opaque mixture is then transferred to a centrifuge tube (50 mL), centrifuged (6000 rpm; for 15 minutes), shaken vigorously, and centrifuged again (6000 rpm; for 15 minutes) to give a phase-separated mixture. The non-polar phase of the phase-separated mixture (top layer) is then isolated and solvent-stripped (rotary evaporator) to give a reaction product comprising an alkoxy-functional organosilicon compound (free-flowing; slightly yellow; viscosity=728 mPa-s (25° C.)). A sample of the reaction product is then analyzed via1H NMR to show ring-opening of the epoxide functionality. The terms “comprising” or “comprise” are used herein in their broadest sense to mean and encompass the notions of “including,” “include,” “consist(ing) essentially of,” and “consist(ing) of. The use of “for example,” “e.g.,” “such as,” and “including” to list illustrative examples does not limit to only the listed examples. Thus, “for example” or “such as” means “for example, but not limited to” or “such as, but not limited to” and encompasses other similar or equivalent examples. The term “about” as used herein serves to reasonably encompass or describe minor variations in numerical values measured by instrumental analysis or as a result of sample handling. Such minor variations may be in the order of ±0-25, ±0-10, ±0-5, or ±0-2.5, % of the numerical values. Further, The term “about” applies to both numerical values when associated with a range of values. Moreover, the term “about” may apply to numerical values even when not explicitly stated. Generally, as used herein a hyphen “-” or dash “−” in a range of values is “to” or “through”; a “>” is “above” or “greater-than”; a “≥” is “at least” or “greater-than or equal to”; a “<” is “below” or “less-than”; and a “≤” is “at most” or “less-than or equal to.” On an individual basis, each of the aforementioned applications for patent, patents, and/or patent application publications, is expressly incorporated herein by reference in its entirety in one or more non-limiting embodiments. It is to be understood that the appended claims are not limited to express and particular compounds, compositions, or methods described in the detailed description, which may vary between particular embodiments which fall within the scope of the appended claims. With respect to any Markush groups relied upon herein for describing particular features or aspects of various embodiments, different, special, and/or unexpected results may be obtained from each member of the respective Markush group independent from all other Markush members. Each member of a Markush group may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims. Further, any ranges and subranges relied upon in describing various embodiments of the present invention independently and collectively fall within the scope of the appended claims, and are understood to describe and contemplate all ranges including whole and/or fractional values therein, even if such values are not expressly written herein. One of skill in the art readily recognizes that the enumerated ranges and subranges sufficiently describe and enable various embodiments of the present invention, and such ranges and subranges may be further delineated into relevant halves, thirds, quarters, fifths, and so on. As just one example, a range “of from 0.1 to 0.9” may be further delineated into a lower third, i.e., from 0.1 to 0.3, a middle third, i.e., from 0.4 to 0.6, and an upper third, i.e., from 0.7 to 0.9, which individually and collectively are within the scope of the appended claims, and may be relied upon individually and/or collectively and provide adequate support for specific embodiments within the scope of the appended claims. In addition, with respect to the language which defines or modifies a range, such as “at least,” “greater than,” “less than,” “no more than,” and the like, it is to be understood that such language includes subranges and/or an upper or lower limit. As another example, a range of “at least 10” inherently includes a subrange of from at least 10 to 35, a subrange of from at least 10 to 25, a subrange of from 25 to 35, and so on, and each subrange may be relied upon individually and/or collectively and provides adequate support for specific embodiments within the scope of the appended claims. Finally, an individual number within a disclosed range may be relied upon and provides adequate support for specific embodiments within the scope of the appended claims. For example, a range “of from 1 to 9” includes various individual integers, such as 3, as well as individual numbers including a decimal point (or fraction), such as 4.1, which may be relied upon and provide adequate support for specific embodiments within the scope of the appended claims. The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. The invention may be practiced otherwise than as specifically described. | 108,968 |
11859055 | DETAILED DESCRIPTION OF THE INVENTION The compound according to the present invention will be described below in further detail. The present compound is a (poly)siloxane monomer represented by formula (1). The compound has a (poly)siloxane structure (represented by A) at the end and a hydrophilic group (Z) as a side chain (represented by -L3-Z) bounded to a linking group which is represented by -L1-CH-L2- in formula (1) and exists between the polysiloxane structure and a terminal polymerizable group. The compound has excellent compatibility with a hydrophilic monomer, and the surface wettability of hydrogel obtained from the compound as a monomer is improved. The hydrophilic group is a side chain of the linking group and, therefore, has high mobility, so that the polysiloxane structure is surrounded with the hydrophilic group. On account of this conformation, the compatibility and the surface wettability are improved. In formula (1), R1is a hydrogen atom or a methyl group. Among these, a methyl group is preferred. In formula (1), L1is a single bond or a divalent hydrocarbon group which has 1 to 6 carbon atoms and may have at least one selected from an ether bond, an ester bond, and a urethane bond, or two or more of these bonds. Examples of the divalent hydrocarbon group having 1 to 6 carbon atoms include a methylene group, an ethylene group, a 1,3-propylene group, a 1-methylpropylene group, a 1,1-dimethylpropylene group, a 2-methylpropylene group, a 1,2-dimethylpropylene group, a 1,1,2-trimethylpropylene group, a 1,4-butylene group, a 2-methyl-1,4-butylene group, a 2,2-dimethyl-1,4-butylene group, a 3-methyl-1,4-butylene group, a 2,2-dimethyl-1,4-butylene group, a 2,3-dimethyl-1,4-butylene group, a 1,5-pentylene group, and a 1,6-hexanylene group. Examples of the group having at least one of ether bond include polyalkylene oxide groups such as a polyethylene oxide group, a polypropylene oxide group, and a polyethylene-propylene oxide group. L1is preferably a single bond. L2is a linear, branched or cyclic divalent or trivalent hydrocarbon group which has 2 to 10 carbon atoms and may have at least one of ether bond. Preferred is a linear or branched hydrocarbon group. Examples of the divalent hydrocarbon group include a 1,7-heptenylene group, a 1,8-octanylene group, a 1,9-rionanylene group, and a 1,10-decanylene group, in addition to the divalent hydrocarbon groups defined for L1in above. Examples of the group having at least one of ether bond include polyalkylene oxide groups such as a polyethylene oxide group, a polypropylene oxide group, and a polyethylene-propylene oxide group. Among these, —CH2OC3H6— is preferred. When L2is trivalent, then L3and L2together form a ring. L3is a divalent or trivalent hydrocarbon group having one carbon atom, i.e., methylene or methine. When L3is a metin group, L3bonds with L2to together form a ring. In formula (1), when L3is divalent, the structure indicated by —CH(L3-)-L2- is represented by the following formula (1a). When L3is trivalent, the ring structure formed by L3and L2is represented by the following formula (1b). In formulas (1a) and (1b), L2is a linear, branched, or cyclic divalent hydrocarbon group having 2 to 10 carbon atoms, and may have an ether bond; L2′is a linear or branched divalent hydrocarbon group having 2 to 10 carbon atoms, and may have an ether bond; the site marked with * bonds to L1; the site marked with ** bonds to Z; and the site marked with *** bonds to A. Examples of the compound having the structure represented by formula (1a) include compounds represented by the following formula. wherein A and Z are as defined above. The structure represented by the above (1b) is preferably represented the following formula. wherein r is an integer of from 0 to 8, preferably an integer of from 0 to 4, h is an integer of from 0 to 6, preferably an integer of from 0 to 2. The following structure is more preferred. wherein h is an integer of from 0 and 6. Examples of the compound having the structure of formula (1b) include compounds represented by the following formula. wherein A and Z are as defined above. Z is a monovalent organic group having 1 to 10 carbon atoms and having a quaternary ammonium group or an amphoteric ion group. The group represented by the following formula (4) or (5) is preferred. In formulas (4) and (5), R2is an alkyl group having 1 to 6 carbon atoms, R3is an alkyl group having 1 to 10 carbon atoms, L4is a divalent hydrocarbon group having 1 to 6 carbon atoms, and X is a halogen atom. In formulas (4) and (5), R2is an alkyl group having 1 to 6 carbon atoms, preferably a methyl group. In formula (5), R3is an alkyl group having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms. In formula (4), L4is a divalent hydrocarbon group having 1 to 6 carbon atoms, preferably 3 or 4 carbon atoms. More preferably, Z is a methyldimethylammonium group, an ethyldimethylammonium group, a propyldimethylammonium group, a butyldimethylammonium group, or a group represented by the following formula (a) or (b), wherein the site marked with ** bonds to a carbon atom. A is a linear or branched organo(poly)siloxanyl group having 1 to 100, preferably 2 to 20, siloxane units, and is preferably represented by the following formula (2) or (3). In formula (2), n is an integer of from 1 to 100, preferably an integer of from 2 to 20. In formula (3), a is an integer of from 0 to 10, preferably an integer of from 1 to 5, b is an integer of from 0 to 10, preferably an integer of from 1 to 5, c is an integer of from 0 to 10, preferably an integer of from 1 to 5, and at least two of a, b, and c are an integer of 1 or more. Preferably, a is 1, b is 1 and c is 0. R is, independently of each other, a monovalent hydrocarbon group having 1 to 10 carbon atoms. R is, independently of each other, a monovalent hydrocarbon group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms. Examples of the monovalent hydrocarbon group include alkyl groups such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group; cycloalkyl groups such as a cyclopentyl group and a cyclohexyl group; and aryl groups such as a phenyl group and a tolyl group. R is preferably an alkyl group having 1 to 6 carbon atoms or a phenyl group, more preferably a methyl group or a butyl group. A method for preparing the compound represented by formula (1) will be described below. The present preparation method comprises a step of reacting a tertiary amino group-containing (poly)siloxane compound represented by the following formula (6): wherein L, L2, L3, A, R1and R2are as defined above, with a halogenated alkyl or alkylsulton compound to obtain the compound represented by formula (1), hereinafter referred to as step III. The present method further comprises a step of reacting a secondary hydroxy group-containing (poly)siloxane compound represented by the following formula (7): wherein L2, L3, A and R2are as defined above, with a (meth)acryl group-containing compound represented by the following formula (8) or (9): wherein L5is a divalent hydrocarbon group having 1 to 6 carbon atoms, and may comprise an ether bond, an ester bond, or a combination thereof; X is a halogen atom, R1is as defined above, and Y is an isocyanato group, to obtain the compound represented by formula (6), hereinafter referred to as step II. In formula (9), L5is a divalent hydrocarbon group having 1 to 6 carbon atoms, and may have an ether bond, an ester bond, or a combination thereof. The present method further includes a step of reacting an epoxy group-containing (poly)siloxane compound with a dialkylamine compound to obtain the compound represented by formula (7), hereinafter, referred to as step I. The each step will be described below in detail. Step I In Step I, an epoxy group-containing (poly)siloxane compound is reacted with a dialkylamine compound to obtain a secondary hydroxyl group-containing (poly)siloxane compound represented by the following formula (7). wherein L2, L3, A and R2are as defined above. In formula (7), L2is a divalent hydrocarbon group having 2 to 10 carbon atoms, and may comprise an ether bond, as described above. If the number of carbon atoms is larger than the aforesaid upper limit, the siloxane content small, and the properties of the siloxane may not be exhibited. As described above, R2is an alkyl group having 1 to 6 carbon atoms, preferably a methyl group. If the number of carbon atoms is larger than the aforesaid upper limit, the reactivity of the tertiary amine is poor in the reaction of Step III, and the desired product may not be obtained. A is as defined above. If a, b, c or n is larger than the aforesaid upper limit, the reactivity of the (meth)acryl group is poor, and the unreacted (meth)acryl group may remain after the polymerization. The reaction of the epoxy group-containing (poly)siloxane compound with the dialkylamine compound may be carried out according to any conventional method. For example, at least a molar equivalent amount of the dialkylamine compound may be added to the epoxy group-containing (poly)siloxane compound to allow to react. Although a reaction temperature is not particularly limited, it is preferred that the reaction temperature does not exceed a boiling point of a solvent used, for instance, from about 0 to about 120 degrees C. The reaction may be carried out in the presence of a solvent and/or a catalyst. Any conventional solvent and catalyst may be used and are not particularly limited. Examples of the epoxy group-containing (poly) siloxane compound include a polydimethylsiloxane having one 3-glycidyloxypropyl group at one terminal and one butyl group at the other terminal, a polydimethylsiloxane having one 3-[2-(3,4-epoxycyclohexyl) ethyl] group at one terminal and one butyl group at the other terminal, (3-glycidyloxypropyl)bis(trimethylsiloxy)methylsilane, and 3-[2-(3,4-epoxycyclohexyl)ethyl]bis(trimethylsiiloxy)methylsilane. The compound whose L3in formula (1) is divalent is obtained by using polydimethylsiloxane having a 3-glycidyloxy group at the terminal, such as polydimethylsiloxane having one 3-glycidyloxypropyl group at one terminal and one butyl group at the other terminal and (3-glycidyloxypropyl)bis(trimethylsiloxy)methylsilane, as a starting material. The compound whose L3in formula (1) is trivalent and forms a ring together with L2is obtained by using polydimethylsiloxane having an epoxycycloalkyl group, such as an epoxycyclohexyl group, at a terminal as a starting material. For example, use is made of a polydimethylsiloxane having one 3-[2-(3,4-epoxycyclohexyl)ethyl] at one terminal and one butyl group at the other terminal, which is represented by the following formula, as a starting material, to obtain a compound represented b the following formulas. Examples of the dialkylamine compound include dimethylamine, diethylamine, dipropylamine, dibutylamine, dipentylamine, dihexylamine, ethylmethylamine, propylmethylamine, butylmethylamine, pentylmethylamine and hexylmethylamine. These may be in a form of solution, such as an aqueous solution, for easy handling. Examples of the catalyst include organophosphorus compounds, tertiary amines, and Lewis acids. Examples of the organophosphorus compounds include tricyclohexylphosphine, tributylphosphine, trioctylphosphine, cyclohexyldiphenylphosphine, dicyclohexylphenylphosphine, butyldiphenylphosphine, dibutylphenylphosphine, octyldiphenylphosphine, dioctylphenylphosphine and triphenylphosphine. Examples of the tertiary amine include trimethylamine, triethylamine, tripropylamine, tributylamine, diazabicycloundecene, diazabicyclononene and 1-methylimidazole. Examples of the Lewis acid include boron trifluoride, aluminum chloride, methyldichloroaluminum, dimethylchloroaluminum, trimethylaluminum, magnesium chloride, magnesium bromide, titanium tetrachloride, dichlorotitanium bistriflate, biscyclopentadienyltitanium bistriflate, dichlorotitanium bisfluorosulfonate, tin tetrachloride and tin (II) bistriflate. Examples of the solvent include glycol ether solvents such as methyl cellosolve, ethyl cellosolve, isopropyl cellosolve, butyl cellosolve, propylene glycol monomethyl ether, diethylene glycol monomethyl ether, triethylene glycol monomethyl ether, polyethylene glycol monomethyl ether, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, and polyethylene glycol dimethyl ether; ester solvents such as ethyl acetate, butyl acetate, amyl acetate, ethyl lactate, and methyl benzoate; aliphatic hydrocarbon solvents such as linear hexane, linear heptane, and linear octane; alicyclic hydrocarbon-based solvents such as cyclohexane and ethyl cyclohexane; ketone solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; aromatic hydrocarbon solvents such as benzene, toluene, and xylene; and petroleum solvents. The solvents may be used alone or in a combination of two or more of them. Step II. Step II is to prepare the compound represented by formula (6) by reacting the secondary hydroxy group-containing (poly)siloxane compound represented by the following formula (7): wherein L2, L3, A and R2are as described above, with a (meth)acryl group-containing compound represented by the following formula (8) or (9), wherein L5, R1and X are as described above, and Y is an isocyanato group. The reaction may be conducted according to any conventional method. For example, the reaction may be conducted by adding at least a molar equivalent amount of the (meth)acryl group-containing compound represented by formula (8) or (9), to the secondary hydroxy group-containing (poly)siloxane compound represented by formula (7). The reaction temperature is not particularly limited and is preferably a temperature which does not exceed a boiling point of a solvent used. For instance, the reaction may be conducted at a temperature of about 0 to 80 degrees C. The reaction may be conducted in the presence of a catalyst or a solvent. Any known solvent and catalyst may be used and are not particularly limited. Any of the solvents described above may be used. The (meth)acryl group-containing compound represented by formula (8) or (9) has a group reactive with a secondary hydroxy group. Examples of the reactive group include a halogenated alkyl group, an acyl halide group and an isocyanato group. Examples of the (meth)acryl group-containing compound represented by formula (8) or (9) include 2-isocyanatoethyl methacrylate, 2-isocyanatoethyl acrylate, methacrylic acid chloride, and acrylic acid chloride, but are not limited to them. Examples of the catalyst include an organic metal catalyst and an amine catalyst. Examples of the organic metal catalyst, but not particularly limited, include organic tin catalysts such as stannous diacetate, stannous dioctoate, stannous dioleate, stannous dilaurate, dibutyltin oxide, dibutyltin diacetate, dibutyltin dilaurate, dibutyltin dichloride, and dioctyltin dilaurate; and acetylacetone metal salts such as acetylacetone aluminium, acetylacetone iron, acetylacetone copper, acetylacetone zinc, acetylacetone beryllium, acetylacetone chromium, acetylacetone indium, acetylacetone manganese, acetylacetone molybdenum, acetylacetone titanium, acetylacetone cobalt, acetylacetone vanadium, and acetylacetone zirconium. Examples of the amine catalyst include pentamethyldiethylenetriamine, triethylamine, N-methylmorpholine bis(2-dimethylaminoethyl)ether, N,N,N′,N″,N″-pentamethyldiethylenetriamine, N,N,N′-trimethylaminoethyl-ethanol amine, bis(2-dimethylaminoethyl)ether, N-methyl-N′,N′-dimethylamino ethyl piperazine, N,N-dimethylcyclohexylamine, diazabicyclo undecene, triethylene diamine, tetramethyl hexamethylene diamine, N-methylimidazole, trimethylaminoethyl piperazine, tripropylamine, a tetramethyl ammonium salt, and a tetraethyl ammonium salt, and a triphenyl ammonium salt. Step III. Step III is to prepare a compound represented by formula (1) by reacting a tertiary amino group-containing (poly)siloxane compound represented by the following formula (6): wherein L1, L2, L3, A, R1and R2are as defined above, with a halogenated alkyl or alkylsulton compound. The reaction may be conducted according to any conventional known method. For example, the reaction may be conducted by reacting one molar equivalent amount or less of the halogenated alkyl compound or the alkyl sultone compound, per mole of the tertiary amino group-containing (poly)siloxane compound represented by formula (6). The reaction temperature is not particularly limited and is preferably a temperature which does not exceed a boiling point of a solvent used. For instance, the reaction may be conducted at a temperature of about 0 to 120 degrees C. The reaction may be conducted in the presence of a catalyst in addition to the solution. Any known solvent and catalyst may be used and are not particularly limited. Any of the solvents described above can be used. Examples of the halogenated alkyl compound, but not particularly limited, include methyl chloride, ethyl chloride, propyl chloride, butyl chloride, pentyl chloride, hexyl chloride, methyl bromide, ethyl bromide, propyl bromide, butyl bromide, pentyl bromide, hexyl bromide, methyl iodide, ethyl iodide, propyl iodide, butyl iodide, pentyl iodide and hexyl iodide. Examples of the alkyl sultone compound, but not particularly limited, include propane sultone and butane sultone. Examples of the catalyst include a metal catalyst and an amine catalyst. Examples of the metal catalyst, but not particularly limited, include alkali metal salt catalysts such as lithium chloride, sodium chloride, potassium chloride, lithium bromide, sodium bromide, potassium bromide, lithium iodide, sodium iodide, and potassium iodide; and alkaline earth metal catalysts such as magnesium chloride, calcium chloride, magnesium bromide, calcium bromide, magnesium iodide and calcium iodide. Any of the amine catalysts described above can be used. In any of the aforementioned reactions, the completion of the reaction can be determined by confirming the disappearance of peaks of the starting compounds according to a conventional method, for instance, using thin-layer chromatography (TCL), high performance liquid chromatography (HPLC), or gas chromatography (GC). After the completion of the reaction, the obtained product may be purified by the conventional method. For example, the obtained product is isolated by washing the organic layer with water and then removing the solvent. Distillation at a reduced pressure and an active carbon treatment may also be used. As an example of the preparation method, one mole of the epoxy group-containing (poly)siloxane compound and two moles of the dialkylamine compound are placed in a reactor, and heated with stirring at 100 degrees C. The reaction completes in about 4 hours. The progress of the reaction can be followed by monitoring the epoxy group-containing (poly)siloxane compound or the produced secondary hydroxy group-containing (poly)siloxane by GC or the like. After the completion of the reaction, one mass equivalent of toluene is added, and an organic layer is washed with water and subjected to distillation to remove any unreacted starting materials at a reduced pressure. Thus, the secondary hydroxy group-containing (poly)siloxane compound represented by formula (7) is obtained. Subsequently, one mole of the secondary hydroxy group-containing (poly)siloxane compound represented by formula (7), one mole of the triethylamine, and one mass equivalent of toluene are placed in a reactor and, then, one molar equivalent of methacrylic acid chloride represented by formula (8) is added. After the addition, stirring is conducted at room temperature. The reaction completes in about 10 hours. The progress of the reaction can be followed by monitoring the methacrylic acid chloride by GC or the like. After the completion of the reaction, an organic phase is washed with water and subjected to distillation to remove the solvent off and the unreacted starting materials remaining in the organic phase at a reduced pressure. Thus, the tertiary amino group-containing (poly)siloxane compound represented by formula (6) is obtained. Subsequently, one mole of the tertiary amino group-containing (poly)siloxane compound represented by formula (6), 1.2 moles of propanesultone, and three mass equivalent of acetonitrile are placed in a reactor and stirred at 80 degrees C. The reaction completes in about 6 hours. The progress of the reaction can be followed by monitoring the tertiary amino group-containing (poly)siloxane compound or propanesultone by GC or the like. After the completion of the reaction, four mass equivalent of n-hexane is added and a lower layer is discarded. One mass equivalent of acetonitrile is added and a lower layer is discarded, which procedure are repeated twice, so that any unreacted sultone compound is removed. The upper layer is subjected to distillation at a reduced pressure to remove the solvent in the upper layer and the unreacted starting materials. Thus, the (poly)siloxane compound of the present invention represented by formula (6) is obtained. The compound according to the present invention can provide a polymer having a repeating unit derived from the addition polymerization at a (meth)acryl group. The compound according to the present invention is well compatible with other compounds having a polymerizable group such as a (meth)acryl group, hereinafter referred to as a polymerizable monomer or a hydrophilic monomer. Therefore, the compound according to the present invention is copolymerizable with the polymerizable monomer to provide a colorless and transparent copolymer. Moreover, the compound according to the present invention can be polymerized alone. In the preparation of the copolymer having the repeating unit derived from polymerization of the present silicone compound and the other polymerizable (hydrophilic) monomer, the proportion of the present silicone compound to be added may be such that the mass proportion of the repeating unit derived from the present silicone compound is 10% or more, relative to the total mass of the copolymer. Specifically, the amount of the present compound is preferably 10 to 80 parts by mass, more preferably 20 to 60 parts by mass, relative to the total 100 parts by mass of the present compound and the polymerizable (hydrophilic) monomer. Examples of the polymerizable monomer include acrylic monomers such as (meth)acrylic acid, methyl(meth)acrylate, ethyl(meth)acrylate, (poly)ethylene glycol dimethacrylate, polyalkylene glycol mono(meth)acrylate, polyalkylene glycol monoalkylether(meth)acrylate, trifluoroethyl(meth)acrylate, 2-hydroxyethyl(meth)acrylate, and 2,3-dihydroxypropyl(meth)acrylate; acrylic acid derivatives such as N,N-dimethylacrylamide, N,N-diethylacrylamide, N-acryloylmorpholine, and N-methyl(meth)acrylamide; N-vinylpyrrolidone, other unsaturated aliphatic compounds or aromatic compounds such as crotonic acid, cinnamic acid, and vinylbenoic acid; and a siloxane monomer having a polymerizable group such as a (meth)acryl group. These polymerizable monomers may be used alone or in a combination of two or more of them. The copolymerization of the present compound and the other polymerizable monomer may be conducted according to a conventional method. For example, the copolymerization can be conducted using a known polymerization initiator such as a thermal polymerization initiator or a photopolymerization initiator. Examples of the polymerization initiator include 2-hydroxy-2-methyl-1-phenyl-propane-1-one, azobisisobutyronitrile, azobis dimethylvaleronitrile, benzoyl peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide. These polymerization initiators may be used alone or in a combination of two or more of them. The amount of the polymerization initiator is 0.001 to 2 parts by mass, preferably 0.01 to 1 part by mass, relative to 100 parts by mass of the total amount of the polymerizable starting materials. The polymer having the repeating unit derived from the present compound has excellent oxygen permeability. The hydrogel obtained from the polymer has excellent surface wettability. Therefore, the present compound is suitable for the production of ophthalmic devices such as contact lenses, intraocular lenses, and artificial corneas. A method for preparing the ophthalmic device using the polymer is not particularly limited and may be according to any conventional one. For example, a cutting means or a template (molding) may be used for forming lenses such as contact lenses and intraocular lenses. EXAMPLES The present invention will be explained below in further detail with reference to a series of the Examples and the Comparative Examples, though the present invention is in no way limited by these Examples. In the following Examples,1H-NMR analysis was performed using ECS400 manufactured by JEOL and deuterated chloroform as a solvent for measurement. Example 1 [Step I] To a 1 L, three-necked flask equipped with a dimroth condenser and a thermometer, were added 140.0 g of an epoxy compound represented by the following formula (10A), 214.0 g of a 50% aqueous solution of N,N-dimethylamine, and 140.0 g of toluene, and the mixture was stirred at 50 degrees C. for 4 hours. After the completion of the reaction, the mixture was washed with deionized water three times, and the unreacted starting materials were distilled off at an inner temperature of 80 degrees C. and a reduced pressure, to obtain a colorless, transparent liquid in a yield of 140.6 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (11A). [Step II] To a 1 L, three-necked flask equipped with a dimroth condenser, a thermometer, and a dropping funnel, were added 130.0 g of the compound represented by formula (11A) obtained in step I, 33.8 g of triethylamine, and 520.0 g of toluene, and the mixture was cooled to 10 degrees C. To this mixture, 35.0 g of methacryloyl chloride was added dropwise, and the mixture was then aged at 20 degrees C. for 4 hours. The mixture after the reaction was washed with deionized water three times to remove a by-producted salt, and the solvent and by-products were distilled off at an inner temperature of 80 degrees C. and a reduced pressure to obtain 127.3 g of a colorless transparent liquid.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (12A). 1H-NMR data are as follows: 0.0 ppm (30H), 0.5 ppm (4H), 0.9 ppm (3H), 1.3 ppm (4H), 1.6 ppm (2H), 2.0 ppm (3H), 2.3 ppm (6H), 2.5 ppm (2H), 3.4 ppm (2H), 3.6 ppm (2H), 5.2 ppm (1H), 5.6 ppm (1H), 6.1 ppm (1H) [Step III] To a 1 L, three-necked flask equipped with a dimroth condenser, a thermometer, and a dropping funnel, were added 60.0 g of the compound represented by formula (12A) obtained in step II, 20.6 g of 1,3-propanesultone, and 180.0 of acetonitrile, and the mixture was aged at 80 degrees C. for 6 hours. To this mixture, 240.0 g of n-hexane was added, washed three times with acetonitrile, and the solvent was distilled off at an internal temperature of 80 degrees C. and a reduced pressure to obtain 51.3 g of a colorless, transparent greasy solid.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (13A). 1H-NMR data are as follows: 0.0 ppm (30H), 0.5 ppm (4H), 0.9 ppm (3H), 1.3 ppm (4H), 1.6 ppm (2H), 2.0 ppm (3H), 2.3 ppm (2H), 2.9 ppm (2H), 3.2 ppm (6H), 3.4 ppm (2H), 3.5-3.6 ppm (2H), 3.8 ppm (2H), 3.9 ppm (2H), 5.5 ppm (1H), 5.7 ppm (1H), 6.2 ppm (1H) Example 2 [Step I] Step I of Example 1 was repeated, except that the epoxy compound represented by formula (10A) in Step I of Example 1 was replaced with an epoxy compound represented by the following formula (10B) to obtain a colorless, transparent liquid. The yield was 143.1 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (11B). [Step II] Step II of Example 1 was repeated, except that the compound represented by formula (11A) in Step II of Example 1 was replaced with a compound represented by formula (11B) to obtain a colorless, transparent liquid. The yield was 129.0 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (12B). 1H-NMR data are as follows: 0.0 ppm (72H), 0.5 ppm (4H), 0.9 ppm (3H), 1.3 ppm (4H), 1.6 ppm (2H), 2.0 ppm (3H), 2.3 ppm (6H), 2.5 ppm (2H), 3.4 ppm (2H), 3.6 ppm (2H), 5.2 ppm (1H), 5.6 ppm (1H), 6.1 ppm (1H) [Step III] Step III of Example 1 was repeated, except that the compound represented by formula (12A) in Step III of Example 1 was replaced with the compound represented by formula (12B) to obtain a colorless, transparent greased solid. The yield was 55.5 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (13B). 1H-NMR data are as follows: 0.0 ppm (72H), 0.5 ppm (4H), 0.9 ppm (3H), 1.3 ppm (4H), 1.6 ppm (2H), 2.0 ppm (3H), 2.3 ppm (2H), 2.9 ppm (2H), 3.2 ppm (6H), 3.4 ppm (2H), 3.5-3.6 ppm (2H), 3.8 ppm (2H), 3.9 ppm (2H), 5.5 ppm (1H), 5.7 ppm (1H), 6.2 ppm (1H) Example 3 [Step I] Step I of Example 1 was repeated, except that the epoxy compound represented by formula (10A) in Step I of Example 1 was replaced with an epoxy compound represented by formula (10C) to obtain a colorless, transparent liquid. The yield was 135.2 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (11C). [Step II] Step II of Example 1 was repeated, except that the compound represented by formula (11A) in Step II of Example 1 was replaced with a compound represented by formula (11C) to obtain a colorless, transparent liquid. The yield was 120.0 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (12C). 1H-NMR data are as follows: 0.0 ppm (21H), 0.4 ppm (2H), 1.6 ppm (2H), 2.0 ppm (3H), 2.3 ppm (6H), 2.5 ppm (2H), 3.4 ppm (2H), 3.6 ppm (2H), 5.2 ppm (1H), 5.6 ppm (1H), 6.1 ppm (1H) [Step III] Step III of Example 1 was repeated, except that the compound represented by formula (12A) in Step III of Example 1 was replaced with a compound represented by formula (12C) to obtain a white solid. The yield was 50.7 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (13C). 1H-NMR data are as follows: 0.0 ppm (21H), 0.5 ppm (2H), 1.6 ppm (2H), 2.0 ppm (3H), 2.3 ppm (2H), 2.9 ppm (2H), 3.2 ppm (6H), 3.4 ppm (2H), 3.5-3.6 ppm (2H), 3.8 ppm (2H), 3.9 ppm (2H), 5.5 ppm (1H), 5.7 ppm (1H), 6.2 ppm (1H) Example 4 To a 1 L, three-necked flask equipped with a dimroth condenser, a thermometer, and a dropping funnel, were added 60.0 g of the compound represented by formula (12A) obtained in Step II of Example 1, 26.6 g of 1-bromobutane, and 180.0 g of acetonitrile, and the mixture was aged at 80 degrees C. for 6 hours. To the solution after the reaction, 240.0 g of n-hexane was added, the mixture was washed with acetonitrile three times, and the solvent was distilled off at an internal temperature of 80 degrees C. and reduced pressure to obtain a colorless, transparent liquid. The yield was 55.2 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (14A). 1H-NMR data are as follows: 0.0 ppm (30H), 0.5 ppm (4H), 0.9 ppm (3H), 1.0 ppm (3H), 1.3-1.4 ppm (8H), 1.6 ppm (2H), 2.0 ppm (3H), 3.2 ppm (6H), 3.3 ppm (2H), 3.4 ppm (2H), 3.5-3.6 ppm (2H), 3.8 ppm (2H), 5.5 ppm (1H), 5.7 ppm (1H), 6.2 ppm (1H) Example 5 The procedures of Example 4 were repeated, except that the compound represented by formula (12A) was replaced with the compound represented by formula (12B) obtained in Step II of Example 2 to obtain a colorless, transparent liquid. The yield was 54.6 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (14B). 1H-NMR data are as follows: 0.0 ppm (72H), 0.5 ppm (4H), 0.9 ppm (3H), 1.0 ppm (3H), 1.3-1.4 ppm (8H), 1.6 ppm (2H), 2.0 ppm (3H), 3.2 ppm (6H), 3.3 ppm (2H), 3.4 ppm (2H), 3.5-3.6 ppm (2H), 3.8 ppm (2H), 5.5 ppm (1H), 5.7 ppm (1H), 6.2 ppm (1H) Example 6 The procedures of Example 4 were repeated, except that the compound represented by formula (12A) was replaced with the compound represented by formula (12C) obtained in Step II of Example 3 to obtain a white solid. The yield was 46.9 g.1H-NMR analysis showed that the obtained one was a compound represented by the following formula (14C). 1H-NMR data are as follows: 0.0 ppm (21H), 0.5 ppm (2H), 1.0 ppm (3H), 1.3-1.4 ppm (4H), 1.6 ppm (2H), 2.0 ppm (3H), 3.2 ppm (6H), 3.3 ppm (2H), 3.4 ppm (2H), 3.5-3.6 ppm (2H), 3.8 ppm (2H), 5.5 ppm (1H), 5.7 ppm (1H), 6.2 ppm (1H) Comparative Examples 1 to 3 Compounds used in Comparative Examples 1 to 3 are as follows.SiGMA: methylbis(trimethylsiloxy)silylpropyl glycerol methacrylatemPDMS: polydimethylsiloxane having a monomethacryloxypropyl radical at one end and a monobutyl group at the other end and having a molecular weight of 800 to 1000.TRIS-PEG2: 2-methyl-2-[2-[3-[3,3,3-trimethyl-1,1-bis[(trimethylsilyl)oxy]-1-disiloxanyl]propoxy]ethoxy]ethyl acrylate [Preparation of Polymer] Each of the compounds obtained in the Examples 1-6 or the Comparative Examples 1-3, N-vinylpyrrolidone (NVP), ethylene glycol dimethacrylate (EGDMA), and IRGACURE 1173 (Irg 1173) were mixed in the amounts shown in Table 1 and stirred until a uniform solution was obtained. After the stirring, N2was bloom into the solution for five minutes. The solution was sufficiently deaerated, and poured in a polypropylene mold. The solution was irradiated by UV with a high pressure mercury lamp to cause curing. The cured product was soaked in isopropanol, a 50% isopropanol aqueous solution, and then deionized water for washing, so that a hydrogel film was obtained. The properties of the obtained hydrogel film were determined according to the following methods. The results are as shown in Table 1. [Equilibrium Water Content] Each of the films was soaked in deionized water at 25 degrees C. for 48 hours and, then, water on the surface of the film was wiped away. Thus, the hydrated film was formed. The hydrated film was weighed. Subsequently, the hydrated film was dried at 50 degrees C. for 48 hours and further at 25 degrees C. for 24 hours in an oven, and the mass of the dried film was weighed. The equilibrium water content was calculated according to the following equation. Equilibrium water content (%)=100×(mass of the hydrated film−mass of the dried film)/mass of the hydrated film [Transparency] A film was soaked in deionized water at 25 degrees C. for 48 hours and, then, water on the surface of the film was wiped away. Thus, the hydrated film was provided. Appearance of the hydrated film was then observed with the naked eye and evaluated according to the following criteria.A: uniform and transparentB: ununiform or clouded [Compatibility with a Hydrophilic Monomer] Compatibility with a hydrophilic monomer, N-vinylpyrrolidone (NVP) widely used in the production of ophthalmic devices was evaluated. Specifically, equal masses of the polysilicone compound of Examples or Comparative Examples and NVP were mixed, and stirred at 25 degrees C. for 10 minutes. After the stirring, the mixture was left still at 25 degrees C. for five hours, and the appearance of the mixture was then observed visually and evaluated according to the following criteria.A: uniform and transparentB: cloudedC: the silicone compound and NVP separated completely [Contact Angle] The contact angle (°) of each of the hydrated films obtained above with water was determined by a sessile drop method using a contact angle meter CA-D (ex. Kyowa Interface Science Co. Ltd.). TABLE 1Com.Com.Com.Ex . 1Ex.2Ex .3Ex . 4Ex. 5Ex.6Ex. 1Ex.2Ex. 3Siloxane monomer13A50————————13B—50——————-—13C——50——————14A———50—————14B————50————14C—————50———ComparativeSIGMA——————50——compoundmPDMS———————50—TRIS-PEG2————————50PolymerizableNVP505050505050505050monomerEGDMA0.50.50.50.50.50.50.50. 50.5Irg11730.040.040.040.040.040.040.040.040.04ResultsEquilibrium water content, %46.445.146.845.944.645.546.843.144.9TransparencyAAAAAAABACompatibility with NVPAAAAAAACBContact angle, °4952515155519010495 As shown in Table 1, the compound according to the present invention has excellent compatibility with a hydrophilic monomer, and the hydrogel obtained by copolymerization of the present compound has high transparency and excellent surface wettability. INDUSTRIAL APPLICABILITY The compound according to the present invention attains an improved surface wettability of an obtained hydrogel. The present compound is useful as a monomer for preparing ophthalmic devices such as a contact lens, an intraocular lens, an artificial cornea, and a lens for eyewears. | 37,202 |
11859056 | DETAILED DESCRIPTION The following description recites various aspects and embodiments of the inventions disclosed herein. No particular embodiment is intended to define the scope of the invention. Rather, the embodiments provide non-limiting examples of various compositions, and methods that are included within the scope of the claimed inventions. The description is to be read from the perspective of one of ordinary skill in the art. Therefore, information that is well known to the ordinarily skilled artisan is not necessarily included. Definitions The following terms and phrases have the meanings indicated below, unless otherwise provided herein. This disclosure may employ other terms and phrases not expressly defined herein. Such other terms and phrases shall have the meanings that they would possess within the context of this disclosure to those of ordinary skill in the art. In some instances, a term or phrase may be defined in the singular or plural. In such instances, it is understood that any term in the singular may include its plural counterpart and vice versa, unless expressly indicated to the contrary. As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to “a substituent” encompasses a single substituent as well as two or more substituents, and the like. As used herein, “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. Unless otherwise expressly indicated, such examples are provided only as an aid for understanding embodiments illustrated in the present disclosure, and are not meant to be limiting in any fashion. Nor do these phrases indicate any kind of preference for the disclosed embodiment. As used herein, “polymer” refers to a substance having a chemical structure that includes the multiple repetition of constitutional units formed from substances of comparatively low relative molecular mass relative to the molecular mass of the polymer. The term “polymer” includes soluble and/or fusible molecules having chains of repeat units, and also includes insoluble and infusible networks. As used herein, the term “polymer” can include oligomeric compounds, which have only a few (e.g., 5-100) constitutional units. As used herein, “silicone polymer” or “siloxane polymer” refer to a polymer that includes a series of repeating constitutional units having unsubstituted or substituted silicon atoms and oxygen atoms, e.g., —Si—O—Si—O—Si—O—Si—O—Si—O—, wherein each silicon atom is additionally attached to two hydrogen atoms, non-hydrogen substituents, or a combination thereof. In general, the silicone polymers or siloxane polymers envisioned herein have a weight-average molecular weight of at least 500 Da, or 1 kDa. Such polymers can also contain additional constitutional units, including additional non-siloxane blocks, thereby resulting in copolymers, such as graft copolymers and block copolymers. As used herein, silicone polymers or siloxane polymers includes oligomeric compounds, which have only a few (e.g., 5-100) constitutional units. As used herein, “natural oil,” “natural feedstock,” or “natural oil feedstock” refer to oils derived from plants or animal sources. These terms include natural oil derivatives, unless otherwise indicated. The terms also include modified plant or animal sources (e.g., genetically modified plant or animal sources), unless indicated otherwise. Examples of natural oils include, but are not limited to, vegetable oils, algae oils, fish oils, animal fats, tall oils, derivatives of these oils, combinations of any of these oils, and the like. Representative non-limiting examples of vegetable oils include rapeseed oil (canola oil), coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybean oil, sunflower oil, linseed oil, palm kernel oil, tung oil, jatropha oil, mustard seed oil, pennycress oil, camelina oil, hempseed oil, and castor oil. Representative non-limiting examples of animal fats include lard, tallow, poultry fat, yellow grease, and fish oil. Tall oils are by-products of wood pulp manufacture. In some embodiments, the natural oil or natural oil feedstock comprises one or more unsaturated glycerides (e.g., unsaturated triglycerides). In some such embodiments, the natural oil feedstock comprises at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of one or more unsaturated triglycerides, based on the total weight of the natural oil feedstock. As used herein, “natural oil derivatives” refers to the compounds or mixtures of compounds derived from a natural oil using any one or combination of methods known in the art. Such methods include but are not limited to saponification, fat splitting, transesterification, esterification, hydrogenation (partial, selective, or full), isomerization, oxidation, and reduction. Representative non-limiting examples of natural oil derivatives include gums, phospholipids, soapstock, acidulated soapstock, distillate or distillate sludge, fatty acids and fatty acid alkyl ester (e.g. non-limiting examples such as 2-ethylhexyl ester), hydroxy substituted variations thereof of the natural oil. For example, the natural oil derivative may be a fatty acid methyl ester (“FAME”) derived from the glyceride of the natural oil. In some embodiments, a feedstock includes canola or soybean oil, as a non-limiting example, refined, bleached, and deodorized soybean oil (i.e., RBD soybean oil). Soybean oil typically comprises about 95% weight or greater (e.g., 99% weight or greater) triglycerides of fatty acids. Major fatty acids in the polyol esters of soybean oil include saturated fatty acids, as a non-limiting example, palmitic acid (hexadecanoic acid) and stearic acid (octadecanoic acid), and unsaturated fatty acids, as a non-limiting example, oleic acid (9-octadecenoic acid), linoleic acid (9, 12-octadecadienoic acid), and linolenic acid (9,12,15-octadecatrienoic acid). As used herein, “metathesis catalyst” includes any catalyst or catalyst system that catalyzes an olefin metathesis reaction. As used herein, “metathesize” or “metathesizing” refer to the reacting of a feedstock in the presence of a metathesis catalyst to form a “metathesized product” comprising new olefinic compounds, i.e., “metathesized” compounds. Metathesizing is not limited to any particular type of olefin metathesis, and may refer to cross-metathesis (i.e., co-metathesis), self-metathesis, ring-opening metathesis, ring-opening metathesis polymerizations (“ROMP”), ring-closing metathesis (“RCM”), and acyclic diene metathesis (“ADMET”). In some embodiments, metathesizing refers to reacting two triglycerides present in a natural feedstock (self-metathesis) in the presence of a metathesis catalyst, wherein each triglyceride has an unsaturated carbon-carbon double bond, thereby forming a new mixture of olefins and esters which may include a triglyceride dimer. Such triglyceride dimers may have more than one olefinic bond, thus higher oligomers also may form. Additionally, in some other embodiments, metathesizing may refer to reacting an olefin, such as ethylene, and a triglyceride in a natural feedstock having at least one unsaturated carbon-carbon double bond, thereby forming new olefinic molecules as well as new ester molecules (cross-metathesis). As used herein, “olefin” or “olefins” refer to compounds having at least one unsaturated carbon-carbon double bond. In certain embodiments, the term “olefins” refers to a group of unsaturated carbon-carbon double bond compounds with different carbon lengths. Unless noted otherwise, the terms “olefin” or “olefins” encompasses “polyunsaturated olefins” or “poly-olefins,” which have more than one carbon-carbon double bond. As used herein, the term “monounsaturated olefins” or “mono-olefins” refers to compounds having only one carbon-carbon double bond. A compound having a terminal carbon-carbon double bond can be referred to as a “terminal olefin,” while an olefin having a non-terminal carbon-carbon double bond can be referred to as an “internal olefin.” As used herein, the term “low-molecular-weight olefin” may refer to any one or combination of unsaturated straight, branched, or cyclic hydrocarbons in the C2-14range. Low-molecular-weight olefins include “alpha-olefins” or “terminal olefins,” wherein the unsaturated carbon-carbon bond is present at one end of the compound. Low-molecular-weight olefins may also include dienes or trienes. Low-molecular-weight olefins may also include internal olefins or “low-molecular-weight internal olefins.” In certain embodiments, the low-molecular-weight internal olefin is in the C4-14range. Examples of low-molecular-weight olefins in the C2-6range include, but are not limited to: ethylene, propylene, 1-butene, 2-butene, isobutene, 1-pentene, 2-pentene, 3-pentene, 2-methyl-1-butene, 2-methyl-2-butene, 3-methyl-1-butene, cyclopentene, 1,4-pentadiene, 1-hexene, 2-hexene, 3-hexene, 4-hexene, 2-methyl-1-pentene, 3-methyl-1-pentene, 4-methyl-1-pentene, 2-methyl-2-pentene, 3-methyl-2-pentene, 4-methyl-2-pentene, 2-methyl-3-pentene, and cyclohexene. Non-limiting examples of low-molecular-weight olefins in the C7-9range include 1,4-heptadiene, 1-heptene, 3,6-nonadiene, 3-nonene, 1,4,7-octatriene. Other possible low-molecular-weight olefins include styrene and vinyl cyclohexane. In certain embodiments, it is preferable to use a mixture of olefins, the mixture comprising linear and branched low-molecular-weight olefins in the C4-10range. In one embodiment, it may be preferable to use a mixture of linear and branched C4olefins (i.e., combinations of: 1-butene, 2-butene, and/or isobutene). In other embodiments, a higher range of C11-14may be used. In some instances, the olefin can be an “alkene,” which refers to a straight- or branched-chain non-aromatic hydrocarbon having 2 to 30 carbon atoms and one or more carbon-carbon double bonds, which may be optionally substituted, as herein further described, with multiple degrees of substitution being allowed. A “monounsaturated alkene” refers to an alkene having one carbon-carbon double bond, while a “polyunsaturated alkene” refers to an alkene having two or more carbon-carbon double bonds. A “lower alkene,” as used herein, refers to an alkene having from 2 to 10 carbon atoms. As used herein, “alpha-olefin” refers to an olefin (as defined above) that has a terminal carbon-carbon double bond. In some embodiments, the alpha-olefin is a terminal alkene, which is an alkene (as defined above) having a terminal carbon-carbon double bond. Additional carbon-carbon double bonds can be present. As used herein, “ester” or “esters” refer to compounds having the general formula: R—COO—R′, wherein R and R′ denote any organic group (such as alkyl, aryl, or silyl groups) including those bearing heteroatom-containing substituent groups. In certain embodiments, R and R′ denote alkyl, alkenyl, aryl, or alcohol groups. In certain embodiments, the term “esters” may refer to a group of compounds with the general formula described above, wherein the compounds have different carbon lengths. In certain embodiments, the esters may be esters of glycerol, which is a trihydric alcohol. The term “glyceride” can refer to esters where one, two, or three of the —OH groups of the glycerol have been esterified. It is noted that an olefin may also comprise an ester, and an ester may also comprise an olefin, if the R or R′ group in the general formula R—COO—R′ contains an unsaturated carbon-carbon double bond. Such compounds can be referred to as “unsaturated esters” or “olefin esters.” Further, a “terminal olefin ester” may refer to an ester compound where R has an olefin positioned at the end of the chain. An “internal olefin ester” may refer to an ester compound where R has an olefin positioned at an internal location on the chain. Additionally, the term “terminal olefin” may refer to an ester or an acid thereof where R′ denotes hydrogen or any organic compound (such as an alkyl, aryl, or silyl group) and R has an olefin positioned at the end of the chain, and the term “internal olefin” may refer to an ester or an acid thereof where R′ denotes hydrogen or any organic compound (such as an alkyl, aryl, or silyl group) and R has an olefin positioned at an internal location on the chain. As used herein, “hydrocarbon” refers to an organic group composed of carbon and hydrogen, which can be saturated or unsaturated, and can include aromatic groups. The term “hydrocarbyl” refers to a monovalent or polyvalent hydrocarbon moiety. As used herein, “alkyl” refers to a straight or branched chain saturated hydrocarbon having 1 to 30 carbon atoms, which may be optionally substituted, as herein further described, with multiple degrees of substitution being allowed. Examples of “alkyl,” as used herein, include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neopentyl, n-hexyl, and 2-ethylhexyl. The number of carbon atoms in an alkyl group is represented by the phrase “Cx-yalkyl,” which refers to an alkyl group, as herein defined, containing from x to y, inclusive, carbon atoms. Thus, “C1-6alkyl” represents an alkyl chain having from 1 to 6 carbon atoms and, for example, includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, isobutyl, n-butyl, sec-butyl, tert-butyl, isopentyl, n-pentyl, neopentyl, and n-hexyl. In some instances, the “alkyl” group can be divalent, in which case the group can alternatively be referred to as an “alkylene” group. Also, in some instances, one or more of the carbon atoms in the alkyl or alkylene group can be replaced by a heteroatom (e.g., selected from nitrogen, oxygen, or sulfur, including quaternary nitrogen atoms, N-oxides, sulfur oxides, and sulfur dioxides, where feasible), and is referred to as a “heteroalkyl” or “heteroalkylene” group, respectively. Non-limiting examples include “oxyalkyl” or “oxyalkylene” groups, which are groups of the following formulas: -[-(alkylene)-O-]v-alkyl, or -[-(alkylene)-O-]v-alkylene-, respectively, where v is 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the like. As used herein, “alkenyl” refers to a straight or branched chain non-aromatic hydrocarbon having 2 to 30 carbon atoms and having one or more carbon-carbon double bonds, which may be optionally substituted, as herein further described, with multiple degrees of substitution being allowed. Examples of “alkenyl,” as used herein, include, but are not limited to, ethenyl, 2-propenyl, 2-butenyl, and 3-butenyl. The number of carbon atoms in an alkenyl group is represented by the phrase “Cx-yalkenyl,” which refers to an alkenyl group, as herein defined, containing from x to y, inclusive, carbon atoms. Thus, “C2-alkenyl” represents an alkenyl chain having from 2 to 6 carbon atoms and, for example, includes, but is not limited to, ethenyl, 2-propenyl, 2-butenyl, and 3-butenyl. In some instances, the “alkenyl” group can be divalent, in which case the group can alternatively be referred to as an “alkenylene” group. As used herein, “halogen” or “halo” refers to a fluorine, chlorine, bromine, and/or iodine atom. In some embodiments, the terms refer to fluorine or chlorine. As used herein, “haloalkyl” or “haloalkoxy” refer to alkyl or alkoxy groups, respectively, substituted by one or more halogen atoms. The terms “perfluoroalkyl” or “perfluoroalkoxy” refer to alkyl groups and alkoxy groups, respectively, where every available hydrogen is replaced by fluorine. As used herein, “aryl” refers to a 6- to 30-membered cyclic, aromatic hydrocarbon, which may be optionally substituted as herein further described, with multiple degrees of substitution being allowed. Examples of “aryl” groups as used herein include, but are not limited to, phenyl and naphthyl. As used herein, the term “aryl” also includes ring systems in which a phenyl or naphthyl group is optionally fused with one to three non-aromatic, saturated or unsaturated, carbocyclic rings. For example, “aryl” would include ring systems such as indene, with attachment possible to either the aromatic or the non-aromatic ring(s). As used herein, “substituted” refers to substitution of one or more hydrogen atoms of the designated moiety with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated, provided that the substitution results in a stable or chemically feasible compound. A stable compound or chemically feasible compound is one in which the chemical structure is not substantially altered when kept at a temperature from about −80° C. to about +40° C., in the absence of moisture or other chemically reactive conditions, for at least a week. As used herein, the phrases “substituted with one or more . . . ” or “substituted one or more times . . . ” refer to a number of substituents that equals from one to the maximum number of substituents possible based on the number of available bonding sites, provided that the above conditions of stability and chemical feasibility are met. As used herein, “mix” or “mixed” or “mixture” refers broadly to any combining of two or more compositions. The two or more compositions need not have the same physical state; thus, solids can be “mixed” with liquids, e.g., to form a slurry, suspension, or solution. Further, these terms do not require any degree of homogeneity or uniformity of composition. This, such “mixtures” can be homogeneous or heterogeneous, or can be uniform or non-uniform. Further, the terms do not require the use of any particular equipment to carry out the mixing, such as an industrial mixer. As used herein, “optionally” means that the subsequently described event(s) may or may not occur. In some embodiments, the optional event does not occur. In some other embodiments, the optional event does occur one or more times. As used herein, “comprise” or “comprises” or “comprising” or “comprised of” refer to groups that are open, meaning that the group can include additional members in addition to those expressly recited. For example, the phrase, “comprises A” means that A must be present, but that other members can be present too. The terms “include,” “have,” and “composed of” and their grammatical variants have the same meaning. In contrast, “consist of” or “consists of” or “consisting of” refer to groups that are closed. For example, the phrase “consists of A” means that A and only A is present. As used herein, “or” is to be given its broadest reasonable interpretation, and is not to be limited to an either/or construction. Thus, the phrase “comprising A or B” means that A can be present and not B, or that B is present and not A, or that A and B are both present. Further, if A, for example, defines a class that can have multiple members, e.g., A1and A2, then one or more members of the class can be present concurrently. As used herein, the various functional groups represented will be understood to have a point of attachment at the functional group having the hyphen or dash (-) or an asterisk (*). In other words, in the case of —CH2CH2CH3, it will be understood that the point of attachment is the CH2group at the far left. If a group is recited without an asterisk or a dash, then the attachment point is indicated by the plain and ordinary meaning of the recited group. In some instances herein, organic compounds are described using the “line structure” methodology, where chemical bonds are indicated by a line, where the carbon atoms are not expressly labeled, and where the hydrogen atoms covalently bound to carbon (or the C—H bonds) are not shown at all. For example, by that convention, the formula represents n-propane. In some instances herein, a squiggly bond is used to show the compound can have any one of two or more isomers. For example, the structure can refer to (E)-2-butene or (Z)-2-butene. As used herein, multi-atom bivalent species are to be read from left to right. For example, if the specification or claims recite A-D-E and D is defined as —OC(O)—, the resulting group with D replaced is: A-OC(O)-E and not A-C(O)O-E. Other terms are defined in other portions of this description, even though not included in this subsection. Siloxane Polymers In a one or more aspects, the disclosure provides siloxane polymers comprising:(a) one or more segments of formula (I): and(b) one or more segments of formula (II): wherein: each R1, R2, R3, and R4is independently a hydrogen atom, a C1-14hydrocarbyl group, or a C1-14hydrocarbyloxy group; each X1and X2is independently an oxygen atom, a sulfur atom, >NH, or >N(C1-6alkyl); each G1and G2is independently a C1-14hydrocarbylene group; each G3is independently a C1-50hydrocarbylene group, which is optionally substituted one or more times by substituents selected from the group consisting of —OH and —O(C1-6alkyl), or a C2-120oxyalkylene group; and each k is independently an integer ranging from 5 to 5000. The segments of formula (II) can have any suitable chemical structure, according to the formula set forth above. These segments can, in some embodiments, be derived from renewable oils. For example, in some embodiments, compounds such as 9-decenoic acid, 10-undecenoic acid, 11-dodecenoic acid, or esters thereof, can be reacted with a polyol, such as ethylene glycol or polyethylene glycols, to form difunctional segments having two terminal carbon-carbon double bonds that have reactive affinity to certain silanes. Thus, in some embodiments, each G1is independently a C9-11alkylene group. In some further such embodiments, each G1is independently —(CH2)9—, —(CH2)10—, or —(CH2)11—. In some further such embodiments, each G1is independently —(CH2)9— or —(CH2)11—. In some further such embodiments, each G1is —(CH2)9—. In some other such embodiments, each G1is —(CH2)10—. In some other such embodiments, each G1is —(CH2)11—. In an analogous manner, in some embodiments according to any of the aforementioned embodiments, each G2is independently a C9-11alkylene group. In some further such embodiments, each G2is independently —(CH2)9—, —(CH2)10—, or —(CH2)11—. In some further such embodiments, each G2is independently —(CH2)9— or —(CH2)11—. In some further such embodiments, each G2is —(CH2)9—. In some other such embodiments, each G2is —(CH2)10—. In some other such embodiments, each G2is —(CH2)11—. The variable G3can have any suitable value. As noted above, the segments of formula (II) can, in some embodiments, be formed by reacting two terminally unsaturated monobasic fatty acids (or esters thereof) with a polyol. Thus, in some embodiments, G3can be any formed from any suitable polyol moiety. In some embodiments, each G3is independently a C1-50hydrocarbylene group, which is optionally substituted one or more times by substituents selected from the group consisting of —OH and —O(C1-6alkyl). In some further such embodiments, each G3is a C1-14alkylene group, which is optionally substituted one or more times by substituents selected from the group consisting of —OH and —O(C1-6alkyl). In some further embodiments, each G3is an unsubstituted C1-14alkylene group. In some further embodiments, each G3is an unsubstituted C1-8alkylene group, such as a methylene group, an ethylene group, a 1,2-propylene group, a 1,3-propylene group, a 1,4-butylene group, a 1,5-pentylene group, a 1,6-hexylene group, and the like. In some embodiments, each G3is an ethylene group, a 1,2-propylene group, or a 1,3-propylene group. In some further such embodiments, each G3is an ethylene group. The variables X1and X2can have any suitable value, according to the embodiments set forth above. As noted, in some embodiments, the segments according to formula (II) can be formed from reacting terminally unsaturated acids with polyols. In such embodiments, X1and X2are both an oxygen atom in each instance. In some other embodiments, however, the compounds besides polyols can be used to connect the terminally unsaturated acids. Thus, in some other embodiments, X1and X2are both a sulfur atom in each instance. In some other embodiments, in each instance, X1and X2are independently >NH or >N(C1-6alkyl), such as >NH or >N(CH3). The segments of formula (I) can have any suitable chemical structure, according to the formula set forth above. In some embodiments, each R1, R2, R3, and R4is independently a hydrogen atom, a C1-14hydrocarbyl group, or a C1-14hydrocarbyloxy group. In some such embodiments, each R1, R2, R3, and R4is independently a C1-14hydrocarbyl group. In some further such embodiments, each R1, R2, R3, and R4is independently a C1-8alkyl group. In some even further embodiments, each R1, R2, R3, and R4is independently selected from the group consisting of methyl, ethyl, propyl, and isopropyl. In some even further embodiments, each R1, R2, R3, and R4is independently selected from the group consisting of methyl and ethyl. In some even further embodiments, each R1, R2, R3, and R4is methyl. In some embodiments, most, but not all, of R1, R2, R3, and R4are independently C1-6alkyl (such as methyl, ethyl, or isopropyl). In some such embodiments, at least 50% by number, or at least 60% by number, or at least 70% by number, or at least 80% by number, or at least 90% by number, of all R1, R2, R3, and R4in each segment of formula (I) are independently C1-6alkyl (such as methyl, ethyl, or isopropyl), and the remaining R1, R2, R3, and R4are independently a hydrogen atom or C1-6alkoxy (such as methoxy, ethoxy, or isopropoxy). In some further such embodiments, at least one of R1, R2, R3, and R4in each segment of formula (I) in the polymer is a hydrogen atom. In some further such embodiments, at least 50% by number, or at least 60% by number, or at least 70% by number, or at least 80% by number, or at least 90% by number, of all R1, R2, R3, and R4in each segment of formula (I) are independently C1-6alkyl (such as methyl, ethyl, or isopropyl), and the remaining R1, R2, R3, and R4are independently a C1-6alkoxy (such as methoxy, ethoxy, or isopropoxy). The siloxane polymers disclosed herein can contain additional segments or constitutional units besides those of formula (I) and formula (II). For example, the siloxane polymers can be copolymers, which have additional constitutional units interspersed with the segments of formula (I) and formula (II). In some other cases, the siloxane polymers can be block copolymers or graft copolymers, where the polymer contains, for example, other non-siloxane segments or blocks. Thus, in some embodiments, in the siloxane polymers disclosed herein, the segments of formula (I) and the segments of formula (II) together make up at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, of the siloxane polymer. In some further embodiments of the foregoing embodiments, in the siloxane polymers disclosed herein, the segments of formula (I) and the segments of formula (II) together make up no more than 80% by weight, or no more than 90% by weight, or no more than 95% by weight, or no more than 97% by weight, or no more than 99% by weight, of the siloxane polymer. The siloxane polymers disclosed herein can contain any suitable amount of the segments of formula (I). Thus, in some embodiments, in the siloxane polymers disclosed herein, the segments of formula (I) make up at least 10% by weight, or at least 20% by weight, or at least 30% by weight, or at least 40% by weight, or at least 50% by weight, of the siloxane polymer. In some further embodiments of any of the foregoing embodiments, in the siloxane polymers disclosed herein, the segments of formula (I) make up no more than 60% by weight, or no more than 70% by weight, or no more than 80% by weight, or no more than 90% by weight, or no more than 95% by weight, of the siloxane polymer. The siloxane polymers disclosed herein can contain any suitable amount of the segments of formula (II). Thus, in some embodiments, in the siloxane polymers disclosed herein, the segments of formula (II) make up at least 30% by weight, or at least 40% by weight, or at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, of the siloxane polymer. In some further embodiments of any of the foregoing embodiments, in the siloxane polymers disclosed herein, the segments of formula (II) make up no more than 30% by weight, or no more than 40% by weight, or no more than 50% by weight, or no more than 60% by weight, or no more than 70% by weight, or no more than 80% by weight, or no more than 90% by weight, or no more than 95% by weight, of the siloxane polymer. The segments of formula (I) and the segments of formula (II) can be present in the siloxane polymer in any suitable relative amounts. For example, in the siloxane polymers disclosed herein, the numerical ratio of segments of formula (I) to segments of formula (II) in the siloxane polymer ranges from 1:10 to 10:1, or from 1:7 to 7:1, or from 1:5 to 5:1, or from 1:4 to 4:1, or from 1:3 to 3:1, or from 1:2 to 2:1. In some other embodiments, the numerical ratio of segments of formula (I) to segments of formula (II) in the siloxane polymer ranges from 1:1 to 10:1, or from 1:1 to 7:1, or from 1:1 to 5:1, or from 1:1 to 4:1, or from 1:1 to 3:1, or from 1:1 to 2:1. In some other embodiments, the numerical ratio of segments of formula (II) to segments of formula (I) in the siloxane polymer ranges from 1:1 to 10:1, or from 1:1 to 7:1, or from 1:1 to 5:1, or from 1:1 to 4:1, or from 1:1 to 3:1, or from 1:1 to 2:1. The siloxane polymers of any of the above embodiments can have any suitable physical properties. For example, in some embodiments, the siloxane polymer has a molecular weight ranging from 1 kDa to 50 kDa. In general, the siloxane polymers disclosed herein contain two or more endcap groups. The endcap groups can have any suitable chemical structure, but are generally silane- or siloxane-based moieties. In some embodiments, the endcap groups are moieties of formula (III): —(O)m—Si(R21)(R22)(R23) (III) wherein: R21, R22, and R23are independently a hydrogen atom, —OH, C1-14hydrocarbyl, or C1-14hydrocarbyloxy; and m is 0 or 1. In general, the value of m will vary on the chemical functionality of the group that the group is capping. For example, if the endcap group is capping a moiety that otherwise terminates in an oxygen atom, then m is typically 0, as the siloxane polymers disclosed herein do not generally contain peroxide functionality in the backbone of the polymer. On the other hand, in embodiments where the endcap group is capping a moiety that otherwise terminates in silicon or carbon, then m is 1. In some such embodiments, at least two of R21, R22, and R23is a hydrogen atom or C1-14hydrocarbyl. In some further such embodiments, at least two of R21, R22, and R23is C1-14hydrocarbyl. In some even further such embodiments, at least two of R21, R22, and R23are selected independently from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or neopentyl. In some even further such embodiments, at least two of R21, R22, and R23are methyl. In some further embodiments of any of the aforementioned embodiments, one of R21, R22, and R23is a —OH or C1-14hydrocarbyloxy. In some further such embodiments, one of R21, R22, and R23is C1-14hydrocarbyloxy. In some even further such embodiments, one of R21, R22, and R23is selected independently from the group consisting of: methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, or neopentoxy. In some even further such embodiments, one of R21, R22, and R23is methoxy. Methods of Making Siloxane Polymers The siloxane polymers disclosed herein may be made by any suitable means. For example, in some cases, polysiloxane segments may be prepared by suitable means, where such polysiloxane segments have Si—H groups at their terminal points, which allows them to be reactive with carbon-carbon double bonds. Organic segments, such as those according to formula (II) can be prepared by suitable means. As described above, in some embodiments, methyl esters of 9-decenoic acid, 10-undecenoic acid, or 11-dodecenoic acid can be reacted with ethylene glycol or a polyethylene glycol, which reacts with the ester groups to form a segment having two terminal carbon-carbon double bonds. This organic segment can then be reacted with the polysiloxane segments having terminal Si—H groups to form polysiloxanes according to certain embodiments disclosed herein. Silicone Compositions In one or more aspects, the present disclosure provides silicone compositions that comprise one or more siloxane polymers according to any of the embodiments set forth above. Such compositions can include the one or more siloxane polymers in any suitable concentration. For example, in some embodiments, the silicone polymer compositions comprise from 0.1 to 99 percent by weight, or from 0.5 to 99 percent by weight, or from 1 to 99 percent by weight, or from 5 to 99 percent by weight, of one or more siloxane polymers, based on the total weight of dry solids in the composition (i.e., the weight excluding the weight of any solvent(s), but including the weight of suspended or solvated non-solvent components). In some embodiments, the silicone compositions disclosed herein include a carrier. Such compositions can include the one or more siloxane polymers in any suitable concentration. For example, in some embodiments, such silicone compositions comprise from 0.1 to 99 percent by weight, or from 0.5 to 99 percent by weight, or from 1 to 99 percent by weight, or from 5 to 99 percent by weight, based on the total weight of the composition. Any suitable carriers can be used. In some embodiments of any of the aforementioned embodiments, the carrier comprises water. In some other embodiments of any of the aforementioned embodiments, the carrier comprises an organic solvent. The silicone compositions disclosed herein can also include one or more additives. For example, in some embodiments, the silicone composition includes one or more additives, such as surfactants, pigments, antimicrobial agents, photostabilizers, and the like. The one or more siloxane polymers in the silicone compositions can have any suitable molecular weight range. For example, in some embodiments, the one or more siloxane polymers in the silicone composition have a weight-average molecular weight ranging from 1 kDa to 50 kDa. Such silicone compositions can be used in any suitable way. For example, in some embodiments, the silicone composition is a surfactant composition, a sizing composition for a matrix reinforcement material (a siliceous material, such as silica or sand; glass, such as glass fiber, glass particles, or glass beads; a metal, such as silver or titanium; a metal oxide, such as zinc oxide or titanium dioxide; carbon, such as carbon nanoparticles, carbon nanotubes, graphite, graphene, diamond, and fullerenes, or any combination of the foregoing), a coating composition, a sealant composition, a grease composition, a defoaming composition, a dry-cleaning composition, a rubber composition, an ophthalmic composition, a personal care composition, a lubricant composition, a personal lubricant composition, a functional fluid, such as a brake fluid, a mold release composition, a gel composition, or an electronics encasement composition. Articles of Manufacture In one or more aspects, the present disclosure provides articles of manufacture formed from the silicone compositions of any of the aforementioned embodiments. The article of manufacture can be any suitable article of manufacture, such as those that may typically be formed using silicone polymers. For example, in some embodiments, the article of manufacture is an electrical insulating article, an electronic device (where, for example, the silicone composition is in a coating or sealing layer), a gasket, a seal, a pad, a mold (such as, for example, a dental mold), a paper article (such as a sheet, where, for example, the silicone composition is in a coating), a textile article (where, for example, the silicone composition is in a coating), a fire stop, a microfluidic device, a bandage, a dressing for a wound, a scar treatment sheet, a breast implant, a testicular implant, a pectoral implant, a contact lens, an ophthalmic tube, an ophthalmic stent, or a nipple, such as a nipple on a baby bottle. Derivation from Renewable Sources The siloxane polymers disclosed herein and used in any of the aspects and embodiments disclosed herein can, in certain embodiments, be derived from renewable sources, such as various natural oils. Any suitable methods can be used to make these compounds from such renewable sources. Suitable methods include, but are not limited to, fermentation, conversion by bioorganisms, and conversion by metathesis. Olefin metathesis provides one possible means to convert certain natural oil feedstocks into olefins and esters that can be used in a variety of applications, or that can be further modified chemically and used in a variety of applications. In some embodiments, a composition (or components of a composition) may be formed from a renewable feedstock, such as a renewable feedstock formed through metathesis reactions of natural oils and/or their fatty acid or fatty ester derivatives. When compounds containing a carbon-carbon double bond undergo metathesis reactions in the presence of a metathesis catalyst, some or all of the original carbon-carbon double bonds are broken, and new carbon-carbon double bonds are formed. The products of such metathesis reactions include carbon-carbon double bonds in different locations, which can provide unsaturated organic compounds having useful chemical properties. A wide range of natural oils, or derivatives thereof, can be used in such metathesis reactions. Examples of suitable natural oils include, but are not limited to, vegetable oils, algae oils, fish oils, animal fats, tall oils, derivatives of these oils, combinations of any of these oils, and the like. Representative non-limiting examples of vegetable oils include rapeseed oil (canola oil), coconut oil, corn oil, cottonseed oil, olive oil, palm oil, peanut oil, safflower oil, sesame oil, soybean oil, sunflower oil, linseed oil, palm kernel oil, tung oil, jatropha oil, mustard seed oil, pennycress oil, camelina oil, hempseed oil, and castor oil. Representative non-limiting examples of animal fats include lard, tallow, poultry fat, yellow grease, and fish oil. Tall oils are by-products of wood pulp manufacture. In some embodiments, the natural oil or natural oil feedstock comprises one or more unsaturated glycerides (e.g., unsaturated triglycerides). In some such embodiments, the natural oil feedstock comprises at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of one or more unsaturated triglycerides, based on the total weight of the natural oil feedstock. The natural oil may include canola or soybean oil, such as refined, bleached and deodorized soybean oil (i.e., RBD soybean oil). Soybean oil typically includes about 95 percent by weight (wt %) or greater (e.g., 99 wt % or greater) triglycerides of fatty acids. Major fatty acids in the polyol esters of soybean oil include but are not limited to saturated fatty acids such as palmitic acid (hexadecanoic acid) and stearic acid (octadecanoic acid), and unsaturated fatty acids such as oleic acid (9-octadecenoic acid), linoleic acid (9,12-octadecadienoic acid), and linolenic acid (9,12,15-octadecatrienoic acid). Metathesized natural oils can also be used. Examples of metathesized natural oils include but are not limited to a metathesized vegetable oil, a metathesized algal oil, a metathesized animal fat, a metathesized tall oil, a metathesized derivatives of these oils, or mixtures thereof. For example, a metathesized vegetable oil may include metathesized canola oil, metathesized rapeseed oil, metathesized coconut oil, metathesized corn oil, metathesized cottonseed oil, metathesized olive oil, metathesized palm oil, metathesized peanut oil, metathesized safflower oil, metathesized sesame oil, metathesized soybean oil, metathesized sunflower oil, metathesized linseed oil, metathesized palm kernel oil, metathesized tung oil, metathesized jatropha oil, metathesized mustard oil, metathesized camelina oil, metathesized pennycress oil, metathesized castor oil, metathesized derivatives of these oils, or mixtures thereof. In another example, the metathesized natural oil may include a metathesized animal fat, such as metathesized lard, metathesized tallow, metathesized poultry fat, metathesized fish oil, metathesized derivatives of these oils, or mixtures thereof. Such natural oils, or derivatives thereof, can contain esters, such as triglycerides, of various unsaturated fatty acids. The identity and concentration of such fatty acids varies depending on the oil source, and, in some cases, on the variety. In some embodiments, the natural oil comprises one or more esters of oleic acid, linoleic acid, linolenic acid, or any combination thereof. When such fatty acid esters are metathesized, new compounds are formed. For example, in embodiments where the metathesis uses certain short-chain olefins, e.g., ethylene, propylene, or 1-butene, and where the natural oil includes esters of oleic acid, an amount of 1-decene and 1-decenoid acid (or an ester thereof), among other products, are formed. Following transesterification, for example, with an alkyl alcohol, an amount of 9-denenoic acid alkyl ester is formed. In some such embodiments, a separation step may occur between the metathesis and the transesterification, where the alkenes are separated from the esters. In some other embodiments, transesterification can occur before metathesis, and the metathesis is performed on the transesterified product. In some embodiments, the natural oil can be subjected to various pre-treatment processes, which can facilitate their utility for use in certain metathesis reactions. Useful pre-treatment methods are described in United States Patent Application Publication Nos. 2011/0113679, 2014/0275595, and 2014/0275681, all three of which are hereby incorporated by reference as though fully set forth herein. In some embodiments, after any optional pre-treatment of the natural oil feedstock, the natural oil feedstock is reacted in the presence of a metathesis catalyst in a metathesis reactor. In some other embodiments, an unsaturated ester (e.g., an unsaturated glyceride, such as an unsaturated triglyceride) is reacted in the presence of a metathesis catalyst in a metathesis reactor. These polyol esters of unsaturated fatty acids may be a component of a natural oil feedstock, or may be derived from other sources, e.g., from esters generated in earlier-performed metathesis reactions. In certain embodiments, in the presence of a metathesis catalyst, the natural oil or unsaturated ester can undergo a self-metathesis reaction with itself. In other embodiments, the natural oil or unsaturated ester undergoes a cross-metathesis reaction with the low-molecular-weight olefin or mid-weight olefin. The self-metathesis and/or cross-metathesis reactions form a metathesized product wherein the metathesized product comprises olefins and esters. In some embodiments, the low-molecular-weight olefin is in the C2-6range. As a non-limiting example, in one embodiment, the low-molecular-weight olefin may comprise at least one of: ethylene, propylene, 1-butene, 2-butene, isobutene, 1-pentene, 2-pentene, 3-pentene, 2-methyl-1-butene, 2-methyl-2-butene, 3-methyl-1-butene, cyclopentene, 1,4-pentadiene, 1-hexene, 2-hexene, 3-hexene, 4-hexene, 2-methyl-1-pentene, 3-methyl-1-pentene, 4-methyl-1-pentene, 2-methyl-2-pentene, 3-methyl-2-pentene, 4-methyl-2-pentene, 2-methyl-3-pentene, and cyclohexene. In some instances, a higher-molecular-weight olefin can also be used. In some embodiments, the metathesis comprises reacting a natural oil feedstock (or another unsaturated ester) in the presence of a metathesis catalyst. In some such embodiments, the metathesis comprises reacting one or more unsaturated glycerides (e.g., unsaturated triglycerides) in the natural oil feedstock in the presence of a metathesis catalyst. In some embodiments, the unsaturated glyceride comprises one or more esters of oleic acid, linoleic acid, linoleic acid, or combinations thereof. In some other embodiments, the unsaturated glyceride is the product of the partial hydrogenation and/or the metathesis of another unsaturated glyceride (as described above). In some such embodiments, the metathesis is a cross-metathesis of any of the aforementioned unsaturated triglyceride species with another olefin, e.g., an alkene. In some such embodiments, the alkene used in the cross-metathesis is a lower alkene, such as ethylene, propylene, 1-butene, 2-butene, etc. In some embodiments, the alkene is ethylene. In some other embodiments, the alkene is propylene. In some further embodiments, the alkene is 1-butene. And in some even further embodiments, the alkene is 2-butene. Metathesis reactions can provide a variety of useful products, when employed in the methods disclosed herein. For example, the polyol esters of unsaturated fatty acids may be derived from a natural oil feedstock, in addition to other valuable compositions. Moreover, in some embodiments, a number of valuable compositions can be targeted through the self-metathesis reaction of a natural oil feedstock, or the cross-metathesis reaction of the natural oil feedstock with a low-molecular-weight olefin or mid-weight olefin, in the presence of a metathesis catalyst. Such valuable compositions can include fuel compositions, detergents, surfactants, and other specialty chemicals. Additionally, transesterified products (i.e., the products formed from transesterifying an ester in the presence of an alcohol) may also be targeted, non-limiting examples of which include: fatty acid methyl esters (“FAMEs”); biodiesel; 9-decenoic acid (“9DA”) esters, 9-undecenoic acid (“9UDA”) esters, and/or 9-dodecenoic acid (“9DDA”) esters; 9DA, 9UDA, and/or 9DDA; alkali metal salts and alkaline earth metal salts of 9DA, 9UDA, and/or 9DDA; dimers of the transesterified products; and mixtures thereof. Further, in some embodiments, multiple metathesis reactions can also be employed. In some embodiments, the multiple metathesis reactions occur sequentially in the same reactor. For example, a glyceride containing linoleic acid can be metathesized with a terminal lower alkene (e.g., ethylene, propylene, 1-butene, and the like) to form 1,4-decadiene, which can be metathesized a second time with a terminal lower alkene to form 1,4-pentadiene. In other embodiments, however, the multiple metathesis reactions are not sequential, such that at least one other step (e.g., transesterification, hydrogenation, etc.) can be performed between the first metathesis step and the following metathesis step. These multiple metathesis procedures can be used to obtain products that may not be readily obtainable from a single metathesis reaction using available starting materials. For example, in some embodiments, multiple metathesis can involve self-metathesis followed by cross-metathesis to obtain metathesis dimers, trimmers, and the like. In some other embodiments, multiple metathesis can be used to obtain olefin and/or ester components that have chain lengths that may not be achievable from a single metathesis reaction with a natural oil triglyceride and typical lower alkenes (e.g., ethylene, propylene, 1-butene, 2-butene, and the like). Such multiple metathesis can be useful in an industrial-scale reactor, where it may be easier to perform multiple metathesis than to modify the reactor to use a different alkene. The conditions for such metathesis reactions, and the reactor design, and suitable catalysts are as described above with reference to the metathesis of the olefin esters. That discussion is incorporated by reference as though fully set forth herein. In the embodiments above, the natural oil (e.g., as a glyceride) is metathesized, followed by transesterification. In some other embodiments, transesterification can precede metathesis, such that the fatty acid esters subjected to metathesis are fatty acid esters of monohydric alcohols, such as methanol, ethanol, or isopropanol. Olefin Metathesis In some embodiments, one or more of the unsaturated monomers can be made by metathesizing a natural oil or natural oil derivative. The terms “metathesis” or “metathesizing” can refer to a variety of different reactions, including, but not limited to, cross-metathesis, self-metathesis, ring-opening metathesis, ring-opening metathesis polymerizations (“ROMP”), ring-closing metathesis (“RCM”), and acyclic diene metathesis (“ADMET”). Any suitable metathesis reaction can be used, depending on the desired product or product mixture. In some embodiments, after any optional pre-treatment of the natural oil feedstock, the natural oil feedstock is reacted in the presence of a metathesis catalyst in a metathesis reactor. In some other embodiments, an unsaturated ester (e.g., an unsaturated glyceride, such as an unsaturated triglyceride) is reacted in the presence of a metathesis catalyst in a metathesis reactor. These unsaturated esters may be a component of a natural oil feedstock, or may be derived from other sources, e.g., from esters generated in earlier-performed metathesis reactions. In certain embodiments, in the presence of a metathesis catalyst, the natural oil or unsaturated ester can undergo a self-metathesis reaction with itself. In other embodiments, the natural oil or unsaturated ester undergoes a cross-metathesis reaction with the low-molecular-weight olefin or mid-weight olefin. The self-metathesis and/or cross-metathesis reactions form a metathesized product wherein the metathesized product comprises olefins and esters. In some embodiments, the low-molecular-weight olefin is in the C2-6range. As a non-limiting example, in one embodiment, the low-molecular-weight olefin may comprise at least one of: ethylene, propylene, 1-butene, 2-butene, isobutene, 1-pentene, 2-pentene, 3-pentene, 2-methyl-1-butene, 2-methyl-2-butene, 3-methyl-1-butene, cyclopentene, 1,4-pentadiene, 1-hexene, 2-hexene, 3-hexene, 4-hexene, 2-methyl-1-pentene, 3-methyl-1-pentene, 4-methyl-1-pentene, 2-methyl-2-pentene, 3-methyl-2-pentene, 4-methyl-2-pentene, 2-methyl-3-pentene, and cyclohexene. In some instances, a higher-molecular-weight olefin can also be used. In some embodiments, the metathesis comprises reacting a natural oil feedstock (or another unsaturated ester) in the presence of a metathesis catalyst. In some such embodiments, the metathesis comprises reacting one or more unsaturated glycerides (e.g., unsaturated triglycerides) in the natural oil feedstock in the presence of a metathesis catalyst. In some embodiments, the unsaturated glyceride comprises one or more esters of oleic acid, linoleic acid, linoleic acid, or combinations thereof. In some other embodiments, the unsaturated glyceride is the product of the partial hydrogenation and/or the metathesis of another unsaturated glyceride (as described above). In some such embodiments, the metathesis is a cross-metathesis of any of the aforementioned unsaturated triglyceride species with another olefin, e.g., an alkene. In some such embodiments, the alkene used in the cross-metathesis is a lower alkene, such as ethylene, propylene, 1-butene, 2-butene, etc. In some embodiments, the alkene is ethylene. In some other embodiments, the alkene is propylene. In some further embodiments, the alkene is 1-butene. And in some even further embodiments, the alkene is 2-butene. Metathesis reactions can provide a variety of useful products, when employed in the methods disclosed herein. For example, terminal olefins and internal olefins may be derived from a natural oil feedstock, in addition to other valuable compositions. Moreover, in some embodiments, a number of valuable compositions can be targeted through the self-metathesis reaction of a natural oil feedstock, or the cross-metathesis reaction of the natural oil feedstock with a low-molecular-weight olefin or mid-weight olefin, in the presence of a metathesis catalyst. Such valuable compositions can include fuel compositions, detergents, surfactants, and other specialty chemicals. Additionally, transesterified products (i.e., the products formed from transesterifying an ester in the presence of an alcohol) may also be targeted, non-limiting examples of which include: fatty acid methyl esters (“FAMEs”); biodiesel; 9-decenoic acid (“9DA”) esters, 9-undecenoic acid (“9UDA”) esters, and/or 9-dodecenoic acid (“9DDA”) esters; 9DA, 9UDA, and/or 9DDA; alkali metal salts and alkaline earth metal salts of 9DA, 9UDA, and/or 9DDA; dimers of the transesterified products; and mixtures thereof. Further, in some embodiments, the methods disclosed herein can employ multiple metathesis reactions. In some embodiments, the multiple metathesis reactions occur sequentially in the same reactor. For example, a glyceride containing linoleic acid can be metathesized with a terminal lower alkene (e.g., ethylene, propylene, 1-butene, and the like) to form 1,4-decadiene, which can be metathesized a second time with a terminal lower alkene to form 1,4-pentadiene. In other embodiments, however, the multiple metathesis reactions are not sequential, such that at least one other step (e.g., transesterification, hydrogenation, etc.) can be performed between the first metathesis step and the following metathesis step. These multiple metathesis procedures can be used to obtain products that may not be readily obtainable from a single metathesis reaction using available starting materials. For example, in some embodiments, multiple metathesis can involve self-metathesis followed by cross-metathesis to obtain metathesis dimers, trimmers, and the like. In some other embodiments, multiple metathesis can be used to obtain olefin and/or ester components that have chain lengths that may not be achievable from a single metathesis reaction with a natural oil triglyceride and typical lower alkenes (e.g., ethylene, propylene, 1-butene, 2-butene, and the like). Such multiple metathesis can be useful in an industrial-scale reactor, where it may be easier to perform multiple metathesis than to modify the reactor to use a different alkene. The metathesis process can be conducted under any conditions adequate to produce the desired metathesis products. For example, stoichiometry, atmosphere, solvent, temperature, and pressure can be selected by one skilled in the art to produce a desired product and to minimize undesirable byproducts. In some embodiments, the metathesis process may be conducted under an inert atmosphere. Similarly, in embodiments were a reagent is supplied as a gas, an inert gaseous diluent can be used in the gas stream. In such embodiments, the inert atmosphere or inert gaseous diluent typically is an inert gas, meaning that the gas does not interact with the metathesis catalyst to impede catalysis to a substantial degree. For example, non-limiting examples of inert gases include helium, neon, argon, and nitrogen, used individually or in with each other and other inert gases. The rector design for the metathesis reaction can vary depending on a variety of factors, including, but not limited to, the scale of the reaction, the reaction conditions (heat, pressure, etc.), the identity of the catalyst, the identity of the materials being reacted in the reactor, and the nature of the feedstock being employed. Suitable reactors can be designed by those of skill in the art, depending on the relevant factors, and incorporated into a refining process such, such as those disclosed herein. The metathesis reactions disclosed herein generally occur in the presence of one or more metathesis catalysts. Such methods can employ any suitable metathesis catalyst. The metathesis catalyst in this reaction may include any catalyst or catalyst system that catalyzes a metathesis reaction. Any known metathesis catalyst may be used, alone or in combination with one or more additional catalysts. Examples of metathesis catalysts and process conditions are described in US 2011/0160472, incorporated by reference herein in its entirety, except that in the event of any inconsistent disclosure or definition from the present specification, the disclosure or definition herein shall be deemed to prevail. A number of the metathesis catalysts described in US 2011/0160472 are presently available from Materia, Inc. (Pasadena, Calif.). In some embodiments, the metathesis catalyst includes a Grubbs-type olefin metathesis catalyst and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes a first-generation Grubbs-type olefin metathesis catalyst and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes a second-generation Grubbs-type olefin metathesis catalyst and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes a first-generation Hoveyda-Grubbs-type olefin metathesis catalyst and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes a second-generation Hoveyda-Grubbs-type olefin metathesis catalyst and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes one or a plurality of the ruthenium carbene metathesis catalysts sold by Materia, Inc. of Pasadena, California and/or one or more entities derived from such catalysts. Representative metathesis catalysts from Materia, Inc. for use in accordance with the present teachings include but are not limited to those sold under the following product numbers as well as combinations thereof: product no. C823 (CAS no. 172222-30-9), product no. C848 (CAS no. 246047-72-3), product no. C601 (CAS no. 203714-71-0), product no. C627 (CAS no. 301224-40-8), product no. C571 (CAS no. 927429-61-6), product no. C598 (CAS no. 802912-44-3), product no. C793 (CAS no. 927429-60-5), product no. C801 (CAS no. 194659-03-9), product no. C827 (CAS no. 253688-91-4), product no. C884 (CAS no. 900169-53-1), product no. C833 (CAS no. 1020085-61-3), product no. C859 (CAS no. 832146-68-6), product no. C711 (CAS no. 635679-24-2), product no. C933 (CAS no. 373640-75-6). In some embodiments, the metathesis catalyst includes a molybdenum and/or tungsten carbene complex and/or an entity derived from such a complex. In some embodiments, the metathesis catalyst includes a Schrock-type olefin metathesis catalyst and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes a high-oxidation-state alkylidene complex of molybdenum and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes a high-oxidation-state alkylidene complex of tungsten and/or an entity derived therefrom. In some embodiments, the metathesis catalyst includes molybdenum (VI). In some embodiments, the metathesis catalyst includes tungsten (VI). In some embodiments, the metathesis catalyst includes a molybdenum- and/or a tungsten-containing alkylidene complex of a type described in one or more of (a) Angew. Chem. Int. Ed. Engl., 2003, 42, 4592-4633; (b) Chem. Rev., 2002, 102, 145-179; and/or (c) Chem. Rev., 2009, 109, 3211-3226, each of which is incorporated by reference herein in its entirety, except that in the event of any inconsistent disclosure or definition from the present specification, the disclosure or definition herein shall be deemed to prevail. In certain embodiments, the metathesis catalyst is dissolved in a solvent prior to conducting the metathesis reaction. In certain such embodiments, the solvent chosen may be selected to be substantially inert with respect to the metathesis catalyst. For example, substantially inert solvents include, without limitation: aromatic hydrocarbons, such as benzene, toluene, xylenes, etc.; halogenated aromatic hydrocarbons, such as chlorobenzene and dichlorobenzene; aliphatic solvents, including pentane, hexane, heptane, cyclohexane, etc.; and chlorinated alkanes, such as dichloromethane, chloroform, dichloroethane, etc. In some embodiments, the solvent comprises toluene. In other embodiments, the metathesis catalyst is not dissolved in a solvent prior to conducting the metathesis reaction. The catalyst, instead, for example, can be slurried with the natural oil or unsaturated ester, where the natural oil or unsaturated ester is in a liquid state. Under these conditions, it is possible to eliminate the solvent (e.g., toluene) from the process and eliminate downstream olefin losses when separating the solvent. In other embodiments, the metathesis catalyst may be added in solid state form (and not slurried) to the natural oil or unsaturated ester (e.g., as an auger feed). The metathesis reaction temperature may, in some instances, be a rate-controlling variable where the temperature is selected to provide a desired product at an acceptable rate. In certain embodiments, the metathesis reaction temperature is greater than −40° C., or greater than −20° C., or greater than 0° C., or greater than 10° C. In certain embodiments, the metathesis reaction temperature is less than 200° C., or less than 150° C., or less than 120° C. In some embodiments, the metathesis reaction temperature is between 0° C. and 150° C., or is between 10° C. and 120° C. The metathesis reaction can be run under any desired pressure. In some instances, it may be desirable to maintain a total pressure that is high enough to keep the cross-metathesis reagent in solution. Therefore, as the molecular weight of the cross-metathesis reagent increases, the lower pressure range typically decreases since the boiling point of the cross-metathesis reagent increases. The total pressure may be selected to be greater than 0.1 atm (10 kPa), or greater than 0.3 atm (30 kPa), or greater than 1 atm (100 kPa). In some embodiments, the reaction pressure is no more than about 70 atm (7000 kPa), or no more than about 30 atm (3000 kPa). In some embodiments, the pressure for the metathesis reaction ranges from about 1 atm (100 kPa) to about 30 atm (3000 kPa). EXAMPLES General Procedure for the Preparation of Terminally Unsaturated Diesters: Into a 1 liter 3-necked round-bottomed flask equipped with a rubber septum fitted with a needle to bubble nitrogen through the reaction mixture, thermocouple, J-Kem controller, heating mantle, stir bar, short path distillation apparatus, and nitrogen outlet was added the 9-decenoic acid methyl ester (Elevance Renewable Sciences, Woodridge, IL) (9-DAME, 507 g, 2.75 mol) and either ethylene glycol (EG, ethane-1,2-glycol) or neopentyl glycol (NPG, 2,2-dimethylpropane-1,3,-diol) (Sigma-Aldrich, St. Louis, MO)(1.52 mol, 1.1 eq.). The nitrogen was bubbled through the reaction mixture (15 min) prior to the addition of sodium methoxide in methanol as a catalyst (25 wt % in methanol) (Sigma-Aldrich, St. Louis, MO) (6.0 g, 27.8 mmol, 0.3 wt %). The now yellow solution was slowly heated to 200° C. while the methanol was collected in the receiving flask (−78° C.). The reaction was judged complete by gas chromatography after 6 h. The crude reaction mixture was subjected to fractional distillation to remove residual starting materials from the desired product. The ethylene glycol-based diester (EGu-C10DE) was collected at 165° C./0.20 Torr (98.2 area % purity by gas chromatography, m/z=366 by mass spectrometry) while the neopentyl glycol-based diester (NPG-uC10DE) was collected at 177° C./0.33 Torr (97.9 area % purity; m/z=408; 1H-NMR (ppm) 5.8 (2H, m), 4.9 (4H, d of d), 3.9 (4H, s), 2.3 (4H, t), 2.0, (4H, m), 1.6 (4H, m), 1.3 (16H, m), 0.9 (6H, s)). Residual 9-DAME was less than 0.5 wt % for each diester product. General Procedure for the Preparation of Segmented Diester-Polysiloxane Copolymers: Into a 50 ml 3-necked round-bottomed flask equipped with a thermocouple, J-Kem controller, heating mantle, glass stopper, and nitrogen inlet was added in the Silmer H Di-10 hydrosilane (Siltech, Lawrenceville, GA) (1.0 eq. MW=875) and the relevant diester produced above (0.50 eq.). The Karstedt's catalyst (Pt(0)-1,3-divinyl-1,1,3,3,-tetramethyl disiloxane complex solution) (Sigma-Aldrich, St. Louis, MO) was added (1 drop, 5 microliters) which resulted in a significant exotherm in the case of the EGuC10DE that required the use of a dry ice/methanol bath to maintain the temperature blow 65° C. Once the temperature stabilized, then the remaining diester (0.50 eq.) was added at a rate to hold the temperature at 65° C. Once the addition was complete, then the reaction was held at 65° C. for about an hour. The resulting segmented polysiloxane copolymers were analyzed without further purification. Brookfield viscosity and number average molecular weights (Mn) by size exclusion chromatography (SEC) of the monomers and segmented polysiloxane copolymers are shown in Table 1. Table 1. Characterization of Monomers and Hybrid Segmented Copolymers. TABLE 1Characterization of monomers andhybrid segmented copolymers.ViscosityMnAppearance(Cp)(Da)MonomersEG-uC10DEClear, colorless11367NPG-uC10DEClear, colorless13409Silmer H Di-10Clear, colorless6.9875CopolymersEG-uC10DE:SilmerClear, colorless783046,800H Di-10 (1:1)EG-uC10DE:SilmerClear, colorless340038,300H Di-10 (1:1.1)EG-uC10DE:SilmerClear, colorless173032,500H Di-10 (1.2:1)EG-uC10DE:SilmerClear, colorless10011,300H Di-10 (1:2)NPG-uC10DE:SilmerClear, colorless691043,600H Di-10 (1:1) Illustrations Illustration 1 is a siloxane polymer comprising:(a) one or more segments of formula (I): and(b) one or more segments of formula (II): wherein:each R1, R2, R3, and R4is independently a hydrogen atom, a C1-14hydrocarbyl group, or a C1-14hydrocarbyloxy group;each X1and X2is independently an oxygen atom, a sulfur atom, >NH, or >N(C1-6alkyl);each G1and G2is independently a C1-14hydrocarbylene group;each G3is independently a C1-14hydrocarbylene group, which is optionally substituted one or more times by substituents selected from the group consisting of —OH and —O(C1-6alkyl); andeach k is independently an integer ranging from 5 to 5000. Illustration 2 is the siloxane polymer of any preceding or subsequent illustration, wherein each G1is —(CH2)9—. Illustration 3 is the siloxane polymer of any preceding or subsequent illustration, wherein each G1is —(CH2)10—. Illustration 4 is the siloxane polymer of any preceding or subsequent illustration, wherein each G1is —(CH2)11—. Illustration 5 is the siloxane polymer of any preceding or subsequent illustration, wherein each G2is —(CH2)9—. Illustration 6 is the siloxane polymer of any preceding or subsequent illustration, wherein each G2is —(CH2)10—. Illustration 7 is the siloxane polymer of any preceding or subsequent illustration, wherein each G2is —(CH2)11—. Illustration 8 is the siloxane polymer of any preceding or subsequent illustration, wherein each G3is a C1-14alkylene group. Illustration 9 is the siloxane polymer of any preceding or subsequent illustration, wherein each G3is a C1-8alkylene group. Illustration 10 is the siloxane polymer of any preceding or subsequent illustration, wherein each G3is —(CH2)1-6—. Illustration 11 is the siloxane polymer of any preceding or subsequent illustration, wherein each G3is —(CH2)2—. Illustration 12 is the siloxane polymer of any preceding or subsequent illustration, wherein each X1is an oxygen atom. Illustration 13 is the siloxane polymer of any preceding or subsequent illustration, wherein each X2is an oxygen atom. Illustration 14 is the siloxane polymer of any preceding or subsequent illustration, wherein each R1, R2, R3, and R4is independently a hydrogen atom or a C1-14hydrocarbyl group. Illustration 15 is the siloxane polymer of any preceding or subsequent illustration, wherein each R1, R2, R3, and R4is independently a C1-14hydrocarbyl group. Illustration 16 is the siloxane polymer of any preceding or subsequent illustration, wherein each R1, R2, R3, and R4is independently a C1-8alkyl group. Illustration 17 is the siloxane polymer of any preceding or subsequent illustration, wherein each R1, R2, R3, and R4is independently selected from the group consisting of methyl, ethyl, propyl, and isopropyl. Illustration 18 is the siloxane polymer of any preceding or subsequent illustration, wherein each R1, R2, R3, and R4is independently selected from the group consisting of methyl and ethyl. Illustration 19 is the siloxane polymer of any preceding or subsequent illustration, wherein each R1, R2, R3, and R4is methyl. Illustration 20 is the siloxane polymer of any preceding or subsequent illustration, wherein at least one of R1, R2, R3, and R4is a hydrogen atom. Illustration 21 is the siloxane polymer of any preceding or subsequent illustration, wherein the siloxane polymer has a molecular weight ranging from 1 kDa to 50 kDa. Illustration 22 is the siloxane polymer of any preceding or subsequent illustration, wherein the segments of formula (I) and the segments of formula (II) together make up at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, of the siloxane polymer. Illustration 23 is the siloxane polymer of any preceding or subsequent illustration, wherein the segments of formula (I) make up at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, of the siloxane polymer. Illustration 24 is the siloxane polymer of any preceding or subsequent illustration, wherein the segments of formula (I) make up no more than 70% by weight, or no more than 80% by weight, or no more than 90% by weight, of the the siloxane polymer Illustration 25 is the siloxane polymer of any preceding or subsequent illustration, wherein the segments of formula (II) make up at least 10% by weight, or at least 20% by weight, or at least 30% by weight, or at least 40% by weight, of the siloxane polymer. Illustration 26 is the siloxane polymer of any preceding or subsequent illustration, wherein the segments of formula (II) make up no more than 10% by weight, or no more than 20% by weight, or no more than 30% by weight, of the siloxane polymer. Illustration 27 is the siloxane polymer of any preceding or subsequent illustration, wherein the numerical ratio of segments of formula (I) to segments of formula (II) in the siloxane polymer ranges from 1:5 to 5:1, or from 1:4 to 4:1, or from 1:3 to 3:1, or from 1:2 to 2:1. Illustration 28 is the siloxane polymer of any preceding or subsequent illustration, having two or more endcap groups. Illustration 29 is the siloxane polymer of any preceding or subsequent illustration, wherein the endcap groups are moieties of formula (III): —(O)m—Si(R21)(R22)(R23) (III) wherein: R21, R22, and R23are independently a hydrogen atom, —OH, C1-14hydrocarbyl, or C1-14hydrocarbyloxy; and m is 0 or 1. Illustration 30 is the siloxane polymer of any preceding or subsequent illustration, wherein at least two of R21, R22, and R23is a hydrogen atom or C1-14hydrocarbyl. Illustration 31 is the siloxane polymer of any preceding or subsequent illustration, wherein at least two of R21, R22, and R23is C1-14hydrocarbyl. Illustration 32 is the siloxane polymer of any preceding or subsequent illustration, wherein at least two of R21, R22, and R23are selected independently from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, or neopentyl. Illustration 33 is the siloxane polymer of any preceding or subsequent illustration, wherein at least two of R21, R22, and R23are methyl. Illustration 34 is the siloxane polymer of any preceding or subsequent illustration, wherein one of R21, R22, and R23is a —OH or C1-14hydrocarbyloxy. Illustration 35 is the siloxane polymer of any preceding or subsequent illustration, wherein one of R21, R22, and R23is C1-14hydrocarbyloxy. Illustration 36 is the siloxane polymer of any preceding or subsequent illustration, wherein one of R21, R22, and R23is selected independently from the group consisting of: methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, or neopentoxy. Illustration 37 is the siloxane polymer of any preceding or subsequent illustration, wherein one of R21, R22, and R23is methoxy. Illustration 38 is the siloxane polymer of any preceding or subsequent illustration, the silicone composition comprising one or more siloxane polymers of any one of illustrations 1 to 37. Illustration 39 is the siloxane polymer of any preceding or subsequent illustration, wherein the silicone composition comprises a carrier, and wherein the one or more siloxane polymers make up from 0.1 to 95 percent by weight of the silicone composition, based on the total weight of the composition. Illustration 40 is the siloxane polymer of any preceding or subsequent illustration, wherein the carrier comprises water. Illustration 41 is the siloxane polymer of any preceding or subsequent illustration, wherein the carrier comprises an organic solvent. Illustration 42 is the siloxane polymer of any preceding or subsequent illustration, further comprising one or more additives, such as surfactants, pigments, antimicrobial agents, photostabilizers, and the like. Illustration 43 is the siloxane polymer of any preceding or subsequent illustration, wherein the one or more siloxane polymers have a weight-average molecular weight ranging from 1 kDa to 50 kDa. Illustration 44 is the siloxane polymer of any preceding or subsequent illustration, wherein the silicone composition is a surfactant composition, a sizing composition for a matrix reinforcement material (a siliceous material, such as silica or sand; glass, such as glass fiber, glass particles, or glass beads; a metal, such as silver or titanium; a metal oxide, such as zinc oxide or titanium dioxide; carbon, such as carbon nanoparticles, carbon nanotubes, graphite, graphene, diamond, and fullerenes, or any combination of the foregoing), a coating composition, a sealant composition, a grease composition, a defoaming composition, a dry-cleaning composition, a rubber composition, an ophthalmic composition, a personal care composition, a lubricant composition, a personal lubricant composition, a functional fluid, such as a brake fluid, a mold release composition, a gel composition, or an electronics encasement composition. Illustration 45 an article of manufacture, which comprises a portion formed from a silicone composition of any preceding or subsequent illustration. Illustration 46 is the article of manufacture which comprises a portion formed from a silicone composition of any preceding or subsequent illustration, and which is an electrical insulating article, an electronic device (where, for example, the silicone composition is in a coating or sealing layer), a gasket, a seal, a pad, a mold (such as, for example, a dental mold), a paper article (such as a sheet, where, for example, the silicone composition is in a coating), a textile article (where, for example, the silicone composition is in a coating), a fire stop, a microfluidic device, a bandage, a dressing for a wound, a scar treatment sheet, a breast implant, a testicular implant, a pectoral implant, a contact lens, an ophthalmic tube, an ophthalmic stent, or a nipple, such as a nipple on a baby bottle. | 76,287 |
11859057 | DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. The disclosures of US patents and patent publications cited herein are to be incorporated by reference to the extent consistent with the present disclosure. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a,” “an” and “the” are intended to include plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements components and/or groups or combinations thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups or combinations thereof. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise. As used herein, the term “and/or” includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and claims and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity. The transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited, and also additional materials or steps that do not materially affect the basic and novel characteristics of the claimed invention as described herein. It will be understood that, although the terms first, second, etc., may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. Rather, these terms are only used to distinguish one element, component, region, layer and/or section, from another element, component, region, layer and/or section. Thus, a first element, component, region, layer or section discussed herein could be termed a second element, component, region, layer or section without departing from the teachings of the present invention. Furthermore, a first, second, third, etc. component or material may be the same as or different from one another. It will also be understood that an “additional” component or material may be the same as or different from the component or material previously used. For example, additional reactive diluent may be the same reactive diluent as used in a prior step, or it may be a different reactive diluent that is added, without departing from the teachings of the present invention. If the prior component or material was optional, the “additional” component or material may be the first or only instance of that component or material. “ABPU” or “reactive blocked polyurethane” as used herein refers to UV-curable, (meth)acrylate blocked, polyurethane/polyurea with blocked isocyanate groups such as described in U.S. Pat. Nos. 9,453,142 and 9,598,606 to Rolland et al. A particular example of a suitable reactive (or UV-curable) blocking agent for the isocyanates of the ABPU is a tertiary amine-containing (meth)acrylate (e.g., t-butylaminoethyl methacrylate, TBAEMA, tertiary pentylaminoethyl methacrylate (TPAEMA), tertiary hexylaminoethyl methacrylate (THAEMA), tertiary-butylaminopropyl methacrylate (TBAPMA), acrylate analogs thereof, and mixtures thereof). In some embodiments, non-reactive blocked polyurethane may be further provided in the composition, inclusive of an ABPU that has at least one end that is blocked by a non-reactive group (while another end is reactive). Such non-reactive blocking groups may include, for example, volatile blocking groups such as 3,5-dimethylpyrazole (DMP), 2-butanone oxime (also called methyl ethyl ketoxime or “MEKO”), etc. See, e.g., WO 2018/226943 to Chen et al. In some embodiments, an excess of the blocking agent (reactive or non-reactive) may be removed from the composition by distillation or chromatography, if desired. Polyisocyanates (including diisocyanates) useful in carrying out the present invention include, but are not limited to, 1,1′-methylenebis(4-isocyanatobenzene) (MDI), 2,4-diisocyanato-1-methylbenzene (TDI), methylene-bis(4-cyclohexylisocyanate) (H12MDI), hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), polymeric MDI, 1,4-phenylene diisocyanate (PPDI), and o-tolidine diisocyanate (TODI). In some embodiments, a preferred diisocyanate is H12MDI, such as Desmodur W, supplied by Covestro AG. Additional examples include but are not limited to those given in U.S. Pat. No. 3,694,389 to Levy. Reactive blocking agents useful in the present invention include agents that reversibly block isocyantes and also contain one or more groups that are UV polymerizable, such as amine (meth)acrylate monomer blocking agents (e.g., tertiary-butylaminoethyl methacrylate (TBAEMA), tertiary pentylaminoethyl methacrylate (TPAEMA), tertiary hexylaminoethyl methacrylate (THAEMA), tertiary-butylaminopropyl methacrylate (TBAPMA), acrylate analogs thereof, and mixtures thereof). See, e.g., US Patent Application Publication No. 20130202392. There are, however, many blocking agents for isocyanate, and those skilled in the art can couple (meth)acrylate groups to other blocking agents to create additional blocking agents that can be used to carry out the present invention. Still further, those skilled in the art can use maleimide, or substitute maleimide on other known blocking agents, for use in the present invention. Reactive capping agents useful in the present invention are generally agents containing one or more groups reactive with polyisocyantes, and one or more groups that are UV polymerizable, including hydroxyl or amine (meth)acrylate monomer capping agents. Examples of suitable capping agents include, but are not limited to, 2-hydroxylethyl acrylate, 2-hydroxylethyl methacrylate (HEMA), hydroxypropyl acrylate, hydroxypropyl methacrylate, tert-butylaminoethyl methacrylate, 4-hydroxybutyl acrylate, 3-phenoxy 2-hydroxypropyl methacrylate, glycerol methacrylate, etc. Additional examples include, but are not limited to, butane monohydroxy monoacrylate, polypropylene glycol monoacrylate, caprolactone monohydroxy monoacrylate, 2-terbutylaminoethylmethacrylate and 2-terbutylaminoethylacrylate. See EP 0525578 A1 to Peiffer, and U.S. Pat. No. 7,279,505 to Phelan et al., which are incorporated by reference herein. Photoinitiators useful in the present invention include, but are not limited to, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO), Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (PPO), 2-isopropylthioxanthone and/or 4-isopropylthioxanthone (ITX), etc. “Diluents” as used herein includes both UV-curable diluents (for example monoacrylates, mono-methacrylates, polyacrylates, polymethacrylates, acrylamides, methacrylamides, etc.), and non-UV-curable diluents (for example, plasticizers such as bis(2-ethylhexyl) phthalate, bis(2-propylheptyl) phthalate, diisononyl phthalate, tri-(2-ethylhexyl) trimellitate, bis(2-ethylhexyl) adipate, diisononyl adipate, dibutyl sebacate, diisobutyl maleate, etc.). “Solvents” as used herein includes, for example, xylenes, diethyl ether, tetrahydrofuran (THF), ethyl acetate, benzene, toluene, N,N-dimethylformamide, propylene carbonate, dimethyl carbonate, etc. Solvents may also include an excess of blocking agent and/or capping agent, such as TBAEMA and/or HEMA. “Crosslinkers” as used herein includes UV reactive crosslinkers, such as polyfunctional acrylates and methacrylates, for example, diethylene glycol dimethacrylate (DEGMA), triethylene glycol dimethacrylate (TEGDMA), tetraethylene glycol dimethracrylate (TeEGDMA), 1,6-hexanediol dimethacrylate (HDDMA), diurethane dimethacrylate (DUDMA), trimethylol propane trimethacrylate (TMPTMA), pol(yethylene glycol) dimethacrylate (PEGDMA), 1,6-hexanediol diacrylate (HDDA), trimethylol propane triacrylate (TMPTA), poly(ethylene glyocol) diacrylate (PEGDA), etc., including mixtures of two or more thereof. A “radical polymerization inhibitor” includes, but is not limited to, 4-methoxyphenol (also known as monomethyl ether hydroquinone (MEHQ), or mequinol), 4-ethoxyphenol, 4-propoxyphenol, 4-butoxyphenol 4-heptoxyphenol, 2,6-di-tert-butyl-4-methylphenol (see, e.g., U.S. Pat. No. 9,796,693), etc., including combinations thereof. “Shipping” and “collecting” as used herein may be performed by any method of transferring, delivering, or providing, whether directly or through an intermediary. “User” refers to a location, such as a manufacturing or production facility at which objects are produced from the resin, remote or separate from the location from which the resin is shipped, or to which an object may be sent for collection, which location may be operated by the same corporate entity, or by a different corporate entity, from the location from which the resin is shipped. Likewise, a “plurality of users” may be different sites or locations operated by the same corporate entity, or operated by different corporate entities. 1. Single-Cure Additive Manufacturing Resins Containing ABPUs. Dual cure additive manufacturing resins containing ABPUs are described in, for example, U.S. Pat. Nos. 9,453,142 and 9,598,606 to Rolland et al., which are incorporated by reference herein. In the present invention, the resin can consist essentially of the same ingredients as found in such a dual cure resin, but with at least one constituent required for the second or subsequent cure (e.g., the chain extender(s)) omitted or substantially omitted. A. Light-polymerizable monomers and/or prepolymers. Sometimes also referred to as “Part A” of a dual cure resin, these are monomers and/or prepolymers that can be polymerized by exposure to actinic radiation or light. This resin can comprise difunctional or polyfunctional monomers, but can also include monofunctional monomers (to act as “chain stoppers” to control molecular weight). In contrast to a dual cure resin, where the amount of this “Part A” corresponds to 50 to 90% by weight of the total resin (polymerizable liquid) composition, in the present invention these light-polymerizable components correspond to more than 90 or 95 percent by weight of the total resin composition. Examples of reactive end groups suitable for Part A constituents, monomers, or prepolymers include, but are not limited to: acrylates, methacrylates, α-olefins, N-vinyls, acrylamides, methacrylamides, styrenics, epoxides, thiols, 1,3-dienes, vinyl halides, acrylonitriles, vinyl esters, maleimides, and vinyl ethers. Note that, in the present invention, the light polymerizable component, once polymerized, is one which can partially degrade/decrosslink upon heating or baking and, in the presence of a reactive blocking or reactive capping agent, regenerate a reactive prepolymer such as an ABPU. The regenerated reactive prepolymer can be separated from the remainder of the polymerized material, which is an insoluble crosslinked material having permanent UV crosslinks that are stable in the presence of the blocking agent or capping agent and/or solvent during the heating and extraction. This is schematically illustrated inFIG.1. In a typical “dual cure” additive manufacturing resin, the additional part B thermally reactive components (e.g., chain extenders) are carried in the green, light cured, object, where they participate in a subsequent cure to impart desired physical properties to the object. In the present invention, these components are left out, and the reactive prepolymer Part A component is regenerated by heating the formed blocked polymer in the presence of additional blocking agents or capping agents, which may be followed by extraction and recovery of the regenerated component. As will be understood, the “regenerated” reactive prepolymer may be the same as or different from the reactive prepolymer use for light polymerization, depending upon the blocking agent or capping agent used. B. Additional resin ingredients. Photoinitiators included in the polymerizable liquid (resin) can be any suitable photoinitiator, including type I and type II photoinitiators and including commonly used UV photoinitiators, examples of which include but are not limited to acetophenones (diethoxyacetophenone for example), phosphine oxides such as diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, phenylbis(2,4,6-trimethylbenzoyl) phosphine oxide (PPO), Irgacure® 369, etc. See, e.g., U.S. Pat. No. 9,453,142 to Rolland et al. The liquid resin or polymerizable material can have solid particles suspended or dispersed therein. Any suitable solid particle can be used, depending upon the end product being fabricated. The particles can be metallic, organic/polymeric, inorganic, or composites or mixtures thereof. The particles can be nonconductive, semi-conductive, or conductive (including metallic and non-metallic or polymer conductors); and the particles can be magnetic, ferromagnetic, paramagnetic, or nonmagnetic. The particles can be of any suitable shape, including spherical, elliptical, cylindrical, etc. The particles can be of any suitable size (for example, ranging from 1 nm to 20 μm average diameter). The particles can comprise an active agent or detectable compound as described below, though these may also be provided dissolved or solubilized in the liquid resin as also discussed below. For example, magnetic or paramagnetic particles or nanoparticles can be employed. The liquid resin can have additional ingredients solubilized therein, including, for example, pigments, dyes, diluents, active compounds or pharmaceutical compounds, detectable compounds (e.g., fluorescent, phosphorescent, radioactive), etc., again depending upon the particular purpose of the product being fabricated. Examples of such additional ingredients include, but are not limited to, proteins, peptides, nucleic acids (DNA, RNA) such as siRNA, sugars, small organic compounds (drugs and drug-like compounds), etc., including combinations thereof. Dyes non-reactive light absorbers. In some embodiments, polymerizable liquids for carrying out the present invention include a non-reactive pigment or dye that absorbs light, particularly UV light. Suitable examples of such light absorbers include, but are not limited to: (i) titanium dioxide (e.g., included in an amount of from 0.05 or 0.1 to 1 or 5 percent by weight), (ii) carbon black (e.g., included in an amount of from 0.05 or 0.1 to 1 or 5 percent by weight), and/or (iii) an organic ultraviolet light absorber such as a hydroxybenzophenone, hydroxyphenylbenzotriazole, oxanilide, benzophenone, thioxanthone, hydroxyphenyltriazine, and/or benzotriazole ultraviolet light absorber (e.g., Mayzo BLS1326) (e.g., included in an amount of 0.001 or 0.005 to 1, 2 or 4 percent by weight). Examples of suitable organic ultraviolet light absorbers include, but are not limited to, those described in U.S. Pat. Nos. 3,213,058; 6,916,867; 7,157,586; and 7,695,643, the disclosures of which are incorporated herein by reference. Fillers. Any suitable filler may be used in connection with the present invention, depending on the properties desired in the part or object to be made. Thus, fillers may be solid or liquid, organic or inorganic, and may include reactive and non-reactive rubbers; siloxanes; acrylonitrile-butadiene rubbers; reactive and non-reactive thermoplastics (including but not limited to: poly(ether imides), maleimide-styrene terpolymers, polyarylates, polysulfones and polyethersulfones, etc.); inorganic fillers such as silicates (such as talc, clays, silica, mica), glass, carbon nanotubes, graphene, cellulose nanocrystals, etc.; including combinations of all of the foregoing. Suitable fillers include tougheners, such as core-shell rubbers, as discussed below. Tougheners. One or more polymeric and/or inorganic tougheners can be used as a filler in the present invention. The toughener may be uniformly distributed in the form of particles in the cured product. The particles could be less than 5 microns (μm) in diameter. Such tougheners include, but are not limited to, those formed from elastomers, branched polymers, hyperbranched polymers, dendrimers, rubbery polymers, rubbery copolymers, block copolymers, core-shell particles, oxides or inorganic materials such as clay, polyhedral oligomeric silsesquioxanes (POSS), carbonaceous materials (e.g., carbon black, carbon nanotubes, carbon nanofibers, fullerenes), ceramics and silicon carbides, with or without surface modification or functionalization. Core-shell rubbers. Core-shell rubbers are particulate materials (particles) having a rubbery core. Such materials are known and described in, for example, US Patent Application Publication No. 20150184039, as well as US Patent Application Publication No. 20150240113, and U.S. Pat. Nos. 6,861,475, 7,625,977, 7,642,316, 8,088,245, and elsewhere. In some embodiments, the core-shell rubber particles are nanoparticles (i.e., having an average particle size of less than 1000 nanometers (nm)). Generally, the average particle size of the core-shell rubber nanoparticles is less than 500 nm, e.g., less than 300 nm, less than 200 nm, less than 100 nm, or even less than 50 nm. Typically, such particles are spherical, so the particle size is the diameter; however, if the particles are not spherical, the particle size is defined as the longest dimension of the particle. Suitable core-shell rubbers include, but are not limited to, those sold by Kaneka Corporation under the designation Kaneka Kane Ace, including the Kaneka Kane Ace 15 and 120 series of products, including Kaneka Kane Ace MX 120, Kaneka Kane Ace MX 153, Kaneka Kane Ace MX 154, Kaneka Kane Ace MX 156, Kaneka Kane Ace MX170, Kaneka Kane Ace MX 257 and Kaneka Kane Ace MX 120 core-shell rubber dispersions, and mixtures thereof. Organic diluents. In some embodiments, diluents for use in the present invention are preferably reactive organic diluents; that is, diluents that will degrade, isomerize, cross-react, or polymerize, with themselves or a light polymerizable component, during the additive manufacturing step. In general, the diluent(s) are included in an amount sufficient to reduce the viscosity of the polymerizable liquid or resin (e.g., to not more than 15,000, 10,000, 6,000, 5,000, 4,000, or 3,000 centipoise at 25 degrees Centigrade). Suitable examples of diluents include, but are not limited to, isobornyl methacrylate, TBAEMA (tert-butyl amino ethyl methacrylate), tetrahydrofurfuryl methacrylate, N,N-dimethylacrylamide, N-vinyl-2-pyrrolidone, and N-vinyl formamide, or a mixture of two or more thereof. The diluent may be included in the polymerizable liquid in any suitable amount, typically from 1, 5 or 10 percent by weight, up to about 30 or 40 percent by weight, or more. 2. Additive Manufacturing. Techniques for additive manufacturing are known. Suitable techniques include bottom-up or top-down additive manufacturing, generally known as stereolithography. Such methods are known and described in, for example, U.S. Pat. No. 5,236,637 to Hull, U.S. Pat. Nos. 5,391,072 and 5,529,473 to Lawton, U.S. Pat. No. 7,438,846 to John, U.S. Pat. No. 7,892,474 to Shkolnik, U.S. Pat. No. 8,110,135 to El-Siblani, U.S. Patent Application Publication No. 2013/0292862 to Joyce, and US Patent Application Publication No. 2013/0295212 to Chen et al. The disclosures of these patents and applications are incorporated by reference herein in their entirety. In some embodiments, the object is formed by continuous liquid interface production (CLIP). CLIP is known and described in, for example, PCT Application Nos. PCT/US2014/015486 (U.S. Pat. No. 9,211,678); PCT/US2014/015506 (U.S. Pat. No. 9,205,601), PCT/US2014/015497 (U.S. Pat. No. 9,216,546), and in J. Tumbleston, D. Shirvanyants, N. Ermoshkin et al., Continuous liquid interface production of 3D Objects,Science347, 1349-1352 (2015). See also R. Janusziewcz et al., Layerless fabrication with continuous liquid interface production,Proc. Natl. Acad. Sci. USA113, 11703-11708 (2016). In some embodiments, CLIP employs features of a bottom-up three-dimensional fabrication as described above, but the irradiating and/or said advancing steps are carried out while also concurrently maintaining a stable or persistent liquid interface between the growing object and the build surface or window, such as by: (i) continuously maintaining a dead zone of polymerizable liquid in contact with said build surface, and (ii) continuously maintaining a gradient of polymerization zone (such as an active surface) between the dead zone and the solid polymer and in contact with each thereof, the gradient of polymerization zone comprising the first component in partially-cured form. In some embodiments of CLIP, the optically transparent member comprises a semipermeable member (e.g., a fluoropolymer), and the continuously maintaining a dead zone is carried out by feeding an inhibitor of polymerization through the optically transparent member, thereby creating a gradient of inhibitor in the dead zone and optionally in at least a portion of the gradient of polymerization zone. Other approaches for carrying out CLIP that can be used in the present invention and obviate the need for a semipermeable “window” or window structure include utilizing a liquid interface comprising an immiscible liquid (see L. Robeson et al., WO 2015/164234), generating oxygen as an inhibitor by electrolysis (see I. Craven et al., WO 2016/133759), and incorporating magnetically positionable particles to which the photoactivator is coupled into the polymerizable liquid (see J. Rolland, WO 2016/145182). Other examples of methods and apparatus for carrying out particular embodiments of CLIP include, but are not limited to, those described in B. Feller, US Patent App. Pub. No. US 2018/0243976 (published Aug. 30, 2018); M. Panzer and J. Tumbleston, US Patent App Pub. No. US 2018/0126630 (published May 10, 2018); K. Willis and B. Adzima, US Patent App Pub. No. US 2018/0290374 (Oct. 11, 2018); Batchelder et al., Continuous liquid interface production system with viscosity pump, US Patent Application Pub. No. US 2017/0129169; Sun and Lichkus, Three-dimensional fabricating system for rapidly producing objects, US Patent Application Pub. No. US 2016/0288376; Willis et al., 3d print adhesion reduction during cure process, US Patent Application Pub. No. US 2015/0360419; Lin et al., Intelligent 3d printing through optimization of 3d print parameters, US Patent Application Pub. No. US 2015/0331402; and D. Castanon, Stereolithography System, US Patent Application Pub. No. US 2017/0129167. 3. Recovery of Part a Components from Additively Manufactured Objects. As shown in the scheme presented inFIG.1, a three-dimensional thermoset object or article may be formed by additive manufacturing such as stereolithography that includes polymer backbones (e.g., poly(methacrylate)) crosslinked with ABPUs and also including permanent (i.e., not heat labile) crosslinks. Alternatively, the material to be recycled may be recovered coating material formed from a first resin and produced by a light-cured coating process. The resin used to form the preformed article or coating may comprise or consist essentially of: (i) a reactive blocked prepolymer (e.g., ABPU); (ii) a UV reactive crosslinker (e.g., polyfunctional (meth)acrylate crosslinker); (iii) a photoinitiator; (iv) optionally a reactive diluent; (v) optionally a light absorber; (vi) optionally pigments and/or dyes; and (vii) optionally at least one filler; said preformed article (or coating) comprised of at least 70, 80 or 90 percent by weight of a crosslinked polymer, said crosslinked polymer consisting essentially of said reactive blocked prepolymer, said crosslinker, and said reactive diluent when included, in light polymerized, cross-linked, thermoset form. The article (or recovered coating) may then be: (b) comminuted into a reactive particulate material (e.g., by grinding such as cryo-milling, shredding, chopping, or pelletizing); and (c) combined (e.g., in an amount of from 1, 2 or 4 percent by weight to 20, 25, or 30 percent by weight, or more) with a reactive blocking agent or reactive capping agent to form a mixture and heating said mixture (e.g., to a temperature of from 40, 60 or 700 Celsius to 90, 100, or 1200 Celsius, or more) for a time sufficient to form a regenerated reactive prepolymer. The regenerated reactive prepolymer may be extracted from the mixture into a solvent (e.g. an organic solvent such as xylenes). Example methods of extracting include, but are not limited to, 1) Batch reaction followed by continuous extraction (detailed in the examples below); 2) Eliminating solvent and using excess of blocking or capping agent in a batch-to-continuous method; 3) Multiple batch extractions to improve yield (blocking or capping agent only, or solvent and blocking or capping agent); and 4) Supercritical CO2extraction at elevated temperatures. The recovered regenerated reactive prepolymer may thereafter be used as a component for a subsequent resin, which may be the same as or different from the original resin. To provide a sustainable manufacturing method, the recyclable light-polymerizable resin as taught herein may be: (a) shipped to at least one user; (b) objects produced from said resin collected back from said at least one user; and (c) objects processed to recover a regenerated reactive prepolymer therefrom. The regenerated reactive prepolymer may then used as a component in a new resin (the same as or different from the original light-polymerizable resin), which may be shipped to at least one user (same or different users). In some embodiments, the objects comprise dental models. In some embodiments, the at least one user comprises a plurality of users (same or different users). The present invention is explained in greater detail in the following non-limiting Examples. EXAMPLES The procedure illustrated inFIG.2is used for extracting the raw material (ABPU) from a recyclable formulation, referred to as ABPU Recovery Resin (ARR), using a so-called ‘batch’ and ‘continuous’ (Soxhlet) extraction method. ARR Formulation: IngredientLoading (wt %)ABPU (with 10 wt % benzyl methacrylate)83.3Benzyl methacrylate (reactive diluent)12.2DEGDMA (crosslinker)2.0TPO (photoinitiator)2.5 After printing or casting ARR, the parts are cut into 1 inch pieces and comminuted by using a freezer mill. This process yields granules of an average particle size of 500-2000 microns. The powder is typically stored in a desiccator overnight to prevent moisture contamination. Batch Extraction The batch extraction procedure is carried out by adding the dried powder, xylenes, and TBAEMA into a 60 mL vial. The TBAEMA is loaded with a 2 eq. amount relative to the chain ends. Butylated hydroxytoluene (BHT) is used as an antioxidant stabilizer. ComponentAmount (g)ARR powder3.00Xylenes (solvent)14.37TBAEMA (500 ppm BHT inhibitor)1.35 The contents of the vial are heated to 120° C. for a period of 4 hours. During this time, the powder will swell in the solvent making stirring with a stir bar impossible. Continuous Extraction After these 4 hours, the contents of the vial are transferred to a Soxhlet cellulose thimble. Roughly 10 g of THF are added to the vial to improve the yield. This THF amount is added to the thimble as well. The thimble is placed in the Soxhlet chamber (together with the 10 g THF). The round bottom flask is filled with 130 g of THF and a stir bar and the Soxhlet extractor is assembled and placed in an oil bath. The bath will be heated to 100° C. for 90 min. ComponentNotesThimbleCellulose-based, tare before useRound bottom250 mL, clean before use and tare before useflask (RBF)Soxhlet chamberClean before useCondenserEnsure condenser is working and clean before use All the solvents are collected in the RBF after the Soxhlet extraction is finished. BHT is added to the solution in a 500 ppm loading. All the solvents are removed in vacuo. Methanol is added to the mixture to remove xylenes via the azeotrope. The remaining powder can be dried in vacuo as well. This is typically done at 60° C. overnight. After removing the solvents from the extract, a GPC trace is taken and a TBAEMA titration is performed. The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. | 29,777 |
11859058 | DESCRIPTION OF EMBODIMENTS The method according to an embodiment of the present invention can produce resin particles through: melt-kneading a water-soluble matrix, containing a water-soluble polyvinyl alcohol-based resin, and a non-water-soluble resin (particularly a meltable resin); and removing the matrix by elution with an aqueous solvent from a pre-molded article (melt-kneaded product), in which the non-water-soluble resin is dispersed in particulate form in the water-soluble matrix, to form resin particles. This method may typically include: collecting the formed resin particles; and controlling moisture to dry or humidify collected particles by controlling temperature and humidity as necessary. Melt-Kneading Water-Soluble Matrix In the melt-kneading, the water-soluble matrix contains a water-soluble polyvinyl alcohol (PVA)-based resin, and this PVA-based resin contains a modified polyvinyl alcohol-based resin that is modified with a hydrophilic modifying group and has a wide usable temperature range of melt-kneading (the modified polyvinyl alcohol-based resin may be hereinafter simply referred to as the modified PVA-based resin). This modified PVA-based resin contains in a side chain thereof (a) an alkyl group or alkyl chain (or a unit containing the alkyl group (or alkyl chain)) including at least one hydroxyl group (one or more hydroxyl groups). Modified PVA-Based Resin The alkyl group or alkyl chain in the side chain of the modified PVA-based resin may be a linear or branched C1-12alkyl group (for example, a C2-8alkyl group), such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group. The number of the hydroxyl group is, for example, from 1 to 7 (for example, from 1 to 5), preferably from 2 to 4, more preferably from approximately 2 to 3, and particularly 2 per alkyl group (or alkyl chain) or side chain. That is, the side chain of the modified polyvinyl alcohol-based resin may include an alkyl group including a plurality of hydroxyl groups. Furthermore, the modified PVA-based resin is often modified with a modifying group (or side chain) including at least a primary hydroxyl group, and the side chain of the modified PVA-based resin often includes, for example, at least a primary hydroxyl group. That is, the modified PVA-based resin often includes in a side chain thereof an alkyl group (or a unit including an alkyl group) (a1) and/or (a2) below.(a1) an alkyl group (or a unit including an alkyl group) including a primary hydroxyl group;(a2) an alkyl group (or a unit including an alkyl group) including a primary hydroxyl group and a secondary hydroxyl group. The number of the primary hydroxyl group is, for example, from 1 to 5 (for example, from 1 to 4), preferably from 1 to 3, and more preferably approximately 1 or 2 (particularly 1) per alkyl group (or alkyl chain) or side chain. More specifically, the modified PVA-based resin often includes in a side chain thereof (or an alkyl group or an alkyl chain in a side chain thereof) an alkyl group substituted with a hydroxyalkyl group, particularly two hydroxyl groups or two hydroxy C1-4alkyl groups (such as hydroxymethyl groups) at the same carbon atom or adjacent carbon atoms (a dihydroxyalkyl group or a dihydroxyalkyl-alkyl group). The modified PVA-based resin often includes, for example, a unit including a dihydroxyalkyl group, represented by Formula (1) below. where R1, R2, R3, R4, R5, and R6are the same or different and represent a hydrogen atom or an organic group; and X represents a single bond or a bonding chain (or a linking group). The organic group may be exemplified by linear or branched C1-4alkyl groups, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a t-butyl group. Preferred alkyl groups are a methyl group or an ethyl group. These alkyl groups may include as necessary a substituent, such as a halogen atom, a hydroxyl group, an alkoxy group, a carboxyl group, an alkoxycarbonyl group, or a sulfonyl group. In Formula (1) above, preferred R1to R6are hydrogen atoms. The bonding chain (linking group) represented by X may be exemplified by hydrocarbon groups (in these hydrocarbon groups, a halogen atom, such as fluorine, chlorine, or bromine, may be substituted), such as alkylene groups (C2-10alkylene groups, such as an ethylene group), alkenylene groups (C2-6alkenylene groups, such as a vinylene group and a propenylene group), alkynylene groups, and arylene groups (such as a phenylene group and naphtylene group); an ether group (—O—); C2-4alkyleneoxy groups [such as —(CH2O)m—, —(OCH2)m—, and —(CH2O)mCH2—]; a carbonyl group (—CO—); a diketo group (—COCO—); alkylenedicarbonyl groups (such as —CO(CH2)mCO—); arenedicarbonyl groups (such as —CO(C6H4)CO—); a thioether group (—S—); a thiocarbonyl group (—CS—); a sulfinyl group (—SO—); a sulfonyl group (—SO2—); an imino group or a substituted imino group (—NR—); urethane groups (—CONR— and —NRCO—); thiourethane groups (—CSNR— and —NRCS—), and —NRCS—); an azo group (—NRNR—); a phosphate ester group (—HPO4—); silicon-containing groups [—Si(OR)2—, —OSi(OR)2—, and —OSi(OR)2O—]; titanium-containing groups [such as —Ti(OR)2— and —OTi(OR)2—]; and aluminum-containing groups [such as —Al(OR)—, —OAl(OR)—, and —OAl(OR)O—]. Here, R is each independently an arbitrary substituent and may be, for example, a hydrogen atom, a C1-12alkyl group, an alkoxy group, and an acyl group; and m each independently represents a natural number. Of these bonding chains (linking groups) X, C2-6alkylene groups (particularly C1-2alkylene groups, such as a methylene group) and —CH2OCH2— are preferred. In particular, a preferred X is a single bond. In particular, the modified PVA-based resin preferably includes in a side chain thereof (or an alkyl group in a side chain thereof) a 1,2-diol structure (or a 1,2-dihydroxyalkyl group), and preferably includes a unit represented by Formula (1-1) below. Such a modified PVA-based resin may be any of: a modified PVA-based resin formed by modifying an unmodified PVA-based resin to introduce a side chain including the unit (a) (for example, a modified PVA-based resin modified by esterification, etherification, acetalization, urethanization, phosphate esterification, or the like with a hydroxycarboxylic acid, such as glycolic acid, lactic acid, and glycerol acid); and a copolymerization-modified PVA-based resin. Preferred modified PVA-based resins can be prepared by at least saponification of a copolymerization-modified PVA-based resin, for example, a copolymer of a vinyl ester-based monomer and a copolymerizable monomer that is copolymerizable with a vinyl ester-based monomer, where the copolymerizable monomer only needs to contain a copolymerizable monomer (a first copolymerizable monomer) into which a side chain including at least the unit (a) can be introduced and may further contain an additional copolymerizable monomer (a second copolymerizable monomer). The vinyl ester-based monomer may be exemplified by vinyl C1-20alkanoates or vinyl C2-20alkenoates, such as vinyl formate, vinyl acetate, vinyl propionate, vinyl valerate, vinyl butyrate, vinyl isobutyrate, vinyl pivalate, vinyl caprylate, vinyl laurate, vinyl stearate, and vinyl versatate; and vinyl arene carbonates, such as vinyl benzoate. In addition, if necessary, a substituted vinyl acetate, such as 1-methoxyvinyl acetate and isopropenyl acetate, can also be used. These vinyl ester-based monomers can be used alone or in combination of two or more types. Of these vinyl ester-based monomers, a vinyl C1-3alkanoate, particularly vinyl acetate is often used in terms of economy and the like. The first copolymerizable monomer may be exemplified by hydroxy group-containing C3-10α-olefins or derivatives thereof (for example, acylated compounds, such as acetylated compounds), such as 3-buten-1-ol, 4-penten-1-ol, and 5-hexen-1-ol; and monomers represented by Formulas (1a) to (1c) below. where R7and R8each independently represent a hydrogen atom or an acyl group R9—CO— (R9represents a hydrogen atom or a C1-4alkyl group); R10and R11each independently represent a hydrogen atom or a C1-4alkyl group; and R1to R6and X are the same as described above. The acyl group may be exemplified by a formyl group; and C1-4alkyl-carbonyl groups, such as an acetyl group and a propionyl group. The acyl group is often a C1-2alkyl-carbonyl group, particularly an acetyl group. The alkyl group is often an C1-4alkyl group, such as a methyl group and an ethyl group (particularly, a methyl group or an ethyl group). R7and R8are typically a hydrogen atom or an acetyl group, and R10and R11are often typically a hydrogen atom or a C1-2alkyl group. Representative compounds represented by Formula (1a) may be exemplified by diacyl C4-10alkenes, for example, such as 1,4-diacyloxy-2-butenes (for example, such as 1,4-diacetyloxy-2-butene), 3,4-diacyloxy-1-butenes (for example, such as 3,4-diacetyloxy-1-butene); representative compounds represented by Formula (1b) may be exemplified by 2,2-dialkyl-4-vinyl-1,3-dioxolanes (for example, 2,2-dimethyl-4-vinyl-1,3-dioxolane); and representative compounds represented by Formula (1c) may be exemplified by vinyl C2-6alkylene carbonates (for example, vinyl ethylene carbonate). These first copolymerizable monomers can be used alone or in combination of two or more types. Of these first copolymerizable monomers, a compound represented by Formula (1a) (for example, 3,4-diacetyloxy-1-butene) is often used in terms of copolymerization reactivity and industrial handleability. For example, copolymerization of vinyl acetate as a vinyl ester monomer and 3,4-diacetyloxy-1-butene is highly copolymerizable with reactivity ratios (r) of each monomer being r=0.710 for vinyl acetate while r=0.701 for 3,4-diacetyloxy-1-butene. On the other hand, in copolymerization of vinyl acetate and vinyl ethylene carbonate as a compound represented by Formula (1c), reactivity ratios (r) are r=0.85 for vinyl acetate while r=5.4 for vinyl ethylene carbonate. In addition, 3,4-diacetyloxy-1-butene is highly polymerizable with a chain transfer constant (Cx) of 0.003 (65° C.), relative to vinyl ethylene carbonate with Cx=0.005 (65° C.) and 2,2-dimethyl-4-vinyl-1,3-dioxolane as a compound represented by Formula (1b) with Cx=0.023 (65° C.). Furthermore, 3,4-diacyloxy-1-butenes (for example, such as 3,4-diacetyloxy-1-butene) have a great industrial advantage as a byproduct formed by saponification thereof is an alkanoic acid (for example, acetic acid), similarly to a byproduct compound formed by saponification of a vinyl ester-based monomer, and thus a treatment and recovery of a solvent after saponification of the copolymer is possible without providing a special apparatus or process. Here, if decarboxylation or deketalization of a copolymer obtained by copolymerizing monomers represented by Formulas (1b) and (1c) is insufficient, the modified PVA-based resin may be crosslinked with the remaining carbonate ring or acetal ring to form a gel-like substance. Compounds represented by Formulas (1a) to (1c) are well known and may be prepared by a well-known method or may be commercially available. For example, the monomer represented by Formula (1a) can be prepared by methods described in WO 2000/24702, U.S. Pat. Nos. 5,623,086, 6,072,079, etc., or methods similar thereto. For example, 3,4-diacetyloxy-1-butene can be produced by a synthesis method via an epoxybutene derivative or by a reaction to isomerize 1,4-diacetyloxy-1-butene, which is an intermediate product of a 1,4-butanediol production process, using a metal catalyst, such as palladium chloride. In addition, the monomer represented by Formula (1a) can be obtained from Across Co., Ltd., etc. A ratio (molar ratio) of the vinyl ester-based monomer and the first copolymerizable monomer is, for example, the former/the latter=approximately from 50/50 to 99.5/0.5 (for example, from 70/30 to 99/1), preferably from 80/20 to 98.5/1.5 (for example, from 85/15 to 98/2), and more preferably from 90/10 to 97.5/2.5 (for example, from 92/8 to 97/3) according to a degree of modification of the PVA-based resin. The second copolymerizable monomer may be exemplified by various vinyl compounds, for example, linear or branched C2-12olefins, such as ethylene, propylene, isobutylene, α-octene, α-dodecene, and α-octadecene; unsaturated carboxylic acids or derivatives thereof (for example, salts, mono- or dialkylesters thereof), such as (meth)acrylic acid, crotonic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid, and undecylenic acid; nitriles, such as (meth)acrylonitrile; amides, such as diacetone acrylamide and (meth)acrylamide; olefin sulfonic acids or salts thereof, such as ethylene sulfonic acid and (meth)allylsulfonic acid; alkyl vinyl ethers; vinyl ketones (such as dimethyl allyl vinyl ketone and N-vinylpyrrolidone); halogen-containing vinyl compounds (such as vinyl chloride and vinylidene chloride); allyl ethers (such as glycerin monoallyl ether); and vinyl carbonates (such as vinylene carbonate). These copolymerizable monomers can be used alone or in combination of two or more types. The second copolymerizable monomer may be used in an amount of, for example, approximately from 0 to 30 mol % (for example, from 1 to 25 mol %), preferably from 0 to 20 mol % (for example, from 3 to 15 mol %), and more preferably from 0 to 10 mol % (for example, from 5 to 10 mol %), based on the entire amount of monomers. The modified PVA-based resin can be prepared, for example, by at least saponification of a copolymer of a vinyl ester-based monomer and a copolymerizable monomer containing at least a monomer corresponding to Formula (1) above (for example, the monomer represented by Formulas (1a) to (1c) above) as a copolymerizable monomer. More specifically, the modified PVA-based resin can be prepared by methods, for example, such as: (i) saponifying a copolymer of a vinyl ester-based monomer and a copolymerizable monomer containing at least the monomer represented by Formula (1a) above as a copolymerizable monomer; (ii) saponifying a copolymer of a vinyl ester-based monomer and a copolymerizable monomer containing at least the monomer represented by Formula (1b) above as a copolymerizable monomer, followed by deketalization; and (iii) saponifying a copolymer of a vinyl ester-based monomer and a copolymerizable monomer containing at least the monomer represented by Formula (1c) above as a copolymerizable monomer, followed by decarboxylation. For these methods, reference can be made to, for example, JP 2006-95825 A. An amount of modification of the modified PVA-based resin (ratio of a unit represented by Formula (1) above relative to all units of the modified PVA-based resin) varies depending on the nature of the modifying group but may typically be, for example, approximately from 1 to 30 mol % (for example, from 1 to 20 mol %), preferably from 1.2 to 12 mol % (for example, from 1.5 to 10 mol %), more preferably from 2 to 10 mol % (for example, from 3 to 8 mol %). If the introduced amount of the modifying group is too small, the melting point increases, thus the molding temperature needs to be increased, which tends to generate insoluble matter due to thermal degradation, and water solubility tends to decrease. On the other hand, if the introduced amount of the modifying group is too large, the melt moldability tends to decrease. The content of the unit represented by Formula (1) can be determined from a1H-NMR spectrum (solvent: DMSO-d6, internal standard: tetramethylsilane) of a completely saponified modified PVA-based resin. Specifically, the content can be calculated from peak areas derived from a proton of a hydroxyl group, a methine proton, and a methylene proton, a methylene proton of the main chain, a proton of a hydroxyl group bonded to the main chain, and the like in the structural unit represented by Formula (1). An average degree of polymerization (measured in accordance with JIS K 6726) of the modified PVA-based resin may be, for example, approximately from 100 to 3000 (for example, from 150 to 2000), preferably from 170 to 1000 (for example, from 200 to 800), and more preferably from 230 to 600 (for example, from 250 to 600). If the average degree of polymerization is too low, the production efficiency of the modified PVA-based resin would likely decrease, and if the average degree of polymerization is too high, the melt viscosity of the modified PVA-based resin would increase, and the melt moldability would likely decrease. In addition, a viscosity of a 4 wt. % aqueous solution (20° C., floppier viscometer) of the modified PVA-based resin may be, for example, approximately from 1 to 100 mPa·s (for example, from 1.5 to 75 mPa·s), preferably from 2 to 70 mPa·s (for example, from 2.3 to 60 mPa·s), and more preferably from 3 to 50 mPa·s (for example, from 5 to 30 mPa·s), or may be approximately from 1.5 to 10 mPa·s (for example, from 2 to 8 mPa·s), and preferably from 2.3 to 5 mPa·s (for example, from 2.5 to 4 mPa·s). When measured in accordance with JIS K 6726, a degree of saponification of the modified PVA-based resin may be for example approximately from 50 to 100 mol %, preferably from 60 to 100 mol % (for example, from 70 to 100 mol %), and more preferably from 80 to 100 mol % (for example, from 90 to 100 mol %), or may be approximately from 95 to 100 mol % (for example, from 98 to 100 mol %). If the degree of saponification is too low, water solubility and thermal stability in the melt molding process may decrease, and an acetic acid odor may occur. A melt viscosity of the modified PVA-based resin at a temperature of 260° C. and a shearing speed of 122 sec−1may be, for example, approximately from 2 to 100 Pa·s (for example, from 5 to 80 Pa·s), preferably from 7 to 75 Pa·s (for example, from 10 to 70 Pa·s), and more preferably from 12 to 65 Pa·s (for example, from 15 to 50 Pa·s), or may be approximately from 20 to 60 Pa·s (for example, from 30 to 50 Pa·s). The melt viscosity can be measured using a flow profile measuring device (“Capilograph 1D” available from Toyo Seiki Seisaku-sho, Ltd.) for a molten polymer with a barrel diameter of 9.55 mm and a total barrel length of 350 mm (effective length of 250 mm). A melt flow rate (MFR) of the modified PVA-based resin at a temperature of 210° C. under a load of 2160 g may be, for example, approximately from 1 to 160 g/10 min (for example, from 10 to 155 g/10 min), preferably from 15 to 150 g/10 min (for example, from 20 to 140 g/10 min), and more preferably from 25 to 120 g/10 min (for example, from 30 to 100 g/10 min), or may be approximately from 10 to 100 g/10 min (for example, from 15 to 80 g/10 min), and preferably from 20 to 70 g/10 min (for example, from 25 to 50 g/10 min). If the MFR is too low, flow moldability and the productivity would likely decrease, and if the MFR is too high, the moldability tends to decrease. The matrix containing the modified PVA-based resin can disperse a non-water-soluble resin in particulate form by a forced emulsification method. Thus, the matrix is useful for dispersing a non-water-soluble resin in particulate form by melt-kneading. In particular, the modified PVA-based resin not only has high heat resistance but also has a wide molding temperature range (molding processing temperature range) in comparison with an unmodified PVA-based resin. Thus, the matrix can be melt-kneaded with a non-water-soluble resin in a wide range of melt-kneading temperature according to the melting point or glass transition temperature of the non-water-soluble resin and can disperse resin particles in the matrix. A melting point of the modified PVA-based resin when the temperature is increased at a rate of 10° C./min under a nitrogen stream by differential scanning calorimetry (DSC) may be, for example, approximately from 120 to 225° C. (for example, from 130 to 220° C.), preferably from 140 to 215° C. (for example, from 145 to 210° C.), and more preferably from 150 to 205° C. (for example, from 160 to 200° C.), or may be approximately from 150 to 225° C. (for example, from 160 to 222° C.), preferably from 165 to 220° C. (for example, from 170 to 220° C.), and more preferably from 175 to 215° C. On the other hand, with regard to a thermal decomposition starting temperature, when the temperature is increased at a rate of 10° C./min under a nitrogen stream by differential scanning calorimeter (DSC), a temperature at which the weight of the modified PVA-based resin decreases by 1 wt. % is 255° C. or higher (for example, approximately from 256 to 260° C.), and a temperature at which the weight of the modified PVA-based resin decreases by 2 wt. % is 275° C. or higher (for example, approximately from 276 to 280° C.). Furthermore, when the temperature is increased from 30° C. to 600° C. at a temperature increase rate of 10° C./min in thermogravimetric analysis (TGA), a thermal decomposition temperature of the modified PVA resin is from 330 to 420° C. (for example, from 350 to 410° C., preferably from 370 to 405° C., and more preferably from 380 to 400° C.) in a nitrogen atmosphere, and approximately from 320 to 410° C. (for example, from 340 to 400° C., preferably from 360 to 395° C., and more preferably from 370 to 390° C.) in an air atmosphere. Thus, the modified PVA-based resin has a wide melt molding processing temperature range and excellent moldability. For example, the molding processing temperature (or melt-kneading temperature) may be approximately from 170 to 230° C., preferably from 175 to 225° C., and more preferably from 180 to 210° C. (for example, from 185 to 210° C.). In addition, a melting point of a completely saponified unmodified PVA resin is, for example, approximately 227° C., a temperature at which the weight decreases by 1 wt. % is, for example, approximately 257.4° C., and a temperature at which the weight decreases by 2 wt. % is, for example, approximately 277.3° C. Furthermore, when the temperature is increased from 30° C. to 600° C. at a temperature increase rate of 10° C./min in thermogravimetric analysis (TGA), a thermal decomposition temperature of a completely saponified unmodified PVA resin is 292° C. in a nitrogen atmosphere and 303° C. in an air atmosphere. Thus, an unmodified PVA resin has a narrow melt molding processing temperature range. A water contact angle of the modified PVA-based resin, which is a film having a thickness of 60 μm (produced by feeding a 5 wt. % aqueous solution into a 10 cm×10 cm mold and drying it in an environment at 23° C. and 50% RH for 2 days), may be approximately from 20 to 80°, preferably from 25 to 80°, more preferably from 30 to 75°, and particularly from 30 to 70°. If the water contact angle is too small, uniformity of particle size and particle shape of hydrophilic resin particles may be impaired, and if the water contact angle is too large, uniformity of particle size and particle shape of hydrophobic resin particles may be impaired. Here, the water contact angle can be determined by dripping 0.2 mL of purified water onto the surface of the film in an environment at 23° C. and 50% RH, and measuring the angle between the water droplet and the film surface. Such a measurement is performed 10 times, and the average value can be determined as the contact angle. For example, a “solid-liquid interface analyzer” available from Kyowa Interface Science Co., Ltd. can be used to measure the contact angle. The water-soluble matrix may include as necessary an additional water-soluble resin, for example, such as a polyethylene glycol-based resin (such as a polyethylene glycol or a polyethylene glycol-polypropylene glycol block copolymer), an unmodified polyvinyl alcohol-based resin, a polyvinylpyrrolidone-based resin, a cellulose ether (such as a hydroxypropyl cellulose, a carboxymethylcellulose, or a salt thereof), a polysaccharide (such as alginic acid or a salt thereof), or an oligosaccharide, a monosaccharide, or a sugar alcohol (such as erythritol, pentaerythritol, xylitol, or sorbitol). Non-Water-Soluble Resin The non-water-soluble resin may be formed of a non-water-soluble resin or a water-insoluble resin of various types that are inmiscible with the matrix, for example, a thermosetting resin (such as a phenolic resin and an epoxy resin), but typically, the resin particles are often formed of a thermoplastic resin (particularly a melt-kneadable resin). The thermoplastic resin may be exemplified by olefin-based resins, acrylate-based resins, styrene-based resins, halogen-containing resins, vinyl ester-based resins or water-insoluble derivatives thereof, polyester-based resins, polyamide-based resins, polycarbonate-based resins, polyurethane-based resins, poly(thio)ether-based resins (for example, polysulfide-based resins, such as polyphenylene ether-based resins and polyphenylene sulfide-based resins), polysulfone-based resins (for example, polysulfone resins and polyethersulfone-based resins), polyether ketone-based resins (such as polyphenylene ether ether ketone-based resins), polyimide-based resins (for example, such as polyetherimide-based resins, polyamide-imide-based resins, and polybenzimidazole-based resins), polyacetal-based resins, and cellulose ester-based resins (such as cellulose acetates), thermoplastic elastomers (for example, polyamide-based elastomers (such as polyamide-polyether block copolymers), polyester-based elastomers, polyurethane-based elastomers, polystyrene-based elastomers, polyolefin-based elastomers, and fluorine-based thermoplastic elastomers). These resins can be used alone or in combination of two or more types. The olefin-based resin may be exemplified by a homopolymer or copolymer of an α-C2-6olefin, such as a polyethylene, a polypropylene, and an ethylene-propylene copolymer; and a copolymer of an olefin and a copolymerizable monomer (such as an ethylene-(meth)acrylate copolymer and an ethylene-(meth)acrylate copolymer; and a homopolymer or copolymer of a cyclic olefin, such as dicyclopentadiene (such as an ethylene-norbornene copolymer). The acrylate-based resin may be exemplified by a poly(meth)acrylate, such as methyl poly(meth)acrylate; a homopolymer or copolymer of a (meth)acrylate-based monomer, such as a methyl methacrylate-(meth)acrylate copolymer, a methyl methacrylate-acrylate-(meth)acrylic acid copolymer, a methyl methacrylate-(meth)acrylate copolymer, and a (meth)acrylate-styrene copolymer (such as an MS resin). Examples of the styrene-based resin include a homopolymer or copolymer of a styrene-based monomer, such as polystyrene, a styrene-vinyltoluene copolymer, and a styrene-α-methylstyrene copolymer; a copolymer of a styrene-based monomer and a copolymerizable monomer, such as a styrene-acrylonitrile copolymer (an AS resin), a (meth)acrylate-styrene copolymer (such as an MS resin), and a styrene-maleic anhydride copolymer; a block copolymer and the like, such as a styrene-butadiene block copolymer; and a graft polymer that is graft-polymerized with at least a styrene-based monomer in the presence of a rubber component, for example, a rubber-containing styrene-based copolymer, such as a high-impact polystyrene (HIPS), an acrylonitrile-butadiene-styrene copolymer (ABS resins), and a methyl methacrylate-butadiene rubber-styrene copolymer (an MBS resin). The halogen-containing resin may be exemplified by polyvinyl chloride-based resins, vinyl chloride-vinyl acetate copolymers, vinylidene chloride-based resins, and fluororesins (melt-flowable fluororesins). The vinyl ester-based resin or the water-insoluble derivative thereof may be exemplified by a homopolymer or copolymer of a vinyl carboxylate ester (such as a polyvinyl acetate and an ethylene-vinyl acetate copolymer), a derivative from a saponified product thereof (for example, a polyvinyl acetal-based resin, such as a polyvinyl formal and a polyvinyl butyral). Examples of the polyester-based resin include poly C2-6alkylene-arylate-based resins, such as a polyethylene terephthalate, a poly(trimethylene terephthalate), a polypropylene terephthalate, a polybutylene terephthalate, a polyethylene naphthalate, and a polybutylene naphthalate; polycyclo C6-10alkylene-arylate-based resins, such as poly(1,4-cyclohexyldimethylene terephthalate); copolyesters containing a C2-6alkylene-arylate unit or a cyclo C6-10alkylene-arylate unit as a main component (for example, 50 wt. % or greater) (for example, the copolymer component is a copolyester, such as an aliphatic dicarboxylic acid, isophthalic acid, and phthalic acid); aromatic polyester-based resins, such as polyarylate-based resins and liquid crystal polyesters; a poly(C2-6alkylene glycol-C2-10aliphatic dicarboxylate), such as a poly C2-6alkylene-succinate and a poly C2-6alkylene-adipate; polyoxycarboxylic acid-based resins (for example, such as polyglycolic acids, polylactic acids, and glycolic acid-lactic acid copolymers); lactone-based resins and the like, such as polycaprolactones), and copolyesters thereof (for example, such as polycaprolactone-polybutylene succinate copolymer resins). The polyester-based resin may contain a urethane bond. Furthermore, the polyester-based resin may have biodegradability. Examples of the polyamide-based resin include aliphatic polyamides, alicyclic polyamides, and aromatic polyamides. The polyamide-based resin may be a homopolyamide (a polyamide-based homopolymer resin) or a copolyamide (a polyamide-based copolymer resin) of a polyamide-forming component. Of the aliphatic polyamide-based resins, the homopolyamides include: a homopolyamide or copolyamide of an aliphatic diamine component [such as an alkanediamine (for example, a C4-16alkylenediamine, such as tetramethylenediamine, hexamethylenediamine, trimethylhexamethylenediamine and dodecanediamine; preferably a C6-14alkylenediamine and more preferably a C6-12alkylenediamine)] and an aliphatic dicarboxylic acid component [for example, such as an alkanedicarboxylic acid (for example, a C4-20alkanedicarboxylic acid, such as adipic acid, sebacic acid, and dodecane diacid; preferably a C5-18alkanedicarboxylic acid and more preferably a C6-16alkanedicarboxylic acid)]; a homopolyamide or copolyamide of a lactam [such as a lactam having approximately from 4 to 20 carbons (preferably from 6 to 16 carbons and more preferably from 8 to 14 carbons), such as ε-caprolactam and ω-laurolactam] or an aminocarboxylic acid (for example, a C4-20aminocarboxylic acid, such as w-aminoundecanoic acid and ω-aminododecanoic acid; preferably a C6-16aminocarboxylic acid and more preferably a C8-14aminocarboxylic acid); a copolyamide of a first amide-forming component including an aliphatic diamine component and an aliphatic dicarboxylic acid component in combination and a second amide-forming component including an lactam and/or an aminocarboxylic acid; and the like. The dicarboxylic acid component of the polyamide-based resin may contain a dimer acid unit. Furthermore, the polyamide-based resin may have biodegradability. The aliphatic polyamide-based resin may contain a unit derived from at least one type of component selected from laurolactam, aminoundecanoic acid, and aminododecanoic acid. Specific examples of the aliphatic polyamide-based resin include polyamide 6, polyamide 11, polyamide 12, polyamide 46, polyamide 66, polyamide 610, polyamide 611, polyamide 612, polyamide 613, polyamide 1010, polyamide 1012, polyamide 1212, polyamide 66/11, polyamide 6/12, polyamide 66/12, polyamide 610/12, and polyamide 6/12/612. Examples of the alicyclic polyamide-based resin include a homopolyamide or copolyamide formed by polymerizing at least one type selected from alicyclic diamine components and alicyclic dicarboxylic acid components as a monomer, and, for example, alicyclic polyamides obtained by using, of the diamine components and the dicarboxylic acid components, an alicyclic diamine and/or an alicyclic dicarboxylic acid as at least a part of the component can be used. In particular, as the diamine component and the dicarboxylic acid component, the aliphatic diamine component and/or the aliphatic dicarboxylic acid component exemplified above are preferably used in combination with the alicyclic diamine component and/or the alicyclic dicarboxylic acid component. Such alicyclic polyamides are highly transparent and are known as so-called transparent polyamides. Examples of the alicyclic diamine components include diaminocycloalkanes (such as diamino C5-10cycloalkanes), such as diaminocyclohexane; bis(aminocycloalkyl)alkanes [such as bis(amino C5-8cycloalkyl)C1-3alkanes], such as bis(4-aminocyclohexyl)methane, bis(4-amino-3-methylcyclohexyl)methane, and 2,2-bis(4′-aminocyclohexyl)propane; and a hydrogenated xylylenediamine. The alicyclic diamine components may include a substituent, such as an alkyl group (a C1-6alkyl group, such as a methyl group and an ethyl group; preferably a C1-4alkyl group and more preferably a C1-2alkyl group. In addition, examples of the alicyclic dicarboxylic acids include cycloalkane dicarboxylic acids (such as C5-10cycloalkane-dicarboxylic acids), such as 1,4-cyclohexanedicarboxylic acid and 1,3-cyclohexanedicarboxylic acid. Examples of representative alicyclic polyamide-based resins include condensates of an alicyclic diamine component [for example, such as bis(aminocyclohexyl)alkanes] and an aliphatic dicarboxylic acid component [for example, such as an alkanedicarboxylic acid (for example, such as a C4-20alkane-dicarboxylic acid component)]. The aromatic polyamide-based resin include polyamides in which at least one component of the aliphatic diamine component or the aliphatic dicarboxylic acid component in the aliphatic polyamide is an aromatic component, for example, such as polyamides in which the diamine component is an aromatic diamine component [for example, a condensate (for example, such as MXD-6) of an aromatic diamine (such as metaxylylene diamine) and an aliphatic dicarboxylic acid] and polyamides in which the dicarboxylic acid component is an aromatic component [for example, a condensate of an aliphatic diamine (such as trimethylhexamethylenediamine) and an aromatic dicarboxylic acid (such as terephthalic acid and isophthalic acid)]. In addition, the aromatic polyamide-based resin may be a wholly aromatic polyamide (aramid) [such as poly(m-phenylene isophthalamide)], a polyamide in which the diamine component and the dicarboxylic acid component are aromatic components. These polyamide-based resins may be used alone or in combination of two or more types. In addition, the polyamide-based resin may be an aromatic polyamide-based resin in which one component of the aliphatic diamine component and the aliphatic dicarboxylic acid component is an aromatic component, but typically, the polyamide-based resin is often an aliphatic polyamide and/or an alicyclic polyamide. Also, polyamide-based resin (such as a copolyamide) often includes a long-chain alkylene group, for example, such as a C8-16alkylene group and preferably a C10-14alkylene group, deriving from a dicarboxylic acid, a lactam, and/or an aminocarboxylic acid (for example, at least one type of component selected from lactams and aminoalkanecarboxylic acids). Furthermore, the polyamide-based resin may be a copolyamide (polyamide-based copolymer resin) whose properties can be adjusted according to the application. Examples of the polycarbonate-based resin include aromatic polycarbonates based on bisphenols, such as bisphenol A type polycarbonate resins; and aliphatic polycarbonates, such as diethylene glycol bisallyl carbonates. The polyurethane-based resin can be exemplified by polyurethane-based resins obtained by a reaction of an aliphatic, alicyclic, or aromatic diisocyanate; a polyol (for example, such as a polyester polyol; a polyether polyol, such as a polytetramethylene ether glycol; and a polycarbonate polyol); and a chain extender as necessary. Of these thermoplastic resins, for example, an olefin-based resin, an acrylate-based resin, a styrene-based resin, a polyester-based resin, a polyamide-based resin, or the like is often used, and a biodegradable resin, for example, a biodegradable polyester-based resin, such as a polyester-based resin (for example, such as a polylactic acid-based resin, a polylactone-based resin, and a polyesteramide) is often used. The non-water-soluble resin (for example, a thermoplastic resin) may be a non-polar or inert resin that does not include a functional group, or may be a resin including a functional group. For example, a thermoplastic resin, such as an olefin-based resin or a styrene-based resin, does not necessarily have to include a functional group. In an embodiment of the present invention, even if the non-water-soluble resin (for example, a thermoplastic resin) includes a functional group (for example, such as a hydroxyl group, a carboxyl group, an alkoxycarbonyl group, and an amino group), the resin particles can be formed while maintaining the concentration of the functional group. Thus, concentrations of these functional groups are not particularly limited, and in the resin including a plurality of different functional groups, the ratio of concentrations of the different functional groups is not particularly limited either. Such a non-water-soluble resin including a functional group may be, for example, a thermoplastic resin including a hydroxyl group, a carboxyl group, and/or an alkoxycarbonyl group [for example, such as a modified olefin-based resin, an acrylate-based resin, a styrene-based resin into which a functional group is introduced), a thermoplastic resin including a carboxyl group and/or a hydroxyl group (representatively, such as a polyester-based resin and a polycarbonate-based resin), a thermoplastic resin including an amino group and/or a carboxyl group (representatively a polyamide-based resin)]. In particular, unlike with polyethylene glycol, a concentration of the amino group of the resin is not reduced and a carboxyl group concentration of the resin is not increased when melt-kneaded with the water-soluble matrix. Thus, the present invention is useful for applying to a non-water-soluble resin (for example, a thermoplastic resin) including at least an amino group as a functional group to produce resin particles. More specifically, even if the water-soluble matrix and the polyamide-based resin including an amino group and a carboxyl group are melt-kneaded to form polyamide resin particles, fluctuations in concentrations of these functional groups can be prevented and the amino group concentration can be maintained in the polyamide resin particles. Thus, a ratio (molar ratio) of the amino group (terminal amino group) and the carboxyl group (terminal carboxyl group) in the polyamide-based resin is the former/the latter=approximately from 1/99 to 99/1 (for example, from 2/98 to 98/2), preferably from 5/95 to 95/5 (for example, from 10/90 to 90/10), more preferably from 20/80 to 80/20 (for example, from 30/70 to 70/30), or may be approximately from 25/75 to 60/40 (for example, from 40/60 to 60/40). In this connection, polyamide resin particles having a high amino group concentration (terminal amino group concentration) relative to the carboxyl group (terminal carboxyl group concentration) can be used for various applications (for example, such as a fiber-reinforced composite material) using high activity of the amino group. Thus, the amino group concentration (terminal amino group concentration, in mmol/kg) in the polyamide-based resin can be selected from a wide range, for example, approximately from 1 to 160 (for example, from 2 to 155 and particularly from 5 to 150), may typically be approximately from 10 to 150 (for example, from 15 to 120), preferably from 20 to 100 (for example, from 25 to 80), more preferably from 30 to 60 (for example, from 35 to 55), or may be approximately from 1 to 150 (for example, from 1 to 100), preferably from 2 to 75 (for example, from 5 to 70), and more preferably from 10 to 60 (for example, from 15 to 50). In addition, the ratio (molar ratio) of the amino group (terminal amino group) to the carboxyl group (terminal carboxyl group) may be the former/the latter=approximately from 20/80 to 95/5 (for example, from 25/75 to 80/20) and preferably from 30/70 to 70/30 (for example, from 35/65 to 60/40) or may be approximately from 30/70 to 50/50 (for example, from 35/65 to 45/55). Concentrations (contents) of these functional groups can be measured using a commonly used method, for example, such as a titration method, a spectroscopic analysis method, or an NMR method according to the type of resin, and concentrations of functional groups of the polyester-based resin or the polyamide-based resin may be measured by a titration method. A number average molecular weight of the non-water-soluble resin (for example, a thermoplastic resin, such as a polyamide-based resin) can be selected from a range approximately from 3000 to 500000 according to the type of resin and may be, for example, approximately from 5000 to 200000, preferably from 7500 to 150000, and more preferably from 10000 to 100000. The number average molecular weight can be measured by a common method, for example, such as gel permeation chromatography using a polystyrene as a standard material, according to the type of resin. Viscosity average molecular weight can be adopted for thermoplastic resins, such as cellulose derivatives (such as cellulose ester-based resins), whose molecular weight is difficult to be measured by gel permeation chromatography. The non-water-soluble resin (for example, a thermoplastic resin) may be amorphous or crystalline. Olefin-based resins, polyester-based resins, polyamide-based resins (such as copolyamides), and the like are often crystalline. A crystallinity of the crystalline thermoplastic resin is often 90% or less (for example, approximately from 1 to 70% and preferably from 5 to 50%), and, for example, a crystallinity of a semicrystalline or crystalline polyamide-based resin may be 40% or less, for example, approximately from 1 to 30% (for example, from 2 to 25%), preferably from 3 to 20% (for example, from 4 to 17%), and more preferably from 5 to 20% (for example, from 5 to 15%). In addition, a melting point of the crystalline thermoplastic resin can be selected, for example, from a range approximately from 50 to 350° C. (for example, from 70 to 300° C.), and may be approximately from 80 to 280° C. (for example, from 90 to 250° C.) and preferably from 100 to 240° C. (for example, from 120 to 230° C.). For example, a melting point of a semicrystalline or crystalline polyamide-based resin (such as an aliphatic polyamide and an alicyclic polyamide) can be selected from a range approximately from 80 to 350° C. (for example, from 100 to 320° C.), and may typically be approximately from 120 to 300° C. (for example, from 150 to 280° C.) and preferably from 160 to 270° C. (for example, from 170 to 260° C.). If the crystallinity or the melting point is too high, the melt-kneadability and the uniform dispersibility may decrease, which could limit the application of the resin particles. The crystallinity and the melting point can be measured by a commonly used method, for example, an X-ray method and a differential scanning calorimetry (DSC) method. A glass transition temperature of the non-water-soluble resin (for example, a thermoplastic resin) can be selected from a range of 25° C. or higher (for example, approximately from 25 to 280° C.) according to the type of resin and may typically be approximately from 30 to 270° C. (for example, from 50 to 250° C.), preferably from 70 to 230° C. (for example, from 100 to 220° C.), and more preferably from 120 to 210° C. A glass transition temperature of the polyamide-based resin can be selected, for example, from a range approximately from 30 to 250° C. (for example, from 35 to 230° C.), and may be approximately from 40 to 200° C. (for example, from 45 to 190° C.), preferably from 50 to 180° C. (for example, from 60 to 170° C.), and more preferably from 70 to 160° C. (for example, from 80 to 150° C.), or may be approximately from 100 to 160° C. (for example, from 105 to 155° C.) and preferably from 120 to 150° C. (for example, from 125 to 150° C.). If the glass transition temperature is too high, the melt-kneadability with the water-soluble matrix and the dispersibility of the non-water-soluble resin (for example, a thermoplastic resin) may decrease, and if the glass transition temperature is too low, uniformity of the form of the resin particles may decrease. The water-soluble matrix (or the modified PVA-based resin) can uniformly disperse the resin in particulate form even if the resin content (or the ratio of the resin to the water-soluble matrix) is high. Thus, a weight ratio of the non-water-soluble resin (for example, a thermoplastic resin) to the water-soluble matrix can be selected in a wide range of the former/the latter=approximately from 1/99 to 70/30 (for example, from 10/90 to 60/40), and may typically be approximately from 20/80 to 50/50 (for example, from 25/75 to 50/50), preferably from 30/70 to 50/50 (for example, from 35/65 to 45/55), and more preferably from 40/60 to 50/50 (for example, from 45/55 to 50/50), or may be approximately from 40/60 to 49/51. If the ratio of the water-soluble matrix is too large, the productivity of the resin particles may decrease, and conversely if the proportion is too small, production of resin particles having a small particle diameter may be difficult. In addition, the water-soluble matrix and/or the non-water-soluble resin (for example, a thermoplastic resin) may contain an additive of various types, for example, such as a filler, a stabilizer (such as a heat-resistant stabilizer, a weather-resistant stabilizer, an antioxidant, and an ultraviolet absorber), a colorant, a plasticizer, a dispersant, a preservative, an anti-foaming agent, a lubricant, a flame retardant, and an antistatic agent. These additives may be used alone or in combination of two or more types. A ratio of each additive or a total ratio of additives may be, for example, 10 parts by weight or less (for example, approximately from 0.01 to 10 parts by weight) relative to 100 parts by weight of the water-soluble matrix or the resin. In the melt-kneading, the non-water-soluble resin (a hot-melt resin, particularly a thermoplastic resin) is forcibly emulsified and can be dispersed in particulate form in the matrix by kneading or melt-kneading with the water-soluble matrix. The kneading or the melt-kneading can be performed by premixing the water-soluble matrix and the resin as necessary, and using a commonly used kneader (for example, such as a single-screw or twin-screw extruder, a kneader, a calendar roll, and a Banbury mixer). In an embodiment of the present invention, resin particles are formed in the matrix by a method (A) and/or (B) below and resin particles are produced.(A) Melt-kneading the water-soluble matrix and the water-insoluble resin at a temperature above 220° C.;(B) Forming resin particles having an average particle size of greater than 5 μm by melt-kneading to produce resin particles having a corresponding average particle size. In the above method (A), a kneading temperature (for example, a cylinder temperature of the extruder) can be selected, for example, from a temperature equal to or higher than the melting point or glass transition temperature of the resin, for example, a temperature above 220° C., for example, a range approximately from 225 to 350° C. (for example, from 230 to 330° C.), and may typically be approximately from 230 to 320° C. (for example, from 240 to 310° C.) and preferably from 250 to 300° C. (for example, from 260 to 280° C.). More specifically, the melt-kneading temperature of the polyamide-based resin can also be selected from a range approximately from 225 to 350° C. (for example, from 230 to 350° C.) according to the type of polyamide-based resin and may be, for example, approximately from 240 to 350° C., preferably from 250 to 320° C., and more preferably from 260 to 300° C. Unlike an aqueous medium containing an oligosaccharide and a saccharide, the water-soluble matrix does not scorch during the kneading process. A kneading time may be, for example, approximately from 10 seconds to 1 hour. In the above method (B) (a method for producing resin particles having an average particle size of 5 μm or less), it is not necessary to perform melt-kneading at the above temperature, and the melt-kneading temperature can be selected from, for example, a range of approximately 90 to 220° C. (for example, 100 to 210° C.) and may typically be approximately from 120 to 210° C. (for example, from 150 to 200° C.) and preferably from 170 to 200° C. (for example, from 180 to 200° C.). The shape and average particle size of the resin particles obtained by the method according to an embodiment of the present invention often correspond to the shape and average particle size of the resin particles dispersed in the matrix, and the average particle size of the resin particles can be adjusted by melt flow characteristics of the water-soluble matrix and the water-insoluble resin, a ratio of the water-soluble matrix and the water-insoluble resin, the melt-kneading temperature, a shearing speed during the melt-kneading process, and the like. In an embodiment of the present invention, a melt-kneaded product (a pre-dispersion or a pre-molded article) can be effectively formed through a kneader probably because a large amount of the melt-kneaded product is entangled with a screw or a blade of the kneader. For example, a large amount of the melt-kneaded product is entangled with a screw of the extruder, and thus the discharge rate from the extruder can be increased. Furthermore, the kneaded product from the extruder can be molded into the form of pellets, which can improve the handleability of the molded article (a pre-molded article in which resin particles are dispersed). Thus, the water-soluble matrix is suitable for dispersing the resin in particulate form by melt-kneading to produce a wide range of resin particles. Matrix Removing The melt-kneaded product is typically cooled (gradually cooled or rapidly cooled), and the water-soluble matrix of the formed pre-molded article (or dispersion) is eluted with an aqueous solvent and removed in the matrix removing. The water-soluble matrix only needs to be eluted or removed by bringing the pre-molded article (or dispersion) into contact with an aqueous solvent and can be typically eluted or removed by mixing and washing the pre-molded article (or dispersion) with an aqueous solvent while applying shear force or stirring force. The aqueous solvent may contain a water-soluble organic solvent, such as an alcohol (such as ethanol) or a water-soluble ketone (such as acetone), but typically water is often used. The water-soluble matrix may be warmed and eluted as necessary. In this matrix removing, the modified PVA-based resin, because of its high water solubility, can improve the elution rate of the matrix, which can also improve the production efficiency of the resin particles. Collecting and Moisture Controlling The resin particles can be formed by removing the matrix from the pre-molded article. The formed resin particles can be used as-is depending on the application or, as necessary, can be collected by a commonly used solid-liquid separation method, such as filtration and centrifugation. For the collected resin particles, the moisture content of the resin particles may be adjusted as necessary in the moisture controlling. That is, the resin particles may be dried, and the moisture content may be adjusted to, for example, approximately 0.1 to 5 wt. % (for example, 0.5 to 3 wt. %) according to the application of the resin particles. Specifically, the moisture content of the polyamide resin particles may be adjusted to, for example, approximately 0.5 to 2.5 wt. % (for example, 0.5 to 2 wt. %), preferably 0.55 to 2 wt. % (for example, 0.8 to 1.5 wt. %), more preferably 0.6 to 1.5 wt. % (for example, 0.65 to 1 wt. %), and particularly 0.7 to 0.8 wt. %. The moisture content can be measured by a commonly used method, such as Karl Fischer method, a thermal analysis method, or a trace moisture measurement device equipped with a moisture vaporizing device. In addition, if the crystalline thermoplastic resin is excessively heated or heated for a long time, crystallization may proceed. Thus, the crystalline thermoplastic resin particles may be subjected to controlled heating or drying conditions (for example, dried at a temperature below the glass transition temperature Tg (for example, a temperature equal to or lower than (Tg−30° C.) and a humidity approximately from 40 to 90% RH) to prevent increase in crystallinity of the resin particles and adjust the crystallinity to the above range. Such resin particles (for example, such as polyamide-based resin particles) whose moisture content or crystallinity is adjusted are useful for improving a toughness of a cured product of a fiber-reinforced composite material, for example, such as an epoxy resin composition containing reinforcing fibers, such as carbon fibers. A shape of the resulting resin particles only needs to be particulate and may be, for example, spherical or odd shape (such as ellipsoidal, polygonal, prismatic, cylindrical, rod-like, or indefinite shape). The resin particles may also be porous particles, coated particles, or the like. The preferred form of the resin particles is spherical. The spherical particles include not only true spherical particles but also particles having a shape similar to the true spherical shape, for example, particles having a shape whose major axis and minor axis are nearly the same, for example, the major axis/the minor axis=approximately from 1.5/1 to 1/1, preferably from 1.3/1 to 1/1 (for example, from 1.2/1 to 1/1), and more preferably from 1.1/1 to 1/1. In addition, a surface of the resin particles may have unevenness, but preferably the surface is smooth, and the surface smoothness is high. An average particle size (volume average particle size) of the resin particles is not particularly limited and may be selected from a range approximately from 0.1 to 1000 μm (for example, from 0.5 to 500 μm) according to the application and may be, for example, approximately from 1 to 300 μm, preferably from 3 to 150 μm, and more preferably 5 μm or greater (for example, from 5 to 100 μm). In addition, as in the above method (B), the average particle size of the resin particles may typically be greater than 5 μm and may be, for example, approximately from 6 to 100 μm (for example, from 7 to 90 μm) and preferably from 9 to 85 μm (for example, from 10 to 80 μm). More specifically, an average particle diameter (average particle size) of the polyamide-based resin particles may be, for example, approximately from 1 to 100 μm (for example, from 3 to 80 μm), preferably from 5 to 100 μm (for example, from 7 to 80 μm), more preferably from 10 to 75 μm (for example, from 15 to 70 μm), and as in the above method (B), the average particle size is often above 5 μm. The average particle size is expressed by the volume average primary particle size and can be measured by laser diffraction scattering method or the like. A form (such as a particle shape and an average particle size) of the resin particles obtained by eluting the matrix typically has the form of the resin particles dispersed in the matrix. A specific surface area of the resin particles according to BET method is not particularly limited and may be, for example, approximately from 0.08 to 12 m2/g (for example, from 0.15 to 6 m2/g) and preferably from 0.2 to 3 m2/g (for example, from 0.3 to 2 m2/g) in accordance with the average particle size. Furthermore, the polyamide-based resin particles may have an exothermic peak in a temperature range between the glass transition temperature and the melting point (for example, at a temperature higher than the glass transition temperature by approximately 1 to 70° C., preferably 1 to 60° C., and more preferably 1 to 50° C. (for example, 1 to 40° C.)) when the temperature is increased at a rate of 10° C./min by differential scanning calorimetry (DSC). The polyamide-based resin particles having such thermal properties (a crystal structure) can improve a toughness of a cured product of a fiber-reinforced composite material and a reinforcing effect of reinforcing fibers. EXAMPLES Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited by these examples. Abbreviations for materials used in the Examples and Comparative Examples, and evaluation items and evaluation methods thereof are as follows. Materials (1) Water-Soluble Matrix Modified PVA-based resin: Modified PVA-based resins 1 and 2 were prepared as follows. Production of Modified PVA-Based Resin 1 To a reactor equipped with a reflux condenser, a dripping device, and a stirrer, 14 parts by weight of vinyl acetate (14 wt. % of the total was initially charged), 29 parts by weight of methanol, and 1.68 parts by weight of 3,4-diacetyloxy-1-butene (14 wt. % of the total was initially charged) were charged, the temperature was increased under a nitrogen stream while stirring, and after the boiling point was reached, 0.093 parts by weight of acetyl peroxide was added to initiate polymerization. Furthermore, 0.04 parts by weight of acetyl peroxide was added over 1.0 hour from the initiation of the polymerization. Moreover, 1.0 hours after the initiation of the polymerization, 86 parts by weight of vinyl acetate and 10.32 parts by weight of 3,4-diacetyloxy-1-butene were added dropwise at the same speed over 19.7 hours. When the polymerization rate of vinyl acetate reached 93%, a predetermined amount of m-dinitrobenzene was added to terminate the polymerization. Next, the unreacted vinyl acetate monomer was removed out of the system by distillation while blowing methanol vapor, and a methanol solution of a copolymer was obtained. Then, the solution of the above copolymer was diluted with methanol and adjusted to a solid concentration of 50 wt. %. This methanol solution was charged in a kneader, and a methanol solution containing sodium hydroxide in an amount of 2 wt. % in terms of sodium therein was added at a ratio of 8 mmol relative to 1 mol of a total amount of the vinyl acetate structural unit and the 3,4-diacetyloxy-1-butene structural unit in the copolymer while the solution temperature was kept at 35° C. to saponify the copolymer. As the saponification proceeded, the saponified product was precipitated to form particles, the above methanol solution containing 2 wt. % of sodium was further added at a ratio of 5 mmol relative to 1 mol of a total amount of the vinyl acetate structural unit and the 3,4-diacetyloxy-1-butene structural unit to saponify the copolymer. Thereafter, acetic acid for neutralization, 0.8 equivalent to sodium hydroxide, was added, and the mixture was filtered, washed adequately with methanol, dried in a hot air dryer, and a PVA-based resin including in a side chain thereof a 1,2-diol structure was obtained. A degree of saponification of the resulting PVA-based resin including in a side chain thereof a 1,2-diol structure was 99 mol % as analyzed by alkali consumption required for hydrolysis of structural units of residual vinyl acetate and 3,4-diacetyloxy-1-butene in the resin. In addition, an average degree of polymerization was 300 as analyzed in accordance with JIS K 6726. A content of the 1,2-diol structural unit represented by Formula (1-1) above was 5.6 mol % as calculated from an integrated value measured by1H-NMR (300 MHz proton NMR, solvent DMSO-d6, internal standard material: tetramethylsilane, 50° C.). A crystallization temperature Tc of this modified PVA-based resin was 152° C., and a melt viscosity (at 260° C. and a shearing speed of 122 sec−1) was 42 Pa·s. The resulting PVA-based resin 1 above was pelletized under the following conditions.Extruder: 15 mm φ L/D=60, available from Technovel CorporationExtrusion temperature: C1/C2/C3/C4/C5/C6/C7/C8/D=100/170/180/190/200/210/220/220/210° C.Rotation number: 200 rpmDischarge rate: 1.5 kg/h Production of Modified PVA-Based Resin 2 To a reactor equipped with a reflux condenser, a dripping device, and a stirrer, 10 parts by weight of vinyl acetate (10 wt. % of the total was initially charged), 40 parts by weight of methanol, and 1.25 parts by weight of 3,4-diacetoxy-1-butene (10 wt. % of the total was initially charged) were charged, the temperature was increased under a nitrogen stream while stirring, and after the boiling point was reached, 0.22 parts by weight of acetyl peroxide was added to initiate polymerization. Furthermore, 0.5 hours after the initiation of the polymerization, 90 parts by weight of vinyl acetate and 11.25 parts by weight of 3,4-diacetoxy-1-butene were added dropwise at the same speed over 22.5 hours. When the polymerization rate of vinyl acetate reached 95%, a predetermined amount of m-dinitrobenzene was added to terminate the polymerization. Next, the unreacted vinyl acetate monomer was removed out of the system by distillation while blowing methanol vapor, and a methanol solution of a copolymer was obtained. Then, the solution of the above copolymer was diluted with methanol and adjusted to a solid concentration of 55 wt. %. This methanol solution was charged in a kneader, and a methanol solution containing sodium hydroxide in an amount of 2 wt. % in terms of sodium therein was added at a ratio of 8.7 mmol relative to 1 mol of a total amount of the vinyl acetate structural unit and the 3,4-diacetoxy-1-butene structural unit in the copolymer while the solution temperature was kept at 35° C. to saponify the copolymer. As the saponification proceeded, the saponified product was precipitated to form particles, the above methanol solution containing 2 wt. % of sodium was further added at a ratio of 4 mmol relative to 1 mol of a total amount of the vinyl acetate structural unit and the 3,4-diacetoxy-1-butene structural unit to saponify the copolymer. Thereafter, acetic acid for neutralization, 0.8 equivalent to sodium hydroxide, was added, and the mixture was filtered, washed adequately with methanol, dried in a hot air dryer, and a PVA-based resin including in a side chain thereof a 1,2-diol structure was obtained. A degree of saponification of the resulting PVA-based resin (a1) including in a side chain thereof a 1,2-diol structure was 99 mol % as analyzed by alkali consumption required for hydrolysis of structural units of residual vinyl acetate and 3,4-diacetoxy-1-butene in the resin. In addition, an average degree of polymerization was 250 as analyzed in accordance with JIS K 6726. A content of the 1,2-diol structural unit represented by Formula (1-1) above was 5.6 mol % as calculated from an integrated value measured by1H-NMR (300 MHz proton NMR, d6-DMSO solution, internal standard material: tetramethylsilane, 50° C.). A crystallization temperature Tc of this modified PVA-based resin 2 was 152° C., and a melt viscosity (at 260° C. and a shearing speed of 122 sec−1) was 18 Pa·s. The resulting PVA-based resin 2 above was pelletized under the following conditions.Extruder: 15 mm φ L/D=60, available from Technovel CorporationExtrusion temperature: C1/C2/C3/C4/C5/C6/C7/C8/D=100/170/180/190/200/210/210/200/190° C.Rotation number: 200 rpmDischarge rate: 2.0 kg/hOligosaccharide: Starch sugar (reduced starch saccharification product PO-10, available from Towa Chemical Industry Co., Ltd.)Sugar alcohol: D-Sorbitol LTS powder 20 M, available from Mitsubishi Shoji Foodtech Co., Ltd.Polyethylene glycol (PEG): “Alkox R150” available from Meisei Chemical Works, Ltd. (2) Thermoplastic Resin(A): Polyamide 12 (PA12)-based resin, “Vestamid L1600” available from Daicel-Evonik Ltd., melting point 178° C., terminal carboxyl group concentration (COOH group concentration) 114 mmol/kg, and terminal amino group concentration (NH2concentration) 1 mmol/kg(B): Polyamide 12 (PA12)-based resin, “Vestamid L2140” available from Daicel-Evonik Ltd., melting point 178° C., terminal carboxyl group concentration (COOH group concentration) 53 mmol/kg, and terminal amino group concentration (NH2concentration) 14 mmol/kg(C): Polyamide 1010 (PA1010)-based resin, “Vestamid DS22” available from Daicel-Evonik Ltd., melting point 192° C., terminal carboxyl group concentration (COOH group concentration) 72 mmol/kg, and terminal amino group concentration (NH2concentration) 31 mmol/kg(D): Alicyclic polyamide-based resin, “Trogamid PACM12” available from Daicel-Evonik Ltd., melting point 247° C., terminal carboxyl group concentration (COOH group concentration) 67 mmol/kg, and terminal amino group (NH2concentration) concentration 45 mmol/kg(E): Polyamide 12 (PA12)-based resin, “Vestamid L1901” available from Daicel-Evonik Ltd., melting point 178° C., terminal carboxyl group concentration (COOH group concentration) 86 mmol/kg, and terminal amino group concentration (NH2concentration) 5 mmol/kg(F): Polyamide 12 (PA12)-based resin, “Daiamid L1640” available from Daicel-Evonik Ltd., melting point 178° C., terminal carboxyl group concentration (COOH group concentration) 130 mmol/kg, and terminal amino group concentration (NH2concentration) 4 mmol/kg. Average Particle Diameter D50 About 1 g of the resin particles was added to 50 mL of pure water, one drop of a cleaning agent containing a surfactant (“Mamalemon (trade name)” available from Lion Corporation) was added and dispersed over 1 minute with an ultrasonic disperser (“US Cleaner” available from AS ONE Corporation). Then, the resin particles were dispersed in water, and an average particle diameter (on a volume basis) D50 was measured using a particle size distribution measuring device (“LA960” available from Horiba, Ltd.). Crystallization Temperature, Melting Point, and Glass Transition Temperature Crystallization temperature Tc of the water-soluble matrix, melting point and glass transition temperature Tg of the polyamide-based resin were measured using a differential scanning calorimeter (“X-DSC7000” available from Seiko Instruments Inc.) as follows. Crystallization temperature Tc: In a nitrogen atmosphere, when the temperature was increased from 20° C. to 230° C. at a temperature increase rate of 10° C./min, then maintained at 230° C. for 1 minute, and cooled at a temperature decrease rate of 10° C./min, a peak top temperature of crystallization observed was taken as the crystallization temperature Tc. Melting point Tm of resin: In a nitrogen atmosphere, the temperature was increased from 20° C. to 280° C. at a temperature increase rate of 10° C./min, then maintained at the same temperature for 1 minute, cooled to 20° C. at a temperature decrease rate of 10° C./min, maintained at the same temperature for 1 minute, then increased at 10° C./min, and a peak top temperature of melting of the crystal observed was taken as the melting point Tm. Glass transition temperature Tg of resin: In a nitrogen atmosphere, the temperature was increased from 20° C. to 280° C. at a temperature increase rate of 10° C./min, then maintained at the same temperature for 1 minute, cooled to 20° C. at a temperature decrease rate of 10° C./min, maintained at the same temperature for 1 minute, then increased at 10° C./min, and the glass transition temperature Tg was measured during this temperature increasing process. Maximum Resin Concentration and Maximum Discharge Rate The water-soluble matrix and the resin in varying weight ratios were melt-kneaded with an extruder (“TEX30” available from The Japan Steel Works, Ltd.). The melt-kneaded product was extruded from a die and cooled, and then each kneaded product was washed with water, thereby determining a maximum resin concentration at which the resin was obtained in the form of resin particles (powder). If the maximum resin concentration is exceeded, the water solubility is extremely lowered, thereby forming oddly shaped resin particles, such as elongated particles, and aggregates of resin particles. Thus, the maximum resin concentration (a maximum concentration at which true spherical resin particles are generated) was determined by judging whether or not oddly shaped resin particles or aggregates of resin particles were formed. A composition corresponding to the maximum resin concentration (the composition containing the water-soluble matrix and the resin) was melt-kneaded with an extruder. The melt-kneaded product was extruded from a die in varying discharge rates and cooled, and then each kneaded product was washed with water, thereby determining a maximum discharge rate at which the resin is obtained in the form of resin particles (powder). If the maximum discharge rate is exceeded, oddly shaped resin particles, such as elongated particles, and aggregates of resin particles are formed. Carboxyl Group Concentration and Amino Group Concentration The terminal carboxyl group concentration (COOH group concentration) and the terminal amino group concentration (NH2concentration) were measured for the polyamide-based resin as the thermoplastic resin and the resulting polyamide-based resin particles by the following titration method. Carboxyl group concentration: A sample of the polyamide resin was dissolved in benzyl alcohol to prepare a 1 wt. % benzyl alcohol solution, and the carboxyl group concentration was measured by neutralization titration with 1/100 N KOH aqueous solution. Amino group concentration: A sample of the polyamide resin was dissolved in a mixed solvent of phenol and ethanol at a volume ratio of 10:1 to prepare a 1 wt. % solution, and the amino group concentration was measured by neutralization titration with 1/100 N HCl aqueous solution. Example 1 A composition containing the modified PVA-based resin 2 and the polyamide 12-based resin (A) at a ratio corresponding to a maximum resin concentration of 49 wt. % (the modified PVA-based resin 2=51 parts by weight and the resin (A)=49 parts by weight) was melt-kneaded (at a cylinder temperature of 230° C.) with an extruder (“TEX30” available from The Japan Steel Works, Ltd.). When extruded from a die, the melt-kneaded product could be discharged at a maximum discharge rate of 30 kg/h. The extruded melt-kneaded product was cooled and cut, and a pre-molded article in the form of pellets was obtained. This pre-molded article was added to water and stirred to elute the water-soluble matrix. The formed resin particles were filtered with a glass filter and collected, then dried naturally at a temperature of 23° C. and a humidity of 50% RH, and polyamide resin particles (powder) were obtained. The resulting polyamide resin particles were true spherical, and the average particle diameter was 1.3 μm. Example 2 When a composition was melt-kneaded and the melt-kneaded product was extruded from a die in the same manner as in Example 1 with the exception that the polyamide 12-based resin (B) was used in place of the resin (A) of Example 1, spherical polyamide resin particles having an average particle diameter of 22.8 μm were obtained at a maximum resin concentration of 49 wt. % and a maximum discharge rate of 30 kg/h. Example 3 When a composition was melt-kneaded and the melt-kneaded product was extruded from a die in the same manner as in Example 1 with the exception that the polyamide 1010-based resin (C) was used in place of the resin (A) of Example 1 and the melt-kneading was performed at 240° C. (a cylinder temperature of 240° C.), spherical polyamide resin particles having an average particle diameter of 12.0 μm were obtained at a maximum resin concentration of 49 wt. % and a maximum discharge rate of 30 kg/h. Example 4 When a composition was melt-kneaded and the melt-kneaded product was extruded from a die in the same manner as in Example 1 with the exception that the alicyclic polyamide resin (D) was used in place of the resin (A) of Example 1 and the melt-kneading was performed at 270° C. (a cylinder temperature of 270° C.), spherical polyamide resin particles having an average particle diameter of 9.8 μm were obtained at a maximum resin concentration of 47 wt. % and a maximum discharge rate of 30 kg/h. Example 5 When a composition was melt-kneaded and the melt-kneaded product was extruded from a die in the same manner as in Example 1 with the exception that the polyamide 12-based resin (E) was used in place of the resin (A) of Example 1, spherical polyamide resin particles having an average particle diameter of 5.7 μm were obtained at a maximum resin concentration of 45 wt. % and a maximum discharge rate of 30 kg/h. Comparative Example 1 Polyamide resin particles were produced in the same manner as in Example 1 with the exception that the polyethylene glycol (PEG) was used in place of the water-soluble matrix of Example 1, and the polyethylene glycol (PEG) and the alicyclic polyamide resin (D) were used at a ratio of 51 parts by weight of the former and 49 parts by weight of the latter. A crystallization temperature Tc of polyethylene glycol was 42° C., and a melt kneaded product was discharged from a die of the extruder at a discharge rate of 25 kg/h, but it became a rice cake-like mass, which was difficult to be collected. In addition, the resulting polyamide resin particles were oddly shaped (thready), and thus the average particle diameter was not able to be accurately measured. Comparative Example 2 A maximum resin concentration of the alicyclic polyamide-based resin (D) was 42 wt. % as examined in the same manner as in Example 1 with the exception that the polyethylene glycol (PEG) was used in place of the water-soluble matrix of Example 1. Thus, polyamide resin particles were produced in the same manner as in Example 2 at a ratio of the resin (D) and polyethylene glycol (PEG) of the former/the latter=42 parts by weight/58 parts by weight. As a result, a melt-kneaded product in a rice cake-like form was discharged from a die of the extruder at a maximum discharge rate of 10 kg/h. The resulting polyamide resin particles were true spherical, and the average particle diameter was 3 μm. Comparative Example 3 A water-soluble matrix prepared by melt-mixing 80 parts by weight of the oligosaccharide and 20 parts by weight of the sugar alcohol was used in place of the water-soluble matrix of Example 1, and the polyamide 12-based resin (F) was used in place of the polyamide 12-based resin (A) of Example 1. A crystallization temperature Tc of the above water-soluble matrix (an oligosaccharide composition) was not measurable. A maximum resin concentration was 33 wt. % as examined in the same manner as in Example 1 with the exception that the water-soluble matrix (the oligosaccharide composition) and the polyamide 12-based resin (F) were used, and the melt-kneading temperature was reduced to 230° C. (a cylinder temperature of 230° C.). Then, in correspondence with the maximum resin concentration of 33 wt. %, 67 parts by weight of the water-soluble matrix (53.6 parts by weight of the oligosaccharide and 13.4 parts by weight of the sugar alcohol) and 33 parts by weight of the resin (F) were melt-kneaded (at a cylinder temperature of 230° C.) with the above extruder. When extruded from a die as in the same manner in Example 2, the melt-kneaded product could be discharged in the form of flakes at a maximum discharge rate of 15 kg/h. The resulting polyamide resin particles were true spherical, and the average particle diameter was 3 μm. Comparative Example 4 When 75 parts by weight of the water-soluble matrix (60 parts by weight of the oligosaccharide and 15 parts by weight of the sugar alcohol) and 25 parts by weight of the alicyclic polyamide-based resin (D) were melt-kneaded (at a cylinder temperature of 270° C.) and extruded in the same manner as in Example 1, the water-soluble matrix was thermally decomposed, and the melt-kneaded product failed to be extruded from a die. That is, the water-soluble matrix containing the oligosaccharide and the sugar alcohol failed to withstand the kneading temperature for the resin (D) (the cylinder temperature of 270° C.), and the resin particles of the resin (D) failed to be produced. The results of Examples 1 to 5 and Comparative Examples 1 to 3 are shown in Table 1 except for Comparative Example 4 in which the water-soluble matrix was decomposed. TABLE 1ExamplesComparative Examples12345123Water-Modified PVA-based100solubleresin 1componentModified PVA-based100100100100resin 2Sugar alcohol20PEG100100Oligosaccharide80Crystallization15215215215215242Nottemperature Tc (° C.)measurableResinType of resin(A)(B)(C)(D)(E)(D)(D)(F)Melting point Tm (° C.)178178192247178247247178COOH group114537267868967130concentrationNH2group1173145519414concentrationMaximum resin concentration4949494745494233(wt. %)Form of extrudatePelletsPelletsPelletsPelletsPelletsRice cake-RiceFlakeslike masscake-likeMaximum discharge rate (kg/h)3030303030251015ResinShapeTrueTrueTrueTrueTrueThreadyTrueTrueparticlessphericalsphericalsphericalsphericalspherical(oddlysphericalsphericalshaped)Average particle size1.322.8129.85.7Not33(μm)measurableCOOH group113517166889089128concentrationNH2group1183143317193concentration In the table, units of the terminal carboxyl group concentration (COOH group concentration) and the terminal amino group concentration (NH2concentration) are mmol/kg. As is clear from the results shown in Table 1, in the examples, polyamide-based resins, which are even polyamide-based resins having a high glass transition temperature, can be melt-kneaded at a high concentration and can be discharged at a high resin concentration and a large discharge rate, achieving high productivity of the spherical resin particles. In addition, the pre-molded article can be obtained in the form of pellets, and thus the handleability thereof and the productivity of the resin particles can be improved. In particular, unlike Comparative Examples 1, the resin particles can be produced without reducing the functional group (particularly the amino group) concentration of the polyamide-based resin. INDUSTRIAL APPLICABILITY The resin particles according to an embodiment of the present invention can be used widely in fields of, for example, cosmetics, coating agents, paints, and molding materials. For example, the resin particles can be used as light diffusing agents, matting agents, lubricants, anti-blocking agents, cosmetics, light blocking agents, toners, fillers, ceramics void forming materials, reinforcing agents of fiber-reinforced composite materials, and the like, according to the type of resin, the average particle size, and the like. For example, resin particles having an average particle size of 5 μm or less (for example, approximately from 0.1 to 3 μm) can be used in the fields of cosmetics, coating agents, paints, molding materials, and the like, resin particles having an average particle size of greater than 5 μm, for example, approximately from 6 to 40 μm (for example, from 10 to 30 μm and preferably from 15 to 25 μm) may be used in the field of fiber-reinforced composite materials (particularly materials containing an epoxy resin), and resin particles having an average particle size approximately from 30 to 100 μm (for example, from 50 to 75 μm) may be used in the field of modeling with a 3D printer or the like. In addition, if a predetermined amount (for example, approximately from 0.1 to 15 volume % and preferably from 0.5 to 5 volume %) of the resin particles (for example, such as crystalline aliphatic or alicyclic polyamide-based resin particles having the moisture content and the crystallinity described above) is added or impregnated as a reinforcing agent to a composition (or a prepreg) for a fiber-reinforced composite material containing an epoxy resin (such as a bisphenol A type epoxy resin), a thermosetting resin, such as a vinyl ester resin, and reinforcing fibers, such as carbon fibers and glass fibers (including fabric reinforcing fibers), and molded by a molding method, such as a hand lay-up molding method, to form a fiber-reinforced composite material FRP, a toughness of a cured product (including an interlaminar toughness of the FRP) can be improved, and the reinforcing effect of the reinforcing fibers (particularly carbon fibers) can be improved. The prepreg may contain a curing agent, such as an aromatic amine-based curing agent, and a curing accelerator, such as a phosphine and a tertiary amine. Molded articles formed of the cured product of the composition (or the prepreg) may be exemplified by structural members (structural materials) in various fields of, for example, vehicles (for example, such as airplanes, helicopters, rockets, automobiles, bikes, bicycles, trains, ships, and wheelchairs), artificial satellites, windmills, sporting goods (golf shafts and tennis rackets), housings (such as housings of notebook personal computers), molded products in the medical field (such as artificial bones), IC trays, fishing rods, and bridge piers. | 81,546 |
11859059 | EXAMPLES Example 1 A superabsorbent was produced analogously to example 1 of WO 2016/134905 A1. The monomer solution used additionally comprised 1.07% by weight of the disodium salt of 1-hydroxyethylidene-1,1-diphosphonic acid. The gas inlet temperature of reaction zone (5) was 167° C., the gas outlet temperature of reaction zone (5) was 107° C., the gas inlet temperature of the internal fluidized bed (27) was 100° C., the product temperature in the internal fluidized bed (27) was 78° C., the gas outlet temperature of the condensation column (12) was 57° C., and the gas outlet temperature of the gas drying unit (37) was 47° C. The superabsorbent produced (base polymer) had a bulk density (ASG) of 0.73 g/ml, a centrifuge retention capacity (CRC) of 49.4 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 10.5 g/g, a residual monomer content of 5200 ppm, an extractables content of 4.5% by weight and a moisture content of 8.0% by weight. The superabsorbent had the following particle size distribution: >1000 μm0.3% by weight850-1000 μm1.1% by weight600-850 μm3.7% by weight500-600 μm9.9% by weight400-500 μm32.8% by weight300-400 μm40.4% by weight250-300 μm6.4% by weight200-250 μm4.1% by weight106-200 μm1.2% by weight<106 μm0.1% by weight The superabsorbent had a median particle size (d50) of 377 μm and an average sphericity (ASPHT) of 0.81. The base polymer was subsequently surface postcrosslinked analogously to examples 11 to 15 of WO 2015/110321 A1. 2.0% by weight of ethylene carbonate, 5.0% by weight of water and 0.3% by weight of aluminum sulfate were used, based in each case on the base polymer. The product temperature was 160° C. and the height of the weir was 75%. In the cooler, after the surface postcrosslinking, first 2.35% by weight of a 0.2% by weight aqueous solution of sorbitan monolaurate and then 2.35% by weight of a dilute aqueous polymer dispersion of Poligen® CE 18 (BASF SE; Ludwigshafen; Germany) were added. Poligen® CE 18 is a 21% by weight aqueous wax dispersion of an ethylene-acrylic acid copolymer composed of 20% by weight of acrylic acid and 80% by weight of ethylene, stabilized with potassium hydroxide. The wax has a glass transition temperature of 80° C. The dilute aqueous polymer dispersion was calculated such that 500 ppm of wax was added in solid form, based on the superabsorbent particle on the polymer. The temperature of the superabsorbent particles at the time of addition was 75° C. The surface postcrosslinked superabsorbent produced had a bulk density (ASG) of 0.794 g/ml, a centrifuge retention capacity (CRC) of 40.1 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 32.9 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 23.3 g/g, a saline flow conductivity (SFC) of 5×10−7cm3s/g, a vortex of 68 s, a moisture content of 3.2% by weight, a residual monomer content of 399 ppm and an extractables content of 3.2% by weight. The surface postcrosslinked superabsorbent had the following particle size distribution: >850 μm0.0% by weight710-850 μm0.4% by weight600-710 μm2.2% by weight500-600 μm9.0% by weight400-500 μm36.4% by weight300-400 μm39.2% by weight250-300 μm7.0% by weight200-250 μm4.0% by weight150-200 μm1.5% by weight<150 μm0.2% by weight The superabsorbent had a median particle size (d50) of 379 μm and an average sphericity (ASPHT) of 0.80. Subsequently, the superabsorbent particles were pneumatically conveyed. The conveying conduit used was a smooth pipeline of aluminum having a length of 164 m and an internal diameter of 100 mm. The conveying conduit consisted of two horizontal and two vertical sections, with the sections connected by bends. The total vertical height gain was 13 m. The conveying conduit had an internal bypass of the Intraflow type (Zeppelin Systems GmbH; Friedrichshafen; Germany). The product was conveyed into the conveying conduit by means of a CFH250 star feeder (Zeppelin Systems GmbH; Friedrichshafen; Germany). The conveying output was 7.5 t/h of superabsorbent particles, the speed of the star feeder was 13.5 rpm, the conveying air rate was 560 m3(STP)/h, and the gas velocity was 11 m/s at the start of the conveying conduit and 11.1 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from +660 to 0 mbar, based on the ambient pressure. During the stable conveying, the pressure fluctuations were ±50 mbar and the average pressure in the conveying was 560 mbar. The conveying material load was 11 kg/kg, and the Froude number at the start of the conveying was 11. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 39.5 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 31.1 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 23.1 g/g, a saline flow conductivity (SFC) of 4×10−7cm3s/g. The starting pressure in the conveying conduit as a function of time is shown inFIG.2. The number of dust particles of example 1 before and after the pneumatic conveying is collated in table 1. Example 2 (Comparative Example) The procedure was as in example 1. The amount of Poligen® CE 18 added was lowered to 125 ppm, based on the superabsorbent particles. The surface postcrosslinked superabsorbent produced had a bulk density (ASG) of 0.761 g/ml, a centrifuge retention capacity (CRC) of 39.3 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 32.7 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 23.1 g/g, a saline flow conductivity (SFC) of 5×10−7cm3s/g, a vortex of 65 s, a moisture content of 3.1% by weight, a residual monomer content of 428 ppm and an extractables content of 3.0% by weight. The surface postcrosslinked superabsorbent had the following particle size distribution: >850 μm0.0% by weight710-850 μm0.4% by weight600-710 μm2.9% by weight500-600 μm8.6% by weight400-500 μm36.4% by weight300-400 μm39.5% by weight250-300 μm6.8% by weight200-250 μm3.8% by weight150-200 μm1.1% by weight<150 μm0.3% by weight The superabsorbent had a median particle size (d50) of 380 μm and an average sphericity (ASPHT) of 0.80. The conveying output was 7.6 t/h of superabsorbent particles, the speed of the star feeder was 13.5 rpm, the conveying air rate was 560 m3(STP)/h, and the gas velocity was now 7.7 m/s at the start of the conveying conduit and 16.4 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from +1600 to 0 mbar, based on the ambient pressure. The conveying material load was 12 kg/kg, and the Froude number at the start of the conveying was 7.7. Uniform operation of the pneumatic conveying with a conveying air rate of 560 m3(STP)/h was not possible. During the unstable conveying, the pressure fluctuations were ±450 mbar and the average pressure in the conveying was 1120 mbar. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 39.3 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 30.7 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 21.6 g/g, a saline flow conductivity (SFC) of 3×10−7cm3s/g. The starting pressure in the conveying conduit as a function of time is shown inFIG.3. The number of dust particles of example 2 before and after the pneumatic conveying is collated in table 1. Example 3 (Comparative Example) The procedure was as in example 1. No Poligen® CE 18 was added. The surface postcrosslinked superabsorbent produced had a bulk density (ASG) of 0.78 g/ml, a centrifuge retention capacity (CRC) of 39.6 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 33.2 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 24.8 g/g, a saline flow conductivity (SFC) of 5×10−7cm3s/g, a vortex of 66 s, a moisture content of 3.5% by weight, a residual monomer content of 386 ppm and an extractables content of 3.0% by weight. The surface postcrosslinked superabsorbent had the following particle size distribution: >850 μm0.0% by weight710-850 μm0.4% by weight600-710 μm2.3% by weight500-600 μm10.0% by weight400-500 μm36.3% by weight300-400 μm39.4% by weight250-300 μm6.0% by weight200-250 μm4.1% by weight150-200 μm1.3% by weight<150 μm0.2% by weight The superabsorbent had a median particle size (d50) of 383 μm and an average sphericity (ASPHT) of 0.79. The conveying output was 7.2 t/h of superabsorbent particles, the speed of the star feeder was 13.5 rpm, the conveying air rate was 560 m3(STP)/h, and the gas velocity was now 7.5 m/s at the start of the conveying conduit and 16.4 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from 2400 to 0 mbar, based on the ambient pressure. During the conveying, the pressure fluctuations were ±1000 mbar. The conveying material load was 11.3 kg/kg, and the Froude number at the start of the conveying was 7.5. The pressure peaks during the conveying were clearly audible in the form of loud banging in the conduit. Uniform operation of the pneumatic conveying with a conveying air rate of 560 m3(STP)/h was not possible. During the very unstable conveying, the pressure fluctuations were ±900 mbar and the average pressure in the conveying was 1160 mbar. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 38.6 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 32.0 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 20.6 g/g, a saline flow conductivity (SFC) of 2×10−7cm3s/g. The starting pressure in the conveying conduit as a function of time is shown inFIG.4. The number of dust particles of example 3 before and after the pneumatic conveying is collated in table 1. Example 4 The base polymer from example 1 was subsequently surface postcrosslinked analogously to examples 11 to 15 of WO 2015/110321 A1. 2.0% by weight of ethylene carbonate, 5.0% by weight of water and 0.05% by weight of aluminum sulfate were used, based in each case on the base polymer. The product temperature was 159° C. and the height of the weir was 75%. In the cooler, after the surface postcrosslinking, first 4.35% by weight of a 0.23% by weight aqueous solution of aluminum lactate and then 1.66% by weight of a dilute aqueous polymer dispersion of Poligen® CE 18 (BASF SE; Ludwigshafen; Germany) and sorbitan monolaurate were added. The dilute aqueous polymer dispersion was calculated such that 500 ppm of wax in solid form and 25 ppm of sorbitan monolaurate were added, based on the superabsorbent particles. The surface postcrosslinked superabsorbent produced had a bulk density (ASG) of 0.75 g/ml, a centrifuge retention capacity (CRC) of 38.1 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 34.4 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 25.9 g/g, a saline flow conductivity (SFC) of 5×10−7cm3s/g, a vortex of 69 s, a moisture content of 4.5% by weight, a residual monomer content of 263 ppm and an extractables content of 2.6% by weight. The surface postcrosslinked superabsorbent had the following particle size distribution: >850 μm0.0% by weight710-850 μm0.1% by weight600-710 μm2.5% by weight500-600 μm12.5% by weight400-500 μm38.2% by weight300-400 μm37.4% by weight250-300 μm5.0% by weight200-250 μm3.0% by weight150-200 μm0.6% by weight<150 μm0.2% by weight The superabsorbent had a median particle size (d50) of 396 μm and an average sphericity (ASPHT) of 0.80. The superabsorbent particles thus obtained were pneumatically conveyed under different conditions (examples 4a to 4d). Example 4 a The conveying output in the first conveying operation was 7.2 t/h of superabsorbent particles, the speed of the star feeder was 13.5 rpm, the conveying air rate was 550 m3(STP)/h, and the gas velocity was now 10.7 m/s at the start of the conveying conduit and 17.3 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from 740 to 0 mbar, based on the ambient pressure. The conveying material load was 10.9 kg/kg, and the Froude number at the start of the conveying was 11. During the stable conveying, the pressure fluctuations were ±50 mbar and the average pressure in the conveying was 580 mbar. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 38.7 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 34.2 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 26.2 g/g, a saline flow conductivity (SFC) of 5×10−7cm3s/g. The number of dust particles of example 4a before and after the pneumatic conveying is collated in table 1. Example 4 b The conveying output in the subsequent second conveying operation was 11.2 t/h of superabsorbent particles, the speed of the star feeder was 20 rpm, the conveying air rate was 660 m3(STP)/h, and the gas velocity was now 11.0 m/s at the start of the conveying conduit and 20.4 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from 910 to 0 mbar, based on the ambient pressure. During the conveying, the pressure fluctuations were ±50 mbar. The conveying material load was 14.2 kg/kg, and the Froude number at the start of the conveying was 11. During the stable conveying, the pressure fluctuations were ±50 mbar and the average pressure in the conveying was 825 mbar. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 38.6 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 33.6 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 25.3 g/g, a saline flow conductivity (SFC) of 4×10−7cm3s/g. Example 4 c The conveying output in the subsequent third conveying operation was 16.4 t/h of superabsorbent particles, the speed of the star feeder was 30 rpm, the conveying air rate was 560 m3(STP)/h, and the gas velocity was now 10.6 m/s at the start of the conveying conduit and 23.1 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from 1230 to 0 mbar, based on the ambient pressure. During the conveying, the pressure fluctuations were ±50 mbar. The conveying material load was 18.3 kg/kg, and the Froude number at the start of the conveying was 10. During the stable conveying, the pressure fluctuations were ±50 mbar and the average pressure in the conveying was 1160 mbar. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 38.6 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 33.0 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 24.2 g/g, a saline flow conductivity (SFC) of 4×10−7cm3s/g. Example 4 d The conveying output in the subsequent fourth conveying operation was 20.8 t/h of superabsorbent particles, the speed of the star feeder was 40 rpm, the conveying air rate was 770 m3(STP)/h, and the gas velocity was now 9.1 m/s at the start of the conveying conduit and 23.2 m/s at the end of the conveying conduit. The pressure in the conveying conduit was from 1800 to 0 mbar, based on the ambient pressure. During the conveying, the pressure fluctuations were ±50 mbar. The conveying material load was 23.1 kg/kg, and the Froude number at the start of the conveying was 9. During the stable conveying, the pressure fluctuations were ±200 mbar and the average pressure in the conveying was 1500 mbar. After the conveying, the superabsorbent had a centrifuge retention capacity (CRC) of 38.6 g/g, an absorption under a pressure of 21.0 g/cm2(AUL) of 32.8 g/g, an absorption under a pressure of 49.2 g/cm2(AUHL) of 23.9 g/g, a saline flow conductivity (SFC) of 3×10−7cm3s/g. The starting pressure in the conveying conduit as a function of time in examples 4a to 4d is shown inFIG.5. TABLE 1Normalized number of particles P discharged from the Heubachdustmeter in 5 l/min of dry air, normalized per min andper g of superabsorbent, classified and as sum totalNormalized particleExam-number [P/min/g]pleSample<1 μm1 μm-10 μm>10 μmSum total1before conveying601541189153189182258after conveying8152515164742322374042*)before conveying1265431974213068327032after conveying26643335387760276263373*)before conveying4195574420393511865107after conveying682969602259437712896054abefore conveying27042712932231100566after conveying526881144165455172559*)comparative example | 16,139 |
11859060 | DETAILED DESCRIPTION Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing. Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, known processes or well-known apparatus or structures, and well known techniques are not described in detail. The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure are not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed. Conventional methods of dyeing the film involve the incorporation of mixtures of blue, red, and yellow dyes in the polyester matrix during extrusion. However, in practice, it is not feasible to produce small quantities of colored films by using this method, as colors cannot be changed in the production runs in a short time. Another method involving waterless dyeing or solvent-assisted dyeing. In an aspect of the present disclosure, there is provided a process for dyeing a hydrolysis resistant polyester film. The process is described in detail. In a first step, a hydrolysis resistant polyester film is subjected to dyeing in a dye bath comprising at least one coloring agent (dye), at least one polyhydric alcohol, and optionally at least one UV absorber, at a temperature in the range of 140° C. to 190° C. to obtain a dyed film. In an embodiment, the hydrolysis resistant polyester film is dyed for a time period in the range of 10 seconds to 120 seconds to obtain a dyed film. In an exemplary embodiment, the hydrolysis resistant polyester film is dyed for 60 seconds. In accordance with an embodiment of the present disclosure, the thickness of the hydrolysis resistant polyester film prior to dyeing is in the range of 12 μm to 150 μm. In accordance with an embodiment of the present disclosure, the coloring agent is a fast dye comprising blue color, yellow colour, and red colour. In an exemplary embodiment, the coloring agent is disperse blue56. In accordance with an embodiment of the present disclosure, the polyhydric alcohol is at least one selected from monoethylene glycol, diethylene glycol, propylene glycol, glyceraldehyde, and polyethylene glycol. In an exemplary embodiment, the polyhydric alcohol is monoethylene glycol. In accordance with an embodiment of the present disclosure, the UV absorber is at least one selected from 2-hydroxybenzophenone, 2-hydroxybenzotriazole, organonickel compound, salicylic ester, cinnamic ester derivative, resorcinol monobenzoate, oxanilide, hydroxybenzoic ester, benzoxazinone, sterically hindered amine, and triazine. In accordance with an embodiment of the present disclosure, the hydrolysis resistant polyester film is passed through the dye bath at a speed in the range of 5 meters/min to 80 meters/min. In an exemplary embodiment, the speed is 50 meters/min. Lower dyeing time is required for making lighter tints, whereas the process can run at higher speeds. Higher dyeing time is required for the production of darker tints. Dye uptake is directly proportional to the dye bath concentration and the dyeing time. The dye bath of the present disclosure is equipped with SS Tank, supporting roller, and winder for passing the hydrolysis resistant polyester film through the bath. The dye bath is further equipped with an individual colour dispenser to maintain the color composition of the dye bath and a second doser to maintain the fluid medium concentration of the bath. The polyester film is passed through the dye bath with the help of the rollers, which are in turn mounted in the dye bath in such a way that the film is completely immersed in the dye bath throughout the dyeing step. After attaining the desired level of the UV stabilization and the color depth, the dyed films are monitored for UV absorbance, and the film is processed till it reaches the absorbance of >95% at 355 nm, preferably above >98% at 355 nm and is further passed to the next step. In a second step, the dyed film is subjected to quenching in a first fluid medium to obtain a quenched film. In accordance with an embodiment of the present disclosure, the first fluid medium is at least one selected from chilled demineralised water, polyhydric alcohol, diethylene glycol, and triethylene glycol. In an exemplary embodiment of the present disclosure, the first fluid medium is chilled demineralised water. The quenching step is required to precipitate the dissolved additives such as undissolved free dust, dyes, UV absorber, and polyester oligomers in the dye bath. The film surface has a thin layer of solvent along with undissolved dust, UV absorber, dissolved dyes, and polyester oligomers. The film is quenched with the first fluid medium that precipitates the dissolved additives onto the film surface. The precipitated free dust is removed by a high velocity solvent spray or an ultrasonic energy. The temperature of the chilled water is maintained between −5° and 10° C. The film surface has a thin layer of the solvent along with undissolved free dust, UV stabilizer, and polyester oligomers. When the film is suddenly quenched in the chilled water, the dissolved additives are precipitated onto the film surface. Thus precipitated free dust can be removed by a high-velocity solvent spray. Ultrasonic energy can also be applied to remove particles from the film. In a third step, the quenched film is cleaned with a second fluid medium followed by scrubbing and rinsing to obtain a cleaned film. In accordance with an embodiment of the present disclosure, the second fluid medium is selected from dimethylformamide, santosol, benzoyl alcohol, 2-vinyl-pyrrolidone, dimethyl sulfoxide, and dimethylacetamide. In an exemplary embodiment, the second fluid medium is dimethyl formamide. The second fluid medium used for cleaning the dyed hydrolysis resistant polyester film has a boiling point in the range of 110° C. to 220° C. The second fluid medium with a boiling point above 110° C. is preferred because the chilled water carried along with the film will accumulate in the fluid media. After a certain frequency, the same can be separated from this fluid media by distillation. Solvents with higher boiling point are preferred since they posses' higher solvation power and offers process safety. Since the film is continuously passing through the fluid media, some quantity of unwanted surface contamination affects the purity of the fluid medium in the tank and therefore it is necessary to remove some quantity of used solvents at predetermined intervals from the tank and simultaneously add an equivalent quantity of pure fluid medium into the tank. The distillation of the used fluid medium is carried out under a high vacuum and at relatively low temperatures and the distilled fraction of used fluid medium is recycled for washing. The residual dye along with fluid medium in the distillation vessel can be recycled. The cleaning by using the second fluid medium is followed by scrubbing the cleaned film and rinsing it with water to obtain a cleaned film. The scrubber assembly is provided with a self-cleaning device in which a high-pressure jet is provided before squeezing the film between nip rollers. A set of eight rollers is mounted on the scrubbing tank, out of which the four rollers are mounted on the top side of the film and remaining four rollers are mounted on the opposite side of the film. Each roller is wound with cloth and lapping movement is also provided to minimize the pressure of the scrubbing material on the film, depending on the type and thickness of the film to be cleaned. In an embodiment, water in the process is continuously recycled and filtered through 0.3 micron cartridge filters. The dyed hydrolysis resistant polyester film is passed through a specially designed tenter chain. The tenter having two parallel chain tracks is provided with digital width indications mounted across the length of the chain to monitor width at various locations. The tenter chain plays a major role in monitoring the shrinkage property of the dyed hydrolysis resistant polyester film. The desired shrinkage values can be monitored by adjusting the width of the web from the entrance and exit ends of the tenter. The cleaned film passes through the tenter where the specially designed clips hold the film tightly in the lower and upper jaws. The jaws are provided with a soft rubber lining, which helps in holding the film tightly in the jaws. In a final step, the cleaned film is subjected to drying in an oven at a temperature in the range of 50° C. to 250° C. In an embodiment, the cleaned film is fed into the oven at temperatures maintained between 100° C. and 200° C. and at a speed of 20 to 80 meters/min. In an embodiment, the cleaned film is dried for a time period in the range of 10 seconds to 70 seconds. In an exemplary embodiment, the cleaned film is dried for 30 seconds. The film can be allowed to shrink in the oven or can be stretched in the oven in the transverse or machine direction. In the first zone, the film is allowed to shrink between 0 and 150 mm across the transverse direction, preferably between 2 mm and 50 mm. The films are stretched in the second zone and the third zone between 0 and 300 mm. The films are stretched in the second zone and the third zone is allowed to stabilize and relax in the fourth zone. The film produced in the tenter process has excellent shrinkage properties. The shrinkage values in the machine direction (MD) and traverse direction (TD) are controlled consistently lot to lot and roll to roll to obtain a dimensionally stabilized film. This dimensionally stabilized film is then cooled with cold air jets. The dyed film is finally wound on a paper or metal core of a winder. The dyed hydrolysis resistant polyester film obtained by using the process of the present disclosure receives an accelerated weathering test and tests for sun control capability, light fastness, and thermal properties. Carboxyl end-groups present in the polyester molecule are primarily responsible for the hydrolytic degradation of polyesters, including polyethylene terephthalate. The polyester resin degrades during the extrusion process followed by an increase in —COOH end groups. Generally, the —COOH content of the polyester resin is reduced by passing through the solid-state polymerization process. However, the polyester resin having —COOH end groups lower than ten that typically has a very high IV (inherent viscosity) that makes processing and extrusion difficult because of processing issues such as high pressure at filters and high torque at extruders. In an embodiment, the hydrolysis resistant polyester film comprises polyester resin, hydrolysis restricting stabilizer, UV stabilizer, and anti-oxidant. In an embodiment, the polyester resin present in an amount in the range of 80 wt % to 97 wt % based on the total weight of hydrolysis resistant polyester film. The polyester resin has an inherent viscosity (IV) of 0.65 to 0.75 wherein the inherent viscosity (IV) lower than 0.65 may result in a high —COOH content that leads to degradation which is too high, and the inherent viscosity (IV) higher than 0.75 is difficult to process into films. In the present disclosure, the incorporation of the hydrolysis restricting stabilizer into the polyester resin is carried out, which acts as an end-group capper by reacting with the carboxyl end-groups of the polyester. The hydrolysis resistant polyester film comprises at least one hydrolysis resistant stabilizer selected from the group consisting of carbodiimide compound and glycidyl ester of branched mono-carboxylic acid. Typically, the content of the hydrolysis stabilizer is in the range from 0.1 to 10.0% by weight, preferably in the range of 1.0 to 6.0% by weight, and more preferably in the range of 0.5 to 4.0% by weight, based on the weight of the film. In an embodiment, the carbodiimide compound is selected from the group consisting of dicyclohexyl carbodiimide, diisopropyl carbodiimide, di-isobutyl carbodiimide, dioctyl carbodiimide, octyl decyl carbodiimide, dibenzyl carbodiimide, diphenyl carbodiimide, N-benzyl-N-phenyl carbodiimide, di-p-toluyl carbodiimide, preferably bis(2,6 di isopropyl phenyl)carbodiimide and 2,6,2′,6′-tetra isopropyl diphenyl carbodiimide. In one embodiment, the hydrolysis resistant stabilizer can be directly added to the extruder during the production of the film. In one embodiment, the hydrolysis resistant stabilizer can also be introduced by way of a masterbatch technology. The masterbatch of Glycidyl ester of branched mono-carboxylic acid can be added to PET chip in the hopper of a twin-screw extruder provided with vacuum to remove moisture. The mixture can be melt-extruded to obtain the hydrolysis-resistant polyester film. The hydrolysis resistant stabilizer reacts with the polyester at an elevated temperature in the range of 140° C. and 300° C. The hydrolysis stabilizer can be introduced at various stages during the filmmaking process. The hydrolysis resistant film can further comprise additives such as anti-oxidant. The anti-oxidant is selected from the group consisting of peroxide-decomposing antioxidants, hindered phenols, secondary aromatic amines, and hindered amines. The peroxide-decomposing antioxidants is selected from the group consisting of trivalent phosphorous compounds, such as phosphonites, phosphites (e.g. triphenyl phosphate and trialkylphosphites), and thiosynergists (e.g. esters of thiodipropionic acid, such as dilauryl thiodipropionate). The hindered phenol, such as tetrakis-(methylene 3-(4′-hydroxy-3′,5′-di-t-butylphenyl propionate) methane; pentaerythritol Tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate); octadecyl-3-(3,5-di-tert.butyl-4-hydroxyphenyl)-propionate; Ethylene bis (oxyethylene) bis(3-tert-butyl-4-hydroxy-5(methylhydrocinnamate); N,N′-Hexamethylene-bis (3,5-di-tert-butyl-4-hydroxyhyrocinnamamide); 3,5-Di-tert-butyl-4-hydroxyhydrocinnamic acid, C7-9 125643-61-0 branched alkyl esters; and bis-(1-Octyloxy-2,2,6,6,tetramethyl-4-piperidinyl) sebacate can be used. The concentration of the antioxidant present in the polyester film is in the range of 50 ppm to 5000 ppm, preferably in the range of 300 ppm to 1200 ppm, more preferably in the range from 450 ppm to 600 ppm. Further, optionally UV stabilizers can be added to the polyester film. UV stabilizers are the chemical compounds that can intervene in the physical and chemical processes of light-induced polymer degradation. The UV stabilizers have an extinction coefficient much higher than that of the polyester such that, most of the time UV light is absorbed by the UV stabilizers rather than the polyester. The UV stabilizers generally dissipate the absorbed energy as heat, thereby avoiding degradation of the polymer chain, and improving the stability of the polyester to UV light. UV stabilizer is at least one selected from the group consisting of 2-hydroxybenzophenones, 2-hydroxybenzotriazoles, organonickel compounds, salicylic esters, cinnamic ester derivatives, resorcinol monobenzoates, oxanilides, hydroxybenzoic esters, benzoxazinones, sterically hindered amines, and triazines, preferably 2-hydroxybenzotriazoles, benzoxazinones, and triazines. In an embodiment, the UV stabilizer is Cyasorb UV 3638, Tinuvin 1577, and their mixture thereof. The concentration of the UV stabilizers is in the range of 0.1 to 5.0% by weight with respect to the total weight of the film, preferably in the range of 0.5 to 3.0% by weight with respect to the total weight of the film. In an embodiment, the dyed hydrolysis resistant polyester film obtained by the process of the present disclosure is characterized by havinga) lightfastness for more than 2000 hours;b) thermal stability for 1000 hours;c) tensile strength greater than 40 kgf/cm2after 72 hours of autoclave;d) elongation at break of at least 40%; ande) a visible light transmittance in the range of 2% to 82%. The dyed hydrolysis resistant polyester film produced by the process of the present disclosure has a visible light transmittance as low as 2% and as high as 82%. The films can be produced in various shades. The concentration of various dyes in the dye bath and the temperature kept during dyeing decide the final color and transmission of the substrate. The speed of the process and the concentration of dyes in the dye bath determines the visible light transmission properties. The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure. The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The following experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial scale. EXPERIMENTAL DETAILS Example 1: Process for Dyeing Hydrolysis Resistant Polyester Films in Accordance with the Present Disclosure Hydrolysis resistant polyester films with a thickness of 12 μm, 23 μm, 36 μm, 50 μm and 100 μm (produced by Garware Hi-Tech Films Ltd) were used for dyeing hydrolysis resistant polyester films. The process was described herein below with the help ofFIG.1. The hydrolysis resistant polyester film10was placed on unwinder11. The polyester film was passed through a dye bath12at a speed of 50 meters/min, followed by squeezing between nip roller16of dyeing bath12to obtain a dyed film. The temperature of the dyeing bath12was maintained at 180° C. The dye bath comprises disperse blue56and monoethylene glycol. The hydrolysis resistant polyester film was dyed for 60 seconds in the dye bath. The shrinkage properties of the film were adjusted by stretching the film. MD (machine direction) stretching was done by monitoring differential speeds of nip rollers14and squeezing nip rollers16. The dyed film is quenched in chilled demineralized water maintained at 5° to 20° C. in quenching apparatus18, to obtain a quenched film. The quenched film was then passed through a cleaning apparatus20, and dimethyl formamide (fluid medium) was sprayed with high velocity on the film surface to remove excess chemicals and free dust adhered to the film. The fluid medium from the cleaning apparatus20was purified in a distillation unit21. Further, the cleaning of the dyed film took place in scrubbing tank24, wherein the film was passed through a mechanical scrubber including a set of 8 rollers (25aand25b). Each roller or bar was provided with a pneumatic cylinder to increase or decrease the abrasion pressure on the film. The water in the scrubbing tank24was mixed with non-ionic surfactant i.e., LISSAPOL-PA (soap solution) and continuously spread on the scrubbing rolls. The mechanical scrubber was a self-cleaning device provided with a high pressure jet27before squeezing the film in the nip rollers. Water was continuously recycled and filtered through 0.3 μm cartridge filters. The treated and cleaned dyed hydrolysis resistant polyester film was then passed through tenter28, as shown inFIG.2. The tenter had two parallel chain tracks. The tenter chain was further equipped with an oven having four separate zones, each zone was provided with hot air nozzles and showers mounted on the top and bottom sides of the film. Temperature and airflow were individually monitored. Further, the film was fed through the oven at temperatures of 100° C. to 200° C. in four zones for a period of 30 seconds. The film was allowed to shrink in a transverse direction in a first zone, width control in a second zone, heat stabilization in a third zone, and transverse direction in a fourth zone, followed by stabilization in the fourth zone. The stabilized film was allowed to cool by using high-velocity cold air jets to obtain the dyed hydrolysis resistant polyester film, which was finally wound on a metal core at rewinder46. Further, the roll produced in the above process was slit to the desired length and width. The dyed hydrolysis resistant film obtained in accordance with the process of the present disclosure has a uniform color and transmittance, throughout the length and width of the film (roll). The transmission variation across the web width is 2% and UV transmittance is below 2%. Characteristics of the dyed hydrolysis-resistant polyester film are given below in TABLE 1FilmOpticalVisibleThick-Oven Temperature ° C.ShrinkageDensityLightnessZoneZoneZoneZonein %atTransmit-in μIIIIIIIVTDMD355 nmtance23185185185180−0.421.820.536185185185180−0.421.820.3PET film width between 40″ to 72″MD = Machine DirectionTD = Transverse Direction It is observed that shrinkage, optical density, visible light transmittance properties of films with a thickness of 23 or 36 microns are similar. The films have uniform dye uptake across web width and length with good optical quality. Example 2 Hydrolysis resistant polyester films having a thickness of 23 and 36 μm were used for obtaining the dyed hydrolysis resistant polyester films. A similar process as described in Example 1 and shown inFIG.1was followed, except that there were modifications in the tenter and the oven settings. The temperature of the oven was set at 185° C. The results are given below in Table 2, with the film stretched in TD direction at various stretch ratios. TABLE 2Stretching23μ film Shrinkage %36μ film Shrinkage %Sr. No.RatioMDTDMDTD10.9972−0.42.2−0.221.0091.801.60.231.0181.60.21.20.6 It is observed that the shrinkage properties in the transverse direction can be adjusted by changing the stretching ratio. The shrinkage behaviour of films with a thickness of 23 or 36 microns is not the same at a given stretching ratio. Example 3: Pressure Cooker Test The pressure cooker test wherein controlled conditions of high temperature and high relative humidity was provided for accelerated conditions of aging, to evaluate the dyed hydrolysis resistant polyester film. The dyed hydrolysis resistant polyester film obtained in Examples 1 and 2 were cut in 10 mm width and length 150 mm. These samples were kept in a pressure cooker at a pressure of 1.0 kg/cm2and a temperature of 121° C. The mechanical properties of the dyed hydrolysis-resistant polyester film were evaluated at regular time intervals. The tensile strength of the dyed hydrolysis-resistant polyester film of the present disclosure (10 mm width samples) was determined according to ASTM D882 at a jaw separation rate of 300 mm/min. The tensile strength was determined according to ASTM D882 by using a material test machine (Instron model no 4411H), using mechanical grips with rubber jaw faces at a temperature 23° C. and relative humidity of 50%. The samples were evaluated for elongation at break of the polymer. The results are provided in table 3 below. TABLE 3Mechanical Property.InitialSr.after24 HRS48 HRS72 HRSNo.Film TypePropertyUnitdyeingAutoclaveAutoclaveAutoclave1Control-1*Tensilekgf/cm21008059BrittleStrengthElongation%1007856Brittleat break2Control-2**Tensilekgf/cm21009074BrittleStrengthElongation%1008410Brittleat break3HydrolysisTensilekgf/cm2100898069ResistanceStrength36μ FilmElongation%100897461at break4HydrolysisTensilekgf/cm2100957964ResistanceStrength100μ FilmElongation%100958444at breakInitial values are considered as 100% and retention values are calculated in terms of percentage.Residence time in Dye bath was 30 seconds at 175° C.Autoclave with temp 121° C. and pressure 1.0 psi.Samples were collected after a regular interval of 24 hours up to 96 hours for testing purpose.*Control-1:-23 micron polyester film manufactured by Garware Hi-Tech Films Ltd without UV and Hydrolysis stabilized.**Control-2:-36 micron polyester film manufactured by Garware Hi-Tech Films Ltd without UV and Hydrolysis stabilized. It is evident from the above table that, the dyeing of the hydrolysis resistant polyester films further enhances hydrolysis resistance as established by using the accelerated aging tests. Whereas the conventional films are brittle after exposure to humidity and temperature. The use of the hydrolysis resistant polyester film provides extended mechanical property retention when exposed to harsh environmental conditions. Therefore, the dyed hydrolysis resistant polyester film of the present disclosure has an improved long-term hydrolytic stability. Example 4: Thermal Stability The thermal stability of the dyed hydrolysis resistant polyester films of the present disclosure was determined according to ASTM D1204-94. The films were placed in an oven at 150° C. for 30 minutes and evaluated. The films are found to have good thermal aging properties for 1000 hrs. Example 5: Light Fastness The lightfastness of the dyed hydrolysis resistant polyester films of the present disclosure was determined by using the accelerated weathering tester of Atlas UV test and Xenon Arc Weatherometer (atlas company). The films were exposed continuously to alternate cycles of light and dark; and monitored for changes. The films are found to withstand exposure for more than 2000 hours. TECHNICAL ADVANCEMENTS The present disclosure described hereinabove has several technical advantages including, but not limited to, the realization of a process for dyeing a hydrolysis resistant polyester film that:is capable of providing the film with improved hydrolysis resistance and elasticity;maintains mechanical properties when exposed to harsh environmental conditions;is a simple and economical process of dying;produces the film in various shades; andproduces the film which has a visible light transmittance as low as 2% and as high as 82%. Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the formulation of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this invention. The numerical values given for various physical parameters, dimensions, and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary. While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation. | 29,225 |
11859061 | DETAILED DESCRIPTION Reference may be made in detail to exemplary embodiments of the present invention, some of which are illustrated, exemplified, and/or described herein. Other embodiments and means of carrying out the invention can be utilized, encompassing various structural, compositional, and/or functional changes known to those having skill in this field, without departing from the intended scope. As such, the following description is presented by way of illustration only and should not limit these alternatives and modifications in any way. As used herein, the words “example” and “exemplary” mean an instance or illustration but do not necessarily indicate a key or preferred aspect or embodiment. The word “or” is intended to be inclusive rather an exclusive, unless context suggests otherwise. As an example, the phrase “A employs B or C,” includes any inclusive permutation (e.g., A employs B; A employs C; or A employs both B and C). As another matter, the articles “a” and “an” are generally intended to mean “one or more” unless context suggest otherwise. As noted above, there is a need for a solid composition that can serve as a platform to create concentrates or masterbatch-type compositions that can used be with equal efficacy in low-melting thermoplastic formulations and in combination with engineered resin systems. Manufacturers would welcome a concentrate system that could be used for all their needs. As used herein, the term colorant or additive concentrate refers to a solidified resin-based carrier system, formed from the pre-mix disclosed below (which may include an optional liquid plasticizer), and subsequently introduced as a solid into any number of low or high temperature resin formulations. To that end, a pre-mix of an acrylate copolymer and a ring-opened polymeric cyclic ester or polymeric ether is created. Generally speaking, the acrylate comprises between 20 to 90 wt. % of the pre-mix, while the ring-opened component is provided at less than 30 wt. % of the pre-mix or in other embodiments at between 0.1 to 20 wt. %. The remainder includes the colorant and additive package, as well as an optional plasticizer that, when present, may form between 0.5 to 35 wt. % of the pre-mix. Particular utility has been found in acrylates made from ethylene butyl-, ethyl-, and methyl-acrylate copolymers. Any combination (or single one) of these acrylates may be employed, although ethyl-methyl acrylate (EMA) copolymers are preferred in certain aspects. Other acrylate copolymers may be used, so long as the resulting component(s) provide relatively high temperature stability (in comparison to the pre-mix's other components). Preferably, this acrylate component (or, in aggregate, combination of components) comprises at least 20 wt. %, at least 30 wt. %, at least 40 wt. %, at least 50 wt. %, at least 60 wt. %, at least 70 wt. %, or at least 80 wt. % of the total pre-mix. Conversely, these components should be no more than 90 wt. %, no more than 80 wt. %, no more than 70 wt. %, no more than 60 wt. %, no more than 50 wt. %, no more than 40 wt. %, or no more than 30 wt. % of the total pre-mix. Additional limitations and parameters are encompassed by the examples appended hereto, all of which form part of this written disclosure. Similarly, polycaprolactone and components based from the polycaprolactone ring structure are preferred as the ring-opened component. Polycaprolactone is particularly useful because of its broad Hansen interaction radius, wide availability, and relatively low cost (in comparison to other ring-opened polymeric esters and polymeric ethers. Certain substituted or functionalized derivatives of polycaprolactone and other cyclic ethers are also contemplated. While a nominal amount of ring-opened component(s) (i.e., at least 0.1 wt. % of the pre-mix) is needed, it should not exceed 30 wt. %. In further embodiments, maximum amounts of 1 wt. %, 5 wt. %, 10 wt. %, 15, 20, and 25 wt. % of polycaprolactone (or other aliphatic linear polyester or ring-opened components) are contemplated, as well as examples identified below. Notably, any of these stated intervals also can serve as the minimum end of an acceptable range. In some aspects, the aliphatic linear polyester might serve as a substitute for some or practically all of the acrylate (e.g., polylactide can replace ethyl-methyl acrylate). While polycarpolactone is expected to have particular utility, it may be possible to substitute or augment its use with certain polycaprolactone derivatives (i.e., a ring-opened cyclic ester or ether derivatives). As noted above, these derivatives may have certain functional groups introduced along the carpolactone ring or cyclic ether ring. Three, four, five, and six member ring structures may be preferred for their availability and cost. Some examples of derivatives could include: polyhydroxyalkonates, polyglycolide, polylactide, and copolymers of lactone and one or more additional monomers. Poly(butylene succinate) is another useful substitute/derivative expressly embraced within this class for purposes of this application. All of the above can be separately characterized as aliphatic linear polyester elements. Polyesters having a melting point of 150° to 160° C. or less (and meeting the other criteria set forth herein) are expected to have particular utility as aliphatic linear polyester elements. Thus, as used herein, “a ring-opened cyclic ester or ether derivative” and/or “aliphatic linear polyester elements” may embrace polymers of functionalized caprolactone and/or poly(butylene succinate), copolymers and one or more monomers, and/or polymers of those elements. In particular, the lactones of interest include ring structures containing 2, 3, 4, 5, or 6 carbons, with functional groups possibly appended to on e or more of these carbons. In certain embodiments, no functional groups are added to the lactone ring. When used, monomers for these derivatives are selected from branched and/or straight chain aliphatic structures having any whole number of carbon atoms between 1 to 20 within the structure. These base monomers may include any number of carboxylic or hydroxyl functional groups, as well as methyl, butyl, ethyl, and isopropyl structures (with or without carboxyl and/or hydroxyl functionalities). The functional groups for monomers can also serve preferred functional groups for polycaprolactone and/or lactone ring structures. One advantage of certain aliphatic linear polyester elements relates to biodegradability. That is, over time, these polymers can degrade into smaller molecules, such as carbon dioxide, water, nitrogen, etc. under aerobic or anaerobic conditions, usually brought on by action of enzymes, microorganisms, or catalysts. Polycaprolactone, polylactide, and poly(butylene succinate) are particularly well known in this regard. As government regulations and general concern for polymeric waste continue to evolve, biodegradable concentrates and additives are expected to have particular utility in formulations and various manufactured products. In some aspects, a plasticizer is provided to the pre-mix to wet the polymer surfaces, thereby lowering the processing temperatures required. For example, epoxidized soybean oil (ESO) can be added in an amount between 0.5 to 35 wt. % of the pre-mix, with additional minimum or maximum levels at 1.0 wt. %, 5 wt. %, 10 wt. %, 15 wt. %, 20 wt. %, 25 wt. %, and 30 wt. % also disclosed. Notably, while ESO and other plasticizers may be liquid when introduced during the manufacture of the pre-mix for the concentrate carrier system, the final concentrate carrier itself will be solid. When used, the ESO may be mixed directly into the pre-mix or additive package blend. In some embodiments, the pre-mix and additive package are combined, although a split stream process could be used to separately melt the polymers and the additive package prior to forming the concentrate. Within this context, it will be understood the plasticizer relates to the processing of the pre-mix, and any desired characteristics to be delivered to the final formulation in which the concentrate is used would be properly considered as part of the property enhancers in the additive package itself. However, formulators may also opt to use a plasticizer, including ESO, in the low or high melting resin formulations enabled by the inventive carrier system. That said, the additive package forms a significant aspect of the invention, insofar as the acrylate base and ring-opened component merely serve as a base resins carrier. Thus, within the confines of creating a stable, solid product, it is desirable to optimize and maximize the weight percentage of the additive package relative to the base resins carrier. In some embodiments, the additive package components comprise at least 0.1 wt. % and, more preferably between 45 wt. % to 55 wt. %, with the remainder of the mass of the pre-mix constituting base resins carrier (and plasticizer, to the extent a plasticizer is used). In some embodiments, the additive package approaches 80 wt. % of the total pre-mix. Additionally, the additive package may be 5 wt. %, 10 wt. %, 15 wt. %, 20, wt. %, 25 wt. %, 30 wt. %, 35 wt. %, 40 wt. %, 50 wt. %, 65 wt. %, 70 wt. %, and 75 wt. % the entire mass of the pre-mix. The additive package itself may include one, two, or all three of: colorants, property enhancers, and non-property fillers. Each are familiar to those of skill in the art, and it will be understood that the items used in the additive package must be compatible with one another without degrading the final, intended purpose of the concentrate (both as a colorant or additive vehicle, as well as remaining in solid form for its universal use in low and high temperature resin formulations). Colorants are expected to have particular utility in certain aspects of the invention, and this disclosure expressly contemplates embodiments where colorants are the sole aim of the additive package or where the pre-mix is optimized for colorants such that colorant is the majority component in the additive package with only small amounts of process modifiers and/or anti-oxidants (less than 10 wt. % and, more preferably, less than 5 wt. % of the mass of the additive package). Generally speaking, the colorants may be any combination of organic and inorganic pigments, dyes, alumina, mica, perlescent effects, laser markers, and/or metallocene polyethylene. To the extent these components are referenced or identified in any of the cited publications herein, such portions of those publications are incorporated by reference to further inform this disclosure. Additionally, specific examples are identified below, although these examples should not be taken as necessarily limiting this disclosure. Property enhancers impart specific characteristics to the final thermoplastic formulations (rather than the pre-mix or concentrate carrier system itself). Thus, to the extent a property enhancer is included in the additive package of any claimed or disclosed embodiment, those properties are delivered to the formulation into which the concentrate is introduced. The properties of interest generally include process modifiers and mold release agents, as well as biocides, UV and light stabilizers, thermal/heat stabilizers, anti-oxidants, radical scavengers, acid scavengers and anti-static or conductive fillers. Combinations of these property enhancers can be included in any given concentrate formulation according to some aspects of the invention. As with the colorants, certain non-limiting examples are provided below. Finally, non-property fillers can be used in the additive package. These fillers are not intended to alter the appearance or otherwise impart specific properties to the concentrate/final resin. Thus, unlike the colorants and property enhancers, these non-property fillers are intended to facilitate the formulation of the concentrate carrier system itself. Examples of non-property fillers appropriate to the invention include calcium carbonate, clays, silica, talcum powder, rice husk ash, and certain other, non-reactive types of ash. Reasons for relying on such non-property fillers may be related to controlling costs, improving the manufacture/processing of the carrier system, and/or ensuring the concentrate is sufficiently solid. In one aspect of the invention, the additive package includes a small amount of metallocene polyethylene for facilitating the processing of certain portions of the additive package itself (in this context, the metallocene could be characterized as a non-property filler). The components of the additive package are pre-compounded in a twin screw extruder before being recombined downstream with the pre-mix polymer/plasticizer blend. In another embodiment, the additive package is mixed dry with the pre-mix polymers and plasticizer(s) and then coagulated in a melt to form the concentrate. The additive package itself may consistent entirely of colorants. In other aspects, colorant(s) form the majority of the package, by weight percentage (i.e., at least 50 wt. %). Property enhancers and/or non property fillers may be added to the colorant(s). In some instances, the non-property fillers may constitute a majority of the package. Property enhancers will typically constitute no greater than 50 wt. % of the package. In preferred embodiments, colorants constitute at least 2.5 wt. %, at least 25.0% wt. %, and up to 95.8 wt. % of the additive package. When provided, non-proprety fillers may be anywhere from 50.0 to 60.0 wt. % of the package. Property enhancers may be from 2.0 wt. % up to 25.0 wt. % of the additive package. In another aspect, the pre-mix polymers, plasticizer(s), and additive package will be combined on a two-roll mill, compounder, single- or twin-screw extruder, or Farrel continuous mixer. Combinations of these mixing approaches can also be employed. After mixing, the mixture is then run through a die or a shower head for pelletizing, or taken as a ribbon to be diced. In this regard, the invention includes methods of making the carrier system, as well as formulations for that system. Still other aspects of the invention relate to the subsequent use of the carrier system in combination with low or high melt processing resins, as noted above. As described, concentrates (including the additive package) formed in this manner provide advantages in comparison to existing so-called “universal” or multifunctional concentrates. In particular, the inventive concentrate can be incorporated into low-temperature resins, such as moisture-cure XLPE, while also being suitable for high temperature engineering resins, especially PC, ABS, and/or Nylon 6. U.S. Pat. No. 7,442,742 describes a masterbatch composition relying on a metallocene polymer, while U.S. Pat. No. 9,969,881 and a currently copending continuing application (filed on Apr. 13, 2018 as U.S. Ser. No. 15/952,926 and now published as United States Patent Publication 2018/0258237) describe a split stream process for making such compositions. Certain aspects of these disclosures, including the formulations and methods of manufacture, may further inform aspects of the invention. As such, these documents are incorporated by reference in their entirety herein. Finally, a range of publications describe polymer blends that might be particularly useful when employed in combination with certain aspects of the invention. These documents include: U.S. Pat. Nos. 3,459,834; 3,524,906; 4,320,212; 4,404,248; and 4,908,397, as well as German publications DE3518538 and DE 3662527 and Patent Cooperation Treaty publication WO 2008/001684. In practice, use of the inventive concentrate could enable two shot manufacturing processes with a combination of differing resin systems (i.e., those whose processing temperatures differ by at least 20° C., at least 50° C., and up to at least 100° C.) while relying on the same concentrate platform. Further, owing to its adaptability over a large range of processing temperatures, the risk of degradation or loss of the concentrate (including the desired additive package) is reduced. One significant aspect of the concentrate carrier system described and claimed herein is its ability to remain effective and viable across a broad range of temperatures. In turn, this means the concentrate can be incorporated into low or high temperature processes without fear of the concentrate degrading or failing to perform as intended. The viability of the concentrate can be verified by way of oxidation induction time and/or melt-separation tests, as well as known standards for thermogravimetric analysis, such as ASTM E1131, E2105, and the like. In general, the concentrate needs to retain its integrity and avoid carbon formation or separation during use. Final formulations exhibiting lumps, speckling, and/or other similar traits are indicative of a concentrate carrier system that has failed to melt within the formulation as intended/needed. In other aspects, the ratios of components within the additive package, as well as the relative ratio of base resins (i.e., acrylate and polycaprolactone) to the entire additive package, are significant. Thus, all of the disclosed weight percentages herein may be further combined to form ratios in certain embodiments. In determining such ratios, the amount of plasticizer introduced to the pre-mix may be disregarded. In that same manner, the relative ratios of plasticizer, base resins, and additive package are contemplated and within these disclosed aspects. EXAMPLES Table 1 shows three exemplary formulations for the pre-mix and additive package according to certain aspects of the invention. All components identified with the exception of plasticizer are selected to be in solid, rather than liquid or gaseous, forms. TABLE 1Concentrate formulations. All valuesexpressed as wt. % of the total pre-mix.ComponentSample 1Sample 2Sample 3AcrylatePoly-EMA, 20% MA4543.8317Ring-openedPolycaprolactone, linear MW ~50 k5.04.873.0PlasticizerEpoxidized soybean oil (ESO)0.7200.5Additive package48.331.379.5(total)(total)(total)Colorantsin/organic pigment12.421a302a2.03adyes———alumina———mica———pearlescent effects———laser markers———Property enhancersProcess modifier0.81b0.30.5Mold release agent———Biocide———UV stabilizer——15.0Heat stabilizer———Anti-oxidant—1.02b3.03bRadical scavengers———Acid scavengers———Anti-static or conductive filler———Metallocene PE———Flame retardants———Non-property fillersCalcium carbonate27.2—41Clay———Silica———Talcum powder———Rice husk and other ash———1acombination of C.I. pigments red (48:2) at 1.0%, blue (15:1) at 8.76%, black (7 at 70 nm) at 0.66%, and white (6) at 2.0%1b0.4% zince stearate and 0.4% calcium-fatty acid2aC.I. pigment black (7)3aC.I. pigment white (6)2band3bIrganox 1010 Sample 1 was mixed by hand and then melt compounded on a two-roll mill, with temperature set to 205° F. for the front roll and 130° F. for the back roll. Sample 1 was shown to provide uniform color at all tested quantities (up to 5 phr) in the following polymer resins: rigid and flexible polyvinyl chloride, XLPE, poly(vinylidene fluoride), high-density polyethylene, polypropylene, polyoxymethylene, ABS, general purpose- and high-impact PS, PC, Nylon 6, and TPE. As a control experiment, a comparable concentrate based upon the teachings of U.S. Pat. No. 6,713,545 was created using a linear diblock copolymer of styrene and ethylene/propylene. This material remained rubbery at lower temperatures and was extremely difficult to compound below 280° F. It could not be compounded with XLPE, and plated when attempting to compound it with PVC. Differential scanning calrimetry (DSC) of poly(ethylene-co-methyl acrylate), trade name Elvaloy AC1820, and polycaprolactone are shown inFIG.1, showing that the melt temperature for this material is 92° C. (198° F.). The embodiment of the invention described above was tested in a melt flow indexer to demonstrate that it could be dispersed at this temperature using ASTM D1248, with the temperature set to 93° C. The melt flow index of this embodiment of the invention was found to be 0.01 g/10 min (2.16 kg, 93° C.). At 190° C., the melt flow of this material was found to be 2.38 g/10 min (2.16 kg). FIG.2shows a dry air TGA thermogram of the above-represented embodiment of the invention. The temperatures of 1% mass loss at 305.6° C. (582° F.) and a minor degradation onset at 325.7° C. (618.3° F.). This is sufficient to allow the concentrate to be compounded with high temperature polymers that require the material to reach 600° F. for short periods, such as PC. Sample 2 was produced using a Farrel continuous mixer and extruder system to produce pelletized industry-scale quantities. Sample 3 was produced using a two-roll mill as described above. Both Samples 1 and 3 maintained sufficient integrity and could be sectioned into uniform pieces from the solid mill sheet. Sample 2 could be pelletized using an underwater-cut pelletizing die. Although specific embodiments have been illustrated, described, and/or exemplified in this specification, it is to be understood that the invention is not to be limited to just the embodiments disclosed, and numerous rearrangements, modifications and substitutions are also contemplated. The exemplary embodiment has been described with reference to the preferred embodiments, but further modifications and alterations encompass the preceding detailed description. These modifications and alterations also fall within the scope of the appended claims or the equivalents thereof. | 21,680 |
11859062 | EXAMPLES Preparation of an Aqueous Polymer P1 Dispersion (Dispersion 1) A 500 L pilot plant reactor equipped with a stirrer, a reflux condenser and metering devices was initially charged at 20 to 25° C. (room temperature) and under a nitrogen atmosphere with 36.5 kg of deionized water, and heated up to 95° C. under atmospheric pressure (1.013 bar absolute) while stirring. On attainment of this temperature, 14.0 kg of a 7% by weight aqueous solution of sodium persulfate were metered in continuously while stirring within 10 minutes. Subsequently, the following were metered continuously into the reaction vessel at constant flow rates while stirring and while maintaining the aforementioned temperature, each commencing at the same time: a mixture of 61.6 kg of acrylic acid, 3.2 kg of methyl methacrylate and 40.5 kg of deionized water within 70 minutes, and a mixture of 14.0 kg of a 40% by weight aqueous solution of sodium hydrogensulfite and 1.4 kg of deionized water, likewise within 70 minutes, and 32.5 kg of a 7% by weight aqueous solution of sodium persulfate within 75 minutes. Subsequently, the polymerization mixture was stirred for a further 5 minutes and then cooled down to 93° C. Thereafter, 13.9 kg of 25% by weight sodium hydroxide solution were metered in while stirring within 10 minutes and hence a pH of 3.3 was established, followed by stirring for a further 5 minutes. Subsequently, feed 1 was metered in within 170 minutes, with addition first of 48% by weight of feed 1 within 20 minutes and then of 52% by weight of feed 1 within 150 minutes—each continuously at constant flow rates. Feed 1 consisted of 21.8 kg of a 7% by weight aqueous solution of sodium persulfate. 5 minutes after the start of feed 1, feed 2 was metered in continuously at constant flow rate while maintaining the aforementioned polymerization temperature within 150 minutes. Feed 2 consisted of a homogeneous emulsion produced from 28.4 kg of deionized water, 3.86 kg of a 28% by weight aqueous solution of sodium lauryl ether sulfate (Disponil® FES 27; product from BASF SE), 2.88 kg of a 15% by weight aqueous solution of sodium dodecylsulfate (Disponil® SDS 15; product from BASF SE), 4.54 kg of glycidyl methacrylate, 1.06 kg of butane-1,4-diol diacrylate, 57.00 g of methyl methacrylate, 86.48 kg of styrene and 2.12 kg of acrylic acid. After the addition of feed 1 had ended, stirring was continued for another 10 minutes. Subsequently, 108 g of a defoamer (TEGO® Foamex 822; product from Evonik Resource Efficiency GmbH) were added. Thereafter, the polymerization mixture was cooled down to 90° C., and feeds 3 and 4, commencing simultaneously, were added continuously at constant flow rates within 30 minutes. Feed 3 consisted of 650 g of a 10% by weight aqueous solution of tert-butyl hydroperoxide, and feed 4 consisted of 820 g of a 13.1% by weight aqueous solution of acetone bisulfite (molar 1:1 addition product of acetone and sodium hydrogensulfite). Thereafter, the polymerization mixture obtained was cooled down to room temperature and filtered through a 125 μm filter. The aqueous polymer dispersion obtained had a solids content of 53.5% by weight. The number-average particle size was determined as 347 nm and the glass transition temperature as 103° C. The solids content was generally determined with a Mettler Toledo moisture analyzer by drying of 0.5 to 1 g of a polymer dispersion or polymer solution obtained to constant weight at 140° C. The glass transition temperature was generally determined with the aid of a TA Instruments Q 2000 differential calorimeter. The heating rate was 10 K per minute. The number-average particle size of the dispersion particles was generally determined by dynamic light scattering on a 0.005 to 0.01% by weight aqueous dispersion at 23° C. using an Autosizer IIC from Malvern Instruments, England. What is reported is the cumulant z-average diameter of the measured autocorrelation function (ISO Standard 13321). The pH values were generally determined by analyzing a sample with a Schott pH electrode at room temperature. Performance Testing The studies were conducted with a 12 inch refiner from Antriz and a blowline connected thereto. The refiner was operated at 160 to 170° C. and an internal pressure of 5 to 6 bar (gauge). The distance between the two grinding plates was 0.3 mm, and one of the grinding plates was operated at 3000 revolutions per minute. The blowline (steel tube) connected to the refiner via a flange had an internal diameter of 3 cm and a tube length of 30 m. The aqueous polymer dispersion P was then injected at 2 bar (gauge) via a 0.2 mm nozzle that was within the blowline at a distance of 50 cm from the refiner outlet/blowline inlet, and the di- or polyisocyanate I was likewise injected at 2 bar gauge via a 0.2 mm nozzle that was within the blowline at a distance of 80 cm from the refiner outlet/blowline inlet. At the end of the blowline was a cyclone separator, by means of which the coated wood fibers were dried further, and cooled down to a temperature of about 80° C. and deposited into an open vessel. For the studies, spruce woodchips that have been pretreated with water/steam at 160 to 170° C. at 5 to 6 bar (gauge) in a so-called boiler were used, with the mass flow rate of woodchips into the refiner (or wood fibers into the blowline) set at 30 kg per hour. The binder used was dispersion 1, and isocyanates used were Lupramat® M 20 R (PMDI), product from BASF Polyurethane GmbH, Lupramat® Ml (MDI), product from BASF Polyurethane GmbH, and Lupramat® MP 100/1, a 40% by weight aqueous PMDI dispersion (E-PMDI), product from BASF Polyurethane GmbH, alone and dispersion 1 in combination with the di- and polyisocyanates mentioned. The binders were injected here into the blowline via the 0.2 mm nozzle(s) by means of an eccentric screw pump at a pressure of 2 bar (gauge), with the mass flow rates adjusted in each case to the respective amount of binder required (calculated as solids) per hour. There was a test for each binder or binder combination over 2 hours in the continuous steady state, with collection of the wood fibers sprayed with the respective binder in an open vessel over the course of this time as well. In this way, the fiber/binder combinations described in table 1 were produced, the stated amounts being parts by weight. It should be noted here that the quantitative data of dispersion 1 and E-PMDI are based on the respective solids contents. TABLE 1Fiber/binder combinations produced (in parts by weight)TypeWood fibersDispersion 1PMDIMDIE-PMDIVD110010———VP1100—5——VM1100——5—VE1100———5EP110055——EM11005—5—EE11005——5 Study of the Mechanical Properties The coated fibers obtained from the blowline according to the aforementioned experimental procedure were used to produce 51×51 cm fiberboards with a thickness of 4.5 mm and a density of 0.8 g/cm3. For this purpose, 936 g of the fibers obtained were scattered homogeneously into a horizontal wood frame having internal dimensions of 51×51×30 cm (L/B/H). Thereafter, a 51×51 cm wooden board was placed horizontally onto the fiber web present within the wooden frame and the fiber web was subjected to preliminary compaction to a height of 10 cm with a ram in the middle. The fiber cake thus obtained was then taken out of the wooden frame, covered with a release paper on both square faces and compacted to a thickness of 4.5 mm between two 10 mm-thick horizontal separation plates at 200° C. under pressure at a compression rate of 10 seconds per mm, with the lower face of the fiber cake being placed onto the lower horizontal separation plate in each case. Thereafter, the fiberboards obtained were left to cool down to room temperature outside the press. The fiberboards thus obtained, depending on the binder used, are called FVD1 (fiberboard with dispersion 1), FVP1 (fiberboard with PMDI), FVM1 (fiberboard with MDI), FVE1 (fiberboard with E-PMDI), FEP1 (fiberboard with dispersion 1 and PMDI), FEM1 (fiberboard with dispersion 1 and MDI) and FEE1 (fiberboard with dispersion 1 and E-PMDI). The aforementioned fiberboards were subjected to a second compaction to a density of 0.9 g/m3, with storage of the fiberboards first in a climate-controlled room at 23° C. and 50% relative humidity for one week. Thereafter, the fiberboards were compressed to a thickness of 4.0 mm, corresponding to a density of 0.9 g/cm3, in a heated press at 160° C., using an embossed plate in the contact press to impress a sharp-edged engraving with a depth of 0.1 to 1.0 mm in each case within 60 seconds. Water absorption and thickness swelling were determined on the fiberboards obtained after this further compression, and the embossment was assessed visually. The water absorption and thickness swelling were determined here in such a way that corresponding 5×5 cm test specimens were punched out of the fiberboards and these were then weighed accurately and their thicknesses were determined exactly. Subsequently, these test specimens were stored vertically in deionized water at 23° C. for 24 hours, then dabbed dry with a cotton cloth and then weighed, and the thickness of the individual test specimens was determined. The water absorption (in % by weight) was determined here from the difference in weight of the test specimens after and before the water storage multiplied by 100, divided by the respective weight before the water storage. In a corresponding manner, the thickness swelling was also determined from the difference in the thickness of the test specimens after and before the water storage multiplied by 100, divided by the thickness of the test specimens before the water storage. 5 test specimens were produced from each fiberboard and used for the tests. The test values reported below are the averages of these 5 measurements. The lower the water absorption and the lower the thickness swelling, the better the assessment of the water resistance. The results obtained for the respective test specimens are listed in table 2. Embossability was assessed in that the edges of the embossment of the respective test specimens were assessed visually, after water storage, by means of a magnifying glass (with 12-fold magnification). Embossability was assessed as good (+) when the edges of the embossments after water storage did not have any visible protruding or loose fibers [=roughness]. By contrast, if the edges of the embossments after water storage had visible protruding or loose fibers, embossability was assessed as inadequate (−). The assessments specified were made when at least 4 of the 5 test specimens met the criteria mentioned. The corresponding results are likewise listed in table 2. TABLE 2Results of the respective test specimens after water storageWater absorptionThickness swellingTest specimen[in % by wt.][in %]EmbossabilityFVD17333+FVP12312−FVM12413−FVE12714−FEP12813+FEM12614+FEE12614+ It is clearly apparent from the results that the test specimens consolidated with dispersion 1 alone did have good embossability, but had high water absorption and high thickness swelling, whereas the test specimens consolidated solely with a di- or polyisocyanate had low water absorption and low thickness swelling, but inadequate embossability. By contrast, the test specimens consolidated both with dispersion 1 and with a di- or polyisocyanate had both good embossability and low water absorption, and also low thickness swelling. | 11,467 |
11859063 | EXAMPLES The present invention is further specifically described with reference to Examples, though the present invention is not limited thereto. The measurement method and the evaluation method in the present Examples are as follows. (1) Measurement of Relaxation Time and Component Ratio of Component A A film sample of the PVA films obtained in Examples and Comparative Examples were laminated to a UV release tape (trade name “SELFA-SE” manufactured by Sekisui Chemical Co., Ltd.) to be fixed using a laminator (laminator HOTDOG Leon 13DX manufactured by Lami Corporation Inc.) set at a temperature of 60° C. and a speed of 5. Subsequently, the fixed PVA film was aged in a thermostat at 23° C. and 50% RH for 48 hours. In order to achieve fixation of the PVA film to the UV release tape by the laminator, both are sandwiched with SUS plate having a thickness of 1 mm or less and a release-treated PET film having a thickness of 50 μm. On the occasion of fixation, the lamination was performed such that the roll width direction of the UV release tape is perpendicularly crossed with the longitudinal direction of the PVA film. The longitudinal direction of the PVA film is the same as the cutting direction of the PVA film when rolled into a cylindrical form for introduction into an NMR tube (longitudinal direction of the film in a cylindrical form). The fixed film sample was exposed to UV rays having a wavelength of 365 nm to receive an energy of 1000 mJ/cm2at the irradiated surface using a UV irradiation apparatus “manufactured by ORC Manufacturing Co., Ltd., apparatus model: JL-4300-3, lamp model: IML-4000”, so that the UV release tape was detached from the film sample. A film sample having a low water content may wrinkle when aged after fixation with an UV release tape. When wrinkles occur after aging, the UV release tape is detached through the irradiation step, the wrinkles of the PVA film are then smoothed once by a laminator. Fixation is performed through lamination using a new SELFA-SE, and the aging step is performed. The operation may be repeated until the wrinkles disappear. A film sample detached from the UV release tape having a weight of about 700 mg was rolled into a cylindrical form and introduced into a sample tube made of glass having a diameter of 10 mm (manufactured by BRUKER, item No. 1824511, diameter: 10 mm, length: 180 mm, flat bottom), such that the height was controlled to 15 mm. The sample was installed in a pulse NMR apparatus (“the Minispec MQ20” manufactured by BRUKER), and the temperature was raised stepwise from 25° C. (retained for 40 minutes), to 40° C. (retained for 40 minutes), and to 60° C. (retained for 10 minutes). The measurement was performed by a solid echo method at 60° C., and the resulting free induction decay curve of 1H spin-spin relaxation was subjected to waveform separation into three curves derived from three components a component A, a component B, and a component C. The waveform separation was performed by fitting to both a Gaussian model and an exponential model. From the curves derived from the three components obtained in each measurement, the ratio of each component was obtained. The same type of measurement was performed twice to obtain the ratio of each component as an average. Using analytical software “TD-NMRA (Version 4.3, Rev. 0.8)” manufactured by Bruker Corporation, the component A was fitted to a Gaussian model and the component B and the component C were fitted to an exponential model according to the product manual. Also, in the analysis, fitting was performed using points up to 0.6 milliseconds in the relaxation curve. Also, the following equation was used in the fitting. Y=A1*exp(−1/w1*(t/T2A){circumflex over ( )}w1)+B1*exp(−1/w2*(t/T2B){circumflex over ( )}w2)+C1*exp(−1/w3*(t/T2C){circumflex over ( )}w3), where w1 to w3 are Weibull coefficients; w1 is 2; w2 and w3 are 1; A1, B1 and C1 are the component ratios of the component A, the component B and the component C, respectively; T2A, T2B and T2C represent the relaxation times of the component A, the component B and the component C, respectively; and t represents time. The component A, the component B and the component C are components defined in order of short relaxation time when the PVA film is measured using pulse NMR. Although each value of the relaxation time is not particularly limited, the normal relaxation time is less than 0.02 milliseconds for the component A, 0.02 milliseconds or more and less than 0.1 milliseconds for the component B, and 0.1 milliseconds or more for the component C. <Solid echo method> Scans: 128 timesRecycle delay: 1 secAcquisition scale: 1 ms (2) Elongation at Break A PVA film having a width of 10 cm obtained in each of Examples and Comparative Examples was immersed in an aqueous solution dissolving iodine (I2) and potassium iodide (KI). In the aqueous solution, the PVA film with a span length of 2 cm was stretched at a stretching rate of 1 cm/second using a stretcher to be broken. The ratio of the film length at the breakage to the film length before stretching was defined as the elongation at break. The aqueous solution comprised 0.4 parts by mass of iodine, 40 parts by mass of potassium iodide, and 1000 parts by mass of water. (3) Measurement of Degree of Polarization The degree of polarization P of a polarizing film obtained in each of Examples and Comparative Examples was evaluated by the following equation using a spectrophotometer “UV-3101PC” manufactured by Shimadzu Corporation. YP (parallel transmittance) is the transmittance of films superimposed to each other in parallel with the stretching direction of the film, and YC (cross transmittance) is the transmittance of films superimposed to each other in orthogonal to the stretching direction of the film. Degree of polarizationP(%)={(YP−YC)/(YP+YC)}1/2×100 [Criteria of Degree of Polarization]A: degree of polarization of 99 or moreB: degree of polarization of 90 or more and less than 99C: degree of polarization of less than 90 SYNTHESIS EXAMPLE 1 [PVA1 (Degree of Saponification: 99.5 mol %, Degree of Polymerization: 2700)] Into a reaction vessel equipped with a thermometer, a stirrer and a cooling tube, 2000 parts by mass of vinyl acetate monomer and 200 parts by mass of methanol were added, and after nitrogen purge with nitrogen gas blown in for 30 minutes, the reaction vessel was heated at 60° C. for 30 minutes. Subsequently, 0.4 parts by mass of 2,2′-azobisisobutyronitrile as polymerization initiator was added thereto and then let the reaction proceed at 60° C. for 4 hours. After completion of the reaction time, the reaction liquid was cooled. The polymerization ratio was 29% based on the measurement of the polymerization ratio after cooling. Subsequently, an operation for removing the residual vinyl acetate monomer together with methanol under reduced pressure was performed along with addition of methanol, so that a methanol solution containing 50 mass % of polyvinyl acetate was obtained. To the methanol solution, a methanol solution of sodium hydroxide was added so that a sodium hydroxide content was 0.08 mol % based on vinyl acetate, and saponification was performed at 40° C. The resulting solid content was pulverized, washed with methanol, and then dried to obtain PVA1. The resulting PVA1 was subjected to measurement of the degree of saponification and the degree of polymerization in accordance with JIS K 6726. The degree of saponification was 99.5 mol % and the degree of polymerization was 2700. Synthesis Example 2 [PVA2 (Degree of Saponification: 99.5 mol %, Degree of Polymerization: 1500)] Into a reaction vessel equipped with a thermometer, a stirrer and a cooling tube, 2000 parts by mass of vinyl acetate monomer and 200 parts by mass of methanol were added, and after nitrogen purge with nitrogen gas blown in for 30 minutes, the reaction vessel was heated at 60° C. for 30 minutes. Subsequently, 0.6 parts by mass of 2,2′-azobisisobutyronitrile as polymerization initiator was added thereto and then let the reaction proceed at 60° C. for 4 hours. After completion of the reaction time, the reaction liquid was cooled. The polymerization ratio was 35% based on the measurement of the polymerization ratio after cooling. Subsequently, an operation for removing the residual vinyl acetate monomer together with methanol under reduced pressure was performed along with addition of methanol, so that a methanol solution containing 50 mass % of polyvinyl acetate was obtained. To the methanol solution, a methanol solution of sodium hydroxide was added so that a sodium hydroxide content was 0.08 mol % relative to vinyl acetate, and saponification was performed at 40° C. The resulting solid content was pulverized, washed with methanol, and then dried to obtain PVA2. The resulting PVA2 was subjected to measurement of the degree of saponification and the degree of polymerization in accordance with JIS K 6726. The degree of saponification was 99.5 mol % and the degree of polymerization was 1500. EXAMPLE 1 (Manufacturing of PVA Film) Into a reaction vessel equipped with a thermometer, a stirrer and a cooling tube, 1000 parts by mass of water was fed at 25° C., and 100 parts by mass of PVA1 and 10 parts by mass of glycerol were fed therein while stirring. The mixture liquid was heated to 95° C. and retained at 95° C. for 120 minutes, so that PVA1 and glycerol were dissolved in water. The mixture liquid (PVA solution) was then cooled down to 35° C. at a temperature lowering rate of 1° C./minute. The PVA solution cooled to 35° C. was applied to a glass plate having a thickness of 7 mm, dried at 80° C. for 1 hour, and then detached from the glass plate to obtain a PVA film having a thickness of 30 μm. From the resulting film, 10 g of the film was cut out to be subjected to 100 cycles of Soxhlet extraction with methanol. The resulting resin was then subjected to measurement of the degree of saponification and the degree of polymerization in accordance with JIS K 6726. The degree of saponification was 99.5 mol % and the degree of polymerization was 2700. (Manufacturing of Polarizing Film) Next, while immersing the resulting PVA film in an aqueous solution dissolving iodine (I2) and potassium iodide (KI) at 25° C. for 60 seconds, stretching was performed at a stretching ratio of 5 times. The aqueous solution comprised 0.4 parts by mass of iodine, 40 parts by mass of potassium iodide and 1000 parts by mass of water. The PVA film was then immersed in a boric acid aqueous solution with a concentration of 4.0 mass % at 25° C. for 5 minutes, washed with water after pulling out from the aqueous solution, and then dried in a drying oven set at 70° C. to obtain a polarizing film. EXAMPLE 2 (Manufacturing of PVA Film) Into a reaction vessel equipped with a thermometer, a stirrer and a cooling tube, 1000 parts by mass of water was fed at 25° C., and 100 parts by mass of PVA1 and 8 parts by mass of polyethylene glycol having an average molecular weight of 400 were fed therein while stirring. The mixture liquid was heated to 95° C. and retained at 95° C. for 120 minutes, so that PVA1 and polyethylene glycol were dissolved in water. The mixture liquid (PVA solution) was then cooled down to 35° C. at a temperature lowering rate of 1° C./minute. The PVA solution cooled at 35° C. was applied to a glass plate having a thickness of 7 mm, dried at 80° C. for 1 hour, and then detached from the glass plate to obtain a PVA film having a thickness of 30 μm. From the resulting film, 10 g of the film was cut out to be subjected to 100 cycles of Soxhlet extraction with methanol. The resulting resin was then subjected to measurement of the degree of saponification and the degree of polymerization in accordance with JIS K 6726. The degree of saponification was 99.5 mol % and the degree of polymerization was 2700. (Manufacturing of Polarizing Film) Next, a polarizing film was manufactured from the resulting film in the same manner as in Example 1. EXAMPLES 3 AND 4 The procedure was performed in the same manner as in Example 1, except that the amount of glycerol fed was changed as described in Table 1. EXAMPLE 5 The procedure was performed in the same manner as in Example 1, except that the drying time of the PVA solution applied to the glass plate was changed to 2 hours. EXAMPLE 6 The procedure was performed in the same manner as in Example 1, except that the temperature lowering rate in cooling of the PVA solution down to 35° C. was changed to 2° C./minute and the PVA solution applied to the glass plate was dried at 90° C. for 30 minutes. COMPARATIVE EXAMPLE 1 A PVA film and a polarizing film were obtained in the same manner as in Example 1, except that the type of PVA was changed to PVA2, the amount of glycerol fed was changed to 0 parts by mass, and the stretching ratio in manufacturing the polarizing film was changed to 3 times. From the resulting PVA film, 10 g of the film was cut out to be subjected to 100 cycles of Soxhlet extraction with methanol. The resulting resin was then subjected to measurement of the degree of saponification and the degree of polymerization in accordance with JIS K 6726. The degree of saponification was 99.5 mol % and the degree of polymerization was 1500. COMPARATIVE EXAMPLE 2 A PVA film and a polarizing film were obtained in the same manner as in Example 1, except that the type of PVA was changed to PVA2, the amount of glycerol fed was changed to 20 parts by mass, and the stretching ratio in manufacturing the polarizing film was changed to 3 times. COMPARATIVE EXAMPLE 3 (Manufacturing of PVA Film) Into a reaction vessel equipped with a thermometer, a stirrer and a cooling tube, 1000 parts by mass of water was fed at 25° C., and 100 parts by mass of PVA1 and 20 parts by mass of glycerol were fed therein while stirring. The mixture liquid was heated to 95° C. and retained at 95° C. for 120 minutes, so that PVA1 and glycerol were dissolved in water. The PVA solution retained at 95° C. was then applied to a PET film having a thickness of 50 um, dried at 90° C. for 30 minutes, and then detached from the glass plate to obtain a PVA film having a thickness of 30 um. (Manufacturing of Polarizing Film) Next, a polarizing film was manufactured from the resulting film as in the same manner as in Example 1. TABLE 1Example 1Example 2Example 3Example 4Example 5PVA solutionPVA100100100100100compositionPlasticizer10820110(parts by mass)Water10001000100010001000PVA propertiesDegree of99.599.599.599.599.5saponification(mol %)Degree of27002700270027002700polymerizationPlasticizerTypeGlycerolPEG400GlycerolGlycerolGlycerolManufacturingTemperatureRT→95° C.RT→95° C.RT→95° C.RT→95° C.RT→95° C.conditionsraisingconditionRetention timeRetained forRetained forRetained forRetained forRetained for2 hours2 hours2 hours2 hours2 hoursTemperatureLoweredLoweredLoweredLoweredLoweredloweringdown to 35° C.down to 35° C.down to 35° C.down to 35° C.down to 35° C.conditionat 1° C./minat 1° C./minat 1° C./minat 1° C./minat 1° C./minApplicationGlass plateGlass plateGlass plateGlass plateGlass plate(support)Drying80° C. for80° C. for80° C. for80° C. for80° C. forcondition1 hour1 hour1 hour1 hour2 hoursComponent ARelaxation time0.00850.000860.00880.00890.0075(milliseconds)Component333520.14833ratio (%)EvaluationElongation at780780850550730resultsbreak (%)Degree ofAAAAApolarizationComparativeComparativeComparativeExample 6Example 1Example 2Example 3PVA solutionPVA100100100100compositionPlasticizer1002020(parts by mass)Water1000100010001000PVA propertiesDegree of99.599.599.599.5saponification(mol %)Degree of2700150015002700polymerizationPlasticizerTypeGlycerol—GlycerolGlycerolManufacturingTemperatureRT→95° C.RT→95° C.RT→95° C.RT→95° C.conditionsraisingconditionRetention timeRetained forRetained forRetained forRetained for2 hours2 hours2 hours2 hoursTemperatureLoweredLoweredLoweredNo loweringloweringdown to 35° C.down to 35° C.down to 35° C.ofconditionat 2° C./minat 1° C./minat 1° C./mintemperatureApplicationGlass plateGlass plateGlass platePET film(support)Drying90° C. for80° C. for80° C. for90° C. forcondition0.5 hours1 hour1 hour0.5 hoursComponent ARelaxation time0.00920.0090.00950.0096(milliseconds)Component33652535ratio (%)EvaluationElongation at800350700900resultsbreak (%)Degree ofABBBpolarization As shown in Examples described above, control of the relaxation time of the component A and the component ratio of the component A to specified ranges resulted in excellent elongation at break, so that the PVA film was unlikely to be broken in manufacturing of a polarizing film. Also, the resulting polarizing film had excellent polarization performance. | 16,755 |
11859064 | DETAILED DESCRIPTION Reference will now be made in detail to embodiments of processes for modifying a deformable substrate, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts. The method described herein may be used to create a deformed region in a deformable substrate using a sessile liquid droplet. The method includes depositing a sessile liquid droplet on a surface of the deformable substrate, such that the sessile liquid droplet forms a deformed region in the deformable substrate via elastocapillary deformation, then curing the deformed substrate to increase the elastic modulus of the deformable substrate such that, upon removal of the sessile liquid droplet, the deformed region remains in the deformable substrate. Before curing, the deformable substrate comprises an initial elastic modulus that is small enough that the elastocapillary force applied by the sessile liquid droplet deforms the deformable substrate. Curing the deformable substrate increases the elastic modulus of the deformable substrate (i.e. increases the elastic modulus from the initial elastic modulus to a cured elastic modulus), such that when the sessile liquid is removed from the deformable substrate, the deformable substrate does not deflect back to its original shape and the deformed region remains in the deformable substrate. The method described herein provides a tunable, low cost process for creating structures directly on the deformable substrate that offers cost and adjustability improvements over previous methods, such as photolithography, laser patterning, printing, convective assembly (e.g., coffee-ring patterning), or the like. Referring now toFIG.1A, an example deformable substrate110is schematically depicted. The deformable substrate110comprises a first surface112, a second surface114opposite the first surface112, and an undeformed plane115, which is the plane of an undeformed portion the first surface112, for example, the plane of the first surface112before the deformable substrate110is deformed. InFIG.1A, the undeformed plane115is co-located with the first surface112, however, when the first surface112includes a deformed region120(FIGS.1C and1D), the undeformed plane115is offset from at least portions of the deformed region120. Further, the deformable substrate110comprises a curable substrate. In some embodiments, the deformable substrate110may comprise a polymer, such as polyvinylsiloxane, polydimethylsiloxane (PDMS), ultraviolet (UV) curable polyacrylate, epoxy polymer, thermosetting polymer, or combinations thereof. In some embodiments, the deformable substrate110may further comprise a curing agent, for example, peroxide, platinum complex solution, a photoinitiator, or combinations thereof. The deformable substrate110comprises an initial elastic modulus and a cured elastic modulus, which is greater than the initial elastic modulus. In particular, the initial elastic modulus is small enough that the deposition of a sessile liquid droplet160(FIG.1B) on the first surface112of the deformable substrate110deforms the deformable substrate110when the deformable substrate110comprises the initial elastic modulus. Further, the cured elastic modulus is large enough that the deposition of the sessile liquid droplet160on the first surface112of the deformable substrate110does not deform the deformable substrate110when the deformable substrate110comprises the cured elastic modulus. Thus, the deformed region120may be formed in the deformable substrate110by first depositing the sessile liquid droplet160on the first surface112of the deformable substrate110when the deformable substrate110comprises the initial elastic modulus and then curing the deformable substrate110, while the sessile liquid droplet160is on the first surface112, to increase the elastic modulus from the initial elastic modulus to the cured elastic modulus. Once the deformable substrate110comprises the cured elastic modulus, the first surface112will not deflect back to its original shape and the deformed region120will remain in the first surface112when the sessile liquid droplet160is removed. In some embodiments, the initial elastic modulus of the deformable substrate110may comprise about 800 kPa or less, for example, about 500 kPa or less, 250 kPa or less, 100 kPa or less, 50 kPa or less, 25 kPa or less, 10 kPa or less, 5 kPa or less, 1 kPa or less, or the like, for example, from about 0.1 kPa to about 800 kPa, from about 0.1 kPa to about 100 kPa, from about 0.1 kPa to about 50 kPa, from about 0.1 kPa to about 25 kPa, from about 0.1 kPa to about 10 kPa, from about 0.1 kPa to about 5 kPa, from about 0.1 kPa to about 2 kPa, from about 0.1 kPa to about 1 kPa, from about 0.5 kPa to about 1 kPa, from about 0.5 kPa to about 2 kPa, from about 0.5 kPa to about 3 kPa, from about 0.5 kPa to about 5 kPa, from about 0.5 kPa to about 10 kPa, from about 0.5 kPa to about 25 kPa, from about 0.5 kPa to about 50 kPa, from about 0.5 kPa to about 100 kPa, or the like. Further, the cured elastic modulus may comprise about 100 kPa or greater, for example, about 200 kPa or greater, about 500 kPa or greater, about 800 kPa or greater, about 1 GPa or greater, about 2 GPa or greater, about 5 GPa or greater, about 10 GPa or greater or the like, such as from about 100 kPa to about 10 GPa, about 500 kPa to about 10 GPa, from about 800 GPa to about 10 GPa, from about 1 GPa to about 10 GPa, from about 1 GPa to about 5 GPa, or the like. Referring now toFIGS.1B and1C, the deformable substrate110is schematically depicted with the sessile liquid droplet160disposed on the first surface112of the deformable substrate110before deformation of the deformable substrate110(FIG.1B) and after deformation of the deformable substrate110(FIG.1C). While no deformation of the deformable substrate110is depicted inFIG.1B, it should be understood that in some embodiments, the sessile liquid droplet160may begin to deform the deformable substrate110immediately upon contact between the sessile liquid droplet160and the deformable substrate110. The sessile liquid droplet160may comprise any liquid having a surface tension of about 40 dynes/cm or greater. In some embodiments, the sessile liquid droplet160comprises a surface tension of about 45 dynes/cm or greater, about 50 dynes/cm or greater, about 55 dynes/cm or greater, about 60 dynes/cm or greater, or the like. Example sessile liquid droplets160include water, a polyol liquid, such as ethylene glycol and glycerol, or a combination thereof. The sessile liquid droplet160may also include a plurality of nanoparticles162. The plurality of nanoparticles162may comprise any particle comprising a higher refractive index than the deformable substrate110. Example nanoparticles162include TiO2, zinc oxide, zirconium oxide, silica, alumina, cerium oxide, or a combination thereof. In some embodiments, when the sessile liquid droplet160comprises a plurality of nanoparticles162, the plurality of nanoparticles162may comprise from about 5 weight percent (wt %) to about 30 wt % of the sessile liquid droplet160, for example 10 wt %, 15 wt %, 20 wt %, 25 wt %, or the like. As depicted inFIGS.1C and1D, the deformed region120comprises a recess122and a perimeter rim124. The recess122extends into the deformable substrate110beyond the undeformed plane115in a direction toward the second surface114(e.g., downward as depicted inFIG.1C). The recess122terminates at a recess floor123and comprises a recess depth d, which is the distance from the undeformed plane115to the recess floor123. In some embodiments, the recess depth d may be from about 10 nm to about 50 μm, for example, from about 50 nm to about 25 μm, from about 50 nm to about 10 μm, from about 50 nm to about 7.5 μm, from about 50 nm to about 5 μm, from about 100 nm to about 5 μm, from about 250 nm to about 5 μm, from about 500 nm to about 5 μm, from about 750 nm to about 5 μm, from about 1 μm to about 5 μm, from about 1 μm to about 2.5 μm, or the like. Further, the perimeter rim124extends outward from the deformable substrate110beyond the undeformed plane115in a direction away from the second surface114. The perimeter rim124terminates at a rim tip126. Further, the perimeter rim124comprises a rim height h, which is the distance from the undeformed plane115to the rim top126. In some embodiments, the rim height h may be from about 50 nm to about 100 μm, for example, from about 100 nm to about 75 μm, from about 100 nm to about 50 μm, from about 100 nm to about 25 μm, from about 100 nm to about 15 μm, from about 100 nm to about 10 μm, from about 100 nm to about 7.5 μm, from about 100 nm to about 5 μm, from about 250 nm to about 5 μm, from about 500 nm to about 5 μm, from about 750 nm to about 5 μm, from about 1 μm to about 5 μm, from about 1 μm to about 4 μm, from about 1 μm to about 4 μm, or the like. A full distance f, which is the sum of the recess depth d and the rim height h is also depictedFIGS.1C and1D. WhileFIGS.1C and1Dshow schematic cross-sections of the deformable substrate110, it should be understood that the deformed region120comprises a circular or otherwise closed and rounded shape, as the sessile liquid droplet160forms a rounded shape on the first surface112of the deformable substrate110. The deformed region120may comprise a maximum cross sectional dimension (e.g., diameter in circular embodiment measured at the rim tip126) of from about 200 μm to about 800 μm, for example about 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, or the like. Referring again toFIGS.1B and1C, the sessile liquid droplet160comprises a contact angle θ measured at a liquid-vapor-solid interface of the sessile liquid droplet160and the deformable substrate110. The contact angle θ is mathematically related to properties of the deformable substrate110via Young's equation: γSL+γLcos θ=γS, where γSLis the solid-liquid interfacial energy between the sessile liquid droplet160and the deformable substrate110, γLis the surface tension of the sessile liquid droplet160, and YSis the surface energy of the deformable substrate110. Without intending to be limited by theory, the vertical component YLsin θ of the liquid surface tension of the sessile liquid droplet160cannot remain unbalanced while maintaining the stationary condition of the solid-liquid-vapor three phase contact line at the interface of the sessile liquid droplet160and the first surface112of the deformable substrate110. Thus, the vertical component YLsin θ of the liquid surface tension of the sessile liquid droplet160pulls the first surface112of the deformable substrate110(e.g., capillary pulling) and creates a tensile deformation (e.g., the deformed region120) in the first surface112having portions that both extend in a direction toward from the second surface114(the perimeter rim124) and in a direction toward the second surface114(the recess122). Further, the sessile liquid droplet160deforms the first surface112of the deformable substrate110until equilibrium is established between the vertical component YLsin θ of the liquid surface tension of the sessile liquid droplet160and the opposing vertical component of the surface energy γSsin θ of the deformable substrate110, which increases as the deformable substrate110is deformed. Thus, while not intending to be limited by theory, the rim height h is related to the vertical component YLsin θ of the liquid surface tension of the sessile liquid droplet160and the initial elastic modulus E of the deformable substrate110, where h=γLsinθE. Referring now toFIG.2, a graph10is depicted showing relationship between the rim height h of the perimeter rim124of the deformed region120formed in the deformable substrate110using the methods described herein, and the initial elastic modulus of the deformable substrate110. Line12shows that as the initial elastic modulus of the deformable substrate110increases, the rim height h decreases and line12shows that the initial elastic modulus of the deformable substrate110has a logarithmic relationship with the rim height h. Further, as shown inFIG.2, when the initial elastic modulus of the deformable substrate110is about 1 GPa or greater, little to no deformation is caused by the elastocapillary forces of the sessile liquid droplet160. Referring again toFIGS.1A-1D, a method of modifying the deformable substrate110includes depositing the sessile liquid droplet160on the first surface112of the deformable substrate110, the sessile liquid droplet160forming the deformed region120in the first surface112in the deformable substrate110. As described above, the deformed region120is formed by elastocapillary deformation when the sessile liquid droplet160is disposed on the first surface112of the deformable substrate110. However, if the sessile liquid droplet160is removed from the deformable substrate110while the deformable substrate110comprises the initial elastic modulus, the first surface112will deflect back to its original shape, removing the deformed region120from the first surface112of the deformable substrate110. Thus, the method next comprises curing the deformable substrate110while the sessile liquid droplet160is in contact with the first surface112of the deformable substrate110, thereby increasing the elastic modulus of the deformable substrate110, for example, from the initial elastic modulus to the cured elastic modulus. In some embodiments, curing the deformable substrate110comprises heating the deformable substrate110, for example, to a curing temperature for a curing period. In other embodiments, curing the deformable substrate110comprises irradiating at least the deformed region120of the deformable substrate110with light, such as ultraviolet light, at a curing wavelength for a curing period. The method next comprises removing the sessile liquid droplet160from the deformable substrate110. As depicted inFIG.1D, upon removal of the sessile liquid droplet160from the deformable substrate110, the deformed region120remains in the first surface112of the deformable substrate110, as the deformable substrate110now comprises the cured elastic modulus. In some embodiments, curing the deformable substrate110removes the sessile liquid droplet160from the deformable substrate110, for example, by evaporating the sessile liquid droplet160. In other embodiments, removing the sessile liquid droplet160from the deformable substrate110may comprise a separate step performed after curing the deformable substrate110. Referring still toFIG.1D, during formation of the deformed region120, the plurality of nanoparticles162of the sessile liquid droplet160may embed into the deformable substrate110(FIG.1D). In some embodiments, the plurality of nanoparticles162may embed into the deformed region120, for example, the perimeter rim124of the deformed region120. When embedded in the deformable substrate110, the plurality of nanoparticles162may alter the refractive index of a portion of the deformable substrate110(e.g., the refractive index of the deformed region120). In some embodiments, the plurality of nanoparticles162may be configured to scatter light propagating through the deformable substrate110. For example, the plurality of nanoparticles162comprise a higher refractive index than the deformable substrate110and thus, when light propagates through the portion of the deformable substrate110having embedded nanoparticles162(e.g., the perimeter rim124), the refractive index difference between the material of the deformable substrate110and the individual nanoparticles162causes light rays that impinge the nanoparticles162to refract in a variety of directions, causing a scattering effect. In some embodiments, mere contact between the sessile liquid droplet160and the deformable substrate110may embed the plurality of nanoparticles162into the deformable substrate110. In other embodiments, curing the deformable substrate110while the sessile liquid droplet160contacts the deformable substrate110may embed the plurality of nanoparticles162into the deformable substrate110. In some embodiments, as depicted inFIGS.1A-1D, the deformable substrate110may be disposed on a second substrate105. For example, the method may further comprise depositing the deformable substrate110on the second substrate105. The second substrate105may comprises any substrate having a higher elastic modulus than the initial elastic modulus of the deformable substrate110. For example, the second substrate105may comprise a glass substrate, glass-ceramic substrate, or a ceramic substrate. While the second substrate105is depicted in each ofFIGS.1A-1D, it should be understood that the second substrate105is an optional component and the methods described herein may be performed with the second substrate105. While the methods described herein may be useful in a variety of applications, one example application is in transparent display technology, which allows a user to observe both a display and objects located behind the display. For example, in a retail setting, a transparent display may be located between an observer and a product and may display information while allowing the observer to see the product. A conventional transparent display may include one or more light guide plates that provide edge illumination at angles parallel or near parallel to the display plate (e.g., a glass substrate). However, traditional, opaque filtering films (such as 3M BEF filters), which alter the angular distribution of light rays propagating parallel or near-parallel to the display plate, are not usable in a transparent display application. Thus, the present application may be used to form a transparent or semi-transparent rim structure (e.g., a rim perimeter of a deformed region of a deformable substrate as described above) configured to alter the angular distribution of light rays (e.g., an angular filter) without degrading the transparency of the display. For example, the angular filter functionality may be achieved by embedding a plurality of nanoparticles into the transparent rim structure, for example, scattering particles and/or particles that alter the refractive index of the transparent rim structure, using the methods described herein, to form and angular filter and provide angular distribution functionality. As used herein, the term “about” means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. When the term “about” is used in describing a value or an end-point of a range, the specific value or end-point referred to is included. Whether or not a numerical value or end-point of a range in the specification recites “about,” two embodiments are described: one modified by “about,” and one not modified by “about.” It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Directional terms as used herein—for example up, down, right, left, front, back, top, bottom—are made only with reference to the figures as drawn and are not intended to imply absolute orientation. Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order, nor that with any apparatus specific orientations be required. Accordingly, where a method claim does not actually recite an order to be followed by its steps, or that any apparatus claim does not actually recite an order or orientation to individual components, or it is not otherwise specifically stated in the claims or description that the steps are to be limited to a specific order, or that a specific order or orientation to components of an apparatus is not recited, it is in no way intended that an order or orientation be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps, operational flow, order of components, or orientation of components; plain meaning derived from grammatical organization or punctuation, and; the number or type of embodiments described in the specification. As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a” component includes aspects having two or more such components, unless the context clearly indicates otherwise. It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spirit and scope of the claimed subject matter. Thus it is intended that the specification cover the modifications and variations of the various embodiments described herein provided such modification and variations come within the scope of the appended claims and their equivalents. EXPERIMENTS In the following experiments, deformed regions were formed on a variety of deformable substrates using the methods described herein. Referring now toFIG.3, in a first experiment, a deformable substrate210comprising a Filtec™ Dental Mold including polyvinylsiloxanes was modified using sessile liquid droplets comprising water to form a plurality of deformed regions220. In this example, the deformable substrate210was spread on a glass surface. Then, water droplets were dispersed in ten second intervals onto the deformable substrate210using the Kruss goniometer. Finally, the water droplets were removed from the deformable substrate210. As shown inFIG.3, deformed regions220were formed and the extent of deformation of each deformed region220is smaller with each successive droplet, because the deformable substrate210cured while the water droplets were dispersed (i.e. cured at a curing temperature of about room temperature for a curing period of about 1 minute). Thus, the first water droplet deposited created a deformed region with the tallest rim perimeter (i.e., the leftmost deformed region depicted inFIG.3) and the last water droplet deposited created the deformed region with the shortest rim perimeter (i.e., the rightmost deformed region depicted inFIG.3). Referring now toFIG.4, in a second experiment, a deformable substrate310comprising PDMS and a curing agent was modified using the method described herein. In particular, the deformable substrate310comprises a Sylgard™ 184 elastomer in a ratio of from about 1:0.03 to about 1:0.05 of prepolymer to curing agent. In this experiment, droplets of water/ethylene glycol and glycerol were dispensed onto the deformable substrate110using an Asymtek Nordson dispenser. The deformable substrate310was then cured at a first curing temperature of about 60° C. for a first curing period of about 2 hours and cured at a second curing temperature of from about 120-150° C. for a second curing period of about 30 minutes.FIG.4shows an optical image20, a zygo 3D image22, and a zygo height profile24of the deformable substrate310having deformed regions320. The deformed regions320depicted inFIG.4comprise a diameter of about 600 μm and a rim height of about 125 μm. Referring now toFIGS.5A and5B, in a third experiment, a deformable substrate410comprising PDMS is modified using the method described herein. In the third experiment, a plurality of nanoparticles comprising scattering elements were embedded into the deformable substrate410by forming deformed regions420using a sessile liquid droplet comprising scattering elements. In particular, the sessile liquid droplets used in the third experiment comprised 15% TiO2(Sigma), 57% ethylene glycol, and 28% water (viscosity 8.8 cPs, ST 76.6 N/m, pH 2.6). The deformable substrate410was then cured using the curing temperatures and curing periods of the second experiment.FIG.5Ashows an optical image30, a zygo 3D image32, and a zygo height profile34of the deformable substrate410having deformed regions420. During curing, TiO2particles (i.e. the plurality of nanoparticles) were embedded in the deformed regions420of the deformable substrate410. Further,FIG.5Bdepicts scattering electron microscope (SEM) images of the deformable substrate and the deformed regions ofFIG.5A. | 24,528 |
11859065 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following definitions are provided for the purpose of understanding the present subject matter and for construing the appended patent claims. Definitions Throughout the application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps. It is noted that, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein. The use of the terms “include,” “includes”, “including,” “have,” “has,” or “having” should be generally understood as open-ended and non-limiting unless specifically stated otherwise. As used herein the term “superhydrophic surface” means a surface having i) a receding static water contact angle (a 50 μl water droplet on a flat surface in an essentially horizontal plane) of more than 135°, preferably more than 140° or more than 145°, more preferably from 145° to 160°, and ii) an advancing static water contact angle of more than 135°, preferably more than 140° or more than 145°, and more preferably from 145° to 160°, as measured by a Drop Shape Krüss Analyser and corresponding protocol and iii) preferably a water roll-off angle also called sliding angle (dynamic measure) of less than 10°, preferably less than 6°. The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term “about” is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. The term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains. Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter. Throughout the application, descriptions of various embodiments use “comprising” language. However, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of”. For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Additionally, as used in this application and unless stated otherwise, “virgin” means polypropylene supplied commercially in the form of granules (unused or firsthand) before being plasticized. “Waste” polymer includes when a virgin polymer is used in preparation of plastic products and some amount of the material is discarded until flowability is achieved; that discarded virgin polymer is considered waste polymer in the context of this application. The terms “coating” and “sheet” and “layer” may be used interchangeably herein unless stated otherwise. “Heated” or “hot” as used herein means above room temperature unless stated otherwise. “Strong” means heated such that porosity of a polypropylene polymer coating is minimized, eliminated or essentially eliminated by melting the polymer and crosslinking the polymeric chains by increasing the intermolecular interactions. The present subject matter is related specifically to upcycling polypropylene (PP) plastic waste into superhydrophobic sheets and a process for making such sheets that uses 20% of total plastic waste to prepare said sheets having contact angles ranging from about 140 to 160 degrees. The as-prepared polymeric material is in the form of superhydrophobic sheets with said high contact angles where such sheets or coatings demonstrate exceptional resilience to various weather conditions and can be applied to glass, rooftops, lamp posts, high-rise walls, etc. In one embodiment, the present subject matter is related to polypropylene (PP) superhydrophobic sheets made from upcycled PP. The composition of such sheets includes base and top layers of PP. The porous top layer provides maximum hydrophobicity with a contact angle ranging from 140 to 160 degrees while the nonporous base layer provides support and strength. In an embodiment the base layer or layers are each heated and annealed to achieve a strong, supportive nonporous characteristic before another base layer(s) is applied and heated, and the top layer is not heated and/or annealed, so that it retains a porous rough surface characteristic and a desired high contact angle. In an embodiment the sheets can be used with and without adhesive tapes which can be pasted on the base layer. The sheets have varied thickness, strength, roughness, and hydrophobicity depending on the end-user requirements and can be applied as a protection from water on rooftops, tabletops, lamp posts, high-rise walls, etc. In an embodiment, the superhydrophobic PP sheets are made from plastic waste, the plastic waste comprises recycled polypropylene and/or waste polypropylene, wherein the waste PP may include virgin or discarded virgin polypropylene, and the polypropylene may comprise isotactic, atactic, syntactic, and/or amorphous polypropylene. No wax is required, and no wax or other additive is added. In an embodiment the superhydrophobic sheet herein will have two layers. One is the base layer, and the other is the top layer. But if an application requires a thicker base and/or a thicker overall sheet, then the base layers can be coated multiple times. In an embodiment, the thickness of the base layer ranges from about 3 μm to about 170 μm. In another embodiment, a top layer of polypropylene can be coated on the base layer while the base layer and the PP top layer solution are hot. Then, the solvent is removed from the top PP layer under vacuum or air. The top PP layer is not additionally heated but uses only the remaining heat of the base layer such that the top layer is not annealed or strengthened, retains micropores, and possesses the high desired contact angle for superhydrophobicity. Further, no heating is required to remove any solvent. In an embodiment, the total thickness of the superhydrophobic sheet coatings can range from about 10 μm to about 200 μm, or about 7 μm to about 80 μm, and the tensile strength of the superhydrophobic coatings can range from about 5 MPa to about 70 MPa. In another embodiment, the present subject matter method relates to a method of making a superhydrophobic sheet, the method comprising: dissolving polypropylene (PP) isolated from plastic waste in a solvent to form a clear polypropylene solution; pre-heating a solid substrate to a temperature below a boiling point of the solvent; applying the clear polypropylene solution onto the solid substrate followed by annealing to obtain a strong base layer; and applying another layer of the clear polypropylene solution onto the strong base layer without additional heating, thereby producing a superhydrophobic sheet. The superhydrophobic sheet can further be separated from the substrate. In an embodiment, the process can utilize about 20% of total plastic waste to prepare sheets having contact angles ranging from about 140 to about 160 degrees. The as-prepared material can demonstrate exceptional resilience to various weather conditions and can be applied to rooftops, lamp posts, high-rise walls, etc. In an embodiment, the plastic waste can come from recycled polypropylene, waste polypropylene, virgin polypropylene, or combinations thereof, and the polypropylene may comprise isotactic, atactic, syntactic, or amorphous polypropylene, or combinations thereof. In an embodiment, the solvent can be selected from the group consisting of p-xylene, m-xylene, o-xylene, an isomeric mixture of xylenes, toluene, decalin, mesitylene, other compatible aromatic hydrocarbons, and mixtures thereof. In one embodiment, the solvent is a laboratory-grade isomeric mixture of xylene. Other, similar organic solvents may be useful in this regard. The organic solvent can be used to dissolve the polymers under reflux conditions. In an embodiment, base and top layers can be formed using different coating application techniques. For example: 1. Base layer using spin coat and top layer using spin coat. 2. Base layer using slot die and top layer using slot die. 3. Base layer using spin coat and top layer using slot die. 4. Base layer using slot die and top layer using spin coat. In an embodiment, the present superhydrophobic sheets can have two layers, one is the base layer and the other is the top layer. But if an application requires a thicker base, then the base layers can be coated multiple times. Each time, the base coat must be heated. Finally, the top layer is coated, which should not be heated. In an embodiment, the process of forming the PP superhydrophobic sheets comprises dissolution of waste polypropylene(s) in an organic solvent at a temperature ranging from about 130° C. to 180° C. but below the boiling point of the solvent to form a polymer solution. In an embodiment, the ratio of the polypropylene to the organic solvent is in the range of about 1 mg/ml to about 300 mg/ml. In another embodiment, the solid substrate may be selected from the group consisting of glass, copper, silica, alumina, and another metal. In an embodiment, the solid substrate is preheated to have a surface temperature before the spin coating process begins ranging from about 0° C. to about 190° C., but below the boiling point of the solvent. In an embodiment, the polymer solution can be poured onto the solid substrate and allowed to spin coat at a speed ranging from about 100 to about 6000 rpm for a period of about 1 minute to about 15 minutes, or until the solvent is removed and a film formed. The excess polymer and solvent can be collected from a drain. Alternatively, the polymer solution can be drop cast and spread into a thin film onto the substrate using a slot die coater or doctors blade technique. After the completion of the base coating, the solid substrate with base coating can be detached from the coating device or platform and allowed to dry under vacuum or air to remove traces of organic solvents present inside the pores, if any. The thickness of the porous unheated base layer can range from about 3 μm to about 170 μm, from about 5 μm to about 100 μm, or from about 7 μm to about 40 μm. Then the base layer can be subjected to heat at a temperature ranging from about 150° C. to about 180° C. for a period ranging from about 10 seconds to about 20 minutes to eliminate pores and achieve a strong base layer. If desired, similar base layer coatings can be repeated until a required base layer thickness is achieved. In this regard, the thickness of the heated base layer can range from about 3 μm to about 170 μm, from about 3 μm to about 150 μm, from about 5 μm to about 80 μm, or from about 7 μm to about 30 μm. Lastly, a top layer of the polypropylene solution can be coated on the base layer while the base layer is hot. Then, the solvent can be removed under vacuum or air. The top layer should not be further heated. The thickness of the top layer can range from about 7 μm to about 170 μm, from about 9 μm to about 100 μm, or from about 12 μm to about 40 μm. The combined stacked layers can be peeled off collectively from the substrate using a blade, a tweezer, or forceps without further heating to achieve the polypropylene (PP) superhydrophobic sheet. The thickness of the PP superhydrophobic coating or sheet of combined base layer and top layer can range from about 10 μm to about 200 μm, from about 12 μm to about 140 μm, from about 15 μm to about 80 μm, or from about 7 μm to about 80 μm. The tensile strength of the superhydrophobic coatings can range from about 5 MPa to about 70 MPa, and the contact angle, including water contact angle, on the superhydrophobic coating surface can be in the range of about 140° to about 160°. In an embodiment, a method of making a PP superhydrophobic coating comprises dissolving polypropylene in a compatible solvent to form a hot polymer solution; stirring the hot polymer solution at an optimum temperature for a time ranging from about 10 minutes to about 40 minutes; pouring the hot polymer solution onto a solid substrate; coating the solid substrate with the hot polymer solution using a spin coating technique for a time ranging from about 2 minutes to about 15 minutes at an rpm in the range of about 100 rpm to about 6000 rpm, or until the solvent is removed from the coated layer; alternatively coating the solid substrate with hot polymer solution using a doctors blade or drop cast or slot die technique for a time ranging from about 3 seconds to about 2 minutes with a drag speed ranging from about 0.1 cm/sec to about 10 cm/sec, collecting the excess polymer and the solvent from a drain; removing the solid substrate having the base coating layer thereon from the coater or coating apparatus; subjecting the coating layer while still on the substrate to heat ranging from about 150° C. to about 180° C. for a time period ranging from about 10 seconds to about 20 minutes to achieve a heated strong base layer; coating a second layer on the strong base layer using a similar or different but suitable coating technique and allowing the solvent to dry from the coating either by vacuum or air; and peeling off the combined layers collectively from the substrate using a blade, a tweezer or forceps to obtain the superhydrophobic coating. In an embodiment, the step of applying the clear polypropylene solution onto the solid substrate followed by annealing is conducted by spin coating and comprises: a first spin coating step at a first speed for about 10 seconds to obtain a first film with a first thickness; a second spin coating step at a second speed which is higher than the first speed for about 60 seconds to obtain a second film with a second thickness; a third spin coating step at a third speed which is higher than the second speed for about 60 seconds to obtain a third film with a third thickness and ensure complete removal of the solvent; and heating the third film to obtain the strong base layer having a thickness of about 3 μm to about 150 μm. In an embodiment, the first speed is about 400 rpm, the second speed is about 1000 rpm, the third speed is about 3000 rpm, and the heating is conducted at about 170° C. In another embodiment, the step of applying another layer of the clear polypropylene solution onto the strong base layer is conducted by spin coating and results in formation of a top layer having a thickness of about 7 μm to about 170 μm. In a further embodiment, the strong base layer and the top layer can be taken together to form the superhydrophobic sheet having a thickness of about 10 μm to about 200 μm and a tensile strength of about 5 MPa to 70 MPa. In certain embodiments, for example, where multiple base layers are formed, the thickness of the superhydrophpbic sheet can be from about 20 μm to about 1 mm. By using multiple coating layers, the mechanical strength of the superhydrophobic sheet can be improved. In certain embodiments, the present methods can overcome the shortcomings of spin-coating for semi-crystalline polymers by optimizing their strong dependency on melting temperature and heating time. In further embodiments, the present methods can comprise an additional step of separating the superhydrophobic sheet from the substrate. According to this embodiment, the superhydrophobic sheet can be peeled from the substrate using a blade, a tweezer or forceps without further heating to achieve freestanding superhydrophobic films. In a further embodiment, the superhydrophobic film can be used for anti-corrosion or anti-wetting applications. Referring to the drawings,FIG.1Ashows a schematic cross-sectional view of an incomplete superhydrophobic coating laid on a solid substrate.FIG.1Arepresents a schematic cross-sectional view of base layer1of a superhydrophobic coating without strength while still porous (having pores or micropores3) and not yet heated laid on a solid substrate A base layer can be prepared from plastic waste comprising recycled polypropylene (PP), waste polypropylene recovered from waste plastic material derived from post-consumer and/or industrial waste and including virgin PP, or combinations thereof. Said plastic is dissolved in a hot suitable solvent to form a solution and formed into a thin layer on a solid substrate by using slot die, drop cast, doctors' blade, or spin coating techniques. The thickness of the porous unheated base layer1can range from about 3 μm to about 170 μm, from about 5 μm to about 100 μm, or from about 7 μm to about 40 μm. FIG.1Bshows a cross-sectional side view ofFIG.1A. FIG.2Ashows a cross-sectional view of a base coating with sufficient strength laid on a solid substrate.FIG.2Arepresents a schematic cross-sectional view of base layer4of a superhydrophobic coating laid on a solid substrate2, with strength. The as-prepared base layer without strength1was subjected to heat to a temperature ranging from about 150° C. to about 180° C. Upon heating, the base layer achieves strength by melting the polymer and crosslinking the polymeric chains by increasing the intermolecular interactions. Thus, a strong base layer4with no pores/micropores is achieved by heating base layer1having pores/micropores. The thickness of the base layer with strength4ranges from about 3 μm to about 150 μm, from about 5 μm to about 80 μm, or from about 7 μm to about 30 μm. FIG.2Bshows a schematic cross-sectional side view ofFIG.2A. FIG.3Ashows a schematic representation of heating a base coat layer laid on the solid substrate.FIG.3Arepresents heating a base coat layer1without strength laid on the solid substrate to achieve a base coat layer4with strength. FIG.3Bshows a schematic cross-sectional side view of a base coat layer and top coat layer of a superhydrophobic coating laid on the solid substrate.FIG.3Brepresents a schematic cross-sectional side view of a superhydrophobic coating6laid on the solid substrate2. The heated strong base layer4is further coated with the hot PP polymer solution comprising recycled polypropylene and/or waste propylene at a temperature ranging between about 130° C. and about 180° C. to form a top layer5. The coating is carried out using slot die, drop cast, doctors' blade, or spin coating techniques. The thickness of top layer5ranges from about 7 μm to about 170 μm, from about 9 μm to about 100 μm, or from about 12 μm to about 40 μm. The thickness of the combined base layer4and top layer5ranges from about 10 μm to about 200 μm, from about 12 μm to about 140 μm, from about 15 μm to about 80 μm, or from about 7 μm to about 80 μm. The combined base layer4and top layer5result in a superhydrophobic coating6which is not yet peeled off from the substrate2. FIG.4shows a schematic cross-sectional view of a base coat layer and top coat layer of a superhydrophobic coating laid on the solid substrate.FIG.4represents a schematic cross-sectional view of a superhydrophobic coating6on the solid substrate2. The combined strong base layer4and top layer5make up total superhydrophobic coating sheet6. SeeFIG.3Bfor a cross sectional side view. FIG.5Ashows a schematic representation of base layer and top layer of a formed and completed superhydrophobic coating sheet separated collectively from the solid substrate.FIG.5Arepresents a schematic cross-sectional view of a superhydrophobic coating6separated from the solid substrate2. The combined strong base layer4and top layer5were peeled off from the substrate using forceps, tweezers, or a blade. The thickness of top layer5ranges from about 7 μm to about 170 μm, from about 9 μm to about 100 μm, or from about 12 μm to about 40 μm. The thickness of the combined base layer4and top layer5ranges from about 10 μm to about 200 μm, from about 12 μm to about 140 μm, from about 15 μm to about 80 μm, or from about 7 μm to about 80 μm. The combined strong base layer4and top layer5resulted in a superhydrophobic coating6peeled off from the substrate2to achieve a superhydrophobic coating. FIG.5Bshows a SEM image of superhydrophobic sheet showing top layer and base layer.FIG.5Brepresents an SEM image of a superhydrophobic sheet with both base layer and top layer. The base layer and top layer comprise polypropylene. The base layer is not porous and the top layer is porous with roughness. FIG.6shows a contact angle of water with superhydrophobic sheets.FIG.6represents the contact angle of water over the surface of superhydrophobic sheets. The angle was measured to be 148°. The contact angle on the superhydrophobic surface is in the range of 140°-160°. FIG.7Shows water droplets on a superhydrophobic coating showing superhydrophobicity.FIG.7represents the superhydrophobicity of the superhydrophobic sheets that do not allow the water to stick to the surface. The water contact angle is in the range of 140°-160°. EXAMPLES Example 1: 100 gm of waste polypropylene was placed in a round bottomed flask and dissolved in one liter of xylene under reflux conditions at a temperature of 130° C. for a time of 20 minutes. A clean glass plate of sides 5 cm each was taken and heated to a temperature of 110° C. and was placed on a spin coater chuck. The hot polymer solution was poured on to the glass plate and spin coated at a gradient speed of 400 rpm for 10 seconds, followed by 1000 rpm for 60 seconds, followed by 3000 rpm for 60 seconds. The excess solvent and polymer solution was collected from the drain. After completion of the spin coating, the substrate was separated from the chuck and the base layer was heated to a temperature of 170° C. for a period of 5 minutes on a Heidolph hot plate. Then the hot glass plate with polymer base layer was placed on the chuck and the hot polypropylene solution was poured on the hot base layer to form a top layer and allowed to spin coat at similar speed and time. After the spin coating, the glass substrate with combined top and base layers were detached from the chuck and the coatings were collectively separated from the substrate resulting in a superhydrophobic sheet. Example 2: If there is a requirement of superhydrophobic sheet with a thickness of 200 μm, one base layer and one top layer is insufficient. To achieve this, multiple base coating layers must be applied to achieve a final thick base layer. Then, finally a top layer is coated. For example, if each layer gives a thickness of 40 μm, the final base layer should have a thickness of 160 μm and top layer thickness as 40 μm. The following steps are followed to obtain such a superhydrophobic coating: (a) a first layer with 40 μm is achieved, followed by heating, (b) a second coating of 40 μm is coated achieving 80 μm thickness, followed by heating, (c) a third coating of 40 μm is coated achieving 120 μm thickness, followed by heating, (d) a fourth coating of 40 μm is coated achieving 160 μm thickness, followed by heating, where the final base layer is formed, and (e) a fifth and last layer, the top layer, of 40 μm is coated achieving a total coating or sheet thickness of 200 μm, thus, achieving a superhydrophobic sheet with a thickness of 200 μm. It is to be understood that the PP superhydrophobic sheets, use, method of making, and properties of said sheets are not limited to the specific embodiments or examples described above, but encompasses any and all embodiments within the scope of the generic language of the following claims enabled by the embodiments described herein, or otherwise shown in the drawings or described above in terms sufficient to enable one of ordinary skill in the art to make and use the claimed subject matter. | 26,435 |
11859066 | DETAILED DESCRIPTION In a first aspect, the invention relates to the use of a mineral of the general formula ABX3, A and B being cations and X being anions, wherein the mineral has perovskite crystal structure (in the following “mineral having perovskite structure”, or “perovskite”) in vinyl aromatic polymer foam. The foam further comprises one or more athermanous additives selected from a) powder inorganic additive selected from powders of silica and calcium phosphate, b) powder carbonaceous additive selected from powders of graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene, and c) powder geopolymer and powder geopolymer composite. According to a first embodiment of the first aspect, the invention relates to the use of mineral having perovskite structure for decreasing the thermal conductivity of the vinyl aromatic polymer foam, the decrease being measured according to ISO 8301. According to a second embodiment of the first aspect, the present invention relates to the use of mineral having perovskite structure for improving the mechanical properties, specifically for increasing compressive strength and bending strength, of vinyl aromatic polymer foam, the increase in compressive strength and in bending strength being measured in accordance with EN 13163. According to a third embodiment of the first aspect, the present invention relates to the use of mineral having perovskite structure for improving the self-extinguishing properties of vinyl aromatic polymer foam, the improvement being measured in accordance with EN ISO 11925-2. Perovskite reduces flame development by the creation of char with higher viscosity and thus reduces dripping and flaming. Preferably, the improvement of the self-extinguishing properties is an improvement as measured in accordance with DIN 4102 B1, B2. The preferred concentration of mineral having perovskite structure in the vinyl aromatic polymer foam, for i) the decrease of the thermal conductivity, ii) the improvement of the specified mechanical properties, and iii) the increase of the self-extinguishing properties, is in a range of from 0.01 to 50 wt. %, based on the weight of vinyl aromatic polymer in the granulate including solid and, if any, liquid additives, but exclusive of propellant, more preferably 0.05 to 25 wt. %, most preferably 0.1 to 15 wt. %, in particular 0.5 to 12 wt. %, such as 1 to 8 wt. %. The mineral having perovskite structure as used in accordance with the invention has a crystalline structure of general formula of ABX3, where A and B are two cations of different sizes and X is an anion that bonds to both, the A atoms are larger than the B atoms, and its ionic radii close to that on the anion X thus they can form together a cubic (orthorhombic) close packing with space group Pm3m. In the structure, the B cation is 6-fold coordinated and A cation 12-fold coordinated with the oxygen anions. A ideal cubic perovskite structure has cations A at the corners of the cube, and cation B in the centre, with oxygen ions in the face-centered positions, as shown inFIG.1(structure of the ideal cubic perovskite (ABO3), where A represents cation, B represents cation, and O represents oxygen anions forming an octahedron). For the stoichiometric oxide perovskites, the sum of the oxidation states of cations A and B should be equal to 6. Preferably, A is selected from the group consisting of Ca, Sr, Ba, Bi, Ce, Fe, and mixtures thereof. Moreover, the A atom can be represented also by hybrid organic-inorganic groups, e.g. (CH3NH3)+. Among the most preferred representatives of perovskite struc-tures are dielectric BaTiO3, high-temperature semiconductor YBa2Cu3O7-x, materials exhibiting magnetoresistance R1-xAxMnO3, where R=La3+, Pr3+or other earth ion, A=Ca2+, Sr2+, Ba2+, Bi2+, Ce2+, and multiferroic materials. The B atom is preferably represented by Ti, Zr, Ni, Al, Ga, In, Bi, Sc, Cr, Pb as well as ammonium groups. The X atom is preferably represented by oxygen or halide ion, or mixtures thereof. Perovskites have large reflectance properties in the broad wavelength and a high optical constant, even in the far-infrared region. Hence, perovskites are infrared reflective materials that reflect infrared rays included in sunlight or the like and reduce the amount of absorbed energy in the infrared range. A preferred perovskite has a BET surface size in a range of from 0.01 to 100 m2/g, as measured according to the standards ASTM C1069 and ISO 9277, as explained below. The method to determine the mineral having perovskite structure's BET surface is preferably based on the standards ASTM C1069 and ISO 9277 and is conducted as follows: in the first step, 2 to 5 g of sample are dried at 105° C. and placed in a desiccator for cooling and further degassing. Subsequently, 0.3 to 1.0 g of the dry material is weighed into a test tube and placed in the degassing unit for about 30 min. Afterwards, the sample is transferred to the measuring unit and is measured using the Micromeritics Tristar 3000 instrument. The BET active surface is preferably in a range of from 0.05 to 50 m2/g and more preferable in a range of from 0.1 to 15 m2/g. Typical perovskites have an average particle size in a range of from 0.01 to 100 μm, as measured according to the standard procedure using a Malvern Mastersizer 2000 apparatus. The average particle size is preferably in a range of from 0.1 to 50 μm, more preferably in a range of from 0.5 to 30 μm. Average particle size in the description of the present invention means median primary particle size, D(v, 0.5) or d(0.5), and is the size at which 50% of the sample is smaller and 50% is larger. This value is also known as the Mass Median Diameter (MMD) or the median of the volume distribution. Furthermore, it is preferred that i) the thermal conductivity, ii) the mechanical and iii) the self-extinguishing properties of the polymer foam are improved by the use of minerals with perovskite structure having:i) an average particle size, as determined by laser diffraction, in the range of from 0.01 to 600 μm. In a further preferred embodiment, the mineral having perovskite structure has a thermal conductivity of less than 10 W/m·K, preferably 5 W/m·K or less (300° C.). It is further preferred that the mineral having perovskite structure has a moisture content in a range of from 0.01 to 3.0 wt. %, preferably in a range of from 0.05 to 1.5 wt. %. The polymer used in accordance with the invention is based on one (or more) vinyl aromatic monomer(s), preferably styrene, and optionally one or more comonomers, i.e. it is a homopolymer or a copolymer. The polymer used in accordance with all aspects of the invention is based on one (or more) vinyl aromatic monomer(s), preferably styrene, and optionally one or more comonomers, i.e. it is a homopolymer or a copolymer. The addition of a co-monomer of a specific styrene comonomer possessing steric hindrance, in particular p-tert-butylstyrene, or alpha-methyl styrene comonomer, or some other sterically hindered styrene comonomer, to styrene, may advantageously increase the glass transition temperature of such a vinyl aromatic copolymer. In such a manner, the addition of a specific styrene comonomer to the styrene monomer improves the thermal stability of vinyl aromatic copolymer, which subsequently leads to better dimensional stability of moulded blocks made thereof. The vinyl aromatic copolymer as used in the present invention is preferably comprised of 1 to 99 wt. % of styrene monomer and correspondingly 99 to 1 wt. % of p-tert-butylstyrene monomer, as follows (amounts in wt. %, based on the total amount of monomer): PreferredMore preferredMost preferredMonomer(wt. %)(wt. %)(wt. %)Styrene1-9950-9970-98p-tert-Butyl styrene99-11-5030-2 Alternatively, the vinyl aromatic copolymer as used in the present invention is preferably comprised of 1 to 99 wt. % of styrene monomer and correspondingly 99 to 1 wt. % of alpha-methyl styrene monomer, as follows (amounts in wt. %, based on the total amount of monomer): PreferredMore preferredMost preferredMonomer(wt. %)(wt. %)(wt. %)Styrene1-9950-9875-95alpha-Methyl styrene99-12-5025-5 In addition to the mineral having perovskite structure, the materials according to the invention (the polymer composition, the granulate, the foam and the masterbatch) may contain further additives, as is set out below. The polymer foam further comprises one or more athermanous additives selected from a) powder inorganic additive (other than mineral having perovskite structure), b) powder carbonaceous additive, and c) powder geopolymer or powder geopolymer composite. The powder inorganic additive is selected from powders of silica and powders of calcium phosphate. The powder carbonaceous additive is selected from powders of graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides and graphene. Silica The silica as preferably used in accordance with the invention is amorphous and has the following specific properties:(i) a BET surface of from 1 to 100 m2/g, and(ii) an average particle size in a range of from 3 nm to 1,000 nm. The method to determine the silica's BET surface is the method for the determination of BET described above. The silica as preferably used according to the invention has a BET surface in a range of from 3 to 80 g/m2, more preferably 5 to 70 m2/g, most preferably 8 to 60 m2/g, such as 10 to 50 m2/g, in particular 13 to 40 m2/g, or 15 to 30 m2/g, such as about 20 m2/g. Moreover, the silica as preferably used according to the present invention is defined by an average particle size, as measured according to the procedure detailed below, of 3 nm to 1000 nm. The method to determine the average particle size is conducted as follows: in the first step, 45 g of distilled water and 5 g of sample are placed into a beaker and stirred to allow the entire sample to be wetted. Subsequently, the sample is dispersed in an external ultrasonic probe for 5 min at 100% amplitude. The measurement is performed automatically using the primary agglomerate program in a Malvern MasterSizer 2000 device. It is preferred that the average particle size of the silica as preferably used according to the present invention is within a range of 20 to 800 nm, preferably 30 to 600 nm, such as 40 to 400 nm, in particular from 100 to 200 nm. According to the present invention, the silica is preferably used in an amount of from 0.01 to less than 2 wt. %, based on the weight of the polymer (inclusive of solid and, if any, liquid additives, but exclusive of propellant). More preferably, the silica is used in an amount of 0.1 to 1.6 wt. %, most preferably 0.5 to 1.5 wt. %, such as about 1.0 wt. %, based on the weight of the vinyl aromatic polymer (inclusive of solid and, if any, liquid additives, but exclusive of propellant). The silica as preferably used according to the invention is amorphous (i.e. non-crystalline) silicon dioxide, and the silica particles are preferably spherically shaped. It is most preferred that the silica comprises Sidistar type of material from ELKEM, typically with an average primary particle size of about 150 nm and a low BET surface area of about 20 m2/g, and most preferred is that a) is Sidistar T120. Calcium Phosphate The calcium phosphate as typically used according to the invention has an average particle size, as measured by laser diffraction, of 0.01 μm to 100 μm. It is preferred that the average particle size is from 0.1 μm to 50 μm, such as 0.5 μm to 30 μm. The calcium phosphate is preferably tricalcium phosphate (specifically a type of hydroxyapatite). According to the present invention, the calcium phosphate, if present, is preferably used in an amount of from 0.01 to 50 wt. %, based on the weight of vinyl aromatic polymer including solid and, if any, liquid additives, but exclusive of propellant, more preferably 0.1 to 15 wt. %, most preferably 0.5 to 10 wt. %, in particular 1 to 8 wt. %. Moreover, b) carbon-based athermanous additives can be present in the foam, such as graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene. Graphite The graphite as preferably used in the present invention has the following properties:(i) a carbon content in a range of from 50 to 99.99 wt. % and(ii) a particle size in a range of from 0.01 to 100 μm. Preferably, the graphite's carbon content is in a range of from 95 to 99.9 wt. % and more preferably over 99.5 wt. %. Preferably, the carbon content is measured according to the method L-03-00A of the company GK. The graphite as preferably used according to the invention has a particle size in a range of from 0.01 to 100 μm, preferably as measured according to method L-03-00 of the company GK, which is a laser diffraction method using a Cilas 930 particle size analyzer equipment. It is preferred that the particle size of the graphite as used according to the invention is from 0.1 to 30 μm. The most preferred particle size range is from 0.5 to 25 μm, in particular from 1 to 10 μm; specifically, for example, a range of from 3 to 8 μm. The graphite's mean particle size is preferably in a range of from 5 to 7 μm, D90 in a range of from 7 to 15 μm, and D100 in a range of from 15 to 20 μm. The sulphur content of the graphite as preferably used according to the invention is in a range of from 10 to 2000 ppm, as measured according to ASTM D1619, more preferably from 100 to 1500 ppm, in particular from 400 to 1000 ppm. The ash content of the graphite as preferably used according to the invention is in a range of from 0.01 to 2 wt. %, preferably from 0.1 to 1 wt. %, in particular below 0.5 wt. %. The ash content is preferably measured according to method L-02-00 of the company GK. The moisture content of the graphite as preferably used according to the invention is in a range of from 0.01 to 1 wt. %, preferably from 0.1 to 0.5 wt. %, in particular below 0.4 wt. %. The moisture content is preferably measured according to a method of the company GK (L-01-00). The graphite is preferably used according to the invention in an amount of 0.01 to 10 wt. %, based on the weight of the vinyl aromatic polymer (inclusive of solid and, if any, liquid additives, but exclusive of propellant), preferably in a range of from 1.0 to 8.0 wt. %, more preferably in a range of from 1.5 to 7.0 wt. %, in particular in a range of from 2.0 to 6.0 wt. %, such as in a range of from 2.5 to 5.0 wt. %, e.g. in a range of from 3 to 4 wt. %. More preferably, a) the silica and b) the graphite are used in a weight ratio a):b) in a range of from 1:1.5 to 1:8, most preferably a) the silica and b) the graphite are used in a weight ratio a):b) in a range of from 1:2 to 1:5, in particular a) the silica and b) the graphite are used in a weight ratio a):b) of about 1:3. The best performance in foams in terms of i) decrease of thermal conductivity, ii) increase in mechanical properties and iii) improvement in self-extinguishing properties is achieved, accompanied by a reduction in the required content of graphite, when (in addition to the mandatory mineral having perovskite structure as athermanous additive) silica and graphite are present, specifically Sidistar T120 from Elkem is used in combination with the natural graphite CR5995 from GK, in a weight ratio of about 1:3, as further athermanous additives. Then it is possible to reduce the graphite content to about 3 wt. %, and to maintain the thermal conductivity at the same level as if 5 to 6% of graphite were used, whilst the mechanical properties are significantly improved, as compared to foam containing from 5 to 6 wt. % of graphite without addition of Sidistar T120 and/or a mineral having perovskite structure. Carbon Black The carbon black as preferably used according to the invention has a BET surface, as measured according to ASTM 6556, of more than 40 to 250 m2/g. It is preferred that the BET surface of the carbon black as used according to the invention is from 41 to 200 m2/g, preferably from 45 to 150 m2/g, in particular from 50 to 100 m2/g. The sulphur content of the carbon black as preferably used according to the invention is in the range of from 50 to 20,000 ppm, as measured according to ASTM D1619, preferably from 3,000 to 10,000 ppm. The carbon black is preferably present in an amount of 0.1 to 12 wt. %, based on the weight of the vinyl aromatic polymer including additives, but exclusive of propellant, preferably 0.2 to 12.0 wt. %, more preferred 0.5 to 9.0 wt. %, such as 1.0 to 8.0 wt. %, in particular 2.0 to 7.0 wt. %, such as 3.0 to 6.0 wt. %, e.g. about 5.0 wt. %. c) Geopolymer and Geopolymer Composite It has further been found that it is possible to maintain the foam's self-extinguishing and mechanical properties in the same range as in an expanded polymer without addition of filler or any other athermanous additive, while at the same time the thermal conductivity can be decreased significantly, namely by addition of c) a geopolymer, or a geopolymer composite prepared from geopolymer and various types of athermanous fillers. This is possible because the geopolymer itself gives fire resistance, and may in the composite encapsulate the particles of athermanous additive, especially those additives based on carbon, and separates them from any interactions with the flame, the polymer or the flame retardant. Geopolymer and geopolymer composite further decrease thermal conductivity, based on a heat radiation scattering effect. Geopolymer synthesis from aluminosilicate materials takes place by the so-called geopolymerization process, which involves polycondensation phenomena of aluminates and silicate groups with formation of Si—O—Al-type bonds. In a preferred embodiment, geopolymers encapsulate carbon-based athermanous fillers in a matrix and limit the contact (interphase) between carbon-based filler and brominated flame-retardants, including those based on polystyrene-butadiene rubbers. This phenomenon allows a significant decrease of the required concentration of brominated flame retardant in expandable vinyl aromatic polymer composites. A preferred geopolymer composite is prepared by a process wherein an athermanous additive component is present during the production of geopolymer composite, so that the geopolymer composite incorporates the athermanous additive component. Preferably, this athermanous additive component comprises one or more athermanous additives selected from the group consisting ofa. carbon black, petroleum coke, graphitized carbon black, graphite oxides, various types of graphite (especially poor and amorphous forms with a carbon content in the range of from 50 to 90%) and graphene, andb. titanium oxides, ilmenite, rutiles, chamotte, fly ash, fumed silica, hydromagnesite/huntite mineral, barium sulfate and mineral having perovskite structure,preferably the athermanous additive component of the geopolymer composite comprises one or more carbon-based athermanous additives selected from the group of heat absorbers and heat reflectors,in particular the athermanous additive component is carbon black, graphite, or a mixture thereof. Further details of the preparation of geopolymer composite may be found in the international application entitled “Geopolymer and composite thereof and expandable vinyl aromatic polymer granulate and expanded vinyl aromatic polymer foam comprising the same”, PCT/EP2016/050594, filed on even date herewith. In the following, the further athermanous fillers that are present, namely one or more of a) powder inorganic additive selected from powders of silica and calcium phosphate, b) powder carbonaceous additive selected from powders of graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene, and c) powder geopolymer and powder geopolymer composite, will be referred to as additional athermanous fillers or additives. The foam also preferably comprises one or more of nucleating agent, flame retardant, synergist, thermal oxidative stabiliser, flame retardant thermal stabiliser, and dispersion aid. For instance, the flame retardant system is, especially in an extrusion process, usually a combination of two types of compounds, namely x) a brominated aliphatic, cycloaliphatic, aromatic or polymeric compound containing at least 50 wt. % of bromine, and a second compound (so called synergistic compound, y) which can be bicumyl (i.e. 2,3-dimethyl-2,3-diphenylbutane) or 2-hydroperoxy-2-methylpropane, or dicumyl peroxide, cumene hydroxide, or 3,4-dimethyl-3,4-diphenylbutane. The total content of flame retardant system, i.e. x) plus y), is typically in a range of from 0.1 to 5.0 wt. % based on the weight of vinyl aromatic polymer including solid and, if any, liquid additives, but exclusive of propellant, preferably between 0.2 and 3 wt. %. The weight-to-weight ratio of bromine compound x) to synergistic compound y) is preferably in a range of from 1:1 to 15:1, usually in a range of from 3:1 to 10:1, in particular from 2:1 to 7:1. In a further aspect, the invention relates to (II) processes for the preparation of expandable polymer granulate. The granulate according to the invention comprises one or more additional athermanous additives selected from a) powder inorganic additive selected from powders of silica and calcium phosphate, b) powder carbonaceous additive selected from powders of graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene, and c) powder geopolymer and powder geopolymer composite. In a first embodiment (IIa), the process is a process for the preparation of expandable polymer granulates comprising the following steps:i) feeding vinyl aromatic polymer into an extruder,ii) adding mineral having perovskite structure and optionally thermal stabiliser and flame suppressant,iii) injecting blowing agent into the melt of polymer,iv) extruding the homogenous blend, andv) pelletizing the blend in an underwater pelletizer, so as to obtain the granulate. Preferably, the extrusion process (IIa) comprises the steps:i) feeding a first polymer component comprising vinyl aromatic polymer into a first mixer;ii) feeding a first additive component a) into the first mixer, to produce a first mixture from the first polymer component and the first additive component;iii) feeding a second polymer component b) comprising vinyl aromatic polymer into a second mixer;iv) feeding a second additive component b) into the second mixer, to produce a second mixture from the second polymer component and the second additive component, wherein the processing conditions in the second mixer are more severe than the processing conditions in the first mixer, by providing higher shear force;v) combining the first and second mixtures, to produce a third mixture;vi) injecting blowing agent c) into the third mixture, to produce a fourth mixture;vii) mixing the fourth mixture; andviii) pelletizing the fourth mixture, to obtain the granulate. The first polymer component can be a vinyl aromatic polymer having a melt index from 4 to 20 g/10 min, as measured according to ISO 1133. The second polymer component can be a vinyl aromatic (e.g. styrene) homopolymer (or preferably copolymer with p-tert butyl styrene or alpha-methyl styrene), having a melt index ranging from 4 to 30 g/10 min, as measured according to ISO 1133. According to this first and preferred embodiment of the second aspect, the invention allows for the separate addition of first and second additive components into a mixture that is ultimately charged with propellant and is pelletized, so as to obtain the expandable granulate. Because of the separate addition of the first and second additive components, the process is highly flexible and allows for the processing of additives that have very different processing requirements, in particular in view of their stability under those processing conditions that are necessary so that the different additive components can best perform their desired function. Typically, at least a part (and preferably all) of the mineral having perovskite structure is introduced as part of the second additive component in this extrusion process, whereas at least a part (and preferably all) of the flame retardant system is introduced as part of the first additive component in this extrusion process. This is advantageous since the flame retardant system typically requires more moderate processing conditions, in particular as compared to mineral having perovskite structure. Thus, according to the invention, a mixture comprising mineral having perovskite structure can be prepared in a dedicated mixer that provides for the high shearing that is preferred for this type of additive, so that it is properly dispersed. As a first alternative, the second additive components (e.g. the mineral having perovskite structure and the additional athermanous filler) can be mixed with polymer, in equipment that provides for high shearing and good dispersion, and the obtained mixture is directly, i.e. as a melt, combined with the mixture containing the first additive components, to give a mixture that is ultimately charged with blowing agent. As a second alternative, the second additive components (e.g. the mineral having perovskite structure and the additional athermanous filler) can be mixed with polymer and be provided as a masterbatch. Such a masterbatch is advantageous in case the plant design does not allow for the processing conditions that are preferable for the mineral having perovskite structure, e.g. high shearing conditions. The masterbatch can for instance be prepared off-site, in dedicated processing equipment, and having to provide such processing equipment on site can be dispensed with. The masterbatch comprising the mineral having perovskite structure and the additional athermanous filler is subject of the fifth aspect of the present invention, and is described below. According to a second embodiment of the second aspect (IIb), expandable polymer granulates is prepared in an aqueous suspension polymerization process comprising the steps:i) adding a vinyl aromatic monomer and optionally one or more comonomers to the reactor, and subsequently addingi1) optional polymeric suspension aid,i2) athermanous fillers (mineral having perovskite structure and additional athermanous additive),i3) flame retardant,i4) at least one peroxide (or the mixture of two or more peroxides) as reaction initiator,ii) adding the demineralised water, andii1) at least one suspending agent which is an inorganic acid salt,ii2) at least one peroxide (or the mixture of two or more peroxides) as reaction initiator,ii3) at least one suspension stabilizer selected from the group of anionic surface active compounds and/or high molecular weight compounds (e.g. hydrophilic and/or amphiphilic polymers), andiii) continuing the polymerization (preferably until the concentration of vinyl aromatic monomer(s) is below 1000 ppm by weight, based on the weight of the polymer),iv) adding the blowing agent during or after the polymerization step,v) cooling, and then separating the granulate from the water. The athermanous filler that is mandatory according to the present invention (namely mineral having perovskite structure) may be added in the form of a masterbatch, it may be introduced at the beginning of the suspension polymerization process, or may be dissolved in the monomer and/or a mixture of the monomer and comonomer. The same applies for the additional athermanous fillers, a), b), and c) as mentioned above. According to the present invention, the mineral having perovskite structure and the additional athermanous filler are introduced as athermanous fillers i2), and they may also be introduced in step ii) and/or in step iii) of this suspension process. The polymer granulate is prepared using well known inorganic salts of phosphoric acid, such as types of calcium phosphate, magnesium phosphate, or a combination of salts as suspending agents. These salts may be added to the reaction mixture in a finely divided form, or as a product of an in situ reaction (for example, between sodium phosphate and magnesium sulphate). The salts are supported in their suspending action by anionic surface-active compounds, such as sodium dodecylobenzene sulfonate or sodium poly(naphthalene formaldehyde) sulfonate. Those surface-active compounds can be also being prepared in situ using their precursors such as sodium metabisulfite and potassium persulfate. The suspension can be also stabilized by high molecular weight organic polymers, such as polyvinyl alcohol or hydroxyethylcellulose or hydroxypropylmethyl-cellulose. To improve the stability of the suspension, up to 30 wt. % of polymer (fresh vinyl aromatic polymer or waste vinyl aromatic polymer from a previous polymerization) may be added as the optional suspension aid, preferably 5 to 15 wt. %, based on the vinyl aromatic monomer amount. It increases the viscosity of the reagent mixture (monomer with all additives), which facilitates the creation of a suspension. The same or similar effect can be achieved by mass pre-polymerization of the monomer or mixture of comonomers and additives until the suitable melt viscosity is obtained (as for 1% to 30% of polymer concentration). In the most preferred process, before start of the polymerization step iii), athermanous fillers in the form of a concentrated masterbatch are added to the styrene and/or its mixture with comonomer, particularly p-tert-butylstyrene. The masterbatch can contain from 10 to 60% of athermanous fillers (i.e. the mineral having perovskite structure, and the additional ones, a), b) and c)), pre-silanized or silanized in the masterbatch compounding process by e.g. triethoxy(phenyl)silane, to decrease its hydrophilic properties. The polymerization is then continued in an aqueous suspension phase, in the presence of the above-mentioned suspending agents, suspension stabilizers, athermanous fillers, flame retardants and flame suppressors, optionally at least in the presence of suspension aid. The polymerization process is triggered by initiators. Normally, two organic peroxides are used as initiators. The first peroxide, with a half-life of about one hour at 80-95° C., is used to start and run the reaction. The other, with a half-life of about one hour at 105-125° C., is used during the following polymerization process continued at higher temperature, in the so called high temperature cycle (HTC). For the above specific process with the presence of carbon black, a composition of three peroxides was used to achieve suitable average molecular weight despite the negative inhibiting effect caused by the carbon black's presence. Preferably were used: dicumyl peroxide and tert-butylperoxy-2-ethyl hexyl carbonate peroxide as high temperature cycle peroxides (120° C.) and tert-butyl 2-ethylperoxyhexanoate as low temperature cycle peroxide (82-90° C.). The end of the process is typically indicated by a concentration of residual vinyl monomer(s) of below 1000 ppm by weight, based on the mass of vinyl aromatic polymer or copolymer. The vinyl aromatic polymer or copolymer which is obtained at the end of the process typically has an average molecular mass (Mw) ranging from 50 to 600 kg/mol, preferably from 150 to 450, most preferably from 100 to 350 kg/mol. The procedure for controlling molecular mass in suspension polymerization is well known and is described in detail in Journal of Macromolecular Science, Review in Macromolecular Chemistry and Physics C31 (263) p. 215-299 (1991). During the polymerization process, conventional additives can be added directly to the monomer(s), their solution with suspension aid, to the pre-polymer, or to the suspension. Additives such as the flame retardant system, nucleating agents, antistatic agents, blowing agents and colorants stay in the polymer drops during the process and are thus present in the final product. The concentrations of conventional additives are the same as for the extrusion process, as set out above. The flame retardant systems suitable for the present suspension process are similar to those used in the extrusion process described above. One suitable system is the combination of two types of compounds, namely a brominated aliphatic, cycloaliphatic, aromatic or polymeric compound containing at least 50 wt. % of bromine (such as hexabromocyclododecane, pentabromomonochlorocyclohexane, or a polymeric bromine compound, specifically brominated styrene-butadiene rubber) and a second compound called synergistic compound which can be e.g. an initiator or peroxide (e.g. dicumyl peroxide, cumene hydroxide, and 3,4-dimethyl-3,4-diphenylbutane). The content of flame retardant system is typically in a range of from 0.1 to 5.0 wt. % with respect to the total weight of vinyl aromatic polymer (weight of monomer(s) plus weight of polymer if added on the start), preferably between 0.2 and 3 wt. %. The ratio between bromine compound and synergistic compound is preferably in a range of from 1:1 to 15:1 weight to weight, usually from 3:1 to 5:1. The blowing agent or agents are preferably added during the polymerization to the suspension phase and are selected from aliphatic or cyclic hydrocarbons containing from 1 to 6 carbons and their derivatives. Typically are used n-pentane, cyclopentane, i-pentane, combination of two of them or their mixture. In addition, the halogenated aliphatic hydrocarbons or alcohols containing from 1 to 3 carbons are commonly used. The blowing agent or agents can also be added after the end of polymerization. At the end of the polymerization, spherical particles of expandable styrenic polymer are obtained as granulate, with an average diameter range of 0.3 to 2.3 mm, preferably from 0.8 to 1.6 mm. The particles can have different average molecular mass distribution, depending on their size, but all contain used additives dispersed homogenously in the polymer matrix. In the final step after the HTC step, the mass is cooled down to e.g. 35° C., and the polymer granulate is separated from the water, preferably in a centrifuging process. The particles are then dried and preferably coated with a mixture of mono- and triglycerides of fatty acids and stearic acid salts. After discharging the particles from the reactor, they are typically washed: first with water, then with non-ionic surfactant in aqueous solution, and finally again with water; they are then desiccated and dried with hot air having a temperature in the range 35-65° C. The final product is typically pre-treated by applying a coating (the same as for the extruded granulate) and can be expanded by the same method as the extrusion product. According to a third embodiment of the second aspect (IIc), expandable polymer granulate is prepared in a continuous mass process comprising the following steps:i) providing continuously to a mass prepolymerization reactor (or the first from a cascade reactor) a stream of:i1) vinyl aromatic monomer and optionally at least one co-monomer (preferably p-tert-butylstyrene),i2) at least one additive solution, andi3) optionally recycled monomer,ii) continuing polymerization in the prepolymerization reactor or the sequence of cascade reactors,iii) adding athermanous fillers (mineral having perovskite structure and additional athermanous additive),iv) degassing the polymer,v) feeding the polymer in molten state into the extruder, preferably directly from the polymerization plant,vi) optionally adding a flame retardant system including synergist and thermal stabilisers,vii) injecting the blowing agent,viii) extruding the homogenous polymer mixture, andix) pelletizing in an underwater pelletizer, so as to obtain the granulate. The reactor or cascade reactor is preferably arranged horizontally. If a cascade reactor is used, then there are preferably up to 5 reactors, in particular up to 4, such as three reactors. The continuous mass polymerization is process congruous to the extrusion process, but the vinyl aromatic polymer or copolymer together with athermanous fillers is used in a molten state and the extruder is fed directly by the polymerization plant. The mass polymerization reactor (or first from cascade reactors) is fed continuously by vinyl aromatic monomer, particularly styrene, and optionally by its vinyl aromatic comonomer, for instance p-tert-butylstyrene. At this stage, athermanous fillers in the form of a masterbatch or in the form of powders are fed into the mass polymerisation reactor, one or more additives and optionally recycled monomer recovered from the process. The athermanous additives (e.g. masterbatches) are preferably dissolved in the vinyl aromatic monomer or before feed to the polymerization reactor. The polymerisation reaction is initiated thermally, without addition of initiators. In order to facilitate heat collection, polymerisation is generally carried out in the presence of for instance monocyclic aromatic hydrocarbon. The prepolymerised mass from the pre-polymerisation reactor is pumped through the sequence of several horizontal reactors, and the polymerisation reaction is subsequently continued. At the end of the mass polymerization stage, the rest of unpolymerized monomer is removed by degassing of the melt. A vinyl polymer in the molten state, produced in mass polymerization and containing athermanous fillers, is fed into an extruder at a temperature in a range of from 100 to 250° C., preferably from 150 to 230° C. In the next stage, the flame retardant system and the nucleating agent are fed to the polymer melt. Again, a combination of two types of flame retarding compounds can be used, namely a brominated aliphatic, cycloaliphatic, aromatic or polymeric compound containing at least 50 wt. % of bromine, and a second compound called synergistic compound, which can be bicumyl (2,3-dimethyl-2,3-diphenylbutane) or 2-hydroperoxy-2-methylpropane. The concentrations of additives are typically the same as for the extrusion process, as set out above. In the following step, the blowing agent is injected into the molten polymer mixture and mixed. The blowing agent or agents are the same as for the suspension process, i.e. selected from aliphatic or cyclic hydrocarbons containing from 1 to 6 carbons and their derivatives. The polymer with all additives and blowing agent is subsequently extruded to give expandable beads. The homogenous polymer mixture comprising additives and blowing agent is pumped to the die, where it is extruded through a number of cylindrical die holes with 0.5-0.8 mm of diameter, immediately cooled by a water stream and cut with a set of rotating knives in a pressurized underwater pelletizer, to obtain micropellets (granulate). The micropellets are transported by water, washed, drained off and fractioned. The final product is pre-treated in the same way as it is in the suspension and extrusion processes. In a further aspect, the invention relates to (III) expandable polymer granulate comprising one or more propellants, x) mineral having perovskite structure and y) polymer of vinyl aromatic monomer and optionally one or more comonomers. Preferably, the expandable polymer granulate is obtainable (and is more preferably obtained) by the process according to the second aspect. The expandable polymer granulate further comprises one or more of the additional athermanous additives a), b) and c) above. Specifically, the expandable polymer granulate further comprises one or more additional athermanous additives selected from a) powders of silica and calcium phosphate, b) powders of graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene, and c) powder geopolymer and powder geopolymer composite. In a further aspect, the invention relates to (IV) expanded vinyl aromatic polymer foam comprising x) mineral having perovskite structure and y) polymer of vinyl aromatic monomer and optionally one or more comonomers. The expanded polymer foam hasa density of 8 to 30 kg/m3, anda thermal conductivity of 25-35 mW/K·m. The foam further comprises one or more athermanous additives selected from a) powder inorganic additive selected from powders of silica and calcium phosphate, b) powder carbonaceous additive selected from powders of graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene, and c) powder geopolymer and powder geopolymer composite. Preferably, the expanded polymer foam is obtainable and is more preferably obtained by expansion of the expandable polymer granulate according to the third aspect. According to the fifth aspect, the invention relates to (V) a masterbatch. The masterbatch comprises x) mineral having perovskite structure and y) vinyl aromatic polymer, and the amount of x) is in a range of from 10 to 70 wt. %, based on the weight of the masterbatch. Preferably, the amount of mineral having perovskite structure x) is in a range of from 10 to 65 wt. %, based on the weight of the masterbatch, more preferably from 20 to 60 wt. %, in particular from 25 to 55 wt. %. In a preferred embodiment, y) is a vinyl aromatic polymer having a melt index in a range of from 4 to 30 g/10 min, as measured according to ISO 1133, and the vinyl aromatic polymer is preferably a homopolymer or copolymer with p-tert butyl styrene or alpha-methyl styrene. The masterbatch, in addition to components x) mineral having perovskite structure and y) vinyl aromatic polymer, further comprises one or more of the additional athermanous additives a) to c). Additional athermanous fillers that are present in the masterbatch are one or more powders of a) silica and calcium phosphate, b) graphite, carbon black, petroleum coke, graphitized carbon black, graphite oxides, and graphene, and c) geopolymer and geopolymer composite. These additional powder athermanous fillers very often require processing conditions that are similar to the conditions required by the mineral having perovskite structure. Moreover, the masterbatch preferably comprises one or more silanes. Preferred silanes are for example aminopropyltriethoxysilane (e.g. Dynasylan AMEO from Evonik), aminopropyltrimethoxysilane (e.g. Dynasylan AMMO from Evonik), and phenyltriethoxysilane (e.g. Dynasylan 9265 from Evonik). Preferably, the amount of silane is in a range of from 0.01 to 1 wt. %, based on the weight of the athermanous additive in the masterbatch. It is noted that, unlike the properties of the additives as starting materials, the properties of additives as contained in granulate or foam are notoriously difficult to determine. It is often considered more appropriate in the art to characterize the additives in granulate and foam with reference to the properties of the additives as initially used. The advantages of the present invention become apparent from the following examples. Unless indicated otherwise, all percentages are given by weight. Moreover, whenever reference is made in the present description of the invention to an amount of additive “by weight of vinyl aromatic polymer”, this refers to the amount of the additive by weight of polymer component inclusive of (solid and, if any, liquid) additives, but exclusive of propellant. EXAMPLES Expandable Polymer Granulate was Prepared in an Extrusion Process, with Addition of Athermanous Fillers in Powder Form (Examples 1 to 11) Example 1 (Comparative) A mixture of vinyl aromatic polymer in the form of granules, and containing 2.5 wt. % of polymeric brominated flame retardant (Emerald 3000), 0.5 wt. % of bicumyl, Irganox 1010 in an amount of 0.125 wt. %, Irgafos 126 in an amount of 0.125 wt. %, Epon 164 in an amount of 0.250 wt. %, XIRAN SZ15170 in an amount of 1 wt. % and F-2200 HM in an amount of 1 wt. % were dosed to the main hopper of the main 32D/40 mm twin-screw co-rotating extruder. The melt temperature in the main extruder was 180° C. The powder of carbon black (Regal 350 from Cabot Corporation with a BET surface of 55.0 m2/g) was dosed to the side arm (54D/25 mm) twin-screw co-rotating extruder via one side feeder, in an amount of 3 wt. %, based on the total weight of granulate, excluding propellant, and the vinyl aromatic polymer (in the form of granules) was dosed to the main hopper of this extruder. The melt, then containing 40 wt. % of concentrated carbon black, was transported to the main extruder. The melt temperature inside the extruder was 190° C. The blowing agent (n-pentane/isopentane mixture 80/20%) was injected to the main 32D/40 mm extruder, downstream from the injection of the melt from the side twin-screw extruder. The concentration of blowing agent was 5.5 wt. %, calculated on total mass of product. The melt of vinyl aromatic polymer containing flame retardant, bicumyl, carbon black and blowing agent was transported to the 30D/90 mm cooling extruder and pumped through a static mixer, melt pump, screen changer, diverter valve, was then and extruded through the die head with 0.75 mm diameter holes, and was finally underwater pelletized by the rotating knifes. Downstream, the rounded product (a granulate with a particle size distribution of 99.9% of the fraction 0.8-1.6 mm) was centrifuged to remove the water, and was finally coated by the suitable mixture of magnesium stearate with glycerine monostearate and tristearate. The melt temperature in the cooling extruder was 170° C. The coated beads were expanded, and the final general properties of the expanded foam composite were then determined:thermal conductivity according to standard ISO 8301.mechanical properties (compressive and bending strength) according to standard EN 13163.flammability according to tests methods: EN ISO 11925-2 and DIN 4102 B1, B2. Example 2 (Comparative) The components according to Example 1 were used. Regal 350 carbon black was replaced with the CSX910 from Cabot Corporation, having a BET surface area of 71.8 m2/g. Example 3 (According to the Invention) The components according to Example 1 were used. 1 wt. % of calcium titanate was added, premixed with 3 wt. % of Regal 350, and dosed to the side arm extruder. The concentration of the two additives in the melt in side arm extruder was 40 wt. %. Example 4 (According to the Invention) The components according to Example 3 were used, and the calcium titanate content was increased to 3 wt. %. The flame retardant concentration was reduced to 2.0 wt. %, bicumyl to 0.4 wt. % and thermal stabilizers subsequently were reduced too. XIRAN SZ15170 and F-2200 HM were absent from the composition. Example 5 (According to the Invention) Again, the components according to Example 4 were used. The calcium titanate content was increased to 5 wt. %. Example 6 (According to the Invention) The components according to Example 5 were used. The Regal 350 was replaced with CSX910. Example 7 (According to the Invention) The components according to Example 6 were used. The calcium titanate was replaced with barium titanate. Example 8 (According to the Invention) The process and components according to Example 1 were used. The flame retardant was added in an amount of 1 wt. %. The thermo-oxidative and thermal stabilizers were excluded, as was XIRAN SZ15170. The calcium titanate was used in an amount of 5 wt. %. Example 9 (According to the Invention) The process and components according to Example 8 were used, and calcium titanate was replaced with barium titanate. Example 10 (According to the Invention) The process and components according to Example 8 were used, and calcium titanate was replaced with strontium titanate. Example 11 (According to the Invention) The process and components according to Example 8 were used, and calcium titanate was replaced with magnesium titanate. TABLE 1Summary of Examples 1 to 11.ComponentsExamples*(wt. %)1234567891011GP585X+++++++++++Regal 3503—333——————CSX 910—3———33————Calcium——1355—5———titanateBarium——————5—5——titanateStrontium—————————5—titanateMagnesium——————————5titanateEmerald2.52.52.52.02.02.02.01.01.01.01.03000Bicumyl0.50.50.50.40.40.40.40.20.20.20.2Irganox 10100.1250.1250.1250.10.10.10.1————Irgafos 1260.1250.1250.1250.10.10.10.1————Epon 1640.2500.2500.2500.20.20.20.2————Polywax0.30.30.30.30.30.30.30.30.30.30.32000XIRAN111————————SZ15170F-2200 HM111————————Pentane/5.55.55.55.55.55.55.55.55.55.55.5Isopentane80/20*Examples 1 and 2 are comparative, examples 3 to 11 are according to the invention. TABLE 2Examples 1 to 11.Final product parameters at a foam density of ca. 19.0 g/l.Examples*1234567891011Thermal conductivity (mW/m · K)/32.531.632.031.030.730.130.032.831.431.230.9ISO 8301/Flammability/+++++++++++EN ISO 11925-2/Flammability/+/+−/++/++/++/++/++/++/++/++/++/+DIN 4102 B1/B2/Compressive strength at 10% def.9298100115122126123125126127128(kPa)/EN 13163/Bending strength (kPa)/179185186198206212210220222219221EN 13163/Passed (+ or B2 or B1);Not passed (−)*Examples 1 and 2 are comparative, examples 3 to 11 are according to the invention. Expandable Polymer Granulate was Prepared in an Extrusion Process, with Addition of Athermanous Fillers in the Form of a Masterbatch (Examples 12 to 22) Examples from 1 to 11 were repeated. The masterbatch was prepared on the same extruder as side arm co-rotating twin-screw extruder—54D/25 mm. Synthos PS 585X was used a the masterbatch's polymer carrier. The results were very similar to those obtained from Examples 1 to 11 (where the athermanous fillers in powder form were used in an extrusion process and were (directly) dosed via the side extruder). Expandable Polymer Granulate was Prepared in a Suspension Process (Examples 23 to 33) Example 23 (Comparative) 20 000 kg of styrene was dosed to the 60 m3reactor. In the next step, the following components (calculated per styrene) were added: 3.0 wt. % of Regal 350 in the form of a 40 wt. % concentrated masterbatch (based on Synthos PS 585X), 0.002 wt. % of divinylbenzene, 2.0 wt. % of Emerald 3000, 0.3 wt. % of Polywax 1000, and 1.0 wt. % of dicumyl peroxide. The mixture was heated relatively quickly to a temperature of 70° C. and mixed at this temperature for 30 min with 275 rpm. Then, the temperature was increased to 90° C. and 30 000 kg of demineralised water (temperature of 60° C.) were added. The mixing force immediately created a suspension of prepolymer and the suspension was heated to 82° C. Immediately, 0.3 wt. % of Peroxan PO and 0.5 wt. % of TBPEHC were added. The radical polymerization was started and the following surfactant composition was introduced:potassium persulfate—0.0001 wt. %Poval 205—0.18 wt. % of 5% concentrated water solutionPoval 217 (alternatively Poval 224)—0.09 wt. % of a 5% concentrated water solutionDCloud 45—0.1 wt. %Arbocel CE 2910HE50LV—0.1 wt. % (hydroxypropylmethyl-cellulose supplied by J. RETTENMAIER & SÖHNE GMBH) The polymerization was then continued for 120 min. at a temperature of 82° C., and the temperature was then increased to 90° C. The suspension was kept at this temperature for 120 min., to achieve particle identity point of suspension. A further portion of Poval 217 (in a concentration of 0.3 wt. % of a 5 wt. % concentrated solution in water) was introduced. In this step, sodium chloride can be added (in an amount of 0.5 wt. % per water phase) to reduce the water content in the polymer. Alternatively, the surfactant (sodium dodecylbenzenesulfonate, SDBS) can be used (in an amount of 0.2 wt. %). The reactor was closed and the n-pentane/isopentane 80/20% mixture was added in an amount of 5.5 wt. % over 60 min. Simultaneously, the temperature was increased to 125° C. Then the polymerization was continued for 120 min. and after that time the suspension slurry was cooled down to 25° C. The product was removed from the reactor and water was removed in a basket centrifuge. The particles were then dried in a fluid bed drier at a temperature of 40° C. for 30 min. and fractionated on 80% of particles fraction 0.8-1.6 mm, 15% of 0.3-1.3, 4% of 1.0-2.5 mm and 1% of higher and lower size. Fractions were then coated the same way as the product as obtained in the extrusion process, and then expanded to foam at 35° C. Then the polymer was centrifuged from water and dried in the fluid bed dryer. Finally, after sieving, the granulate was coated with a mixture of glycerol monostearate and glycerol tristearate. Example 24 (Comparative) The components according to Example 23 were used. Regal 350 carbon black was replaced with CSX910 from Cabot Corporation, having a BET surface area of 71.8 m2/g. Example 25 (According to the Invention) The components according to Example 1 were used. 1 wt. % of calcium titanate (silanized with 0.1 wt. % of Dynasylan 9265), premixed with 3 wt. % of Regal 350, was dosed in the form of a 40 wt. % concentrate to the side arm extruder. Example 26 (According to the Invention) The components according to Example 25 were dosed and the calcium titanate content was increased to 3 wt. %. The flame retardant concentration was reduced to 1.5 wt. %, dicumyl peroxide content to 0.8 wt. %. Example 27 (According to the Invention) Again, the components according to Example 26 were dosed. The calcium titanate content was increased to 5 wt. %. Example 28 (According to the Invention) The components according to Example 27 were used. The Regal 350 was replaced with CSX910. Example 29 (According to the Invention) The components according to Example 28 were used. The calcium titanate was replaced with barium titanate (silanized with 0.1 wt. % of Dynasylan 9265). Example 30 (According to the Invention) The process and components according to Example 23 were used. The flame retardant (in an amount of 0.6 wt. %) and dicumyl peroxide (in an amount of 0.4 wt. %) were dosed. Calcium titanate was used in an amount of 5 wt. %. Example 31 (According to the Invention) The process and components according to Example 30 were used, and calcium titanate was replaced with barium titanate. Example 32 (According to the Invention) The process and components according to Example 31 were used, and calcium titanate was replaced with strontium titanate (silanized with 0.1 wt. % of Dynasylan 9265). Example 33 (According to the Invention) The process and components according to Example 32 were used, and calcium titanate was replaced with magnesium titanate (silanized with 0.1 wt. % of Dynasylan 9265). TABLE 3Summary of Examples 23 to 33.ComponentsExamples*(wt. %)2324252627282930313233GP585X+++++++++++Regal 3503—333——————CSX 910—3———33————Calcium titanate*——1355—5———Barium titanate*——————5—5——Strontium—————————5—titanate*Magnesium——————————5titanate*Emerald 30002.02.02.01.51.51.51.50.60.60.60.6Dicumyl peroxide1.01.01.00.80.80.80.80.40.40.40.4Polywax 10000.30.30.30.30.30.30.30.30.30.30.3Pentane/Isopentane5.55.55.55.55.55.55.55.55.55.55.580/20%*silanized with 0.1 wt. % of Dynasylan 9265*Examples 23 and 24 are comparative, examples 25 to 33 are according to the invention. TABLE 4Examples 23 to 33.Final product parameters at a foam density of ca. 19.0 g/l.Examples*2324252627282930313233Thermal conductivity (mW/m · K)/33.032.132.331.331.030.430.333.132.031.331.0ISO 8301/Flammability/+++++++++++EN ISO 11925-2/Flammability/+/+−/++/++/++/++/++/++/++/++/++/+DIN 4102 B1/B2/Compressive strength at 10% def.889094110119121120124124126129(kPa)/EN 13163/Bending strength (kPa)/168177181191201205208217220215223EN 13163/Passed (+ or B2 or B1);Not passed (−)*Examples 23 and 24 are comparative, examples 25 to 33 are according to the invention. Expandable Polymer Granulate was Prepared in a Continuous Mass Polymerization Process (Examples 34 to 44) Example 34 (Comparative) In this set of experiments, continuous mass polymerization was carried out in a three reactor cascade. The polymerization of styrene was initiated by heating. The powder form of carbon black (Regal 350 from Cabot Corporation with a BET surface of 55.0 m2/g) was added to the first reactor in an amount of 3 wt. % based on the total weight of granulate. After polymerization and degassing of the polymer melt, the flame retardant was added, directly to the extruding raw polystyrene and in an amount of 2.5 wt. %, together with: bicumyl in an amount of 0.5 wt. %, Irganox 1010 in an amount of 0.125 wt. %, Irgafos 126 in an amount of 0.125 wt. %, Epon 164 in an amount of 0.250 wt. % and nucleating agent (Polywax 2000) in an amount 0.3 wt. %. An extrusion was performed in similar like extruder 32D/40 mm attached to the degassing unit. Pentane in admixture with isopentane (80/20%) was dosed into the extruder during the process (in a concentration of 5.5 wt. %). The granulate form was obtained by means of underwater pelletizing. Example 35 (Comparative) The components according to Example 34 were used. Regal 350 carbon black was replaced with the CSX910 from Cabot Corporation, having a BET surface area of 71.8 m2/g. Example 36 (According to the Invention) The components according to Example 34 were used. 1 wt. % of calcium titanate was added (silanized with 0.1 wt. % of Dynasylan 9265), premixed with 3 wt. % of Regal 350 and dosed to the side arm extruder. The concentration in the melt in the side arm extruder was 40 wt. %. Example 37 (According to the Invention) The components according to Example 36 were dosed, and the calcium titanate content was increased to 3 wt. %. The flame retardant concentration was reduced to 2.0 wt. %, bicumyl to 0.4 wt. % and thermal stabilizers subsequently were reduced too. XIRAN SZ15170 and F-2200 HM were absent from the composition. Example 38 (According to the Invention) Again, the components according to Example 36 were dosed. The calcium titanate content was increased to 5 wt. %. Example 39 (According to the Invention) The components according to Example 38 were used. The Regal 350 was replaced with CSX910. Example 40 (According to the Invention) The components according to Example 39 were used. The calcium titanate was replaced with barium titanate (silanized with 0.1 wt. % of Dynasylan 9265). Example 41 (According to the Invention) The process and components according to Example 34 were used. The flame retardant was used in an amount of 1 wt. %. The thermo-oxidative and thermal stabilizers were absent, as was XIRAN SZ15170. Calcium titanate was used in an amount of 5 wt. %. Example 42 (According to the Invention) The process and components according to Example 41 were used and calcium titanate was replaced with barium titanate. Example 43 (According to the Invention) The process and components according to Example 42 were used and calcium titanate was replaced with strontium titanate (silanized with 0.1 wt. % of Dynasylan 9265). Example 44 (According to the Invention) The process and components according to Example 43 were used and calcium titanate was replaced with magnesium titanate (silanized with 0.1 wt. % of Dynasylan 9265). TABLE 5Summary of Examples 34 to 44.ComponentsExamples*(wt. %)3435363738394041424344GP585X+++++++++++Regal 3503—333——————CSX 910—3———33————Calcium——1355—5———titanate*Barium——————5—5——titanate*Strontium—————————5—titanate*Magnesium——————————5titanate*Emerald2.52.52.52.02.02.02.01.01.01.01.03000Bicumyl0.50.50.50.40.40.40.40.20.20.20.2Irganox 10100.1250.1250.1250.10.10.10.1————Irgafos 1260.1250.1250.1250.10.10.10.1————Epon 1640.2500.2500.2500.20.20.20.2————Polywax0.30.30.30.30.30.30.30.30.30.30.32000XIRAN111————————SZ15170F-2200 HM111————————Pentane/5.55.55.55.55.55.55.55.55.55.55.5Isopentane80/20*silanized with 0.1 wt. % of Dynasylan 9265*Examples 34 and 35 are comparative, examples 36 to 44 are according to the invention. TABLE 6Examples 34 to 44.Final product parameters at a foam density of ca. 19.0 g/l.Examples*3435363738394041424344Thermal conductivity (mW/m · K)/32.731.532.231.130.630.230.132.531.731.230.8ISO 8301/Flammability/+++++++++++EN ISO 11925-2/Flammability/+/+−/++/++/++/++/++/++/++/++/++/+DIN 4102 B1/B2/Compressive strength at 10% def.9096101114120125124120126128130(kPa)/EN 13163/Bending strength (kPa)/175181183199205211205215218220222EN 13163/Passed (+ or B2 or B1);Not passed (−)*Examples 34 and 35 are comparative, examples 36 to 44 are according to the invention. | 60,388 |
11859067 | DETAILED DESCRIPTION Subject matter is described throughout this Specification in detail and with specificity in order to meet statutory requirements. The aspects described throughout this Specification are intended to be illustrative rather than restrictive, and the description itself is not intended necessarily to limit the scope of the claims. Rather, the claimed subject matter might be practiced in other ways to include different elements or combinations of elements that are equivalent to the ones described in this Specification and that are in conjunction with other present technologies or future technologies. Upon reading the present disclosure, alternative aspects may become apparent to ordinary skilled artisans that practice in areas relevant to the described aspects, without departing from the scope of this disclosure. It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by, and is within the scope of, the claims. The subject matter described in this Specification generally relates to, among other things, tooling and components of an injection molding system configured to mold various articles. For example, the tooling and components of the present disclosure may be configured to mold foam articles having properties (e.g., size, thickness, density, energy return, consistency, uniformity, etc.) that may be difficult to achieve using conventional systems. For example, according to this disclosure, parameters (e.g., pressure, temperature, shot size, shot location, dwell time, etc.) may be controlled to influence the foaming activity of a single phase solution (SPS), which may ultimately affect the thickness, consistency, density, surface texture and appearance, etc. of the part, and when the foam polymer product is a component of a footwear article (e.g., a midsole) or other apparel or equipment item (e.g., athletic foam padding), these properties may affect the performance and usability. One type of injection molding system that both foams and molds polymeric materials uses a microcellular injection molding process, in which one or more supercritical fluids (SCFs)—e.g., supercritical nitrogen, supercritical carbon dioxide, etc.—are used as physical blowing agents. For example, the SCF may be injected into a polymer melt contained in an injection barrel of the injection molding system, where the SCF dissolves in the polymer melt to form the SPS. The SPS may then be flowed into the mold cavity, at which point conditions are adjusted to cause the SCF to transition to a gas (e.g., nucleation to a gas) and the polymer to solidify. This transition of the polymer composition in the mold cavity may cause the polymer composition to expand (e.g., due to foaming) to fill the mold cavity, and once solidified, retain the shape of the foam polymer product. These types of injection molding systems that use a microcellular injection molding process are often configured to control system parameters, which may affect properties of the foam polymer product. System parameters may be controlled at various stages of the injection molding process. For example, parameters may be controlled at the melting and mixing components forming the SPS, as well as among the components that transport the SPS to the mold. In addition, parameters may be controlled among the mold tooling. Theses stages may independently and/or collectively influence the melt, mixing, and molding processes. Conventional injection molding systems have been configured to execute a microcellular injection molding process. However, the attributes of parts typically molded with these systems is often limited, such that the operating parameters and tooling of a conventional system are often not calibrated to mold parts having different attributes. For example, the part thickness or wall thickness of a part typically molded in a conventional system may be relatively thin, such that conventional systems may not produce thicker parts having desired properties. In some instances, operating parameters and tooling of a conventional system may not be calibrated to mold a foam part having the properties and characteristics for a footwear article (or similar item). For example, under conventional systems, when the mold cavity size is increased (e.g., to include the thickness of a midsole) the SPS may experience undesirable or unwanted changes when deposited into the larger mold cavity, and these changes may affect foaming (e.g., nucleation and bubble formation) and properties of the foamed product (e.g., strength, surface quality, density, elasticity, skin thickness, bubble-size consistency, weak front interfaces, etc.). Part size and thickness is an example of one property that is different between shoe components and parts typically molded using conventional microcellular injection molding processes. However, there may be other properties (e.g., molded-product density, amount of SCF, amount of desired foaming, molded-product strength, molded-product compressibility, etc.) that are also different and that may contribute to conventional systems experiencing disadvantages when used to manufacture shoe parts. In contrast, the present disclosure controls parameters within the mold cavity to achieve foaming in a desired manner. For example, the temperature of the tooling (e.g., mold cavity walls) of the present disclosure may be controlled (e.g., temperature conditioned) to affect the temperature of the SPS (and the viscosity as a result) upon deposit into the mold cavity and during the foaming process. In one aspect, the mold temperature may be conditioned by positioning the mold on a temperature-conditioning rack (e.g., shelf) prior to the mold receiving the SPS, and the rack may include a cooling system or a heating system that conductively or actively moderates the temperature of the mold when the mold is placed on the rack. As such, when the conditioned mold is then engaged with the injector, the temperature of the mold cavity may be in a range that reduces the likelihood that the SPS will experience undesirable or unwanted changes. Furthermore, when the system is configured such that the temperature-conditioning rack conductively moderates the mold temperature, the mold may be constructed of materials (e.g., aluminum) that more efficiently transfer heat. The present disclosure may include other aspects that reduce a likelihood that the SPS may experience undesirable or unwanted changes in the mold cavity. For example, a gas counter pressure (GCP) system may affect a pressure within the mold cavity during the molding process (e.g., prior to distribution of the SPS into the cavity; dwell of SPS in the mold cavity; foaming of SPS in the mold cavity; etc.). Among other things, the GCP system may include one or more vents in the mold cavity that allow pressurized gas to pass into or from the mold cavity, and in one aspect, a property of the one or more vents (e.g., size, location, etc.) may relate to a viscosity of the SPS. For example, with a lower viscosity SPS, a size of the one or more vents may be increased (relative to systems molding higher viscosity SPS), which may increase the efficiency of the GCP system. In other aspects, the present disclosure describes a system of molds that may be used to manufacture a range of sizes of footwear articles. For example, the system of molds might include a first mold for a first sole size and a second mold for a second sole size (different from the first sole size), which each include a respective interface for fluidly communicating with a universal (shared) runner plate (e.g., a universal hot-runner plate or universal cold-runner plate affixed to injection nozzles). The interface of each of the first mold and the second mold may be constructed similarly (e.g., similar port size, shape, position, and number). However, the first mold may include a first set of runners and gates that communicate with the universal runner plate, and the second mold may include a second set of runners and gates that communicate with the universal runner plate. The first and second sets of runners and gates may be configured differently from one another (e.g., different runner paths/numbers and gate positions/numbers) in order to effectively distribute the SPS to a respective mold cavity having a respective size. Using a universal plate affixed to the injection nozzles, instead of a separate plate for each mold may, among other things, reduce costs associated with constructing and maintaining the tooling. Aspects of the present disclosure may include other features as well. For example, the mold plates may include a series of pins and stops that permit the plates to be moved and spaced with respect to one another at different stages of the molding process, such as at the unloading station. In addition, the tooling may include carrier plates that permit the mold plates to be manipulated, transported, and properly aligned at different stages of the molding process. Furthermore, the tooling may include a latch assembly that releasably connects portions of the tooling together. Also presented herein are manufacturing systems, processes and control logic for forming foamed thermoplastic polymer articles incorporating recycled thermoplastic materials, methods for operating such systems, shoe structure segments fabricated from such articles, and footwear assembled with such segments. In a general sense, the present technology enables the waste from an injection molding operation (e.g., runner waste, flashing, reused foam, etc.) to be reincorporated/integrated into a subsequently formed midsole such that the net waste from the molding operation is greatly reduced and/or eliminated. By way of example, there is presented a manufacturing process for fabricating a single-piece foamed midsole of an athletic shoe using scrap and/or waste (collectively “recycled”) thermoplastic, such as a regrind thermoplastic polyester elastomer (TPE-E) composition. The midsole is a foam component with a foam volume, which includes a foam core and an integrally formed skin that encases the foam core. In an injection molding application, spent scrap and waste thermoplastic material, such as foamed and/or unfoamed TPE-E composition, is ground into granular form and mixed into a composition containing virgin polymer. The mixture of ground/pelletized recycled material and virgin material is heated into a polymer melt composition, which is then passed, under pressure, through an injection barrel. While in the injection barrel, a supercritical fluid (SCF), such as supercritical nitrogen or supercritical carbon dioxide, may be injected into the polymer melt composition contained in the barrel, where the SCF dissolves in the melt to form a molten single-phase solution (SPS). The injection molding system foams and molds the ground virgin and recycled polymer using a microcellular molding process in which the SCF is employed as a physical blowing agent. The SPS may then be flowed into the mold cavity, at which point system conditions are modulated to activate transition of the SCF to a gas (e.g., nucleation to a gas) and the polymer to solidify. This transition of the polymer composition in the mold cavity may cause the polymer composition to expand (e.g., by foaming) to fill the mold cavity and, once solidified, retain the shape of the foam polymer product. The tooling and components of the injection molding system, as well as the calibrated parameters for operating the molding system, may be specifically tailored to mold foamed polymer articles using recycled TPE-E composition. Recombination of regrind and virgin polymer material may occur, as mentioned above, inside an injection barrel via a dry blend process; alternatively, regrind and virgin material recombination may occur on a separate extrusion line and, once combined, the pre-blended pellets may then be fed into the injection molder. Aspects of this disclosure are also directed to manufacturing systems and processes for fabricating footwear, apparel, and sporting goods from scrap and waste plastic. In an example, a method is presented for manufacturing foamed polymer articles from recycled TPE-E or TPE-E composition. This representative method includes, in any order and in any combination with any of the above or below disclosed features and options: inputting a batch of recycled thermoplastic polyester elastomer composition; grinding the recyclate batch into a ground recyclate material; combining a metered amount of the ground recyclate material with ground or pelletized virgin thermoplastic polyester elastomer composition into a mixed batch, the metered amount being about 20% by mass or less of a total mass of the mixed batch; melting the mixed batch into a polymer melt composition; adding a physical foaming agent to the polymer melt composition; injecting the polymer melt composition with the physical foaming agent into an internal cavity of a mold tool; forming the foamed polymer article by activating the physical foaming agent such that the physical foaming agent causes the polymer melt composition to expand and fill the mold tool's internal cavity; and extracting the foamed polymer article from the mold tool. In another example, a method of manufacturing a foamed polymer article includes, in any order and in any combination with the above and/or below concepts: grinding a recyclate batch of recycled thermoplastic polyester elastomer composition into a ground recyclate material; combining a metered amount of the ground recyclate material and a virgin polymer material of virgin thermoplastic polyester elastomer composition into a mixed batch; melting the ground recyclate material and the virgin polymer material into a polymer melt composition; adding a physical foaming agent to the polymer melt composition; injecting the polymer melt composition with the physical foaming agent into an internal cavity of a mold tool; activating the physical foaming agent such that the physical foaming agent causes the polymer melt composition to expand and fill the internal cavity of the mold tool to form the foamed polymer article; and removing the formed foamed polymer article from the mold tool. In yet another example, a method of manufacturing a foamed polymer article includes, in any order and in any combination with the above and/or below concepts: adding a physical foaming agent to a polymer melt composition, the polymer melt composition including a blend of a recyclate polymer material and a virgin polymer material, both of virgin thermoplastic polyester elastomer compositions, the recyclate polymer material being about 20% by mass or less of a total mass of the polymer melt composition; injecting the polymer melt composition with the physical foaming agent into an internal cavity of a mold tool; activating the physical foaming agent such that the physical foaming agent causes the polymer melt composition to expand and fill the internal cavity of the mold tool to form the foamed polymer article; and removing the formed foamed polymer article from the mold tool. Further aspects of this disclosure are directed to control logic and algorithms for operating manufacturing systems that fabricate footwear, apparel, and sporting goods from scrap and waste plastic. In an example, a method is presented for operating a manufacturing system to reduce waste during production of a foamed polymer article, such as a sole component of a shoe. This representative method includes, in any order and in any combination with any of the above or below disclosed features and options: injecting a mixed thermoplastic composition resin into a mold, the mixed thermoplastic composition resin including a mixture of virgin thermoplastic composition resin and recycled thermoplastic composition resin, and the mold comprising an internal mold cavity that is fluidly connected to one or more filling portions, such as a sprue, runner, and/or gate; and foaming the mixed thermoplastic composition resin within the internal mold cavity to form the foamed polymer article. In this method, the mass of the recycled thermoplastic composition resin within the internal mold cavity is greater than or equal to a mass of the mixed thermoplastic composition resin within the filling portion of the mold. As such, it may be possible for the entirety of thermoplastic composition within the filling portion of the mold to be fully incorporated into subsequently formed soles. In another example, a method of reducing waste during production of a foamed sole component of a shoe includes, in any order and in any combination with any of the above or below disclosed features and options: injecting a mixed thermoplastic composition resin into a mold, the mixed thermoplastic composition resin comprising a mixture of a virgin thermoplastic composition resin and a recycled thermoplastic composition resin, and the mold comprising a sole cavity portion fluidly coupled to a filling portion; and foaming the mixed thermoplastic composition resin within the sole cavity portion to form the foamed sole component of the shoe, wherein a mass of the recycled thermoplastic composition resin within the sole cavity portion is greater than or equal to a mass of the mixed thermoplastic composition resin within the filling portion. Further aspects of the present disclosure are directed to sporting goods, apparel, footwear, and segments of footwear fabricated from any of the disclosed processes and materials. For instance, an article of footwear, such as an athletic shoe, includes an upper that receives and attaches to the user's foot. A single-piece or multilayered sole structure, which is attached to a lower portion of the upper, supports thereon the user's foot. This sole structure includes an outsole that defines the ground-engaging portion of the footwear. The sole structure is fabricated with one or more foamed sole components, each of which includes a metered amount of a (ground or pelletized) recycled thermoplastic polyester elastomer composition and a (ground or pelletized) virgin thermoplastic polyester elastomer composition. The metered amount of recyclate TPE-E composition is about 20% by mass or less of a total mass of the mixed batch. Further aspects of this disclosure are directed to a method of manufacturing a foamed polymer article. In this instance, the method includes: grinding a recyclate batch of recycled thermoplastic polyester elastomer composition into a ground recyclate material; combining a metered amount of the ground recyclate material and a virgin polymer material of virgin thermoplastic polyester elastomer composition into a mixed batch; prior to or after combining, melting the ground recyclate material and the virgin polymer material into a polymer melt composition; adding a physical foaming agent to the polymer melt composition; injecting the polymer melt composition with the physical foaming agent into an internal cavity of a mold tool; activating the physical foaming agent such that the physical foaming agent causes the polymer melt composition to expand and fill the internal cavity of the mold tool to form the foamed polymer article; and removing the formed foamed polymer article from the mold tool. The formed foamed polymer article has: a ratio of energy efficiency to energy intensity that is greater than about 1.3; a ratio of energy efficiency to the product of energy intensity and density that is greater than about 5.9; a ratio of energy return to energy intensity that is greater than about 7,225; and/or a ratio of energy return to the product of energy intensity and density that is greater than about 38,250. Additional aspects of this disclosure are directed to method of reducing waste during production of a foamed polymer article. In this instance, the method includes: injecting a mixed thermoplastic composition resin into a mold, the mixed thermoplastic composition resin comprising a mixture of a virgin thermoplastic composition resin and a recycled thermoplastic composition resin; and foaming the mixed thermoplastic composition resin within an internal mold cavity of a molding system to form the foamed polymer article, wherein a mass of the recycled thermoplastic composition resin within the mixed thermoplastic composition resin is at least about 20% by mass of a total mass of the mixed thermoplastic composition resin. For any of the disclosed systems, methods, articles, and footwear, the recycled TPE-E composition in the recyclate batch includes scrap material or waste material, or both, that was recovered from an un-foamed batch of extruded TPE-E composition and/or a foamed batch of injection molded TPE-E composition. As yet a further option, the recycled and virgin TPE-E compositions may be derived from a dihydroxy-terminated polydiol material, such as a poly(alkylene oxide)diol, or a C2-C8 diol material, such as an ethanediol, propanediol, butanediol, pentanediol, or an aromatic dicarboxylic acid material, such as a C5-C16 dicarboxylic acid, or any combination thereof. In addition, the physical foaming agent may be added by injecting the physical foaming agent into the polymer melt composition while the polymer melt composition is contained in an injection barrel of an injection molding system. The physical foaming agent may be an SCF, such as supercritical nitrogen and/or supercritical carbon dioxide. For any of the disclosed systems, methods, articles, and footwear, the mixed batch of ground recyclate material and virgin polymer material may have a set point temperature of at least about 150° C. or, in some embodiments, ranging from about 190° C. to about 265° C. In this regard, the mixed batch of recyclate and virgin materials may have an average peak crystallization temperature of at least about 90° C. or, in some embodiments, ranging from about 135° C. to about 165° C. A resultant foamed polymer article may have a cell size average, e.g., by volume of a longest cell dimension, of less than about 0.68 mm or, in some embodiments, about 0.18 mm to about 0.58 mm. Creating the polymer melt composition may comprise melting then mixing the recyclate and virgin materials or melting a mixed batch already containing the recyclate and virgin materials. For any of the disclosed systems, methods, articles, and footwear, the resultant foamed polymer article exhibits: a ratio of energy efficiency to energy intensity that is between about 1.1 and about 1.9; a ratio of energy efficiency to the product of energy intensity and density that is between about 4.8 and about 9.1; a ratio of energy return to energy intensity that is between about 6,000 and about 11,000; and/or a ratio of energy return to the product of energy intensity and density that is greater than about 45,000. The recycled and virgin thermoplastic polyester elastomer compositions may be derived from a block copolymer, a segmented copolymer, a random copolymer, and/or condensation copolymer, and may have a weight average molecular weight (Mw) of at least about 30,000 Daltons or, in some embodiments, about 50,000 Daltons to about 200,000 Daltons. For any of the disclosed systems, methods, articles, and footwear, the ground recyclate material may be processed prior to melting the mixed batch. This processing may include adding a filler, pigment, and/or processing aid to the ground recyclate material (before or after incorporation into the mixed batch). As yet a further option, adding the physical foaming agent to the polymer melt composition may include dissolving a supercritical inert fluid into the polymer melt composition under pressure to form a single-phase solution. Moreover, activating the physical foaming agent may include releasing the pressure to expand the supercritical inert fluid. Receiving the recyclate batch of recycled TPE-E composition may include obtaining, from a sprue, a runner, and/or a gate of an injection molding system, scrap segments of a prior-foamed polymer article formed from a prior mixed batch of ground recyclate material and virgin polymer material. For any of the disclosed systems, methods, articles, and footwear, a resultant foamed article formed with recycled polymer material may have an energy return measurement that is within a predefined tolerance of an energy return measurement of a comparable foamed article formed entirely or almost entirely from virgin polymer material. For example, the predefined tolerance of a foamed sole component formed with recyclate is about 75% to about 99% of the energy return measurement of a comparable shoe sole component formed from virgin material. A shoe sole component may be considered “comparable” to another sole component if the two articles share an equivalent or nearly equivalent common shape, size, and/or method of molding. A percent by mass of the recycled thermoplastic resin within the mixed thermoplastic resin may be less than about 30% or, in some embodiments, between about 1% and about 20%. For any of the disclosed systems, methods, articles, and footwear, the filling portion of the mold comprises one or more cold runners. In this instance, the filling portion may include one or more hot runners disposed within one or more runner plates, which may be stacked on and fluidly coupled to one or more mold plates that define therein the internal mold cavity. Moreover, the filling portion may consist of one or more channels that direct a flow of mixed thermoplastic resin from a nozzle or hot runner of an injection molding apparatus to the internal mold cavity portion of the mold. As yet a further option, the ground recyclate material may have an irregular shape with a largest measurement of about 1-10 mm, and the virgin polymer material has a pellet size of about 1-10 mm. A foamed sole component may have a melting temperature of at least about 190° C. and an average peak crystallization temperature of at least about 135° C. These and other aspects are described in more detail in the below parts of this Detailed Description. Foamed Thermoplastic Elastomer Composition A disclosed thermoplastic elastomer foam (i.e., a foam formed by expanding a thermoplastic elastomer composition as disclosed herein), including thermoplastic polyester foams, can exhibit various beneficial properties. For example, the thermoplastic elastomer foam can exhibit a beneficial split tear, for example a high split tear value for a sole component in an article of footwear. In some aspects, the thermoplastic elastomer foam can have a split tear value of greater than about 1.5 kilogram/centimeter (kg/cm), or greater than about 2.0 kg/cm, or greater than about 2.5 kg/cm, when determined using the Split Tear Test Method described herein. In some aspects, the thermoplastic elastomer foam can have a split tear value of 1.0 kg/cm to 4.5 kg/cm, or 1.0 kg/cm to 4.0 kg/cm, or 1.5 kg/cm to 4.0 kg/cm, or 2.0 kg/cm to 3.5 kg/cm, or 2.5 kg/cm to 3.5 kg/cm, when determined using the Split Tear Test method described herein. The thermoplastic elastomer foam can have a split tear value of 0.8 kg/cm to 4.0 kg/cm, or 0.9 kg/cm to 3.0 kg/cm, or 1.0 to 3.0 kg/cm, or of 1.0 kg/cm to 2.5 kg/cm, or 1 kg/cm to 2 kg/cm. In some aspects, the thermoplastic elastomer foam is injection molded, and has a split tear value of 0.7 kg/cm to 2.5 kg/cm, or 0.8 kg/cm to 2.0 kg/cm, or 0.9 to 1.5 kg/cm, or 1.0 kg/cm to 2.5 kg/cm, or of 1.0 kg/cm to 2.2 kg/cm. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the product of physically foaming a thermoplastic elastomer composition as disclosed herein, i.e., a foam formed using a physical foaming agent (i.e., a physical blowing agent). As used herein, a thermoplastic elastomer foam is understood to refer to a foamed material which has thermoplastic and elastomeric properties. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. In some aspects, the thermoplastic elastomer foam is injection molded (i.e., is not exposed to a separate compression molding step after being formed by injection molding and removed from the injection mold). In other aspects, the thermoplastic elastomer foam is injection molded and subsequently compression molded in a separate compression mold having different dimensions than the mold used in the injection molding step. The density or specific gravity of a disclosed thermoplastic elastomer foam, including a thermoplastic polyester foam, is also an important physical property to consider when using a foam for an article of apparel, footwear or athletic equipment. As discussed above, the thermoplastic elastomer foam of the present disclosure exhibits a low density or specific gravity, which beneficially reduces the weight of midsoles or other components containing the thermoplastic elastomer foam. The thermoplastic elastomer foams of the present disclosure, including thermoplastic polyester foams, can have a specific gravity of from 0.02 to 0.22, or 0.03 to 0.12, or 0.04 to 0.10, or 0.11 to 0.12, or 0.10 to 0.12, or 0.15 to 0.20, or 0.15 to 0.30, when determined using the Specific Gravity Test Method described herein. In some aspects, the thermoplastic elastomer foams can have a specific gravity of from 0.15 to 0.22, such as from 0.17 to 0.22 or from 0.18 to 0.21, when determined using the Specific Gravity Test Method described herein. Alternatively or in addition, the thermoplastic elastomer foam can have a specific gravity of from 0.01 to 0.10, or 0.02 to 0.08, or 0.03 to 0.06, or 0.08 to 0.15, or 0.10 to 0.12, when determined using the Specific Gravity Test Method described herein. For example, the specific gravity of the thermoplastic elastomer foam can be from 0.15 to 0.2, or 0.10 to 0.12. The thermoplastic elastomer foam can be injection molded, or can be injection molded and subsequently compression molded. In some aspects, the thermoplastic elastomer foam has a specific gravity of about 0.7 or less, or 0.5 or less, or 0.4 or less, or 0.3 or less, when determined using the Specific Gravity Test Method described herein. In some aspects, the thermoplastic elastomer foam, including the thermoplastic elastomer foam present in midsoles and midsole components, can have a specific gravity of 0.05 to 0.25, or 0.05 to 0.2, or 0.05 to 0.15, or 0.08 to 0.15, or 0.08 to 0.20, or 0.08 to 0.25, or 0.1 to 0.15, when determined using the Specific Gravity Test Method described herein. In some aspects the thermoplastic elastomer foam has a specific gravity of about 0.15 to about 0.3, or about 0.2 to about 0.35, or about 0.15 to about 0.25, when determined using the Specific Gravity Test Method described herein. The thermoplastic elastomer foam article or article component can be formed by injection molding without a subsequent compression molding step. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The thermoplastic elastomer foams of the present disclosure, including thermoplastic polyester foams, can have a density of from 0.02 grams per cubic centimeter (g/cc) to 0.22 g/cc, or 0.03 g/cc to 0.12 g/cc, or 0.04 g/cc to 0.10 g/cc, or 0.11 g/cc to 0.12 g/cc, or 0.10 g/cc to 0.12 g/cc, or 0.15 g/cc to 0.2 g/cc, or 0.15 g/cc to 0.30 g/cc, when determined using the Density Test Method described herein. In some aspects, the thermoplastic elastomer foams can have a density of from 0.15 g/cc to 0.22 g/cc, such as from 0.17 g/cc to 0.22 g/cc, or from 0.18 g/cc to 0.21 g/cc, when determined using the Density Test Method described herein. Alternatively or in addition, the thermoplastic elastomer foam can have a density of from 0.01 g/cc to 0.10 g/cc, or 0.02 g/cc to 0.08 g/cc, or 0.03 g/cc to 0.06 g/cc, or 0.08 g/cc to 0.15 g/cc, or 0.10 g/cc to 0.12 g/cc, when determined using the Density Test Method described herein. For example, the density of the thermoplastic elastomer foam can be from 0.15 g/cc to 0.2 g/cc, or 0.10 g/cc to 0.12 g/cc. The thermoplastic elastomer foam can be injection molded, or can be injection molded and subsequently compression molded. In some aspects, the thermoplastic elastomer foam has a density of about 0.7 g/cc or less, or 0.5 g/cc or less, or 0.4 g/cc or less, or 0.3 g/cc or less, or 0.2 g/cc or less, when determined using the Density Test Method described herein. In some aspects, the thermoplastic elastomer foam, including the thermoplastic elastomer foam present in midsoles and midsole components, can have a density of 0.05 g/cc to 0.25 g/cc, or 0.05 g/cc to 0.2 g/cc, or 0.05 g/cc to 0.15 g/cc, or 0.08 g/cc to 0.15 g/cc, or 0.08 g/cc to 0.20 g/cc, or 0.08 g/cc to 0.25 g/cc, or 0.10 g/cc to 0.15 g/cc, when determined using the Density Test Method described herein. In some aspects the thermoplastic elastomer foam has a density of about 0.15 g/cc to about 0.30 g/cc, or about 0.20 g/cc to about 0.35 g/cc, or about 0.15 g/cc to about 0.25 g/cc, when determined using the Density Test Method described herein. The thermoplastic elastomer foam article or article component can be formed by injection molding without a subsequent compression molding step. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The thermoplastic elastomer foam portion of the article or component of an article, including thermoplastic polyester foam portion, can have a stiffness of about 200 kPa to about 1000 kPa, or about 300 to about 900 kPa, or about 400 to about 800 kPa, or about 500 to about 700 kPa, when determined using the Cyclic Compression Test for a Sample with a 45 millimeter diameter cylindrical sample. The thermoplastic elastomer foam portion of the article or component of an article can have a stiffness of about 100 N/mm to about 400 N/mm, or about 150 N/mm to about 350 N/mm, or about 200 N/mm to about 300 N/mm, or about 225 N/mm to about 275 N/mm, when determined using the Cyclic Compression Test for a Foot Form with the foot form sample. The thermoplastic elastomer foam article or article component can be formed by injection molding without a subsequent compression molding step. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The thermoplastic elastomer foam portion of the article or component of an article, including a thermoplastic polyester portion, can have an Asker C durometer hardness of from about 30 to about 50, or from about 35 to about 45, or from about 30 to about 45, or from about 30 to about 40, when determined using the Durometer Hardness Test described herein. The thermoplastic elastomer foam article or article component can be formed by injection molding without a subsequent compression molding step. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The energy input of a foam is the integral of the force displacement curve during loading of the foam during the Cyclic Compression test. The energy return of a foam is the integral of the force displacement curve during unloading of the foam during the Cyclic Compression test. The thermoplastic elastomer foam portion of the article or component of an article, including a thermoplastic polyester foam portion, can have an energy return of about 200 millijoules (mJ) to about 1200 mJ, or from about 400 mJ to about 1000 mJ, or from about 600 mJ to about 800 mJ, when determined using the Cyclic Compression Test for a Sample with a 45 millimeter diameter cylindrical sample. The thermoplastic elastomer foam portion of the article or component of an article (e.g., footwear sole for a Men's US Size 10) can have an energy input of about 2000 millijoules (mJ) to about 9000 mJ, or from about 3000 mJ to about 8000 mJ, or from about 4500 mJ to about 6500 mJ, when determined using the Cyclic Compression Test for a Foot Form with the foot form sample. The thermoplastic elastomer foam article or article component can be formed by injection molding without a subsequent compression molding step. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The energy efficiency (EE), a measure of the percentage of energy of the thermoplastic elastomer foam portion of the article or component, including a thermoplastic polyester foam portion, returns when it is released after being compressed under load, which can provide improved performance for athletic footwear, e.g., for reducing energy loss or dissipation when running. This is especially true for running and other athletic footwear. In some aspects, the thermoplastic elastomer foam portion of the articles and components provided herein have an energy efficiency of at least 50 percent, or at least 60 percent, or at least 70 percent, or at least about 75 percent, or at least about 80 percent, or at least about 85 percent, when determined using the Cyclic Compression Test for a Sample with a 45 millimeter diameter cylindrical sample. The thermoplastic elastomer foam portion of the articles and components provided herein can have an energy efficiency of at about 50 percent to about 97 percent, or about 60 percent to about 95 percent, or about 60 percent to about 90 percent, or about 60 percent to about 85 percent, or about 65 percent to about 85 percent, or about 70 percent to about 85 percent, or about 70 percent to about 90 percent, or about 70 percent to about 95 percent, when determined using the Cyclic Compression Test for a Sample with a 45 millimeter diameter cylindrical sample. The thermoplastic elastomer foam article or article component can be formed by injection molding without a subsequent compression molding step. The thermoplastic elastomer foam can have an open-cell foam structure. The thermoplastic elastomer foam can be the foamed product of foaming a thermoplastic elastomer composition comprising less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The resulting foams can have a multicellular closed-cell or open-cell foam structure. Cells are the hollow structures formed during the foaming process, in which bubbles are formed in the thermoplastic elastomeric composition by the foaming agents. The cell walls are generally defined by the thermoplastic elastomeric composition. “Closed cells” form an individual volume that is fully enclosed and that is not in fluid communication with an adjoining individual volume. “Closed-cell structures” refer to foam structures in which at least 50 percent or more of the cells are closed cells, or at least 60 percent or more of the cells are closed cells, or at least 80 percent of the cells are closed cells, or at least 90 percent of the cells are closed cells, or at least 95 percent of the cells are closed cells. “Open-cell structures” refer to foam structures in which less than 50 percent, or less than 40 percent, or less than 20 percent, or less than 10 percent, or less than 5 percent or less than 4 percent, or less than 3 percent or less than 1 percent of the cells are closed cells. The disclosed open-cell and closed-cell thermoplastic elastomer foams may have an average cell size (e.g., maximum width or length) linearly measured from one side of the cell to an opposing side of the cell. For example, in some aspects of this disclosure, open-cell and closed-cell thermoplastic elastomer foams may have an average cell size of from about 50 micrometers to about 1000 micrometers, or from about 80 micrometers to about 800 micrometers, or from about 100 micrometers to about 500 micrometers. These are example cell sizes of one aspect of this disclosure in which foams form portions of a footwear article, and in other aspects the cell sizes may be larger or smaller when foams form other footwear articles. In addition, open-cell and closed-cell thermoplastic elastomer foams may form all or a portion of a non-footwear article, and in those instances, the foams may have a cell diameter including these example cell sizes, smaller than these example cell sizes, larger than these example cell sizes, or any combination thereof. For both open-cell and closed-cell structures, the proportion of cells in the thermoplastic elastomer foam having a cell diameter of about 50 micrometers to about 1000 micrometers is preferably not less than 40 percent relative to all the cells, or not less than 50 percent or not less than 60 percent relative to all the cells. If the proportion of cells is less than 40 percent, the cell structure will tend to be nonuniform and/or have a coarse cell structure. As used herein, a “coarse cell structure” refers to a foam structure in which the average cell diameter is greater than 1 millimeter, and/or for greater than 20 percent of the cells, a 1 millimeter line drawn across the largest dimension of the cell, will not cross a cell wall or a strut (i.e., an open cell wall or portion thereof). The number of open cells and/or closed cells and cell diameter of the cells of the foam can be determined visually, for example by capturing an image of a cut surface with a camera or digital microscope, determining the number of cells, number of open cells and/or number of closed cells, and determining an area of a cell, and converting it to the equivalent circle diameter. Methods of Manufacturing Disclosed Foams In some examples, the disclosed foamed thermoplastic elastomer compositions can be prepared by various methods as disclosed herein and as known in the art. That is, disclosed articles or components of articles such as midsoles, midsole components, inserts and insert components can be prepared by injection molding a melt composition comprising a thermoplastic elastomer composition as described herein using a physical foaming agent, using a combination of a physical foaming agent and a chemical foaming agent, or using only a chemical foaming agent. A disclosed foam component, e.g., a disclosed foam article or component, can be prepared by the methods disclosed herein below. Disclosed herein are methods for making a foam article or component, the method comprising: forming a mixture of a molten thermoplastic elastomer composition and a foaming agent; injecting the mixture into a mold cavity; foaming the thermoplastic elastomer composition, thereby forming a foamed thermoplastic elastomer composition; solidifying the foamed thermoplastic elastomer composition, thereby forming a foam article having a multicellular foam structure; and removing the foam article from the mold cavity. In some aspects, forming the mixture of the thermoplastic elastomer composition and the foaming agent comprises forming a single-phase solution of a liquid, gas or supercritical fluid foaming agent and the molten thermoplastic elastomer composition. In some aspects, the mixture is a single-phase solution of supercritical nitrogen or supercritical carbon dioxide and the thermoplastic elastomer composition. In a particular example, the mixture is a single-phase solution of supercritical nitrogen in a thermoplastic polyester composition. In some aspects, the thermoplastic elastomer composition comprises less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. In such aspects, injecting the mixture into a mold cavity can comprise injecting the single-phase solution into a mold cavity, then cooling the single-phase solution in the mold cavity prior to decreasing pressure in the mold cavity to a level at which the supercritical fluid phase transitions to a gas, and the gas drops out of solution in the molten polymer, forming gas bubbles in the molten polymer and foaming the molten polymer. In some aspects, the foaming forms a foam having an open-cell foam structure. Also disclosed are methods for making a foam article or component, the method comprising: forming a mixture of a molten thermoplastic elastomer composition and a foaming agent; injecting the mixture into a mold cavity; foaming the molten thermoplastic elastomer composition in the mold cavity, thereby forming a thermoplastic elastomer foam; solidifying the thermoplastic elastomer foam in the mold cavity, thereby forming a molded foam article comprising a thermoplastic elastomer composition having a multicellular foam structure; and removing the molded foam article from the mold cavity. In some aspects, the temperature of the mixture at the point that it is foamed in the mold cavity is from about the melting temperature of the thermoplastic elastomer composition to about 50 degrees C. above the tail temperature of the thermoplastic elastomer composition. In some aspects, the melting temperature of the thermoplastic elastomer composition is the melting temperature of a polymeric component of the thermoplastic elastomer composition. In other aspects, the melting temperature of the thermoplastic elastomer composition is the melting temperature of a thermoplastic elastomer present in the thermoplastic elastomer composition. In yet other aspects, the melting temperature of the thermoplastic elastomer present in the thermoplastic elastomer composition is the melting temperature of the thermoplastic elastomer having the highest melting temperature of all polymers present in the polymeric component of the thermoplastic elastomer composition. In yet other aspects, the melting temperature is the melting temperature of a thermoplastic polyester, such as a polyester elastomer, present in the thermoplastic elastomer composition. The foaming can occur when the mixture is at a foaming temperature, wherein the foaming temperature is a temperature from about the melting temperature of the thermoplastic elastomer to about 50 degrees C. above the tail temperature of the thermoplastic elastomer. In some aspects, forming the mixture of the thermoplastic elastomer composition and a foaming agent comprises forming a single-phase solution of a supercritical fluid and the molten thermoplastic elastomer composition. The thermoplastic elastomer composition can comprise less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. If more than one thermoplastic elastomer is present in the thermoplastic elastomer composition, the melting temperature can be the highest melting temperature of the thermoplastic elastomers present in the composition. In such aspects, injecting the mixture into a mold cavity can comprise injecting the single-phase solution into a mold cavity, then cooling the single-phase solution in the mold cavity prior to decreasing pressure in the mold cavity to a level at which the supercritical fluid phase transitions to a gas, and the gas drops out of solution in the thermoplastic elastomer composition, forming gas bubbles in the thermoplastic elastomer composition and foaming the thermoplastic elastomer. The foaming can form a foam having an open-cell foam structure. Dynamic scanning calorimetry (DSC) is used to determine the melting temperature and the tail temperature of the thermoplastic elastomer composition, or of the polymeric component of the thermoplastic elastomer composition, or of an individual thermoplastic elastomer present in the thermoplastic elastomer composition, and an exemplary method is described herein below. Briefly, 10-30 mg pieces of undried resin pellets are cycled from −90 degrees C. to 225 degrees C. at 20 degrees C./min and cooled to −90 degrees C. at 10 degrees C./min. In some instances, experiments are run using a heat-cool-heat profile with a ramp rate of 10 degrees C. per min, minimum temperature of 0 degrees C. and maximum temperature of 250 degrees C. Analyses should be determined in duplicate. The melting temperature and glass transition temperature values are recorded from the second cycle. The melt “peak” is identified as the local maximum of the second heating cycle. If there is more than one peak in the DSC curve, the peak occurring at hotter temperatures is chosen as the temperature reference. The tail is identified as the intersection of the tangent of the line of the higher temperature side of the melt peak with the extrapolated baseline. The disclosed foamed thermoplastic elastomer compositions can be prepared using a suitable injector. The injector can have a motor to turn a screw inside the injector. The injector may include a single screw or twin screws, and may include individual elements of various sizes and pitches appropriate for mixing or kneading the specific materials used. The various components included in the foamed thermoplastic elastomer compositions described herein can be added into the injector through one or more ports. The various components can be added as a melt or as appropriately-sized solid particles, for example chips or pellets, which may be melted as they are mixed in the barrel of the injector. The contents of the injector can be heated to melt the composition. A physical foaming agent such as, for example, a supercritical fluid can be added into the melt while it is present in the barrel of the injector. In one example, thermoplastic polyester foam is prepared by using a physical foaming agent which foams the composition in the mold cavity, and the resulting thermoplastic elastomer foam is thus substantially free of unreacted chemical blowing agents or a decomposition or degradation product of a chemical blowing agent. The thermoplastic elastomer composition can be added to the injector as a melt at a temperature close to the melting temperature of the polymeric component of the composition. If a chemical foaming agent is used, the processing (melting) temperature used can be sufficiently below the temperature that would trigger the chemical foaming agent. In order to foam the composition, the temperature near the exit of the injector or within the mold cavity can be increased to a temperature close to or at the triggering temperature of the chemical foaming agent, thereby producing a chemically foamed thermoplastic polyester foam as the composition exits the injector (e.g., as the composition is injected into a mold cavity), or within the mold cavity. Additionally or alternatively, the temperature of the runners leading to the mold cavity or the mold cavity or both can be a temperature at or above the triggering temperature of the chemical foaming agent, thereby producing a chemically foamed thermoplastic elastomer foam within the runners and/or the mold cavity. Alternatively or in addition, a physical foaming agent can be used to foam the thermoplastic elastomer composition to form a physically foamed thermoplastic elastomer foam, or a physically and chemically foamed thermoplastic elastomer foam. For example, a supercritical fluid such as supercritical carbon dioxide or supercritical nitrogen can be mixed with the molten thermoplastic elastomer composition in the barrel of the injector to form a single-phase solution. A pressure drop can be used to cause the supercritical fluid to transition to the gas phase and foam the thermoplastic elastomer composition. In one aspect, a gas counter-pressure can be applied to the mold cavity and to the runners leading to the mold cavity. The counter pressure can be a pressure sufficiently high to keep the supercritical fluid in solution within the runners and the mold cavity. Once a dose of the single-phase solution is in the mold cavity, the counter-pressure within the mold cavity can be decreased to a level at which the supercritical fluid phase transitions to a gas and drops out of solution in the molten thermoplastic elastomer composition, forming gas bubbles in the thermoplastic elastomer composition and foaming the thermoplastic elastomer composition in the mold cavity. In one aspect the thermoplastic elastomer composition comprises less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition, and the multicellular foam has an open-cell structure. The articles, cushioning elements, or components of articles such as midsoles, midsole components, inserts and insert components can be prepared by injection molding a thermoplastic elastomer composition described herein using a physical foaming agent. The injection molding process can use a screw-type injector that allows for maintaining and controlling the pressure in the injector barrel. The injection molding machine can allow metering and delivering a supercritical fluid such as carbon dioxide or nitrogen into the thermoplastic elastomer composition prior to injection. The supercritical fluid can be mixed into the thermoplastic elastomer composition within the injection barrel and then injected into the mold cavity. When the temperature and/or pressure is altered to the point that the solubility of the supercritical fluid in the molten thermoplastic elastomer composition is altered and the supercritical fluid transitions to the gas phase, these physical processes will cause expansion (foaming) of the molten thermoplastic elastomer composition. The injection molding process can include physical foaming of the compositions described herein using an injection molding process which forms a multicellular foam structure, such as, for example the “MUCELL” process (Trexel Inc., Wilmington, Mass., USA). The thermoplastic elastomer foams described herein can be made using a process that involves impregnating a thermoplastic elastomer composition (e.g., at or above a softening temperature of the composition) with a physical foaming agent at a first concentration or first pressure. As used herein, the term “impregnating” generally means dissolving or suspending a physical foaming agent in a composition. The impregnated composition can then be foamed, or can be cooled (when applicable) and re-softened (when applicable) for foaming at a later time. In some aspects, the impregnated molten thermoplastic elastomer composition forms a single-phase solution comprising a supercritical fluid (e.g., carbon dioxide or nitrogen) dissolved in the molten thermoplastic elastomer composition. In one aspect, the thermoplastic elastomer composition comprises less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on a total weight of the thermoplastic elastomer composition. The impregnated thermoplastic elastomer composition (e.g., the single-phase solution) is foamed by reducing the solubility of the physical foaming agent in the thermoplastic elastomer composition through pressure and/or temperature changes. The pressure and/or temperature change can occur immediately after the impregnated composition exits the injector or the injection barrel, or can occur in the runners leading to the mold cavity, or can occur in the mold cavity. For example, the system can include hot runners or gas counter-pressure or both, which control the temperature and pressure under which the impregnated composition is held, up to and including the point at which the composition enters the mold cavity. In some aspects, the temperature and pressure under which the impregnated composition is held are controlled such that the impregnated composition remains a single-phase solution up to and including the point it enters the mold cavity. Once the single-phase solution has flowed into the mold cavity, the temperature or the pressure or both can be altered to reduce the solubility of the supercritical fluid in the molten thermoplastic elastomer composition, causing the molten thermoplastic elastomer composition to expand into a foam, including a foam having an open-cell foam structure. The reduction in solubility of the physical foaming agent can release additional amounts of gas (e.g., to create a secondary expansion of a partially-foamed thermoplastic elastomer composition), to further expand the composition, forming a foam structure (e.g., a foam having a multicellular structure). Alternatively or additionally, a chemical blowing agent can be activated in the thermoplastic elastomer composition in the mold cavity to create a secondary expansion of a partially-foamed thermoplastic elastomer composition. Chemical foaming agents may be endothermic or exothermic, which refers to a type of decomposition or degradation they undergo to produce the gas used to produce the foam. The decomposition or degradation may be triggered by thermal energy present in the molding system. Endothermic foaming agents absorb energy and typically release a gas, such as carbon dioxide, upon decomposition. Exothermic foaming agents release energy and generate a gas, such as nitrogen, when decomposed. Regardless of the chemical foaming agent used, thermal variables of the thermoplastic elastomer composition being foamed and thermal variables of the foaming agent to be decomposed or degraded are coupled together such that process parameters are selected so that the thermoplastic elastomer composition can be foamed and molded and the foaming agent can decompose or degrade at an appropriate phase of the foaming and molding process. Thermoplastic Elastomer Composition Thermoplastic elastomer compositions disclosed herein include one or more thermoplastic elastomers. The one or more thermoplastic elastomers can be one or more thermoplastic polyester elastomers. In some aspects, the thermoplastic elastomer composition includes at least 90 percent, or at least 95 weight percent, or at least 99 weight percent of a thermoplastic resin component, based on the total weight of the thermoplastic elastomer composition, where thermoplastic resin component includes all the polymers present in the composition. Thermoplastic resin component comprises one or more thermoplastic elastomers. Thermoplastic resin component can comprise at least one thermoplastic polyester elastomer. Thermoplastic resin component can comprise more than one thermoplastic polyester elastomer. Thermoplastic resin component can comprise one or more thermoplastic polyester elastomer, and one or more thermoplastic polyester which is not an elastomer. In some aspects, thermoplastic resin component comprises the one or more thermoplastic polyester, and further comprises one or more thermoplastic polymers each of which is not a polyester. The one or more thermoplastic polymers each of which is not a polyester can each be a thermoplastic elastomer. Alternatively, in other aspects, thermoplastic resin component consists essentially of the one or more thermoplastic elastomer. Optionally, thermoplastic resin component can consist essentially of one or more thermoplastic polyester elastomer. In some aspects, the thermoplastic elastomer composition comprises less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of non-polymeric ingredients based on the total weight of the thermoplastic elastomer composition. In some aspects, the thermoplastic elastomer composition is substantially free of non-polymeric nucleating agents, or is substantially free of non-polymeric fillers, or is substantially free of coloring agents, or is substantially free of non-polymeric processing aids, or is substantially free of both non-polymeric nucleating agents and non-polymeric fillers, or is substantially free of non-polymeric nucleating agents, non-polymeric fillers, coloring agents, and non-polymeric processing aids. In some such aspects, the thermoplastic elastomer composition comprises less than 10 weight percent, or less than 5 weight percent, or less than 1 weight percent of solid coloring agents, based on the total weight of the thermoplastic elastomer composition. In one aspect, the thermoplastic elastomer composition consists essentially of one or more thermoplastic elastomers. In another aspect, the thermoplastic elastomer composition consists essentially of one or more thermoplastic polyester elastomers. It should be understood that a thermoplastic polyester elastomer can refer to a thermoplastic polyester homopolymer elastomer, a thermoplastic copolyester elastomer, or both. In aspects, the thermoplastic copolyester elastomer can include copolyesters having two or more types of polyester monomeric segments, or copolyesters comprising polyester monomeric segments and one or more non-polyester monomeric segments. In some aspects, the resin component of the thermoplastic elastomer composition, which is comprised of all the polymeric materials present in thermoplastic polyester composition, consists essentially of the one or more thermoplastic elastomers, or consists essentially of the one or more thermoplastic polyesters. Thermoplastic polyesters can include chain units derived from one or more olefins and chain units derived from one or more ethylenically-unsaturated acid groups, in aspects. The thermoplastic elastomer compositions can have a melt flow index of from about 5 to about 40, or about 10 to about 20, or about 20 to about 30 as determined at 210 degrees C. using a 2.16 kilogram weight. Alternatively or additionally, the thermoplastic elastomer compositions can have a melt flow index of from about 5 to about 40, or about 10 about 20, or about 20 to about 30 as determined at 220 degrees C. using a 2.16 kilogram weight. Alternatively or additionally, the thermoplastic elastomer compositions can have a melt flow index of from about 5 to about 40, or about 10 to about 20, or about 20 to about 30 as determined at 230 degrees C. using a 2.16 kilogram weight. The thermoplastic elastomer, including thermoplastic polyester, can have a weight average molecular weight of about 50,000 Daltons to about 1,000,000 Daltons; or about 50,000 Daltons to about 500,000 Daltons; or about 75,000 Daltons to about 300,000 Daltons; or about 100,000 Daltons to about 250,000 Daltons; or about 100,000 Daltons to about 500,000 Daltons. The thermoplastic elastomers, including thermoplastic copolyesters, can be terpolymers. In some aspects, thermoplastic copolyesters can be terpolymers of moieties derived from ethylene, acrylic acid, and methyl acrylate or butyl acrylate. In some aspects, a ratio of a total parts by weight of the acrylic acid in thermoplastic copolyesters to a total weight of thermoplastic copolyesters is about 0.05 to about 0.6, or about 0.1 to about 0.6, or about 0.1 to about 0.5, or about 0.15 to about 0.5, or about 0.2 to about 0.5. The thermoplastic elastomers can be terpolymers comprising a plurality of first segments, a plurality of second segments, and a plurality of third segments. In some aspects, the thermoplastic elastomer is a thermoplastic copolyester comprising: (a) a plurality of first segments, each first segment derived from a dihydroxy-terminated polydiol; (b) a plurality of second segments, each second segment derived from a diol; and (c) a plurality of third segments, each third segment derived from an aromatic dicarboxylic acid. In various aspects, thermoplastic copolyester is a block copolymer. In some aspects, thermoplastic copolyester is a segmented copolymer. In further aspects, thermoplastic copolyester is a random copolymer. In still further aspects, thermoplastic copolyester is a condensation copolymer. The thermoplastic elastomer, including thermoplastic copolyester, can have a ratio of first segments to third segments from about 1:1 to about 1:5 based on the weight of each of the first segments and the third segments; or about 1:1 to about 1:4 based on the weight of each of the first segments and the third segments; or about 1:1 to about 1:2 based on the weight of each of the first segments and the third segments; or about 1:1 to about 1:3 based on the weight of each of the first segments and the third segments. The thermoplastic elastomer, including thermoplastic copolyester, can have a ratio of second segments to third segments from about 1:1 to about 1:2 based on the weight of each of the first segments and the third segments; or about 1:1 to about 1:1.52 based on the weight of each of the first segments and the third segment. The thermoplastic elastomer, including thermoplastic copolyester, can have first segments derived from a poly(alkylene oxide)diol having a number-average molecular weight of about 250 Daltons to about 6000 Daltons; or about 400 Daltons to about 6,000 Daltons; or about 350 Daltons to about 5,000 Daltons; or about 500 Daltons to about 3,000 Daltons; or about 2,000 Daltons to about 3,000 Daltons. The thermoplastic elastomer, including thermoplastic copolyester, can have first segments derived from a poly(alkylene oxide)diol such as poly(ethylene ether)diol; poly(propylene ether)diol; poly(tetramethylene ether)diol; poly(pentamethylene ether)diol; poly(hexamethylene ether)diol; poly(heptamethylene ether)diol; poly(octamethylene ether)diol; poly(nonamethylene ether)diol; poly(decamethylene ether)diol; or mixtures thereof. In a still further aspect, thermoplastic copolyester can have first segments derived from a poly(alkylene oxide)diol such as poly(ethylene ether)diol; poly(propylene ether)diol; poly(tetramethylene ether)diol; poly(pentamethylene ether)diol; poly(hexamethylene ether)diol. In a yet further aspect, thermoplastic copolyester can have first segments derived from a poly(tetramethylene ether)diol. The thermoplastic elastomer, including thermoplastic copolyester, can have second segments derived from a diol having a molecular weight of less than about 250. The diol from which the second segments are derived can be a C2-C8 diol. In a still further aspect, the second segments can be derived from ethanediol; propanediol; butanediol; pentanediol; 2-methyl propanediol; 2,2-dimethyl propanediol; hexanediol; 1,2-dihydroxy cyclohexane; 1,3-dihydroxy cyclohexane; 1,4-dihydroxy cyclohexane; and mixtures thereof. In a yet further aspect, the second segments can be derived from 1,2-ethanediol, 1,3-propanediol, 1,4-butanediol, 1,6-hexanediol, and mixtures thereof. In an even further aspect, the second segments can be derived from 1,2-ethanediol. In a still further aspect, the second segments can be derived from 1,4-butanediol. The thermoplastic elastomer, including the copolyester, can have third segments derived from an aromatic C5-C16 dicarboxylic acid. The aromatic C5-C16 dicarboxylic acid can have a molecular weight less than about 300 Daltons; about 120 Daltons to about 200 Daltons; or a value or values of molecular weight within any of the foregoing ranges or a molecular weight range encompassing any sub-range of the foregoing ranges. In some instances, the aromatic C5-C16 dicarboxylic acid is terephthalic acid, phthalic acid, isophthalic acid, or a derivative thereof. In a still further aspect, the aromatic C5-C16 dicarboxylic acid is a diester derivative of the terephthalic acid, phthalic acid, or isophthalic acid. In a yet further aspect, the aromatic C5-C16 dicarboxylic acid is terephthalic acid or the dimethyl ester derivative thereof. Thermoplastic copolyester can comprise: (a) a plurality of first copolyester units, each first copolyester unit of the plurality comprising the first segment derived from a dihydroxy-terminated polydiol and the third segment derived from an aromatic dicarboxylic acid, wherein the first copolyester unit has a structure represented by a Formula 1: wherein R1is a group remaining after removal of terminal hydroxyl groups from the poly(alkylene oxide) diol of the first segment, wherein the poly(alkylene oxide) diol of the first segment is a poly(alkylene oxide) diol having a number-average molecular weight of about 400 to about 6000; and wherein R2is a group remaining after removal of carboxyl groups from the aromatic dicarboxylic acid of the third segment; and (b) a plurality of second copolyester units, each second copolyester unit of the plurality comprising the second segment derived from a diol and the third segment derived from an aromatic dicarboxylic acid, wherein the second copolyester unit has a structure represented by a Formula 2: wherein R3is a group remaining after removal of hydroxyl groups from the diol of the second segment derived from a diol, wherein the diol is a diol having a molecular weight of less than about 250; and wherein R2is the group remaining after removal of carboxyl groups from the aromatic dicarboxylic acid of the third segment. Thermoplastic copolyester can comprise a plurality of first copolyester units having a structure represented by a Formula 3: wherein R is H or methyl; wherein y is an integer having a value from 1 to 10; wherein z is an integer having a value from 2 to 60; and wherein a weight average molecular weight of each of the plurality of first copolyester units is from about 300 Daltons to about 7,000 Daltons. In some aspects, in the foregoing formula, y can be an integer having a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or y can be any set or range of the foregoing integer values. In some aspects, in the foregoing formula, z is an integer having a value from 5 to 60; an integer having a value from 5 to 50; an integer having a value from 5 to 40; an integer having a value from 4 to 30; an integer having a value from 4 to 20; an integer having a value from 2 to 10; or z can be any set or range of the foregoing integer values. In some aspects, R is hydrogen. In a still further aspect, R is methyl. In some instances, R is hydrogen and y is an integer having a value of 1, 2, or 3. Alternatively, in other instances, R is methyl and y is an integer having a value of 1. Thermoplastic copolyester can comprise a plurality of first copolyester units having a structure represented by a Formula 4: wherein z is an integer having a value from 2 to 60; and wherein a weight average molecular weight of each of the plurality of first copolyester units is from about 300 Daltons to about 7,000 Daltons. In some aspects, in the foregoing formula, y can be an integer having a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or y can be any set or range of the foregoing integer values. In some aspects, in the foregoing formula, z is an integer having a value from 5 to 60; or an integer having a value from 5 to 50; or an integer having a value from 5 to 40; or an integer having a value from 4 to 30; or an integer having a value from 4 to 20; or an integer having a value from 2 to 10. Thermoplastic copolyester can comprise a plurality of first copolyester units having a weight average molecular weight from about 400 Daltons to about 6,000 Daltons; or about 400 Daltons to about 5,000 Daltons; or about 400 Daltons to about 4,000 Daltons; or about 400 Daltons to about 3,000 Daltons; or about 500 Daltons to about 6,000 Daltons; or about 500 Daltons to about 5,000 Daltons; or about 500 Daltons to about 4,000 Daltons; or about 500 Daltons to about 3,000 Daltons; or about 600 Daltons to about 6,000 Daltons; or about 600 Daltons to about 5,000 Daltons; or about 600 Daltons to about 4,000 Daltons; or about 600 Daltons to about 3,000 Daltons; or about 2,000 Daltons to about 3,000 Daltons. Thermoplastic copolyester can comprise a plurality of second copolyester units, each second copolyester unit of the plurality having a structure represented by a Formula 5: wherein x is an integer having a value from 1 to 20; wherein the foam article has a multicellular closed-cell or open-cell foam structure. In some aspects, in the foregoing formula, x is an integer having a value from 2 to 18; 2 to 17; 2 to 16; 2 to 15; 2 to 14; 2 to 13; 2 to 12; 2 to 11; 2 to 10; 2 to 9; 2 to 8; 2 to 7; 2 to 6; 2 to 5; 2 to 4; or x can be any integer value or set of integer values within the foregoing ranges or values, or any range of integer values encompassing a sub-range of the foregoing integer value ranges. In a further aspect, x is an integer having a value of 2, 3, or 4. Thermoplastic copolyester can comprise a plurality of second copolyester units, each second copolyester unit of the plurality having a structure represented by a Formula 6: Thermoplastic copolyester can comprise a weight percent range of the plurality of first copolyester units based on total weight of thermoplastic copolyester such that the weight percent range is about 30 weight percent to about 80 weight percent; or about 40 weight percent to about 80 weight percent; or about 50 weight percent to about 80 weight percent; or about 30 weight percent to about 70 weight percent; or about 40 weight percent to about 70 weight percent; or about 50 weight percent to about 70 weight percent; or about 40 weight percent to about 65 weight percent; or about 45 weight percent to about 65 weight percent; or about 50 weight percent to about 65 weight; or about 55 weight percent to about 65 weight percent; or about 40 weight percent to about 60 weight percent; or about 45 weight percent to about 60 weight percent; or about 50 weight percent to about 60 weight percent; or about 55 weight percent to about 60 weight percent. In some aspects, the thermoplastic elastomer, including thermoplastic copolyester, can comprise phase separated domains. For example, a plurality of first segments derived from a dihydroxy-terminated polydiol can phase-separate into domains comprising primarily the first segments. Moreover, a plurality of second segments derived from a diol can phase-separate into domains comprising primarily the second segments. In other aspects, thermoplastic copolyester can comprise phase-separated domains comprising primarily of a plurality of first copolyester units, each first copolyester unit of the plurality comprising the first segment derived from a dihydroxy-terminated polydiol and the third segment derived from an aromatic dicarboxylic acid, wherein the first copolyester unit has a structure represented by a Formula 1: wherein R1is a group remaining after removal of terminal hydroxyl groups from the poly(alkylene oxide) diol of the first segment, wherein the poly(alkylene oxide) diol of the first segment is a poly(alkylene oxide) diol having a number-average molecular weight of about 400 to about 6000; and wherein R2is a group remaining after removal of carboxyl groups from the aromatic dicarboxylic acid of the third segment; and other phase-separated domains comprising primarily of a plurality of second copolyester units, each second copolyester unit of the plurality comprising the second segment derived from a diol and the third segment derived from an aromatic dicarboxylic acid, wherein the second copolyester unit has a structure represented by a Formula 2: wherein R3is a group remaining after removal of hydroxyl groups from the diol of the second segment derived from a diol, wherein the diol is a diol having a molecular weight of less than about 250; and wherein R2is the group remaining after removal of carboxyl groups from the aromatic dicarboxylic acid of the third segment. In other aspects, thermoplastic copolyester can comprise phase-separated domains comprising primarily of a plurality of first copolyester units, each first copolyester unit of the plurality having a structure represented by a Formula 3: wherein R is H or methyl; wherein y is an integer having a value from 1 to 10; wherein z is an integer having a value from 2 to 60; and wherein a weight average molecular weight of each of the plurality of first copolyester units is from about 300 Daltons to about 7,000 Daltons. In some aspects, in the foregoing formula, y can be an integer having a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or y can be any set or range of the foregoing integer values. In some aspects, in the foregoing formula, z is an integer having a value from 5 to 60; an integer having a value from 5 to 50; an integer having a value from 5 to 40; an integer having a value from 4 to 30; an integer having a value from 4 to 20; an integer having a value from 2 to 10; or z can be any set or range of the foregoing integer values. In some aspects, R is hydrogen. In a still further aspect, R is methyl. In some instances, R is hydrogen and y is an integer having a value of 1, 2, or 3. Alternatively, in other instances, R is methyl and y is an integer having a value of 1. In other aspects, thermoplastic copolyester can comprise phase-separated domains comprising primarily of a plurality of first copolyester units, each first copolyester unit of the plurality having a structure represented by a Formula 4: wherein z is an integer having a value from 2 to 60; and wherein a weight average molecular weight of each of the plurality of first copolyester units is from about 300 Daltons to about 7,000 Daltons. In some aspects, in the foregoing formula, y can be an integer having a value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; or y can be any set or range of the foregoing integer values. In some aspects, in the foregoing formula, z is an integer having a value from 5 to 60; or an integer having a value from 5 to 50; or an integer having a value from 5 to 40; or an integer having a value from 4 to 30; or an integer having a value from 4 to 20; or an integer having a value from 2 to 10. Thermoplastic copolyester can comprise phase-separated domains comprising primarily of a plurality of first copolyester units having a weight average molecular weight from about 400 Daltons to about 6,000 Daltons; or about 400 Daltons to about 5,000 Daltons; or about 400 Daltons to about 4,000 Daltons; or about 400 Daltons to about 3,000 Daltons; or about 500 Daltons to about 6,000 Daltons; or about 500 Daltons to about 5,000 Daltons; or about 500 Daltons to about 4,000 Daltons; or about 500 Daltons to about 3,000 Daltons; or about 600 Daltons to about 6,000 Daltons; or about 600 Daltons to about 5,000 Daltons; or about 600 Daltons to about 4,000 Daltons; or about 600 Daltons to about 3,000 Daltons; or about 2,000 Daltons to about 3,000 Daltons In other aspects, thermoplastic copolyester can comprise phase-separated domains comprising a plurality of second copolyester units, each second copolyester unit of the plurality having a structure represented by a Formula 5: wherein x is an integer having a value from 1 to 20; wherein the foam article has a multicellular closed-cell or open-cell foam structure. In some aspects, in the foregoing formula, x is an integer having a value from 2 to 18; or 2 to 17; or 2 to 16; or 2 to 15; or 2 to 14; or 2 to 13; or 2 to 12; or 2 to 11; or 2 to 10; or 2 to 9; or 2 to 8; or 2 to 7; or 2 to 6; or 2 to 5; or 2 to 4. In other aspects, thermoplastic copolyester can comprise phase-separated domains comprising a plurality of second copolyester units, each second copolyester unit of the plurality having a structure represented by a Formula 6: Thermoplastic copolyester can comprise phase-separated domains comprising a weight percent range of the plurality of first copolyester units based on total weight of thermoplastic copolyester such that the weight percent range is about 30 weight percent to about 80 weight percent; or about 40 weight percent to about 80 weight percent; or about 50 weight percent to about 80 weight percent; or about 30 weight percent to about 70 weight percent; or about 40 weight percent to about 70 weight percent; or about 50 weight percent to about 70 weight percent; or about 40 weight percent to about 65 weight percent; or about 45 weight percent to about 65 weight percent; or about 50 weight percent to about 65 weight percent; or about 55 weight percent to about 65 weight percent; or about 40 weight percent to about 60 weight percent; or about 45 weight percent to about 60 weight percent; or about 50 weight percent to about 60 weight percent; or about 55 weight percent to about 60 weight percent. In various aspects, the thermoplastic elastomer composition can include one or more thermoplastic polyester homopolymer, where the thermoplastic polyester homopolymer comprises any of the polyester monomeric segments or units disclosed herein or modifications thereof. In the same or alternative aspects, the thermoplastic elastomer composition can include one or more thermoplastic polyester homopolymer, where the thermoplastic polyester homopolymer comprises any polyester homopolymer exhibiting any or all of the properties and parameters discussed herein with respect to thermoplastic elastomers and/or the thermoplastic elastomer composition. The disclosed thermoplastic elastomer composition, the polymeric component of the composition or an individual thermoplastic elastomer in neat form can be characterized by one or more properties. In some aspects, the thermoplastic elastomer composition or the polymeric component, or the polymer has a maximum load of about 10 newtons to about 100 newtons, or from about 15 newtons to about 50 newtons, or from about 20 newtons to about 40 newtons, when determined using the Cyclic Tensile Test method described herein. The tensile strength of the thermoplastic elastomer composition or of the polymeric component of the thermoplastic elastomer composition or of a thermoplastic elastomer in neat form is another important physical characteristic. The thermoplastic elastomer composition or polymeric component or elastomer can have a tensile strength of from 5 kilograms per square centimeter to 25 kilograms per square centimeter, or of from 10 kilograms per square centimeter to 23 kilograms per square centimeter, or of from 15 kilograms per square centimeter to 22 kilograms per square centimeter, when determined using the Cyclic Tensile Test method described herein. The thermoplastic elastomer composition or polymeric component of the thermoplastic elastomer composition or a thermoplastic elastomer in neat form can have a tensile modulus of about 2 megapascals to about 20 megapascals or from about 5 megapascals to about 15 megapascals when determined using the Cyclic Tensile Test method described herein. Exemplary, but non-limiting, thermoplastic elastomers, including thermoplastic polyesters, that can be used in the disclosed methods, foams, and articles include “HYTREL” 3078, “HYTREL” 4068, and “HYTREL” 4556 (DuPont, Wilmington, Del., USA); “PELPRENE” P30B, P40B, and P40H (Toyobo U.S.A. Inc., New York, N.Y., USA); “TRIEL” 5300, “TRIEL” 5400, and blends thereof (Samyang Corporation, Korea); “KEYFLEX” BT1028D, BT1033D, BT1035D, BT1040D, BT1045D, and BT1047D (LG Chem, Korea); and “KOPEL” KP3340, KP3346, KP3347 (Kolon Plastics, Inc., Korea). The disclosed thermoplastic elastomer compositions can further include one or more ionomers, such as any of the “SURLYN” polymers (DuPont, Wilmington, Del., USA). Foams as described herein can be made by a process/method including receiving a composition described herein, and physically foaming the composition to form a thermoplastic elastomer foam having a density of about 0.7 gram per cubic centimeter or less, or 0.5 gram per cubic centimeter or less, or 0.4 gram per cubic centimeter or less, or 0.3 gram per cubic centimeter or less. The disclosed thermoplastic elastomer compositions can further include one or more thermoplastic polyurethanes, such as “FORTIMO” (Mitsui Chemicals, Inc., Tokyo, Japan); “TEXIN” (Covestro LLC, Pittsburgh, Pa., USA); and “BOUNCELL-X” (Lubrizol Advanced Materials, Inc., Brecksville, Ohio, USA). The disclosed thermoplastic elastomer compositions can further include one or more olefinic polymers. Olefinic polymers can include ethylene-based copolymers, propylene-based copolymers, and butene-based copolymers. In some aspects, the olefinic polymer is an ethylene-based copolymer such as a styrene-ethylene/butylene-styrene (SEBS) copolymer; an ethylene-propylene diene monomer (EPDM) copolymer; an ethylene-vinyl acetate (EVA) copolymer; an ethylene alkyl acrylate (EAA) copolymer; an ethylene alkyl methacrylate (EAMA) copolymer; any copolymer thereof, and any blend thereof. In some aspects, a ratio V of a total parts by weight of the olefinic polymers present in the composition to a total parts by weight of thermoplastic polyesters in the composition is about 0.0 to about 0.6, or about 0.0 to about 0.4, or about 0.01 to about 0.4, or about 0.01 to about 0.6, or about 0.1 to about 0.4. The disclosed thermoplastic elastomer compositions can further include an ethylene-vinyl acetate (EVA) copolymer. The ethylene-vinyl acetate (EVA) copolymer can have a range of vinyl acetate contents, for example about 50 percent to about 90 percent, or about 50 percent to about 80 percent, or about 5 percent to about 50 percent, or about 10 percent to about 45 percent, or about 10 percent to about 30 percent, or about 30 percent to about 45 percent, or about 20 percent to about 35 percent, based on the weight of the copolymer. Thermoplastic Elastomer Composition Characterization Component Sampling Procedure This procedure can be used to obtain a sample of a foam composition or material when the composition or material is incorporated into a component such as a sole structure or midsole or outsole of an article of footwear. A sample of the component which includes the composition or material is obtained as formed into the component, or cut from the article of footwear using a blade. This process is performed by separating the component from an associated footwear upper, if present, and removing any materials from the article's top surface (e.g., corresponding to the top surface). For example, the article's top surface can be skinned, abraded, scraped, or otherwise cleaned to remove any upper adhesives, yarns, fibers, foams, and the like that could potentially interfere with the test results. The resulting component sample includes the composition or material. As such, any test using a Component Sampling Procedure can simulate how the composition or material will perform as part of an article of footwear. As specified by the test method, the component may be tested as a full component (e.g., full midsole component), or it can be extracted as a sample having a certain geometry. A sample of a component is taken at a location along the component that provides a substantially constant thickness for the component (within plus or minus 10 percent of the average thickness), such as in a forefoot region, mid-foot region, or a heel region of the article. Unless otherwise specified, the desired harvested geometry is a cylindrical puck with a 45-millimeter diameter and a cylinder height of at least about 10 millimeters, preferably from about 20 to 25 millimeters. Density Test The density is measured for samples taken using the Component Sampling Procedure, using a digital balance or a Densicom Tester (Qualitest, Plantation, Fla., USA). For each sample a sample volume is determined in cubic centimeters, and then each sample is weighed (g). The density of the sample is the mass divided by the sample volume, given in grams/cubic centimeters. Specific Gravity Test This test is appropriate for testing closed-cell foams, and samples of open-cell foams having a substantially uniform closed skin. The specific gravity (SG) is measured for samples taken using the Component Sampling Procedure, using a digital balance or a Densicom Tester (Qualitest, Plantation, Fla., USA). Each sample is weighed (g) and then is submerged in a distilled water bath (at 22 degrees C. plus or minus 2 degrees C.). To avoid errors, air bubbles on the surface of the samples are removed, e.g., by wiping isopropyl alcohol on the sample before immersing the sample in water, or using a brush after the sample is immersed. The weight of the sample in the distilled water is recorded. The specific gravity is calculated with the following formula: S.G.=Weightofthesampleinair(g)Weightofsampleinair(g)-Weightofsampleinwater(g) Force/Displacement Test (Cyclic Compression Test for a Foot Form) Force/displacement behavior for the foams and the foamed articles may be measured using a full midsole sample, a full outsole sample, a split midsole and/or a split midsole, tested using a foot form for impact to accurately simulate full gate loading. For these tests, a US men's size 10 midsole is tested, and a men's size 9 foot form used for impact, with a load of 2000N being applied to the midsole with the foot form at a loading rate of 5 Hz with a cyclic compression testing device such an Instron Electropuls E10000 (Instron, Norwood, Mass., USA). Each sample is compressed to the peak load at 5 Hz for 100 cycles. Energy input (J), energy return (J), energy efficiency (energy return/energy input), energy efficiency percentage (100*(energy return/energy input)) and maximum displacement (mm) are measured from the force vs. displacement curves generated. Stiffness of a particular foam sample is the maximum load divided by the displacement at the maximum load, giving a value in N/mm. The reported value for each metric is the average of the metrics from the 60th, 70th, 80th, and 90thcycles. Cyclic Compression Test for a Sample Force/displacement behavior for the foams and the foamed articles may also, or alternatively, be measured using samples harvested from a larger component (e.g., cylindrical pucks harvested from a footwear midsole), and a method for obtaining a sample is described in the “Component Sampling Procedure” portion of this disclosure. In one testing methodology, when testing a sample (e.g., a cylindrical puck harvested from a larger component), the sample is tested along the length axis of the part using compression platens that are at least 2× the diameter (e.g., of the cylindrical puck). Furthermore, the sample is compressed to the peak load (e.g., 50% strain) at 5 Hz for 500 cycles. Stiffness, efficiency, and energy return are measured from the force vs. displacement curves for cycles 200, 300, 400, and 500, and the reported value for each metric is the average of each metric between cycles 200, 300, 400, and 500. Stiffness, efficiency, and energy return are defined in the following ways, with example property ranges (possibly dependent on sample geometries) provided in parentheses. Stiffness is the stress at the maximum strain divided by the maximum strain (e.g., 200-1000 kPa). Efficiency is the integral of the unloading force-displacement curve divided by the integral of the loading force-displacement curve (e.g., 0.50-0.97). Energy return is the integral of the unloading curve (e.g., 200-1200 mJ). Cyclic Tensile Test The cyclic tensile testing is carried out on solid samples prepared using the Component Sampling Procedure, having a dog-bone shape as described in ASTM D638 with a 2 mm thickness. In the test, the specimen is placed under a pre-load of 5 N. Strain is controlled to extend the sample to an extension 6 percent at a strain rate of 5 Hz. The stiffness is the load at 6 percent strain divided by the extension at 6 percent strain, giving a value in N/mm. The maximum load (N) observed over the test cycle of 500 cycles is also recorded. Durometer Hardness Test—Shore A The test used to obtain the hardness values for the foam articles is as follows. A flat foam sample is prepared using the Component Sampling Procedure, where the sample has a minimum of 6 mm thick for Shore A durometer testing. If necessary, samples are stacked to make up the minimum thickness. Samples are large enough to allow all measurements to be performed at a minimum of 12 mm from the edge of the sample and at least 12 mm from any other measurement. Regions tested are flat and parallel with an area at least 6 mm in diameter. A minimum of five hardness measurements are taken and tested using a 1 kilogram head weight. Split Tear Test The split tear test can determine the internal tear strength for a foam material. A sample may be provided using the Component Sampling Procedure. The sample is die cut into a rectangular shape having a width of 1.54 centimeters and a length of 15.24 centimeters (1 inch by 6 inches), and having a thickness of 10 millimeters, plus or minus 1 millimeter. On one end, a cut is made into the sample that bisects the thickness, the cut extending the full width of the sample, and 3 centimeters from the end of the sample. Starting from the end of the cut, 5 marks are placed along the length of the sample spaced 2 centimeters apart. The cut ends of the sample are placed in the clamps of a tensile tester. Each section of the sample is held in a clamp in such a manner that the original adjacent cut edges form a straight line joining the centers of the clamps. The crosshead speed is set to 50 millimeters per minute. The tear strength is measured throughout the separation of the crossheads. If necessary, a sharp knife may be used to keep separating the foam in the center of the sample, discarding the readings caused by cutting of the knife. The lowest split tear strength values are recorded for each of the five marked segments of the sample (between each of the 2-centimeter markings). An average split tear strength value is recorded for each sample. If a segment of a sample has an air bubble measuring more than 2 millimeters, the tear strength for the segment is discarded, and the air bubble recorded as a test defect. If more than one segment of a sample has an air bubble measuring more than 2 millimeters, the entire sample is discarded. Energy Intensity Energy intensity is a measure of the energy used in forming a particular foam article in kilowatt hours (kW-h). To obtain the energy intensity, the energy required (in kW-H) to produce a run, or batch, of articles, such as cushioning elements (such as pairs of the midsole122) is first calculated, determined or measured (from pellet to finished component). For example, for a physical foaming process the measured energy may include the energy required for all energy consuming steps, such as: preheating the molds and hot runners (if utilized), melting the pellets, generating gas counter-pressure, injecting the molten plastic, introducing the supercritical fluid, cooling the molds and/or work-pieces and ejecting the work-pieces from the mold. The overall energy required to produce the run of cushioning element pairs is then divided by the number of cushioning element pairs produced in the run. Zero Shear Viscosity The zero shear viscosity is determined using a flow curve obtained on a rotational rheometer. Zero shear viscosity is determined as the apparent viscosity of the polymer melt measured at a shear rate of 1×10−21/s when the polymer is heated to 10° C. above its melting temperature. Apparent viscosity is measured under continuous flow using a cone and plate rotational fixture. The temperature of the rotational fixture is maintained at the polymer melt temperature. The gap and geometry of the cone are selected such that the measured torque is well within the measuring limits of a rheometer. Melt Flow Index Test The melt flow index is determined using a sample prepared using Component Sampling Procedure, according to the test method detailed in ASTM D1238-13 Standard Test Method for Melt Flow Rates of Thermoplastics by Extrusion Plastometer, using Procedure A described therein. Briefly, the melt flow index measures the rate of extrusion of thermoplastics through an orifice at a prescribed temperature and load. In the test method, approximately 7 grams of the sample is loaded into the barrel of the melt flow apparatus, which has been heated to a specified temperature of 210 degrees C., 220 degrees C., or 230 degrees C. A weight of 2.16 kilograms is applied to a plunger and the molten sample is forced through the die. A timed extrudate is collected and weighed. Melt flow rate values are calculated in g/10 min, and are reported with the specified temperature (i.e., 210, 220 or 230 degrees C.) and the weight applied to the plunger (i.e., 2.15 kilograms). Injection Molding System for Forming a Foam Article As indicated above, some aspects of the present disclosure include an injection-molding system and components for forming an article comprising a foamed thermoplastic elastomer composition. For example, referring toFIG.1A, a tooling assembly110is illustrated in combination with a first hot-runner plate112and a second hot-runner plate114. The tooling assembly110includes one or more injection molds coupled to a first carrier plate116and a second carrier plate118, which may support the one or more molds during one or more stages of an injection-molding process (e.g., when the molds are being transported from one station to another station, when the part(s) are being unloaded, etc.). For instance, referring toFIG.2, the hot-runner plates112and114and the carrier plates116and118have been omitted to unobstructedly illustrate a first mold120and a second mold122. The first hot-runner plate112interfaces with the first mold120, and the second hot-runner plate114interfaces with the second mold122to distribute one or more deposits or shots of thermoplastic elastomer composition (e.g., single-phase solution or other composition having a foaming agent) into mold cavities. For purposes of this disclosure, various elements of the first mold120are described, and it is understood that the second mold122may have similar elements, although not explicitly described. For example, the first mold includes a first mold plate220, a mold ring plate320, and a second mold plate420, and the second mold122might also include a respective first mold plate221, a respective mold ring plate321, and a respective second mold plate421having similar elements. The first mold plate220, the mold ring plate320, and the second mold plate420are layerable (e.g., stackable) to form one or more cavities for receiving a deposit of a thermoplastic elastomer composition. For example,FIG.3depicts a partially exploded view including the first mold plate220and the mold ring plate320separated from one another. In this partially exploded view ofFIG.3, a portion of a first mold cavity and a portion of a second mold cavity can be seen, each of which is shaped to form at least a portion of a footwear sole. Although the first mold120includes two mold cavities, in other aspects, a mold may include fewer or more mold cavities. In this disclosure, various elements of the first mold cavity are described, and it is understood that the second mold cavity may have similar elements, although not explicitly described. For example, the first mold cavity may be at least partially enclosed by a mold core224, and the second mold cavity may include a similar mold core226(although a mirror image). In one aspect of this disclosure, the mold core224is nestable in a mold-ring cavity328when the mold ring plate320is layered against or abutting the first mold plate220. Referring toFIG.4, the mold ring plate320is illustrated as layered directly against and abutting the first mold plate220, and the mold core224is depicted nested in the mold-ring cavity328, such that the mold core224forms a first mold cavity wall. In addition,FIG.4depicts a partially exploded view with the second mold plate420being separated from the mold ring plate320. The second mold plate420may be positioned directly against (e.g., abutting) the mold ring plate320to more fully enclose the mold-ring cavity328. The second mold plate420may also include one or more runners and gates through which the thermoplastic elastomer composition may flow when being deposited into the cavities. For example,FIG.4depicts a plurality of gate outlets434,436,438,440,442and444, andFIG.2depicts a plurality of runner inlets426,428,430, and432and runners that are fluidly coupled to the plurality of gate outlets434,436,438,440,442and444. Some details of the first mold120have been described above, and these details provide context for other components of the system (e.g., the hot-runner plate112). Additional details of the first mold120will be described in other parts of this disclosure below. Referring toFIG.5, a partially exploded view depicts the hot-runner plate112that interfaces with the second mold plate420, and the hot-runner plate112may include one or more hot-runner outlets134a-134hthat align with, and fluidly connect to, the plurality of runner inlets426,428,430, and432of the second mold plate420. For example,FIG.1Adepicts the hot-runner plate112interfacing with the second mold plate420and also depicts a plurality of nozzle-receiving openings136a-136hinto which injection nozzles may be inserted for distributing material through the hot-runner plate112and the second mold plate420and into the mold cavity or cavities. Referring toFIG.6, an example of a set of injection nozzles (e.g.,138) is shown as part of an injection manifold140. That is, the injection manifold140includes an injection port142that connects to an injector of an injection molding system. The injection manifold140also includes a series of internal components (not shown) that receive a deposit/shot of thermoplastic elastomer composition from the injector and divide the shot into a number of deposits for separately distributing through the nozzles. The injection manifold140may also include one or more sensors (e.g., thermocouples) for monitoring conditions of the manifold (e.g., temperature, pressure, etc.) that may affect the thermoplastic elastomer composition, as well as a manifold temperature conditioning unit for maintaining, increasing, or decreasing a temperature of the manifold. For example, the manifold temperature conditioning unit may include conditioned-fluid lines150for holding and transporting coolant or heated conditioning fluid. As such, a state of the thermoplastic elastomer composition (e.g., single-phase solution) may be maintained while a deposit is distributed from each nozzle into the hot-runner plates112and114. For example, the manifold140may maintain the thermoplastic elastomer composition at conditions conducive to maintaining the foaming agent in a supercritical-fluid phase and to reducing the likelihood of transition to a gas. In a further aspect, the manifold includes an injector-pin assembly (e.g.,152) for each nozzle, which may selectively insert a pin (or other obstruction) into a tip of each nozzle to impede a flow of material. In a further aspect of the present disclosure, the nozzles are arranged in groups of nozzles, including between two and six nozzles. For example, inFIG.6, the nozzles are arranged in four groups of four linearly aligned nozzles, and in other aspects the groups may include two, three, five, or six linearly aligned nozzles. Each group of nozzles is positioned to collectively inject material into a single mold cavity. For example, in one aspect of the present disclosure, each group of four nozzles is configured to inject material into a single mold cavity having a three-dimensional shape of a footwear component (e.g., footwear sole). In one aspect, four nozzles optimizes the available footprint and injection-system real estate operable to distribute material in to the mold cavity having the 3D shape of a footwear sole. That is, the 3D shape of an average size footwear sole includes a length and four nozzles may optimally use that length to evenly distribution shots of material into the mold cavity in a way that each shot foams and solidifies in a desired manner InFIG.6, the manifold140includes sixteen nozzles, each of which is insertable into a respective nozzle-receiving opening (e.g.,136a-136h) of the hot-runner plates112and114depicted inFIG.1A. Referring toFIGS.7A and7B, some of the walls of the hot-runner plate112are omitted to illustrate some interior components of the hot-runner plate112in more detail. For example, the hot-runner plate112includes eight nozzle-receiving sleeves156a-156h, each of which receives a respective nozzle of the injection manifold140. Each nozzle-receiving sleeve includes a nozzle-receiving opening136a-136h(seeFIG.1A and136ainFIG.7B) and a sleeve outlet (e.g.,158in the cross-sectional view ofFIG.7B). The sleeve outlet158includes a perimeter rim160that forms a nozzle seat162against which a tip of the nozzle biases when then nozzle is fully inserted into the nozzle-receiving sleeve. The hot-runner plate112also includes hot runners (e.g.,164) that transport material from each nozzle after being dispensed. For example, each hot runner (e.g.,164) includes a hot-runner inlet (e.g.,166inFIG.7B) that fluidly connects with the sleeve outlet (e.g.,158in the cross-sectional view ofFIG.7B) and includes a hot-runner outlet134a-134h(see alsoFIG.5showing the hot-runner outlets). In an aspect of the present disclosure, the hot-runner inlet is spaced apart from the hot-runner outlet by a distance in a range of about 1 cm to about 3 cm. As such, when material is dispersed from the nozzle, a sprue is formed in the hot runner, the sprue having a length in a range of about 1 cm to about 3 cm. In an aspect of the present disclosure, this sprue length provides a grasping region at which a tool can grip the sprue for removing solidified material from the runners in the second mold plate420. The hot-runner plate112includes various components to help control conditions related to the injection-molding system. For example, the hot-runner plate112includes conditioned-fluid lines170for transporting conditioned fluid throughout the hot-runner plate112. The conditioned fluid may be conditioned to include a temperature for maintaining, increasing, or decreasing a temperature of components of the hot-runner plate, including the hot runners (e.g.,164), the nozzle-receiving sleeves156a-156h, and the nozzles when inserted in the sleeves (see alsoFIG.1Edepicting a conditioned-fluid inlet172and a conditioned fluid outlet174positioned on an exterior wall of the hot-runner plate112). As such, when the thermoplastic elastomer composition (e.g., single-phase solution with supercritical fluid as physical foaming agent) is dispensed from a nozzle, the temperature in the hot runner may be maintained high enough to delay transition of the supercritical fluid to a gas. In a further aspect, the hot-runner plate112includes a gas conduit176having a first gas port178(see e.g.,FIGS.1A and7A) for fluidly communicating with a gas counter pressure (GCP) system and a second gas port180or “gas-conduit port” (see e.g.,FIG.5) for fluidly communicating with a gas port181(also referred to as “fluid-channel port”) of the second mold plate420. In addition, the side of the hot-runner plate112facing towards the mold includes a seal assembly for sealingly mating with the second mold plate420. For example,FIG.5depicts a seal groove184that receives a resilient seal182. The second mold plate420may additionally, or alternatively, include a seal groove. In some aspects of the present disclosure, the GCP system is fluidly connected from the gas port178on the hot-runner plate112to the sleeve outlet (e.g.,158inFIG.7B). As such, when the thermoplastic elastomer composition (e.g., single-phase solution with supercritical fluid as physical foaming agent) is dispensed from a nozzle, the pressure in the mold cavity and runners may be maintained high enough to delay transition of the supercritical fluid to a gas. In one aspect of the present disclosure, the hot-runner plate112is a universal hot-runner plate that is coupled directly to the manifold. For example, one or more fasteners may couple the hot-runner plate112to the manifold140. Some conventional injection molding systems may, in contrast to the present disclosure, have separate hot-runner plates that each interfaces with a different cold-runner plate (or other plate that is not temperature conditioned) and that are connected and disconnected to the manifold or nozzles in each injection cycle. This aspect of the present disclosure includes a universal hot-runner plate that is mountable to the nozzles and that can interface with an array of different molds, each of which includes a different mold cavity, a different gate scheme, or any combination thereof. For example, the mold cavities may differ in volume and or shape as being used to mold parts of different sized shoes, and the gate scheme may differ by including different gate positions and/or quantities of gates. In addition, the hot-runner plate112is plumbed with all of the components used to control various aspects of the molding process, including the conditioned-fluid lines170and the gas conduit176. Hot-runner plates are often associated with higher costs (e.g., added plumbing for temperature conditioning elements). Among other things, a universal hot-runner plate may reduce costs across multiple sets of molds, since only a single, common hot-runner plate may be used across the multiple sets, as opposed to having to make a hot-runner plate for each mold. In addition, it can reduce costs over time since there are fewer parts to store, maintain, repair, move, handle, etc. Although the figures of this present disclosure illustrate the hot-runner plates112and114, which provide an interface between the injector nozzles and the first and second molds120and122, in other aspects of this disclosure, universal cold-runner plates or other types of universal runner plates may provide the interface between the injector nozzles and the first and second molds120and122. Mold for Forming a Foam Article Referring back toFIGS.2-4, and also referring toFIGS.8A-8C, some additional aspects of the first mold120are depicted, including the first mold plate220, the mold ring plate320, and the second mold plate420. For example, the second mold plate420includes a first side422that faces towards the mold ring plate320and a second side424that faces away from the mold ring plate320. When the first mold120interfaces with the hot-runner plate112(e.g.,FIG.1A), the second side424faces towards and abuts the hot-runner plate112. The second side424includes a plurality of runner inlets426,428,430, and432that align with, and receive material from, the hot-runner outlets of the hot-runner plate112. Furthermore, each runner inlet may fluidly connect to one or more runners. For example, referring toFIG.8Cthe runner inlet426fluidly connects with a first runner426aand a second runner426b, and the runner inlet428fluidly connects with a first runner428aand a second runner428b. In addition, the runner inlet430connects to a single runner, including a runner430a, and the runner inlet432also connects to a single runner, including a runner432a. In addition,FIG.8Cdepicts the inlets of a plurality of secondary runners427a,427b,429a,429b,431, and433that fluidly connect each runner to a respective gate and gate outlet of the second mold plate420. That is, only the inlet of each secondary runner is viewable inFIG.8Cbecause the secondary runner extends from the second side424, through a mold body of the second mold plate, towards the second side422, the gates, and the gate outlets. The plurality of runner inlets426,428,430, and432may include various features. For example, each runner inlet is positioned and arranged to align with a hot-runner outlet134d,134c,134b, and134a(respectively) of the hot-runner plate112. As such, when nozzles inserted into the hot-runner plate112inject material through the hot-runner outlets134d,134c,134b, and134a, the material may flow into the runner inlets426,428,430, and432to be directed to the mold cavity by way of the runners. In a further aspect, the relative positioning of the runner inlets426,428,430, and432represents a universal positioning and arrangement configured to align with the hot-runner outlets of the hot-runner plate. That is, in aspects in which the hot-runner plate112is a universal hot-runner plate used to interface with an array of different molds, each of the molds in the array may include a mold plate having runner inlets positioned similarly to the runner inlets426,428,430, and432, regardless of the arrangement of the runners. In one aspect, this universal positioning and arrangement includes four runner inlets that may be linearly aligned and that may be evenly spaced apart from one another. For example,FIG.8Cdepicts a longitudinal reference line435marking a position of a longitudinal reference plane that intersects each of the runner inlets (the longitudinal reference plane extends orthogonal to the view shown inFIG.8Cat longitudinal reference line435). In another aspect, the runners may also include various features. For example, the runners may extend in various directions from a respective runner inlet to a secondary runner. That is, in some instances, a runner (e.g.,428a) may extend from a runner inlet in a direction aligned with the longitudinal reference plane, such that the secondary runner (e.g.,429a) and terminal gate outlet (e.g.,438) is also aligned with the longitudinal reference plane. In other examples, a runner (e.g.,428b) may extend from a runner inlet in a direction away from the longitudinal reference plane, and in this sense, the runner428bis incongruent from the runner428a. In that case, the runner (e.g.,428b) may curve or bend back into the longitudinal reference plane, such that the secondary runner (e.g.,429b) and terminal gate outlet is still aligned with the longitudinal reference plane. Alternatively, the runner (e.g.,426a,426b,430a, and432a) may extend away from the longitudinal reference plane and terminate at a secondary runner (e.g.,427a,427b,431, and433) not aligned with the longitudinal reference plane. In a further aspect of the disclosure, the second mold plate420includes a mold cavity wall425on the first side422that encloses at least a portion of the mold cavity of the first mold120. The mold cavity wall425includes a plurality of gate outlets through which material flows when injected into the mold cavity. That is, each secondary runner is fluidly connected with a respective gate outlet on the first side422of the second mold plate. For example,FIG.8Cdepicts a plurality of gate outlets434,436,438,440,442, and444from which material may flow after being injected into the runners. The plurality of gate outlets434,436,438,440,442, and444include various features. For example, in one aspect of the present disclosure, each secondary runner tapers from a larger diameter near a respective runner to a smaller diameter near each gate. As such, in one aspect, each gate outlet is an aperture having a diameter in a range of about 1.00 millimeters to about 5.00 millimeters, or 2.00 millimeters to 3.50 millimeters. In at least some instances, a diameter in this range is operable to affect a flow, condition, and/or foaming activity of a material injected through the gate outlet, and is related to a viscosity of the thermoplastic elastomer composition (e.g., single-phase solution). In addition, each gate outlet includes a position that is relative to other gate outlets and that is relative to the three-dimensional shape of the mold cavity. For example,FIG.8Cdepicts a longitudinal reference line437that is coplanar with the longitudinal reference line435(e.g., both being positioned in the same longitudinal reference plane) and that represents a position of the longitudinal reference plane. In one aspect of the present disclosure, one or more gate outlets (e.g.,438and440) may be aligned with the longitudinal reference plane. Further, one or more gate outlets (e.g.,434,436,442, and444) may be offset from the long longitudinal reference plane. In a further aspect, a length of the mold cavity wall425may be evenly divided into a forefoot third446, a midfoot third448, and a heel third450, and the gate outlets may be apportioned in various manners among the thirds. For example, in the illustrated example, the heel third450includes three gate outlets434,436, and438; the midfoot third448includes two gate outlets440and442; and the forefoot third includes a single gate outlet444. In some instances, this arrangement of gate outlets may contribute to dispersion and foaming of the thermoplastic elastomer composition in the mold cavity in a manner resulting in a foamed product having material properties conducive to a footwear sole. For example, it may be advantageous to position three gate outlets434,436, and438in the heel portion (with gate outlets434and436being laterally offset from the longitudinal reference plane), since the heel portion may be thicker, whereas it may be advantageous to position fewer gate outlets in the forefoot portion (which may be thinner than the heel portion) and closer to the longitudinal reference plane. However, in other aspects, the gate outlets may be positioned in different arrangements. In one aspect, the variability of the runner orientations may permit runners to both include the universal runner inlet configuration and be positioned in a customized manner to mold a particular shape and/or size of foamed article. For example, a mold configured to form a footwear sole for a first size (e.g., US men's size 10) footwear article may have a first set of runners, and a mold configured to form a footwear sole for a second size (e.g., US men's size 6) may have a second set of runners. Both the first set of runners and the second set of runners may each include a runner-inlet pattern matching the universal runner-inlet pattern depicted inFIG.8C, and may also each include a different runner pattern configured to distribute material differently into a respective mold cavity based on the respective 3D mold-cavity shapes and sizes. One aspect of the present disclosure includes a mold system having a universal runner plate (e.g., universal hot-runner plate or universal cold-runner plate) and an array of two or more molds, each of which is configured to interface with the universal runner plate and includes a 3D mold-cavity size. Furthermore, the 3D mold-cavity size of a first mold of the array is for a footwear component of a first shoe size, such that the first mold includes a first runner configuration. The 3D mold-cavity size of the second mold of the array is for a footwear component of a second shoe size, such that the second mold includes a second runner configuration that is different from the first runner configuration (e.g., different positions of gate outlets). In one aspect, the first shoe size and the second shoe size are each in a range of US Men's 3.5 to US Men's 15, or US Men's 5 to US Men's 12, or US Men's 6 to US Men's 11, or US Men's 7 to US Men's 10. For example, the first shoe size might be in a range of US Men's 3.5 to U.S. Men's 8, or US Men's 5 to US Men's 7.5, or US Men's 6 to US Men's 7; and the second shoe size might be in a range of US Men's 8.5 to US Men's 15, or US Men's 9 to US Men's 12, or US Men's 9 to US Men's 10. Referring toFIG.4, the second mold plate420includes a perimeter wall452forming a boundary along sides of the mold cavity wall425. In addition, the mold ring plate320includes a mold cavity wall322at least partially enclosing the mold-ring cavity328, and the mold cavity wall322includes a first perimeter ridge324traversing the perimeter of the mold-ring cavity328. When the second mold plate420is layered next to or abuts the mold ring plate320, such as when the first mold120is assembled, the perimeter wall452nests inside the mold cavity wall322to at least partially enclose a portion of the mold-ring cavity328. In addition, the perimeter wall452abuts the first perimeter ridge324to at least partially seal the mold cavity. For example, referring toFIG.9, a cross-sectional view taken along the reference line9-9inFIG.2is provided, showing the first mold plate220, the mold ring plate320, and the second mold plate420, assembled into the first mold120and enclosing a mold cavity C. In addition,FIG.9depicts the perimeter wall452abutting the first perimeter ridge324. In a further aspect, a seal assembly is positioned between the mold ring plate320and the second mold plate420. For example, a seal groove326may be positioned on a side330of the mold ring plate320facing towards the second mold plate420and a resilient seal may be positioned in the seal groove326. A seal groove may also, or alternatively, be positioned on the first side422of the second mold plate420. Moreover, in another aspect, the first side422of the second mold plate420may include a gas port454that aligns with, and fluidly connects with, a gas port332of the mold ring plate320, and this alignment and fluid connection is also depicted in the cross-sectional view ofFIG.9. Furthermore, the second mold plate420may include a gas channel458fluidly connecting the gas port181on the second side424with the gas port454on the first side422. In another aspect of the disclosure, the mold ring plate320includes at least a first pin-receiving aperture334(see e.g.,FIG.4) and the second mold plate includes at least a second pin-receiving aperture456, which aligns with the first pin-receiving aperture334when the first mold120is assembled. The second pin-receiving aperture456is also shown inFIGS.8A and8B, including an opening on the first side422, and an opening on the second side424—the opening on the second side including a width (e.g., diameter). Furthermore, the first pin-receiving aperture334may have a pin engager (e.g., threaded interior wall) to securely receive a pin (e.g.,335depicted inFIG.5) that is coupled in the first pin-receiving aperture334and that protrudes out of the first pin-receiving aperture334and into, or through, the second pin-receiving aperture456. In one aspect, the pin335includes a head337or other stop on a portion of the pin335positioned on the second side424of the second mold plate420. Furthermore, the hot-runner plate112may also have pin-receiving apertures186a-186dinto which a head of a pin (such as head337of pin335) may be inserted when the mold interfaces with the hot-runner plate112. When the mold is not coupled to the hot-runner plate112, the second mold plate420may be moved on the pin(s) away from the mold ring plate320to a pre-determined distance, based on a position of the head or stop on the pin (e.g., the length of the pin between the portion affixed to the mold ring plate320and the head). For example, after material has been injected into the mold cavity and has set (e.g., foamed, solidified, etc.), the second mold plate420may be moved away from the mold ring plate320along the pins in order to separate the molded part from the material in the gates and runners. Referring now toFIG.3, the mold ring plate320includes a first side338that faces towards a second side230of the first mold plate220, and the first side338is opposite a second side330(FIG.4) of the mold ring plate320facing towards the second mold plate420. The mold cavity wall322of the mold ring plate320is depicted inFIG.3, together with a second perimeter ridge340traversing at least a portion of the perimeter of the mold cavity wall322. As such, the mold cavity wall322of the mold ring plate320may include the first perimeter ridge324and the second perimeter ridge340, which divide the mold cavity wall322of the mold ring plate320into a first wall segment323, a second wall segment325, and a third wall segment327(see e.g.,FIG.9). The second wall segment325is between the first wall segment323and the third wall segment327, and when the first mold120is assembled (see e.g.,FIG.9), the first wall segment323interfaces with the perimeter wall452of the second mold plate420; the second wall segment325faces the mold cavity C; and the third wall segment327interfaces with the mold core224. When the mold ring plate320is layered next to or abuts the first mold plate220, such as when the first mold120is assembled (as shown inFIG.2), the mold core224nests inside the mold cavity wall322to at least partially enclose a portion of the mold-ring cavity328. In addition, the mold core224is positioned adjacent the second perimeter ridge340to at least partially enclose the mold cavity. For example, referring toFIG.9, a cross-sectional view is provided, showing the first mold plate220, the mold ring plate320, and the second mold plate420, assembled into the first mold and enclosing a mold cavity C. In addition,FIG.9depicts the mold core224adjacent the second perimeter ridge340, and the third wall segment327is spaced apart from the mold core224. In one aspect of the disclosure, the mold core224is adjacent to, and spaced apart from, the second perimeter ridge340by a distance large enough to permit gas to flow through, and small enough to impede material flowing from the cavity. For example, inFIG.9, dashed circles342and344depict a location at which a perimeter edge of the mold core224(e.g., an edge at which the mold cavity wall formed by the mold core transitions to a side wall231) and the second perimeter ridge340are slightly spaced apart to form a vent. In one aspect of the present invention, the vent extends around at least a portion of the mold cavity C. For example, the vent may extend around 50%, 60%, 70%, 80%, 90%, or 100% of the perimeter of the mold cavity C. In a further aspect, the mold cavity C has a first cross-sectional profile as taken along a first plane that is perpendicular to an axis of the fluid channel348, and the vent includes a second cross-sectional profile as taken along a second plane that is parallel to the first plane. For example,FIG.9depicts a longitudinal reference line235amarking a position of a first plane that is perpendicular to an axis of the fluid channel348, and a longitudinal reference line235bmarking a position of a second plane parallel to the first plane (the first plane and the second plane extend orthogonal to the view shown inFIG.9at longitudinal reference lines235aand235brespectively). The first cross-sectional profile and the second cross-sectional profile may be substantially congruent. In another aspect, the distance between the perimeter edge of the mold core224and the second perimeter ridge340(e.g., the vent width) is in a range of about 0.5 millimeters to about 2.5 millimeters, or about 1.0 to about 2.0 millimeters. The vent identified by the dashed circles342and344fluidly connects with the open space between the third wall segment327and the side wall231of the mold core. In this sense, the side wall231may form a first vent wall and the third wall segment327may form a second vent wall. Furthermore, as depicted inFIGS.3and9, the mold ring plate320includes a surface channel346in the first side338that extends from the third wall segment327(i.e., the second vent wall) to an internal gas channel348, which extends entirely through a plate body of the mold ring plate and fluidly connects with the gas port332. As such, in an aspect of the present disclosure, when the first mold120is assembled, pressurized gas may be supplied from a GCP system connected to gas port178of the hot-runner plate112, through the fluidly connected channels458,348, and346and through the vents, to the mold cavity C. The pressure of the gas may vary, and in one aspect, the pressure is in a range of about 550 psi to about 1500 psi. As previously mentioned, the positive pressure may further extend from the mold cavity C, through the runners of the second mold plate420and the hot-runner plate112, and up to a nozzle tip. Likewise, the pressurized gas may be actively pulled (e.g., via negative pressure or vacuum) from, or may passively flow from, the mold cavity C out of the gas port178. Among other things, this pressurized gas may help maintain a state of material flowed into the mold cavity and control a foaming activity. For example, the runners and the mold cavity C may be pressurized prior to receiving shots of material, and the pressure may be maintained after the shots have been received in the cavity to effectuate a dwell time without the material foaming. After the dwell time, the pressure may be released to trigger a phase transition, including a transition of the supercritical fluid to a gas. In one aspect, the dwell time includes a duration operable to bring a temperature of the thermoplastic elastomer composition in a range conducive to foaming a foam article having properties for a footwear article (e.g., sole). In a further aspect of the present disclosure, the first mold120may include features that contribute to controlling a temperature of the thermoplastic elastomer composition when dispersed into the mold cavity C. For example, the first mold plate220may include a first side229(seeFIG.1B) that is positionable on a cooling shelf or in a cooling rack to conductively lower a temperature of the first mold plate220(e.g., lower a temperature of the mold core224and resulting cavity wall that contact the thermoplastic elastomer composition when initially dispersed into the mold cavity). The first mold plate220may, in-turn, conductively lower a temperature of the mold ring plate320, the second mold plate420, the ambient or pressurized air in the mold cavity C, or any combination thereof. Furthermore, the first mold plate220, the mold ring plate320, and the second mold plate420may each have a respective plate body constructed of aluminum, which may include conduction properties conducive to lowering a temperature of the first mold120. As such, the first mold plate220, the mold ring plate320, and the second mold plate420may be brought to a temperature in a range between 15 degrees Celsius and 90 degrees Celsius prior to receiving the thermoplastic elastomer composition (e.g., by placing the first mold120on the cooling rack), such that when the thermoplastic elastomer composition is dispersed into the mold cavity C, a temperature of the thermoplastic elastomer composition may be reduced during the dwell time. In one aspect, the combination of pressure control and temperature control contributes to molding a foam article having properties desirable for a footwear article. For example, the foam article may have a thickness corresponding to a distance between the mold cavity wall426and the core224(which also provides a wall to enclose the mold cavity). In one aspect, this distance is in a range of about 10 millimeters to about 50 millimeters, or 12 millimeters to 40 millimeters, or 14 millimeters to 30 millimeters, or 16 millimeters to 22 millimeters, or 16 millimeters to 40 millimeters. As such, when a thermoplastic elastomer composition is disposed into the mold cavity from the gate outlets, the composition may initially be deposited on the core224. As described above the mold may include a temperature and a pressure that are within a particular range, which may reduce the likelihood that the supercritical fluid will prematurely transition to a gas in a manner that generates undesired properties of the foam article. The pressure of the mold cavity may be held for a dwell time in a range of between about 0.5 sec. to about 20 sec. During this time, the single-phase solution may more fully disperse among the mold cavity, prior to foaming. In addition to the pressure control and temperature control, the location, number, and relative spacing of the gate outlets may also contribute to the foam article having desirable properties. For example,FIG.8Cdepicts one aspect in which the six gate outlets are positioned to evenly distribute the single-phase solution in a manner conducive to relatively uniform foaming (e.g., bubble size). Carrier Plates for Manipulating the Mold Referring toFIGS.1A-1E, an aspect of the present disclosure includes the first carrier plate116coupled to the first mold plate220and the second carrier plate118coupled to the second mold plate420. The first carrier plate116and the second carrier plate118are operable to manipulate the first mold120and the second mold122throughout the injection molding process. For example, the first carrier plate116and/or the second carrier plate118may be used by a plate manipulator (e.g., manual or automated transport device, robot, robotic arm, etc.) to transport the first mold120and the second mold122from one station to another station during the injection molding process. In other aspects, the first carrier plate116and the second carrier plate118may be used by a plate manipulator to move mold plates towards one another when assembling the first mold120and the second mold122or to separate mold plates from one another when molded parts are being unloaded. In a further aspect, the first carrier plate116includes one or more plate-manipulator interfaces configured to mate with or receive a protruding key of a plate manipulator. Furthermore, the one or more plate-manipulator keyways may be asymmetrical in some respect to increase the likelihood that the first carrier plate116is properly aligned with a plate manipulator. For example,FIG.1Adepicts a first plate-manipulator keyway124that is rectangular and a second plate-manipulator keyway126that is circular. As such, a plate manipulator (e.g., robotic arm or other lifting equipment used to grasp, clamp, engage, lift, etc. the first carrier plate116) may include a rectangular protruding key to mate with the keyway124and a circular protruding key to mate with the keyway126. The asymmetry may help reduce the likelihood that the first carrier plate116is engaged while positioned or aligned improperly (e.g., backwards), since the keys of the plate manipulator would not fit in the misaligned keyways. AlthoughFIG.1Adepicts two plate-manipulator keyways, in other aspects the first carrier plate116may include a single keyway that is asymmetrical (e.g., irregular rectangle) or may include more than two keyways. Referring toFIG.1B, a bottom side of the first carrier plate116is depicted. In one aspect of the present disclosure, the first carrier plate116includes a cutout for receiving the first mold plate220. For example, the first side229of the first mold plate220is exposed on the bottom side of the first carrier plate116. In some instances, exposing the first side229of the first mold plate220may allow the first side229to directly contact a temperature conditioning system, such as a cooling shelf or cooling rack, to facilitate conductive cooling. In another aspect of the present disclosure, the bottom side of the first carrier plate116includes one or more plate-alignment interfaces (e.g., keyways) configured to mate with or receive a protruding key of a shelf or other support surface (e.g., cooling rack, injection-molding station, unloading station, etc.). Furthermore, the one or more plate-alignment keyways may be asymmetrical in some respect to increase the likelihood that the first carrier plate116is properly aligned on the shelf or support surface. For example,FIG.1Bdepicts a first plate-alignment keyway128that is rectangular and a second plate-manipulator keyway130that is circular. As such, a shelf or other support surface may include a rectangular protruding key to mate with the keyway128and a circular protruding key to mate with the keyway130. The asymmetry may help reduce the likelihood that the first carrier plate116is misaligned or backwards when placed on a shelf or support surface, since the keys of the shelf or support surface would not fit in the misaligned keyways. AlthoughFIG.1Bdepicts two keyways, in other aspects the first carrier plate116may include a single plate-alignment keyway that is asymmetrical (e.g., irregular rectangle) or may include more than two keyways. With continued reference toFIG.1B, the bottom side of the first carrier plate116may include an RFID port132for retaining an RFID device (not shown). The RFID device may be used to track the first mold120and the second mold122as the molds are moved through different stations of an injection molding system. In addition, inFIG.1Ba side of the first mold plate220facing away from the mold ring plate320can be seen seated in the first carrier plate116. As such, a perimeter ledge227(see e.g.,FIG.2) of the first mold plate220may rest atop the first carrier plate116, and as shown inFIG.3, one or more mechanical fasteners (e.g.,223) may couple the perimeter ledge227to the first carrier plate116. Referring toFIGS.1A and1E, in a further aspect, the first carrier plate116includes one or more clamp zones131aand131b. For example, each clamp zone131aand131bis configured to mate with, and be engaged by, a respective clamp at one or more stations of the injection molding system. For example, when the tooling assembly110is located at a part-unloading station, one or more clamps may lock onto the first carrier plate at the clamp zones131aand131b. As such, when the second carrier plate118is lifted, the first carrier plate116remains stationary on a support surface, and one or more of the mold plates are permitted to separate. In another aspect of the present disclosure, the second carrier plate118includes one or more plate-manipulator keyways configured to mate with or receive a protruding key of a plate manipulator. Furthermore, the one or more plate-manipulator keyways may be asymmetrical in some respect to increase the likelihood that the second carrier plate118is properly aligned with a plate manipulator. For example,FIG.1Adepicts a first plate-manipulator keyway125that is rectangular and a second plate-manipulator keyway127that is circular. As such, a plate manipulator (e.g., manual or automated lift device used to lift the second carrier plate118) may include a rectangular protruding key to mate with the keyway125and a circular protruding key to mate with the keyway127. The asymmetry may help reduce the likelihood that the second carrier plate118is engaged while positioned or aligned improperly (e.g., backwards), since the keys of the plate manipulator would not fit in the misaligned keyways. AlthoughFIG.1Adepicts two plate-manipulator keyways, in other aspects the second carrier plate118may include a single keyway that is asymmetrical (e.g., irregular rectangle) or may include more than two keyways. Referring toFIG.4, the second carrier plate118includes a ring-plate cutout sized and shaped to receive the mold ring plate320. As such, the second carrier plate118may extend around at least a portion of the mold ring plate320. However, in one aspect of the disclosure, the second carrier plate118is not affixed directly to the mold ring plate320, such as with a mechanical faster; rather, the second mold plate420may be positioned atop, and coupled directly to, the second carrier plate118. For example,FIG.5depicts mechanical fasteners (e.g.,421) that may couple the second mold plate420to the second carrier plate118. Tooling Latch Assembly In another aspect of the present disclosure, referring toFIGS.1D,10, and11A-11D, a tooling latch assembly500may releasably couple the first carrier plate116to the second carrier plate118. The tooling latch assembly500may include a first latch base510coupled to the first carrier plate116(e.g., by one or more mechanical fasteners), and a second latch base512coupled to the second carrier plate118(e.g., by one or more mechanical fasteners). For example,FIGS.11C and11Ddepict a first fastener hole514and a second fastener hole516for receiving a first and second fastener (respectively) for attaching the first latch base510to the first carrier plate116. In addition,FIGS.11A and11Bdepict a third fastener hole518and a fourth fastener hole520for receiving a third and fourth fastener (respectively) for attaching the second latch base512to the second carrier plate118. In addition, the first latch base510includes a pin (see e.g.,522inFIGS.10,11B, and11D) that is biased toward the second latch base512. For example,FIGS.10and11Billustrate a spring or other resilient member524that applies a force on the pin522in a direction towards the second latch base512. In addition, the second latch base512includes a pin-receiving through-hole526having a first end528towards the first latch base510and a second end530opposite the first end528. In one aspect of the disclosure, when the through-hole526is axially aligned with the pin522, the biasing force of the resilient member524thrusts the pin522into the first end528of the through-hole526to latch or couple the first carrier plate116to the second carrier plate118. The tooling latch assembly500may be coupled and decoupled in various manners. For example, when the tooling latch assembly500is in a coupled arrangement and the pin522is biased into the through-hole526, then another pin (not shown) may be inserted into the second end530of the through-hole526to push the pin522against the biasing member524, thereby causing the biasing member524to compress, at least until the pin522clears the first end528. Once the pin522clears the first end528, the second latch base512may be separated from the first latch base510. For example, a robot arm configured to lift the second carrier plate118may include keys to engage the keyways125and127and a pin for insertion into the through-hole526. As such, when the robot arm engages the second carrier plate118, it may near simultaneously insert keys into the keyways and the pin into the through-hole, at which time, the robot arm may separate the second carrier plate118from the first carrier plate116. In a further aspect, the second latch base512includes an angled cam surface532that is alignable with an end of the pin522. As such, to reconnect the tooling latch assembly500(e.g., when the second latch base512and the angled cam surface532are above the protruding pin), the second latch base512may be moved downward, thereby causing the angled cam surface532to contact the protruding end of the pin522and push the pin522into the first latch base510and compress the biasing member524. Once the second latch base512is moved to a position aligning the pin522with the first end528of the through-hole526, the force from the biasing member524pushes the pin into the through-hole526. The first carrier plate116and the second carrier plate118may be exposed to various operations, manipulation, and engagements with plate manipulators. As such, the first carrier plate116and the second carrier plate118may be constructed of steel to improve durability and reduce damage susceptibility when repeatedly engaged during various injection molding cycles. Single Gate Aspects In other aspects, rather than the use of multiple gate outlets434,436,438,440,442and444within each mold cavity wall425(see, e.g.FIG.4) as described above with respect toFIGS.1A-11D, it has been found that a single gate system, having only a single gate outlet within each mold cavity wall425may offer advantages in some aspects. Such a single gate system and components for forming an article comprising a foamed thermoplastic elastomer composition is depicted inFIGS.12A-21E. Many of the features and components of the injection molding single gate system depicted inFIGS.12A-21Eare the same, or similar, to those depicted and described above with respect toFIGS.1A-11D, and as such, they are not further described below. Those features and components of the injection molding single gate system depicted inFIGS.12A-21Ethat are the same as, or similar to, the description above with respect toFIGS.1A-11Dare depicted with the same reference numerals, and share the same above-described features. As shown inFIG.12A, the single gate system includes a tooling assembly1110that is similar in many respects to tooling assembly110described above. Tooling assembly1110is illustrated in combination with a first hot-runner plate1112and a second hot runner plate1114. The tooling assembly1110includes one or more injection molds coupled to a first carrier plate116and a second carrier plate118, which may support the one or more molds during one or more stages of an injection-molding process (e.g., when the molds are being transported from one station to another station, when the part(s) are being unloaded, etc.). For instance, referring toFIG.13, the hot-runner plates1112and1114and the carrier plates116and118have been omitted to unobstructedly illustrate a first mold1120and a second mold1122. The first hot-runner plate1112interfaces with the first mold1120, and the second hot-runner plate1114interfaces with the second mold1122to distribute one or more deposits or shots of thermoplastic elastomer composition (e.g., single-phase solution or other composition having a foaming agent) into mold cavities. For purposes of this disclosure, various elements of the first mold1120are described, and it is understood that the second mold1122may have similar elements, although not explicitly described. For example, the first mold1120includes a first mold plate220, a mold ring plate320, and a second mold plate1420, and the second mold1122might also include a respective first mold plate221, a respective mold ring plate321, and a respective second mold plate1421having similar elements. The first mold plate220, the mold ring plate320, and the second mold plate1420are layerable (e.g., stackable) to form one or more cavities for receiving a deposit of a thermoplastic elastomer composition. For example,FIG.14depicts a partially exploded view including the first mold plate220and the mold ring plate320separated from one another. In this partially exploded view ofFIG.14, a portion of a first mold cavity and a portion of a second mold cavity can be seen, each of which is shaped to form at least a portion of a footwear sole. Although the first mold1120includes two mold cavities, in other aspects, a mold may include fewer or more mold cavities. In this disclosure, various elements of the first mold cavity are described, and it is understood that the second mold cavity may have similar elements, although not explicitly described. For example, the first mold cavity may be at least partially enclosed by a mold core224, and the second mold cavity may include a similar mold core226(although a mirror image). In one aspect of this disclosure, the mold core224is nestable in a mold-ring cavity328when the mold ring plate320is layered against or abutting the first mold plate220. Referring toFIG.15, the mold ring plate320is illustrated as layered directly against and abutting the first mold plate220, and the mold core224is depicted nested in the mold-ring cavity328, such that the mold core224forms a first mold cavity wall. In addition,FIG.15depicts a partially exploded view with the second mold plate1420being separated from the mold ring plate320. The second mold plate1420may be positioned directly against (e.g., abutting) the mold ring plate320to more fully enclose the mold-ring cavity328. The second mold plate1420may also include one or more runners and gates through which the thermoplastic elastomer composition may flow when being deposited into the cavities, as further described below. To deliver the one or more deposits or shots of thermoplastic elastomer composition into mold cavities, the hot-runner plates1112and1114have at least one nozzle receiving opening1136for each mold cavity. As shown inFIG.12A, the hot-runner plate1112has two nozzle receiving openings1136, one for each mold cavity as further described below. Similarly, the hot-runner plate1114has two nozzle receiving openings1136, one for each mold cavity. As shown inFIGS.18A and18B, some of the walls of the hot-runner plate1112are omitted to illustrate some interior components of the hot-runner plate1112in more detail. Each nozzle receiving opening1136is the entry point for a nozzle receiving sleeve1156, similar to the nozzle receiving sleeve156described above with respect toFIGS.7A and7B. More specifically, each nozzle-receiving sleeve1156also includes a sleeve outlet (e.g.,158in the cross-sectional view ofFIG.18B). The sleeve outlet158includes a perimeter rim160that forms a nozzle seat162against which a tip of a nozzle biases when the nozzle is fully inserted into the nozzle-receiving sleeve156. The hot-runner plate1112also includes hot runners (e.g.,164) that transport material from each nozzle after being dispensed. For example, each hot runner (e.g.,164) includes a hot-runner inlet (e.g.,166inFIG.18B) that fluidly connects with the sleeve outlet (e.g.,158in the cross-sectional view ofFIG.18B) and includes a hot-runner outlet1134(see alsoFIG.16showing the hot-runner outlets1134). In an aspect of the present disclosure, the hot-runner inlet166is spaced apart from the hot-runner outlet1134by a distance in a range of about 1 cm to about 3 cm. As such, when material is dispersed from the nozzle, a sprue is formed in the hot runner164, the sprue having a length in a range of about 1 cm to about 3 cm. In an aspect of the present disclosure, this sprue length provides a grasping region at which a tool can grip the sprue for removing solidified material from the runners in the second mold plate1420. Like hot-runner plate112, the hot-runner plate1112includes various components to help control conditions related to the injection-molding system. For example, the hot-runner plate1112includes conditioned-fluid lines170for transporting conditioned fluid throughout the hot-runner plate1112. The conditioned fluid may be conditioned to include a temperature for maintaining, increasing, or decreasing a temperature of components of the hot-runner plate1112, including the hot runners (e.g.,164), the nozzle-receiving sleeves1156, and the nozzles1138(seeFIG.17) when inserted in the sleeves (see alsoFIG.12Edepicting a conditioned-fluid inlet172and a conditioned fluid outlet174positioned on an exterior wall of the hot-runner plate1112). As such, when the thermoplastic elastomer composition (e.g., single-phase solution with supercritical fluid as physical foaming agent) is dispensed from a nozzle1138, the temperature in the hot runner164may be maintained high enough to delay transition of the supercritical fluid to a gas. In a further aspect, the hot-runner plate1112includes a gas conduit176having a first gas port178(see e.g.,FIGS.12A and18A) for fluidly communicating with a gas counter pressure (GCP) system and a second gas port180or “gas-conduit port” (see e.g.,FIG.16) for fluidly communicating with a gas port181(also referred to as “fluid-channel port”) of the second mold plate1420. In addition, the side of the hot-runner plate1112facing towards the mold includes a seal assembly for a sealing and mating contact with the second mold plate1420. For example,FIG.16depicts a seal groove184that receives a resilient seal182. The second mold plate1420may additionally, or alternatively, include a seal groove. In some aspects of the present disclosure, the GCP system is fluidly connected from the gas port178on the hot-runner plate1112to the sleeve outlet (e.g.,158inFIG.18B). As such, when the thermoplastic elastomer composition (e.g., single-phase solution with supercritical fluid as physical foaming agent) is dispensed from a nozzle1138, the pressure in the mold cavity and runners may be maintained high enough to delay transition of the supercritical fluid to a gas. In one aspect of the present disclosure, the hot-runner plate1112is a universal hot-runner plate that is coupled directly to the manifold. For example, one or more fasteners may couple the hot-runner plate1112to the manifold1140shown inFIG.17. Some conventional injection molding systems may, in contrast to the present disclosure, have separate hot-runner plates that each interfaces with a different cold-runner plate (or other plate that is not temperature conditioned) and that are connected and disconnected to the manifold or nozzles in each injection cycle. This aspect of the present disclosure includes a universal hot-runner plate that is mountable to the nozzles and that can interface with an array of different molds, each of which includes a different mold cavity, a different gate scheme, or any combination thereof. For example, the mold cavities may differ in volume and or shape as being used to mold parts of different sized shoes, and the gate scheme may differ by including different gate positions. In addition, the hot-runner plate1112is plumbed with all of the components used to control various aspects of the molding process, including the conditioned-fluid lines170and the gas conduit176. Hot-runner plates are often associated with higher costs (e.g., added plumbing for temperature conditioning elements). Among other things, a universal hot-runner plate may reduce costs across multiple sets of molds, since only a single, common hot-runner plate may be used across the multiple sets, as opposed to having to make a hot-runner plate for each mold. In addition, it can reduce costs over time since there are fewer parts to store, maintain, repair, move, handle, etc. Although the figures of this present disclosure illustrate the hot-runner plates1112and1114, which provide an interface between the injector nozzles1138and the first and second molds1120and1122, in other aspects of this disclosure, universal cold-runner plates or other types of universal runner plates may provide the interface between the injector nozzles and the first and second molds1120and1122. Referring toFIG.17, an example of a set of injection nozzles (e.g.,1138) is shown as part of an injection manifold1140. That is, the injection manifold1140includes an injection port142that connects to an injector of an injection molding system. The injection manifold1140also includes a series of internal components (not shown) that receive a deposit/shot of thermoplastic elastomer composition from the injector and divides the shot into a number of deposits for separately distributing through the nozzles1138. The injection manifold1140may also include one or more sensors (e.g., thermocouple) for monitoring conditions of the manifold (e.g., temperature, pressure, etc.) that may affect the thermoplastic elastomer composition, as well as a manifold temperature conditioning unit for maintaining, increasing, or decreasing a temperature of the manifold. For example, the manifold temperature conditioning unit may include conditioned-fluid lines150for holding and transporting coolant or heated conditioning fluid. As such, a state of the thermoplastic elastomer composition (e.g., single-phase solution) may be maintained while a deposit is distributed from each nozzle into the hot-runner plates1112and1114. For example, the manifold1140may maintain the thermoplastic elastomer composition at conditions conducive to maintaining the foaming agent in a supercritical-fluid phase and to reducing the likelihood of transition to a gas. In a further aspect, the manifold includes an injector-pin assembly (e.g.,152) for each nozzle1138, which may selectively insert a pin (or other obstruction) into a tip of each nozzle1138to impede a flow of material. In a further aspect of the present disclosure, the nozzles1138are arranged to correspond to the number and placement of the nozzle receiving sleeves1156. For example, inFIG.17, there are four nozzles1138, one for each nozzle receiving sleeve1156. In one aspect of the present disclosure, each nozzle1138is configured to inject material into a single mold cavity having a three-dimensional shape of a footwear component (e.g., footwear sole). In one aspect, each nozzle1138and nozzle receiving sleeve1156are optimally positioned relative to the three-dimensional shape of the footwear component in a way that each shot foams and solidifies in a desired manner. The material exiting the hot-runner outlet1134leaves hot-runner plate1112or hot-runner plate1114and is deposited on and in a respective runner inlet1426or1427on second mold plate1420or second mold plate1421as seen inFIG.13.FIG.13shows a one-to-one correspondence between the nozzle1138, nozzle receiving sleeve1156and the runner inlet1426or1427. In some aspects, there may be two or more nozzles1138for each runner inlet1426or runner inlet1427. The following description details first mold1120, but applies equally to second mold1122. As best seen inFIGS.19A,19C and20A-20C, the runner inlet1426(or runner inlet1427) directs the material to a sprue1437that extends from a hole in the runner inlet1426or runner inlet1427to a gate outlet1438. While runner inlet1426and runner inlet1427show two different runner inlet configurations, other runner inlet configurations are also possible, in other aspects, such as any of the configurations426,428,430and/or432shown inFIG.8A. As seen inFIGS.20A-20C, the geometry of sprue1437may vary. In some aspects, as shown inFIG.20A, the sprue1437includes tapered walls, with a larger opening at runner inlet1426and tapering to a smaller gate outlet1438. In one aspect, the sprue1437ofFIG.20Atakes the form of a truncated cone. In other aspects, as shown inFIG.20B, the sprue1437includes straight walls, such that the runner inlet1426is the same size as the gate outlet1438. In one aspect, the sprue1437ofFIG.20Btakes the form of a cylinder. In yet another aspect, as shown inFIG.20C, the sprue1437may be formed with a constricted top portion and a wider bottom portion. As shown inFIG.20C, the sprue1437may be formed with a runner inlet1426having a diameter X and a first section with straight walls. The sprue1437may then include a first outwardly-tapered section, with a taper at an angle A in a first tapered portion and may also include a second outwardly-tapered section with a taper at an angle B. In some aspects, the angle A is greater than the angle B, such that the first outwardly tapered section has a greater taper than the second outwardly tapered section. As shown inFIG.20C, the sprue1437has a gate outlet1438that has a diameter Y that is greater than the diameter X of the runner inlet1426, in some aspects. In some aspects, the diameter X is 2.5 mm and the diameter Y is between 8 mm and 12 mm. In some aspects, the angle A is 60 degrees and the angle B is 1.5 degrees. While three different configurations are shown and described for sprue1437, other configurations are contemplated by, and within the scope of, this disclosure. As shown inFIG.20D, the sprue1437may have an axis1439. In some aspects, the axis1439is oriented perpendicularly to the surface of the mold ring cavity328, such that the material leaving the gate outlet1438is deposited within the mold ring cavity328directly below the gate outlet1438. In other aspects, as shown inFIG.20D, the axis1439may be oriented at an angle (shown as angle D inFIG.20D). In this aspect, the material leaving the gate outlet1438may be deposited on the surface of the mold ring cavity328in front of (when angled forwardly) or in back of (when angled rearwardly) the gate outlet1438. Any of the sprue1437depicted inFIGS.20A through20Ccould have an axis1439that is angled forwardly or rearwardly. In some aspects, the angle D is between zero and forty-five degrees. FIGS.21A-21Eshow different geometries or configurations of the gate outlet1438. As shown inFIG.21A, gate outlet1438may be circular. In some aspects, the gate outlet1438inFIG.21Amay have a diameter of between 0.07 inches to 0.6 inches. In some aspects, the larger diameter circular gate outlet ofFIG.21Awith a diameter of 0.5 inches produced a footwear component with desirable properties. Other geometric configurations of gate outlet1438may also be used, in some aspects. As additional examples, and without limitation, gate outlet1438could have a “carrot” or “tear-drop” shape, as shown inFIG.21B; a “racetrack” shape as shown inFIG.21Cwith two spaced-apart semi-circular ends; a narrow “fan” slit as shown inFIG.21D; or an elongated slit or “spine” configuration as shown inFIG.21E. In some aspects, the mold plate1420may be fitted with a removable gate outlet plate1429, as shown inFIG.20A, having the desired geometry for gate outlet1438. In some aspects, the gate outlet1438may be changed in the second mold plate1420by changing the gate outlet plate1429, allowing the shape of the gate outlet1438to change without requiring an entirely new second mold plate1420. The gate outlet1438, in some aspects, is located just forward of the heel area of the footwear component (e.g. footwear sole), as best seen inFIG.19B. It has been found that this location produces a foamed footwear component with desirable properties. In other aspects, the gate outlet1438could be located farther toward the heel area, and in some aspects, could be located in the forefoot or toe area. In some aspects, the carrier plate116and carrier plate118and the tooling latch assembly500of the single gate system ofFIGS.12A-21Ehave similar features as described above with respect toFIGS.1A-11D, and are not repeated here. Similarly, the features of the first mold plate220and the mold ring plate320are common between the multiple gate system ofFIGS.1A-11Dand the single gate system ofFIGS.12A-21E, and are not repeated here. In some aspects, the single gate system and method described above with respect toFIGS.12A-21Eoffers advantages over the multiple-gate system ofFIGS.1A-11D. It has been found that the single gate system results in less waste associated with the runners, because there are fewer of them. Additionally, the single gate system produces parts with fewer gate “vestiges” (the small indicator left at each gate outlet) because the single gate system has only one gate outlet1438per mold cavity (as compared to six gate vestiges associated with the multiple gate system described with reference toFIGS.1A-11D). Because the single gate system uses only one “shot” of material per mold cavity, in some aspects, the expanding foam of the single “shot” may take longer to expand and fill the mold cavity. The single gate system also avoids any issues associated with material flow and/or any issues associated with material boundaries between different regions of the article of footwear. For example, after shots of thermoplastic elastomer composition (e.g., single-phase solution or other composition having a foaming agent) enter the mold cavity ofFIG.4, through gate outlets434,436,438,440,442and444, the expanding foam from the regions around each gate outlet expands, forming material boundaries. In the single gate system described above with reference toFIGS.12A-21E, no such material boundaries exist. It has also been found, in some aspects, that a part with lower overall weight is achievable (for example, a men's size 10 midsole with a part weight of 112 grams), and part variability is lower with the single gate system, with a resultant foam that is more consistent with fewer large “voids” or bubbles in the foamed footwear sole, along with an energy efficiency of between about 65 percent and 80 percent. Recyclate With reference next to the flowchart ofFIG.22, an improved method or control strategy for manufacturing a foamed polymer article, using a tooling assembly, such as tooling assembly110ofFIG.1A, is generally described at2200in accordance with aspects of the present disclosure. Some or all of the operations illustrated inFIG.22and described in further detail below may be representative of an algorithm that corresponds to processor-executable instructions that may be stored, for example, in main or auxiliary or remote memory, and executed, for example, by a local or remote controller, processing unit, control logic circuit, or other module or device or network of devices, to perform any or all of the above or below described functions associated with the disclosed concepts. One or more of the illustrated operations may be performed manually or assisted manually by an onsite technician. It should be recognized that the order of execution of the illustrated operation blocks may be changed, additional blocks may be added, and some of the blocks described may be modified, combined, or eliminated. Method2200ofFIG.22is initialized at block2201, e.g., responsive to input of an activation command signal received from a human machine interface (HMI) of a central control terminal. Initial stages of the manufacturing process may comprise supplying, accessing, and/or utilizing (collectively “receiving”) the various materials, tools, and machines needed to manufacture foamed polymer articles. At process block2203, for example, a batch of recycled plastic material is accessed from an available store of polymer recyclate. As used herein, the term “recycled plastic” may encompass used or excess or scrapped plastic that is put into a recycling stream, including wholesale recycling of entire products, disassembly of products and recycling only selected parts thereof, recycling of manufacturing byproduct, all of which may require sorting and cleaning of any collected materials. For at least some embodiments, scrap and waste thermoplastic polyester elastomer (TPE-E) composition may be recovered (e.g., reclaimed from foamed or unfoamed virgin TPE-E material and/or virgin TPE-E compositions), and then incorporated into foamed articles produced with at least some virgin TPE-E and/or virgin TPE-E compositions. The recycled TPE-E and/or virgin TPE-E compositions may be derived from one or more reactants, such as a poly(alkylene oxide)diol material and/or an aromatic dicarboxylic acid material. The recycled thermoplastic polyester elastomer and/or virgin TPE-E compositions may have a weight average molecular weight ranging from about 50,000 Daltons to about 200,000 Daltons. Once the batch of recycled plastic is received and any attendant sorting, cleaning, and other pre-processing is complete at process block2203, the method2200shreds, chops, cuts, and/or grinds (collectively “grind”) the batch of recycled plastic at process block2205. By way of non-limiting example, a dedicated recycling station may be responsible for grinding recycled TPE-E into granular or pelletized form; ground recycled material may be produced in real-time or stored in inventory and reused when desired. Alternatively, “grinding” may comprise feeding a hot compound of recyclate into an extruder fitted with a perforated die; a cutter immediately in front of the die slices extruded strings of compound into granulized pellets. Cut pellets are then cooled as they are transported to a sieve grader to separate out irregularly sized pellets. A “regrind” thermoplastic polymer and/or virgin TPE-E composition may originate from re-extruded material, such as unfoamed, mold-runner derived TPE-E and/or virgin TPE-E composition waste that is put through an extruder, pelletized, and turned back into resin. Regrind may also originate from injected foam material, such as virgin TPE-E and/or virgin TPE-E composition resin that is injected and foamed during normal processing, scrapped, then shredded and re-introduced as regrind. The ground recyclate material may have an irregular shape with a major length size of about 1-10 mm, and the virgin polymer material has a pellet size of about 1-10 mm. At process block2207, the ground recycled material is mixed with a composition of virgin polymer material. As used herein, the terms “mixing” and “blending” may be used interchangeably and synonymously to mean to combine or intermingle, where the resultant mixed batch may or may not be homogenous throughout the mixture. A recycled material may be contrasted with a virgin material in that a raw “virgin” material has neither been injected into a mold assembly nor expanded through activation of an intermixed foaming agent and formed into an end product. The virgin polymer composition may be the same or similar general polymer composition as the recyclate or, alternatively, may be a distinguishable polymer composition from the recyclate. To properly calibrate the operating parameters of the injection molding system and control the functional properties of the resultant foamed polymer article, a metered amount of the ground recyclate material is mixed with a predetermined amount of virgin polymer material to form a mixed batch of virgin and recycled material. In at least some implementations, the metered amount is limited to about 20% by mass or less of a total mass of the mixed batch. It may be desirable, depending on an intended application, that about 10 to about 50 parts of recycled TPE-E composition per about 80 to about 100 parts virgin TPE-E composition be incorporated into newly foamed TPE-E articles by the methods described herein. With continuing reference toFIG.22, method2200continues to process block2209with instructions to treat the recycled material, either before, during, or after admixture with the virgin material. Processing the recyclate may include the addition of blowing/foaming agents, fillers, pigments, and/or processing aids. In at least some implementations, a foaming agent is incorporated as a separate ingredient into the mixture of recycled and virgin polymer material for invoking the expansion of the mixture during molding. The foaming agent may comprise a suitable stimulant that, alone or in combination with other substances, is capable of producing a cellular structure in a plastic. Foaming agents may include fluids that expand when pressure is released. It may be desirable, for at least some applications, to add a physical foaming agent to the mixture of recycled and virgin material during the melting of the mixture or after the mixture has melted. When injection molding a midsole cushioning element with the injection molding system described above with respect toFIG.1AthroughFIG.21E, it may be desirable to inject a physical foaming agent into the polymer melt composition while the polymer melt composition is contained in one or more of the injection barrels inside of the hot-runner plates112and114or the hot-runner plates1112and1114. The physical foaming agent may be composed of one or more supercritical fluids (SCF), such as supercritical nitrogen or carbon dioxide, which is/are dissolved into the polymer melt composition under pressure to form a single-phase solution (SPS). As a further option, the method2200may be characterized by a lack of a chemical foaming agent for the forming of the foamed polymer article. SCF concentration may be dictated by, among other things, a desired solubility and a desired density. For some embodiments, a chemical blowing agent may be utilized in addition to, or as a substitute for, the physical foaming agent. Numerous other additives may be incorporated into the recyclate batch prior to introduction into the final mold for forming the foamed polymer article, including fillers, activators, homogenizing agents, pigments, fire retardants, lubricants, and other suitable additives. Non-limiting examples of filler materials include talcum powder, mica silicate, bearing sulfate, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium carbonate, and other commercially available fillers. The polymer compositions can also contain rubber fillers, such as ethylene propylene rubber (EPR), styrene isoprene styrene (SIS) copolymer rubber, styrene butadiene rubber, as well as other polyolefin resins, in addition to ethylene-vinyl acetate (EVA) or TPE-based materials. In other examples, polyethylene wax may be used as a processing agent, stearic acid may be used as a lubricant, dicumyl peroxide may be used as a polymerization initiator, zinc oxide may be used as an activator for the foaming agent, while titanium dioxide may be used as a white pigment or carbon black may be used as a black pigment. Process block2211ofFIG.22includes memory-stored, processor-executable instructions to melt the ground recyclate material and the virgin polymer material into a polymer melt composition. It should be appreciated that the ground recyclate and virgin polymer materials may be separately melted and then flowed into a mixed polymer melt composition. Otherwise, the mixed batch of recyclate and virgin polymer materials produced at process block2207may be heated into the polymer melt composition. For at least some embodiments, the mixture of ground recyclate and virgin polymer materials has a set point temperature ranging from about 190° C. to about 215° C. Moreover, the mixed batch of the ground recyclate material and the virgin polymer material may have an average peak crystallization temperature ranging from about 135° C. to about 165° C. Once the polymer composition is complete and ready for molding, the processed recycled and virgin material is pressurized and injected—colloquially “shot”—into the internal cavity or cavities of a mold assembly to form the foamed polymer article, as indicated at process block2213. After the SCF is injected into the polymer melt composition contained in the barrel(s) of hot-runner plates112and114ofFIG.1A, for example, where the SCF dissolves in the melt to form a molten SPS, the molten SPS is flowed into the internal mold cavities of the first mold120and the second mold122. The SCF is employed as a physical blowing agent to expand the melted TPE-E composition and thereby fill the mold cavities. The pressure within the mold cavities is reduced or eliminated to release the SCF from the SPS, and the expanded melt is allowed to cool and solidify. To provide a “closed loop” molding system with circular sustainability that eliminates most if not all manufacturing scrap and waste, the mass of recycled thermoplastic resin within the internal mold cavities may be greater than or equal to a mass of the mixed thermoplastic resin within any filling portions fluidly coupled to the cavities. To ensure the integrity and desired performance characteristics of the resultant foamed polymer article, one or more operating parameters of the injection molding system may be modulated to accommodate the percent mass of recyclate being incorporated into the polymer mixes. For instance, the injection molding system may be set to a molding melt temperature of between about 210° C. and about 215° C. with a batch melt temperature of approximately 190° C. and a crystallization temperature of approximately 147° C. In addition to the selective control of mold temperatures, gas counter-pressure release rates and hold times may be recalibrated to a TPE-E polymer melt composition with approximately 20% by mass recycled TPE-E composition, e.g., to regulate cooling rates within the mold cavities (e.g., higher pressure drop provides faster cooling rate with less cooling time). System operating parameters may be selectively modified to ensure that the polymer melt composition stays within a pre-calculated melt temp-crystallization temp sweet spot for a selected timeframe within the processing cycle. The foamed polymer article is ejected from the internal mold cavity at process block2215. For at least some embodiments, the formed foamed polymer article has a cell size average, e.g., by volume of a longest cell dimension, of less than about 0.68 mm or, in some embodiments, about 0.18 mm to about 0.58 mm. For at least some implementations, the foamed polymer article may exhibit some and/or all of the following characteristics: (1) an energy efficiency of about 55% to about 95% or, in some preferred configurations, a target efficiency of 70% to 85%; (2) an energy return of about 1000 millijoules (mJ) to about 7000 mJ or, in some preferred configurations, a target return of 4500 mJ to 5500 mJ (e.g., assuming a standard midsole geometry); and/or (3) a density of about 0.15 grams/cubic centimeter (g/cc) to about 0.25 g/cc or, in some preferred configurations, a target density of 0.18 g/cc to 0.20 g/cc. As yet a further option, a formed foamed polymer article may exhibit a ratio of energy efficiency to energy intensity (EE/EI) that is greater than about 1.125 or, for some embodiments, greater than about 1.35 or, for some desired embodiments, greater than about 1.5 or, optionally, between about 1.6 and 2.1. Likewise, a formed foamed polymer article may exhibit a ratio of energy efficiency to the product of energy intensity and density (EE/(EI*ρ) that is greater than about 5.25 or, for some embodiments, greater than about 6.3 or, for some desired embodiments, greater than about 7.0 or, optionally, between about 8.8 and 11.2. Moreover, a formed foamed polymer article may exhibit a ratio of energy return to energy intensity (ER/EI) that is greater than about 6,375 or, for some embodiments, greater than about 7,650 or, for some desired embodiments, greater than about 8,500 or, optionally, between about 9,900 and 11,300. A formed foamed polymer article may exhibit a ratio of energy return to the product of energy intensity and density (ER/(EI*ρ) that is greater than about 33,750 or, for some embodiments, greater than about 40,500 or, for some desired embodiments, greater than about 45,000 or, optionally, between about 55,400 and 62,500. As used herein, the term “energy intensity” may be defined to include a measure of the energy used in forming a particular foam article, for example, in kilowatt hours (kW-h). To obtain the energy intensity of a foam article, the energy expended to produce a run, or batch, of articles, such as pairs of footwear midsoles, is first calculated, determined or measured (e.g., from pellet to finished component). For a physical foaming process, the measured energy may include the energy required for all energy consuming steps, such as: preheating the molds and hot runners (if utilized), melting the pellets, generating gas counter-pressure, injecting the molten plastic, introducing the supercritical fluid, cooling the molds and/or work-pieces, and ejecting the work-pieces from the mold. The overall energy required to produce the run of cushioning element pairs is then divided by the number of cushioning element pairs produced in the run. For at least some embodiments, a foamed polymer sole component fabricated from both recyclate and virgin thermoplastic materials may have an energy return measurement that is within a predefined tolerance of an energy return measurement of a comparable shoe sole component formed solely from virgin thermoplastic materials. This predefined tolerance may be about 75% to about 99% of the energy return measurement of the comparable shoe sole component. The foamed sole component and the comparable shoe sole component may share a comparable shape, size, and/or method of molding. At this juncture, the method2200may terminate or may loop back to block2201and run in a repeatable or continuous loop. It is envisioned that disclosed manufacturing systems and processes may utilize any logically relevant source of recycled plastic material in order to conserve natural resources, minimize use of raw materials, and divert waste from landfills with the aspiration of reaching a “circular economy”. In this regard, aspects of this disclosure are directed to “closed-loop” manufacturing processes that limit usable recyclate sources to manufacturing byproducts (e.g., gate or runner trimmings) and reground defective articles (e.g., visually or mechanically flawed foamed polymer footwear sole elements). Implementing such “closed-loop” manufacturing processes may desirably optimize material use efficiencies by achieving, for example, a zero-waste or near-zero-waste of polymer materials in the manufacture of foamed polymer articles. As an extension of, a modification to, or a standalone process from the method2200ofFIG.22, a method of producing foamed polymer articles may be composed of a series of controlled manufacturing steps, including executing one or more production runs to form one or more types of foamed polymer articles. A “production run” may be typified by a predefined number of articles (e.g., 220-260 articles/hr) of a designated design/model (e.g., NIKE® REACT FLYKNIT™) having a preset shape, size and material composition (e.g., single-piece TPE-E midsole for women's size 7 running shoe) produced substantially contiguously by a particular production line. Individual runs may exhibit different quantifiable production variables, including: an average article mass mAAof the foamed polymer articles (e.g., average total mass of all articles per run or average individual article mass or all articles per run), and an average article defect rate {dot over (D)}A(e.g., ratio of total defective articles to total articles produced per run). Because the process may produce multiple envelopes of products, e.g., distinguishable from each other in quantity and geometry, the tooling for each geometry may consume a distinct volume of raw materials and generate a distinct volume of manufacturing byproduct. As will be explained in further detail below, a production line may generate a baseline average byproduct value (e.g., unfoamed byproduct generated upstream of tooling and/or foamed byproduct generated downstream of tooling). For a particular production run, an average byproduct mass amount may be calculated as the sum of: (1) an amount of byproduct generated for each geometry produced in a run divided by the quantity of each geometry in the run; and (2) a remnant upstream byproduct mass per run. By way of non-limiting example, a run size for a production run may include 100 total articles, including twenty of a first geometry, twenty of a second geometry, and sixty of a third geometry. In this instance, byproduct mass may be calculated as: (total byproduct mass for first geometry)/20+(total byproduct mass fir second geometry)/20+(total byproduct mass for third geometry)/60+upstream and/or downstream byproduct mass. For at least some implementations, a production run may be limited to a single run for fabricating a preset number of a singular article design having a predefined shape and size. Alternatively, a mass production run may include multiple batch runs of different types of polymer articles, with each type having a respective shape and size. These batch production runs may be performed simultaneously or sequentially, with each run producing the same number of articles or a distinct number of articles. When carrying out multiple batch runs as part of a larger mass production run, the average article mass mAAfor the mass run may be calculated as the arithmetic sum of the individual average article masses for all of the discrete runs, namely: mAA-1+mAA-2+ . . . +mAA-n. Likewise, the average article defect rate {dot over (D)}Afor the mass run may be calculated as the arithmetic mean of the individual average article defect rates for all of the discrete batch runs, namely: ({dot over (D)}A-1+{dot over (D)}A-2+ . . . +{dot over (D)}A-n)/n. After completing a single production run or a group of discretized batch runs of foamed polymer articles, the method may include reclaiming and recycling one or more batches of manufacturing byproduct incidental to the run or runs. Recyclate byproduct material may be recovered from sections of the molding system upstream from the mold tool (e.g., from hot-runner or cold runner plates), downstream from the mold tool (e.g., mold flash and trimmings), and/or from within the mold tool itself (e.g., inlet and outlet gates to the mold-ring cavities). In this example, the manufacturing byproduct may have an average byproduct mass mAB(e.g., average total byproduct mass per run or average byproduct mass per article per run). When carrying out multiple batch runs, the average byproduct mass for the entire mass production run may be calculated as the arithmetic sum of the individual average byproduct masses, namely: mAB-1+mAB-2+ . . . +mAB-n. Alternatively, the average byproduct mass may be calculated as the arithmetic sum of: (1) a first byproduct mass incidental to a first batch run divided by a first number of first polymer articles in that run; (2) a second byproduct mass incidental to a second batch run divided by a second number of second polymer articles in that run; . . . and (n) an nthbyproduct mass incidental to an nthbatch run divided by an nthnumber of polymer articles in that run. Prior to, contemporaneous with, or after retrieving the batch of manufacturing byproducts, the method may also include reclaiming and recycling one or more lots of defective articles incidental to the production run(s). In accord with the abovementioned footwear example, recycled defect material may be recovered from pre-consumer footwear and, if desired, from post-consumer footwear. For pre-consumer products, a defective foamed article may be identified through any commercially available technique for identifying manufacturing defects. For instance, the injection molding system may incorporate a system-automated visual inspection station and a system-automated mechanical testing station downstream from the tooling assembly ofFIG.1AthroughFIG.21E. The visual inspection station may utilize a high-definition digital camera and a machine-learning algorithm to search for and flag any of a multitude of predefined visual defects (e.g., dimensional flaws, superficial blemishes, contour defects, etc.). Moreover, the mechanical testing station may be in the nature of an impact-testing machine with a linear force transducer operatively coupled to a motor-driven, last-shaped plunger. The plunger and transducer collectively measure each foam article's stiffness, energy efficiency, energy return, etc., and flag the article as defective if any of these measurements fall outside of corresponding manufacturing tolerance ranges. Continuing the discussion of pre-consumer defective products, there will be an associated average defect mass mAD(per run) in the manufacturing system. This average defect mass mADmay be calculated as the arithmetic product of the article defect rate {dot over (D)}Aand the average article mass mAA, or mAD={dot over (D)}A*mAA. For implementations that execute multiple batch runs as part of a larger mass production run, the average defect mass mADmay be the arithmetic mean of the individual average defect masses incidental to the various production runs, namely: (mAD-1+mAD-2+ . . . +mAD-n)/n. To achieve a “closed-loop” manufacturing process, the system may be restricted as follows: (mAB+mAD)/mAA≤0.2 During a closed-loop manufacturing process, foam polymer waste—the manufacturing byproducts and defective articles—may be added directly into the injection barrel for subsequent injection into the mold tool cavity. The foam polymer waste may be crushed or shredded, mixed with virgin pellets, and fed together into the same injection barrel. In this instance, a power-screw type “crammer” feeder may be used to force the waste material back into the tooling assembly. Prior to re-feeding the material, the foam polymer waste may be shredded at least once or, in at least some applications, two or more times to ensure that the discretized waste elements are generally uniform in shape and size. If it determined that the foam polymer waste cannot be added directly to the injection barrel, the foam waste may need to be processed, melted down, and re-pelletized. In this case, the waste material would be shredded a single time or multiple times, fed into a separate extrusion line where it is melted and extruded, and thereafter pelletized to form pellets akin in geometry and density to virgin pellets. These “new” waste material pellets may then be combined with virgin pellets in the injection barrel. An injection molding system's operating parameters will potentially change depending on the type and volume of recyclate being used to form the foamed polymer articles. For instance, the melt temperatures will likely be modified to successfully process recycled material: when foamed, the recyclate material's crystallization temperature may increase (i.e., crystallization temperature gets closer to the melt temperature). As such, the melt composition may need to be processed at higher temperatures compared to processing temperatures that would typically be used for pure virgin material. For at least some footwear midsole embodiments, the production variables per run may be based on the following parameters: about 0.2 kg/pair, about two pair (four midsoles)/minute, eight hour shift, about 10% to about 15% runner waste relative to midsole weight per pair. Aspects of this disclosure may be implemented, in some embodiments, through a computer-executable program of instructions, such as program modules, generally referred to as software applications or application programs executed by any of a controller or the controller variations described herein. Software may include, in non-limiting examples, routines, programs, objects, components, and data structures that perform particular tasks or implement particular data types. The software may form an interface to allow a computer to react according to a source of input. The software may also cooperate with other code segments to initiate a variety of tasks in response to data received in conjunction with the source of the received data. The software may be stored on any of a variety of memory media, such as CD-ROM, magnetic disk, and semiconductor memory (e.g., various types of RAM or ROM). Moreover, aspects of the present disclosure may be practiced with a variety of computer-system and computer-network configurations, including multiprocessor systems, microprocessor-based or programmable-consumer electronics, minicomputers, mainframe computers, and the like. In addition, aspects of the present disclosure may be practiced in distributed-computing environments where tasks are performed by resident and remote-processing devices that are linked through a communications network. In a distributed-computing environment, program modules may be located in both local and remote computer-storage media including memory storage devices. Aspects of the present disclosure may therefore be implemented in connection with various hardware, software or a combination thereof, in a computer system or other processing system. Any of the methods described herein may include machine readable instructions for execution by: (a) a processor, (b) a controller, and/or (c) any other suitable processing device. Any algorithm, software, control logic, protocol or method disclosed herein may be embodied as software stored on a tangible medium such as, for example, a flash memory, a solid-state memory, a CD-ROM, a hard drive, a digital versatile disk (DVD), or other memory devices. The entire algorithm, control logic, protocol, or method, and/or parts thereof, may alternatively be executed by a device other than a controller and/or embodied in firmware or dedicated hardware in an available manner (e.g., implemented by an application specific integrated circuit (ASIC), a programmable logic device (PLD), a field programmable logic device (FPLD), discrete logic, etc.). Further, although specific algorithms are described with reference to flowcharts depicted herein, many other methods for implementing the example machine-readable instructions may alternatively be used. From the foregoing, it will be seen that this subject matter is well adapted to attain all the ends and objects hereinabove set forth together with other advantages which are obvious and which are inherent to the structure. It will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the subject matter without departing from the scope of this disclosure, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense. | 193,339 |
11859068 | DETAILED DESCRIPTION The present invention accordingly had for its object to provide halogen-free flame-retarded thermoplastic molding materials which achieve efficient flame retardancy—especially glow wire resistance—and better mechanical properties by addition of Al phosphites of various compositions to red phosphorus. The molding materials defined in the introduction have accordingly been found. Preferred embodiments may be found in the dependent claims. The molding materials of the invention comprise as component A) 10 to 98.5 wt %, preferably 20 to 97.5 wt % and in particular 30 to 80 wt % of at least one polyamide. The polyamides of the molding materials according to the invention generally have an intrinsic viscosity of 90 to 350 ml/g, preferably 110 to 240 ml/g, determined in a 0.5 wt % solution in 96 wt % sulfuric acid at 25° C. according to ISO 307. Preference is given to semicrystalline or amorphous resins with molar mass Mw (weight average) at least 5000 of the type described by way of example in the U.S. Pat. Nos. 2,071,250, 2,071,251, 2,130,523, 2,130,948, 2,241,322, 2,312,966, 2,512,606 and 3,393,210. Examples thereof are polyamides which derive from lactams having 7 to 13 ring members, such as polycaprolactam, polycaprylolactam and polylaurolactam, and also polyamides obtained by reaction of dicarboxylic acids with diamines. Employable dicarboxylic acids include alkanedicarboxylic acids having 6 to 12 carbon atoms, in particular 6 to 10 carbon atoms, and aromatic dicarboxylic acids. These only include the acids adipic acid, azelaic acid, sebacic acid, dodecanedioic acid and terephthalic and/or isophthalic acid. Particularly suitable diamines are alkanediamines having 6 to 12 carbon atoms, in particular 6 to 8 carbon atoms, and also m-xylylenediamine (e.g. Ultramid® X17 from BASF SE, a 1:1 molar ratio of MXDA to adipic acid), di(4-aminophenyl)methane, di(4-aminocyclohexyl)methane, 2,2-di(4-aminophenyl)propane, 2,2-di(4-aminocyclohexyl)propane or 1,5-diamino-2-methylpentane. Preferred polyamides are polyhexamethyleneadipamide, polyhexamethylenesebacamide and polycaprolactam, and also 6/66 copolyannides, in particular having a proportion of from 5 to 95 wt % of caprolactam units (for example Ultramid® C31 from BASF SE). Suitable polyamides further include those obtainable from w-aminoalkyl nitriles, for example aminocapronitrile (PA 6) and adipodinitrile with hexamethylenediamine (PA 66) by so-called direct polymerization in the presence of water, as described for example in DE-A 10313681, EP-A 1198491 and EP 922065. Also included are polyamides obtainable for example by condensation of 1,4-dianninobutane with adipic acid at elevated temperature (polyamide 4,6). Production processes for polyamides having this structure are described for example in EP-A 38094, EP-A 38582 and EP-A 39524. Also suitable are polyamides obtainable by copolymerization of two or more of the abovementioned monomers or mixtures of a plurality of polyamides in any desired mixing ratio. Particular preference is given to mixtures of polyamide 66 with other polyamides, in particular 6/66 copolyamides. Semiaromatic copolyamides such as PA 6/6T and PA 66/6T having a triamine content of less than 0.5 wt %, preferably less than 0.3 wt %, (see EP-A 299444) have also proven particularly advantageous. Further high-temperature-resistant polyamides are disclosed in EP-A 1994075 (PA 6T/61/MXD6). Production of the preferred semiaromatic copolyamides having a low triamine content may be carried out by the processes described in EP-A 129195 and 129196. The following nonexhaustive list comprises the recited polyamides and other polyamides A) in the context of the invention and also the monomers present. AB Polymers: PA 6ε-caprolactamPA 7enantholactamPA 8caprylolactamPA 99-aminopelargonic acidPA 1111-aminoundecanoic acidPA 12laurolactam AA/BB Polymers PA 46tetramethylenediamine, adipic acidPA 66hexamethylenediamine, adipic acidPA 69hexamethylenediamine, azelaic acidPA 610hexamethylenediamine, sebacic acidPA 612hexamethylenediamine, decanedicarboxylic acidPA 613hexamethylenediamine, undecanedicarboxylic acidPA 12121,12-dodecanediamine, decanedicarboxylic acidPA 13131,13-diaminotridecane, undecanedicarboxylic acidPA 6Thexamethylenediamine, terephthalic acidPA 9T1,9-nonanediamine, terephthalic acidPA MXD6m-xylylenediamine, adipic acidPA 6Ihexamethylenediamine, isophthalic acidPA 6-3-Ttrimethylhexamethylenediamine, terephthalic acidPA 6/6T(see PA 6 and PA 6T)PA 6/66(see PA 6 and PA 66)PA 6/12(see PA 6 and PA 12)PA 66/6/610(see PA 66, PA 6 and PA 610)PA 6I/6T(see PA 6I and PA 6T)PA PACM 12diaminodicyclohexylmethane, dodecanedioic acidPA 6I/6T/PACMsuch as PA 6I/6T + diaminodicyclohexylmethanePA 12/MACMIlaurolactam, dimethyldiaminodicyclohexylmethane, isophthalic acidPA 12/MACMTlaurolactam, dimethyldiaminodicyclohexylmethane, terephthalic acidPA PDA-Tphenylenediamine, terephthalic acidPA4101,4-tetramethylenediamine, sebacic acidPA5101,5-pentamethylenediamine, sebacic acidPA10T1,10-decanediamine, terephthalic acid The molding materials of the invention comprise as component B) 1 to 50 wt %, in particular 1 o 20 wt %, preferably 1 to 10 wt % and in particular 2 to 8 wt % of red phosphorus. A preferred halogen-free flame retardant B), in particular in combination with glass-fiber-reinforced molding materials, is elemental red phosphorus which may be employed in untreated form. Particularly suitable, however, are preparations in which the phosphorus is surface-coated with low molecular weight liquid substances such as silicone oil, paraffin oil or esters of phthalic acid (in particular dioctyl phthalate, see EP 176836) or adipic acid or with polymeric or oligomeric compounds, for example with phenol resins or aminoplasts and also polyurethanes (see EP-A 384232, DE-A 19648503). Such so-called phlegmatizers are generally present in amounts of 0.05 to 5 wt % based on 100 wt % of B). Concentrates of red phosphorus, for example in a polyamide A) or elastomer E), are also suitable flame retardants. Polyolefin homopolymers and copolymers in particular are suitable concentrate polymers. However, if no polyamide is used as the thermoplastic the proportion of the concentrate polymer should not exceed 35 wt % based on the weight of components A) to E) in the molding materials according to the invention. Preferred concentrate compositions are 30 to 90 wt %, preferably from 45 to 70 wt %, of a polyamide A) or elastomer (E), 10 to 70 wt %, preferably from 30 to 55 wt %, of red phosphorus (B). The employed polyamide for the batch may be distinct from A) or preferably identical to A) so that incompatibilities or melting point differences do not have a negative effect on the molding material. The average particle size (d50) of the phosphorus particles distributed in the molding materials is preferably in the range from 0.0001 to 0.5 mm; in particular from 0.001 to 0.2 mm. The molding materials of the invention comprise as component C) 0.5 to 15 wt %, preferably 0.5 to 13 wt % and in particular 1 to 10 wt % of at least one aluminum salt of a phosphonic acid. Phosphonic acid is to be understood as meaning the compound having the empirical formula H3PO3 [CAS Nro. 13598-36-2]. The salts of phosphonic acid are known as phosphonates. Phosphonic acid may be in the form of two tautomeric forms of which the tribonded form has a free electron pair on the phosphorus atom and the tetrabonded form has an oxygen double bonded to the phosphorus (P═O). The tautomeric equilibrium is entirely on the side of the form having the double bonded oxygen. According to A. F. Holleman, E. Wiberg: Lehrbuch der Anorganischen Chemie, 101st edition, Walter de Gruyter, Berlin/New York 1995, ISBN 3-11-012641-9, page 764 the terms “phosphorous acid” and “phosphites” should only be used for the tribonded forms. Even in current literature the terms “phosphorous acid” and “phosphites” are also used for the tetrabonded forms having oxygen double bonded to the phosphorus (P═O) and the terms phosphonic acid and phosphorous acid and phosphonates and phosphites are therefore used syn-onymously with one another. Preferred components C) are constructed from [Al2(HPO3)3.x(H2O)q(formula I) where q=0 to 4 or Al2Ma(HPO3)b(OH)cx(H2O)d(formula II) where M represents alkali metal ions a=0.01 to 1.5 b=2.63 to 3.5 c=0 to 2 d=0 to 4 or Al2(HPO3)e(H2PO3)fx(H2O)g(formula III) where e=2 to 2.99 f=2 to 0.01 g=0 to 4 or mixtures of aluminum phosphites and aluminum oxide of the type Al2(HPO3)3x0.1 to 30Al2O3x0 to 50H2O (formula IV) or primary aluminum phosphonate[Al(H2PO3)3] (formula V) or basic aluminum phosphonate[Al(OH)(H2PO3)x·2H2O] (formula VI) or mixtures thereof. Preferred molding materials comprise as component C) compounds of formula II, in which M represents sodium and/or potassium. Preferred compounds of formula I are secondary aluminum phosphonate[Al2(HPO3)3] (formula Ia) or aluminum phosphonate tetrahydrate[Al2(HPO3)3·4H2O] (formula Ib) or mixtures thereof. Preferred compounds of formula IV are constructed from mixtures of aluminum phosphites and aluminum oxide of the type Al2(HPO3)3x 0.2 to 20 Al2O3x 0 to 50 H2O (formula IV) and very particularly preferably Al2(HPO3)3x1 to 3 Al2O3x 0 to 50 H2O. Preferred compounds C) of formula II are those in which a represents 0.15 to 0.4 and b represents 2.80 to 3 and c represents 0.01 to 0.1. Further preferred components C) are constructed from compounds of formula III in which e represents 2.834 to 2.99 and f represents 0.332 to 0.03 and g represents 0.01 to 0.1. Especially preferred compounds C) are those of formula II or III, wherein a, b and c and also e and f can only assume numbers such that the corresponding aluminum salt of phosphonic acid is uncharged overall. Aluminum phosphites having the CAS numbers 15099-32-8, 119103-85-4, 220689-59-8, 56287-23-1, 156024-71-4 (secondary aluminum phosphonate tetrahydrate), 71449-76-8 (secondary aluminum phosphonate) and 15099-32-8 are particularly preferred. The described aluminum salts of phosphonic acid may be employed individually or in admixture. It is preferable when the aluminum phosphites have particle sizes of 0.2 to 100 pm, the particle size distribution thereof being determinable by customary analytical methods of laser diffraction Production of the preferred aluminum phosphites is typically carried out by reaction of an aluminum source with a phosphorus source in a solvent at 20° C. to 200° C. over a time span of up to 4 days. To this end the aluminum source and the phosphorus source are mixed, heated under hydrothermal conditions or under reflux, filtered off, washed and dried. The preferred solvent here is water. The production of the aluminum salts of phosphonic acid used as component C) according to the invention is derivable for example from WO 2013/083247. Examples of fibrous or particulate fillers D) include carbon fibers, glass fibers, glass beads, amorphous silica, calcium silicate, calcium metasilicate, magnesium carbonate, kaolin, chalk, pulverulent quartz, mica, barium sulfate and feldspar, which may be employed in amounts of from 0 to 55 wt %, preferably from 1 to 50 wt %, in particular 5 to 40 wt %. Preferred fibrous fillers include carbon fibers, aramid fibers and potassium titanate fibers, wherein glass fibers in the form of E glass are particularly preferred. These may be employed as rovings or chopped glass in the commercially customary forms. The fibrous fillers may have been surface-pretreated with a silane compound in order to improve compatibility with the thermoplastics. Suitable silane compounds are those of general formula (X—(CH2)n)k—Si—(O—CmH2mH2m+1)4-k in which the substituents are defined as follows: n an integer from 2 to 10, preferably from 3 to 4 m an integer from 1 to 5, preferably from 1 to 2 k an integer from 1 to 3, preferably 1. Preferred silane compounds are aminopropyltrimethoxysilane, aminobutyltrimethoxysilane, aminopropyltriethoxysilane, aminobutyltriethoxysilane and also the corresponding silanes which comprise a glycidyl group as substituent X. The silane compounds are generally used for surface coating in amounts of from 0.01 to 2 wt %, preferably 0.025 to 1.0 wt % and in particular 0.05 to 0.5 wt % (based on D). Acicular mineral fillers are also suitable. In the context of the invention acicular mineral fillers are to be understood as meaning a mineral filler having distinctly acicular character. One example is acicular wollastonite. The L/D (length to diameter) ratio of the mineral is preferably 8:1 to 35:1, preferably from 8:1 to 11:1. The mineral filler may optionally have been pretreated with the abovementioned silane compounds but pretreatment is not an essential requirement. Examples of further fillers include kaolin, calcined kaolin, wollastonite, talc and chalk, precipitat-ed calcite and also lamellar or acicular nanofillers, preferably in quantities of from 0.1 to 10%. Materials preferably used for this purpose are mica, böhmite, bentonite, montmorillonite, ver-miculite, zinc oxide in acicular form and hectorite. In order to obtain good compatibility between the lamellar nanofillers and the organic binder the lamellar nanofillers are subjected to organic modification according to the prior art. Addition of the lamellar or acicular nanofillers to the nanocomposites of the invention leads to a further increase in mechanical strength. As component E) the molding materials may comprise further additives in amounts of 0 to 30 wt %, preferably 0 to 25 wt %. Contemplated here in amounts of 1 to 15 wt %, preferably 1 to 10 wt %, in particular 1 to 8 wt %, are elastomeric polymers (often also referred to as impact modifiers, elastomers or rubbers). These are very generally copolymers preferably constructed from at least two of the following monomers: ethylene, propylene, butadiene, isobutene, isoprene, chloroprene, vinyl acetate, styrene, acrylonitrile and (meth)acrylate having from 1 to 18 carbon atoms in the alcohol component. Such polymers are described by way of example in Houben-Weyl, Methoden der organischen Chemie [Methods of organic chemistry], vol. 14/1 (Georg-Thieme-Verlag, Stuttgart, 1961), pag-es 392 to 406, and in the monograph “Toughened Plastics” (Applied Science Publishers, Lon-don, 1977) by C. B. Bucknall. Some preferred types of these elastomers are set out below: Preferred components E) are impact modifiers based on ethylene copolymers constructed from: E1) 40 to 98 wt %, preferably 50 to 94.5 wt %, of ethylene, E2) 2 to 40 wt %, preferably 5 to 40 wt %, of a (meth)acrylate having 1 to 18 carbon atoms, or/and E3) 0 to 20 wt %, preferably 0.05 to 10 wt %, of functional monomers selected from the group of ethylenically unsaturated mono- or dicarboxylic acidsor of carboxylic anhydrides or epoxy groups or mixtures thereof, wherein the percentages by weight of E1) to E3) sum to 100%,or an ethylene-(meth)acrylic acid copolymer which has been up to 72% zinc-neutralized. Particular preference is given to ethylene copolymers constructed from: E1) 50 to 69.9 wt % of ethylene E2) 30 to 40 wt % of a (meth)acrylate having 1 to 18 carbon atoms, E3) 0.1 to 10 wt % of functional monomers according to claim1, wherein the weight percentages E1) to E3) sum to 100%. The proportion of functional groups E3) is 0.05 to 5 wt %, preferably 0.2 to 4 wt %, and in particular 0.3 to 3.5 wt % based on 100 wt % of E). Particularly preferred components E3) are constructed from of an ethylenically unsaturated mono- or dicarboxylic acid or from a functional derivative of such an acid. In principle, any of the primary, secondary and tertiary C1-C18-alkyl esters of acrylic acid or methacrylic acid D2is suitable, but preference is given to esters having 1-12 carbon atoms, in particular having 2-10 carbon atoms. Examples thereof include methyl, ethyl, propyl, n-butyl, isobutyl and tert-butyl, 2-ethylhexyl, oc-tyl and decyl acrylates and the corresponding esters of methacrylic acid. Among these, particular preference is given to n-butyl acrylate and 2-ethylhexyl acrylate. In addition to the esters the olefin polymers may also comprise acid-functional and/or latently acid-functional monomers of ethylenically unsaturated mono- or dicarboxylic acids or may comprise epoxy-containing monomers. Further examples of monomers E3) include acrylic acid, methacrylic acid, tertiary alkyl esters of these acids, in particular butyl acrylate, and dicarboxylic acids such as maleic acid and fumaric acid or anhydrides of these acids and also the monoesters thereof. “Latently acid-functional monomers” is to be understood as meaning compounds which form free acid groups under the polymerization conditions or during incorporation of the olefin polymers into the molding materials. Examples include anhydrides of dicarboxylic acids having up to 20 carbon atoms, in particular maleic anhydride, and tertiary C1-C12-alkyl esters of the abovementioned acids, in particular tert-butyl acrylate and tert-butyl methacrylate. The production of the above-described ethylene copolymers may be effected by processes known per se, preferably by random copolymerization at high pressure and elevated temperature. The melt flow index of the ethylene copolymers is generally in the range from 1 to 80 g/10 min (measured at 190° C. under a load of 2.16 kg). The molecular weight of these ethylene copolymers is from 10000 to 500000 g/mol, preferably from 15000 to 400000 g/mol (Mn determined by GPC in 1,2,4-trichlorobenzene with PS cali-bration). Commercially available products preferably used are Fusabond® A 560, Lucalen® A 2910, Lucalen® A 3110, Nucrel 3990, Nucrel 925, Lotader AX9800, and Igetabond FS 7M. The above-described ethylene copolymers may be produced by processes known per se, preferably by random copolymerization at high pressure and elevated temperature. Corresponding processes are well known. Other preferred elastomers are emulsion polymers whose production is described for example by Blackley in the monograph “Emulsion Polymerization”. The emulsifiers and catalysts that may be used are known per se. Copolymers comprising no units E2) but where the acid component E3) has been neutralized with Zn are especially preferred. Preference is given here to ethylene-(meth)acrylic acid copolymers which have been up to 72% zinc-neutralized (commercially available as Surlyn® 9520 from DuPont). It will be appreciated that it is also possible to use mixtures of the rubber types listed above. Further additives E) may be present in amounts up to 30 wt %, preferably up to 20 wt %. As component E) the molding materials according to the invention may comprise 0.05 to 3 wt %, preferably 0.1 to 1.5 wt % and in particular 0.1 to 1 wt % of a lubricant. Preference is given to Al salts, alkali metal salts, alkaline earth metal salts or esters or amides of fatty acids having from 10 to 44 carbon atoms, preferably having from 12 to 44 carbon atoms. The metal ions are preferably alkaline earth metal and Al, wherein Ca or Mg are particularly preferred. Preferred metal salts are Ca stearate and Ca montanate and also Al stearate. It is also possible to use mixtures of different salts in any desired mixing ratio. The carboxylic acids may be mono- or dibasic. Examples include pelargonic acid, palmitic acid, lauric acid, margaric acid, dodecanedioic acid, behenic acid, and particularly preferably stearic acid, capric acid and montanic acid (mixture of fatty acids having from 30 to 40 carbon atoms). The aliphatic alcohols may be mono- to tetrahydric. Examples of alcohols include n-butanol, n-octanol, stearyl alcohol, ethylene glycol, propylene glycol, neopentyl glycol, and pentaerythritol, preference being given here to glycerol and pentaerythritol. The aliphatic amines may be mono- to trifunctional. Examples thereof are stearylamine, ethylenediamine, propylenediamine, hexamethylenediamine, and di(6-aminohexyl)amine, wherein ethylenediamine and hexamethylenediamine are particularly preferred. Preferred esters or amides are correspondingly glyceryl distearate, glyceryl tristearate, ethylenediamine distearate, glyceryl monopalmitate, glyceryl trilaurate, glyceryl monobehenate and pentaerythrityl tetrastearate. It is also possible to use mixtures of different esters or amides or esters combined with amides in any desired mixing ratio. As component E) the molding materials according to the invention may comprise 0.05 to 3, preferably 0.1 to 1.5 and in particular 0.1 to 1 wt % of a Cu stabilizer, preferably of a copper(I) halide, in particular in admixture with an alkali metal halide, preferably KI, in particular in a ratio of 1:4. Suitable salts of monovalent copper preferably include copper(I) complexes with PPh3, copper(I) acetate, copper(I) chloride, bromide and iodide. These are present in amounts of 5 to 500 ppm of copper, preferably 10 to 250 ppm, based on polyamide. The advantageous properties are in particular obtained when the copper is in the form of a molecular dispersion in the polyamide. This is achieved when a concentrate comprising polyamide, a salt of monovalent copper and an alkali metal halide in the form of a solid homogeneous solution is added to the molding material. A typical concentrate consists for example of 79 to 95 wt % of polyamide and 21 to 5 wt % of a mixture of copper iodide or bromide and potassium iodide. The concentration of copper in the solid homogeneous solution is preferably between 0.3 and 3, in particular between 0.5 and 2, wt % based on the total weight of the solution and the molar ratio of copper(I) iodide to potassium iodide is between 1 and 11.5, preferably between 1 and 5. Suitable polyamides for the concentrate are homopolyamides and copolyamides, in particular polyamide 6 and polyamide 6.6. Suitable sterically hindered phenols E) include in principle all compounds having a phenolic structure and having at least one sterically demanding group on the phenolic ring. Preferably contemplated compounds are for example compounds of formula in which: R1and R2represent an alkyl group, a substituted alkyl group or a substituted triazole group, wherein the radicals R1and R2may be identical or different and R3represents an alkyl group, a substituted alkyl group, an alkoxy group or a substituted amino group. Antioxidants of the recited type are described for example in DE-A 2702661 (U.S. Pat. No. 4,360,617). Another group of preferred sterically hindered phenols derives from substituted benzenecarbox-ylic acids, in particular from substituted benzenepropionic acids. Particularly preferred compounds from this class are compounds of the formula wherein R4, R5, R7and R8independently of one another represent C1-C8alkyl groups which may themselves be substituted (at least one thereof being a sterically demanding group), and R6represents a divalent aliphatic radical which has from 1 to 10 carbon atoms and which may also have C—O bonds in the main chain. Preferred compounds of this formula are Sterically hindered phenols altogether include for example: 2,2′-methylenebis(4-methyl-6-tert-butylphenol), 1,6-hexanediol bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate], pentaerythrityl tetrakis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate], distearyl 3,5-di-tert-butyl-4-hydroxybenzylphosphonate, 2,6,7-trioxa-1-phosphabicyclo[2.2.2]oct-4-ylmethyl 3,5-di-tert-butyl-4-hydroxyhydrocinnamate, 3,5-di-tert-butyl-4-hydroxyphenyl-3,5-distearylthiotriazylamine, 2-(2′-hydroxy-3′-hydroxy-3′,5′-di-tert-butylphenyl)-5-chlorobenzotriazole, 2,6-di-tert-butyl-4-hydroxymethylphenol, 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl)benzene, 4,4′-methylenebis(2,6-di-tert-butylphenol), 3,5-di-tert-butyl-4-hydroxybenzyldimethylamine. Compounds that have proven particularly effective and are therefore used with preference are 2,2′-methylenebis(4-methyl-6-tert-butylphenyl), 1,6-hexanediol bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] (Irganox® 259), pentaerythrityl tetrakis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate] and also N,N′-hexamethylenebis-3,5-di-tert-butyl-4-hydroxyhydrocinnamide (Irganox® 1098) and the above-described Irganox® 245 from BASF SE which is particularly suitable. The antioxidants E) which may be employed individually or as mixtures are present in an amount of 0.05 up to 3 wt %, preferably from 0.1 to 1.5 wt %, in particular 0.1 to 1 wt %, based on the total weight of the molding materials A) to E). In some cases, sterically hindered phenols having not more than one sterically hindered group in the ortho-position to the phenolic hydroxy group have proven particularly advantageous, in particular when colorfastness is assessed during storage in diffuse light for prolonged periods. As component E) the molding materials according to the invention may comprise 0.05 to 5, preferably 0.1 to 2 and in particular 0.25 to 1.5 wt % of a nigrosin. Nigrosins are generally understood to refer to a group of black or gray phenazine dyes (azine dyes) in various embodiments (water-soluble, liposoluble, gasoline-soluble), and are related to the indulines, and are used in wool dyeing and printing, for providing black color to silks, and for dyeing leather and for shoe polishes, varnishes, plastics, heat-cured coatings, inks and the like, and also as microscopy dyes. Nigrosins are obtained industrially by heating nitrobenzene, aniline and aniline hydrochloride with metallic iron and FeCl3(name derives from the Latinniger=black). Component E) may be used as the free base or else as a salt (for example hydrochloride). Further details relating to nigrosins may be found for example in the electronic encyclopedia Rompp Online, Version 2.8, Thieme-Verlag Stuttgart, 2006, keyword “Nigrosin”. As component E) the thermoplastic molding materials according to the invention may comprise customary processing aids such as stabilizers, oxidation retarders, agents to counteract thermal degradation and ultraviolet light degradation, lubricants and release agents, colorants such as dyes and pigments, nucleating agents, plasticizers, etc. Examples of oxidation retarders and heat stabilizers are sterically hindered phenols and/or phosphites and amines (e.g. TAD), hydroquinones, aromatic secondary amines such as diphe-nylamines, various substituted representatives of these groups and mixtures thereof in concen-trations of up to 1 wt % based on the weight of the thermoplastic molding materials. Examples of UV stabilizers, which are generally employed in amounts of up to 2 wt % based on the molding material, include various substituted resorcinols, salicylates, benzotriazoles and benzophenones. Colorants that may be added include inorganic pigments, such as titanium dioxide, ultramarine blue, iron oxide and carbon black, and organic pigments, for example phthalocyanines, quinacridones, perylenes, and also dyes, for example anthraquinones. Employable nucleating agents include sodium phenylphosphinate, aluminum oxide, silicon dioxide and preferably talc. The thermoplastic molding materials according to the invention may be produced by processes known per se by mixing the starting components in customary mixing apparatuses such as screw extruders, Brabender mills or Banbury mills and then extruding the resulting mixture. After extrusion the extrudate may be cooled and comminuted. It is also possible to premix individual components and then add the remaining starting materials individually and/or likewise in the form of a mixture. Mixing temperatures are generally in the range from 230° C. to 320° C. In a further preferred procedure, components B) to E) may be mixed with a prepolymer, formu-lated and pelletized. The pelletized material obtained is then condensed to the desired viscosity continuously or batchwise under inert gas in the solid phase at a temperature below the melting point of component A). The thermoplastic molding materials according to the invention feature an improved flame retardancy, especially glow wire test, and better mechanics. They are therefore suitable for the production of fibers, films and moldings of any type. Exam-pies include: plug connectors, plugs, plug parts, cable harness components, circuit mounts, cir-cuit-mount components, three-dimensionally injection-molded circuit mounts, electrical connec-tion elements and mechatronic components. The moldings or semifinished products to be produced according to the invention from the thermoplastic molding materials can be used by way of example in the motor vehicle, electrical, electronics, telecommunications, information technology, entertainment or computer industry, in vehicles and other means of transportation, in ships, spacecraft, in the household, in office equipment, in sport, in medicine, and also generally in products and parts of buildings requiring increased flame retardancy. Possible applications of polyamides with improved flowability for the kitchen and household sectors are production of components for kitchen appliances, for example fryers, smoothing irons, knobs/buttons, and also applications in the garden and leisure sector. EXAMPLES The following components were used: Component A1: Polyamide 66 having an intrinsic viscosity IV of 150 ml/g, measured as a 0.5 wt % solution in 96 wt % sulfuric acid at 25° C. according to ISO 307 (Ultramid® A27 from BASF SE was employed.) Component B: 50% concentrate of red phosphorus having an average particle size (d50) of 10 to 30 μm in an olefin polymer E1): 59.8 wt % ethylene, 35 wt % n-butyl acrylate, 4.5 wt % acrylic acid and 0.7 wt % maleic anhydride having a melt index MFI (190/2.16) of 10 g/10 min. The copolymer was produced by copolymerization of the monomer at elevated temperature and elevated pressure. Component C: Aluminum salt of phosphonic acid (produced according to WO 2013/083247 A1, example 4) Aluminum phosphite of formula (II): 2958 g of water are initially charged into a 16 l high-pressure stirred vessel, heated to 155° C. and stirred. 3362 g of the aluminum sulfate solution and 2780 g of sodium phosphite solution are then added simultaneously over 30 min. The resulting suspension is discharged and at 80° C. filtered, washed with hot water, redispersed and washed once again. The filtercake was dried in a dryer at 220° C. An alkali metal-aluminum mixed phosphite according to the invention having very high thermal stability was obtained in 85% yield. By atomic spectrometry the reaction product comprises 18.3% Al, 32.0% P, 0.3% S and 0.07% Na. The residual moisture content of 0.1% water was determined by Karl-Fischer titration. Component C1V: Aluminum diethylphosphinate (Exolit® OP1230 from Clariant Produkte GmbH). Component D: Standard chopped glass fibers for polyamides, length=4.5 mm, diameter=10 μm Component E2: In all examples in each case 0.35 wt % Irganox® 1098 and 0.55 wt % commercially available calcium stearate as lubricant and 0.70 wt % of commercially available zinc oxide Component E3: 30% concentrate of a gas black having a specific BET surface area (measured according to DIN 66131) of 180 m2/g in polyamide 6. Production of Molding Materials To demonstrate the improvements described according to the invention corresponding plastic molding materials were manufactured by compounding. The individual components were mixed in a twin-screw extruder (Berstorff ZSK 26) at a throughput of 20 kg/h and about 270° C. at a flat temperature profile, discharged as a strand, cooled until pelletizable and pelletized. The test specimens for the investigation set out in table 1 were injection molded on an Arburg 420C injection molding machine at a melt temperature of about 270° C. and a mold temperature of about 80° C. The test specimens for the stress tests were produced according to ISO 527-2:/1993 and the test specimens for the impact strength measurements were produced according to ISO 179-2/1 eA. The MVR measurements were performed according to ISO 1133. The flame retardancy of the molding materials was on the one hand determined by the UL94-V method (Underwriters Laboratories Inc. Standard of Safety, “Test for Flammability of Plastic Materials for Parts in Devices and Appliances”, p. 14 to p. 18, Northbrook 1998). The glow wire resistance GWFI (glow wire flammability index) on sheets was performed according to IEC 60695-2-12. The GWFI is a general suitability test for plastics in contact with voltage-conducting parts. The highest temperature at which one of the following conditions has been met in 3 consecutive tests was determined: (a) no ignition of the sample or (b) afterburn time or afterglow time 30 s after termination of the glow wire exposure time and no ignition of the sub-strate. The proportions of the components A) to E) in table 1 sum to 100 wt %. TABLE 1C1#6842(405694C2C3Ex1Ex2Ex3Components (wt %)67J0)#8048#8179#5212#5074#2268A60.457.449.7357.450.246.87B + E1121210.210.2E166D262626263636C93322C1V13.67E21.61.61.61.61.61.6E33.33Analytical resultsViscosity number/[cm3/g]154159158145——Elastic modulus/[MPa]83258415819084541115411675Tensile stress at break/137117120136158162[MPa]Elongation at break/[%]3.53.23.03.43.12.9Charpy impact strength/695757707874[kJ/m2]Charpy notched impact8.27.06.87.7——strength/[kJ/m2]MVR 275° C./5 kg/302614291820[cm3/10 min]UL94/0.8 mmV-0n.c.n.c.V-0V-0V-0UL94/1.6 mmV-0n.c.n.c.V-0V-0V-0GWFI960/1.0 mm -metnot metnot metmetmetmetburn times on 3 test58/57/55>60/>60/>60>60/>60/>6039/38/4739/38/4739/38/47speciments/secC1 to C3: Comparative examplesEx 1 to Ex 3: inventive examples It is apparent from the data of table 1 that the inventive, synergistic composition exhibits mark-edly shorter burn times in the glow wire test compared to the prior art—especially for thin wall thicknesses. | 33,945 |
11859069 | SUMMARY OF THE INVENTION This problem is solved by the resins described herein and the reactive amine accelerators used in said resins. A reactive resin containing the amine accelerator according to the invention, and the reactive resin component (A) according to the invention that contains this reactive resin, and the reactive resin system according to the invention that comprises this reactive resin component as a component, are characterized in particular in that the amine accelerator according to the invention is almost completely incorporated into the polymer network during radical curing, due to the olefinic groups. This largely or completely prevents a diffusion of the amine accelerators onto the surface of the cured materials. Another positive effect of using the reactive amine accelerators, which are described herein, as a constituent of a reactive resin can be a slowed sedimentation rate and therefore an improved shelf life in comparison to conventional reactive resins. DETAILED DESCRIPTION OF THE INVENTION Although a reactive amine accelerator according to the invention is added to the reactive resin as an additive, said accelerator is covalently incorporated into the cured resin during the curing thereof. This is made possible by the reactive amine accelerator according to the invention being synthesized by reacting a primary or secondary aromatic amine with an epoxide and an α,β-unsaturated carboxylic acid. The reactive amine accelerator according to the invention that results from this synthesis contains either one or two terminal α,β-unsaturated carboxylic acid esters. These then react with the other monomers during the curing of a reactive resin, and, as a result, the reactive amine accelerator according to the invention is incorporated into the resin backbone. As a result, the reactive amine accelerator according to the invention is less harmful to health than the tertiary amines which were used previously. The resin mixture according to the invention that is prepared in this manner (hereinafter also referred to as “reactive resin”) cures at room temperature by mixing with a radical initiator, such as dibenzoyl peroxide, which has a high maximum reactivity temperature Tmax, even without the addition of further accelerators. In contrast with WO 12/164020 A1, the tertiary structure of the amine accelerator is formed in a resin according to the invention during the reaction of a primary or secondary aromatic amine with an epoxide and an α,β-unsaturated carboxylic acid. As a result, asymmetrical structures and structures which don't have chain lengthening, for example, are also possible via secondary amines. The amine accelerator according to the invention—also referred to as “reactive amine accelerator” in the following and as “accelerator” further below—is formed by reacting a primary or secondary aromatic amine with a diglycidyl ether and an α,β-unsaturated carboxylic acid. In this synthesis, (1) an aromatic primary or secondary amine is reacted with (2) a diglycidyl ether of the formula shown in the following reaction scheme and (3) an α,β-unsaturated carboxylic acid. The reaction typically takes place in the presence of (4) a catalyst. (5) an inhibitor can optionally be present in the reaction mixture. A schematic representation of the reaction is as follows: The phenyl ring in this case is a placeholder for an aromatic functional group. In a preferred embodiment, this aromatic functional group is a phenyl ring or naphthyl ring, more preferably a phenyl ring. The meaning of placeholders A, R1, R2and n is described further below. An exemplary synthesis according to the invention with a primary aromatic amine (here: para-toluidine) takes place as follows: The di-iso-propanol-p-toluidine (DiPpT) which is also shown here for the purpose of comparison is a typical tertiary amine, such as is used in the prior art as an accelerator and in WO 12/164020 A1, for example, as part of the UMA-bound DiPpT described therein. For the synthesis of the amine accelerator, which synthesis is according to the invention, the starting substances are preferably selected from the following groups: 1) The aromatic primary or secondary amine is preferably selected from the group of aromatic primary or secondary amines, in which the aromatic functional group is either unsubstituted or substituted with one or more substituents R1selected from the group consisting of halogen, pseudohalogen, C1-C20alkyl, hydroxy-C1-C20alkyl, C2-C20alkenyl, hydroxy-C2-C20alkenyl, C2-C20alkynyl, hydroxy-C2-C20alkynyl and phenyl. R1is preferably selected from the group consisting of halogen, hydroxy-C1-C20alkyl and C1-C20alkyl. R1is particularly preferably selected from the group consisting of halogen and C1-C20alkyl. R1is very particularly preferably selected from the group consisting of chlorine, bromine and C1-C6alkyl, in particular from the group consisting of chlorine, bromine and C1-C4alkyl. The aromatic functional group of the aromatic primary or secondary amine is substituted with no, one or more substituents R1. The aromatic functional group of the aromatic primary amine is preferably substituted with one, two or three substituents R1, more preferably with one or two substituents R1. The aromatic functional group of the aromatic secondary amine is preferably substituted with no, one, two or three substituents R1, more preferably with no, one or two substituents R1, more preferably with no or one substituent R1. The aromatic functional group in the aromatic primary or secondary amine is a phenyl functional group or a naphthyl functional group, particularly preferably a phenyl functional group. In a preferred embodiment, the aromatic primary amine is an alkyl aniline, i.e. it has a phenyl ring as an aromatic functional group, and this carries an R1, which is an alkyl group. Said R1is preferably a C1-C4alkyl; more preferably, R1is methyl and the alkyl aniline is therefore toluidine. In addition, other substituents R1selected from the groups given above for R1may also be present. In a preferred embodiment, there is no further R1. In a further preferred embodiment, one or two further R1are present, more preferably only one further R1is present. In a preferred embodiment, the further R1is selected from the group consisting of halogen and C1-C20alkyl. R1is very particularly preferably selected from the group consisting of chlorine, bromine and C1-C6alkyl, in particular from the group consisting of chlorine, bromine and C1-C4alkyl, and very particularly from the group consisting of chlorine and bromine. In a preferred embodiment, the aromatic primary amine is a toluidine halogenated at the aromatic that does not have further substituents R1, or a toluidine halogenated at the aromatic, that carries another C1-C4alkyl group, preferably another methyl group, at the aromatic. A toluidine halogenated at the aromatic that does not have further substituents R1is particularly preferred. In a preferred embodiment, the aromatic secondary amine is an aniline or an alkyl aniline, i.e. it has a phenyl ring as an aromatic functional group, and this carries either no R1or an R1which is an alkyl group. In one embodiment, the aromatic secondary amine is an aniline. In another embodiment, the aromatic secondary amine is an alkyl aniline, i.e. it carries an R1, which is an alkyl group. Said R1is preferably a C1-C4alkyl: more preferably, R1is methyl and the alkyl aniline is therefore toluidine. In addition, other substituents R1selected from the groups given above for R1may also be present. In a preferred embodiment, there is no further R1. In a further preferred embodiment, one or two further R1are present, more preferably only one further R1is present. In a preferred embodiment, the further R1is selected from the group consisting of halogen and C1-C20alkyl. The further R1is very particularly preferably selected from the group consisting of chlorine, bromine and C1-C6alkyl, in particular from the group consisting of chlorine, bromine and C1-C4alkyl, and very particularly from the group consisting of chlorine and bromine. In a preferred embodiment, the aromatic secondary amine is a toluidine or aniline halogenated at the aromatic that does not have further substituents R1, or is a toluidine or aniline halogenated at the aromatic that carries another C1-C4alkyl group, preferably another methyl group, at the aromatic. A toluidine or aniline halogenated at the aromatic that does not have further substituents R1is particularly preferred. If only one R1is present, it is preferably in the meta or para position in relation to the amino group. The same applies to the presence of a plurality of R1. If two R1are present, one of them is preferably in the meta position and the other in the para position. If three R1are present, at least one of them is preferably in the meta position and one in the para position. In the secondary amines, the substituent R2is on the nitrogen, which substituent is present in addition to the aromatics bound to the nitrogen, preferably selected from the group consisting of C1-C20alkyl, hydroxy-C1-C20alkyl, C2-C20alkenyl, hydroxy-C2-C20alkenyl, C2-C20alkynyl and hydroxy-C2-C20alkynyl. R2is preferably selected from the group consisting of hydroxy-C1-C20alkyl and C1-C20alkyl. R2is particularly preferably selected from the group consisting of hydroxy-C1-C12alkyl and C1-C12alkyl. R2is very particularly preferably selected from the group consisting of hydroxy-C1-C4alkyl and C1-C4alkyl. 2) The diglycidyl ether is preferably selected from the group consisting of diglycidyl ethers of diols of hydrocarbons having 2 to 20 C atoms, preferably having 4 to 15 C atoms. The hydrocarbons can be branched or unbranched. The hydrocarbons can be aromatic or aliphatic, or a combination thereof. The diols are preferably selected from the group consisting of bisphenols, in particular bisphenol A, bisphenol F, and bisphenol S, neopentyl glycol, ethylene glycol, phenol novolac resin, cresol novolac resin, and 1,4-butanediol. The diglycidyl ether is preferably selected from the group consisting of diglycidyl ether of bisphenol A, diglycidyl ether of bisphenol F, and diglycidyl ether of bisphenol S. The diglycidyl ether is more preferably the diglycidyl ether of bisphenol A. It is also possible to use oligomeric or polymeric diols. When using secondary amines, it is also possible to use a glycidyl ether from a polyol instead of a glycidyl ether from a diol, i.e. a compound having more than two hydroxyl groups, in particular a triol and a tetraol, i.e. a compound having three or four hydroxyl groups, since there is no risk of highly viscous polymeric compounds forming. 3) The α,β-unsaturated carboxylic acid is preferably selected from the group consisting of branched and unbranched C2-C10α,β-unsaturated carboxylic acids, more preferably from the group consisting of branched and unbranched C2-C6α,β-unsaturated carboxylic acids. It is particularly preferably selected from the group consisting of tiglic acid ((E)-2,3-dimethylacrylic acid), sorbic acid (hexadienoic acid), crotonic acid (trans-butenoic acid), methacrylic acid and acrylic acid. The α,β-unsaturated carboxylic acid is more preferably selected from the group consisting of methacrylic acid and acrylic acid. Said α,β-unsaturated carboxylic acid is even more preferably methacrylic acid. 4) The catalyst can be any catalyst conventionally used to catalyze the reaction of an epoxide with an α,β-unsaturated carboxylic acid to form the corresponding carboxylic acid ester. The catalyst is preferably a tetraalkylammonium halide, more preferably selected from the group consisting of tetraalkylammonium bromide and tetraalkylammonium chloride. The catalyst is more preferably selected from the group consisting of tetraethylammonium bromide, tetrabutylammonium bromide, tetraethylammonium chloride and tetrabutylammonium chloride. The catalyst is particularly preferably a catalyst used in the examples. 5) The inhibitor which is optionally and preferably actually used can be any inhibitor conventionally used in the synthesis of epoxy (meth)acrylate resins. Suitable inhibitors are described in more detail below. The preparation process for a reactive amine accelerator according to the invention and for a reactive resin according to the invention that contains said accelerator typically proceeds as follows: 1. Preparation of Reactive Amine Accelerators A diglycidyl ether (2) is reacted with an α,β-unsaturated carboxylic acid, for example with (meth)acrylic acid (3) in the presence of a catalyst (4) and one or more primary or secondary aromatic amine(s) (1) (typically at a temperature of 80° C. to 120° C.). The reaction mixture optionally (and preferably) also contains one or more inhibitors (5). In addition, the reaction mixture preferably does not contain any further ingredients. The reactive amine accelerator according to the invention is formed in this reaction. Exemplary reactions are described in the examples. If a secondary amine is used, an ether of higher valence can also be used. In order to lower the viscosity when using primary anilines such as para-toluidine, the diglycidyl ether can be partially reacted with part of the α,β-unsaturated carboxylic acid (for example (meth)acrylic acid) before adding the aromatic amine. In the case of para-toluidine, this is described by way of example in the examples. The equivalent ratio of diglycidyl ether:α,β-unsaturated carboxylic acid:primary aromatic amine is typically in the range of from 1:0.3:0.8 to 1:2.09:0.01, preferably from 1:0.6:0.7 to 1:2.05:0.05, particularly preferably is approximately 1:1.1:0.5. The equivalent ratio of diglycidyl ether:α,β-unsaturated carboxylic acid:secondary aromatic amine is typically in the range of from 1:0.1:2 to 1:2.09:0.01, preferably from 1:0.5:1.6 to 1:2.05:0.05, particularly preferably is approximately 1:1.1:1. When a mixture of primary and secondary amine is used, the equivalent value for the mixture of primary and secondary aromatic amine is typically in a range between the equivalent value for the secondary amine (lower limit) and the equivalent value for the primary amine (upper limit). The value for the mixture in this case results from the molar ratio between the primary and secondary amine. The reaction mixture obtained after the end of the reaction is not further processed, i.e. the reactive amine accelerator is not isolated. Optionally, one or more inhibitors and/or one or more reactive diluents are added to the reaction mixture after completion of the reaction to the reactive amine accelerator. 2. Preparation of the Backbone Resin/Reactive Resin Masterbatch A diglycidyl ether, for example bisphenol A diglycidyl ether, and an α,β-unsaturated carboxylic acid, for example methacrylic acid, are reacted in the presence of a catalyst and an inhibitor (which is used to stabilize the backbone resin formed by the polymerization). The backbone resin was created hereby. The reaction mixture obtained after completion of the reaction is referred to as a reactive resin masterbatch. This is not further processed, i.e. the backbone resin is not isolated. 3. Preparation of the Reactive Resin The reaction mixture from the preparation of the reactive amine accelerator is mixed with a backbone resin or reactive resin masterbatch, for example the reaction mixture from the preparation of the backbone resin/reactive resin masterbatch described under section 2, one or more reactive diluents and one or more inhibitors. Two or more reactive amine accelerators are optionally used. The order in which the individual components of the reactive resin are mixed together is not relevant. Typically, the reactive amine accelerator is initially provided and then the reactive resin masterbatch, the reactive diluent, and the inhibitor are added in succession. Typically, the reactive amine accelerator and the backbone resin or the reactive resin masterbatch are prepared separately from one another and are mixed with one another to prepare the reactive resin. This applies in particular to the preparation of urethane (meth)acrylate-based reactive resins. In a further embodiment, however, the reactive resin is prepared by first preparing the reactive amine accelerator and then the backbone resin in succession in the same vessel, i.e. by carrying out a multi-stage one-pot reaction. This applies in particular to the preparation of epoxy (meth)acrylate-based reactive resins. In a further embodiment, the reactive resin is prepared by first preparing the reactive amine accelerator and part of the backbone resin in a one-step one-pot reaction, and then subsequently preparing the further part of the backbone resin in the same vessel. This also applies to the preparation of epoxy(meth) acrylate-based reactive resins. In yet another embodiment, individual components of the reactive resin, in particular thermally stable and non-reactive components, are added to the reaction mixture for preparation of the reactive amine accelerator, even before the reaction to the reactive amine accelerator begins. This produces the epoxy (meth)acrylate or urethane (meth)acrylate reactive resin with the reactive amine accelerator according to the invention. A first subject matter of the invention is a reactive amine accelerator, in particular an accelerator which has the generic formula (I) or (II). prepared by means of the preparation process described herein, i.e., by (1) an aromatic primary or secondary amine, or a mixture of two or more of said amines, being reacted with (2) a diglycidyl ether of a diol having 2 to 30 C atoms, and (3) an α,β-unsaturated carboxylic acid, preferably an α,β-unsaturated carboxylic acid selected from the group consisting of tiglic acid, sorbic acid, crotonic acid, methacrylic acid and acrylic acid. The reaction typically takes place in the presence of (4) a catalyst. (5) an inhibitor can optionally be present in the reaction mixture. The opening of the epoxy groups present in the diglycidyl ether leads to the formation of glycerol bridges between the amine and the diol and between the α,β-unsaturated carboxylic acid and the diol. An amine accelerator prepared in this way can also be a mixture of different compounds which are formed in this reaction. A second subject matter of the invention is a reactive resin containing this reactive amine accelerator. A third subject matter is a reactive resin component (A) containing a reactive resin according to the invention. A fourth subject matter is a reactive resin system comprising a reactive resin component (A) according to the invention and a hardener component (B) containing an initiator (such as a peroxide) for curing the backbone resin contained in the reactive resin. The components (A) and (B) are packaged so as to be spatially separated from each other until the reactive resin system is used; a reaction therefore takes place only when the two components are brought into contact with one another. A fifth subject matter of the invention is a method for preparing the reactive amine accelerator according to the invention. A sixth subject matter of the invention is the use of a reactive amine accelerator according to the invention, in particular a compound which has the general formula (I) or (II), as an accelerator in a reactive resin. A seventh subject matter of the invention is the use of a reactive resin system according to the invention for chemically fastening anchoring means in boreholes or for structural bonding. Generic Formulas of Reactive Amine Accelerators According to the Invention A reactive amine accelerator according to the invention, which was prepared using a primary aromatic amine which contains a phenyl functional group as an aromatic functional group, has the following idealized generic formula (I): Formula (I) is idealized because it is not only secondary alcohols that are formed during the opening of the epoxide (typically approximately 80% are secondary), and because a certain irregular distribution of the monomers in the formula is possible. R1is optional in the formula (I), i.e. the phenyl functional group in formula (I) can also be unsubstituted. As already described above, in the case of a primary aromatic amine, the aromatic functional group, in this case the phenyl functional group, is preferably substituted with one, two or three functional groups R1. In the generic formula (I) shown, the phenyl ring is a placeholder for an aromatic functional group. In formula (I), the substituent R1which is optionally present on the aromatic functional group is in the ortho-, meta- or para-position relative to the nitrogen N, specifically in the same position as in the free amine which was used for the preparation. The meta- and para-positions are preferred. R1is the same substituent in the bound amine as in the free primary amine which was used to prepare the bound amine. In the generic formula (I) shown, n is a whole number from 1 to 20, preferably from 1 to 10, more preferably from 1 to 7, and n is particularly preferably a whole number from 1 to 5, very particularly preferably from 1 to 3. It is clear from the preparation method for the amine accelerator that, in the amine accelerator resulting from the synthesis according to the invention (which can be a mixture of molecules having different values n), the value n is an average value calculated from the individual values for all molecules and therefore can also be a non-whole number. The average value of n for the amine accelerator is preferably a value from approximately 0.9 to approximately 10, more preferably from approximately 1 to approximately 7, and particularly preferably from approximately 1 to approximately 5. Very particularly preferably, n is a value from approximately 2 to approximately 3, for example approximately 2.7. The value of n is determined by means of gel permeation chromatography (GPC; column (Polymer Standard Service; modified styrene-divinylbenzene copolymer network): PSS 5 μm SDV 50 Å 100 Å 1000 Å; eluent: THF; calibration standard: polystyrene) and the following formula (Mw: mass average molecular weight; M: molecular weight, calculated using n=1): 〈n〉=Mw(GPC)M(calculatedusingn=1) For reasons of process technology, higher molecular weights and molecular weight distributions are advantageous, which also has advantages for REACH registration. In the generic formula (I) shown, A represents the functional group of the diol contained in the diglycidyl ether. The diols used according to the invention are defined above. In the generic formula (II) shown, R1has the meaning given above. A reactive amine accelerator according to the invention, which was prepared using a secondary aromatic amine which contains a phenyl functional group as an aromatic functional group, has the following generic formula (II): In formula (II), the substituent R1which is optionally present on the aromatic functional group is in the ortho-, meta- or para-position relative to the nitrogen N, specifically in the same position as in the free amine which was used for the preparation. The meta- and para-positions are preferred. R1and R2are the same substituent in the bound amine as in the free secondary amine which was used to prepare the bound amine. In the generic formula (II) shown, n is a whole number from 1 to 10, preferably from 1 to 5, more preferably from 1 to 2. Particularly preferably, n is a whole number from 1 to 5, very particularly preferably from 1 to 2. It is clear from the preparation method for the glycidyl ether that the value n from the synthesis of the glycidyl ether (which can be a mixture of molecules having different values n) is an average value calculated from the individual values for all molecules and can therefore also be a non-whole number. The average value of n for the amine accelerator is preferably a value from approximately 0.9 to approximately 10, more preferably from approximately 1 to approximately 5, and particularly preferably from approximately 1 to approximately 2. Very particularly preferably, n is a value from approximately 1 to approximately 1.5. In the generic formula (II) shown, A represents the functional group of the diol contained in the diglycidyl ether. The diols used according to the invention are defined above. In the generic formula (II) shown, R1and R2have the meaning given above. In the generic formula (II) shown, the phenyl ring is a placeholder for an aromatic functional group. A reactive amine accelerator according to the invention, which was prepared using neopentyl glycol and a secondary aromatic amine, has the following formula, for example, for n=1: For a better understanding of the invention, the following explanations of the terminology used herein are considered useful. Within the meaning of the invention:“backbone resin” means a typically solid or high-viscosity radically polymerizable resin which cures by means of polymerization (e.g. after addition of an initiator in the presence of an accelerator, which according to the invention is the reactive amine accelerator);“reactive resin masterbatch” means the reaction product of the reaction for preparing the backbone resin, i.e. a mixture of backbone resin, an inhibitor and other constituents (e.g. a catalyst) of the reaction mixture:“reactive resin” means a mixture of a reactive resin masterbatch, one or more inhibitors, a reactive diluent and optionally further additives; the reactive resin is typically liquid or viscous and can be further processed to form a reactive resin component; the reactive resin is also referred to herein as a “resin mixture;”“inhibitor” means a substance which suppresses unwanted radical polymerization during the synthesis or storage of a resin or a resin-containing composition (these substances are also referred to in the art as “stabilizers”), or which delays the radical polymerization of a resin after addition of an initiator, usually in conjunction with an accelerator (these substances are also referred to in the art as “inhibitors”—the relevant meaning of the term is apparent from the context);“initiator” means a substance which (usually in combination with an accelerator) forms reaction-initiating radicals;“accelerator” means a reagent which reacts with the initiator such that, even at low temperatures, larger amounts of radicals are produced by the initiator, or means a reagent which catalyzes the decomposition reaction of the initiator;“amine accelerator,” means an accelerator based on an amine, in particular an aromatic amine;“reactive amine accelerator” means an amine accelerator that contains one or two α,β-unsaturated carboxylic acid ester groups;“co-accelerator” means a reagent which intervenes in the acceleration reaction either catalytically or stoichiometrically, for example, to rebuild the accelerator, moderate radical production per unit of time, further lower the acceleration temperature, or effect a combination of these or other effects;“reactive diluents” means liquid or low-viscosity monomers and backbone resins which dilute other backbone resins or the reactive resin masterbatch and thereby impart the viscosity necessary for application thereof, which contain functional groups capable of reacting with the backbone resin, and which for the most part become a constituent of the cured composition (e.g. of the mortar) in the polymerization (curing); reactive diluents are also referred to as co-polymerizable monomers;“gel time,” tg25° C., means the time (t) of the curing phase of a reactive resin (thg25° C.), as defined herein, or a reactive resin component (tmg25° C.), as defined herein, in which the temperature is increased from a starting temperature of 25° C. at a gel time measurement to 50° C.; a method for determining the gel time is described in the examples;“maximum reactivity temperature Tmax” means the temperature at which the temperature profile passes through a maximum during a reactivity measurement (for example the gel time measurement described in the examples);“completion of the reaction” or “reaction end” or “reaction completion” mean the point in time at which a reaction was completely executed; this is generally recognizable in the case of a chemical reaction, such as the reaction for preparing the backbone resin, because the exothermicity related to the reaction has ended;“reactive resin component” means a liquid or viscous mixture of reactive resin and fillers and optionally further components, e.g. additives; typically, the reactive resin component is one of the two components of a two-component reactive resin system for chemical fastening;“hardener component” means a composition containing an initiator for the polymerization of a backbone resin; the hardener component may be solid or liquid and may contain, in addition to the initiator, a solvent and fillers and/or additives; typically the hardener component, in addition to the reactive resin component, is the other of the two components of a two-component reactive resin chemical fastening system;“two-component system” or “two-component reactive resin system” a reactive resin system comprising two separately stored components, a reactive resin component (A) and a hardener component (B), so that a curing of the backbone resin contained in the reactive resin component only takes place after the two components are mixed;“multi-component system” or “multi-component reactive resin system” a reactive resin system comprising a plurality of separately stored components, including a reactive resin component (A) and a hardener component (B), so that curing of the backbone resin contained in the reactive resin component only takes place after all of the components are mixed;“(meth)acrylic . . . / . . . (meth)acrylic . . . ” means both the “methacrylic . . . / . . . methacrylic” and the “acrylic . . . / . . . acrylic . . . ” compounds; “methacrylic . . . / . . . methacrylic” compounds are preferred in the present invention;“epoxy (meth)acrylate” means an epoxy resin which has acrylate or methacrylate groups and is essentially free of epoxy groups;“alkyl” means a saturated hydrocarbon functional group that can be branched or unbranched; preferably a C1-C20alkyl, particularly preferably a C1-C4alkyl, i.e. an alkyl selected from the group consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and tert-butyl; methyl, ethyl and tert-butyl are particularly preferred and methyl is very particularly preferred;“hydroxyalkyl” means an alkyl carrying at least one hydroxyl group as a substituent; preferably a hydroxyl group;“alkenyl” means an unsaturated hydrocarbon functional group having at least one and at most five double bonds, preferably one, which can be branched or unbranched; preferably a C2-C20alkenyl, particularly preferably a C2-C6alkenyl, i.e. an alkenyl selected from the group consisting of ethenyl, propenyl, butenyl, pentenyl and hexenyl; ethenyl, propenyl and butenyl are particularly preferred, and ethenyl is very particularly preferred;“hydroxy-alkenyl” means an alkenyl which carries at least one hydroxyl group as a substituent, preferably a hydroxyl group;“alkynyl” means an unsaturated hydrocarbon functional group having at least one and at most five triple bonds, preferably one which can be branched or unbranched; preferably a C2-C20alkynyl, particularly preferably a CrCe alkynyl, i.e. an alkynyl selected from the group consisting of ethynyl, propynyl, butynyl, pentynyl and hexynyl; ethynyl, propynyl and butynyl are particularly preferred, and ethenyl is very particularly preferred:“hydroxy-alkynyl” means an alkynyl which carries at least one hydroxyl group as a substituent; preferably a hydroxyl group;“cold curing” means that a resin mixture or a reactive resin system can cure completely at room temperature;“a,” “an,” “any,” as the article preceding a class of chemical compounds, e.g. preceding the word “epoxy methacrylate,” means that one or more compounds included in this class of chemical compounds, e.g. various epoxy methacrylates, may be intended. In a preferred embodiment, this article means only a single compound;“at least one” numerically means “one or more.” In a preferred embodiment, the term numerically means “one;”“contain,” “comprise,” and “include” mean that further constituents may be present in addition to those mentioned. These terms are intended to be inclusive and therefore also encompass “consist of.” “Consist of” is intended to be exclusive and means that no further constituents may be present. In a preferred embodiment, the terms “contain,” “comprise,” and “include” mean the term “consist of;”“approximately” or “approx.” before a numerical value means a range of ±5% of this value, preferably ±2% of this value, more preferably ±1% of this value, particularly preferably ±0% of this value (i.e. exactly this value);a range limited by numbers, e.g. “from 80° C. to 120° C.,” means that the two extreme values and any value within this range are disclosed individually. All standards cited in this text (e.g. DIN standards) were used in the version that was current on the filing date of this application. The reactive amine accelerator according to the invention is prepared as described above, by (1) an aromatic primary or secondary amine being reacted with (2) a diglycidyl ether and (3) an α,β-unsaturated carboxylic acid. The reaction typically takes place in the presence of (4) a catalyst. (5) an inhibitor can optionally be present in the reaction mixture. The starting compounds are mixed and reacted with one another. Typically, all preparation steps are carried out while stirring, but other types of mixing are also conceivable. After the reaction for preparing the reactive amine accelerator has finished, further components, in particular the backbone resin, are added for the subsequent preparation of the reactive resin. Alternatively, individual components of the reactive resin, in particular thermally stable and non-reactive components, can be added to the reaction mixture for preparation of the reactive amine accelerator, even before the reaction to the reactive amine accelerator begins. However, it is preferable for these other components to be added to the amine accelerator after completion of the reaction to the backbone resin. A reactive amine accelerator according to the invention is preferably a compound of formula (I) or (II), as shown above. If both a primary and secondary aromatic amine were used to prepare the reactive amine accelerator according to the invention, the reactive amine accelerator according to the invention contains both compounds of formula (I) and of the formula (II). A reactive resin according to the invention contains at least one reactive amine accelerator according to the invention, at least one backbone resin, at least one reactive diluent and at least one inhibitor. Since the reactive amine accelerator and the backbone resin are used, typically without isolation, for preparing the reactive resin after their preparation, further constituents are also generally present in the reactive resin according to the invention, which further constituents are contained in the reaction mixture, in addition to the reactive amine accelerator, and are contained in the reactive resin masterbatch, in addition to the backbone resin. In a preferred subject matter of the invention, the reactive resin according to the invention contains a mixture of two or more, preferably two, reactive amine accelerators according to the invention. The accelerator combinations described in the examples characterize a preferred embodiment of the invention. According to the invention, suitable backbone resins are ethylenically unsaturated compounds, compounds which have carbon-carbon triple bonds, and thiol-yne/ene resins, as are known to the person skilled in the art. Of these compounds, the group of ethylenically unsaturated compounds is preferred, which group comprises styrene and derivatives thereof, (meth)acrylates, vinyl esters, unsaturated polyesters, vinyl ethers, allyl ethers, itaconates, dicyclopentadiene compounds and unsaturated fats, of which unsaturated polyester resins and vinyl ester resins are particularly suitable and are described, for example, in the applications EP 1 935 860 A1, DE 195 31 649 A1 and WO 10/108939 A1. Vinyl ester resins are in this case most preferred due to the hydrolytic resistance and excellent mechanical properties thereof. Examples of suitable unsaturated polyesters which can be used in the resin mixture according to the invention are divided into the following categories, as classified by M. Malik et al. in J. M. S.—Rev. Macromol. Chem. Phys., C40 (2 and 3), p. 139-165 (2000): (1) ortho-resins: these are based on phthalic anhydride, maleic anhydride or fumaric acid and glycols, such as 1,2-propylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, 1,3-propylene glycol, dipropylene glycol, tripropylene glycol, neopentyl glycol or hydrogenated bisphenol-A; (2) iso-resins: these are prepared from isophthalic acid, maleic anhydride or fumaric acid and glycols. These resins can contain higher proportions of reactive diluents than the ortho resins; (3) bisphenol A fumarates: these are based on ethoxylated bisphenol A and fumaric acid; (4) HET acid resins (hexachloroendomethylene tetrahydrophthalic acid resins): are resins obtained from chlorine/bromine-containing anhydrides or phenols during the preparation of unsaturated polyester resins. In addition to these resin classes, what are referred to as dicyclopentadiene resins (DCPD resins) can also be distinguished as unsaturated polyester resins. The class of DCPD resins is either obtained by modifying one of the abovementioned resin types by means of a Diels-Alder reaction with cyclopentadiene, or said resins are alternatively obtained by means of a first reaction of a diacid, for example maleic acid, with dicyclopentadiene and then by means of a second reaction of the usual preparation of an unsaturated polyester resin, the latter being referred to as a DCPD male resin. The unsaturated polyester resin preferably has a molecular weight Mn in the range of 500 to 10,000 daltons, more preferably in the range of 500 to 5000 and even more preferably in the range of 750 to 4000 (according to ISO 13885-1). The unsaturated polyester resin has an acid value in the range of 0 to 80 mg KOH/g resin, preferably in the range of 5 to 70 mg KOH/g resin (according to ISO 2114-2000). If a DCPD resin is used as the unsaturated polyester resin, the acid value is preferably 0 to 50 mg KOH/g resin. In the context of the invention, vinyl ester resins are oligomers or polymers having at least one (meth)acrylate end group, what are referred to as (meth)acrylate-functionalized resins, which also include urethane (meth)acrylate resins and epoxy (meth)acrylates, which are particularly preferred. Vinyl ester resins which have unsaturated groups only in the end position are obtained, for example, by reacting epoxy oligomers or polymers (for example bisphenol A digylcidyl ether, phenol novolac-type epoxides or epoxy oligomers based on tetrabromobisphenol A) with (meth)acrylic acid or (meth)acrylamide, for example. Preferred vinyl ester resins are (meth)acrylate-functionalized resins and resins which are obtained by reacting epoxy oligomers or polymers with methacrylic acid or methacrylamide, preferably with methacrylic acid. Examples of compounds of this kind are known from the applications U.S. Pat. Nos. 3,297,745 A, 3,772,404 A, 4,618,658 A, GB 2217722 A1, DE 3744390 A1 und DE 4131457 A1. In this context, reference is made to the application US 2011071234 A1, the content of which is hereby incorporated into this application. The vinyl ester resin preferably has a molecular weight Mn in the range of 500 to 3000 daltons, more preferably 500 to 1500 daltons (according to ISO 13885-1). The vinyl ester resin has an acid value in the range of 0 to 50 mg KOH/g resin, preferably in the range of 0 to 30 mg KOH/g resin (according to ISO 2114-2000). Ethoxylated bisphenol A di(meth)acrylate having a degree of ethoxylation of 2 to 10, preferably of 2 to 4, difunctional, trifunctional or higher functional urethane (meth)acrylate oligomers, or mixtures of these curable constituents are particularly suitable as vinyl ester resin. The known reaction products of di- or polyisocyanates and hydroxyalkylmethylacrylates, as described, for example, in DE 2 312 559 A1, adducts of (di)isocyanates and 2,2-propane bis[3-(4-phenoxy)-1,2-hydroxypropane-1-methacrylate] according to US-PS 3 629 187, and the adducts of isocyanates and methacryloyl alkyl ethers, alkoxybenzenes or alkoxycycloalkanes, as described in EP 44352 A1, are very particularly suitable. In this context, reference is made to DE 2312559 A1, DE 19902685 A1, EP 0684906 A1, DE 4111828 A1 and DE 19961342 A1. Of course, mixtures of suitable monomers can also be used. All of these resins that can be used according to the invention can be modified according to methods known to a person skilled in the art, for example to achieve lower acid numbers, hydroxide numbers or anhydride numbers, or can be made more flexible by introducing flexible units into the backbone, and the like. In addition, the resin may contain other reactive groups that can be polymerized with a radical initiator, such as peroxides, for example reactive groups derived from itaconic acid, citraconic acid and allylic groups and the like, as described, for example, in WO 2010/108939 A1 (itaconic acid ester). The percentual proportion (in wt. % of the reactive resin) of backbone resin in the reactive resin according to the invention is advantageously greater than approximately 5%, preferably greater than approximately 15%, and particularly preferably greater than approximately 20%. The percentual proportion (in wt. % of the reactive resin) of backbone resin in the reactive resin is advantageously approx. 5% to approx. 90%, preferably approx. 8% to approx. 80%, more preferably approx. 10% to approx. 60%, more preferably approx. 20% to approx. 55%, even more preferably approx. 25% to approx. 55%, particularly preferably approx. 25% to approx. 50%, and very particularly preferably approx. 28% to approx. 45%. The proportion (in mmol amine per 100 g of the reactive resin) of reactive amine accelerator according to the invention in the reactive resin according to the invention is 0.5 to 50, preferably 1 to 20, particularly preferably 5 to 15 mmol amine/100 g of reactive resin. If a mixture of a plurality of reactive amine accelerators according to the invention is used in the reactive resin according to the invention, the proportion relates to the mixture. One or more inhibitors are present in the reactive resin according to the invention, both to stabilize the reactive resin or the reactive resin component (A) containing the reactive resin, or other compositions containing the reactive resin, and for adjusting the resin reactivity. The inhibitors which are conventionally used for radically polymerizable compounds, as are known to a person skilled in the art, are suitable for this purpose. These inhibitors are preferably selected from phenolic inhibitors and non-phenolic inhibitors, in particular phenothiazines. Phenols, such as 2-methoxyphenol, 4-methoxyphenol, 2,6-di-tert-butyl-4-methylphenol, 2,4-di-tert-butylphenol, 2,6-di-tert-butylphenol, 2,4,6-trimethylphenol, 2,4,6-tris(dimethylaminomethyl)phenol, 4,4′-thio-bis(3-methyl-6-tert-butylphenol), 4,4′-isopropylidenediphenol, 6,6′-di-tert-butyl-4,4′-bis(2,6-di-tert-butylphenol), 1,3,5-trimethyl-2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl)benzene, 2,2′-methylene-di-p-cresol, catechols such as pyrocatechol, and catechol derivatives such as butylpyrocatechols such as 4-tert-butylpyrocatechol and 4,6-di-tert-butylpyrocatechol, hydroquinones such as hydroquinone, 2-methylhydroquinone, 2-tert-butylhydroquinone, 2,5-di-tert-butylhydroquinone, 2,6-di-tert-butylhydroquinone, 2,6-dimethylhydroquinone, 2,3,5-trimethylhydroquinone, benzoquinone, 2,3,5,6-tetrachloro-1,4-benzoquinone, methylbenzoquinone, 2,6-dimethylbenzoquinone, naphthoquinone, or mixtures of two or more thereof, are suitable as phenolic inhibitors. These inhibitors are often a constituent of commercial radically-curing reactive resin components. Phenothiazines such as phenothiazine and/or derivatives or combinations thereof, or stable organic radicals such as galvinoxyl and N-oxyl radicals, in particular of the piperidinyl-N-oxyl or tetrahydropyrrole-N-oxyl type, are preferably considered as non-phenolic inhibitors, such as aluminum-N-nitrosophenylhydroxylamine, diethylhydroxylamine, oximes such as acetaldoxime, acetone oxime, methyl ethyl ketoxime, salicyloxime, benzoxime, glyoximes, dimethylglyoxime, acetone-O-(benzyloxycarbonyl)oxime, TEMPOL, TEMPO and the like. Furthermore, pyrimidinol or pyridinol compounds substituted in para-position to the hydroxyl group, as described in the patent DE 10 2011 077 248 B1, can be used as inhibitors. Examples of stable N-oxyl radicals which can be used are those described in DE 199 56 509 A1 and DE 195 31 649 A1. Stable nitroxyl radicals of this kind are of the piperidinyl-N-oxyl or tetrahydropyrrole-N-oxyl type, or are a mixture thereof. Preferred stable nitroxyl radicals are selected from the group consisting of 1-oxyl-2,2,6,6-tetramethylpiperidine, 1-oxy-2,2,6,6-tetramethylpiperdin-4-ol (also referred to as TEMPOL), 1-oxyl-2,2,6,6-tetramethylpiperidin-4-one (also referred to as TEMPON), 1-oxyl-2,2,6,6-tetramethyl-4-carboxyl-piperidine (also referred to as 4-carboxy-TEMPO), 1-oxyl-2,2,5,5-tetramethylpyrrolidine, 1-oxyl-2,2,5,5-tetramethyl-3-carboxylpyrrolidine (also referred to as 3-carboxy-PROXYL) and mixtures of two or more of said compounds, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-ol (TEMPOL) being particularly preferred. The inhibitor or inhibitors are preferably selected from the group consisting ofN-oxyl radicals, catechols, catechol derivatives and phenothiazines and a mixture of two or more thereof. The inhibitor or inhibitors selected from the group consisting of tempol, catechols and phenothiazines are particularly preferred. The further inhibitors used in the examples are very particularly preferred, preferably approximately in the amounts stated in the examples. The inhibitors can be used either alone or as a combination of two or more thereof, depending on the desired properties of the reactive resin. The combination of phenolic and non-phenolic inhibitors is preferred. The inhibitor or inhibitor mixture is added in conventional amounts known in the art, preferably in an amount of approximately 0.0005 to approximately 2 wt. % (based on the reactive resin, which is ultimately prepared therewith), more preferably from approximately 0.01 to approximately 1 wt. % (based on the reactive resin), even more preferably from approximately 0.05 to approximately 1 wt. % (based on the reactive resin). The reactive resin according to the invention contains at least one reactive diluent. Suitable reactive diluents are low-viscosity, radically co-polymerizable compounds, preferably compounds free of labeling. Suitable reactive diluents are described in the applications EP 1 935 860 A1 and DE 195 31 649 A1. The reactive resin preferably contains, as the reactive diluent, a (meth)acrylic acid ester, aliphatic or aromatic C5-C15-(meth)acrylates being particularly preferably selected. Suitable examples include: 2-, 3-hydroxypropyl(meth)acrylate (HP(M)A), 1,2-ethanediol di(meth)acrylate, 1,3-propanediol di(meth)acrylate, 1,3-butanediol di(meth)acrylate, 1,4-butanediol di(meth)acrylate, trimethylolpropane tri(meth)acrylate, phenethyl (meth)acrylate, tetrahydrofurfuryl (meth)acrylate, ethyltriglycol (meth)acrylate, N,N-dimethylaminoethyl (meth)acrylate, N,N-dimethylaminomethyl (meth)acrylate, acetoacetoxyethyl (meth)acrylate, isobornyl (meth)acrylate, 2-ethylhexyl(meth)acrylate, diethyleneglycol di(meth)acrylate, methoxypolyethylene glycol mono(meth)acrylate, trimethylcyclohexyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, dicyclopentenyloxyethyl (meth)acrylate and/or tricyclopentadienyl di(meth)acrylate, bisphenol-A-(meth)acrylate, novolac epoxy di(meth)acrylate, di-[(meth)acryloyl-maleoyl]-tricyclo-5.2.1.0.26-decane, dicyclopentenyl oxy ethyl crotonate, 3-(meth)acryloyl-oxymethyl-tricylo-5.2.1.0.26-decane, 3-(meth)cyclopentadienyl (meth)acrylate, and decalyl-2-(meth)acrylate; PEG-di(meth)acrylate such as PEG200 di(meth)acrylate, tetraethylene glycol di(meth)acrylate, solketal (meth)acrylate, cyclohexyl (meth)acrylate, phenoxyethyl di(meth)acrylate, methoxyethyl (meth)acrylate, tert-butyl (meth)acrylate and norbomyl (meth)acrylate. Methacrylates are preferred over acrylates. Particularly preferred are 2- and 3-hydroxypropyl methacrylate (HPMA), 1,2-ethanediol dimethacrylate, 1,4-butanediol dimethacrylate (BDDMA), 1,3-butanediol dimethacrylate, trimethylolpropane trimethacrylate, acetoacetoxyethyl methacrylate, isobornyl methacrylate, bisphenol A methacrylate, trimethylcyclohexyl methacrylate, 2-hydroxyethyl methacrylate, PEG200 dimethacrylate and norbomyl methacrylate. 1,4-butanediol dimethacrylate and a mixture of 2- and 3-hydroxypropyl methacrylate (HPMA), or a mixture of these three methacrylates are very particularly preferred. A mixture of 2- and 3-hydroxypropyl methacrylate (HPMA) is most preferred. In principle, other conventional radically polymerizable compounds, alone or in a mixture with the (meth)acrylic acid esters, can also be used as reactive diluents, e.g. styrene, α-methylstyrene, alkylated styrenes, such as tert-butylstyrene, divinylbenzene and vinyl and allyl compounds, of which the representatives that are not subject to labeling are preferred. Examples of vinyl or allyl compounds of this kind are hydroxybutyl vinyl ether, ethylene glycol divinyl ether, 1,4-butanediol divinyl ether, trimethylolpropane divinyl ether, trimethylolpropane trivinyl ether, mono-, di-, tri-, tetra- and polyalkylene glycol vinyl ether, mono-, di-, tri-, tetra- and polyalkylene glycol allyl ether, divinyl adipate, trimethylolpropane diallyl ether and trimethylolpropane triallyl ether. The reactive diluents used in the examples are very particularly preferred, preferably approximately in the amounts stated in the examples. The reactive diluent(s) is/are preferably present in the reactive resin according to the invention in an amount of from 0 to approx. 80 wt. %, particularly preferably from approx. 10 to approx. 60 wt. %, even more preferably from approx. 20 to approx. 50 wt. %, based on the reactive resin. The curing of the reactive resin is expediently initiated using a peroxide as an initiator. Any of the peroxides known to a person skilled in the art that are used to cure epoxy (meth)acrylate resins can be used. Peroxides of this kind include organic and inorganic peroxides, either liquid or solid, it also being possible to use hydrogen peroxide. Examples of suitable peroxides are peroxycarbonates (of the formula —OC(O)OO—), peroxyesters (of the formula —C(O)OO—), diacyl peroxides (of the formula —C(O)OOC(O)—), dialkyl peroxides (of the formula —OO—), hydroperoxides (of the formula —OOH), and the like. These may be present as oligomers or polymers. A comprehensive set of examples of suitable peroxides is described, for example, in application US 2002/0091214 A1, paragraph [0018]. The peroxides are preferably selected from the group of organic peroxides. Suitable organic peroxides are: tertiary alkyl hydroperoxides such as tert-butyl hydroperoxide and other hydroperoxides such as cumene hydroperoxide, peroxyesters or peracids, such as tert-butyl peresters (e.g. tert-butyl peroxybenzoate), benzoyl peroxide, peracetates and perbenzoates, lauroyl peroxide, including (di)peroxyesters, perethers, such as peroxy diethyl ethers, and perketones, such as methyl ethyl ketone peroxide. The organic peroxides used as curing agents are often tertiary peresters or tertiary hydroperoxides, i.e. peroxide compounds having tertiary carbon atoms which are bonded directly to an —O—O-acyl or —OOH-group. However, mixtures of these peroxides with other peroxides can also be used according to the invention. The peroxides may also be mixed peroxides, i.e. peroxides which have two different peroxide-carrying units in one molecule. In a preferred embodiment, benzoyl peroxide (BPO) or tert-butyl peroxybenzoate is used for curing. The peroxide can be used in its pure form or as a constituent of a mixture. It is typically used as a constituent of a mixture, in particular as a constituent of a hardener component (B) of a reactive resin system. The hardener component used in the examples, or a hardener component having the same constituents is particularly preferred. The present invention also relates to a reactive resin system consisting of a reactive resin component (A) and a hardener component (B). The reactive resin component (A) alone is also a subject of the present invention. Said reactive resin component (A) contains the reactive resin according to the invention. The reactive resin component (A) according to the invention may contain fillers and/or additives in addition to the reactive resin of the invention. It should be noted that some substances can be used as a filler and, can be used, optionally in a modified form, as an additive. For example, fumed silica is preferably used as a filler in the polar, non-after-treated form thereof, and is preferably used as an additive in the non-polar, after-treated form thereof. In cases in which exactly the same substance can be used as a filler or an additive, the total amount thereof should not exceed the upper limit for fillers that is established herein. In order to produce a reactive resin component for construction applications, in particular chemical fastening, conventional fillers can be added to the reactive resin according to the invention. These fillers are typically inorganic fillers, as described below for example. The proportion of the reactive resin according to the invention in the reactive resin component is preferably from approx. 10 to approx. 70 wt. %, more preferably from approx. 30 to approx. 60 wt. %, even more preferably from approx. 35 to approx. 50 wt. %, based on the reactive resin component. Accordingly, the total proportion of the fillers and additives is preferably from approx. 90 to approx. 30 wt. %, more preferably from approx. 70 to approx. 40 wt. %, even more preferably from approx. 75 to approx. 50 wt. % based on the reactive resin component. The fillers used are conventional fillers, preferably mineral or mineral-like fillers, such as quartz, glass, sand, quartz sand, quartz powder, porcelain, corundum, ceramics, talc, silicic acid (e.g. fumed silica, in particular polar, non-after-treated fumed silica), silicates, aluminum oxides (e.g. alumina), clay, titanium dioxide, chalk, barite, feldspar, basalt, aluminum hydroxide, granite or sandstone, polymeric fillers such as thermosets, hydraulically curable fillers such as gypsum, quicklime or cement (e.g. aluminate cement (often referred to as alumina cement) or Portland cement), metals such as aluminum, carbon black, further wood, mineral or organic fibers, or the like, or mixtures of two or more thereof. The fillers may be present in any desired forms, for example as powder or flour, or as molded bodies, for example in cylindrical, annular, spherical, platelet, rod, saddle or crystal form, or else in fibrous form (fibrillar fillers), and the corresponding base particles preferably have a maximum diameter of approximately 10 mm and a minimum diameter of approximately 1 nm. This means that the diameter is approximately 10 mm or any value less than approximately 10 mm, but more than approximately 1 nm. Preferably, the maximum diameter is a diameter of approximately 5 mm in diameter, more preferably approximately 3 mm, even more preferably approximately 0.7 mm. A maximum diameter of approximately 0.5 mm is very particularly preferred. The more preferred minimum diameter is approximately 10 nm, more preferably approximately 50 nm, very preferably approximately 100 nm. Diameter ranges resulting from combination of this maximum diameter and minimum diameter are particularly preferred. However, the globular, inert substances (spherical form) have a preferred and more pronounced reinforcing effect. Core-shell particles, preferably in spherical form, can also be used as fillers. Preferred fillers are selected from the group consisting of cement, silicic acid, quartz, quartz sand, quartz powder, and mixtures of two or more thereof. For the reactive resin component (A), fillers selected from the group consisting of cement, fumed silica, in particular untreated, polar fumed silica, quartz sand, quartz powder, and mixtures of two or more thereof are particularly preferred. For the reactive resin component (A), a mixture of cement (in particular aluminate cement (often also referred to as alumina cement) or Portland cement), fumed silica and quartz sand is very particularly preferred. For the hardener component (B), fumed silica is preferred as the sole filler or as one of a plurality of fillers; one or more further fillers are particularly preferably present in addition to the fumed silica. Conventional additives are used as the additives in the reactive resin components (A), i.e. thixotropic agents, such as optionally organically or inorganically after-treated fumed silica (if not already used as a filler), in particular non-polarly after-treated fumed silica, bentonites, alkyl- and methylcelluloses, castor oil derivatives or the like, plasticizers, such as phthalic or sebacic acid esters, antistatic agents, thickening agents, flexibilizers, rheological aids, wetting agents, coloring additives, such as dyes or in particular pigments, for example for different staining of the components for improved control of the mixing thereof, or the like, or mixtures of two or more thereof. Non-reactive diluents (solvents) can also be contained, preferably in an amount of up to 30 wt. %, based on the total amount of the reactive resin component, such as low-alkyl ketones, e.g. acetone, di lower alkyl lower alkanoyl amides, such as dimethylacetamide, lower alkyl benzenes, such as xylenes or toluene, phthalic acid esters or paraffins, water or glycols. Furthermore, metal scavengers in the form of surface-modified fumed silicas can be contained in the reactive resin component. Preferably, at least one thixotropic agent is present as an additive, particularly preferably an organically or inorganically after-treated fumed silica, very particularly preferably a fumed silica after-treated in a non-polar manner, for example, fumed silica after-treated with polydimethylsiloxane (PDMS), particularly preferably the fumed silica used in the examples which is after-treated in a non-polar manner. In this regard, reference is made to the patent applications WO 2002/079341 A1 and WO 2002/079293 A1 as well as WO 2011/128061 A1, the relevant content of which is hereby incorporated into this application. In one embodiment, the reactive resin component may additionally contain an adhesion promoter. By using an adhesion promoter, the cross-linking of the borehole wall with the dowel mass is improved such that the adhesion increases in the cured state. This is important for the use of a two-component dowel mass, for example in boreholes drilled using a diamond drill, and increases the failure bond stress. Suitable adhesion promoters are selected from the group of silanes which are functionalized with further reactive organic groups and can be incorporated into the polymer network. This group includes, for example, 3-(meth)acryloyloxypropyttimethoxysilane, 3-(meth)acryloyloxypropyltriethoxysilane, 3-(meth)acryloyloxymethytrimethoxysilane, 3-(meth)acryloyloxymethyltiethoxysilane, vinyltrimethoxysilane, vinyltrimethoxysilane, functionalized tetraethoxysilane, functionalized tetramethoxysilane, functionalized tetrapropoxysilane, functionalized ethyl or propyl polysilicate, and mixtures of two or more thereof. In this regard, reference is made to the application DE 10 2009 059210 A1, the relevant content of which is hereby incorporated into this application. The adhesion promoter is expediently contained in amounts of from approximately 1 to approximately 10 wt. %, based on the total weight of the reactive resin component (A). The present invention also relates to a reactive resin system. The reactive resin system according to the invention is a two- or multi-component system, preferably a two-component system. One of the components is the reactive resin component (A) according to the invention, the other a hardener component (B). The latter contains an initiator by means of which the polymerization of the reactive resin is initiated when the components are mixed. In a preferred embodiment of the reactive resin system according to the invention, the reactive resin system is a two-component system and the reactive resin component (A) also contains, in addition to the reactive resin according to the invention, a hydraulically setting or polycondensable inorganic compound, in particular cement, and the hardener component (B) also contains, in addition to the initiator for the polymerization of the reactive resin, water. Hybrid mortar systems of this kind are described in detail in DE 4231161 A1. In this case, component (A) preferably contains, as a hydraulically setting or polycondensable inorganic compound, cement, for example Portland cement or alumina cement, cements which are free of transition metal oxide or have a low level of transition metal being particularly preferred. Gypsum can also be used as such or in a mixture with the cement, as a hydraulically setting inorganic compound. Component (A) may also comprise silicatic, polycondensable compounds, in particular soluble, dissolved and/or amorphous silica-containing substances, such as polar, non-after-treated fumed silica, as the polycondensable inorganic compound. Furthermore, it is preferred that component (A) also contains a thixotropic agent, preferably fumed silica which is after-treated in a non-polar manner, particularly preferably fumed silica after-treated with polydimethylsiloxane (PDMS), very particularly preferably the fumed silica used in the examples which is after-treated in a non-polar manner. The reactive resin component (A) according to the invention contains:the reactive resin according to the invention, as defined above, preferably a preferred embodiment thereof as described above;at least one hydraulically setting or polycondensable inorganic compound, preferably cement;at least one further filler, preferably quartz sand; andat least one thixotropic agent, preferably fumed silica after-treated in a non-polar manner. In a preferred embodiment, the reactive resin component (A) contains:the reactive amine accelerator according to the invention;at least one backbone resin as defined above, preferably urethane (meth)acrylate;at least one reactive diluent, preferably HPMA and/or BDDMA;at least one inhibitor as defined above, preferably an inhibitor of the piperidinyl-N-oxyl or tetrahydropyrrole-N-oxyl type, preferably TEMPOL;at least one hydraulically setting or polycondensable inorganic compound, preferably cement;at least one further filler, preferably quartz sand; andat least one thixotropic agent, preferably fumed silica after-treated in a non-polar manner. In an even more preferred embodiment, the reactive resin component (A) contains:the reactive amine accelerator according to the invention:at least one urethane (meth)acrylate, as defined above;HPMA and/or BDDMA;at least one inhibitor as defined above of the piperidinyl-N-oxyl or tetrahydropyrrole-N-oxyl type, preferably TEMPOL;at least one further inhibitor selected from the group consisting of catechols and phenothiazines;cement; andat least one thixotropic agent, preferably fumed silica after-treated in a non-polar manner. In an even more preferred embodiment, the reactive resin component (A) contains:the reactive amine accelerator according to the invention;at least one urethane (meth)acrylate, as defined above;HPMA and/or BDDMA;TEMPOL;at least one further inhibitor selected from the group consisting of catechols and phenothiazines;cement;fumed silica after-treated in a non-polar manner; andquartz sand. The hardener component (B) required for a reactive resin system according to the invention, in addition to the reactive resin component (A), typically contains:at least one initiator for initiating the polymerization of the reactive resin, preferably benzoyl peroxide (BPO) or tert-butyl peroxybenzoate; andwater. In a preferred embodiment, the hardener component (B) contains:at least one initiator for initiating the polymerization of the reactive resin, preferably benzoyl peroxide (BPO) or tert-butyl peroxybenzoate;at least one filler, preferably fumed silica; andwater. In a more preferred embodiment, the hardener component (B) contains:benzoyl peroxide (BPO) or tert-butyl peroxybenzoate for initiating the polymerization of the reactive resin;fumed silica; andwater. The reactive resin components (A) and the hardener components (B) in each of these embodiments can be combined with one another as desired. In a particularly preferred embodiment, the constituents of the reactive resin according to the invention or of the reactive resin component according to the invention are one or more of the constituents which are mentioned in the examples according to the invention. Reactive resins or reactive resin components which contain the same constituents or consist of the same constituents as are mentioned in the individual examples according to the invention, preferably approximately in the proportions stated in said examples, are very particularly preferred. The reactive resin according to the invention that contains the amine accelerator according to the invention, the reactive resin component (A) according to the invention that contains said reactive resin, and the reactive resin system according to the invention that comprises said reactive resin component as a component are characterized in that the amine accelerator according to the invention is almost completely incorporated into the polymer network during radical curing, due to the olefinic groups. This largely or completely prevents a diffusion of the amine accelerators onto the surface of the cured materials. The reactive resins according to the invention can be used in many fields in which unsaturated polyester resins, vinyl ester resins or vinyl ester urethane resins are otherwise conventionally used. They can be used in particular for preparing reactive resin mortars for structural applications, such as chemical fastening. The reactive resin according to the invention is usually used as a resin constituent in the reactive resin component of a multi-component system, typically a two-component system consisting of a reactive resin component (A) and a hardener component (B). This multi-component system may be in the form of a shell system, a cartridge system or a film pouch system. In the intended use of the system, the components are either ejected from the shells, cartridges or film pouches under the application of mechanical forces or by gas pressure, are mixed together, preferably by means of a static mixer through which the constituents are passed, and inserted into the borehole, after which the devices to be fastened, such as threaded anchor rods and the like, are introduced into the borehole which is provided with the hardening reactive resin, and are adjusted accordingly. A reactive resin system of this kind is used primarily in the construction sector, for example for the repair of concrete, as polymer concrete, as a coating material based on synthetic resin or as cold-curing road marking. It is particularly suitable for chemically fastening anchoring means, such as anchors, reinforcing bars, screws and the like, in boreholes, in particular in boreholes in various substrates, in particular mineral substrates, such as those based on concrete, aerated concrete, brickwork, sand-lime brick, sandstone, natural stone, glass and the like, and metal substrates such as those made of steel. In one embodiment, the substrate of the borehole is concrete, and the anchoring means is made of steel or iron. In another embodiment, the substrate of the borehole is steel, and the anchoring means is made of steel or iron. Another subject matter of the invention is the use of the reactive resin according to the invention as a constituent of a curable binder or as a curable binder, in particular for fastening anchoring means in boreholes of different subsurfaces and for structural bonding. In one embodiment, the substrate of the borehole is concrete, and the anchoring means is made of steel or iron. In another embodiment, the substrate of the borehole is steel, and the anchoring means is made of steel or iron. The steel borehole preferably has grooves. The invention is explained in greater detail in the following with reference to a number of examples. All examples and drawings support the scope of the invention. However, the invention is not limited to the specific embodiments shown in the examples and drawings. EXAMPLES Unless stated otherwise, all constituents of the compositions that are listed here are commercially available and were used in the usual commercial quality. Unless stated otherwise, all % and ppm data given in the examples relate to the total weight of the composition described, as a calculation basis. Preparation Example 1: Preparation of the Reactive Amine Accelerator From primary anilines and monomeric bisphenol A diglycidyl ether: 1 eq. bisphenol A diglycidyl ether (Epilox® A 19-03; epoxy equivalent weight 183 g/mol; LEUNA-Harze GmbH) was placed completely into the round bottom flask, mixed with 0.5 eq. of a primary aniline. 1.1 eq. methacrylic acid (BASF SE), 0.4 wt. % tetraethylammonium bromide (Merck KGaA), 230 ppm tempol (Evonik Industries AG) and 160 ppm phenothiazine (Allessa GmbH), and temperature-controlled to 100° C. It was stirred until, after approximately 4 h, complete conversion was indicated by thin layer chromatography (stationary phase: silica gel plate; eluent:petroleum ether:ethyl acetate 1:1), i.e. no free amine was detectable. It was diluted with 20 wt. % of hydroxypropyl methacrylate (HPMA, Evonik Industries AG), post-stabilized with 400 ppm tempol and cooled. In order to reduce the viscosity, when using para-toluidine as the primary aniline, the diglycidyl ether was partially reacted with 0.5 eq. methacrylic acid at 80° C. for one hour, before the addition of the aniline. Abbreviation forthe resultingPrimary aniline usedSourceamine acceleratormeta-toluidineAlfa AesarmTpara-toluidineSigma-Aldrich Chemie GmbHpTpara-bromanilineTCl Deutschland GmbHpBrApara-chloranneTCl Deutschland GmbHpClAmeta-chloro-TCl Deutschland GmbH3Cl4MeApara-methylanilinepara-tert-butylanilineTCl Deutschland GmbHptBuA From Secondary Anilines and Monomeric Bisphenol a Diglycidyl Ether: 1 eq. bisphenol A diglycidyl ether (Epilox® A 19-03; epoxy equivalent weight 183 g/mol; LEUNA-Harze GmbH) was placed completely into the round bottom flask, mixed with 1 eq. of a secondary aniline, 1.1 eq. methacrylic acid (BASF SE), 0.4 wt. % tetraethylammonium bromide (Merck KGaA), 230 ppm tempol (Evonik Industries AG) and 160 ppm phenothiazine (Allessa GmbH), and temperature-controlled to 100° C. It was stirred until, after approximately 4 h, complete conversion was indicated by thin layer chromatography (stationary phase: silica gel plate; eluent:petroleum ether:ethyl acetate 1:1), i.e. no free amine was detectable. It was diluted with 20 wt. % HPMA (Evonik Industries AG), post-stabilized with 400 ppm tempol, and cooled. From Secondary Anilines and Polymeric Bisphenol a Diglycidyl Ether: 0.5 eq. bisphenol A diglycidyl ether (Epilox® A 19-03; epoxy equivalent weight 183 g/mol; LEUNA-Harze GmbH) and 0.4 eq. bisphenol A diglycidyl ether (Epilox® A 50-02; epoxy equivalent weight 485 g/mol; LEUNA-Harze GmbH) were placed completely into the round bottom flask, mixed with 0.9 eq. of a secondary aniline, 1 eq. Methacrylic acid (BASF SE), 0.4 wt. % tetraethylammonium bromide (Merck KgaA), 230 ppm tempol (Evonik Industries AG) and 160 ppm phenothiazine (Allessa GmbH), and temperature-controlled to 100° C. It was stirred until, after approximately 4 h, complete conversion was indicated by thin layer chromatography. It was diluted with 20 wt. % HPMA (Visiomer® HPMA 98, Evonik Industries AG), post-stabilized with 400 ppm tempol, and cooled. Abbreviation forthe resultingamine accelerator(“poly” representsaccelerators that wereprepared usingpolymeric bisphenolSecondary aniline usedSourceA diglycidyl ether)N-methyl-p-toluidineTCl Deutschland GmbHNMepT or polyNMepTN-ethyl-p-toluidineTCl Deutschland GmbHNEtpT or polyNEtpTN-ethyl-m-toluidineTCl Deutschland GmbHNEtmT or polyNEtmTN-ethylanilineTCl Deutschland GmbHNEtAN-(2-hydroxyethyl)TCl Deutschland GmbHNHOEtAanilineN-dodecyl anilineTCl Deutschland GmbHNdodecAN-ethyl-para-TCl Deutschland GmbHNEtpClAchloroaniline Preparation Example 2: Preparing a Reactive Resin An amine accelerator prepared according to example 1, or DiPpT, as a comparison, was combined, in an amount corresponding to 7.8 mmol amine/100 g of resin mixture (clot=7.8 mmol/100 g resin), with 15.13 wt. % HPMA (Visiomer® HPMA 98, Evonik Industries AG), 32.75 wt. % 1,4-butanediol dimethacrylate (Visiomer® 1,4-BDDMA, Evonik Industries AG), 0.25 wt. % TBC (tert-butylcatechol, Rhodia), 0.015 wt. % tempol (Evonik Industries AG) and (the amount was adjusted such that the sum of all resin contents was 100%), to make up to 100% UMA/HPMA reactive resin masterbatch (prepared analogously to EP 0 713 015 A1, example A3, in which the amounts were adjusted such that 65 wt. % urethane methacrylate resin was obtained in 35 wt. % HPMA). Test Example 1: Gel Time and Maximum Reactivity Temperature The gel time was determined as follows: The gel time (denoted by th25° C.for the reactive resin) is measured, which time is expressed as the period of time from the time of the addition of an initiator to initialize the curing, to the time when the composition has reached a temperature of 50° C. The measurement was as follows: The gel time after the addition of the initiator (Perkadox® 20S (Akzo), weight ratio of reactive resin:initiator 100:30) to the reactive resin prepared according to preparation example 2 was determined using a conventional apparatus (Geltimer, WKS Informatik) at a starting temperature of 25° C. For this purpose, the mixture was filled into a test tube after the addition of the initiator, up to a height of 4 cm below the rim, the test tube being kept at a temperature of 25° C. (DIN 16945, DIN EN ISO 9396). A glass rod or spindle was moved up and down in the mixture at 10 strokes per minute. The gel time corresponds to the time period after the addition of the initiator, after which a temperature of 50° C. was measured in the mixture. The maximum reactivity temperature Tma corresponds to the maximum of the temperature curve in the gel time measurement. In order to determine this maximum, the gel time measurement was continued after reaching the temperature of 50° C. until the maximum of the temperature curve was exceeded. The results are shown in the following table: Gel time:Maximum reactivityAccelerator made of aminethg, 25° C.temperature: TmaxNMepT2.40min168° C.NEtpT3.70min162° C.NEtmT9min164° C.mT21min162° C.pT5.4min163° C.NEtA22min162° C.NHOEtA33min159° C.pBrA100min156° C.ptBuA11min157° C.NdodecA20min158° C.pClA72min155° C.3Cl4MeA36min155° C.NEtpCl42min158° C.(1:1) NMepT + pBrA7.25min165-167° C.(2:1) NMepT + pBrA4.15min165° C.(1:1) NEtpT + pBrA21min156° C.(1:1:1)5.7min159-163° C.NMepT + NEtmT + pBrA(50%, i.e. half the amount ofaccelerator) NMepT6.8min167-168° C.(1:1) NMepT + NEtA6.0min163-165° C.(1:1) NMepT + NEtmT5.3min164° C.(2:1) NMepT + NEtmT3.92min160° C.(3:2) NMepT + NEtmT4.16min158° C.NMepT + pBrA (3:2)6.22min158° C.polyNMepT2.30min157° C.polyNEtpT3.60min161° C.polyNEtmT9min162° C.polyNMepT + NEtA (1:1)5.08min159° C.polyNMepT + NEtmT (3:2)3.30min164° C.monomer DiPpT (reference)4.9min160° C.UMA-bound DiPpT29min154° C.(prepared according to WO12/164020) Conclusions from these Tests: Structural changes to the nitrogen or the aromatic ring of the aniline or toluidine can greatly change the accelerating effect of the aniline or toluidine. Regardless of the gel time, all reactive amine accelerators showed high peak temperatures (maximum reactivity temperatures), which indicates very good curing. Preparation Example 3: Reactive Resin Components (A) In 39.3 wt. % of a reactive resin prepared according to preparation example 2, 37.7 wt. % quartz sand F32 (Quarzwerke Frechen), 20.5 wt. % aluminate cement Secar® 80 (Kerneos) and 2.5 wt. % fumed silica after-treated in a non-polar manner Cab-O-Sil® 720 (Cabot Rheinfelden) were dispersed in a dissolver under a vacuum. The gel time of the reactive resin component, tmg,25° C., was measured using the same method as described in test example 1, the reactive resin component described here being tested instead of the reactive resin from preparation example 2. Accelerator (molar ratio)with ctot= 7.8 mmol/Gel time: tmg, 25° C.Description100 g resinminComparative example 1DiPpT4.50Example 1NMepT/NEtmT (3/2)4.10Example 2NMepT/pBrA (3/2)6.60Example 3NMepT/pBrA (2/1)4.20Example 4polyNMepT/NEtmT (3/2)3.52 Test Example 2: Measurement of Bond Stress A reactive resin system consisting of a reactive resin component (A) prepared according to preparation example 3, and the commercial hardener component HY-110 B (Hilti) used as a hardener component (B) was filled into a plastic cartridge (Ritter GmbH; volume ratio A:B=3:1) having the inside diameter 47 mm (component (A)) or 28 mm (component (B)), and tested as follows: In order to determine the shear strength (synonym: bond stress) achieved by means of the reactive resin system according to comparative example 1 and according to examples 1 to 4, the mixed reactive resin system (i.e. the mixture of reactive resin component (A) and hardener component (B) in a volume ratio of A:B=3:1) is introduced into a steel sleeve having a defined geometry and a defined fill level of the mortar (embedding depth). An anchor rod was then placed in the center of the steel sleeve filled with the mixture, using a centering aid. After curing at 25° C. and for at least 12 hours, the sample was screwed into a tensile testing machine using a thread adapter (Zwick Roell Z050, 50 kN). The sample was loaded with tensile force at a defined speed until failure. The corresponding load-displacement dependency was continuously recorded. Five individual measurements were carried out in each case, and the mean value of the maximum force upon failure was calculated. Anchor rods having an M8 thread and steel sleeves having the following geometry were used to carry out the measurements:Undercut depth: 0.35+/−0.02 mmUndercut width: 2 mmEmbedding depth: 36 mmInner diameter: 12 mm The shear strength determined from these measurements is defined as the quotient of the maximum force upon failure and the shear surface of the anchor rod used (anchor rod M8: 904.3 mm2). The results of the measurements are shown in the following table: ComparativeExampleExampleExampleExampleexample 11234Bond stress16.5 ± 1.314.2 ± 2.215.2 ± 2.012.5 ± 1.014.3 ± 1.3[N/mm2] Conclusion: Cured reactive resin systems having different reactive amine accelerators (examples 1 to 4) showed a comparable bond stress to reactive resin systems which contained DiPpT as an accelerator (comparative example 1). Test Example 3: Sedimentation A comparison of the sedimentation properties was carried out using an accelerated test. The Lumifuge® instrument from LUM GmbH was used for this purpose. The following parameters were used for the method: Light factor0.5Rotation speed2055 rpmPolyamide cuvette10 mmTemperature35° C.8 channels (parallel measurements)Time255 measurements every 20 sModel1120-28Cuvette fill amount1.3 mL The following reactive resin components (A) were tested: Accelerator (molar ratio)with ctot= 7.8 mmol/Example100 g resinComparative example 1DiPpTExample 5NEtpT/NEtmT/pBrA (1/1/1)Example 6pBrA The following measurement results (mean values from 8 parallel measurements) were achieved: Comparative example 1Example 5Example 6Sedimentation0.477 ± 0.040.328 ± 0.030.338 ± 0.02speed [μm/s] Conclusion: Example 5 and example 6 showed a sedimentation rate which is slower by a factor of 1.4, and therefore have an improved shelf life in comparison with comparative example 1. | 81,248 |
11859070 | DETAILED DESCRIPTION OF THE EMBODIMENTS In the description of the invention, it should be noted that, unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be adopted. Reagents or instruments employed are of conventional type and can be procured from the market. The present invention is further described in detail below in combination with embodiments, to help those skilled in the art have a more complete, accurate, and in-depth understanding of the inventive concept and the technical solution of the invention. The scope of protection of the invention includes, but is not limited to, the following embodiments. Any modifications made to the details and form of the technical solution of the invention, without departing from the spirit and scope of the application, fall within the scope of protection of the invention. In the embodiments, a method for preparing the tea pectin cellulose comprises the steps of drying and sterilizing on at least one of the tea stems, tea leaves, and the tea residues, crushing and grinding into powder, cooking at 70° C., squeezing; water extracting to obtain the tea pectin cellulose, filtering, and drying. Embodiment 1 The embodiment provides a method for preparing a tea-based composite board, wherein the method comprises the steps of: Step 1: Weighing the raw materials in the following proportions: 60 parts of waste tea residues, 35 parts of tea stems, 20 parts of straw powder, 20 parts of tea pectin cellulose, 40 parts of coffee residues, 3 parts of a compatibilizer, 1.5 parts of a lubricant, 2 parts of a colorant, 1 part of an antioxidant, and 1 part of stabilizer, wherein the water content of the coffee residues is not higher than 10%; Step 2: Soaking the tea stems in water at 70° C., performing through cleaning, performing drying in the air, and grinding to obtain tea powder with a mesh size of 10-20 meshes; Step 3: Mixing the waste tea residues, the tea powder, the straw powder, the tea pectin cellulose, the coffee residues, the compatibilizer, and the lubricant at 140° C. for 20 min; Step 4: Lowering the temperature to 55° C., further adding the colorant, the antioxidant and the stabilizer, performing uniform mixing, and performing hydro-molding at 80° C. to obtain the tea-based composite board. The tea-based composite board of Embodiment 1 is tested, with the testing result of a compressive strength being 418 MPa, a bacteriostatic time being over 10 hours, a folding resistance being up to 1985 times, and strong moisture absorption ability. Embodiment 2 The embodiment provides a method for preparing a tea-based composite board, wherein the method comprises the steps of: Step 1: Weighing the raw materials in the following proportions: 40 parts of waste tea residues, 35 parts of tea stems, 20 parts of straw powder, 20 parts of tea pectin cellulose, 30 parts of coffee residues, 3 parts of a compatibilizer, 1 part of a lubricant, 3 parts of a colorant, 3 parts of an antioxidant, 1 part of stabilizer, and 20 parts of reinforced resin glass fiber powder, wherein the water content of the coffee residues is not higher than 10%; Step 2: Soaking the tea stems in water at 80° C., performing through cleaning, performing drying in the air, and performing grinding to obtain tea powder with a mesh size of 10-20 meshes; step 3: mixing the waste tea residues, the tea powder, the straw powder, the tea pectin cellulose, the coffee residues, the reinforced resin glass fiber powder, the compatibilizer, and the lubricant at 140 DEG C for 25 minutes; Step 4: Lowering the temperature to 50° C., further adding the colorant, the antioxidant and the stabilizer, performing uniform mixing, and performing hydro-molding at 80° C. to obtain the tea-based composite board. The tea-based composite board of Embodiment 1 is tested, with the testing result of a compressive strength being 429 MPa, a bacteriostatic time being 8.5 hours, a folding resistance being up to 1922 times, and strong moisture absorption ability. Embodiment 3 The embodiment provides a method for preparing a tea-based composite board, wherein the method comprises the steps of: Step 1: Weighing the raw materials in the following proportions: 30 parts of waste tea residues, 50 parts of tea stems, 15 parts of straw powder, 30 parts of tea pectin cellulose, 30 parts of coffee residues, 4 parts of a compatibilizer, 1 part of a lubricant, 1 part of a colorant, 2 parts of an antioxidant, 2 parts of stabilizer, and 35 parts of industrial wood powder, wherein the water content of the coffee residues is not higher than 10%; Step 2: Soaking the tea stems in water at 70° C., performing through cleaning, performing drying in the air, and grinding to obtain tea powder with a mesh size of 10-20 meshes; Step 3: Mixing the waste tea residues, the tea powder, the straw powder, the tea pectin cellulose, the coffee residues, the industrial wood powder, the compatibilizer, and the lubricant at 120° C. for 30 minutes; Step 4: Lowering the temperature to 55° C., further adding the colorant, the antioxidant and the stabilizer, performing uniform mixing, and performing hydro-molding at 80° C. to obtain the tea-based composite board. The tea-based composite board of Embodiment 1 has been tested and found to possess a compressive strength being 408 MPa, a bacteriostatic time of greater than 10 hours, a folding resistance up to 1943 times, and strong moisture absorption capabilities. Embodiment 4 The embodiment provides a method for preparing a tea-based composite board, wherein the method comprises the steps of: Step 1: Weighing the raw materials in the following proportions: 30 parts of waste tea residues, 40 parts of tea stems, 30 parts of straw powder, 10 parts of tea pectin cellulose, 40 parts of coffee residues, 5 parts of a compatibilizer, 0.5 part of a lubricant, 3 parts of a colorant, 3 parts of an antioxidant, 0.5 part of stabilizer, 20 parts of industrial wood powder, and 20 parts of reinforced resin glass fiber powder, wherein the water content of the coffee residues is not higher than 10%; Step 2: Soaking the tea stems in water at 70° C., performing through cleaning, performing drying in the air, and grinding to obtain tea powder with a mesh size of 10-20 meshes; Step 3: Mixing the waste tea residues, the tea powder, the straw powder, the tea pectin cellulose, the coffee residues, the reinforced resin glass fiber powder, the industrial wood powder, the compatibilizer, and the lubricant at 140° C. for 20 minutes; Step 4: Lowering the temperature to 55° C., further adding the colorant, the antioxidant and the stabilizer, performing uniform mixing, and performing hydro-molding at 80° C. to obtain the tea-based composite board. The tea-based composite board of Embodiment 1 has been tested and found to possess a compressive strength being 421 MPa, a bacteriostatic time of greater than 10 hours, a folding resistance up to 1962 times, and strong moisture absorption capabilities. The tea-based composite board thus prepared can be utilized as a substitute for the high-grade woods currently available in the market for any furniture products. The above embodiments are merely illustrative of several implementations of the invention, and the description thereof is more specific and detailed. However, these embodiments may not be construed as a limitation to the patentable scope of the invention. It should be pointed out that several variations and improvements may be made by those of ordinary skill in the art without departing from the conception of the invention, but such variations and improvements should fall within the protection scope of the invention. Therefore, the scope of protection of the invention patent should be subjected to the appended claims. | 7,845 |
11859071 | EXAMPLES Example 1 Example 1 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. The modified graphene is a mixture of piperazine modified graphene and octadecylamine modified graphene. The piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:2. The modified graphene has a content of 0.5 wt % of the ultra-high molecular weight polyethylene. The modified silicon carbide whisker is silicon carbide whisker modified with a silane coupling agent. The modified silicon carbide whisker has a content of 5 wt % of the ultra-high molecular weight polyethylene. The ultra-high molecular weight polyethylene has a weight-average molecular weight of 2 million, and is purchased from Yuyao Jiuding Chemical Material Co., Ltd. The preparation method for the silicon carbide whisker modified with the silane coupling agent comprises the following steps: firstly ultrasonically dispersing the silicon carbide whisker in deionized water for 30 minutes; adding the silane coupling agent; stirring for 5 hours at a controlled temperature of 70° C.; and finally drying, to obtain the silicon carbide whisker modified with the silane coupling agent. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:0.1; the silane coupling agent is a mixture of dodecylsilane coupling agent and 3-aminopropyltrimethoxysilane, and the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 2:3; the dodecylsilane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-312; the 3-aminopropyltrimethoxysilane is purchased from Qingdao Hengda Zhongcheng Technology Co., Ltd., CAS No. KH-540. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker is of β crystal form and has a diameter of 200-500 nm and a length of 10-50 μm, which is purchased from Qinhuangdao Eno High-tech Material Development Co., Ltd., CAS No. SiCW-80. The preparation process for the cut resistant and creep resistant fiber is specifically as follows:(1) the silicon carbide whisker modified with the silane coupling agent prepared above is dispersed in ultra-high molecular weight polyethylene powder by means of high-shear blending, and the prepared powder premix is ultrasonically dispersed uniformly in mineral oil;(2) the mineral oil mixture is slowly heated in a reaction kettle with stirring and shearing, until a uniform solution is formed, wherein the speed of the high-shear stirring is 1000 r/min and the stirring time is 4 hours, and a spinning solution with a mass concentration of 3% is obtained after mixing uniformly;(3) the spinning solution is spun by gel spinning and is subjected to extraction and thermal drawing, to obtain a composite fiber, wherein the spinning temperature is 230° C. Example 2 Example 2 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. The modified graphene is a mixture of piperazine modified graphene and octadecylamine modified graphene. The piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:3. The modified graphene has a content of 5 wt % of the ultra-high molecular weight polyethylene. The modified silicon carbide whisker is silicon carbide whisker modified with a silane coupling agent. The modified silicon carbide whisker has a content of 0.5 wt % of the ultra-high molecular weight polyethylene. The ultra-high molecular weight polyethylene has a weight-average molecular weight of 2.1 million, and is purchased from Shanghai Chuhao Import and Export Co., Ltd. The preparation method for the silicon carbide whisker modified with the silane coupling agent comprises the following steps: firstly ultrasonically dispersing the silicon carbide whisker in deionized water for 30 minutes; adding the silane coupling agent; stirring for 5 hours at a controlled temperature of 70° C.; and finally drying, to obtain the silicon carbide whisker modified with the silane coupling agent. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:0.3; the silane coupling agent is a mixture of dodecylsilane coupling agent and 3-aminopropyltrimethoxysilane, and the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 3:2; the dodecylsilane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-313; the 3-aminopropyltrimethoxysilane is purchased from Qingdao Hengda Zhongcheng Technology Co., Ltd., CAS No. KH-540. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker is of β crystal form and has a diameter of 200-500 nm and a length of 10-50 μm, which is purchased from Qinhuangdao Eno High-tech Material Development Co., Ltd., CAS No. SiCW-80. The preparation process for the cut resistant and creep resistant fiber is specifically as follows:(1) the silicon carbide whisker modified with the silane coupling agent prepared above is dispersed in ultra-high molecular weight polyethylene powder by means of high-shear blending, and the prepared powder premix is ultrasonically dispersed uniformly in mineral oil;(2) the mineral oil mixture is slowly heated in a reaction kettle with stirring and shearing, until a uniform solution is formed, wherein the speed of the high-shear stirring is 3000 r/min and the stirring time is 2 hours, and a spinning solution with a mass concentration of 5% is obtained after mixing uniformly;(3) the spinning solution is spun by gel spinning and is subjected to extraction and thermal drawing, to obtain a composite fiber, wherein the spinning temperature is 280° C. Example 3 Example 3 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. The modified graphene is a mixture of piperazine modified graphene and octadecylamine modified graphene. The piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:2.2. The modified graphene has a content of 1 wt % of the ultra-high molecular weight polyethylene. The modified silicon carbide whisker is silicon carbide whisker modified with a silane coupling agent. The modified silicon carbide whisker has a content of 4 wt % of the ultra-high molecular weight polyethylene. The ultra-high molecular weight polyethylene has a weight-average molecular weight of 2.4 million, and is purchased from Shanghai Chuhao Import and Export Co., Ltd. The preparation method for the silicon carbide whisker modified with the silane coupling agent comprises the following steps: firstly ultrasonically dispersing the silicon carbide whisker in deionized water for 30 minutes; adding the silane coupling agent; stirring for 5 hours at a controlled temperature of 70° C.; and finally drying, to obtain the silicon carbide whisker modified with the silane coupling agent. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:0.2; the silane coupling agent is a mixture of dodecylsilane coupling agent and 3-aminopropyltrimethoxysilane, and the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 1:1; the dodecylsilane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-312; the 3-aminopropyltrimethoxysilane is purchased from Qingdao Hengda Zhongcheng Technology Co., Ltd., CAS No. KH-540. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker is of β crystal form and has a diameter of 100-600 nm and a length of 10-50 μm, which is purchased from Qinhuangdao Eno High-tech Material Development Co., Ltd., CAS No. SiCW-90. The preparation process for the cut resistant and creep resistant fiber is specifically as follows:(1) the silicon carbide whisker modified with the silane coupling agent prepared above is dispersed in ultra-high molecular weight polyethylene powder by means of high-shear blending, and the prepared powder premix is ultrasonically dispersed uniformly in mineral oil;(2) the mineral oil mixture is slowly heated in a reaction kettle with stirring and shearing, until a uniform solution is formed, wherein the speed of the high-shear stirring is 2000 r/min and the stirring time is 3 hours, and a spinning solution with a mass concentration of 4% is obtained after mixing uniformly;(3) the spinning solution is spun by gel spinning and is subjected to extraction and thermal drawing, to obtain a composite fiber, wherein the spinning temperature is 250° C. Example 4 Example 4 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. The modified graphene is a mixture of piperazine modified graphene and octadecylamine modified graphene. The piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:2.8. The modified graphene has a content of 3 wt % of the ultra-high molecular weight polyethylene. The modified silicon carbide whisker is silicon carbide whisker modified with a silane coupling agent. The modified silicon carbide whisker has a content of 2 wt % of the ultra-high molecular weight polyethylene. The ultra-high molecular weight polyethylene has a weight-average molecular weight of 2.1 million, and is purchased from Shanghai Chuhao Import and Export Co., Ltd. The preparation method for the silicon carbide whisker modified with the silane coupling agent comprises the following steps: firstly ultrasonically dispersing the silicon carbide whisker in deionized water for 30 minutes; adding the silane coupling agent; stirring for 5 hours at a controlled temperature of 70° C.; and finally drying, to obtain the silicon carbide whisker modified with the silane coupling agent. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:0.4; the silane coupling agent is a mixture of dodecylsilane coupling agent and 3-aminopropyltrimethoxysilane, and the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 1:1.2; the dodecylsilane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-312; the 3-aminopropyltrimethoxysilane is purchased from Qingdao Hengda Zhongcheng Technology Co., Ltd., CAS No. KH-540. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker is of β crystal form and has a diameter of 100-600 nm and a length of 10-50 μm, which is purchased from Qinhuangdao Eno High-tech Material Development Co., Ltd., CAS No. SiCW-90. The preparation process for the cut resistant and creep resistant fiber is similar to that of Example 3. Example 5 Example 5 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. The modified graphene is a mixture of piperazine modified graphene and octadecylamine modified graphene. The piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:2.5. The modified graphene has a content of 2.2 wt % of the ultra-high molecular weight polyethylene. The modified silicon carbide whisker is silicon carbide whisker modified with a silane coupling agent. The modified silicon carbide whisker has a content of 3 wt % of the ultra-high molecular weight polyethylene. The ultra-high molecular weight polyethylene has a weight-average molecular weight of 2.1 million, and is purchased from Shanghai Chuhao Import and Export Co., Ltd. The preparation method for the silicon carbide whisker modified with the silane coupling agent comprises the following steps: firstly ultrasonically dispersing the silicon carbide whisker in deionized water for 30 minutes; adding the silane coupling agent; stirring for 5 hours at a controlled temperature of 70° C.; and finally drying, to obtain the silicon carbide whisker modified with the silane coupling agent. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:0.3; the silane coupling agent is a mixture of dodecylsilane coupling agent and 3-aminopropyltrimethoxysilane, and the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 1:1.6; the dodecylsilane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-312; the 3-aminopropyltrimethoxysilane is purchased from Qingdao Hengda Zhongcheng Technology Co., Ltd., CAS No. KH-540. In the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker is of β crystal form and has a diameter of 200-500 nm and a length of 10-50 μm, which is purchased from Qinhuangdao Eno High-tech Material Development Co., Ltd., CAS No. SiCW-80. The preparation process for the cut resistant and creep resistant fiber is similar to that of Example 3. Comparative Example 1 Comparative example 1 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 1 is different from Example 5 in that, the modified graphene has a content of 0.1 wt % of the ultra-high molecular weight polyethylene. Comparative Example 2 Comparative example 2 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 2 is different from Example 5 in that, the modified graphene has a content of 20 wt % of the ultra-high molecular weight polyethylene. Comparative Example 3 Comparative example 3 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 3 is different from Example 5 in that, the piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:0.2. Comparative Example 4 Comparative example 4 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 4 is different from Example 5 in that, the piperazine modified graphene and the octadecylamine modified graphene have a weight ratio of 1:15. Comparative Example 5 Comparative example 5 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 5 is different from Example 5 in that, the modified silicon carbide whisker has a content of 0.1 wt % of the ultra-high molecular weight polyethylene. Comparative Example 6 Comparative example 6 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 6 is different from Example 5 in that, the modified silicon carbide whisker has a content of 20 wt % of the ultra-high molecular weight polyethylene. Comparative Example 7 Comparative example 7 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 7 is different from Example 5 in that, the ultra-high molecular weight polyethylene has a weight-average molecular weight of 0.9 million, and is purchased from Yuyao Jiuding Chemical Material Co., Ltd. Comparative Example 8 Comparative example 8 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 8 is different from Example 5 in that, the ultra-high molecular weight polyethylene has a weight-average molecular weight of 5 million, and is purchased from Yuyao Jiuding Chemical Material Co., Ltd. Comparative Example 9 Comparative example 9 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 9 is different from Example 5 in that, in the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:0.02. Comparative Example 10 Comparative example 10 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 10 is different from Example 5 in that, in the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker and the silane coupling agent have a weight ratio of 1:2. Comparative Example 11 Comparative example 11 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 11 is different from Example 5 in that, the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 1:5. Comparative Example 12 Comparative example 12 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 12 is different from Example 5 in that, the dodecylsilane coupling agent and the 3-aminopropyltrimethoxysilane have a weight ratio of 5:1. Comparative Example 13 Comparative example 13 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 13 is different from Example 5 in that, in the silicon carbide whisker modified with the silane coupling agent, the silicon carbide whisker is of β crystal form and has a diameter of 100-600 nm and a length of 100 μm, which is purchased from Qinhuangdao Eno High-tech Material Development Co., Ltd., CAS No. SiCW-98. Comparative Example 14 Comparative example 14 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 14 is different from Example 5 in that, the silane coupling agent is a mixture of octadecyl silane coupling agent and 3-aminopropyltrimethoxysilane; the octadecyl silane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-318. Comparative Example 15 Comparative example 15 provides a cut resistant and creep resistant fiber, which is prepared from an ultra-high molecular weight polyethylene composition comprising the following components: modified graphene, modified silicon carbide whisker, and ultra-high molecular weight polyethylene. Comparative example 15 is different from Example 5 in that, the silane coupling agent is a mixture of methyl silane coupling agent and 3-aminopropyltrimethoxysilane; the methyl silane coupling agent is purchased from Nanjing Youpu Chemical Co., Ltd., CAS No. UP-302. Performance Evaluation 1. Cut Resistance Test on the Product: The tests can be carried out according to National Standard GB24541-2009 or European Standard EN388. According to European Standard EN388, the cut resistance of yarns is tested by using a cut resistance tester, which is a testing machine produced by Sodemat manufacturer according to European Standard EN388. A test sample is placed on a table of the cut resistance tester, with aluminum foil underneath, and is moved horizontally. A spare circular blade is placed on the sample and rotated while advancing, and the advancing direction and the moving direction of the test sample are reversed. Once the test sample is completely cut off, the circular blade contacts with the aluminum foil, and thus a circuit is energized. At this time, the circuit informs a counter that the cutting is finished. The counter is always recording throughout the process, so the cut resistance data of the test sample can be obtained. After the testing, the cut resistance level is evaluated by comparing with the cutting level of a standard sample (a 200 g/m2plain cotton fabric) under the same conditions. The test starts from the standard sample, and the test sample and the standard sample are alternatively tested. After the test sample has been tested for 3 times, the standard sample is finally tested the fourth time. This round of testing is ended. The evaluation value is calculated according to the following equation and is referred to as a cut resistance index:N=(Counter Reading of Standard Sample Before Test Sample Testing+Counter Reading of Standard Sample After Test Sample Testing)/2;Index=(Counter Reading of Test Sample+N)/NIndex classification IndexCut Resistance Level2.0-2.512.5-5.025.0-10310.0-20.04>20.05 In addition, the circular blade used in the tests is a Rotary Cutter L-shaped produced by OLFA, with a diameter of 45 mm, a material of SKS-7 tungsten steel, and a blade thickness of 0.3 mm. 2. Tear Strength Test: The tests are carried out according to JB/T1040-92. 3. Creep Resistance Test: Test conditions for creep elongation: the temperature is 20±2° C. and the relative humidity is 63±3%, the applied stress is 50% of the fiber breaking strength, and the creep time is 1d. TABLE 1CutTear StrengthCreep Resistance (%)Example 1527.521.416Example 2528.351.432Example 3529.871.284Example 4529.581.029Example 5531.670.916Comparative example 1219.822.646Comparative example 2430.152.429Comparative example 3225.351.525Comparative example 4323.555.518Comparative example 5219.436.543Comparative example 6428.221.416Comparative example 7225.911.517Comparative example 8324.615.587Comparative example 9221.384.942Comparative example 10326.486.514Comparative example 11224.611.548Comparative example 12226.189.624Comparative example 13322.312.056Comparative example 14319.676.314Comparative example 15222.468.464 | 24,748 |
11859072 | DETAILED DESCRIPTION OF THE INVENTION A discovery has been made that provides a solution to at least some of the aforementioned problems associated with polypropylene compositions used for thermoforming. In one aspect, the discovery can include a polymeric composition containing at least 95 wt. % of a polypropylene copolymer and 50 ppm to 2000 ppm of an aryl amide containing clarifying agent or a phosphate ester salt containing clarifying agent or a combination thereof. As illustrated in the non-limiting examples polypropylene containing polymeric compositions of the present invention have low haze values and show relatively less increase in haze even after multiple extrusion passes. This can increase the recyclability of the compositions of the present invention, which can help to reduce waste typically associated with thermoforming processes. These and other non-limiting aspects of the present invention are discussed in further detail in the following sections. A. Polymeric Composition The polymeric composition of the present invention can contain, i) at least 95 wt. %, such as 95 wt. % to 99.9 wt. % or at least any one of, equal to any one of, or between any two of 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, and 99.9 wt. % of the polypropylene copolymer and ii) 50 ppm to 2000 ppm or at least any one of, equal to any one of, or between any two of 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, and 2000 ppm of the aryl amide containing clarifying agent or phosphate ester salt containing clarifying agent or a combination thereof. In some aspects, the polymeric composition of the present invention can contain, at least 95 wt. %, such as 95 wt. % to 99.9 wt. %, or 96 wt. % to 99.9 wt. %, or 97 wt. % to 99.9 wt. %, or 98 wt. % to 99.9 wt. %, or 99 wt. % to 99.9 wt. %, of the polypropylene copolymer and 50 ppm to 400 ppm or 100 ppm to 300 ppm of the aryl amide containing clarifying agent. In some aspects, the polymeric composition of the present invention can contain, at least 95 wt. %, such as 95 wt. % to 99.9 wt. %, or 96 wt. % to 99.9 wt. %, or 97 wt. % to 99.9 wt. %, or 98 wt. % to 99.9 wt. %, or 99 wt. % to 99.9 wt. %, of the polypropylene copolymer and 500 ppm to 1500 ppm or 800 ppm to 1200 ppm of the phosphate ester salt containing clarifying agent. The polymeric composition can have a haze value of A after being extruded once and a haze value of B after being extruded 5 times, wherein the ratio of A to B can be 1 to 1.35 or at least any one of, equal to any one of, or between any two of 1, 1.03, 1.05, 1.07, 1.1, 1.15, 1.2, 1.25, 1.3, 1.32, 1.33 and 1.35 and A can be less than 25%, less than 20%, less than 15%, less than 14% such as 13%, or 15% or 13% to 20%. In some aspects, the polymeric composition can have a haze value of C after being extruded thrice, and the ratio of A to C can be 1 to 1.15 or 1 to 1.01. In some aspects, the ratio of C to B can be 1 to 1.15 or 1 to 1.01. The haze values can be determined in accordance with ASTM D1003 (by HazeGard) at a thickness of about 40 mil with molded plaques containing the polymeric composition. A, B and/or C can be determined with the extrusion pass parameters set at, and/or performed at conditions similar to (e.g. within ±5%) the respective conditions provided in Table 4 and 5. 1. Polypropylene Copolymer The polypropylene copolymer can be propylene-ethylene random copolymer. In some particular aspects, the polypropylene copolymer can be an isotactic propylene-ethylene random copolymer. In some aspects, the propylene-ethylene random copolymer, such as the isotactic propylene-ethylene random copolymer can include 0.1 wt. % to 5 wt. %, or 0.1 wt. % to 3 wt. % or 0.1 wt. % to 2 wt. % or at least any one of, equal to any one of, or between any two of 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 and 5 wt. % of ethylene units and 95 wt. % to 99.9 wt. %, or 97 wt. % to 99.9 wt. %, or 98 wt. % to 99.9 wt. % or at least any one of, equal to any one of, or between any two of 95, 96, 97, 98, 99, 99.2, 99.4, 99.6, 99.8 and 99.9 wt. % of propylene units, based on the total weight of the copolymer. In some aspects, the polypropylene copolymer such as the propylene-ethylene random copolymer can have a xylene soluble content of less than 8 wt. % such as 1 wt. % to 4 wt. % or at least any one of, equal to any one of, or between any two of 1, 2, 3 and 4 wt. %. In some aspects, the polypropylene copolymer such as the propylene-ethylene random copolymer can have a polydispersity (Mw/Mn) of 3 to 15 or at least any one of, equal to any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15, wherein the polydispersity is measured using gel permeation chromatography (GPC). In some aspects, the polypropylene copolymer such as the propylene-ethylene random copolymer can have a melt flow rate (MFR) of 0.1 g/10 min to 150 g/10 min, or 1 to 60 g/10 min, or 1 to about 30 g/10 min, or 1 to about 10 g/10 min, or 1 to about 7 g/10 min, or at least any one of, equal to any one of, or between any two of 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 and 160 g/10 min at 230° C., 2.16 kg measured in accordance with ASTM D-1238. In some aspects, the polypropylene copolymer such as the propylene-ethylene random copolymer can have a density of 0.90 g/cc to 0.93 g/cc or 0.90 g/cc to 0.92 g/cc or 0.90 g/cc to 0.91 or at least any one of, equal to any one of, or between any two of 0.9, 0.902, 0.904, 0.906, 0.908, 0.91, 0.915, 0.92, 0.925 and 0.93 g/cc as measured in accordance with ASTM D792. In some aspects, the polypropylene copolymer such as the propylene-ethylene random copolymer can have a combination of, or all of the properties mentioned herein. The polypropylene copolymer can be prepared via conventional polymerization processes such as those known in the art. Examples of such polymerization processes include slurry, liquid-bulk and gas-phase polymerizations. In slurry polymerization processes, polymerization occurs in the presence of a solvent, e.g. hexane, within a loop or continuous stirred tank reactor. Polymerization may also be carried out by bulk-phase polymerization, where liquid propylene and ethylene serve as both monomer and diluent. In a typical bulk process, one or more loop reactors are generally employed. In other aspects, the copolymer may be produced by gas phase polymerization of propylene and ethylene, which is typically carried out in a fluidized bed reactor. Polymer fluff or powder produced from the polymerization reaction can be removed from the reactor and can then be processed via conventional techniques, such as by extrusion, to produce the desired copolymer pellets. The amount of ethylene monomer used during polymerization of the copolymer is desirably in proportion to the desired final ethylene content of the target propylene copolymer. In some embodiments the ethylene content during polymerization can range from 0.1 to 5 wt. %, or 0.1 to about 3 wt. % or 0.1 to about 2 wt. %, based on the total weight of the monomers, e.g. ethylene and propylene, present during polymerization. In some aspects, the polypropylene copolymer, such as propylene-ethylene random copolymer can be prepared using metallocene catalysts or Ziegler-Natta catalyst. Ziegler-Natta catalysts, which are well known in the art, useful in the preparation of isotactic polypropylene can be derived from a halide of a transition metal, such as titanium, chromium or vanadium with a metal hydride and/or metal alkyl, typically an organoaluminum compound, as a co-catalyst. In some aspects, the catalyst is can contain a titanium halide supported on a magnesium compound. Ziegler-Natta catalysts, such as titanium tetrachloride (TiCl4) supported on an active magnesium dihalide, such as magnesium dichloride or magnesium dibromide, as disclosed, for example, in U.S. Pat. Nos. 4,298,718 and 4,544,717, both to Mayr et al., and which are herein incorporated by reference, are supported catalysts. Silica may also be used as a support. The supported catalyst may be employed in conjunction with a co-catalyst or electron donor such as an alkylaluminum compound, for example, triethylaluminum (TEAL), trimethyl aluminum (TMA) and triisobutyl aluminum (TIBAL). 2. Clarifying Agent The polymeric composition of the present invention includes a phosphate ester salt containing clarifying agent and/or an aryl amide containing clarifying agent. Non-limiting examples of phosphate ester salt containing clarifying agent include 2,2-methylene-bis(4,6-ditertbutylphenyl)phosphate, and/or aluminum hydroxybis(2,4,8,10-tetrakis(1,1-dimethyl) 6-hydroxy-12H-dibenzo[d,g][1,2,3][dioxaphophocin 6-oxidato]. In some particular aspect the clarifying agent can be 2,2-methylene-bis(4,6-ditertbutylphenyl)phosphate. Examples of commercially available phosphate ester salts containing clarifying agents include, without limitation, ADK STABILIZER NA-71 and ADK STABILIZER NA-21, both available from Amfine Chemical Corp., Allendale, N.J. Non-limiting examples of the aryl amide containing clarifying agent can be a 1,3,5-benzenetrisamide amide derivative. In some aspects, the aryl amide containing clarifying agent can be 1,3,5-tris(2,2-dimethyl propanamido)benzene. Examples of commercially available aryl amide containing clarifying agents include, without limitation, IRGACLEAR XT 386 available from BASF. The polymeric compositions of the present invention can be free of, or essentially free of, such as contain less than 100 ppm, or less than 50 ppm or less than 10 ppm of clarifying agents containing sorbitol or sorbitol derivative, nonitol or nonitol derivative, and/or xylitol or xylitol derivative. 3. Additives In some aspects, the polymeric composition, can further contain one or more additives selected from antioxidants, stabilizers, neutralizers, processing aids, peroxides, slip agents and/or antistatics. In some aspects, the polymeric composition can contain iii) 50 ppm to 500 ppm or at least any one of, equal to any one of, or between any two of 50, 100, 200, 300, 400 and 500 ppm of an antioxidant, iv) 200 ppm to 2000 ppm or at least any one of, equal to any one of, or between any two of 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 ppm of a stabilizer v) 200 ppm to 2000 ppm or at least any one of, equal to any one of, or between any two of 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 and 2000 ppm of a antistatic, or iv) 100 ppm to 1000 ppm or at least any one of, equal to any one of, or between any two of 100, 200, 400, 600, 800, and 1000 ppm of a neutralizer or any combination thereof. The antioxidant can be a sterically hindered phenol and/or a phosphite containing antioxidant. A combination of antioxidants can be used. In some aspects, the sterically hindered phenol antioxidant can be pentaerythritol tetrakis[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionate, octadecyl-3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionate], pentaerythritol tetrakis[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionate, or 1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione, or any combinations thereof. In some aspects, the phosphite containing antioxidant can be tris(2,4-di-tert.-butylphenyl)phosphite, bis (2,4-dicumylphenyl) pentaerythritol diphosphate, or bis (2,4-di-t-butylphenyl) pentraerythritol diphosphate or any combination thereof. In some particular aspects, the antioxidant can be pentaerythritol tetrakis[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionate. The stabilizer can be a phosphite containing stabilizer and/or oligomeric hindered amine containing stabilizer. In some aspects, the phosphite containing stabilizer can be tris(2,4-di-tert.-butylphenyl)phosphite. In some aspects, the oligomeric hindered amine containing stabilizer can be butanedioic acid, dimethylester, polymer with 4-hydroxy-2,2,6,6-tetramethyl-1-piperidine ethanol. In some particular aspects, the stabilizer can be tris(2,4-di-tert.-butylphenyl)phosphite. In some aspects, the antistatic can be glycerol monostearate. The glycerol monostearate can have a monoester content of 45 to 90 wt. % or at least any one of, equal to any one of, or between any two of 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 wt. %. The neutralizer can be a stearate containing neutralizer, hydrotalcite, zinc oxide or sodium benzoate or any combinations thereof. The stearate containing neutralizer can be calcium stearate, and/or zinc stearate. In some particular aspects, the neutralizer can be a stearate containing neutralizer such as calcium stearate, and/or zinc stearate. In some aspects, the polymeric composition can contain 50 ppm to 500 ppm of a sterically hindered phenol, such as pentaerythritol tetrakis[3-[3,5-di-tert-butyl-4-hydroxyphenyl]propionate, 200 ppm to 2000 ppm of a phosphite containing stabilizer such as tris(2,4-di-tert.-butylphenyl)phosphite, 200 ppm to 2000 ppm of an ester containing antistatic, such as glycerol monostearate, or 100 ppm to 1000 ppm of a stearate such as calcium stearate, and/or zinc stearate or any combinations thereof. B. Properties of the Polymeric Compositions In some aspects, the polymeric composition can have a melt flow rate (MFR) of 0.1 g/10 min to 150 g/10 min, or 1 to 60 g/10 min, or 1 to 30 g/10 min, or 1 to 20 g/10 min, or 1 to 10 g/10 min, or 1 to 7 g/10 min at 230° C., 2.16 kg measured in accordance with ASTM D-1238. In some aspects, the polymeric composition can have a flexural modulus of 100 Kpsi to 300 Kpsi at 4-8 N as determined by ASTM D790-97. In some aspects, the polymeric composition can have a notched Izod impact strength greater than 0.9 ft-lb/in, such as 1 ft-lb/in to 1.5 ft-lb/in at 23° C., as measured in accordance with D638. In some aspects, the polymeric composition can have a tensile modulus greater than 210 KPsi, such as 211 KPsi to 300 KPsi at 23° C., as measured in accordance with D 638. In some aspects, the polymeric composition can have an elongation at break greater than 180%, such as 200% to 250% at 23° C., as measured in accordance with D-638. In some aspects, the polymeric composition can have a crystallization temperature of from about 100° C. to 135° C. or 115° C. to 130° C. or 120° C. to 125° C., as determined by Differential Scanning calorimetry (DSC) in accordance with ASTM D-3418D. In some aspects, prior to any extrusion of the polymeric composition in an extruder, the polymeric composition can exhibit a yellowness index (YI) of less than 1, or −5 to 0 or −3 to −1.5, or −2.7 to −2, as measured in accordance with ASTM D-6290. In certain embodiments, after 1, 2, 3, 4, or 5 extrusions of the polymeric composition through a slot or die of an extruder at a temperature of about 545° F., the YI may increase. For example and without limitation, after 1, 2, 3, 4, or 5 extrusions of the polymeric composition through a slot or die of an extruder at a temperature of about 545° F., the YI may range −2 to 2.5, or −1.8 to 2, as measured in accordance with ASTM D-6290. In some aspects, the polymeric composition can exhibit a “Color L” of 60 to 85, 70 to 80, 72 to 78, about 74, as measured in accordance with ASTM D-6290. In some aspects, the polymeric composition can exhibit a “Color a” of less than 1, less than 0, −2 to 0, −1 to 0, or −0.7 to −0.2, as measured in accordance with ASTM D-6290. In some aspects, the polymeric composition can exhibit a “Color b” of less than 1, less than 0, −2 to 0, −1 to 0, or −0.9 to −0.3 as measured in accordance with ASTM D-6290. In some aspects, prior to any extrusion of the polymeric composition in an extruder, the polymeric composition can exhibit an initial % haze value (e.g., initial haze value being the haze value without being subjected to an extrusion pass) of less than 20%, less than 18%, less than 16%, or less than 15%, as measured in accordance with ASTM D1003, at a thickness of about 40 mm. In certain aspects, after 1, 2, 3, 4, or 5 extrusions of the polymeric composition through a slot or die of an extruder at a temperature of about 545° F., the % haze value as measured in accordance with ASTM D1003, at a thickness of about 40 mil, may change by no more than about 30%, 20%, 10%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less, relative to the initial % haze value. In some aspects, the % change can be an increase in haze value. In some aspects, the % change can be a decrease in haze value. In some aspects, the polymeric composition can have a combination of, or all of the properties mentioned herein. C. Methods for Making the Polymeric Compositions and Methods of Thermoforming The polymeric composition of the present invention can be made by various methods known in the art such as extrusion, injection molding, thermoforming or like. For example the components, such as the polypropylene copolymer, aryl amide containing clarifying agent, and/or phosphate ester salt containing clarifying agent, and one or more additives can be mixed such as dry blended and then melt-blended such as extruded to form the polymeric composition. The extruder used can be any type of extruder known in the art. The extrusion can be performed at a temperature high enough to melt the composition, but as low as possible to avoid excessive thermal degradation of the components. In certain aspects, the thermoplastic composition can be subjected to multiple, sequential passes through an extruder. Without being bound by theory, it is believed that subjecting a polymer resin to 1 or more passes through an extruder simulates accelerated aging of the polymer resin due to the elevated pressures and temperatures encountered by the polymer resin during extrusion. Also, without being bound by theory, it is believed that subjecting a polymer resin to passes through an extruder simulates reprocessing of regrind trim of the polymer in sheet extrusion thermoforming. In each pass, the thermoplastic composition may be extruded through a slot or die. The extruded material can be quenched if desired. For each extrusion, the final melt temperature prior to extrusion through a die can be independently 302 to about 600° F. (150-315° C.), with pressures independently ranging from about 100 to about 30,000 psi (0.7-207 mPa). One aspect is directed to a method for forming a thermoformed article containing the polymeric composition. The method can include melt extruding the components, such as the polypropylene copolymer, aryl amide containing clarifying agent and/or phosphate ester salt containing clarifying agent, and one or more additives to form an initial article, and thermoforming the initial article to form a thermoformed article. The initial article can be an extruded sheet or film containing the polymeric composition. Thermoforming the initial article may include subjecting the initial article to heat, vacuum or pressure, or combinations thereof to convert the initial article into the thermoformed article. For example and without limitation, the initial article can be thermoformed by placing the initial article into a portiotool. The initial article within the too may be subjected to heat, vacuum or pressure, or combinations thereof, causing the initial article to conform to the shape of interior walls of the tool. In some aspects, the initial article may be heated prior to being placed in the tool. The heated initial article may then be placed into the tool, the tool may be closed onto the initial article, and a vacuum or pressure may then be applied to the tool. Application of the vacuum or pressure to the heated initial article within the tool causes the initial article to conform to the shape of the interior of the tool, thus forming the thermoformed article. The formed thermoformed article of desired shape can be removed from the tool. The non-removed portion of the initial article e.g. web of the sheet and/or, not thermoformed waste portion of the sheet can be grinded, reused and/or recycled to make a second initial article. The second initial article can be thermoformed to form another thermoformed article. The second initial article and the another thermoformed article can have haze values that are comparable to the initial article and the first thermoformed shape. Stated another way, the polymeric composition of the present invention and/or articles containing the polymeric composition have stable haze values even after being subjected to 2, 3, 4, 5, or more extrusion passes, which allows for articles containing the polymeric composition to be recycled with limited to no loss of clarity for the recycled portions or articles of manufacture made from at least a portion of the recycled portions. In some aspects, the initial article can be formed by extrusion of the molten polymeric composition through a slot or die and cooling e.g. quenching the extrudate to form the initial article e.g., the extruded sheet. Extrusion of the molten polymeric composition can occur at a temperature ranging from 150° C. to 315° C. or at least any one of, equal to any one of, or between any two of 150° C., 160° C., 170° C., 180° C., 190° C., 200° C., 210° C., 220° C., 230° C., 240° C., 250° C., 260° C., 270° C., 280° C., 290° C., 300° C., 310° C., and 315° C. In some aspects, the extruded sheet can have a thickness of 0.5 to 100 mm, 12 to 20 mm, 12 to 16 mm, or 16 to 20 mm or at least any one of, equal to any one of, or between any two of 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mm. In some aspects, the initial article can be a multilayer extruded sheet and each layer of the multilayer extruded sheet can independently have a thickness of 0.5 to 100 mm, 12 to 20 mm, 12 to 16 mm, or 16 to 20 mm or at least any one of, equal to any one of, or between any two of 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mm. In some aspects, the initial article can be a cast sheet or an oriented sheet. In some aspects, thermoforming the initial article subjects the initial article to solid-state stretching. In some aspects, the extruded sheet can be reheated and thermoformed within a tool. During the conforming of the extruded sheet to the shape of the interior of the tool, the extruded sheet may undergo solid-state stretching in one or more directions, thus forming a solid-state stretched, thermoformed article from the extruded sheet. D. Articles Containing the Polymeric Compositions The polymeric compositions of the present invention can be comprised in an article of manufacture. The article of manufacture can be an extruded, a blow-molded, an injection-molded, and/or thermoformed article. In some aspects, the article of manufacture can be transparent. Non-limited examples of the article of the articles of manufacture include housewares, food storage containers, cooking utensils, plates, cups, measuring cups, drinking cups, strainers, turkey basters, non-food storage containers, filing cabinets and particularly clear drawers used in such cabinets, general storage devices, such as organizers, totes, sweater boxes, films, coatings and fibers, bags, adhesives, yarns, fabrics, bottles, jars, plates and cups, clamshell and the like. Article of manufacture can be rigid packaging, such as deli containers and lids including those used for dips, spreads, and pasta salads, dairy containers and lids including those used for storing cottage cheese, butter and yogurt, personal care products, and bottles and jars. In these and other uses the resins may be combined with other materials, such as particulate materials, including talc, calcium carbonate, wood, and fibers, such as glass or graphite fibers, to form composite materials. Examples of such composite materials include components for furniture, automotive components and building materials, particularly those used as lumber replacement. EXAMPLES The present invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1 Polymeric compositions C-1 to C-6, with compositions as shown in table 1 were made. Compositions C-1 to C-6 were injection molded into ASTM specification. Extrusion parameters used are provided in table 2. The optical and mechanical properties of the compositions are provided in table 3. TABLE 1CompositionsComponentsC-1C-2C-3C-4C-5C-6Propylene-ethylene99.799.599.599.799.6random copolymer (wt. %)Propylene homo polymer99.7(wt. %)Pentaerythritol tetrakis[3-250250250250250250[3,5-di-tert-butyl-4-hydroxyphenyl]propionate(ppm)Tris(2,4-di-tert.-100010001000100010001000butylphenyl)phosphite(ppm)Sodium Benzoate (ppm)800800Glycerol mono stearate800800800800800800(ppm)Calcium stearate (ppm)500500500500Bis(3,4-1800dimethylbenzylidene)sorbitol (ppm)NX8000 (ppm)21001,3,5-tris(2,2-dimethyl200propanamido)benzene(ppm)2,2′-methylenebis (4,6,-di-1000tertbutylphenyl) phosphate(ppm) TABLE 2Extrusion parametersZone 1Zone 1Zone 1Zone 1Die 1Die 2Die 3360° F.370° F.370° F.385° F.390° F.410° F.410° F. TABLE 3Composition propertiesPropertiesC-1C-2C-3C-4C-5C-6Haze20 mil16.510.19.87.610.020.9Plaques40 mil33.121.321.915.019.030.660 mil44.022.624.617.926.652.880 mil62.440.742.431.636.769.2Haze20 mil45.112.91212.111.748.1Plaques40 mil65.228.627.224.721.973.8after60 mil72.435.634.932.732.279.7autoclave80 mil79.353.550.944.843.984.4ColorL74.773.772.77474.176.2Pelletsa−0.55−0.582.72−0.52−0.54−0.52b−1.01−0.98−10.06−0.77−0.64−1.62YI−3.05−3.03−22.15−2.46−2.15−4.38GlossGloss, 4550.451.652.35051.650.1Melt4.14.64.13.94.24.4FlowRateDSCFirst Melt158.2158.6158.4158.3159.2166.2Peak (° C.)First Melt87.8180.5484.890.5984.4293.09Delta H (J/g)Recrystallization125.2125.4125121.1124.1129.6Peak (° C.)Recrystallization99.5492.5795.2296.8993.08105.6Delta H (J/g)Second Melt158.4159.8159.2158.5159.6166Peak (° C.)Second Melt102.693.8998.55100.496.98112.8Delta H (J/g)Crystallinity (%)49.144.947.24846.454HDTUnannealed heat213196198201217245distortion(66 psi) (° F.)TensileTensile218825221163220358219472211280261828BarsModulus (Psi)Tensile Strength497350005039503348895509at Yield (Psi)Tensile Strength323127173119278927352120at Break (%)Elongation8.78.48.58.38.76.1at YieldElongation29410321920422264.1at Break (%)FlexFlexural196200212217213284Modulus(chord 4-8N)(KPsi)IzodIzod-Notched1.90.80.91.01.10.8(ft-lb/in)Break typeCompleteCompleteCompleteCompleteCompleteCompleteInstrum.Impact87.9187.8388.0587.8287.9788.10ImpactEnergy (ft-lb)73° F.Maximum186.06179.61174.73187.15176.93151.95Load (lbf)Total4.513.082.372.422.852.60Energy (ft-lb)Energy to Max1.931.420.840.990.831.57Load (ft-lb)Impact Velocity11.1311.1311.1411.1311.1411.15(ft/sec)Energy after Max2.581.661.531.432.021.03Load (ft-lb)GPCMn (kDa)550165365454378533635439354295Mw (kDa)373893356498364416353378359849361063Mz (kDa)165563914519321518802145651514648511572202Polydispersity6.86.66.76.66.66.7Peak Mw (kDa)171417169182171417169182171417169182 The compositions, C-1 to C-6, have similar stiffness and Izod impact strength. The crystallization temperature (Tc) of the compositions are in line with their nucleating agent/clarifying agent content. Inventive compositions C-4 and C-5, display impressive haze values, while using much lower amount of clarifying agent, 1,3,5-tris(2,2-dimethyl propanamido)benzene and 2,2′-methylenebis (4,6,-di-tertbutylphenyl) phosphate respectively. The haze values of all samples increased after autoclaving at 130° C. for 30 minutes. Composition C-5 shows the lowest “after/before haze” ratio.FIG.1shows percentage haze increase after autoclaving the compositions C-5 to C-6. Properties of the inventive compositions C-4 and C-5 were studied for multi-pass extrusion. Extrusion conditions used for C-4 and C-5 are provided in Tables 4 and 5 respectively. Properties of compositions C-4 and C-5 after each pass were measured and were compared with those of C-6 (Table 6). TABLE 4Multi-pass extrusion parameters for composition C-4SetTemperaturePointsPass 0Pass 1Pass 2Pass 3Pass 4Pass 5Zone 1390386386385392386Zone 2485489479489480488Zone 3545542540548519549Die545543545546545532Melt500506496500500499Extruder PSI795690654595498RPM148.5148.6148.3148.3148.4AMPS6.96.56.36.06.1Pelletizer475497497495530Setting TABLE 5Multi-pass extrusion parameters for composition C-5SetTemperaturePointsPass 0Pass 1Pass 2Pass 3Pass 4Pass 5Zone 1390390385385393386Zone 2485489490481484480Zone 3545549543548545541Die545543548543543543Melt500498501500501499Extruder PSI788712651583546RPM148.4148.3148.2148.2148.2AMPS6.96.56.46.15.9Pelletizer480492500500530Setting TABLE 6Properties after pass 0, 1, 3, and 5SamplesC-4C-5C-6Haze Plaques,Pass 014.62039.340 milPass 113.719.639.8(%)Pass 313.822.541.7Pass 513.925.844.4Melt FlowPass 04.24.34.1RatePass 16.86.85.6(g/10 min)Pass 312.012.29.1Pass 518.718.513YIPass 0−2.66−2.12−3.41Pass 1−1.72−1.01−2.14Pass 30.20.63−1.07Pass 50.991.871.38 The melt flow rate (MFR) and yellowness Index (YI) of the compositions increased after each pass. For composition C-5 the increase of haze value with the multiple extrusion passes were moderate, but for composition C-4 the haze value remained almost same, at around 15%, even after 5 extrusion passes. Over all the inventive compositions C4 and C5 display excellent low haze values and composition C-4 shows minimum change in haze values with multiple regrinding and extrusion. Although embodiments of the present application and their advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the embodiments as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the above disclosure, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. | 30,651 |
11859073 | DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to a foamable composition that provides low density closed-cell foams with substantially higher stiffness than a polypropylene foam produced with commercially available high melt strength polypropylene. It was surprisingly found that the closed-cell content and the stiffness of the foam can be significantly increased by tailoring the polymer composition. Accordingly, one aspect of the invention relates to a foamable composition containing: (a) up to about 20 wt % of a polypropylene-based copolymer, based on a total weight of the foamable composition, (b) about 80 wt % or more of a polyolefin, based on the total weight of the foamable composition. The foamable composition can also include modifications, as well as other components one skilled in the art would typically include in a foamable composition. The foamable composition can be a polymer blend. As understood by one skilled in the art, the term “polymer” refers to a polymeric compound prepared by polymerizing monomers, whether of the same or a different kind. The generic term “polymer” thus includes the term “homopolymer,” which refers to polymers prepared from only one type of monomer, as well as the term “copolymer” which refers to polymers prepared from two or more different monomers. As used herein, the term “blend” or “polymer blend” generally refers to a physical mixture of two or more polymers which are not chemically combined. Such a blend may be miscible and may not be phase separated. The polymer blend may contain one or more domain configurations, which are created by the morphologies of the polymers. The domain configurations can be determined by X-ray diffraction, transmission electron microscopy, scanning transmission electron microscopy, scanning electron microscopy, and atomic force microscopy, or other methods known in the art. The foamable composition contains the polypropylene-based copolymer in an amount of up to about 20 wt %, or from about 5 wt % to about 20 wt %, including any ranges in between. In some embodiments, the amount of the polypropylene-based copolymer ranges from 1 wt % to about 15 wt %, from about 7 wt % to about 12 wt %, or from about 8 wt % to about 11 wt %, including any ranges in between. Conversely, the foamable composition can contain about 80 wt % or more of the polyolefin, or from about 80 wt % to about 95 wt %, including any ranges in between, based on the total weight of the foamable composition. In some embodiments, the amount of the polyolefin ranges from about 85 wt % to about 99 wt %, from about 88 wt % to about 95 wt %, or from about 89 wt % to 92 wt %, including any ranges in between. The foamable composition has a zero-shear viscosity of about 12,000 Pa·s or less at 190° C., or from about 8,000 Pa·s to about 11,500 Pa·s. In some embodiments, the zero-shear viscosity is about 10,000 Pa·s or less. As understood by one skilled in the art, the term “zero-shear viscosity” refers to the viscosity of the melt at a shear rate approaching to zero, and can be determined by methods known in the art such as creep recovery experiments. A melt flow rate of the foamable composition can be about 11.5 g/10 min or less, about 10 g/10 min or less, or ranges from 5.9 g/10 min to about 9 g/10 min, including any ranges in between. A foamable composition with a melt flow rate outside these ranges may also form a foam with the desirable properties. In some embodiments, the melt flow rate of the foamable composition is measured after one pass in the extruder. As used herein, the term “melt flow rate” (MFR) (units of g/10 min) is described according to and measured per ASTM D1238 using a load of 2.16 kg at 230° C. A melt strength of the foamable composition is at least 11.5 cN, or ranges from 11.5 cN to about 20 cN. A foamable composition with a melt strength outside these ranges may also form a foam with the desirable properties. As used herein, the term “melt strength” is an engineering measure of the extensional viscosity and is defined as the maximum tension that can be applied to the melt without breaking. The foamable composition has a velocity at break of at least about 120 mm/s, at least about 140 mm/s, or at least about 160 mm/s. As used herein, the term “velocity at break” refers to the maximum velocity before the melt breaks in extensional flow experiments. Polypropylene-Based Copolymer As used herein, the polypropylene-based copolymer refers to copolymers containing at least 50 wt % propylene monomer units, based on the weight of the copolymer. Polypropylene-based copolymers are typically prepared by polymerizing propylene and at least one other linear a-olefin, branched α-olefin, or cyclic olefin. The a-olefin and the cyclic olefin may have 2 to 20 carbon atoms, 2 to 16 carbon atoms, or 2 to 12 carbon atoms, including but not limited to ethylene, 1-butene, 2-butene, 1-pentene, 3-methyl-1-butene, 1-hexene, 4-methyl-1-pentene, 3-methyl-1-pentene, 4,6-dimethyl-1-heptene, 1-octene, 1-decene, 1-undecene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-octadecene, 1-eicocene, norbornene, tetracyclododecene, and combinations thereof. These olefins may each contain one or more heteroatoms such as an oxygen, nitrogen, and/or silicon atom. The polypropylene-based copolymer has a melt flow rate of about 1 g/10 min or less, about 0.8 g/10 min or less, or about 0.6 g/10 min or less. The polypropylene-based copolymer has a melt strength at 190° C. ranging from about 20 cN to about 100 cN, from about 20 cN to about 80 cN, or from about 20 cN to about 60 cN. A velocity at break of the polypropylene-based copolymer is at least about 100 mm/s, or ranges from about 100 mm/s to about 150 mm/s, including any ranges in between. The polypropylene-based copolymer can be made up of linear and/or branched polymer chains. Exemplary polypropylene-based copolymer includes an alternating copolymer, a periodic copolymer, a block copolymer, a random copolymer, or an impact copolymer. In some embodiments, the polypropylene-based copolymer is a random copolymer or an impact copolymer optionally containing long chain branches. As used herein, the term “random copolymer” refers to a copolymer in which the different types of monomer units are statistically distributed in the polymer molecules. The polypropylene-based copolymer can be a polypropylene-polyethylene random copolymer in which the content of the ethylene monomer units can be up to about 7 wt %, up to about 5 wt %, or in a range of about 0.5 wt % to about 5 wt %, including any ranges in between, based on a total weight of the copolymer. As used herein, the term “impact copolymer” refers to a heterophasic polyolefin copolymer where one polyolefin is the continuous phase (i.e., the matrix) and an elastomeric phase is uniformly dispersed therein. The impact copolymer includes, for instance, a heterophasic polypropylene copolymer where polypropylene homopolymer is the continuous phase and an elastomeric phase, such as ethylene propylene rubber (EPR), is uniformly distributed therein. The polypropylene matrix can make up from about 75 wt % to about 90 wt % of the weight of the impact copolymer. The amount of the elastomeric phase, such as EPR, can be up to about 25 wt %, up to about 20 wt %, up to about 12 wt %, from about 8 wt % to about 12 wt %, or from about 8 wt % to about 10 wt %, including any ranges in between. The elastomeric phase contains ethylene monomer units in an amount of at least about 5 wt %, at least about 10 wt %, at least about 25 wt %, or not more than about 60 wt %, or in a range of about 25 wt % to about 60 wt %, including any ranges in between. The amount of ethylene in the impact copolymer is typically not more than about 12 wt %. The impact copolymer may have a xylenes solubles content of greater than 8 wt % as determined by acetone precipitation. The impact copolymer results from an in-reactor process rather than physical blending. The impact copolymer may or may not be a coupled polymer, which is a rheology-modified polymer resulting from a coupling reaction. Accordingly, the impact copolymer may or may not contain long chain branches. Each long chain branch may be as long as the polymer backbone to which it is attached. Methods for detecting long chain branches are known to one skilled in the art, for example,13C NMR spectroscopy, and gel permeation chromatography coupled to a low angle laser light scattering detector or a differential viscometer detector. In one embodiment, the impact copolymer contains long chain branches, and can be prepared by reacting a coupling agent with a polymeric precursor such as an impact copolymer without long chain branches. The polymeric precursor and the coupling agent can be admixed, or otherwise combined, under conditions which allow for sufficient mixing before or during the coupling reaction. Admixing of the polymeric precursor and the coupling agent can be accomplished by any means known to one skilled in the art. For example, the mixing of the polymeric precursor and the coupling agent can occur in any equipment, such as V-blenders, ribbon or paddle blenders, tumbling drums, or extruders (e.g., twin screw extruders). The polymeric precursor and the coupling agent may be physically mixed by simultaneously introducing the polymeric precursor resin and the coupling agent into the feed section of an extruder, such as through a main feed hopper or through multiple feeders. Alternatively, the polymeric precursor and the coupling agent can be added to the extruder from separate feeders. Optionally, the coupling agent may be pre-blended (e.g., dry blended, melt-mixed) with the polymeric precursor in a first extrusion step at a temperature below the reaction temperature of the coupling agent to form a masterbatch. In a second extrusion step, the masterbatch is fed via the feed section to an extruder either separately from or together with the polymeric precursor. In some embodiments, the coupling agent is added in the form of a molecular melt with other ingredients, such as an antioxidant, to the extruder. During the admixing/combining, it is desirable to have as homogeneous a distribution as possible, to achieve solubility of the coupling agent in the polymer melt, and to avoid uneven amounts of localized reactions. The coupling reaction is implemented via reactive extrusion or any other method which is capable of mixing the coupling agent with the polymeric precursor and adding sufficient energy to cause a coupling reaction between the coupling agent and the polymeric precursor. It may be necessary to activate a coupling agent with heat, sonic energy, radiation or other chemical activating energy, for the coupling agent to be effective for coupling polymer chains. For example, the resulting admixture can be subjected to a heating step to initiate the coupling reaction. The processing conditions (the reaction temperature, the type of reaction vessels, the concentration of the coupling agent, and residence times, etc.) can be varied depending on the characteristics of the polymeric precursor and the coupling agent. For example, the reaction temperature can range from about 190° C. to about 280° C., and the residence time at the reaction temperature can range from 15 seconds to 90 seconds. One skilled in the art understands that a polymer (or mixtures thereof) typically melts over a temperature range rather than sharply at one temperature. Thus, alternatively, it may be sufficient that the polymeric precursor be in a partially molten state. The melting or softening temperature ranges can be approximated from the differential scanning calorimeter (DSC) curve of the polymeric precursor (or mixtures thereof). As used herein, the coupling agent is capable of insertion reactions into C-H bonds of polymers. The C-H insertion reactions and the coupling agents capable of such reactions are known to one skilled in the art. The coupling agent is capable of generating reactive species (e.g., free radicals, carbenes, or nitrenes) that couple the coupling agent with the polymeric precursor. In some embodiments, the coupling agent is a poly(sulfonyl azide) that encompasses a compound having multiple sulfonyl azide groups (—SO2N3). The poly(sulfonyl azide) is any compound having at least two sulfonyl azide groups (—SO2N3) reactive with the polymeric precursor. Preferably the poly(sulfonyl azide)s have a structure represented by X—R—X, where each X is SO2N3and R represents an unsubstituted or inertly substituted hydrocarbyl, hydrocarbyl ether, or silicon-containing group, preferably having sufficient carbon, oxygen, or silicon atoms to separate the sulfonyl azide groups sufficiently to permit a facile reaction between the polymeric precursor and the poly(sulfonyl azide). For example, there can be at least one, at least two, or at least three carbon, oxygen, or silicon atoms between the sulfonyl azide groups. While there is no critical limit to the length of R, each R can have less than about 50, less than about 20, or less than about 15 carbon, oxygen, or silicon atoms. Silicon containing groups include, without limitation, silanes and siloxanes. Examples of a suitable poly(sulfonyl azide) include but are not limited to 1,5-pentane bis(sulfonyl azide), 1,8-octane bis(sulfonyl azide), 1,10-decane bis(sulfonyl azide), 1,10-octadecane bis(sulfonyl azide), 1-octyl-2,4,6-benzene tris(sulfonyl azide), 4,4′-diphenyl ether bis(sulfonyl azide), 1,6-bis(4′-sulfonazidophenyl)hexane, 2,7-naphthalene bis(sulfonyl azide), and mixed poly(sulfonyl azide)s of chlorinated aliphatic hydrocarbons containing an average of from 1 to 8 chlorine atoms and from about 2 to 5 sulfonyl azide groups per molecule, and mixtures thereof In some embodiments, the poly(sulfonyl azide) is oxy-bis(4-sulfonylazidobenzene), 2,7-naphthalene bis(sulfonyl azido), 4,4′-bis(sulfonyl azido)biphenyl, 4,4′-diphenyl ether bis(sulfonyl azide), 1,3-benzenedisulfonyl azide, 1,4-benzenedisulfonyl azide, and bis(4-sulfonyl azidophenyl)methane, a mixture or any combination thereof It is believed that other coupling agents can be used, and the coupling reaction would proceed as intended. These coupling agents include peroxides, such as di(4-tert-butylcyclohexyl) peroxydicarbonate, di(tert-butylperoxyisopropyl)benzene, di(tert-butylperoxyisopropyl)benzene, di(4-methylbenzoyl) peroxide, dicetyl peroxydicarbonate, dimyristyl peroxydicarbonate, dicumyl peroxide, dibenzoyl peroxide, diisopropyl peroxydicarbonate, tert-butyl monoperoxymaleate, didecanoyl peroxide, dioctanoyl peroxide, 2,5-dimethyl-2,5-di(tert-butylperoxy) hexane, tert-butylperoxy-2-ethylhexyl carbonate, tert-amyl peroxy-2-ethylhexanoate, tert-amyl peroxyneodecanoate, tert-amyl peroxypivalate, tert-amyl peroxybenzoate, tert-amyl peroxyacetate, di-sec-butyl peroxydicarbonate, di(2-ethylhexyl) peroxydicarbonate, tert-butyl cumyl peroxide or combinations of these non-limiting examples; an alkyl borane, such as triethylborane, trimethylborane, tri-n-butylborane, triisobutylborane, diethylborane methoxide, diethylborane isopropoxide, or combinations of these non-limiting examples; azo compounds, such as azobisisobutyronitrile (AIBN), 1,1′-azobis(cyclohexanecarbonitrile) (ABCN), 1,1′-azodi(hexahydrobenzonitrile), 2,2′-azodi(hexahydrobenzonitrile), 2,2′-azodi(2- methylbuttyronitrile), or combinations of these non-limiting examples. Polyolefin As used herein, the polyolefin generally embraces a homopolymer prepared from a single type of olefin monomer as well as a copolymer prepared from two or more olefin monomers. A specific polyolefin referred to herein shall mean polymers comprising greater than 50% by weight of units derived from that specific olefin monomer, including homopolymers of that specific olefin or copolymers containing units derived from that specific olefin monomer and one or more other types of olefin comonomers. For instance, polypropylene shall mean polymers comprising greater than 50 wt % of units derived from propylene monomer, including polypropylene homopolymers or copolymers containing units derived from propylene monomer and one or more other types of olefin comonomers. The polyolefin used herein can be a copolymer wherein the comonomer(s) is/are randomly distributed along the polymer chain, a periodic copolymer, an alternating copolymer, or a block copolymer comprising two or more homopolymer blocks linked by covalent bonds. Exemplary polyolefins include those prepared from at least one of a linear α-olefin, a branched α-olefin, and a cyclic olefin, all of which have been described herein. Typical polyolefins include polyethylene, polypropylene, a copolymer of polyethylene and polypropylene, and a polymer blend containing polyethylene, polypropylene, and/or a copolymer of polyethylene and polypropylene. For example, the polyolefin can be a polypropylene homopolymer that contains more than about 99 wt % of propylene monomer. The polyolefin may or may not be a coupled polymer. In some embodiments, the polyolefin is a coupled polymer, being the reaction product of a semi-crystalline polyolefin, such as a polypropylene homopolymer, and a coupling agent such as a poly(sulfonyl azide). The polyolefin may be prepared by methods described herein for the coupled impact copolymer (e.g, by replacing the polymeric precursor with the semi-crystalline polyolefin) or by methods described in U.S. patent application Ser. No. 15/010,099, filed on Jan. 29, 2016, and assigned to Braskem America, Inc., which is incorporated herein by reference in its entirety. The semi-crystalline polyolefin can be a homopolymer that does not contain long chain branches. Examples of a suitable semi-crystalline polyolefin include, but are not limited to, a polypropylene, or a polyethylene, or combinations thereof. Examples of polypropylene include but are not limited to a polypropylene homopolymer. For example, a polypropylene homopolymer having a melt flow rate of at least 1.8 g/10 min, or a melt flow rate ranging from about 15 g/10 min to about 40 g/10 min, or from about 15 g/10 min to about 25 g/10 min, including any ranges in between, can be used in the present disclosure. The crystallinity of the semi-crystalline polyolefin can be at least 50%, or ranges from about 60% to about 90%, or from about 70% to about 80%, including any ranges in between. Crystallinity can be measured by methods known in the art such as DSC. The content of the coupling agent, such as the poly(sulfonyl azide), is at least 500 ppm, or ranges from about 500 ppm to about 6,500 ppm, or from about 3,000 ppm to about 6,500 ppm, including any ranges in between, based on the total weight of the polyolefin. The polyolefin has a melt flow rate of about 2 g/10 min or more, or from about 2 g/10 min to about 20 g/10 min. The polyolefin has a melt strength of about 30 cN or more at 190° C., or from about 30 cN to about 100 cN, or from about 30 cN to about 80 cN. The polyolefin has a melting temperature of at least 140° C., at least about 160° C., or from about 160° C. to about 170° C. A crystallization temperature of the polyolefin is at least about 120° C., or at least about 130° C. The melting and crystallization temperatures of the polyolefins can be measured by methods known in the art such as differential scanning calorimetry (DSC). The polyolefin has a melt drawability of at least about 170 mm/s, or about 170 mm/s to about 250 mm/s. A flexural modulus of the polyolefin is greater than about 240,000 psi, or ranges from about 240,000 psi to about 350,000 psi. As used herein, the term “flexural modulus” is described according to and measured per ASTM D790. The polyolefin has a heat distortion temperature under load of 66 psi (DTUL@ 66 psi) greater than 101° C. and typically not more than 120° C. As used herein, the term “heat distortion temperature under load (DTUL)” is described according to and can be measured per ASTM D-648. The polyolefin has a ratio of melt strength to melt flow rate of greater than 18 and typically not more than 100. The foamable composition typically further comprises a filler (e.g., wood, silica, glass, clay, and other polymers), an additive (e.g., a nucleating agent), or both. The foamable compositions of the present invention can include any conventional plastics additives in any combination. The amount should not be wasteful of the additive. Those skilled in the art of thermoplastics compounding, with reference to such treatises as Plastics Additives Database (2004) from Plastics Design Library (www.elsevier.com), can select from many different types of additives for inclusion into the compounds of the present invention. Non-limiting examples of additives or oligomers are adhesion promoters; antioxidants (e.g., antioxidants containing thioether, phosphite, or phenolic units); flame retardants; biocides (antibacterials, fungicides, and mildewcides); anti-fogging agents; anti-static agents; bonding, blowing and foaming agents; dispersants; fillers (e.g., glass fibers) and extenders; smoke suppressants; expandable char formers; impact modifiers; initiators; acid scavengers; lubricants; micas; pigments, colorants and dyes; plasticizers; processing aids; other polymers; release agents; silanes, titanates and zirconates; additional slip agents; anti-blocking agents; stabilizers such as hindered amine light stabilizers; stearates (e.g., calcium stearate); ultraviolet light absorbers; viscosity regulators; waxes; antiozonants; organosulfur compounds; nucleating agents (e.g., talc); and combinations thereof Antiblock additives are often used together with slip additives and for their complementary functions. Antiblock additives reduce adhesion or the “stickiness” between polymer layers (usually layers of the same polymer), which is created by blocking forces inherent to many polymers. Whereas slip additives decrease friction caused from moving across the surface of a polymer, antiblock additives create a microrough surface that lessens the adhesion caused by these blocking forces. Antiblock additives, like slip additives, are commonly used to improve the handling of a polymer for applications such as packaging. For instance, a non-migratory antiblock additive, such as crosslinked poly(methyl methacrylate) or inorganic silica, can be used. Method for Producing the Foamable Composition The polypropylene-based copolymer and the polyolefin can be dry-blended in a gravimetric mixing feeder. The resultant mixture can be fed to an extruder and then extruded by means known in the art using the extruder (or other apparatus). Alternatively, the polypropylene-based copolymer and the polyolefin can be added separately to the extruder and the mixing of the polymers occurs in the extruder. The term “extruder” takes on its broadest meaning and includes any machine suitable for polyolefin extrusion. For instance, the term includes machines that can extrude polyolefins in the form of powder or pellets, sheets, fibers, films, blow molded articles, foams, or other desired shapes and/or profiles. Generally, an extruder operates by feeding material through the feed throat (an opening near the rear of the barrel) which comes into contact with one or more screws. The rotating screw(s) forces the material forward into one or more heated barrels (e.g., there may be one screw per barrel). In many processes, a heating profile can be set for the barrel in which three or more independent proportional-integral-derivative controller (PID)-controlled heater zones can gradually increase the temperature of the barrel from the rear (where the plastic enters) to the front. The vessel can be, for instance, a single-screw or a twin-screw extruder, or a batch mixer. Further descriptions about extruders and processes for extrusion can be found in U.S. Pat. Nos. 4,814,135; 4,857,600; 5,076,988; and 5,153,382; all of which are incorporated herein by reference. After extrusion, the foamable composition can be solidified, optionally pelletized and stored, transported, and then re-heated with a blowing agent and foamed at any time after the composition is produced. Foam Another aspect of the invention relates to a foam, comprising a polymer composition, containing: (i) up to about 20 wt % of the polypropylene-based copolymer, based on a total weight of the polymer composition, and (ii) about 80 wt % or more of the polyolefin, based on the total weight of the polymer composition, where the foam has a density ranging from about 0.01 g/cc to about 0.20 g/cc, and a closed-cell content of more than about 80%. Foams have a cellular core structure created by the expansion of a blowing agent. A blowing agent is a substance which is capable of creating voids in a polymer matrix thereby producing a foam. The blowing agent can be a physical blowing agent, a chemical blowing agent, or both. Exemplary physical blowing agents include liquefied hydrocarbons (e.g., pentane, isopentane, cyclopentane, butane), liquid carbon dioxide, nitrogen, hydrochlorofluoroolefins (e.g., 1-chloro-3,3,3-trifluoropropene, 2-chloro-3,3,3-trifluoropropene, and dichloro-fluorinated propene), hydrofluoroolefins (3,3,3-trifluoropropene, 1,2,3,3,3-pentafluoropropene, cis- and/or trans-1,3,3,3-tetrafluoropropene, and 2,3,3,3-tetrafluoropropene), and combinations thereof. An amount of the physical blowing agent can be up to 1 wt %, including any fraction ranges in between, based on a total weight of the polymer composition. Chemical foaming agents (CFAs) release gasses upon thermal decomposition. Exemplary chemical blowing agents include azo compounds (e.g., azodicarbonamide), hydrazine derivatives (e.g., p-toluenesulfonylhydrazide, p,p′-oxybis (benzenesulfonylhydrazide), benzenesulfonyl hydrazide, p-toluenesulfonyl acetone hydrazone), carbazides (e.g., p-toluenesulfonylsemicarbazide, p,p′-oxybis (benzenesulfonylsemicarbazide)), tetrazoles (e.g., 5-phenyltetrazole), nitroso compounds (e.g., N,N′-dinitroso-pentamethylenetetramine), carbonates (e.g., sodium bicarbonate), those sold under trade names of SAFOAM®, HYDROCEROL®, and ECOCELL®, or any combinations thereof. An amount of the chemical blowing agent can be up to 0.10 wt %, including any fraction ranges in between, based on the total weight of the polymer composition. The foam structure has at least two phases, a polymer matrix and voids. The foam described herein has a closed-cell structure. In closed-cell foams, the voids are completely enclosed by cell walls and the voids are not interconnected with other voids by open passages. The foam has a closed-cell content of more than about 80%, more than about 85%, or more than about 90%. The closed-cell structure can be determined qualitatively by dipping a sample of the foam strand into a solution of isopropanol and dye. Closed cells are indicated if the alcohol/dye solution does not penetrate the foamed sample. Alternatively, the content of closed-cell can be determined using a pycnometer or indirectly from the ASTM D6226 method. The foam has a cell count ranging from about 1.0 million cells/inch3to about 2.0 million cells/inch3, or from about 1.2 million cells/inch3to about 1.7 million cells/inch3. The cell count can be obtained by methods known in the art, for example, by calculating the number of cells in an unit area in an optical micrograph. The polymer composition is capable of being used to make a low density foam having any suitable density, which may be in the range from about 0.005 g/cc to about 0.60 g/cc, from about 0.01 g/cc to about 0.20 g/cc, or from about 0.10 g/cc to about 0.20 g/cc. The foam has a shear modulus (i.e., foam modulus) of about 12,000 Pa or more and typically not more than 20,000 Pa at 20° C. In some embodiments, the foam has a shear modulus of 2,500 Pa or more and typically not more than 5,000 Pa at100° C. As understood by one skilled in the art, the term “shear modulus” describes the response of the material to shear stress, and is defined as the ratio of shear stress to the shear strain. Shear modulus is also commonly referred to as the modulus of rigidity. The shear modulus is measured by methods known to one skilled in the art, for example, dynamic mechanical analysis and ASTM D4065. The foam disclosed herein exhibit unexpected improved properties including but not limited to high closed-cell content and high rigidity. Method for Producing the Foam The foam can be prepared with a single extrusion in an extruder such as one or more single-screw extruders or a twin-screw extruder. In some embodiments, the pelletized foamable composition is blended with any of the fillers and/or additives listed herein, and/or melted at an increased temperature before the blowing agent is added. In other embodiments, the copolymer, polyolefin, and the blowing agent can be pre-blended in a gravimetric feeder and then fed to the extruder. In some embodiments, the extruder has a cooling barrel extension and an integrated static mixer. The total L/D ratio can be at least 40, although other ratios are possible. The extrusion throughput can be at least 22 kg/hr, although higher or slower throughputs are possible. The resultant mixture can be combined with a physical blowing agent, such as liquefied carbon dioxide, in an extruder fitted with a die. The extruder melts the mixture and mixes it with the physical blowing agent. The resulting melt mixture would then be extruded through the die. A pressure drop at the die would provide for expansion of the blowing agent(s), and the polymer composition would form a foam. Optionally, the die geometry could provide for a foamed strand or a foamed sheet to be produced. In the case of an annular die, the foam could be drawn over a mandrel, then cooled and slit. The diameter of the annular die can be at least 50 mm, although larger or smaller diameters are possible, depending on the desired size of the foam. A ratio of the die diameter to the mandrel diameter can be at least 2:1, although other ratios are possible. A sheet or a fabricated article comprising the polymer composition having a resulting foam structure can be made following the foaming step. The sheet or the fabricated article can be used in packaging, automotive, and insulation applications. Examples of a fabricated article include but are not limited to thermoformable, foamed films and sheets, lightweight packaging trays, beakers and containers, microwaveable food packaging, technical foams for automotive applications such as headliners, carpet backing, door liners, parcel shelves, water shields, under-the-hood acoustic panels, cushioning and protective packaging, and thermal and acoustic insulation, and any other suitable article or combination thereof Another aspect of the invention relates to a foamable composition, containing: (i) about 1 wt % to about 15 wt % of an impact copolymer, based on a total weight of the foamable composition, and (ii) about 85 wt % to about 99 wt % of a polyolefin, based on the total weight of the foamable composition, where the impact copolymer has a melt flow rate of about 1 g/10 min or less, the polyolefin has a melt flow rate of about 2 g/10 min or more, a melt strength of about cN or more at 190° C., and the foamable composition is a blend. A foam containing such a composition can have a density ranging from about 0.01 g/cc to about 0.20 g/cc and a closed-cell content of more than about 90%. Another aspect of the invention relates to a foamable composition, containing: (i) up to about 20 wt % of a long-chain branched impact copolymer, based on a total weight of the foamable composition, and (ii) about 80 wt % or more of a polyolefin, based on the total weight of the foamable composition, where the long-chain branched impact copolymer has a melt flow rate of about 1 g/10 min or less, the polyolefin has a melt flow rate of about 2 g/10 min or more, a melt strength of about 30 cN or more at 190° C., and the foamable composition has a zero-shear viscosity of about 10,000 Pa·s or less at 190° C. A foam containing such a composition can have a density ranging from about 0.01 g/cc to about 0.20 g/cc and a closed-cell content of more than about 90%. Additional aspects, advantages and features of the invention are set forth in this specification, and in part will become apparent to those skilled in the art on examination of the following, or may be learned by practice of the invention. The inventions disclosed in this application are not limited to any particular set of or combination of aspects, advantages and features. It is contemplated that various combinations of the stated aspects, advantages and features make up the inventions disclosed in this application. EXAMPLES The following examples are given as particular embodiments of the invention and to demonstrate the practice and advantages thereof. It is to be understood that the examples are given by way of illustration and are not intended to limit the specification or the claims that follow in any manner. Foamable Composition The foamable compositions containing polymer A and polymer B are summarized in Table 1. Polymer A is a high melt strength modified polypropylene homopolymer produced by reacting polypropylene with a poly(sulfonyl azide). Polymer B is a polypropylene-based copolymer, which can be a random copolymer (RCP), an impact copolymer (ICP), or a long- chain branched impact copolymer (LCB-ICP). Following the weight percentages listed in Table 1, appropriate amounts of the polymer pellets were dry-blended with additives in a gravimetric mixing feeder. The resultant mixture was melted in an extruder and then extruded. Zero-Shear Viscosity The zero-shear viscosity of the foamable composition was measured using an Anton Paar MCR 501 rheometer with a 25-mm 6° cone and plate fixture. Samples were tested using a 0.445-mm gap and allowed to equilibrate at 200° C. for 10 minutes. A creep recovery experiment was run in stress controlled mode. A force of 100 Pa was applied for 500 s and the sample strain was recorded. The force was stopped and sample allowed to recover for 1200 s. The zero-shear viscosity was calculated from the creep phase using: η=t*τσ where η is the viscosity of the material, t is time, τ is the shear stress, and a is the strain on the material taken when τ/σ reached steady state. Melt Flow Rate As used herein, the term “melt flow rate” (MFR) (units of g/10 min or dg/min) is described according to and measured per ASTM D1238 using a load of 2.16 kg at 230° C. Rheotens Melt Strength The extensional flow of the polymer melt was characterized using a Gottfert Rheotester 2000 capillary rheometer equipped with a Rheotens 71.97 set-up. The analysis determines the resistance of the polymer melt to stretching (i.e., melt strength) and its extensibility in a given test condition. A 12-mm capillary barrel was used at a barrel temperature of 190° C. The molten polymer was soaked at the test temperature for 5 minutes prior to the test. A polymer strand was pushed through a 20-mm/2-mm L/D capillary die with a 180° entrance angle at an apparent wall shear rate of about 86 s−1. The polymer strand was then fed into the Rheotens unit and grabbed by two sets of two wheels. The wheel speed was adjusted to reduce the acting force on the polymer strand to approximately zero. Once steady-state was achieved, the speed of the counter- rotating wheels was continuously increased, which deformed the polymer strand until fracture and/or slippage. The polymer strand resistance force to deformation was measured by the Rheotens unit. The peak force recorded during the drawing process is referred to as “melt strength.” The peak velocity is referred to as “velocity at break.” Foam Extrusion Polymer pellets were dry-blended with other additives using a 4-component, gravimetric mixing feeder. ECOCELL° 20 P, supplied by Polyfil, was added as a chemical blowing agent (0-0.10 wt %). The foam extrusion system consists of a single screw extruder with a cooling barrel extension and an integrated static mixer to provide a total extruder L/D=40. Extrusion throughput rate was 22 kg/hr. Liquid carbon dioxide (1 wt %) was used as a physical blowing agent and added directly to the extruder barrel through a positive displacement pump. The extruder was fitted with a 50-mm annular die, and the extrudate was stretched over a mandrel and then slit. The ratio of die diameter to mandrel diameter (blow up ratio) was 2:1. Density Measurement Density, ρ, was determined by using a 100-mm 2 square piece of sample. The mass (m) measured on an analytical balance was divided by the volume calculated from the dimensions, length (l), width (w) and height (h) of the sample measured with a caliper, according to the equation: ρ=mlwh Closed-Cell Content The closed-cell content was measured using a Quantichrome Ultrafoam 1200 e pycnometer (V5.04). Each sample was cut into three pieces with an approximate area of 3-inch2. The exact dimensions of the pieces were measured by a caliper and entered into the equipment to calculate the total volume, V(geometric). First, the mass of N2gas required to stabilize the pressure of an empty cylinder of known volume to 3 psig was measured. This was the calibrating amount of gas. Then, the sample was placed in the cylinder, the cylinder sealed the calibrating amount of gas was introduced, and the resulting pressure was measured. The pressure difference between the empty cylinder and the cylinder holding the sample is proportionally related to the volume occupied by the closed cells of sample present, V(pycnometer), because the gas diffuses into the open cells. The closed-cell content was calculated according to the expression: %Closedcell=V(pycnometer)V(geometric)×100 Cell Count Foam cell morphology was analyzed through high definition images acquired using an optical microscope (Hirox) with magnification at 35X or 50X, depending on the cell density. A small sample of foamed sheet was cut with a surgical blade along a diagonal relative to the machine direction. The cut surface was colored using a blue ink marker to enhance visual contract, and the sample was placed on the microscope stage. The micrograph area, A (μm2), and cell count, NA, were recorded. The number of cells per unit area NA, was used to calculate the number of cells per volume, N, using this equation: N=(NAA)32 Modulus of Foam The mechanical responses of polymer foams were measured over a broad range of temperatures using dynamic mechanical analysis (DMA). Samples were tested in shear mode using a DMA Q800 instrument produced by TA Instruments. Foamed sheets were cut to test pieces with an area of 10 mm2. Two equal-size pieces of the same material were sheared between a fixed and moveable plate at a strain of 0.1% as temperature was increased from −100° C. to +150° C. The shear modulus, G′, was reported as a function of temperature. The dynamic mechanical properties of the foamed PP sheets were measured in shear using an RDA III rheometer (TA Instruments) outfitted with a torsion/rectangular fixture. All of the samples were die-cut parallel to the machine direction (MD). In addition, a sample was analyzed in the transverse direction (TD). Data were collected over the temperature range using a 3° C/minute heating rate and a 1 Hz deformation frequency. All measurements were made in a dry nitrogen environment. While a formal error analysis was not performed on these samples, based on historical data on homogeneous samples, the dynamic moduli are estimated to be accurate to within ±10%. Properties of the Foamable Composition and the Foam The experimental results for the foamable composition and the foam are presented in Table 1. The properties of each polymer are shown in Table 2. Comparative Example 1 is polymer A, which is a high melt strength polypropylene. Inventive Example 1 is a blend of polymer A with 10% of a random copolymer (RCP). Comparative Example 2 is a blend of polymer A with 20% RCP. Inventive Example 2 is a blend of polymer A with 10% of a long-chain branched impact copolymer (LCB-ICP). Inventive Example 3 is a blend of polymer A with 20% LCB-ICP. Inventive Example 4 is a blend of polymer A with 10% of an impact copolymer (ICP). Comparative Example 3 is a blend of polymer A with 20% ICP. Table 1 shows that all polymer blends (Inventive Examples 1-4 and Comparative Examples 2 and 3) have higher melt strengths than Comparative Example 1. Inventive Examples 1-4 have lower zero-shear viscosities than Comparative Examples 2 and 3. All foams containing the polymer blends are stiffer (as shown by the higher foam moduli) than Comparative Example 1 at 20° C. Further, Inventive Examples 2 and 4 are stiffer than Comparative Example 1 at 100° C. Inventive Examples 1-4 have closed-cell contents of more than 80%, which are higher than the closed-cell contents of Comparative Examples 2 and 3. Further, Inventive Examples 2 and 4 have closed-cell contents of at least 90%, which are higher than the closed-cell contents of Inventive Examples 1 and 3. TABLE 1Experimental data for inventive and comparative examples that include foamable compositions and foamsInvention Ex 1Comp Ex 2Invention Ex 2Invention Ex 3Invention Ex 4Comp Ex 3ExampleComp Ex 1Polymer A +Polymer A +Polymer A +Polymer A +Polymer A +Polymer A +PolymerPolymer A10% RCP20% RCP10% LCB-ICP20% LCB-ICP10% ICP20% ICPCompo-wt % Polymer A100908090809080sitionwt % Polymer B0102010201020Velocity at break224166169173167169165(mm/s)Final melt11.312.314.713.212.411.712.1strength (cN)Zero-shear viscosity62501118017294819210685870712335at 190° C. (Pa · s)Melt flow rate11.46.23.78.15.97.75.8after blending*(g/10 min)FoamFoam density, g/cc0.170.200.270.190.190.180.21propertiesFoam cell count,1.01.43.11.31.71.31.06million cells/in3% closed cells94833592849033in foamFoam modulus G′8276103451379313793124141241417241at 20° C. (Pa)Foam modulus G′2069206932072759193127593793at 100° C. (Pa)CommentsBaselineLow closed-Low closed-cell contentcell content*Melt flow rate measured after one pass in the extruder TABLE 2Properties of each polymer prior to blendingPolymer BPolymerPolymer ARCPLCB-ICPICPMelt flow rate (g/10 min)2.50.50.50.5wt % ethylene propylene001010rubberMelt strength (cN)484959.523.3Velocity at break (mm/s)178106133110 | 42,450 |
11859074 | DETAILED DESCRIPTION In the present disclosure, it has been found that a partially fluorinated fluoropolymer can be cured with a fluorinated polyol compound of Formula I and the conjugate base thereof. The fluoropolymers of the present disclosure are partially fluorinated polymers. As disclosed herein, an amorphous partially fluorinated polymer is a polymer comprising at least one carbon-hydrogen bond and at least one carbon-fluorine bond on the backbone of the polymer. In one embodiment, the partially fluorinated polymer is highly fluorinated, wherein at least 60, 70, 80, or even 90% of the polymer backbone comprises C—F bonds. The fluoropolymer of the present disclosure also comprises carbon-carbon double bonds and/or is capable of forming carbon-carbon double bonds along the polymer chain. In one embodiment, the partially fluorinated fluoropolymer comprises carbon-carbon double bonds along the backbone of the partially fluorinated fluoropolymer or is capable of forming carbon-carbon double bonds along the backbone of the partially fluorinated fluoropolymer. In another embodiment, the partially fluorinated fluoropolymer comprises carbon-carbon double bonds or is capable of forming carbon-carbon double bonds in a pendent group off of the backbone of the partially fluorinated fluoropolymer. The fluoropolymer capable of forming carbon-carbon double bonds means that the fluoropolymer contains units capable of forming double bonds. Such units include, for example, two adjacent carbons, along the polymer backbone or pendent side chain, wherein a hydrogen is attached to the first carbon and a leaving group is attached to the second carbon. During an elimination reaction (e.g., thermal reaction, and/or use of acids or bases), the leaving group and the hydrogen leave forming a double bond between the two carbon atoms. An exemplary leaving group includes: a fluoride, an alkoxide, a hydroxide, a tosylate, a mesylate, an amine, an ammonium, a sulfide, a sulfonium, a sulfoxide, a sulfone, and combinations thereof. Those fluoropolymers capable of forming carbon-carbon bonds generally have the structure ˜CH—CX˜, where the tilde is a bond and X is a leaving groups such that when treated with base will provide the requisite unsaturation. In many embodiments the polymer has ˜CH—CF˜ in the backbone, which may be dehydrofluorinated. The fluoropolymer comprises a plurality of these groups (carbon-carbon double bonds or groups capable of forming double bonds) to result in a sufficient cure. Generally, this means at least 0.1, 0.5, 1, 2, or even 5 mol %; at most 7, 10, 15, or even 20 mole % (i.e., moles of these carbon-carbon double bonds or precursors thereof per mole of polymer). In one embodiment, the amorphous partially fluorinated polymer is derived from at least one hydrogen containing monomer such as vinylidene fluoride. In one embodiment, the amorphous fluoropolymer comprises adjacent copolymerized units of vinylidene fluoride (VDF) and hexafluoropropylene (HFP); copolymerized units of VDF (or tetrafluoroethylene) and a fluorinated comonomer capable of delivering an acidic hydrogen atom to the polymer backbone, such as trifluoroethylene; vinyl fluoride; 3,3,3-trifluoropropene-1; pentafluoropropene (e.g., 2-hydropentafluoropropylene and 1-hydropentafluoropmpylene); 2,3,3,3-tetrafluoroproperie; and combinations thereof. In some embodiments, small amounts (e.g., less than 10, 5, 2, or even 1 wt %) of additional monomers may be added so long as the amorphous fluoropolymer is able to be cured using the curing agent disclosed herein. In one embodiment, the amorphous fluoropolymer is additionally derived from a hydrogen containing monomer including: pentafluoropropylene (e.g., 2-hydropentafluropropylene), propylene, ethylene, isobutylene, and combinations thereof. In one embodiment, the amorphous fluoropolymer is additionally derived from a perfluorinated monomer. Exemplary perfluorinated monomers include: hexafluoropropene; tetrafluoroethylene; chlorotrifluoroethylene; perfluoro(alkylvinyl ether) such as perfluoromethyl vinyl ether, CF2═CFOCFCF2CF2OCF3, CF2═CFOCF2OCF2CF2CF3, CF2═CFOCF2OCF2CF3, CF2═CFOCF2OCF3, and CF2═CFOCF2OC3F7, perfluoro(alkylallyl ether) such as perfluoromethyl allyl ether, perfluoro(alkyloxyallyl ether) such as perfluoro-4,8-dioxa-1-nonene (i.e., CF2═CFCF2O(CF2)3OCF3, and combinations thereof. Exemplary types of polymers include those comprising interpolymerized units derived from (i) vinylidene fluoride, tetrafluoroethylene, and propylene; (ii) vinylidene fluoride, tetrafluoroethylene, ethylene, and perfluoroalkyl vinyl ether, such as perfluoro(methyl vinyl ether); (iii) vinylidene fluoride with hexafluoropropylene; (iv) hexafluoropropylene, tetrafluoroethylene, and vinylidene fluoride; (v) hexafluoropropylene and vinylidene fluoride, (vi) vinylidene fluoride and perfluoroalkyl vinyl ether; (vii) vinylidene fluoride, tetrafluoroethylene, and perfluoroalkyl vinyl ether, (viii) vinylidene fluoride, perfluoroalkyl vinyl ether, hydropentafluoroethylene and optionally, tetrafluoroethylene; (ix) tetrafluoroethylene, propylene, and 3,3,3-trifluoroproperte; (x) tetrafluoroethylene, and propylene; (xi) ethylene, tetrafluoroethylene, and perfluoroalkyl vinyl ether, and optionally 3,3,3-trifluoropropylene; (xii) vinylidene fluoride, tetrafluoroethylene, and perfluoroalkyl allyl ether, (xiii) vinylidene fluoride and perfluoroalkyl allyl ether; (xiv) ethylene, tetrafluoroethylene, and perfluoroalkyl vinyl ether, and optionally 3,33-trifluoropropylene; (xv) vinylidene fluoride, tetrafluoroethylene, and perfluoroalkyl allyl ether, (xvi) vinylidene fluoride and perfluoroalkyl allyl ether; (xvii) vinylidene fluoride, tetrafluoroethylene, and perfluoroalkyloxyallyl ether, (xviii) vinylidene fluoride and perfluoroalkyloxyallyl ether; (xiv) vinylidene fluoride, tetrafluoroethylene, and perfluoroalkyloxyallyl ether, (xv) vinylidene fluoride and perfluoroalkyloxyallyl ether; and (xvi) combinations thereof. Advantageously, by using the curing agent disclosed herein, the amorphous fluoropolymers of the present disclosure can be cured without the need for pendent bromine, iodine, or nitrile cure sites along the polymer backbone. Often, the iodine and bromine-containing cure site monomers, which are polymerized into the fluoropolymer and/or the chain ends, can be expensive among other things. The amorphous fluoropolymer of the present disclosure is substantially free of I, Br, and nitrile groups, wherein the amorphous fluoropolymer comprises less than 0.1, 0.05, 0.01, or even 0.005 mole percent relative to the total polymer. In one embodiment, the amorphous fluoropolymers of the present disclosure are non-grafted, meaning that they do not comprise pendant groups including vinyl, allyl, acrylate, amido, sulfonic acid salt, pyridine, carboxylic ester, carboxylic salt, hindered silanes that are aliphatic or aromatic tri-ethers or tri-esters. In one embodiment, the amorphous fluoropolymer does not comprise a monophenol graft. The above described amorphous fluoropolymers may be blended with one or more additional crystalline fluoropolymers. With the instant curing compounds, the crystalline fluoropolymers may be cured into the matrix of the amorphous fluoropolymer Commercially available crystalline fluoropolymers include, for example, those fluoropolymers having the trade designation “THV” (e.g., “THV 200”, “THV 400”, “THV 500”, “THV 610”, or “THV 800”) as marketed by Dyneon, St. Paul, Minn.; “KYNAR” (e.g., “KYNAR 740”) as marketed by Atofina, Philadelphia, Pa.; “HYLAR” (e.g., “HYLAR 700”) as marketed by Ausimont USA, Morristown, N.J.; and “FLUOREL” (e.g., “FLUOREL FC-2178”) as marketed by Dyneon. Useful fluoropolymers also include copolymers of HFP, TFE, and VDF (i.e., THV). These polymers may have, for example, VDF monomeric units in a range of from at least about 2, 10, or 20 percent by weight up to 30, 40, or even 50 percent by weight, and HFP monomeric units in a range of from at least about 5, 10, or 15 percent by weight up to about 20, 25, or even 30 percent by weight, with the remainder of the weight of the polymer being TFE monomeric units. Examples of commercially available THV polymers include those marketed by Dyneon, LLC under the trade designations “3M DYNEON FLUOROPLASTIC THV 221GZ”, “3M DYNEON FLUOROPLASTIC THV 221AZ”, “3M DYNEON FLUOROPLASTIC THV 415GZ”, “3M DYNEON FLUOROPLASTIC THV 815GZ”, “3M DYNEON FLUOROPLASTIC THV 500GZ”, “3M DYNEON FLUOROPLASTIC THV 610GZ”, “3M DYNEON FLUOROPLASTIC ET 6218Z”, “3M DYNEON FLUOROPLASTIC ET 6235Z”, “DYNEON FLUOROTHERMOPLASTIC THV 2030G”, “DYNEON FLUOROTHERMOPLASTIC THV 220”, “DYNEON FLUOROTHERMOPLASTIC THV 415”, “DYNEON FLUOROTHERMOPLASTIC THV 500A”, “DYNEON FLUOROTHERMOPLASTIC THV 610G”, or “DYNEON FLUOROTHERMOPLASTIC THV 810G”. Useful fluoropolymers also include copolymers of ethylene, TFE, and HFP. These polymers may have, for example, ethylene monomeric units in a range of from at least about 2, 10, or 20 percent by weight up to 30, 40, or even 50 percent by weight, and HFP monomeric units in a range of from at least about 5, 10, or 15 percent by weight up to about 20, 25, or even 30 percent by weight, with the remainder of the weight of the polymer being TFE monomeric units. Such polymers are marketed, for example, under the trade designation “DYNEON FLUOROTHERMOPLASTIC HTE” (e.g., “DYNEON FLUOROTHERMOPLASTIC HTE X 1510” or “DYNEON FLUOROTHERMOPLASTIC HTE X 1705”) by Dyneon LLC. Useful fluoropolymers also include copolymers of tetrafluoroethylene and propylene (TFE/P). These copolymers may have, for example, TFE monomeric units in a range of from at least about 20, 30 or 40 percent by weight up to about 50, 65, or even 80 percent by weight, with the remainder of the weight of the polymer being propylene monomeric units. Such polymers are commercially available, for example, under the trade designations “AFLAS” (e.g., “AFLAS TFE ELASTOMER FA 100H”, “AFLAS TFE ELASTOMER FA 150C”, “AFLAS TFE ELASTOMER FA 150L”, or “AFLAS TFE ELASTOMER FA 150P”) as marketed by Dyneon, LLC, or “VITON” (e.g., “VITON VTR-7480” or “VITON VTR-7512”) as marketed by E.I. du Pont de Nemours & Company, Wilmington, Del. Useful fluoropolymers also include copolymers of ethylene and TFE “ETFE”). These copolymers may have, for example, TFE monomeric units in a range of from at least about 20, 30 or 40 percent by weight up to about 50, 65, or even 80 percent by weight, with the remainder of the weight of the polymer being propylene monomeric units. Such polymers may be obtained commercially, for example, as marketed under the trade designation “DYNEON FLUOROTHERMOPLASTIC ET 6235” by Dyneon LLC. VDF-containing fluoropolymers can be prepared using emulsion polymerization techniques as described, for example, in U.S. Pat. No. 4,338,237 (Sulzbach et al.) or U.S. Pat. No. 5,285,002 (Grootaert), the disclosures of which are incorporated herein by reference. The curable composition further comprises a fluorinated sulfonamide polyol curing agent of the formula: wherein Rfis a fully or partially fluorinated group; each R1is independently a (hetero)hydrocarbyl group selected from alkylene, ether or polyether (oxyalkylene or poly(oxyalkylene)), and each R2is R1—OH, H, alkyl or aryl; and subscript c is 1 or 2, with the proviso that the crosslinking component has at least two R1—OH groups. In some embodiments R1and/or R2may comprise an ether or a polyether of the formula —(R5O)yR5—, where R5is an alkylene group having from 2 to about 4 carbon atoms, such as —CH2CH2—, —CH2CH2CH2—, —CH(CH3)CH2—, and —CH(CH3)CH(CH3)—, and y is a number between about 1 and about 15 inclusive. The oxyalkylene units in said polyether can be the same, such as in poly(oxypropylene) or poly(oxyethylene), or can be present as a mixture, such as in a straight or branched chain of randomly distributed oxyethylene and oxypropylene units, i.e., poly(oxyethylene-co-oxypropylene), or as in straight or branched chain blocks of oxypropylene units. The ether group is terminated for example with a hydroxyl to provide the R1—OH, or the R2may be terminated with a with a C1-C15alkyl group, an aryl group or combination thereof. Specific examples of ether/polyether groups include:—(CH2CH2O)yC8H17,—(CH2CH2O)yH,—(CH2CH2O)yCH[CH2CH(CH3)2][CH2CH(CH3)CH2CH(CH3)2],—(CH2CH2O)yC10H21,—(CH2CH2OyC6H4—C(CH3)2—CH2—C(CH3)3,—(CH2CH(CH3)O)yCH2CH3], and—(CH2CH(CH3)O)yH, wherein y is 1-15. It will be appreciated that Formula I include compounds of the formula; where Rfis a fully or partially fluorinated group; each R1is independently a (hetero)hydrocarbyl group selected from alkylene, ether or polyether; and subscript c is 1 or 2. Compounds of Formula I will also include compounds of the formula Rfis a fully or partially fluorinated group; each R4is a (hetero)hydrocarbyl group selected from H, monovalent aryl, alkyl or ether, polyether or —R1—OH. The Rfgroups can contain straight chain, branched chain, or cyclic polyvalent perfluorinated groups in any combination and are of the general formula: —CnF2n−for divalent groups, —CnF2n-1— for trivalent groups, —CnF2n-2— for tetravalent groups, etc. Divalent groups with n=3 to 8 being more preferred and with n=2 to 5 being the most preferred. The compounds of Formula I will include the corresponding salts, or conjugate bases. Minor amounts of hydrogen or chlorine atoms can also be present as substituents, provided that no more than one atom of either is present for every two carbon atoms. Preferably the Rfgroup is entirely perfluorinated. The perfluoroalkylene groups may comprise 1 to 10 carbon atoms, preferably 2 to 6 carbon atoms. A typical divalent perfluoroalkylene is —CF2—CF2—, —CF2—CF2—CF2—, —CF(CF3)—CF2—, —CF2—, —CF2—CF2—CF2—CF2—CF2—CF2—, cyclic —C6F12— or CF(CF3)—. In some embodiments the Rfgroup may be selected from perfluoroether groups:[Rf13—O—Rf14—(Rf15)q]—[(CH2)y—OH]x, IV where[Rf13—O—Rf14—(Rf15)q] has a valence of x from abstraction of two or more F atoms from any of the Rf13, Rf14, or Rf15groups,Rf13represents a perfluoroalkylene group,Rf14represents a perfluoroalkyleneoxy group,Rf15represents a perfluoroalkylene group and q is 0 or 1subscript y is 1 to 8; andsubscript xis 2 to 4. Compounds of Formulas I-III are generally prepared by alkylation of the corresponding fluorinated sulfonamides, which in turn are prepared by reaction of the fluorinated sulfonyl fluorides with ammonia. Reference may be made to U.S. Pat. Nos. 2,803,656, 2,732,398 (Brice), U.S. Pat. No. 4,533,713 (Howells) and U.S. Pat. No. 9,725,683 (Lamanna et al.), each incorporated herein by reference. Compounds of Formula I in which R2is not R1—OH may be prepared by a first reaction of a fluorinated sulfonyl fluoride with an amine of the formula R2—NH2, followed by a second alkylation of the sulfonamide product with a compound of the formula X—R1—OH, where X is a leaving groups such as halide or tosylate. Alternatively, a sulfonamide may be sequentially alkylated with R2—X and X—R1—OH, in either order. It will be understood that a mixture of products of Formula I will result. The curing agent should be used in quantities substantial enough to cause the amorphous fluoropolymer to cure, as indicated by a rise in torque on a moving die rheometer. For example, at least 0.5-20 parts of the crosslinking agent per 100 parts of the amorphous fluoropolymer is used. If too little curing agent is used, the amorphous fluoropolymer will not cure. If too much curing agent is used, the amorphous fluoropolymer can become brittle. For example, no more than 20 millimoles of the curing agent per 100 parts of the amorphous fluoropolymer is used. One or a blend of polyol compounds with Formula I may be used. In addition to the polyol curing agents of Formula I, the curable composition may optionally include a second, optional crosslinking agent. Examples of the optional crosslinking agent include polyol compounds, polythiol compounds, polyamine compounds, amidine compounds, bisaminophenol compounds, oxime compounds, and the like. In some embodiments, the second crosslinking agent may comprise a non-fluorinated hydrocarbyl polyol analogous to Formula I. Generally, examples are not restricted for selecting the specific combination of the sulfonamides of Formula I and secondary crosslinking agent and/or crosslinking promoter, depending on the type of polymer, but typical examples are presented below. For example, with a vinylidene fluoride system (binary system or ternary system), a polyol compound, polyamine compound, polythiophen compound is preferable. With a tetrafluoroethylene-propylene-vinylidene fluoride-based fluorine rubber (ternary) system, polyol compound, polyamine compound, polythiol compound, or the like is preferable. Examples of preferable polyol compounds include 2,2-bis(4-hydroxyphenyl) hexafluoropropane, 4,4′-dihydroxy diphenyl sulfone, 4,4′-diisopropylidene diphenol, and the like. Examples of preferable polythiol compounds include 2-dibutyl amino-4,6-dimercapto-s-triazine, 2,4,6-trimercapto-s-triazine, and the like. Examples of preferable polyamine compounds include hexamethylene diamine carbamate, N,N′-dicinnamylidene-1,6-hexanediamine, 4,4′-methylene bis(cyclohexylamine) carbonate, and the like. Examples of preferable amidine compounds include p-toluene sulfonate salts of 1,8-diazabicyclo[5.4.0]undec-7-ene, and the like. Examples of preferable bisaminophenol compounds include 2,2-bis(3-amino-4-hydroxyphenyl))-hexafluoropropane, 2,2-bis[3-amino-4-(N-phenylamino) phenyl]hexafluoropropane, and the like. In some embodiments, a combination of polyols of Formula I may be combined with a secondary fluorinated compounds of the Formula Z-Q-Rf—O—(Rfo)Rf-Q-Z, as described in U.S. Pat. Nos. 5,384,374 and 5,266,650, Guerra et al, each incorporated herein by reference. If using an optional second crosslinking agent, the molar ratios of the polyol curing agent of Formula I to the second crosslinking agent may be from 5:1 to 1:1. The curable composition may further comprise an acid acceptor including organic, inorganic, or blends of thereof. Examples of inorganic acceptors include magnesium oxide, lead oxide, calcium oxide, calcium hydroxide, dibasic lead phosphate, zinc oxide, barium carbonate, strontium hydroxide, calcium carbonate, hydrotalcite, etc. Organic acceptors include amines, epoxies, sodium stearate, and magnesium oxalate. Particularly suitable acid acceptors include calcium hydroxide, magnesium oxide and zinc oxide. Blends of acid acceptors may be used as well. The amount of acid acceptor will generally depend on the nature of the acid acceptor used. If the presence of an extractable metal compound is not desirable (such as semiconductor applications), the use of inorganic acid acceptors should be minimized, and these preferably should not be used at all. For example, a hardening composition with a formula that does not use an inorganic acid acceptor is particularly useful for sealing materials and gaskets for manufacturing semiconductor elements, sealing materials that are in contact with water, hot water, or the like, and sealing materials for high temperature areas such as automotive applications. Examples of preferred acid acceptors that are commonly used include zinc oxide, calcium hydroxide, calcium carbonate, magnesium oxide, hydrotalcite, silicon dioxide (silica), lead oxide, and the like. These compounds are generally used in order to bond with HF and other acids. These acids are possibly produced at high temperatures that can be encountered during the hardening process when molding a molded article using the fluoropolymer composition, or at temperatures that demonstrate the function of fluoropolymers and the like. In one embodiment, at least 0.5, 1, 2, 3, or even 4 parts of the acid acceptor per 100 parts of the amorphous fluoropolymer are used. In one embodiment, no more than 10, 7, or even 5 parts of the acid acceptor per 100 parts of the amorphous fluoropolymer are used. The curable composition may further comprise an organo onium compound added to the composition as a phase transfer catalyst to assist with the crosslinking of the amorphous fluoropolymer and/or may be used to generate the double bonds on the fluoropolymer through dehydrofluorination. Such organo onium compounds include quaternary ammonium hydroxides or salts, quaternary phosphonium hydroxides or salts, and ternary sulfonium hydroxides or salts. Briefly, a phosphonium and ammonium salts or compounds comprise a central atom of phosphorous or nitrogen, respectively, covalently bonded to four organic moieties by means of a carbon-phosphorous (or carbon-nitrogen) covalent bonds and is ionically associated with an anion. The organic moieties can be the same or different. Briefly, a sulfonium compound is a sulfur-containing organic compound in which at least one sulfur atom is covalently bonded to three organic moieties having from 1 to 20 carbon atoms by means of carbon-sulfur covalent bonds and is ionically associated with an anion. The organic moieties can be the same or different. The sulfonium compounds may have more than one relatively positive sulfur atom, e.g. [(C6H5)2S+(CH2)4S+(C6H5)2Cl−, and two of the carbon-sulfur covalent bonds may be between the carbon atoms of a divalent organic moiety, i.e., the sulfur atom may be a heteroatom in a cyclic structure. Many of the organo-onium compounds useful in this invention are described and known in the art. See, for example, U.S. Pat. No. 4,233,421 (Worm), U.S. Pat. No. 4,912,171 (Grootaert et al.), U.S. Pat. No. 5,086,123 (Guenthner et al.), and U.S. Pat. No. 5,262,490 (Kolb et al.), U.S. Pat. No. 5,929,169, all of whose descriptions are herein incorporated by reference. Another class of useful organo-onium compounds include those having one or more pendent fluorinated alkyl groups. Generally, the most useful fluorinated onium compounds are disclosed by Coggio et al. in U.S. Pat. No. 5,591,804. Exemplary organo onium compounds include: C3-C6symmetrical tetraalkylammonium salts, unsymmetrical tetraalkylammonium salts wherein the sum of alkyl carbons is between 8 and 24 and benzyltrialkylammonium salts wherein the sum of alkyl carbons is between 7 and 19 (for example tetrabutylammonium bromide, retrabutylammonium chloride, benzyltributylammonium chloride, benzyltriethylammonium chloride, tetrabutylammonium hydrogen sulfate and tetrabutylammonium hydroxide, phenyltrimethylammonium chloride, tetrapentylammonium chloride, tetrapropylammonium bromide, tetrahexylammonium chloride, and tetraheptylammonium bromidetetramethylammonium chloride); quaternary phosphonium salts, such as tetrabutylphosphonium salts, tetraphenylphosphonium chloride, benzyltriphenylphosphonium chloride, tributylallylphosphonium chloride, tributylbenzyl phosphonium chloride, tributyl-2-methoxypropylphosphonium chloride, benzyldiphenyl(dimethylamino)phosphonium chloride, 8-benzyl-1,8-diazobicyclo[5.4.0]7-undecenium chloride, benzyltris(dimethylamitio)phosphonium chloride, and bis(benzyldiphenylphosphine)iminium chloride. Other suitable organo onium compounds include 1,8-diazabicyclo[5.4.0]undec-7-ene and 1,5-diazabicyclo[4.3.0]non-5-ene. Phenolate is a preferred anion for the quaternary ammonium and phosphonium salts. In one embodiment, the organo onium compound is used between 1 and 5 millimoles per 100 parts of the amorphous fluoropolymer (mmhr). The fluoropolymer composition can also contain various additives in addition to the aforementioned components. Examples of additives include crosslinking auxiliary agents and/or crosslinking promoting auxiliary agents that combine favorably with the crosslinking agent and/or crosslinking promoter used, fillers (such as carbon black, flowers of zinc, silica, diatomaceous earth, silicate compounds (clay, talc, wollastonite, and the like), calcium carbonate, titanium oxide, sedimentary barium sulfate, aluminum oxide, mica, iron oxide, chromium oxide, fluoropolymer filler, and the like), plasticizers, lubricants (graphite, molybdenum disulfide, and the like), release agents (fatty acid esters, fatty acid amides, fatty acid metals, low molecular weight polyethylene, and the like), colorants (cyanine green and the like), and processing aids that are commonly used when compounding fluoropolymer compositions, and the like. However, these additives are preferably sufficiently stable under the intended conditions of use. Furthermore, the carbon black can be used to achieve a balance between fluoropolymer composition properties such as tensile stress, tensile strength, elongation, hardness, wear resistance, conductivity, processability, and the like. Preferable examples include MT blacks under the product numbers N-991, N-990, N-908, and N-907 (medium thermal black); FEF N-550; and large diameter furnace black, and the like. If carbon black is used, the amount is preferably from approximately 0.1 to approximately 70 mass parts (phr) based on 100 mass parts of the total amount of polymer containing fluorinated olefin units and the additional polymer. This range is particularly preferable for the case where large particle furnace black is used The curable amorphous fluoropolymer compositions may be prepared by mixing the amorphous fluoropolymer, the curing agent, along with the other components (e.g., the acid acceptor, the onium compound, and/or additional additives) in conventional rubber processing equipment to provide a solid mixture, i.e. a solid polymer containing the additional ingredients, also referred to in the art as a “compound”. This process of mixing the ingredients to produce such a solid polymer composition containing other ingredients is typically called “compounding”. Such equipment includes rubber mills, internal mixers, such as Banbury mixers, and mixing extruders. The temperature of the mixture during mixing typically will not rise above about 120° C. During mixing the components and additives are distributed uniformly throughout the resulting fluorinated polymer “compound” or polymer sheets. The “compound” can then be extruded or pressed in a mold, e.g., a cavity or a transfer mold and subsequently be oven-cured. In an alternative embodiment curing can be done in an autoclave. Curing is typically achieved by heat-treating the curable amorphous fluoropolymer composition. The heat-treatment is carried out at an effective temperature and effective time to create a cured fluoroelastomer. Optimum conditions can be tested by examining the cured fluoroelastomer for its mechanical and physical properties. Typically, curing is carried out at temperatures greater than 120° C. or greater than 150° C. Typical curing conditions include curing at temperatures between 160° C. and 210° C. or between 160° C. and 190° C. Typical curing periods include from 3 to 90 minutes. Curing is preferably carried out under pressure. For example, pressures from 10 to 100 bar may be applied. A post curing cycle may be applied to ensure the curing process is fully completed. Post curing may be carried out at a temperature between 170° C. and 250° C. for a period of 1 to 24 hours. The partially fluorinated amorphous fluoropolymer in the curable composition has a Mooney viscosity in accordance with ASTM D1646-06 TYPE A by a MV 2000 instrument (available from Alpha Technologies, Ohio, USA) using large rotor (ML 1+10) at 121° C. Upon curing, using the curing agent disclosed herein, the amorphous fluoropolymer becomes an elastomer, becoming a non-flowing fluoropolymer, and having an infinite viscosity (and therefore no measurable Mooney viscosity). The above curable compositions can be compounded or mixed in one or several steps, and then the mixture can be processed and shaped, for example, by extrusion (for example, in the form of a hose or hose lining) or molding (for example, in the form of an O-ring seal). The shaped article can then be heated to cure the composition and form a cured elastomer article. In some embodiments the desired amounts of conventional additives adjuvants or ingredients are added to the uncured compositions and intimately admixed or compounded therewith by employing any of the usual rubber mixing devices such as Banbury mixers, roll mills, or any other convenient mixing device. The temperature of the mixture on the mill typically will not rise above about 120° C. During milling the components and adjuvants are distributed uniformly throughout the gum. The curing process typically comprises extrusion of the compounded mixture or pressing the compounded mixture in a mold, e.g., a cavity or a transfer mold, and subsequent oven-curing. Pressing of the compounded mixture (press cure) is usually conducted at a temperature between about 95 and about 230° C., preferably between about 150° C. and about 205° C. for a period of from 1 minute to 15 hours, typically from 5 minutes to 30 minutes. A pressure of between about 700 kPa and about 20,600 kPa is usually imposed on the compounded mixture in the mold. The molds first may be coated with a release agent, such as a silicone oil, and prebaked. The molded vulcanizate is then usually post-cured (oven-cured) at a temperature usually between about 150° C. and about 315° C. for a period of from about 2 hours to 50 hours or more depending on the cross-sectional thickness of the article. The compositions of this invention can be used to form seals, O-rings and gaskets. The cured fluorocarbon elastomer mixture has excellent low-temperature flexibility while retaining the desired physical properties, for example tensile strength and elongation, of conventionally compounded and cured compositions. Particularly useful articles that can be fabricated from the fluorocarbon elastomer compositions of this invention are particularly useful as seals, gaskets, and molded parts in automotive, chemical processing, semiconductor, aerospace, and petroleum industry applications, among others. EXAMPLES Unless otherwise noted, all parts, percentages, ratios, etc. in the Examples and the rest of the specification are by weight. Unless otherwise indicated, all other reagents were obtained, or are available from fine chemical vendors such as Sigma-Aldrich Company, St. Louis, Mo., or may be synthesized by known methods. Table 1 (below) lists materials used in the examples and their sources. TABLE 1Materials ListDESIGNATIONDESCRIPTIONFC 2145A low-viscosity copolymer of hexafluoropropylene and vinylidenefluoride that does not include an incorporated curative, availableunder the trade designation “3M DYNEON FLUOROELASTOMERFC 2145” from 3M Company, Maplewood, MN, USA.TPBPCTriphenylbenzyl phosphonium chloride, available from Sigma-Aldrich(St. Louis, MO, USA), diluted to 50% by weight in methanol, alsoavailable from Sigma-Aldrich.C1Bisphenol-AF, 2,2-Bis(4-hydroxyphenyl) hexafluoropropane,available from Sigma-Aldrich.C5FBSEE Diol, C4F9—SO2—N(C2H4—OH)2, prepared as described in USPatent application 2010/0160458, page 3, [0058].C313EO Diol, C4F9—SO2—N(C2H4—OH)(C4H4OC2H4—OH), prepared asdescribed in Synthesis 4 of US Pat. No. 9725683.C324EO Diol, C4F9—SO2—N(C2H4OC2H4—OH)2, prepared as described inSynthesis 3 of US Pat. No. 9725683.Ca(OH)2An acid acceptor. Calcium hydroxide commercially available underthe trade designation “HALLSTAR CALCIUM HYDROXIDE HP-XL”from The Hallstar Company, Chicago, IL, USAMgOAn acid acceptor. Magnesium oxide powder commercially availableunder the trade designation “ELASTOMAG 170” from AkrochemCorp., Akron, OH, USAN990Carbon black, available under the trade designation “N990” fromCancarb, Medicine Hat, Alberta, Canada Compounding 150 g batches of fluoropolymer were compounded with 0.78 parts per hundred resin (phr) of TPBPC, various amounts of curing agent as indicated in Table 2, 6 phr of Ca(OH)2, 3 phr of MgO, and 20 phr of N990 carbon black, using a two-roll mill. Milling continued until a homogeneous blend formed. Ca(OH)2, MgO, and N990 were added as a mixture. The ratios of each component in examples and comparative examples are indicated as phr in Table 2, below. TABLE 2CompoundingEXAMPLECE-1EX-1EX-2EX-3EX-4EX-5EX-6FC 2145, g100100100100100100100TPBPC, phr0.780.780.780.780.780.780.78C1, phr2.02——————C5, phr—2.324.65————C31, phr———2.595.18——C32, phr—————2.855.70Ca(OH)2, phr6666666MgO, phr3333333N990, phr20202020202020 Cure Rheology Cure rheology tests were carried out using uncured, compounded samples using a moving die rheometer (MDR) marketed under the trade designation RPA 2000 by Alpha technologies, Akron, Ohio, in accordance with ASTM D 5289-93a at 177° C. (pre-heated), 12 minute elapsed time, and a 0.5 degree arc. The minimum torque (ML), maximum torque (Mu), the time for the torque to reach a value equal to ML+0.5(MH−ML), (t′50), and the time for the torque to reach ML+0.9(MH−ML), (t′90), the scorch time (Ts2), and Tan delta at maximum torque were measured and their values are listed in Table 3. Press-Cure Molding and Physical Property Test The compound was press-cured using a mold (size: 75 millimeters (mm)×150 mm×2 mm or 150 mm×150 mm×2 mm) at 6.5×103kilopascals (kPa) and 177° C. for 10 minutes. Then the elastomer sheets were removed, cooled to room temperature, and then used for physical property test and post-cure. The dog bone specimens were cutout from the sheets with ASTM Die D and subjected to physical property testing similar to the procedure disclosed in ASTM D412-06a (2013). The typical tensile strength deviation is +/−1.4 MPa (200 psi). The typical elongation deviation is +/−25%. Hardness is +/−2. Since this is a destructive test, it doesn't follow a normal bell shape distribution. It is Weibull distribution. The test results are summarized in Table 3. Post-Cure and Physical Property Test The press-cured elastomer sheet was post cured at 232° C. for 16 hours in a circulating air oven. The samples were then removed from the oven, cooled to room temperature, and physical properties determined. The dog bone specimens were cutout from the sheets with ASTM Die D and subjected to physical property testing similar to the procedure disclosed in ASTM D412-06a (2013). The test results are summarized in Table 3. Heat-Aging and Physical Property Test The dog bone specimens of post cured samples were placed in a circulating air oven for 70 hours at 270° C. The samples were then removed from the oven and cooled to room temperature for measurement of physical properties according to ASTM D412-06a (2013). The test results are summarized in Table 3. O-Ring Molding and Compression Set Test O-rings having a cross-section thickness of 0.139 inch (3.5 mm) were molded at 6.5×103kPa and 177° C. for 10 minutes and then post-cured at 232° C. for 16 hours. The O-rings were subjected to compression set testing similar to the procedure disclosed in ASTM 395-89 method B (2008), with 25% initial deflection. Results of compression tests are reported in Table 3. The typical deviation is +/−2% to 3%. TABLE 3Curable fluoropolymer curing characteristics,and properties of cured fluoropolymersCE-1*EX-1**EX-2EX-3EX-4EX-5EX-6Curing CharacteristicsMoving Die Rheometer, 0.5° at 177° C. (350° F.) 12 minute motorMinimum torque, ML, N · m0.0610.2420.3570.1000.0690.0680.040Maximum torque, MH, N · m1.7930.5680.8490.5090.6690.6100.727MH-ML, N · m1.7320.3260.4930.4090.6000.5410.687Ts2, minutes0.620.870.60.650.590.831t′50, minutes0.730.4790.670.570.70.941.38t′90, minutes1.062.822.793.353.163.745.53Tan delta at maximum torque0.0420.2010.0930.1890.1060.1430.11Properties of Cured GumstocksPhysical Properties, press cure 10 minutes at 177° C. (350° F.)Durometer, shore A66*6357605857Tensile, MPa10.2*9.710.29.811.69.7Elongation, %239*206558405504480100% Modulus, MPa3.3*3.41.52.11.71.6Physical Properties, post cure 16 hours at 232° C. (450° F.)Durometer, shore A67*6363686266Tensile, MPa14.1*11.013.115.113.214.4Elongation, %191**190321214311244100% Modulus, MPa4.5**4.02.54.82.54.2Physical Properties, heat aged 70 hours at 270° C. (518° F.)Durometer, shore A74*6865796978Tensile, MPa7.4*9.710.111.411.512.6Elongation, %151*218324134222123100% Modulus, MPa4.5*4.12.58.44.311.0Compression Set 70 hours at 200° C. (392° F.)Compression Set, %21.1**4562.7626770.5*Did not form a good molded product due to scorching;**Processable to form a molded sample, but some scorching observed All cited references, patents, and patent applications in the above application for letters patent are herein incorporated by reference in their entirety in a consistent manner. In the event of inconsistencies or contradictions between portions of the incorporated references and this application, the information in the preceding description shall control. The preceding description, given in order to enable one of ordinary skill in the art to practice the claimed disclosure, is not to be construed as limiting the scope of the disclosure, which is defined by the claims and all equivalents thereto. | 37,031 |
11859075 | DESCRIPTION OF EMBODIMENTS The invention is specifically described below. The particle of the invention relates to a core-shell particle including a core containing a perfluoropolymer and a shell containing a non-fluorine resin, the core having a periphery coated with the non-fluorine resin at a coverage of 90% or more. The particle of the invention has a core-shell, structure including a core containing a perfluoropolymer and a shell containing a non-fluorine resin. The “core-shell structure” is a conventionally known structure and is the structure of a primary particle in an aqueous dispersion producible by the method disclosed in U.S. Pat. No. 6,841,594 B, for example. In conventional core-shell particles including a core containing a perfluoropolymer and a shell containing a non-fluorine resin, the core is unfortunately insufficiently coated with the shell. The particle of the invention, including a core coated with a non-fluorine resin at a coverage of 90% or more, shows very good dispersibility and thus can be suitably used in various applications. The coverage can be calculated as follows. First, element mapping images of carbon and fluorine for a target particle are prepared with a transmission electron microscope. The element mapping images of carbon and fluorine are superimposed with each other. Using image analyzing software, the superimposed images are binarized and separated into a core particle portion containing fluorine and a carbon portion circumferentially present on the periphery of the core particle portion. Then, the length of the periphery of the core particle portion containing fluorine is determined by selecting 50 or more points and measuring the lengths therebetween by section length measurement in a manual manner. The coverage Z (%) can be calculated by the following formula: Z=(Y/(X+Y))×100 wherein X represents the length (nm) of a part where the inner periphery of the particle containing fluorine and the outer periphery of the particle containing carbon are not superimposed (a part where the core particle is not coated with the shell) and Y represents the length (nm) of a part where the periphery of the particle containing fluorine and the periphery of the particle containing carbon are superimposed with each other (a part where the core particle is coated with the shell). A specific method for calculating the coverage is described in the examples below. The particle of the invention has the aforementioned core-shell structure and thus has excellent dispersibility. Such a core-shell structure can also improve the dispersibility of a dispersion containing the particles of the invention (primary particles) and a powder (secondary particles) formed from the particles of the invention. Furthermore, the presence of the core containing a perfluoropolymer enables the powder or dispersion formed from the particles of the invention to have excellent slidability, excellent non-stickiness, and low dielectricity when the dispersion or powder is used with a different material. The coverage of the core coated with a non-fluorine resin in the particle of the invention is preferably 95% or more. More preferably, the core is completely coated with the shell (coverage: 100%). An example of the particle of the invention is an emulsion particle prepared through emulsion polymerization of a monomer composition that constitutes a core part (a monomer composition containing a monomer such as tetrafluoroethylene, perfluoro(alkyl vinyl ether), or hexafluoropropylene) and a monomer composition that constitutes a shell part (a monomer composition used for producing the non-fluorine resin). The “perfluoropolymer” as used herein means a polymer mainly containing a polymerized unit based on a perfluoro monomer, optionally containing a polymerized unit based on a monomer other than the perfluoro monomer. The polymerized unit based on a perfluoro monomer is preferably present in an amount of 90 mol % or more relative to all the polymerized units, for example. The perfluoropolymer is preferably polytetrafluoroethylene (PTFE), a tetrafluoroethylene (TFE)/perfluoro(alkyl vinyl ether) (PAVE) copolymer (PFA), or a TFE/hexafluoropropylene (HFP) copolymer (FEP). The PTFE may be melt-fabricable PTFE having a low molecular weight or non-melt-fabricable PTFE. When the particle is used for a dripping inhibitor, for example, the PTFE is preferably non-melt-fabricable PTFE. Non-melt-fabricable PTFE allows the particle of the invention to more significantly exhibit its dispersibility. The PTFE may be a tetrafluoroethylene (TFE) homopolymer or a copolymer of a TFE unit based on TFE and a modifying monomer unit based on a monomer other than TFE (hereinafter, also referred to as a “modifying monomer”). The modified PTFE preferably contains the modifying monomer unit in an amount of 0.001 to 1.0% by mass, more preferably 0.01 to 0.50% by mass, still more preferably 0.02 to 0.30% by mass, of all the monomer units. The modifying monomer may be any modifying monomer copolymerizable with TFE, and examples thereof include perfluoroolefins such as hexafluoropropylene (HFP); chlorofluoroolefins such as chlorotrifluoroethylene (CTFE); hydrogen-containing fluoroolefins such as trifluoroethylene and vinylidene fluoride (VDF); fluoro alkyl vinyl ether; fluoroalkyl ethylene; ethylene; and fluorine-containing vinyl ethers containing a nitrile group. One or more of these modifying monomers may be used. An example of the fluoroalkyl vinyl ether is, but not limited to, a fluoromonomer represented by the following formula (1): CF2═CF—ORf1(1) (wherein Rf1is a perfluoro organic group). The “perfluoro organic group” as used herein means an organic group in which hydrogen atoms binding to any carbon atom are all substituted with fluorine atoms. The perfluoro organic group optionally contains ether oxygen. An example of the fluoroalkyl vinyl ether is a fluoromonomer represented by the formula (1) wherein Rf1is a C1-C10 perfluoroalkyl group. The perfluoroalkyl group preferably has a carbon number of 1 to 5. Examples of the perfluoroalkyl group in the fluoroalkyl vinyl ether include a perfluoromethyl group, a perfluoroethyl group, a perfluoropropyl group, a perfluorobutyl group, a perfluoropentyl group, and a perfluorohexyl group. Preferred is perfluoro(propyl vinyl ether) (PPVE) in which the perfluoroalkyl group is a perfluoropropyl group. Examples of the fluoroalkyl vinyl ether further include a fluoroalkyl vinyl ether represented by the formula (1) wherein Rf1is a C4-C9 perfluoro(alkoxy alkyl) group; a fluoroalkyl vinyl ether represented by the formula (1) wherein Rf1is a group represented by the following formula: (wherein m is an integer of 0 or 1 to 4); and a fluoroalkyl vinyl ether represented by the formula (1) wherein Rf1is a group represented by the following formula: (wherein n is an integer of 1 to 4). A preferred example of the fluoroalkyl ethylene is, but not limited to, (perfluoroalkyl)ethylene. Examples thereof include (perfluorobutyl)ethylene (PFBE) and (perfluorohexyl)ethylene. The PTFE preferably has a standard specific gravity (SSG) of 2.13 to 2.30, more preferably 2.14 to 2.29, still more preferably 2.15 to 2.28. SSG refers to a value obtained by a method in conformity with ASTM D-4895-89. The “high molecular weight PTFE” as used herein means a PTFE having a standard specific gravity that falls within the above range. The PTFE preferably has a melt viscosity at 380° C. of 1×102to 7×105Pa·s. The “low molecular weight PTFE” as used herein means a PTFE having a melt viscosity that falls within the above range. The melt viscosity can be measured in conformity with ASTM D 1238. Specifically, the melt viscosity of a 2-g sample previously heated at a measurement temperature (380° C.) for five minutes is measured at the temperature and a load of 0.7 MPa using a flow tester and a 2ϕ-8 L die. The PFA is a copolymer containing a TFE unit and a PAVE unit. Examples of the PAVE constituting the PFA include at least one selected from the group consisting of a PAVE represented by the formula (1): CF2═CFO(CF2CFY1O)p—(CF2CF2CF2O)q—Rf(1) (wherein Y1is F or CF3; Rfis a C1-C5 perfluoroalkyl group; p is an integer of 0 to 5; and q is an integer of 0 to 5); and a PAVE represented by the formula (2): CFX═CXOCF2OR1(2) (wherein Xs are the same as or different from each other and are each F or CF3; and R1is a linear or branched C1-C6 perfluoroalkyl group or a C5 or C6 cyclic perfluoroalkyl group). Specific examples thereof include perfluoro(methyl vinyl ether) (PMVE), perfluoro(ethyl vinyl ether) (PEVE), perfluoro(propyl vinyl ether) (PPVE), and perfluoro(butyl vinyl ether) (PBVE). The PAVE is preferably a PAVE having a bulky side chain, specifically preferably PPVE. The PFA preferably contains a polymerized unit based on PAVE in an amount of 1.0 to 10% by mass relative to all the polymerized units. The amount of the polymerized unit based on PAVE is more preferably 2.0% by mass or more, still more preferably 3.5% by mass or more, particularly preferably 4.0% by mass or more, while more preferably 8.0% by mass or less, still more preferably 7.0% by mass or less, particularly preferably 6.5% by mass or less, relative to all the polymerized units. The amount of the polymerized unit based on PAVE is measured by19F-NMR. The PFA preferably contains the polymerized unit based on TFE and the polymerized unit based on PAVE in a total amount of 90 mol % or more, more preferably 95 mol % or more, relative to all the polymerized units. The PFA also preferably consists only of the polymerized unit based on TFE and the polymerized unit based on PAVE. The PFA preferably has a melting point of 280° C. to 322° C. The melting point is more preferably 290° C. or higher, while more preferably 315° C. or lower. The melting point is the temperature corresponding to the maximum value on a heat-of-fusion curve drawn using a differential scanning calorimeter (DSC) at a temperature-increasing rate of 10° C./min. The PFA can be produced by a conventionally known method, such as a method including mixing monomers to constitute the units of the PFA and an additive such as a polymerization initiator as appropriate and performing emulsion polymerization or suspension polymerization. The FEP includes a TFE unit and a HFP unit. The FEP preferably gives a mass ratio between the TFE unit and the HFP unit (TFE/HFP) of (70 to 99)1(1 to 30) (% by mass), more preferably (85 to 95)/(5 to 15) (% by mass). The FEP also preferably contains a PAVE unit in addition to the TFE unit and the HFP unit, namely, is preferably a TFE/HFP/PAVE copolymer. Examples of the PAVE unit contained in the FEP include the same as those described for the PAVE unit constituting PFA. More preferred among those is PPVE. The FEP preferably contains the polymerized unit based on TFE, the polymerized unit based on HFP, and the polymerized unit based on PAVE in a total amount of 90 mol % or more, more preferably 95 mol % or more, relative to all the polymerized units. The FEP may consist only of the polymerized unit based on TFE and the polymerized unit based on HFP or of the polymerized unit based on TFE, the polymerized unit based on HFP, and the polymerized unit based on PAVE. In the case of a TFE/HFP/PAVE copolymer, the FEP preferably has a mass ratio (TFE/HFP/PAVE) of (70 to 99.8)/(0.1 to 25)/(0.1 to 25) (% by mass). A mass ratio within the above range can lead to better heat resistance. The mass ratio (TFE/HFP/PAVE) is more preferably (75 to 98)/(1.0 to 15)1(1.0 to 10) (% by mass). The TFE/HFP/PAVE copolymer contains the HFP unit and the PAVE unit in a total amount of 1% by mass or more. The TFE/HFP/PAVE copolymer preferably contains the HFP unit in an amount of 25% by mass or less of all the monomer units. The HFP unit in an amount within the above range can lead to better heat resistance. The amount of the HFP unit is more preferably 20% by mass or less, still more preferably 18% by mass or less, particularly preferably 15% by mass or less, while preferably 0.1% by mass or more, more preferably 1% by mass or more, particularly preferably 2% by mass or more. The amount of the HFP unit can be measured by19F-NMR. The TFE/HFP/PAVE copolymer contains the PAVE unit in an amount of more preferably 20% by mass or less, still more preferably 10% by mass or less, particularly preferably 3% by mass or less, while preferably 0.1% by mass or more, more preferably 1% by mass or more. The amount of the PAVE unit can be measured by19F-NMR. The FEP may further contain an ethylenic monomer (a) unit. The ethylenic monomer (a) unit may be any monomer unit copolymerizable with a TFE unit and a HFP unit, and further a PAVE unit in the case of a TFE/HFP/PAVE copolymer. Examples thereof include fluorine-containing ethylenic monomers such as vinyl fluoride (VF), vinylidene fluoride (VdF), and chlorotrifluoroethylene (CTFE) and non-fluorinated ethylenic monomers such as ethylene, propylene, and alkyl vinyl ether. In the case of a TFE/HFP/PAVE/ethylenic monomer (a) copolymer, the polymer preferably has a mass ratio (TFE/HFP/PAVE/ethylenic monomer (α)) of (70 to 98)/(0.1 to 25)7(0.1 to 25)/(0.1 to 10) (% by mass), more preferably (70 to 98)1(0.1 to 25)/(0.1 to 20)/(0.1 to 5) (% by mass), still more preferably (70 to 98)/(0.1 to 20)/(0.1 to 10)/(0.1 to 3) (% by mass). The TFE/HFP copolymer contains the polymerized unit(s) other than the TFE unit in a total amount of 1% by mass or more. The FEP preferably has a melting point of 200° C. to 280° C. The melting point is more preferably 275° C. or lower, still more preferably 270° C. or lower. The melting point is the temperature corresponding to the maximum value on a heat-of-fusion curve drawn using a differential scanning calorimeter (DSC) at a temperature-increasing rate of 10° C./min. The FEP can be produced by a conventionally known method, such as a method including mixing monomers to constitute the units of the FEP and an additive such as a polymerization initiator as appropriate and performing emulsion polymerization, solution polymerization, or suspension polymerization. The PFA and FEP each preferably has a melt flow rate (MFR) of 0.1 to 100 g/10 min, more preferably 0.1 to 50 g/10 min. The MFR is the mass (g/10 min) of the polymer flowed out of a nozzle having an inner diameter of 2 mm and a length of 8 mm per 10 minutes in conformity with ASTM D1238 at 372° C. and a load of 5 kg. The PFA and FEP each preferably has an initial pyrolysis temperature (1% mass reduction temperature) of 360° C. or higher. The lower limit is more preferably 370° C. The upper limit of the initial pyrolysis temperature may be 410° C., for example, as long as it falls within the above range. The initial pyrolysis temperature refers to the temperature at which 1% by mass of the copolymer under a heating test is decomposed. It is obtained by determining the temperature at which 1% by mass of the mass of the copolymer under a heating test is reduced using a thermogravimetric-differential thermal analyzer (TG-DTA). The non-fluorine resin is a polymer containing a polymerized unit based on a non-fluorine monomer. Preferred examples of the non-fluorine monomer include, but are not limited to, at least one selected from the group consisting of acrylic esters, methacrylic esters, acrylonitrile, vinyl chloride, vinylidene chloride, vinyl acetate, styrene monomers, urethane monomers, and silicone monomers. The non-fluorine resin may be a homopolymer of one of these or a copolymer of two or more thereof. The non-fluorine resin contains substantially no polymerized unit based on a fluorine-containing monomer. In order to achieve excellent dispersibility of the particle of the invention and excellent dispersibility of the powder formed from the particles of the invention, preferred among these non-fluorine monomers is at least one selected from the group consisting of acrylic esters, methacrylic esters, vinyl chloride, vinyl acetate, styrene monomers, urethane monomers, and silicone monomers. More preferred is at least one selected from the group consisting of acrylic esters, methacrylic esters, and styrene. The acrylic ester is preferably an alkyl acrylate containing a C1-C10 alkyl group, and more preferably includes at least one alkyl acrylate selected from the group consisting of methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, cyclohexyl acrylate, and glycidyl acrylate, and still more preferably includes at least one alkyl acrylate selected from the group consisting of butyl acrylate and 2-ethylhexyl acrylate. The methacrylic ester is preferably an alkyl methacrylate containing a C1-C10 alkyl group, more preferably includes at least one alkyl methacrylate selected from the group consisting of methyl methacrylate, ethyl methacrylate, propyl methacrylate, isopropyl methacrylate, butyl methacrylate, 2-ethylhexyl methacrylate, cyclohexyl methacrylate, and glycidyl methacrylate, and still more preferably includes at least one alkyl methacrylate selected from the group consisting of methyl methacrylate and cyclohexyl methacrylate. When the non-fluorine resin contains one or both of a polymerized unit based on an acrylic ester and a polymerized unit based on a methacrylic ester, the non-fluorine resin preferably contains the polymerized unit based on an acrylic ester and the polymerized unit based on a methacrylic ester in a total amount of 20 mol % or more of the entire non-fluorine resin. The total amount of the polymerized unit based on an acrylic ester and the polymerized unit based on a methacrylic ester may be 50 mol % or more or 70 mol % or more. Examples of the styrene monomer include styrene, α-methyl styrene, α-ethyl styrene, α-propyl styrene, and α-butyl styrene. When the non-fluorine resin contains a polymerized unit based on a styrene monomer, the amount of the polymerized unit based on a styrene monomer is preferably 20 mol % or more of the entire non-fluorine resin. The amount of the polymerized unit based on a styrene monomer may be 50 mol % or more or 70 mol % or more. Examples of the urethane monomer include an addition reaction product of a (meth)acrylic monomer having a hydroxyl group at the β position and a diisocyanate compound such as toluene diisocyanate or diphenylmethane diisocyanate. When the non-fluorine resin contains a polymerized unit based on a urethane monomer, the amount of the polymerized unit based on a urethane monomer is preferably 20 mol % or more of the entire non-fluorine resin. The amount of the polymerized unit based on a urethane monomer may be 50 mol % or more or 70 mol % or more. Examples of the silicone monomer include alkoxysilanes such as methyltrimethoxysilane, dimethyldimethoxysilane, dibutyldimethoxysilane, diisopropyldipropoxysilane, and diphenyldibutoxysilane. When the non-fluorine resin contains a polymerized unit based on a silicone monomer, the amount of the polymerized unit based on a silicone monomer is preferably 20 mol % or more of the entire non-fluorine resin. The amount of the polymerized unit based on a silicone monomer may be 50 mol % or more or 70 mol % or more. The particles of the invention preferably have an average particle size of 10 to 500 nm, more preferably 20 to 400 nm. The average particle size of the particles refers to the value obtained by diluting the particle dispersion by a factor of about 1000 with water and determining the average particle size with FPAR-1000 (available from Otsuka Electronics Co., Ltd., dynamic light scattering). The cores of the particles of the invention preferably have an average particle size of 5 to 495 nm, more preferably 50 to 400 nm, still more preferably 100 to 300 nm. The average particle size of the cores refers to the average particle size of perfluoropolymer dispersion, which is the value obtained by diluting the perfluoropolymer dispersion by a factor of about 1000 with water and determining the average particle size with FPAR-1000 (available from Otsuka Electronics Co., Ltd., dynamic light scattering). The shells of the particles of the invention each preferably have a thickness of 1 to 300 nm, more preferably 2 to 200 nm, still more preferably 3 to 150 nm. The thickness of each shell refers to the value obtained by subtracting the average particle size of the perfluoropolymer particles as cores from the average particle size of a dispersion containing the particles of the invention. In order to achieve much better dispersibility, the particle of the invention preferably has a mass ratio between the core and the shell (core/shell) of 99.9/0.1 to 10/90, more preferably 99/1 to 30/50, still more preferably 95/5 to 80/20. The particle of the invention preferably has a spherical shape. The expression “spherical” as used herein involves not only a true sphere but also shapes whose cross section has a curved outline such as a circle, an ellipse, a substantial circle, or a substantial ellipse. A spherical particle can be obtained by the production method below. The particle of the invention preferably has an aspect ratio of 1.4 or less, more preferably 1.2 or less. The aspect ratio refers to the value obtained by dividing the longer diameter of a particle by the shorter diameter thereof. The longer diameter and the shorter diameter can be measured on an image of the particle taken with a transmission electron microscope, for example. The particle of the invention can be produced by adding a non-fluorine monomer to a perfluoropolymer dispersion and polymerizing the non-fluorine monomer. The polymerization of the non-fluorine monomer causes formation of a shell of the non-fluorine resin. The perfluoropolymer dispersion can be prepared by a known method. The perfluoropolymer dispersion preferably contains particles containing a perfluoropolymer and an aqueous medium. The perfluoropolymer in the perfluoropolymer dispersion is the same as the perfluoropolymer constituting the core of the particle of the invention. The aqueous medium contains water. The aqueous medium may contain a polar organic solvent in addition to water. Examples of the polar organic solvent include nitrogen-containing solvents such as N-methylpyrrolidone (NMP); ketones such as acetone; esters such as ethyl acetate; polar ethers such as diglyme and tetrahydrofuran (THF); and carbonates such as diethylene carbonate. One of these may be used or two or more thereof may be used in mixture. The perfluoropolymer dispersion preferably contains particles containing a perfluoropolymer in an amount of 1 to 70% by mass, more preferably 10 to 30% by mass, relative to the entire weight. The perfluoropolymer dispersion preferably contains an emulsifier represented by the following formula (A): R2(—O—(CH2CH2O)mH)p(A) (wherein R2is a linear or branched C5-C50 hydrocarbon group with a valence of p, m is an integer of 5 to 30, and p is an integer of 1 to 3) in an amount of 1 ppm or less relative to the entire weight. The presence of the emulsifier in the perfluoropolymer dispersion tends to cause generation of different particles derived only from a non-fluorine monomer in addition to the core-shell particles of the invention. The perfluoropolymer dispersion more preferably contains substantially no emulsifier. The hydrocarbon group in the formula (A) is a group containing a carbon atom and a hydrogen atom and optionally contains a carbon-carbon double bond, a carbon-carbon triple bond, a phenylene group, or the like, but contains no metal atoms such as Si. Examples of the emulsifier represented by the formula (A) include an emulsifier represented by the following formula (A1): CnH2n+1—(Ph)p—O—(CH2CH2O)mH (A1) (wherein n is an integer of 5 to 50, p is 0 or 1, m is an integer of 5 to 30, and Ph is a phenylene group), and an emulsifier represented by the following formula (A2): H(OCH2CH2)r—O—CR3R4—C≡C—CR3R4—O—(CH2CH2O)rH (A2) (wherein R3and R4are each a linear or branched C1-C50 hydrocarbon group, and r is an integer of 5 to 30). In the above formula, CnH2n+1— may be linear or branched. The non-fluorine monomer is preferably polymerized by seed polymerization in which a particle containing a perfluoropolymer is used as a seed particle and a non-fluorine monomer is emulsion polymerized in water. As described, the seed polymerization, which is a kind of emulsion polymerization and in which polymerization is performed using a perfluoropolymer dispersion for a core as a seed and adding a non-fluorine monomer for a shell to the dispersion, can suitably form a particle having a double structure, i.e., a core-shell particle, in which the entire surface of the spherical core particle is coated with a shell polymer while keeping the shape of the spherical core particle. The non-fluorine monomer(s) is/are preferably added in a total amount of 1.0 to 50 parts by mass, more preferably 5.0 to 20 parts by mass, relative to 100 parts by mass of the particles containing a perfluoropolymer. The polymerization of the non-fluorine monomer is preferably initiated by adding a polymerization initiator. The polymerization initiator may be added while the non-fluorine monomer is polymerized. The polymerization initiator used for polymerization of the non-fluorine monomer may be any polymerization initiator usable for free radical reaction in water. The polymerization initiator may be used in combination with a reducing agent in some cases. Examples of a water-soluble polymerization initiator used include persulfates and hydrogen peroxide. Examples of the reducing agent include sodium pyrobisulfite, sodium bisulfite, sodium L-ascorbate, and rongalit. Examples of an oil-soluble polymerization initiator include diisopropyl peroxydicarbonate (IPP), benzoyl peroxide, dibutyl peroxide, and azobisisobutyronitrile (AIBN). The polymerization initiator is preferably used in an amount of 0.05 to 5.0 parts by mass for each 100 parts by mass of the non-fluorine monomer. In order to produce the core-shell particle of the invention, the production method preferably includes adding a non-fluorine monomer and a polymerization initiator simultaneously to a perfluoropolymer dispersion and initiating polymerization. In the production method, the non-fluorine monomer may be added to the reaction system collectively, intermittently, or continually. The non-fluorine monomer is preferably added intermittently or continually, more preferably continually. The production method includes adding a non-fluorine monomer to a perfluoropolymer dispersion and polymerizing the non-fluorine monomer to produce a core-shell particle. The non-fluorine monomer is preferably continually added from the starting of the polymerization, not from the initial stage (before starting of polymerization). The non-fluorine monomer is preferably polymerized by adding a specific emulsifier in addition to the non-fluorine monomer. In other words, the production method preferably includes adding the non-fluorine monomer and a specific emulsifier to a perfluoropolymer dispersion and polymerizing the non-fluorine monomer. The specific emulsifier may be added to a reaction system collectively at the initial stage, intermittently, or continually. The specific emulsifier is preferably an emulsifier that tends to preferentially adsorb on the particle surface of a perfluoropolymer dispersion, and a preferred example is an anionic emulsifier having a high effect of giving polymerization stability. A silicone emulsifier is also preferred because it has an SP value (solubility parameter) close to that of a perfluoropolymer dispersion particle and thus increases the miscibility with the particle surface, which expectedly gives an adsorption effect. Examples of the anionic emulsifier include alkyl sulfonate, alkyl aryl sulfonic acid, alkyl sulfosuccinic acid, a polyoxyethylene aryl sulfate, and an ammonium salt of polyoxyethylene polycyclic phenyl ether sulfate. Preferred among these are alkyl sulfonate, alkyl aryl sulfonic acid, alkylsulfosuccinic acid, and an ammonium salt of a polyoxyethylene polycyclic phenyl ether and an organosulfate. One emulsifier may be used or two or more emulsifiers may be used in combination. The silicone emulsifier is preferably selected from silicone derivatives containing a polyoxyalkylene group in which a polyether group such as a polyethylene oxide or a polypropylene oxide is introduced into each terminal or a side chain with polyether-modified silicone oil. Such a silicone emulsifier can reduce the surface tension of the particle surface and increase the wettability to particles. Examples of the polyether-modified silicone include polyether-modified siloxane, polyether-modified dimethyl siloxane, polyether-modified polydimethylsiloxane, polyether-modified polymethylalkylsiloxane, polyester-modified polydimethylsiloxane, polyester-modified polymethylalkylsiloxane, aralkyl-modified polymethylalkylsiloxane, and polyester-modified hydroxyl-group-containing polydimethylsiloxane. In consideration of polymerization stability, preferred is one having a molecular weight of 1000 to 5000 and an HLB (Si) of 7 to 15. One emulsifier may be used or two or more emulsifiers may be used in combination. Furthermore, an anionic emulsifier and a silicone emulsifier may be used in combination. Addition of the specific emulsifier can prevent formation of particles (different particles) consisting of the non-fluorine monomer and enables production of a dispersion containing a particle that includes a core containing a perfluoropolymer and a shell containing a non-fluorine resin. The absence of the specific emulsifier may cause formation of the different particles and fail in forming a shell. Use of an emulsifier other than the specific emulsifier may reduce the compatibility with the core containing a perfluoropolymer and cause incomplete polymerization in which the surface of the core particle is partly polymerized. Specific examples of the emulsifier include anionic emulsifiers such as Newcol 707SF and silicone emulsifiers such as BYK-348, KF-6013, and KF-6204. The specific emulsifier(s) is/are preferably added in a total amount of 0.1 to 5.0 parts by mass, more preferably 1.0 to 2.0 parts by mass, relative to 100 parts by mass of the particles containing a perfluoropolymer. In polymerization of the non-fluorine monomer, additives such as a chain transfer agent, a chelating agent, and a pH adjuster may be added in addition to the non-fluorine monomer, the specific emulsifier, and the polymerization initiator. Examples of the chain transfer agent include halogenated hydrocarbons such as chloroform and carbon tetrachloride; and mercaptans such as n-dodecyl mercaptan, tert-dodecyl mercaptan, and n-octyl mercaptan. The chain transfer agent is preferably used in an amount of 0 to 5.0 parts by mass, more preferably 0.1 to 3.0 parts by mass, for each 100 parts by mass of the non-fluorine monomer. The polymerization temperature and polymerization duration of the non-fluorine monomer may be appropriately set in accordance with the target non-fluorine resin. For example, the polymerization temperature may be 5° C. to 80° C., and the polymerization duration may be 10 to 300 min. The dispersion of the invention contains the particles of the invention. Conventionally, primary particles of a perfiuoropolymer tend to have poor dispersibility in solvents and poor miscibility with other dispersions. In contrast, the dispersion of the invention, containing the particles of the invention, allows excellent dispersibility in solvents and improved miscibility with other dispersions. The dispersion of the invention preferably contains the particles of the invention in an amount of 1 to 70% by mass, more preferably 5 to 50% by mass, still more preferably 10 to 30% by mass. The dispersion of the invention preferably contains an aqueous medium in addition to the particles of the invention. The aqueous medium contains water. The aqueous medium may contain a polar organic solvent in addition to water. Examples of the polar organic solvent include nitrogen-containing solvents such as N-methylpyrrolidone (NMP); ketones such as acetone; esters such as ethyl acetate; polar ethers such as diglyme and tetrahydrofuran (THF); and carbonates such as diethylene carbonate. One of these may be used or two or more thereof may be used in mixture. The dispersion of the invention preferably contains the emulsifier represented by the formula (A) in an amount of 1 ppm or less, more preferably 0.1 ppm or less. Still more preferably, the dispersion of the invention contains substantially no emulsifier. The amount of the emulsifier can be determined by ion chromatography. The dispersion of the invention preferably contains particles consisting only of a non-fluorine resin in an amount of 1% or less, more preferably 0.1% or less. The dispersion of the invention still more preferably contains substantially no particles consisting only of a non-fluorine resin. The amount of the particles consisting only of a non-fluorine resin can be determined with a transmission electron microscope by taking a STEM image (representing the total amount of the non-fluorine resin and the perfluoropolymer) and an EDS mapping of fluorine (representing the perfluoropolymer only) and comparing the area proportion occupied by both of the non-fluorine resin and the perfluoropolymer with the area proportion occupied by the perfluoropolymer only. The dispersion of the invention may contain components such as a water soluble polymer (e.g., polyvinyl alcohol), an organic solvent, and various additives in addition to the particles of the invention and the aqueous medium. The dispersion of the invention can be obtained by the method for producing the particle of the invention. Specifically, the dispersion of the invention can be produced by a production method including adding a non-fluorine monomer to a perfluoropolymer dispersion and polymerizing the non-fluorine monomer. The dispersion of the invention may be used in combination with a dispersion containing a different polymer other than the particle of the invention. The dispersion of the invention, containing the particles of the invention, allows excellent dispersibility in and miscibility with a dispersion containing a different polymer, and thus can provide a dispersion with the particles of the invention and the different polymer being uniformly mixed. As described, a liquid composition containing the particles of the invention and a different polymer is also an aspect of the invention. The liquid composition preferably contains a liquid medium such as water or an organic solvent. Examples of the different polymer include, but are not limited to, epoxy resin, silicone resin, and polyimide. In the case of preparing such a mixed dispersion, the ratio between the particles of the invention and the different polymer may be appropriately set according to the application. For example, the mass ratio (particles of the invention/different polymer) may be 10/90 to 90/10. The powder of the invention contains the particles of the invention. The powder of the invention contains secondary particles which are aggregates of the particles of the invention. Conventionally, secondary particles of a perfluoropolymer tend to have poor dispersibility in and miscibility with solvents and other resins. In contrast, the powder of the invention, formed from the particles of the invention, achieves significantly improved dispersibility in and miscibility with solvents and other resins. The average particle size of the powder of the invention may be appropriately set according to the intended use thereof, for example, and is preferably 1 to 1000 μm, more preferably 3 to 500 μm, for example. The average particle size is determined by measurement using a laser diffraction particle size distribution analyzer (HELOS & RODOS) available from JEOL Ltd. at a dispersion pressure of 1.0 bar without cascade impaction and taking the particle size corresponding to 50% of the cumulative volume in the particle size distribution as the average particle size. The powder of the invention preferably contains the emulsifier represented by the formula (A) in an amount of 1 ppm or less, more preferably 0.1 ppm or less. Still more preferably, the powder of the invention contains substantially no emulsifier. The amount of the emulsifier can be determined by ion chromatography. The powder of the invention preferably contains spherical particles. Spherical particles can achieve much better dispersibility with other materials. Whether the particles have spherical shapes or not can be confirmed by observing the particles with a scanning electron microscope or a video microscope. The powder of the invention can be obtained by coagulating a dispersion containing the particles of the invention and separating, recovering, and drying the coagulated matter, for example. Alternatively, the powder of the invention can be obtained by spray drying a dispersion containing the particles of the invention. Spray drying enables easy production of spherical secondary particles, and a powder obtained by such a method has much better dispersibility. The spray drying is preferably performed under the conditions of a disk rotation speed of 8000 to 12000 rpm, a stock solution supply speed of 1 to 4 Kg/h, an inlet temperature of 100° C. to 150° C., and an outlet temperature of 50° C. to 80° C. The powder of the invention, having excellent dispersibility in and miscibility with other materials, can be suitably used as an additive to polymers such as resin and rubber. Use of the powder of the invention as an additive can efficiently impart the original properties of a perfluoropolymer, such as excellent slidability, non-stickiness, and low dielectricity. A composition containing the powder and a polymer other than a perfluoropolymer is also an aspect of the invention. An example of the polymer other than a perfluoropolymer is, but not limited to, a thermoplastic resin. Examples of the thermoplastic resin include polyolefin-based resins (e.g., polyethylene-based resins, polypropylene-based resins, polymethylpenten-based resins), polyvinyl chloride-based resins, polystyrene-based resins (e.g., polystyrene, AS, ABS), polycarbonate (PC)-based resins (e.g., PC, PC-based alloy resins such as PC/ABS), polyamide-based resins (e.g., nylon, semi-aromatic polyamide), polyester-based resins (e.g., polybutylene terephthalate, polyethylene terephthalate), acrylic resins (e.g., polymethyl methacrylate, polyacrylonitrile), polyacetal, polyether ether ketone, modified polyphenylene ether, polyarylene sulfide resins, polysulfone resins, polyvinylidene fluoride resins, and various polymer alloys. The polymer other than a perfluoropolymer is preferably a non-fluorine resin. In the case of combination use with a non-fluorine resin, the excellent dispersibility of the powder of the invention is much more improved. The polymer other than a perfluoropolymer preferably includes at least one resin selected from the group consisting of polyvinyl chloride-based resins, polyolefin-based resins (especially, polyethylene-based resins and polypropylene-based resins), nylon-based resins, polyester-based resins, and polycarbonate-based resins, more preferably at least one resin selected from the group consisting of polycarbonate-based resins and nylon-based resins, particularly preferably a polycarbonate-based resin. The composition preferably contains the powder in an amount of 0.01 to 10 parts by mass, more preferably 0.03 to 2 parts by mass, still more preferably 0.1 to 0.5 parts by mass, relative to 100 parts by mass of the polymer other than a perfluoropolymer. The powder of the invention may be added to a medium such as water or an organic solvent and thereby used as a dispersion containing the powder. The particle of the invention, the dispersion containing the particles of the invention, and the powder containing the particles of the invention can be used in various applications. The powder containing the particles of the invention can be used as a dripping inhibitor, an additive for coating material, a slidability-imparting agent, a low-dielectricity-imparting agent, a water- and oil-repellent agent, a release agent, an anti-fluttering agent, or a melt tension regulator, for example. EXAMPLES The invention is described with reference to examples, but the examples are not intended to limit the invention. The following are details of the compounding agents mentioned in the examples, comparative examples, and tables. (Perfluoropolymer Aqueous Dispersion) Low molecular weight PTFE aqueous dispersion: average primary particle size 209 nm, solid concentration 29.0%, melt viscosity 3×104Pa·s High molecular weight PTFE aqueous dispersion: average primary particle size 244 nm, solid concentration 27.4%, SSG 2.17 PFA aqueous dispersion: aqueous dispersion of TFE/PPVE copolymer, TFE/PPVE=97/3 (wt %), average particle size 297 nm, solid concentration 15.2% (Emulsifier) Newcol 707SF (anionic emulsifier, available from Nippon Nyukazai Co., Ltd.): ammonium salt of polyoxyethylene polycyclic phenyl ether sulfate, active constituents 28% KF-6204 (silicone emulsifier, available from Shin-Etsu Chemical Co., Ltd.), polyether-modified silicone, active constituents 100%, molecular weight 1600, HLB(Si) 11 Latemul E-118B (anionic emulsifier, available from KAO Corp.), sodium polyoxyethylene alkyl ether sulfate: 26% Neopelex G-15 (anionic emulsifier, available from KAO Corp.), sodium dodecylbenzene sulfonate: 20% Latemul ASK (anionic emulsifier, available from KAO Corp.), dipotassium alkenyl succinate: 28% OS soap (anionic emulsifier, available from KAO Corp.), fatty acid potassium: 16% (Polymerization Initiator) Ammonium persulfate (available from ADEKA Corp.) Potassium persulfate (available from ADEKA Corp.) Ferrous sulfate (available from Wako Pure Chemical Corporation) L-ascorbic acid (available from Fuso Chemical Co., Ltd.) Perbutyl H-69 (available from NOF Corporation), t-butyl hydroperoxide concentration 69% (Chain Transfer Agent) Thiokalcol 20 (available from KAO Corp.), n-dodecyl mercaptan The evaluations described in the examples and comparative examples were performed as follows. (1) Evaporation Residue Content A 1-g portion of the resulting dispersion was weighed on an aluminum plate and dried at 105° C. for one hour. The evaporation residue was weighed and the evaporation residue content was calculated. (2) PH The resulting dispersion was controlled to have a liquid temperature of 25° C. and the PH of the dispersion was measured with a PH meter. (3) Viscosity The resulting dispersion was controlled to have a liquid temperature of 25° C. and the viscosity of the dispersion was measured with a B-type rotational viscometer (6 rpm, No. 1 rotor). (4) Average Particle Size of Dispersion (Primary Particles) The resulting dispersion was diluted and dispersed by a factor of 1000 with water, and the average particle size of the particles was determined with FPAR-1000 (available from Otsuka Electronics Co., Ltd., dynamic light scattering). (5) Shell Thickness The thickness of the shell was determined as the half of the value obtained by subtracting the average particle size of PTFE particles corresponding to cores from the average particle size of the dispersion (primary particles) determined in (4). The average particle sizes were each determined by diluting and dispersing the dispersion by a factor of 1000 with water and determining the average particle size with FPAR-1000 (available from Otsuka Electronics Co., Ltd., dynamic light scattering). (6) Average Particle Size and Shape of Powder (Secondary Particles) The average particle size was determined by measurement using a laser diffraction particle size distribution analyzer (HELOS & RODOS) available from JEOL Ltd. at a dispersion pressure of 1.0 bar without cascade impaction and taking the particle size corresponding to 50% of the cumulative volume in the particle size distribution as the average particle size. (7) Determination of Core-Shell Particles and Shell Coverage First, 1 part by weight of the dispersion containing particles (primary particles) was diluted with 1000 parts by weight of pure water. The diluted dispersion was sprayed to a filmed sheet mesh for transmission electron microscopic observation and then dried, which allowed the particles for shell thickness measurement to attach to the sheet mesh. In order to prevent charge up, the sheet mesh was coated with osmium having a thickness of about 5 nm at a vacuum of 2 Pa for 10 seconds using an osmium coater (Neoc-Pro Neo osmium coater available from Meiwafosis Co., Ltd.). This sheet mesh sample was subjected to STEM observation of the particles using a scanning transmission electron microscope (Talos F200X available from FEI Company Japan Ltd.) at an observation magnification of 225000 times or 320000 times and an image size of 1024×1024 pixels. Scanning was performed 18 times for 189 seconds at an acceleration voltage of 200 kV. Simultaneously with the STEM observation, element mapping was performed by scanning 9 times for 189 seconds with an energy dispersive X-ray detector (Super-X available from FEI Company Japan Ltd.). The obtained element mapping images of carbon and fluorine were superimposed with each other. An image in which only the carbon atoms circumferentially present on the periphery of the particle containing fluorine are mapped was prepared with image analyzing software (ESPRIT 1,9 available from Bruker BioSpin GmbH), and whether the particles were core-shell particles or not was determined. At the same time, the shell coverage was determined. The shell coverage was determined as follows. Using image analyzing software, the superimposed images were binarized and separated into a core particle portion containing fluorine and a carbon portion circumferentially present on the periphery of the core particle portion. Then, the length of the periphery of the core particle portion containing fluorine was determined by selecting 50 or more points and measuring the lengths therebetween by section length measurement in a manual manner. The coverage Z (%) was calculated by the following formula: Z=(Y/(X+Y))×100 wherein X represents the length (nm) of a part where the inner periphery of the particle containing fluorine and the outer periphery of the particle containing carbon were not superimposed (a part where the core particle was not coated with the shell) and Y represents the length (nm) of a part where the periphery of the particle containing fluorine and the periphery of the particle containing carbon were superimposed with each other (a part where the core particle was coated with the shell). (8) Amount of Different Particles Generated The amount C (%) of different particles generated was calculated from the following formula using the STEM image (black part represents the total of the non-fluorine resin and the perfluoropolymer and this area is referred to as A) and the element mapping image of fluorine (representing the perfluoropolymer only and this area is referred to as B) used in the core-shell particle observation. C=(A−B)/A×100 (9) Dispersibility of Powder (Secondary Particles) A labo plast mill mixer (100C100 available from Toyo Seiki Co., Ltd.) having an inner capacity of 60 ml was charged with 40.5 g of polyethylene pellets (Novatec LD LC500 available from Japan Polyethylene Corporation) and 4.5 g of powder containing core-shell particles (the weight ratio between polyethylene and powder was 90:10 (%)). The mixture was kneaded for 10 minutes with the blade being turned at 100 rpm at 160° C. The kneaded product was pre-heated at 180° C. for 15 minutes and compression-molded at a pressure of 2 MPa using a heat press, whereby a 1-mm-thick press sheet was obtained. This sheet was punched to provide five specimens for a tensile test, each having an ASTM D638 V dumbbell shape. Each specimen was evaluated through a tensile test in accordance with ASTM D638 using an autograph (AGS-J 5kN available from Shimadzu Corporation). The tensile strength and the elongation were each determined by averaging the values of the five specimens. Simple polyethylene (without powder) had a tensile strength of 14.9 MPa and an elongation value of 694%. The tensile strength and elongation value of the powder-added composition were determined, and the reduction percentages from those of the simple polyethylene were calculated. A powder having a higher dispersibility has smaller reduction percentages. Example 1 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 52 parts by mass of the low molecular weight PTFE aqueous dispersion and 0.5 parts by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 15 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.02 parts by mass of ammonium persulfate as a catalyst was dissolved in 3.0 parts by mass of water were simultaneously added dropwise. After four-hour reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. FIG.1is an image in which an image of a core-shell particle in the dispersion obtained in Example 1 taken with a scanning transmission electron microscope is superimposed with element mapping images of carbon and fluorine. The figure demonstrates that the resulting core-shell particle had a structure in which the entire surface of the low molecular weight PTFE particle was completely coated with polymethyl methacrylate. Example 2 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 84 parts by mass of the low molecular weight PTFE aqueous dispersion and 1.0 part by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 6 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.01 parts by mass of ammonium persulfate as a catalyst was dissolved in 1.8 parts by mass of water were simultaneously added dropwise. After one-hour reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Example 3 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 84 parts by mass of the low molecular weight PTFE aqueous dispersion and 0.5 parts by mass of KF-6204, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 6 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.01 parts by mass of ammonium persulfate as a catalyst was dissolved in 1.8 parts by mass of water were simultaneously added dropwise. After one-hour reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Example 4 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 89 parts by mass of the high molecular weight PTFE aqueous dispersion and 1.5 parts by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 6 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.01 parts by mass of ammonium persulfate as a catalyst was dissolved in 1.1 parts by mass of water were simultaneously added dropwise. After one-hour reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Example 5 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 93 parts by mass of the high molecular weight PTFE aqueous dispersion and 1.5 parts by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 1.5 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.004 parts by mass of ammonium persulfate as a catalyst was dissolved in 1.2 parts by mass of water wore simultaneously added dropwise. After 30-minute reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Example 6 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 95 parts by mass of the high molecular weight PTFE aqueous dispersion and 1.5 parts by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 0.3 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.004 parts by mass of ammonium persulfate as a catalyst was dissolved in 1.4 parts by mass of water were simultaneously added dropwise. After 30-minute reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Example 7 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 93 parts by mass of the high molecular weight PTFE aqueous dispersion and 1.5 parts by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 1.5 parts by mass of styrene as an acrylic monomer and a liquid in which 0.004 parts by mass of ammonium persulfate as a catalyst was dissolved in 1.2 parts by mass of water were simultaneously added dropwise. After 30-minute reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Example 8 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 93 parts by mass of the PFA aqueous dispersion and 0.8 parts by mass of Newcol 707SF, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 75° C., 4 parts by mass of methyl methacrylate as an acrylic monomer and a liquid in which 0.005 parts by mass of ammonium persulfate as a catalyst was dissolved in 2.4 parts by mass of water were simultaneously added dropwise. After one-hour reaction under stirring at 75° C. and two-hour aging at 80° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The dispersions prepared in Examples 1 to 8 were each controlled to have an evaporation residue content of 20 to 30% in the formulation. Table 1 shows the evaporation residue content, PH, viscosity, average particle size, shell thickness, shell coverage, and generation amount of different particles of each resulting dispersion. TABLE 1Materials fedExample(parts by mass)12345678Low molecular weight528484PTFE aqueous dispersionHigh molecular weight89939593PTFE aqueous dispersionPFA aqueous dispersion93Methyl methacrylate156661.50.34Styrene1.5Newcol 707SF0.51.01.51.51.51.50.8KF-62040.5Ammonium persulfate0.020.010.010.010.0040.0040.0040.005Water32.599.53.543.242.4Evaporation residue29.729.72930.227.527.828.419.7content (%)PH3.13.43.94.94.74.94.74.3Viscosity (mPa · s)2335510510Average particle size (nm)331281277306256249258337Shell thickness (nm)6136343163720Shel coverage (%)100100100100100100100100Amount of different00000000particles generated (%) Dispersions of Comparative Examples 1 to 3 were prepared with reference to the examples of a prior patent of a third party company. Table 2 shows the properties of the resulting dispersions, such as the evaporation residue and average particle size. Comparative Example 1 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with a liquid mixture containing 40.7 parts by mass of water, 45.4 parts by mass of the low molecular weight PTFE aqueous dispersion, 0.25 parts by mass of Latemul ASK, and 10 parts by mass of methyl methacrylate and 2.5 parts by mass of butyl acrylate as acrylic monomers, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 60° C., a liquid in which 0.025 parts by mass of potassium persulfate as a catalyst was dissolved in 1.25 parts by mass of water was added dropwise over 60 minutes. After two-hour reaction under stirring at 60° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. Comparative Example 2 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with a liquid mixture containing 36.3 parts by mass of water, 51.0 parts by mass of the high molecular weight PTFE aqueous dispersion, and 0.25 parts by mass of Latemul ASK and 9.0 parts by mass of methyl methacrylate and 2.3 parts by mass of butyl acrylate as acrylic monomers, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. After the inner temperature reached 60° C., a liquid in which 0.025 parts by mass of potassium persulfate as a catalyst was dissolved in 1.25 parts by mass of water was added dropwise over 60 minutes. After two-hour reaction under stirring at 60° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki. Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. FIG.2is an image in which an image of a particle in the dispersion obtained in Comparative Example 2 taken with a scanning transmission electron microscope is superimposed with element mapping images of carbon and fluorine. The figure demonstrates that the resulting core-shell particle had a structure in which the surface of the high molecular weight PTFE particle was not completely but only partly coated with acrylic resin. Comparative Example 3 A reactor provided with tools such as a stirrer, a nitrogen inlet tube, and a thermometer was charged with 33.9 parts by mass of water, 43.6 parts by mass of the high molecular weight PTFE aqueous dispersion, and 1.1 parts by mass of OS soap, and the components were stirred for 30 minutes. The reactor was then purged with nitrogen and the temperature was increased. Then, an emulsifying container was charged with a liquid mixture (hereinafter, referred to as monomer liquid mixture) containing 8.2 parts by mass of styrene, 3.5 parts by mass of acrylonitrile, and 0.05 parts by mass of Thiokalcol 20. After the inner temperature reached 60° C., 15% by mass of the monomer liquid mixture was added to the reacter, followed by addition of 0.22 parts by mass of Perbutyl H-69, 0.005 parts by mass of L-ascorbic acid, and 0.0003 parts of ferrous sulfate and aging for 30 minutes. Then, a liquid in which the rest 85% of the monomer liquid mixture and 0.03 parts by mass of L-ascorbic acid were dissolved in 9.4 parts by mass of water was added dropwise over 60 minutes. After two-hour reaction under stirring at 60° C., the reaction product was cooled to room temperature, whereby a dispersion was obtained. The resulting dispersion was treated with an L-8 type spray dryer (available from Ohkawara Kakohki Co., Ltd.) at a disk rotation speed of 10000 rpm, a stock solution supply speed of 4 Kg/h, an inlet temperature of 135° C., and an outlet temperature of 80° C., whereby a powder was obtained. FIG.3is an image in which an image of a particle in the dispersion obtained in Comparative Example 3 taken with a scanning transmission electron microscope is superimposed with element mapping images of carbon and fluorine. The figure demonstrates that the resulting core-shell particle had a structure in which the surface of the high molecular weight PTFE particle was not completely but only partly coated with a styrene/acrylonitrile copolymer. TABLE 2Materials fedComparative Example(parts by mass)123Low molecular weight45.4PTFE aqueous dispersionHigh molecular weight51.043.6PTFE aqueous dispersionMethyl methaetylate109.0Butyl acrylate2.52.3Styrene8.2Acrylonitrile3.5Thiokalcol 200.05Latemul E-118BNeopelex G-15Latemul ASK0.250.25OS soap1.1Ammonium persulfatePotassium persulfate0.0250.025L-ascorbic acid0.035Ferrous sulfate0.0003Perbutyl H-690.22Water41.937.543.1Evaporation residue24.318.321.8content (%)PH6.76.48.2Viscosity (mPa•s)555Average particle size (nm)294248294Shell thickness (nm)43250Shel coverage (%)782942Amount of different2568particles generated (%) Tables 3 and 4 show the particle size and the dispersibility (tensile strength and elongation reduction percentage) in polyethylene of each perfluoropolymer powder (secondary particles) obtained in the examples and comparative examples. A powder obtained from The high molecular weight PTFE aqueous dispersion without shells had a tensile strength reduction percentage of 43% and an elongation reduction percentage of 65%. Each perfluoropolymer powder of Examples 1 to 7, having a shell coverage of as high as 100%, had a low tensile strength reduction percentage of 9 to 14% and a low elongation reduction percentage of 20 to 25%, while each perfluoropolymer powder of Comparative Examples 1 to 3, having a shell coverage of as low as 29 to 78%, had a high tensile strength reduction percentage of 35 to 42% and a high elongation reduction percentage of 51 to 64%. This indicates that powders containing the core-shell particles of Examples 1 to 7 have good dispersibility in polyethylene. TABLE 3PowderExample(secondary particles)1234567Particle size (μm)14.414.813.412.417.315.816.6Tensile strength reduction109121410911percentage (%)Elongation reduction20252221202423percentage (%) TABLE 4Powder (secondaryComparative Exampleparticles)123Particle size (μm)12.311.512.2Tensile strength354142reduction percentageElongation reduction515864percentage (%) | 65,525 |
11859076 | DETAILED DESCRIPTION As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a rheology modifier” or “at least one rheology modifier” may independently include a plurality of rheology modifiers, including mixtures thereof. As used herein, the term “about” is meant to encompass deviation of ±10% from the specifically mentioned value of a parameter, such as temperature, concentration, time, etc. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” or any lingual variations thereof, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any integer or step or group of integers and steps. Generally, it is noted that the term “. . . at least one . . . ” as applied to any component of a composition of the disclosure should be read to encompass one, two, three, four, five, or more different occurrences of said component in a composition or process of this disclosure. The term “derivative” as used herein refers to a chemically modified compound derived from the parent compound, that differs from the parent compound by one or more elements, substituents and/or functional groups such that the derivative has the same or similar properties/activities as the parent compound. As used herein, the term “room temperature” refers to a range of temperature from about 20° C. to about 30° C., for example, about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C. The present disclosure provides bread-based compositions of playdough having stable and reproduceable properties, manufactured in small scale (for example lab scale) or in an industrial scale from bakery residual with variable qualities. The playdough of the present application advantageously has uniform and robust properties, and has a shelf life of at least 12 months from production. The playdough composition is based on biodegradable and compostable components, making it potentially suitable for use as a hydroponic or detached growth medium after disposal. Thus, unlike most commercial playdough products that contain synthetic ingredients, eliminating or substantially reducing the potential for composability, the playdough disclosed herein is based on environment friendly ingredients. Further, the process for production of the playdough composition enables utilization of a variety of the types of bakery residual, without the need for sorting. The process is designed to process all types of bread, sorted or unsorted, regardless of their composition or state, such that the final playdough product has robust and repeatable properties. In addition, as bread is a substrate for proliferation of various mold, fungi and/or yeast, the process permits neutralization or elimination of such contaminants from the bread, as to delay staling of the playdough for a period of at least a few months. In some embodiments, there is provided a biodegradable playdough composition, comprising at least about 20 wt % bakery residual, at most about 50 wt % water, and at least one functional additive, the biodegradable playdough containing at least about 75 wt % of biodegradable components based on the total weight of the playdough, the biodegradable playdough having a complex viscosity of between about 1×104and about 1×106Pa·s (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). In some embodiments, there is provided a process of manufacturing a biodegradable playdough containing at least about 75 wt % of biodegradable components based on the total weight of the playdough. The process comprises the steps of: (a) particulating unsorted bakery residual to an average particle size of at most about 200 μm, thereby obtaining particulated bakery residual; (b) mixing the particulated bakery residual with at least one functional additive and water to obtain a mixture, and maintaining the mixture at a temperature of at least about 40° C. for a predefined period of time; and (c) cooling the mixture to a room temperature to obtain said biodegradable playdough. Playdough is a modeling paste, typically soft and pliable, that can temporarily retain its shape once molded, and can be returned to a non-molded form by slight application of pressure (i.e. pressure that can be applied manually). The term biodegradable refers to a substance or a composition that is capable of being degraded or decomposed by action of microorganisms, e.g. bacteria. The playdough disclosed herein is rendered biodegradable by containing at least 75 wt % of biodegradable components out of its total weight, as defined by at least one of International Standards EN-13432 (“Packaging: requirements for packaging recoverable through composting and biodegradation”), ASTM D6400-19 (“Standard specification for labeling of plastics designed to be aerobically composted in municipal or industrial facilities”), and ISO 17088 (“Specifications for compostable plastics”). In some embodiments, the biodegradable playdough meets the requirements of one or more of International Standards EN-13432, ASTM D6400-19 and ISO 17088. In some embodiments, the biodegradable playdough comprises at least 77 wt %, at least 80 wt %, at least 82 wt %, or at least 85 wt % of biodegradable components out of its total weight. According to some embodiments, the biodegradable playdough is also compostable. The term “compostable” as used herein refers to the ability of at least the majority of the playdough's components to be transformed into compost through composting processes. Thus, the playdough of this disclosure can be repurposed as a soil additive or as a detached substrate for growth of vegetation. The process disclosed herein utilizes unsorted bakery residual from various sources and types, to produce therefrom a playdough, as disclosed herein, that has repeatable and robust properties. Therefore, the process disclosed herein enables recycling or repurposing the bakery residuals or bakery byproducts into a different biodegradable product, for example the playdough, in an industrial scale. In the compositions and processes of this disclosure, the bakery residuals or bakery byproducts are utilized as a source of proteins and carbohydrates, forming a structural matrix of the playdough. The term “bakery residual” or “bakery byproduct” throughout the present disclosure may be used interchangeably. This term may refer to “bread waste” such as any baked goods that were not consumed and can be at any state (either in edible or inedible condition). Thus, in some embodiments, this term may also refer to “recycled bread”. The term bakery residual, bakery byproduct or bread waste also refers to baked products throughout their production and marketing processes (for example manufacturing leftovers or rejects, production surplus, unsold products, and so forth). The bakery residual can vary, inter alia, in flour type, fiber content, sugars, fats and other additives which are present as baking improvers. In addition, the quality and quantity of gluten proteins typically varies between different types of flours. The term means to encompass all types of baked products which are flour-based, regardless of the types of flour utilized—for example baked products that contains at least one of wheat flour (plain or whole wheat), rye flour, barley flour, spelt flour, rice flour, amaranth flour, buckwheat flour, cassava flour, chickpea flour, corn flour, potato flour, tapioca flour, teff flour, and any mixture thereof The term bakery residual, bakery byproduct or bread waste means to also encompass, as noted, various types of baked goods, for example bread loaves, bread-rolls, cookies, cakes, crackers, wafers, sweet pastries, enriched pastry, savory pastries, and so forth, as well as unbaked or partially baked dough. In some embodiments, the bakery residual is unsorted bakery residual. In some embodiments, the bakery residual comprises one or more types of bread. In some embodiments, the process disclosed herein further comprises removal of packaging or wrapping means from the bakery residual prior to further processing. That is to say, the bakery residual can be received from different sources, either in non-packed form or in packed form; such packaging needs to be separated from the bakery residual prior to the particulating step of the process. As the playdough of this disclosure is predominantly based on the bakery residual as a major component thereof, the mixture, in some embodiments, comprises at least 20 wt % bakery residual. For clarity, the bakery residual may be a mixture of partially or fully baked dough (or baked goods) and one or more types of flours defined above. Non-limiting examples of such mixture include bread loaves (being considered as recycled bread) and corn flour, bread-rolls (as the recycled bread) and corn flour, bread loaves (the recycled bread) and tapioca flour, and bread-rolls (as the recycled bread) and tapioca flour. In some embodiments, the biodegradable playdough (and/or the mixture at step (b)) comprises at least 20 wt % bakery residual. In some embodiments, the playdough (and/or the mixture at step (b)) comprises at least 30 wt %, at least 40 wt %, at least 50 wt % or at least 60 wt % of bakery residual. According to some embodiments, the biodegradable playdough (and/or the mixture at step (b)) comprises between about 20 wt % and 80 wt % of bakery residual. In some embodiments, the biodegradable playdough (and/or the mixture at step (b)) comprises between about 30 wt % and 75 wt % of bakery residual. In some embodiments, the biodegradable playdough (and/or the mixture at step (b)) comprises between about 45 wt % and 80 wt % of bakery residual. The biodegradable playdough further comprises up to 60 wt % water. In some embodiments, the playdough comprises between about 10 wt % and about 60 wt % water. By way of example, the playdough of the present disclosure comprises about 10 wt %, 20 wt %, 30 wt %, 40 wt %, 50 wt %, or 60 wt % water. According to some embodiments, the weight ratio of bakery residual to water in the playdough is between about 0.5:1 and 8:1. According to some other embodiments, the weight ratio of bakery residual to water in the playdough is between about 1:1 and 8:1. In some embodiments, the weight ratio of bakery residual to water in the playdough is about 2:1, 3:1, 4:1, 5:1 or 6:1. The bakery residual is particulated at step (a) of the process to obtain particulated bakery residual. In some embodiments, the particulated bakery residual has an average particle size of no more than about 200 μm, for example between about 1 μm and about 100 μm, or between about 1 μm and about 50 μm. The term average particle size refers to the arithmetic mean of measured diameters of the bread particles. As typically the bread particles can be spherical or non-spherical, the average particle size is calculated on the basis of diameter or the longest dimension, respectively, of the particle. In some embodiments, the particulated bakery residual may be made of bakery residual having an average particle size of 100 μm. In some embodiments, the particulated bakery residual may be made of bakery residual having an average particle size of 200 μm. In some embodiments, the particulated bakery residual may be made of a mixture of the bakery residual having an average particle size of 100 μm and the bakery residual having an average particle size of 200 μm. In some embodiments, the weight ratio of the bakery residual having an average particle size of 100 μm and the bakery residual having an average particle size of 200 μm may be adjusted accordingly for example 2:1, 1:1, or 1:2. Other suitable ratio may also be used. According to some embodiments, the particulating can be carried out by a suitable method that permits size diminution of the waste bread. Non-limiting examples of such size reduction method include grinding, milling, ball-milling, crushing, grating, pulverizing, shaving, flaking, granulating, shredding and so forth. According to some embodiments, the process further comprises a step (a0), prior to step (a), comprising drying the unsorted bakery residual to a moisture quantity of less than about 10 wt % (for example about 1 wt %, 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, 8 wt %, or 9 wt %) to obtain substantially dried unsorted bakery residual. The dried unsorted bakery residual can then be particulated. Drying the unsorted bakery residual permits better control over the process parameters in spite of utilizing various and differing types of baked goods constituting the bakery residual. However, when the bakery residual is received at a proper water content (for example below 10 wt % including 9 wt %, 7 wt %, 5 wt % or lower) no further drying may be required. In some embodiments, step (a) of the process comprises wet-particulation of the unsorted bakery residual. In such embodiments, the unsorted bakery residual is first mixed with an initial quantity of water, and then ground under wet conditions in order to obtain a slurry of bread particles dispersed in water. It is noted that the initial quantity of water can be a portion of or the entire amount of water in the composition. According to some embodiments, steps (a) and (b) of the process can be carried out sequentially or concomitantly. In some embodiments, wherein step (b) comprises (b1) mixing said particulated bakery residual with at least one functional additive to obtain a blend; and followed by (b2) mixing said blend with said water to obtain said mixture. In some embodiments, step (b2) comprises mixing said blend with a solution containing at least one functional additive. In some embodiments, said solution comprises the at least one functional additive substantially dissolved in water. In some embodiments, the solution comprises one or more salts. In some embodiments, the one or more salts is NaCl, CaCl2or mixture thereof. In some embodiments, water added in step (b) or (b2) of the process is water heated to a temperature of between about 40° C. and about 100° C., for example about 40° C., 50° C., 60° C., 70° C., 80° C., 90° C., or 100° C. Any other values between this range may also be used. The biodegradable playdough comprises at least one functional additive. Within the context of this disclosure, the term functional additive refers to a compound or composition added to the playdough composition (in step (b) of the process) and bestows one or more properties, or modifies one or more properties, of the playdough. According to some embodiments, said at least one functional additive is selected from the group consisting of rheology modifiers, mycotoxin scavengers, retrogradation preventing agents, colorants, odorants, antioxidants, odor masking agents, preservatives, and any mixture or combination thereof The one or more functional additives can, in some embodiments, be by-products of industrial food processing, for example recycled components. In some embodiments of the disclosure, the one or more functional additives may have more than one functionality (or use). In an exemplary embodiment, a functional additive may act as both rheology modifier and retrogradation preventing agent. In another exemplary embodiment, a functional additive may act as both rheology modifier and preservative. The quality of the playdough is often defined by its rheology properties; namely, it should be elastic enough to enable ease of handling while molding, and plastic enough to prevent the dough from tearing during use. The ratio between elasticity and plasticity determines the rheological properties of the playdough. Typically, changes in composition of the playdough can have a dramatic effect on its rheological properties, hence making the use of non-homogenous bakery residual with variable compositions, challenging. In addition, baking processes of the bread cause starch and flour derived protein (gluten) in bread to undergo gelatinization and coagulation, respectively, to eventually function as water binding agents. Hence, unlike starches utilized in some commercial types of playdough, the bread-originating starches and gluten do not function as elasticity providers, but rather as water-binding agents, that can reduce elasticity of the biodegradable playdough. As disclosed herein, the processes and compositions of this disclosure are designed to overcome this challenge and provide playdough with robust properties even though utilizing variable bakery residual. Thus, according to some embodiments, at least one of the functional additives in the playdough is a rheology modifier. The term rheology modifier refers to a compound or composition that changes (typically improves) the rheological properties of the playdough. The rheology modifier can change one or more of elasticity of the playdough, plasticity of the playdough, viscosity of the playdough, hardness of the playdough, and other properties. The rheology modifier(s) may also function to provide soft and smooth sensorial properties, which are important for playdough intended to be used by infants and children. In some embodiments, said rheology modifiers are selected from the group consisting of one or more of gluten and gluten derivatives, starch and starch derivatives, vegetable oils, mineral oils, waxes, polysaccharides, and any mixture thereof. In some embodiments, to ensure sustainability, the rheology modifiers used in the present disclosure are obtained from renewable resources. In an exemplary embodiment, vegetable oils are preferred over mineral oils. In some embodiments, the rheology modifiers are added to the mixture in an amount of between about 1 and about 20 wt % (for example about 1, 2, 3, 5, 8, 10, 12, 15, or 20 wt %). In some embodiments, the biodegradable playdough composition comprises between about 1 and about 20 wt % of rheology modifier(s). According to some embodiments, gluten is added to the mixture during production of the playdough as a rheology modifier. In such embodiments, the content of gluten added to the mixture is between about 1 wt % and about 8 wt % for example 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, or 8 wt %. Without wishing to be bound by theory, the added gluten provides the playdough with higher elasticity, while reducing dependence on the variable protein quality in the recycled bread. In some embodiments, when the recycled bread is a non-gluten bread (or a gluten free bread), the gluten may be substituted by one or more polysaccharides, for example, amylopectin. Advantageously, when gluten is substituted by amylopectin, the playdough composition of the present disclosure may exhibit better or desirable properties including less firm thus easier to play. When amylopectin is used to substitute gluten, amylopectin added to the mixture is typically between about 1 wt % and about 8 wt % for example 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, or 8 wt %. According to some embodiments, elasticity of the playdough disclosed hereinmay be increased by adding one or more starches and/or modified starches. In such embodiments, starch and/or starch derivatives are added to the mixture as rheology modifiers in an amount of between about 0.5 wt % and about 20 wt % for example about 1 wt %, 2 wt %, 5 wt %, 10 wt % or 15 wt %. In some embodiments, said starch and starch derivatives may be selected from the group consisting of acid tread starch, alkaline modified starch, bleached starch, oxidized starch, mono-starch phosphate, di-starch phosphate, 16hosphate di-starch phosphate, acetylated di-starch phosphate, acetylated starch, mono-starch acetate, acetylated di-starch adipate, di-starch glycerin, hydroxypropyl-starch, hydroxypropyl di-starch glycerin, hydroxypropyl di-starch phosphate, starch sodium octenyl succinate, acetylated oxidized starch, dextrin, and any mixture thereof In some embodiments, the starch or starch derivatives may be supplemented (used in conjunction) with waxy-maize-starch or amylopectin. The starch, in some embodiments, can be of any source, typically a plant-based source, for example corn starch, potato starch, rice starch, wheat starch, cassava starch, and so forth. The starch, in some embodiments, is recycled starch that is recycled from food processing and/or food products. According to some embodiments, one or more mineral oils, vegetable oils and/or waxes can additionally be used as rheology modifiers. In such embodiments, the vegetable oil is selected from a group consisting of soybean oil, rapeseed oil, safflower oil (canola), sunflower oil, peanut oil, cottonseed oil, coconut oil, palm oil, olive oil, rice-bran oil, grapeseed oil, avocado oil, sesame oil, hemp oil, almond oil, and any mixture thereof. In some embodiments, the wax can be selected from the group consisting of bees' wax, soy wax, carnauba wax, candelilla wax, jojoba wax, rice bran wax, and any mixture thereof. In some embodiments, a biodegradable emulsifier, alone or in combination with other rheology modifier, can be used as rheology modifier. In some embodiments, the biodegradable emulsifiers are selected from the group consisting of one or more of mono- and di-glycerides of fatty acids, sodium stearoyl lactylate, calcium stearoyl lactylate, diacetyl tartaric acid ester of mono- and di- glycerides of fatty acids, glycerol monostearate (GMS), and any mixture thereof. In some embodiments, the emulsifier may be selected from an anionic surfactant, a cationic surfactant or a nonionic surfactant. Bakery residual typically varies in its microbial quality depending on shelf-life and storage conditions of the bakery residual, as well as the types of preservatives used and their efficiency against molds. Hence, recycling of bakery residual is challenging not only because of its poor visual appearance and non-homogenous properties, but also due to its ability to produce mycotoxins. Typical molds that grow on bread include, inter alia, various species ofAspergillus, Penicillium, Fusarium, Mucor, Rhizopus,and others. In order to reduce or eliminate such contaminants from the playdough, as well as to prevent growth thereof for a substantial period of time (e.g. at least a few months), the bakery residual, sorted or unsorted, may be dried to remove or at least reduce the moisture thus reducing the growth of the molds or mold spores present in the bread as described above. In order to facilitate the mixing of the bakery residual, the at least one functional additive as well as water and to facilitate the formation of the playdough , the mixture is maintained at a temperature of at least 40° C. (for example 50° C., 55° C., 60° C., 65° C., 70° C. or higher) in step (b) of the process of this disclosure, for a period of time. In some embodiments, the mixture is maintained in step (b) at a temperature of between about 40° C. and about 90° C. (for example 40° C., 60° C., 70° C., 80° C., or 90° C.). According to some embodiments, the mixture is maintained at such temperatures for a period of time of at least about 5 minutes, for example at least about 10 minutes, about 20 minutes or about 30 minutes. In addition, one or more mycotoxin scavengers can be added as functional additives, to prevent or suppress formation of mycotoxins in the playdough. According to some embodiments, said mycotoxin scavengers are selected from the group consisting of active carbon, aluminosilicates, zeolites, phyllosilicates, cellulose, cellulose derivatives (for example hydroxyethyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, etc.), and any combination thereof Use of such scavengers can also reduce or eliminate any foul odors that may develop upon growth of undesired mold contaminants in the playdough. According to some embodiments, said one or more mycotoxin scavengers are added to the mixture in an amount of between about 0.5 wt % and about 5 wt %, preferably between about 0.5 wt % and about 3 wt %. According to some embodiments, the biodegradable playdough of the present disclosure has mycotoxins stability (i.e. does not substantially develop mycotoxins) for a period of at least 3 months from its manufacture, typically for at least 6 moths from manufacture, or even for at least 9 months from manufacture. Another challenge in recycling bread waste or bakery residual is that bread undergoes staling or retrogradation over time. Staling or firming causes baked goods to lose their freshness. Although providing a sense of dryness and associated mistakenly to moisture loss, staling is caused by changes in starch structure. The starch in wheat flour is made up of straight and branched chains contained in granules; during baking, the starch granules swell, and the straight chains diffuse out. As the bread cools, the straight chains link together to provide the loaf's initial shape and strength. The branched chains of starch remain in the granules during baking and link together slowly during storage to make the crumb increasingly firmer with time. Such retrogradation is undesired in the biodegradable playdough, as retrogradation can cause the playdough to harden over time due to starch recrystallization. Hence, in some embodiments, retrogradation preventing agents are added to the playdough as a functional additive. In such embodiments, the retrogradation preventing agent is selected from the group consisting of one or more of biodegradable emulsifiers, biodegradable polysaccharides, oils (for example vegetable oils), and any combination thereof. In some embodiments, the biodegradable emulsifiers are selected from the group consisting of one or more of mono- and di-glycerides of fatty acids, sodium stearoyl lactylate, calcium stearoyl lactylate, diacetyl tartaric acid ester of mono- and di-glycerides of fatty acids, glycerol monostearate (GMS), and any mixture thereof. In some embodiments, the emulsifier may be selected from an anionic surfactant, a cationic surfactant or a nonionic surfactant. Advantageously, the emulsifier may function as a compatibilizer of oil and water phases present in the mixture when ingredients to make the playdough composition are mixed. When the emulsifier is added, a more stable and thus homogeneous mixture may be obtained. In some embodiments, the emulsifier including the biodegradable emulsifier may have a dual functionality. In an exemplary embodiment, the emulsifier including the biodegradable emulsifier may act as both rheology modifier and retrogradation preventing agent. In embodiments, the biodegradable polysaccharides are selected from the group consisting of one or more of algin, pectin, carrageenan, xanthan gum, guar gum, agar, cellulose, locust bean gum, gellan gum, gelatin, amylopectin, starch (different than the starch utilized as a rheological modifier), and any mixture thereof In some embodiments, the retrogradation preventing agents can be one or more oils (for example vegetable oils and non-vegetable oils including mineral oils) In some embodiments, the vegetable oil can be selected from the group consisting of soybean oil, rapeseed oil, safflower oil (canola), sunflower oil, peanut oil, cottonseed oil, coconut oil, palm oil, olive oil, rice-bran oil, grapeseed oil, avocado oil, sesame oil, hemp oil, almond oil, and any mixture thereof In an exemplary embodiment, the oil (for example vegetable oils) may have a dual functionality as both retrogradation preventing agent and rheology modifier. According to some embodiments, the total content of said one or more retrogradation preventing agents is between about 1 wt % and about 12 wt %. In some embodiments, the one or more retrogradation preventing agents is between about 2 wt % and about 10 wt % of the total weight of the playdough. According to some embodiments, the biodegradable playdough has staling stability (i.e. does not undergo substantial staling) for a period of at least 3 months from its manufacture, typically for at least 6 moths from manufacture, or even for at least 9 months from manufacture. In some embodiments, one or more antioxidants may be added as a functional additive to the playdough. Without wishing to be bound by theory, the antioxidants prevent or delay oxidation of lipids in the bread, which can result in degradation of the mechanical properties of the playdough as well as to formation of off-odors. According to some embodiments, the antioxidant is selected from the group consisting of tocopherol or α-tocopherol (vitamin E), phytic acid, rosemary extract, ascorbic acid, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), propyl gallate, and any mixture thereof. In some embodiments, step (b) further comprises adding at least one salt into the mixture. Typically, said salt is added in an amount of between about 5 and about 30 wt % (for example about 8 wt %, 10 wt %, 15 wt %, 20 wt %, or 25 wt %). In some embodiments, the salt may be inorganic or organic salt. In some embodiments, the salt can be selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl2), and mixtures thereof It can be appreciated that other suitable salts may also be added as a substitute of the salt mentioned above or used in conjunction with NaCl and/or CaCl2. In some embodiments, when NaCl is used in conjunction with CaCl2as co-salt, the addition of the two salts may alter the mechanical properties of the resulting playdough composition (for example, to provide a smooth surface). CaCl2may advantageously maintain the moisture or hydration level of the playdough composition as it prevents or at least reduces the water evaporation. In an exemplary embodiment, when both NaCl and CaCl2is used, the ratio (w/w) of NaCl and CaCl2may be 20:80, 40:60, 50:50, 60:40 or 80:20. Further functional additives can be added for aesthetic purposes or for bestowing the biodegradable playdough with a desired fragrance. According to some embodiments, one or more colorants are added as a functional additive, which can typically be natural pigments (for example pigments from a natural source). The colorant can, in some embodiments, be selected from the group consisting of carotenoids, anthocyanins, betalains, chlorophyll, calcium carbonate, clay, bentonite, earth minerals, titanium (IV) oxide or titanium dioxide and any possible mixture thereof In some embodiments, the concentration of the one or more colorants may be between about 0.5 wt % to about 5 wt %. One or more odorants, in some embodiments, can be selected from natural essential oils, natural spices, synthetic (for example biotechnology derived) aromatic compounds, and others. One or more odor masking agents can also be used. Non-limiting examples of the odor masking agents include biodegradable fragrance, zeolites, biochar, mineral powders, and so forth. The playdough can also contain textural additives, such as fine colored particles, glitter particles, shimmer particles, to provide further textural and visual effect. Preservatives can be added to the playdough to prevent or delay proliferation of undesired microbial contamination caused by molds, yeast or bacteria. The preservatives, in some embodiments, are selected from the group consisting of benzoic acid and salts thereof, sorbic acid and salts thereof, caprylic acid, glyceryl caprylate, sorbitan caprylate, methylparaben, ethylhexylglycerin, phenethyl alcohol, maltol, or any mixture thereof. As noted, the biodegradable playdough has a complex viscosity of between about 1×104and about 1×106Pa·s (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). The term complex viscosity is the frequency-dependent viscosity of a material when subjecting this material to oscillatory shear stress. In other words, the complex viscosity is the total resistance of a material to flow when oscillating sheer forces are applied thereon. Unless specifically noted otherwise, the complex viscosity values in this disclosure are provided via measurement by parallel plates rheometer, at 25° C., at 0.1 Hz. In some embodiments, the complex viscosity of the playdough disclosed herein is between about1×104and about 1×106Pa·s. The process, in some embodiments, further comprising a step (d), following step (c), comprises adding at least one secondary additive to the mixture. The secondary additive is different from the at least one functional additive of step (b). In some embodiments, the secondary additive is selected from the group consisting of rheology modifiers, mycotoxin scavengers, retrogradation preventing agents, colorants, odorants, antioxidants, odor masking agents, preservatives, and any mixture or combination thereof. The addition of the secondary additives in step (d) is carried out by mixing the secondary additives into the mixture obtained in step (c). In some embodiments, the mixing in step (d) is carried out by extrusion. In some embodiments, in order to adjust the texture and/or viscosity of the resulting mixture (i.e. after mixing the secondary additives into the mixture obtained in step (c), step (d) further comprises adding water to the mixture. To obtain uniformity of mechanical and elasticity properties to the non-homogenous bakery residual, one or more enzymes can be added to the mixture at or before step (b) of the process. Hence, in some embodiments, the process comprises a step (a1) between steps (a) and (b) that comprises adding one or more enzymes to the mixture to permit at least partial enzymatic decomposition of at least starch and/or gluten present in the bakery residual. Addition of the enzyme(s) causes at least partial enzymatic decomposition of at least starch and/or gluten present in the bakery residual, thereby enabling better control over the properties of the mixture during and post production. According to some embodiments, said one or more enzymes are selected from the group consisting of amylases, proteases, and any mixture thereof. As some types of bread contain whole grains, nuts, seeds, herbs, and so forth, which can hinder the texture of the playdough, removal of such undesired particles can be carried out. Therefore, according to some embodiments, the process further comprises step (b′), between steps (b) and (c), step (b′) comprises filtering (or sieving) said mixture to remove contaminants therefrom. Filtering (or sieving) can be carried out using any suitable separation technique. After obtaining the biodegradable playdough, the process can comprise further processing step, such as portioning, shaping, wrapping, packaging, and others. In some embodiments, this disclosure provides a biodegradable playdough obtained by the process described herein. In some embodiments of the disclosure, there is provided a biodegradable playdough composition, comprising at least about 20 wt % bakery residual, at most about 50 wt % water and at least one functional additive, the biodegradable playdough containing at least 75 wt % of biodegradable components based on the total weight of the playdough, the biodegradable playdough composition having a complex viscosity of between about 1×104and about 1×106Pa·s (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). In some embodiments, the complex viscosity of the playdough disclosed herein is between about 1×104and about 5×105Pa·s (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). In some embodiments, the complex viscosity of the playdough disclosed herein is between about 1×104and about 8×105Pa·s (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). In some embodiments, the complex viscosity of the playdough disclosed herein is between about 2×104and about 8×105Pa·s (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). In some embodiments, this disclosure provides a biodegradable playdough composition, comprising at least about 20 wt % bakery residual, at most about 50 wt % water, at least one mycotoxins scavenger, at least one rheology modifier, and optionally at least one functional additive (different from said mycotoxins scavenger and rheology modifier), the biodegradable playdough containing at least 75 wt % of biodegradable components based on the total weight of the playdough, the biodegradable playdough composition having a complex viscosity of between about 1×104and about 1×106Pa's (as measured by parallel plates rheometer, at 25° C., at 0.1 Hz). In some embodiments, the disclosure provides a kit comprising one or more portioned quantities of playdough as defined herein, and at least one organoleptic additive. The term “portioned quantity” as used herein refers to a portion of playdough quantity for single or multiple use by a user. The portioned amount can, for example, be 50 grams, 100 grams, 200 grams, 300 grams, and so forth. The kit can comprise a single portioned quantity, two or more portioned quantities. Each of the portioned quantities can be individually packaged within the kit, e.g. to maintain separation therebetween. As used herein, the term “organoleptic additive” refers to one or more materials or compositions that can be added to the playdough during use in order to provide one or more textural, visual or odorant effect. For example, the organoleptic additive can be any one of a colorant, a pigment, a perfume, a fragrance concentrate, particulate additive (e.g. fine particles to provide a textural effect), and so forth. The kit can comprise at least such organoleptic additive, two or more different organoleptic additives. In some embodiments, the disclosure provides a kit comprising two or more portioned quantities of playdough, said two or more portioned quantities differing from one another by at least one property. In some embodiments, said property can be any one of color, scent, texture, viscosity, hardness, softness, smoothness, etc. The kits of this disclosure can further comprise instructions for use. The kits of this disclosure can further comprise one or more molds, templates, cutters, shaping tools, and so forth. EXAMPLES Reference Commercial Product The Playdoh® commercial product was used as a reference for assessing the rheological properties of compositions of the present disclosure. The rheological properties of a sample of Playdoh® were measured by a parallel plate rheometer (Discovery HR-1, T.A. Instruments, USA), with plates' diameter of 25 mm, dynamic mode. All measurements were carried out at 25° C. The frequency sweep of oscillation was over the range of 0.1-100 Hz. The rheological properties of Playdoh® are shown inFIG.1. As can be seen, the rheological behavior of Playdoh® is of a pseudoplastic, sheer-thinning liquid, in which the complex viscosity is decreasing with increase of shear rate applied onto the sample. Example 1 Unsorted bakery residual (FIG.2A) was dried and particulated (FIG.2B) to a particle size of at most 400 μm. The dried and particulated bakery residual was mixed with rheology modifiers, retrogradation preventing agents, and preservatives according to the compositions shown in Table 1A, until obtaining a homogenous initial mixture. Sodium chloride salt was dissolved in warm water)(˜70° to obtain a salt solution. The initial mixture was placed in a mixing bowl in a warm water bath, and the salt solution was added gradually, under mixing conditions. The mixture was mixed for at least 10 minutes, until the temperature of the composition reached about 55-60° C. The playdough was then left to cool to room temperature. The rheological properties for compositions 1 and 2 are shown inFIGS.3A and3B, respectively and Table 1B. The test method and parameters were identical to those of the Reference. As can be seen, both Compositions 1 and 2 behave as pseudoplastic liquids, however with a complex viscosity larger in an order of magnitude, as compared to the Reference. Compositions 1 and 2 were, accordingly, somewhat harder to manually knead. TABLE 1ACompositions of Example 1Concentration wt %ComponentComp. 1Comp. 2FunctionalityUnsorted48.7747.3Dough matrix, rheologybakeryresidualWater23.4718.5Dough matrix, rheologyGMS4.394.5Elasticity, retrogradation preventionGluten4.013.1Rheology modifierVegetable oil1.431.5Elasticity, retrogradation preventionPotato starch1.281.5Dough elasticityMethylparaben0.250.2Preservative (food grade)NaCl16.4123.7* Glyceryl monostearate, 40% pre-melted TABLE 1BRheological test resultsComplex viscosity [log(Pa · sec)]Composition0.1 Hz1 Hz10 Hz15.584.844.0125.564.814.01Reference4.73.853.06 Example 2 Unsorted, dried and particulated bakery residual was mixed with rheology modifiers, retrogradation preventing agents, preservatives and salt until obtaining a homogenous mixture, according to the composition shown in Table 2A. The mixture was placed in a mixing bowl in a warm water bath (˜90° C.), and water was added gradually under mixing conditions for at least 10 minutes, until the temperature of the composition reached about 55-60° C. The playdough was then left to cool to room temperature, covered. The following day, 10 ml of warm water (55-60° C.) were mixed into the playdough until a homogenous paste was obtained. The rheological properties for composition 3 are shown inFIG.4and Table 2B. As can be seen, Composition 3 also behaves as pseudoplastic liquids, however with a complex viscosity larger in an order of magnitude, as compared to the Reference. TABLE 2AComposition of Example 2 (composition 3)ComponentConcentration wt %FunctionalityUnsorted bakery35.5Dough matrix, rheologyresidualWater40.3Dough matrix, rheologyGMS4.5Elasticity, retrogradationpreventionGluten3.1Rheology modifierVegetable oil1.5Elasticity, retrogradationpreventionPotato starch1.5Dough elasticityMethylparaben0.2Preservative (food grade)Sodium chloride23.7* Glyceryl monostearate, 40% pre-melted TABLE 2BRheological test resultsComplex viscosity [log(Pa · sec)]Composition0.1 Hz1 Hz10 Hz35.684.833.99Reference4.73.853.06 Example 3 Unsorted, dried and particulated bakery residual was mixed with rheology modifiers, retrogradation preventing agents, preservatives and salt until obtaining a homogenous mixture, according to the composition shown in Table 3A. The mixture was placed in a mixing bowl in a warm water bath (˜90° C.), and water was added gradually under mixing conditions for at least 10 minutes, until the temperature of the composition reached about 60° C. The playdough was then kneaded for at least 5 minutes, and then left to cool to room temperature, covered. Representative pictures of the playdough on this Example are shown inFIGS.5A-5B. The rheological properties for Compositions 4-8 are shown inFIGS.6A-6E, respectively, and Table 3B. TABLE 3ACompositions of Example 3Concentration wt %ComponentComp. 4Comp. 5Comp. 6Comp. 7Comp. 8Unsorted bakery34.134.736.034.235.4residualWater (total)38.739.540.938.940.2GMS4.34.44.64.44.5Gluten3.05.0———Vegetable oil1.41.51.51.51.5Potato starch3.61.21.21.21.2Methylparaben0.20.20.20.20.2Sodium chloride14.714.915.514.712.0Borax—0.5———Amylopectin———5.05.0* Glyceryl monostearate, 40% pre-melted TABLE 3BRheological test resultsComplex viscosity [log(Pa · sec)]Composition0.1 Hz1 Hz10 Hz45.514.683.8155.574.73.8565.414.633.7775.444.63.6685.454.63.74Reference4.73.853.06 As can be seen, while the complex viscosity of Compositions 4-8 is somewhat higher than, the compositions showed pseudoplastic behavior similar to those of the Reference commercial product, with very similar sensorial properties compared to the Reference commercial product. Example 4 Unsorted, dried and particulated bakery residual was mixed with coconut oil, potato starch (dry), methylparaben, a mixture of salt consisting of calcium chloride and sodium chloride, amylopectin, titanium oxide and emulsifier (e.g. Tween 80) until a homogeneous mixture was obtained, according to the compositions shown in Table 4A (i.e. Compositions 9, 10 and 11). Hot or warm water was added to the homogeneous mixture under mixing conditions. The resulting composition was heated for about 20 minutes until the temperature of the suspension is about 55° C. The playdough composition was kneaded until the temperature of the composition is lowered to about 35° C. Following this, the playdough composition was left to cool down further to room temperature, covered. TABLE 4ACompositions of Example 4Concentration wt %ComponentComp. 9Comp. 10Comp. 11FunctionalityRecycled bread36.837.137.1Dough matrix,rheology(100 μm)Water3333.333.3Dough matrix,rheologyGMS*4.14.14.1Elasticity,retrogradationpreventionCoconut oil5.05.05.0Elasticity,retrogradationpreventionPotato starch1.21.31.3Elasticity(dry)Methylparaben0.20.20.2Preservative(food grade)Calcium chloride4.54.52.3SaltSodium chloride6.76.89.0SaltAmylopectin5.25.35.3Titanium Oxide1.70.90.9ColorantTween 801.51.61.6Emulsifier*Glyceryl monostearate, 40% pre-melted TABLE 4BRheological test resultsComplex viscosity [log(Pa · sec)]Composition0.1 Hz1 Hz10 Hz95.374.553.74105.44.573.74115.44.573.73Reference4.73.853.06 As can be seen from Table 4B, although the complex viscosity of inventive Compositions 9-11 is higher than that of the Reference commercial product, the inventive compositions showed pseudoplastic behavior similar as well as similar sensorial properties compared to the Reference commercial product. Rheological properties of Compositions 9-11 are shown inFIGS.7A-7C. Example 5 The compositions shown in Table 5A were prepared using the same method as the compositions prepared in Example 4, except that the coconut oil was replaced with mineral oil. There are three distinct compositions prepared in this example and they differ in the average particle size of the unsorted bakery residual used. Composition 12 was made from the unsorted bakery residual consisting of 50 wt % unsorted bakery residual having an average particle size of 100 μm and 50 wt % unsorted bakery residual having an average particle size of 200 μm. Composition 13 was made from the unsorted bakery residual having a particle size in a range of from 100 μm to 200 μm. Composition 14 was made from the unsorted bakery residual having an average particle size of 100 μm. TABLE 5ACompositions of Example 5ComponentConcentration wt %FunctionalityRecycled36.6Dough matrix, rheologybread (dry)(Composition 12,Composition 13 andComposition 14** )Water33.8Dough matrix, rheologyGMS*4.7Elasticity, retrogradationpreventionMineral oil3.6Elasticity, retrogradationpreventionPotato starch (dry)1.2ElasticityMethylparaben0.2Preservative (food grade)Calcium chloride4.4Sodium chloride6.7Amylopectin5.2Titanium Oxide2.1Tween 801.5*Glyceryl monostearate, 40% pre-melted**Compositions 12, 13 and 14 differ in the average particle size of the unsorted recycled bread used It is appreciated that certain features detailed in this disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment disclosed herein. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is rendered inoperative without those elements. | 48,154 |
11859077 | DETAILED DESCRIPTION Overview Various embodiments are based on a realization by the inventors of an improved epoxy chemistry that limits VOC emissions—e.g., at low vapor pressure while retaining the broad applicability of current systems—to dramatically reduce processing costs and mitigate associated health hazards. The present disclosure relates generally to techniques and mechanisms that, according to different embodiments, variously provide a system of reactive ionic liquids that, when combined, react to form high-strength, versatile and/or added-functionality epoxy-based thermosets. These epoxy systems solve the aforementioned issue of VOC outgassing. Some embodiments include synthesizing ionic liquids that, for example, incorporate anions substituted with epoxides (glycidyl groups) on the anion. Another such ionic liquid can contain cations of both diamines and triamines. Still another such ionic liquids can contain methylated-DABCO cation catalysts. Room-temperature ionic liquids are organic salts that melt below standard conditions and form solvent-less liquids with a number of unique physical properties, including zero vapor pressure. There are estimated to be ˜106 likely ion-pair combinations that form ionic liquids. Synthesizing organic salts that incorporate reactive moieties enable solvent-free and volatile-free chemistry. What follows are a set of example reductions to practice. To illustrate certain features of various embodiments, solvent-less ionic liquid epoxy systems are variously described with respect to an ionic moiety group R1and an ionic portion B having respective positive charges, and further with respect an ionic moiety group R2and an ionic portion A having respective negative charges. For example, scheme 1 shows a positive R1+substituent in a hardener ionic liquid (IL) and the negative R2−in the resin IL as one illustrative embodiment. However, in other embodiments, the respective charge signs of ionic moiety groups R1, R2could be reversed (i.e., wherein the respective charge signs of ionic portions A, B are also reversed). DESCRIPTION OF SEVERAL EMBODIMENTS Disclosed herein is an epoxy system that includes a hardener compound (H) and an epoxy compound (E). Typically, the hardener compound and the epoxy compound are provided separately and then mixed to form a polymer when used. In embodiments, the hardener compound has the molecular structure according to: Y1—R1—Y2, wherein R1is an ionic moiety and Y1and Y2are bonded to R1. In certain embodiments Y1is, or includes, a nucleophilic group. In certain embodiments Y2is, or includes, a nucleophilic group. In certain embodiments, Y1and Y2are identical. In certain embodiments, Y1and Y2are non-identical. In specific examples, Y1and Y2comprise a nucleophile independently selected from: a NH2group, a SH group, an OH group, a SeH group, and a PH2group. In certain embodiments, the hardener compound (H) is part of, such as a component of, a solvent-less ionic liquid, for example as a molecular complex with an ionic moiety A acting as a counter ion to R1. Examples of Y1—R1—Y2are shown in Table 1 andFIGS.4,5,6,7,8,10A-10I and17A-17D. Examples of ionic counter ions are shown inFIGS.4,5,6,7,8,9,10A-10I,13A-17D. The disclosed epoxy system further incudes an epoxy compound E. In embodiments the epoxy compound has the molecular structure according to: Z1—R2—Z2, where R2is an ionic moiety, Z1is or includes an epoxide group, and Z2is or includes an epoxide group. In certain embodiments, Z1and Z2are identical. In certain embodiments, Z1and Z2are non-identical. In certain embodiments, the epoxy compound (E) is part of, such as a component of a solvent-less ionic liquid, for example as a molecular complex with an ionic moiety B acting as a counter ion to R2. Examples of Z1—R1—Z2are shown in Table 1 andFIGS.4,5,6,7,8,10A-10I and17A-17D. Examples of ionic counter ions are shown inFIGS.4,5,6,7,8,9,10A-10I,13A-17D. In certain embodiments the epoxy system further includes one or more of an accelerator, a crosslinker, a plasticizer, or an inhibitor, The accelerator, crosslinker, plasticizer, and/or inhibitor can be included with the hardener compound, the epoxy compound, or even as a separate component of the system. Examples of accelerators, crosslinkers, plasticizers, and inhibitors ions are shown inFIGS.10A-10I and16A-16F. In certain embodiment, the epoxy system further includes an ionic hydrophobic and/or super-hydrophobic compound. In embodiments, the ionic hydrophobic and/or super-hydrophobic compound can be provided with either or both of the epoxy and hardener compound, for example as counter ion A, the epoxy compound, for example as a counter ion B, or both for example as a counter ion A and a counter ion B. In embodiments, the ionic hydrophobic and/or super-hydrophobic compound is released as an ionic liquid upon polymerization of hardener compound H and epoxy compound E to modify the properties of a polymer produced. Such ionic hydrophobic and/or super-hydrophobic compounds are known in the art and representative examples can found inFIGS.5and6. In certain embodiment, the epoxy system further includes an ionic hydrophilic compound. In embodiments, the ionic hydrophilic compound can provided with either or both of the hardener compound, for example as counter ion A, the epoxy compound, for example as a counter ion B, or both for example as a counter ion A and a counter ion B. In embodiments, the ionic hydrophilic compound is released as an ionic liquid upon polymerization of hardener compound H and epoxy compound E to modify the properties of a polymer produced. Such ionic hydrophilic compounds are known in the art. In certain embodiment, the epoxy system further includes an ionic transitional hydrophobic/hydrophilic compound. In embodiments, the ionic transitional hydrophobic/hydrophilic compound can provided with either or both of the hardener compound, for example as counter ion A, the epoxy compound, for example as a counter ion B, or both for example as a counter ion A and a counter ion B. In embodiments, the ionic transitional hydrophobic/hydrophilic compound is released as an ionic liquid upon polymerization of hardener compound H and epoxy compound E to modify the properties of a polymer produced. Such ionic transitional hydrophobic/hydrophilic compounds are known in the art and representative examples can found inFIG.7. In certain embodiment, the epoxy system further includes a biological active (BAIL, Biological Active Ionic Liquid) compound. In embodiments, the biological active (BAIL, Biological Active Ionic Liquid) compound can provided with either or both of the hardener compound, for example as counter ion A, the epoxy compound, for example as a counter ion B, or both for example as a counter ion A and a counter ion B. In embodiments, the biological active (BAIL, Biological Active Ionic Liquid) compound is released as an ionic liquid upon polymerization of hardener compound H and epoxy compound E to modified the properties of a polymer produced. Such biological active (BAIL, Biological Active Ionic Liquid) compounds are known in the art and representative examples can found inFIGS.8,9A-9F, and15A-15F. In certain embodiment, the epoxy system further includes a plasticizer compound. In embodiments, the plasticizer compound can provided with either or both of the hardener compound, for example as counter ion A, the epoxy compound, for example as a counter ion B, or both for example as a counter ion A and a counter ion B. In embodiments, the plasticizer compound is released as an ionic liquid upon polymerization of hardener compound H and epoxy compound E to modify the properties of a polymer produced. Such plasticizer compounds are known in the art and representative examples can found inFIGS.16A-16F. In certain embodiments, the plasticizer compound has a low to zero volatility. Scheme 1 Scheme 1 shows examples of polymerization reactions between a first compound and a second compound each including a respective ionic moiety group and a corresponding counter-ion, in accordance with disclosed embodiments. More particularly, scheme 1 illustrates examples of a disclosed epoxy system according to an embodiment. As shown, the epoxy system includes a hardener compound H and an epoxy compound E. As depicted, the hardener compound H includes a cationic molecular structure (Y1—R1—Y2) containing an ionic moiety group R1and the Y1and Y2groups bonded, for example chemically bonded to R1. As shown in the first reaction, the hardener compound H further includes an anionic portion A−, for example, a counter ion, in conjunction with the cationic molecular structure (Y1—R1—Y2) at R1. As shown, the epoxy compound E has an anionic molecular structure (Z1—R2—Z2) that includes an ionic moiety group R2and two epoxide/electrophilic (represented herein by “Z”) groups bonded to R2. In addition, the epoxy compound E includes a cationic portion B+in conjunction with the anionic molecular structure (Z1—R2—Z2), for example, acting as a counter ion to at R2. As shown in the second reaction, the hardener compound H further includes an anionic portion A+, for example, a counter ion, in conjunction with the anionic molecular structure (Y1—R1—Y2) at R1. As shown, the epoxy compound E has a cationic molecular structure (Z1—R2—Z2) that includes an ionic moiety group R2and two epoxide/electrophilic (represented herein by “Z”) groups bonded to R2. In addition, the epoxy compound E includes an anionic portion B−in conjunction with the anionic molecular structure (Z1—R2—Z2), for example, acting as a counter ion to at R2. As shown in the third reaction, the hardener compound H further includes an anionic portion A−, for example, a counter ion, in conjunction with the cationic molecular structure (Y1—R1—Y2) at R1. As shown, the epoxy compound E has a cationic molecular structure (Z1—R2—Z2) that includes an ionic moiety group R2and two epoxide/electrophilic (represented herein by “Z”) groups bonded to R2. In addition, the epoxy compound E includes an anionic portion B−in conjunction with the anionic molecular structure (Z—R2—Z), for example, acting as a counter ion to at R2. As shown in the fourth reaction, the hardener compound H further includes a cationic portion A+, for example, a counter ion, in conjunction with the cationic molecular structure (Y1—R1—Y2) at R1. As shown, the epoxy compound E has a cationic molecular structure (Z1—R2—Z2) that includes an ionic moiety group R2and two epoxide/electrophilic (represented herein by “Z”) groups bonded to R2. In addition, the epoxy compound E includes a cationic portion B+in conjunction with the anionic molecular structure (Z1—R2—Z2), for example, acting as a counter ion to at R2. Unless otherwise indicated, “anionic”—as used as used in the particular context of “anionic molecular structure,” “anionic portion,” “anionic moiety group,” or the like—refers to the characteristic of an atom or molecular structure (e.g., a molecule or portion thereof) providing a negative charge to facilitate bonding with a positive charge of a counterpart “cationic” structure/portion/group. For example an anionic portion A−can be bonded to ionic moiety group R1by an ionic bond (e.g., where A−is a single atom) or by an intermolecular bond, for example. Alternatively or in addition a cationic portion B+can be bonded to ionic moiety group R2by an ionic bond (e.g., where B+is a single atom) or by an intermolecular bond. In another example an cationic portion A+can be bonded to ionic moiety group R1by an ionic bond (e.g., where A+is a single atom) or by an intermolecular bond, for example. Alternatively or in addition a anionic portion B−can be bonded to ionic moiety group R2by an ionic bond (e.g., where B−is a single atom) or by an intermolecular bond. In the example reaction pathway shown in scheme 1, Y1and/or Y2can be a nucleophilic group—e.g., including but not limited to, —NH2, —SH, —OH, —SeH, —PH2or other nucleophilic substituent. In a molecular structure (Y1—R1—Y2), at least one such Y group can be reactive with an epoxide group of molecular structure (Z1—R2—Z2) to for a stable chemical bond—e.g., a dimer formation—in a completed polymerization reaction. Table 1 shows examples of molecular structures that can be variously utilized in respective ionic liquid epoxy systems. It is noted that superscripted numbers (e.g., R1, R2, R3, R4, etc.) are used herein to indicate component structure of a moiety group that, for example, is instead identified using subscripted numbers (e.g., R1, R2). TABLE 1Examples of possible structures for R1and R2in scheme 1Possible (Y1-R1-Y2) structuresPossible (Z1-R2-Z2) structures R1, R2, R3R4and R5could be any suitable chain, Y1and/or Y2could be a nucleophilic group—e.g., including but not limited to —NH2, —SH, —OH, —SeH, —PH2. Y1and/or Y2and epoxy moieties (epoxy group is an example of Z group that could be any electrophilic group suitable to react with Y1and/or Y2and form a permanent chemical bond) could be exchanged between R1and R2. Anionic moieties could be any suitable anionic substituent. As illustrated by the embodiment shown in scheme 1, the Y1and/or Y2groups bonded to ionic moiety group R1can be amine groups (e.g., where Y1and/or Y2is a primary amine group). The hardener compound H can function as a hardener to react with the epoxy compound E. A reaction of compounds H, E can result in at one of the epoxide groups forming a chain with one of the Y1and/or Y2groups—e.g., wherein a separate by-product molecule is formed by anionic portion A−and cationic portion B+. Certain embodiments variously facilitate a wide variety of combinations of R1, R2, Z1and/or Z2, Y1and/or Y2, A−, A+, and B−, and B+to be chosen from to achieve desired material characteristics, while providing significantly reduced VOC byproducts. In the example embodiments shown in scheme 1, the first compound includes an ionic moiety group R1and a corresponding counter-ion A, while the second compound includes an ionic moiety group R2and a corresponding counter-ion B. The illustrative reaction pathway shown in scheme 1 represents examples of dimer formation from a polymerization reaction. Various combinations of ionic moieties R1and R2groups are possible, and if the corresponding counter-ions (A and B) are carefully selected, the two compounds can form a secondary ionic liquid (A−B+), limiting or even avoiding the possibility of VOC emissions from an ionic liquid epoxy system. Also is possible to use same charge ionic liquid resin and ionic liquid hardener where a secondary ionic liquid will not be produce but permanent charges remains in the polymeric chains compensate for the corresponding counter ions, as is shown in the last two examples in scheme 1. Aspect of the present disclosure concern a polymer produced by the polymerization of the epoxide system disclosed herein. In embodiments, a polymer produced upon polymerization of hardener compound H and epoxy compound E comprises self-healing properties due to the presence of stable electrical charges along to the polymeric chains that drive the healing process through electrostatic attraction. In embodiments, a polymer produced upon polymerization of hardener compound H and epoxy compound E forms a highly and regular porous system, which could be used but not limited to as filtration membrane, solid electrolyte after replacing the secondary ionic liquid, exchange membrane, etc. In embodiments, a polymer comprises a solid electrolyte. An electronic component comprising the polymers disclosed herein. In embodiments, the electronic component is a component of a battery, a capacitor, a piezoelectric material and/or an electro-actuator. Synthetic Methods Scheme 2 Scheme 2 shows an example reaction to synthesize a hardener compound of an epoxy system according to embodiments disclosed herein. Such reactions can contribute to the manufacture of some or all of the hardener compounds H, for example, as shown in scheme 1. As shown in scheme 2, the class of diamine imidazolium ionic liquids provide amine chemistry that can be used as a hardener in an epoxy polymer system, such as those disclosed herein. For example, the illustrative reactions of scheme 2 provide for synthesis of 1,3-di(2′-aminoethylene)-2-methylimidazolium bromide. The first step of the synthesis is the protection of the amino group in bromo-ethylamine (1) using tritylchloride (2), and substituting the resulting compound (3) in 2-methylimidazole (4) under basic conditions (refluxing in DMF for 12 h) in order to obtain the bi-substituted intermediate (5), deprotection of amine groups is carried out in acidic media in dioxane to obtain the hydrochloride derivative (6), careful neutralization using NaOH is required in order to obtain the target compound (7). Full proton NMR spectroscopic characterization was obtained for the target compound (7) (seeFIG.1) showing proper peaks that correlate with expected characteristics. The material obtained is a highly viscous brown liquid. Additional studies indicate that stability of this hardener in a time window of 6 months (storage without inert atmosphere in a lab shelf, closed container) without signs of decomposition. An ionic liquid hardener including compound (7) was tested against commercially available resins (1:1 mass ratio), without accelerators or modifiers of the polymerization reaction. The testing revealed that the hardener was effective with a curing temperature of 120° C. for two hours producing a brown solid material. Scheme 3 Scheme 3 shows an example of a reaction in a process to synthesize an epoxy compound including the anionic molecular structure (Z1—R2—Z2) as shown in scheme 1 according to embodiments disclosed herein. As shown, synthesis of phosphinate di-epoxy acid can be produced using a modified Arbuzov reaction. In the example reaction shown in scheme 3, acidic compound (9) is neutralized with tetraakyl phosphonium hydroxide in order to obtain the corresponding phosphonium ionic liquid, where R5can be an alkyl, such as an alkyl having between 1 and 16 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. Scheme 4 Scheme 4 shows another example of a reaction in a process to synthesize an epoxy compound including the anionic molecular structure (Z1—R2—Z2) as shown in scheme 1 according to embodiments disclosed herein. More particularly, scheme 4 shows a synthesis of a bisphenol A diglycidyl ether (2,2-bis[4-(glycidyloxy)phenyl]propane) analog by addition of an ionic moiety into the monomer structure (scheme 4). Scheme 5 Scheme 5 shows another example of a reaction in a process to synthesize an epoxy compound including the anionic molecular structure Z1—R2—Z2) as shown in scheme 1 according to embodiment disclosed herein. In the illustrative di-glycidylation reaction of scheme 5, 4-hydroxy-γ-(4-hydroxyphenyl)-γ-methyl-methyl ester benzenebutanoic acid (10) reacts with epichlorohydrin (11) in basic conditions at 100° C. for 15 minutes. Such a reaction can result in a yield above 90% of γ-methyl-4-(2-oxiranylmethoxy)-γ-[4-(2-oxiranylmethoxy) phenyl]-, methyl ester benzenebutanoic acid (12). A proton NMR analysis of a material resulting from one such reaction is shown inFIG.2.FIG.2shows characteristic peaks indicating that compound (12) is the main component. Scheme 6 Scheme 6 shows another example of a reaction in a process to synthesize an epoxy compound including the anionic molecular structure (Z1—R2—Z2) as shown in scheme 1 according to embodiment disclosed herein. The reactions shown in scheme 6 can be continued from those shown in scheme 5, for example. As shown in scheme 6, the —OMe (oxygen/methyl group) moiety can be hydrolyzed—e.g., without requiring further purification—using a NaOH (3 eq)/acetone/water mixed at 0° C. and allowed to warm up to room temperature for 1.5 h, (scheme 6). Extended reaction time does not show deviation from the desired product when the reaction was followed by TLC. The free acid derivative (13), γ-methyl-4-(2-oxiranylmethoxy)-γ-[4-(2-oxiranylmethoxy)phenyl]-benzenebutanoic acid was obtained in a quantitative yield and fully characterized by proton NMR in CDCl3. An example of a typical spectra obtained for compound (13) is shown inFIG.3.FIG.3reveals all the characteristic features of compound (13). The NMR of the reaction product also shows the presence of the solvent (ethyl acetate) used during the purification process. Scheme 7 Scheme 7 shows another example of a reaction in a process to synthesize an epoxy compound including the anionic molecular structure (Z1—R2—Z2) as shown in scheme 1 according to embodiment disclosed herein. The reaction shown in scheme 7 can be continued from those shown in scheme 6, for example. In order to mitigate the possible of damaging the epoxy groups in compound (13), the ionic liquid formation can be carried out in methanol, using equimolar amounts of tetrabutyl phosphonium hydroxide (14) to neutralized the benzenebutanoic acid proton (scheme 7), and quickly removing the MeOH (15 minutes mixing time) and produced water under vacuum (30 mmHg) at 45° C. during 4 h and dried at room temperature and full vacuum for 24 h. In a test run of such a process, a dark yellow viscous liquid was obtained. In embodiments, equimolar amounts of the compound (15) ionic liquid resin and the compound (7) ionic liquid hardener can be combined—e.g., mixed manually at room temperature and poured into a 1.5 ml silicon mold, and placed overnight in a vacuum oven at 120° C. for 12 h. Reaction of the combined compounds (7) and (15) result in a solid material with a greasy feature and rubber-like toughness. It was theorized that such properties might be related to relatively low amounts of crosslinking agents in the epoxy system. In order to probe this assumption, a new ionic liquid hardener was prepared. Secondary ionic liquid produce during the polymerization process is tetrabutylphosphonium bromide Scheme 8 Scheme 8 shows an example of a reaction in a process to synthesize an epoxy compound of aliphatic nature: tetrabutylphosphonium salt of 2,2-bis(glycidyloxymethyl)propionic acid (21). The synthetic route includes 3 steps: alkylation of commercially available 2,2-bis(hydroxymethyl)propionic acid (16) with allyl bromide (17) in toluene with NaOH. This reaction requires overnight reflux for completion and produce diallyl intermediate (18) in 90% yield. The product is quite pure and does not require further purification for the next step. Oxidation the olefinic intermediate (18) to epoxide (20) was conducted by a standard method with m-chloroperbenzoic acid (19) at room temperature overnight. This method requires tedious column purification, but is safe and gives 90% yield of epoxidized product (20). Formation of the target ionic liquid epoxy resin (21) was carried out in methanol with equimolar amounts of tetrabutylphosphonium hydroxide (14), by a similar method described for compound (15) on Scheme 7. Scheme 9 Scheme 9 shows an example of a reaction in a process to synthesize an epoxy compound with positively charged heterocyclic core. Such epoxy ionic resin can react either with a negatively charged hardener (second line in Scheme 1) or with similarly positive hardener (third line in Scheme 1). In the case of both positively charged components (third line) no additional ionic liquid of AB type is formed, which can be useful for certain properties. The synthetic route includes 2 steps: alkylation and quaternization of commercially available imidazole (22) with 4-bromo-1-butene (23) in usual alkylation conditions (NaHCO3-acetonitrile, reflux overnight). The quaternized intermediate (24) was obtained in 99%. The crude product was pure enough and was used for the next step without additional purification. Epoxidation of the olefinic quaternized intermediate (24) was conducted under a standard method with m-chloroperbenzoic acid (19) at room temperature overnight. As in the analogous case with aliphatic epoxy ionic resin (Scheme 8, compound 20), the product required tedious column purification. The final yield was about 50%. Scheme 10 Scheme 10 shows an example of reactions in a process to synthesize a hardener compound of an epoxy system according to an embodiment. In this example embodiment, the new hardener is intended to have a multi-branch structure in order to promote crosslinking between the polymeric chains. N1, N1-bis(2-aminoethyl)-1,2-ethanediamine (compound 26, scheme 10) was protected using a BOC (e.g., tert-butyloxycarbonyl) protecting group under room temperature conditions and overnight stirring. Protected compound (28) was then alkylated using methyl iodide at 120° C. in acetonitrile reflux with overnight stirring, the alkylation reaction was followed by TLC until the complete consumption of (28), solvent and Mel (methyl iodide) excess were remove by rotary evaporation at 45°-50° C. and 30 mmHg during 4 h, followed by drying at room temperature and full vacuum. It is important to mention that Mel alkylation agent was selected due to facile access to the reagent, but there are several options to choose from and the final selection could be used to modify the properties of the whole epoxy resin system. BOC protection was removed using HCl-dioxane solution and the remaining acid was neutralized using NaOH. After this step the final ionic liquid was obtained by metathesis of the ionic liquid in an aqueous solution of LiTFSI, inorganic salts were removed by several washes with nanopure water and rotary evaporation at 50° C. and 15 mmHg for 4 h. Compound (30) 2-amino-N, N-bis(2-aminoethyl)-N-methyl-ethanaminium bis(trifluoromethane)sulfonamide was obtained as a viscous white liquid, dried for 24 h at room temperature and full vacuum. Example results of a test reaction with compound (15) and (30) are described herein. More particularly, one gram of 2-amino-N, N-bis(2-aminoethyl)-N-methyl-ethanaminium bis(trifluoromethane)sulfonamide (30) was mixed manually with one gram of tetrabutylphosphonium γ-methyl-4-(2-oxiranylmethoxy)-γ-[4-(2-oxiranylmethoxy)phenyl]-benzenebutanoate (15) (molar ratio 1.5:1) and cured at 120° C. for 12 h in a silicone mold, resulting in a hard solid material, pale yellow in color, where the secondary ionic liquid produced is tetrabutylphosphonium bis(trifluoromethane)sulfonamide. Scheme 11 Scheme 11 shows an example of a reaction to facilitate synthesis of a modifier (e.g., an accelerant or catalyst) for the epoxy system according to an embodiment. Such an accelerant/catalyst can expedite a reaction such as that shown in scheme 1. It is possible to synthesize a modifier of the polymerization reaction as an ionic liquid or ionic compound that will mitigate or even void the possibility of VOC emissions. One of the most commonly used reaction modifiers is DABCO, whose catalytic effect in the polymerization reaction helps to accelerate the process of curing. Although synthesis of ionic DABCO compounds is known, its ionic form has been tested as an anti-microbial agent, but not as polymerization modifier. In one illustrative embodiment, a dabconium compound can be synthesized, for example, by direct alkylation of 1,4-diazabicyclo[2.2.2]octane with 1-Bromo octane in dichloromethane under reflux conditions and overnight stirring. Octyl Dabconium bromide can be obtained in quantitative yield. One advantage of this approach to epoxy technology is the possibility of tuning the properties of the ionic liquid produced during the polymerization reaction in order to give to the final product different characteristics according to the specific use of each material. This in-situ modifier could be designed to be hydrophobic or hydrophilic, to act as a plasticizer of the polymer network and/or to be solidified to act as filler. Alternatively or in addition, such an in-situ modifier can be adapted for use in providing an antibacterial ionic liquid for medical use. Example Compounds and Epoxy Systems FIG.4shows an example of an epoxy system, according to an embodiment, that includes some or all of the features of that shown in scheme 1. More particularly,FIG.4shows one example of a system including a solvent-less epoxy resin (diepoxy phosphinate tetrabutylphosphonium) and hardener (dimethyl amine imidazolium bromide). When a polymerization reaction of such a system is complete, a resulting ionic liquid obtained as a by-product can include tetrabutylphosphonium bromide, which in turn can be used—for example—as plasticizer of a polymerized phosphinate/dimethylamine imidazolium network. FIG.5shows an example of an epoxy system, according to an embodiment, that includes some or all of the features of that shown in scheme 1. More particularly,FIG.5shows one example of a possible solvent-less ionic liquid epoxy system. If, for example, a user requires a polymer with a super hydrophobic surface it is possible to design the hardener and resin to produce a super hydrophobic ionic liquid after the polymerization reaction happens, as the case of imidazolium bis[bis(pentafluoroethyl)phosphinyl]imide ionic liquids, where the anionic portion is the hydrophobic part of the ionic liquid. One such ionic liquid epoxy system is shown inFIG.5. FIG.6shows an example of an epoxy system, according to an embodiment, that includes some or all of the features of that shown in scheme 1. More particularly,FIG.6illustrates an alternative use of super-hydrophobic cations such as Tri(n-hexyl)[2-ethoxy-2-oxoethyl]ammonium. FIG.7shows an example of an epoxy system, according to an embodiment, that includes some or all of the features of that shown in scheme 1. In the case of the example embodiment shown inFIG.7, the hydrophobic-hydrophilic character of the final product can be tuned and can be modified after the polymerization process using ionic liquids with a transitional hydrophobicity. In this case the hydrophobicity is modified by the presence of carbon dioxide. In CO2free environments this kind of ionic liquid has hydrophobic behavior. When the material is exposed to CO2the ionic liquid suffers a transition to a hydrophilic condition. This phenomenon is reversible and could provide a tunable material even after the curing of the epoxy resins. The same behavior has been observed in anionic portions derived from pyrazole, imidazole and triazole. FIG.8shows an example of an epoxy system, according to an embodiment, that includes some or all of the features of that shown in scheme 1. The production of a secondary ionic liquid, after the curing process, can be useful in various medical, pharmaceutical and/or other important fields of application for ionic liquid epoxy resins. Some embodiments variously provide a long term release system for medication—e.g., using pharmacologically active ionic liquids such as the ones derived from ibuprofenate and lidocainium. Several combinations can be obtained from these ionic liquids, according to various embodiments, to open—for example—the possibility of pain-killer releasing ferules (FIG.8). The secondary ionic liquid thus produced would be lidocainium ibuprofenate. FIGS.9A-9Fshow various examples of anionic portions and cationic portions—e.g., each to variously function as a respective one of anionic potion A−or cationic portion B+of scheme 1, respectively—each of an epoxy system according to an embodiment. Some embodiments variously blend epoxy polymer technology with the emerging field of pharmaceutical active ionic liquids.FIGS.9A-9Fshow some examples of useful therapeutic materials that can be adapted for use according to various embodiment. FIGS.10A-10Ishow various examples of hardener compounds, epoxy compounds and modifiers each of an epoxy system according to a respective embodiment. Some or all of the compounds shown inFIGS.10A-10Ican each be a component of a respective system having, for example, some of all of the features of the system shown in scheme 1. It is important to remark that the existence of a large number of possible counter-ions permits the design of a final polymer that is to meet any of a wide variety of specifications required by the end user of a solvent-less ionic liquid epoxy system. Combination of the proper ions could tune polymer properties such as flexibility, hardness, hydrophobicity, curing time, curing temperature, set up secondary reactions, ionic conductivity, etc. Also, the design of ionic liquid crosslinking agents, accelerators, and catalysts (examples shown inFIGS.10A-10I) would guaranty that the whole epoxy system is composed of zero vapor pressure components. Due at least in part to some or all such characteristics, it can be possible, as an example, to produce thermoset solid state electrolytes, important in the development of batteries for the storage of electrical energy. A solvent-less ionic liquid epoxy system according to some embodiments allows the injection of an electrolyte into the battery structure, setting up a polymerization reaction to provide a fully polymerized, ionic liquid filled, solid state electrolyte. FIG.11shows various examples of an anionic portion—e.g., the anionic portion A-shown in scheme 1—each of a respective epoxy system according to an embodiment.FIG.12shows various examples of cationic portions—e.g., the cationic portions B+shown in scheme 1—each of a respective epoxy system according to an embodiment. As mentioned above, hydrophobic materials could be produced from ionic liquids epoxies with selection of the corresponding counter ions to the hardener and epoxy ionic liquids. A wide variety of hydrophobic anions (FIG.11) and hydrophobic cations (FIG.12) are available to facilitate selection of a combination that, according to different embodiments, precisely accommodates a particular desired level of hydrophobicity for a final material. FIG.13shows various examples of an anionic portion—e.g., the anionic portion A-shown in scheme 1—each of a respective epoxy system according to an embodiment. As illustrated by the examples shown inFIG.11, it can be possible to synthesized epoxides ionic liquids where the secondary ionic liquid has a prominent hydrophilic character. Many inorganic anions are highly hydrophilic (FIG.13) and require bulky anions to produce ionic liquids. FIG.14shows various examples of a cationic portions—e.g., the cationic portion B+shown in scheme 1—each of a respective epoxy system according to an embodiment.FIG.14illustrates inorganic cations and organic cations with hydrogen bond donor moieties that are also highly hydrophilic. FIGS.15A-15Fshow various examples of an ionic liquid epoxy compound—e.g., such as that shown in scheme 1—each of a respective epoxy system according to an embodiment. There is a wide range of biologically active ionic liquids (BAILs), from ionic liquids with herbicidal properties to ionic liquids with antitumor activity. Some examples are shown inFIGS.15A-15F. New BAILs are being introduced regularly, and many of these BAILs can be used as a secondary ionic liquid in the ionic liquid epoxy systems providing a drug-eluding material after the proper curing process. Other examples are the ionic liquids derived from flufenamic acid (non-steroidal anti-inflammatory drugs) and ampicillin (anti-tumor activity). FIGS.16A-16Fshow various examples byproduct compounds each to be formed by a reaction of a respective epoxy system according to an embodiment. The compounds shown inFIGS.16A-16Fcan each be formed, for example, by the reaction of the anion A−with the cation B+shown in scheme 1. Plasticizers are used to modify the mechanical properties of different polymers—e.g., changing the rigidity, deformability, elongation; toughness, process viscosity, service temperature and/or the like. Traditionally, there are two types of plasticizers: inner and external plasticizers. Inner plasticizers are structural modifications to the polymers that affect its mechanical properties, i.e. copolymerization moieties, addition of substituent groups, etc. External plasticizers are additives incorporated during the polymers processing, that have effect on the crystallinity of the polymers. Organic solvents are usually utilized as plasticizers but their efficiency is typically related to the permanence of the solvent in the polymer structure. Many common plasticizers dissipate over time—e.g., at a rate depending on parameters such as volatility, boiling point, osmotic pressure and solvent power. Due to such problems, ionic liquids—which have relatively very low vapor pressure—can be used as a new class of plasticizers, in some embodiments. Such use can take advantage of better solvent powers, osmotic pressures and low volatility. Some of the ionic liquids used as plasticizers are shown inFIGS.16A-16Fand all of them can be used as the secondary ionic liquid in the ionic liquid epoxides systems. FIGS.17A-17Dshow various examples of an epoxy compound—e.g., such as that shown in scheme 1—each of a respective epoxy system according to an embodiment. In recent years it has been discovered that the presence of Bis Phenol A (BPA) in various polymer formulations presents a health hazard concern. BPAs have been associated/correlated to problems in the reproduction systems of women and men, birth defects in children, metabolic diseases and immune system affectation. For these and/or other reasons, it is important for manufacturers to have BPA-free options in polymer production. Since solvent-less ionic liquid epoxide system according to various embodiments have low intrinsic vapor pressure and the risk of volatile BPAs is relatively low, they can be important in mitigating the possibility of BPA contamination in polymer-based products intended for human use. Aliphatic systems are one example of an implementation that can mitigate BPA problems. Some proposed structures to mitigate the possibility of BPA byproducts are show inFIGS.17A-17D. FIGS.18A-18Bshow an example of devices each including a respective epoxy material according to an embodiment. For example, the devices ofFIGS.18A and18Bcan each include a respective epoxy material such as one formed by a reaction such at that shown in scheme 1. Solid electrolytes and electrochemical actuators are closely related—e.g., both systems are generally compromised of a polymeric matrix containing an electrolyte (organic or inorganic salt) between two electronic conductors (electrodes). The main difference is that in solid electrolytes the corresponding chemistries are typically designed to minimize a volume change in the electrodes, the volume change provoked by ion migration due to an applied potential (FIG.18A), where the electrolyte concentration is to be constant during the charge and discharge cycles. On the other hand, in an electrochemical actuator, a different effect is desired—e.g., wherein electrode volume and electrolyte concentration are to change. Accordingly, a different chemistry can be needed in order to provoke a differential volume change in the electrodes (FIG.18B), resulting in compression in one side of the cell and expansion in the opposite side, this phenomena is used to produce a movement proportional to the potential difference applied to the cell. Ionic liquid epoxide systems according to different embodiments can be variously adapted for the production of respective ones of solid electrolytes and electrochemical actuators. Such an epoxide system can facilitate synthesis of a polymeric matrix (epoxide polymer) with the production of a secondary ionic liquid as a byproduct of the polymerization reaction. A transition between an electrochemical cell with a solid state electrolyte and an electrochemical actuator can be based on design-time selection of the secondary ionic liquid ions and the composition of the electrodes. Also, the presence of these electromechanical properties can allow an ionic liquid epoxide system to provide improved design and development of piezoelectric materials—e.g., due to a strong correspondence between the mechanical stress in a polymer and an applied electrochemical potential. One possible use for this technology is the construction of a wide variety of sensors. Self-Healing Polymer FIG.19shows an example of a self-healing polymer including an epoxy material according to a disclosed embodiment, for example, the epoxy material formed by a reaction such at that shown in scheme 1. Self-healing polymers are materials capable of repair themselves from mechanical damage, as scratches, punctures, or cracking. There are several mechanisms that provide the polymers with the self-healing properties being the most used the formation of micro-capsules filled with the monomeric material and catalysts that react after the formation of the mechanical damage. However, there are also polymeric materials that consist of ionomeric chains, where the healing process is drive for the electrostatic attraction of the charges present in the polymers structure.FIG.19, shows a cross-sectional illustration of a healing process for this kind of system. The nature of ionic liquid epoxide systems according to some embodiments can variously enable polymeric chains with fixed charges that are suited to promote self-healing properties of a material, for example, wherein a secondary ionic liquid produced during the polymerization reaction is to act as a plasticizer improving the mechanical behavior of the final product. Polymer Films FIG.20shows an example of a film including an epoxy material according to disclosed embodiments, for example, the epoxy material formed by a reaction such at that is shown in scheme 1. Modification of epoxide polymers using an ionic liquid can be performed to change curing reaction conditions, such as temperature, time, hardener/resin ratio and/or the like. For example, ionic liquid content in an epoxy system can be in a range of 2 to 5 parts per hundred rubber (phr) when utilized as a modifier. Ionic liquids can be used in a range of 5 to 10 phr to modify the viscosity of some epoxide components during a curing process. However, with higher ionic liquids contents (around 30 to 70% w/w of the total mass), the ionic liquid tends to produce void space in the final material. After washing out this ionic liquid, the resulting material is a highly porous solid with porous size in the order of 10-20 μm (SeeFIG.20SEM image of a Jeffamine-BPA system with 50% tetrabutyl phosphonium TFSI ionic liquid). An ionic liquid epoxide system according to some embodiments can produce similar results, with a final product that could be used as a filter structure with a highly regular porous size. By modifying the ionic liquid content, it can be possible to selectively design (“tune”) the resulting porous size and selectivity of the filter system. Although certain embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a wide variety of alternate and/or equivalent embodiments or implementations calculated to achieve the same purposes may be substituted for the embodiments shown and described without departing from the scope. Those with skill in the art will readily appreciate that embodiments may be implemented in a very wide variety of ways. This application is intended to cover any adaptations or variations of the embodiments discussed herein. Therefore, it is manifestly intended that embodiments be limited only by the claims and the equivalents thereof. | 43,619 |
11859078 | DESCRIPTION OF EMBODIMENTS Hereinafter, an embodiment of the present invention is described in detail with reference to the accompanying drawings as necessary. [Treated Yam] Examples of a fiber (reinforcing fiber) forming a cord (tension member) include a natural fiber (cotton, hemp, etc.), a regenerated fiber (rayon, acetate, etc.), a synthetic fiber (a polyolefin fiber such as polyethylene and polypropylene, a fluororesin fiber such as polyfluoroethylene, an acrylic fiber, a vinyl alcohol fiber such as polyvinyl alcohol, a polyamide fiber, a polyester fiber, a fully aromatic polyester fiber, an aramid fiber, etc.), and an inorganic fiber (a carbon fiber, a glass fiber, etc.). These fibers may be used alone or in combination of two or more. Preferred examples of the fiber (reinforcing fiber) include a fiber having a high elastic modulus, for example, a high strength polyethylene fiber, a polyparaphenylene benzobisoxazole (PBO) fiber, a polyester fiber [a C2-4alkylene C6-14arylate fiber such as a polyethylene terephthalate (PET) fiber, a polypropylene terephthalate (PPT) fiber, a polytrimethylene terephthalate (PTT) fiber, a polybutylene terephthalate (PBT) fiber, or a polyethylene naphthalate (PEN) fiber, a polyarylate fiber, etc.], and a polyamide fiber (an aromatic polyamide fiber such as an aramid fiber, etc.). These fibers may be used alone or in combination of two or more. Among these fibers (reinforcing fibers), the polyester fiber (the PET fiber, the PEN fiber, the polyarylate fiber, and the like) and/or the aramid fiber or the aromatic polyamide fiber (a para-aramid fiber, a meta-aramid fiber, etc.) are often used. In particular, in a power transmission belt on which a high load acts, the aramid fiber (in particular, the para-aramid fiber) is preferable. Furthermore, according to the present invention, since the adhesiveness to the reinforcing fiber can be improved, the fraying resistance and the bending fatigue resistance can be effectively improved even for a poorly adhesive fiber having poor adhesiveness. Therefore, the present invention is effective for application to an aramid fiber (particularly, a para-aramid fiber having high strength and high elastic modulus). Examples of the para-aramid fiber include a polyparaphenylene terephthalamide fiber (e.g., “Twaron (registered trademark)” manufactured by Teijin Limited, “Kevlar (registered trademark)” manufactured by Du Pont-Toray Co., Ltd.) and a copolymer fiber having a paraphenylene terephthalamide unit and a 3,4′-oxydiphenylene terephthalamide unit (e.g., “Technola (registered trademark)” manufactured by Teijin Limited). The tensile elastic modulus of the fiber (reinforcing fiber) can be selected from a range of, for example, about 0.01 GPa to 500 GPa (e.g., 0.1 GPa to 300 GPa), and in order to impart high tensile strength to the power transmission belt, the tensile elastic modulus of the fiber (reinforcing fiber) is preferably high. The tensile elastic modulus of the fiber (reinforcing fiber) may be, for example, about 1 GPa to 500 GPa (e.g., 5 GPa to 400 GPa), preferably about 10 GPa to 300 GPa (e.g., 25 GPa to 250 GPa), may be about 50 GPa or more (e.g., 60 GPa to 500 GPa, preferably 70 GPa to 400 GPa, and more preferably 100 GPa to 300 GPa), and may be usually about 60 GPa to 150 GPa (e.g., 65 GPa to 120 GPa). The fiber having such high elastic modulus includes, for example, an aramid fiber. The average fineness of the fiber (reinforcing fiber or monofilament yarn) may be, for example, about 0.1 dtex to 10 dtex (e.g., about 0.3 dtex to 7 dtex), preferably about 0.5 dtex to 5 dtex (e.g., about 0.7 dtex to 3 dtex), and more preferably about 1 dtex to 2.5 dtex (e.g., about 1.2 dtex to 2 dtex). The fiber (para-aramid fiber, etc.) can be used in a form of a multifilament yarn (raw yarn) including a plurality of monofilaments, and from the viewpoint of durability of the power transmission belt, the number of monofilaments in the multifilament yarn (raw yarn) can be selected from a range of, for example, about 100 to 50000 (e.g., 200 to 10000), and may be about 250 to 5000 (e.g., 300 to 3000), and more preferably about 350 to 2000 (e.g., 400 to 1500). The multifilament yarn (raw yarn) preferably contains at least an aramid fiber, and may contain a monofilament yarn of another fiber (e.g., a polyester fiber). The ratio of the aramid fiber is 50% by mass or more (particularly 80% by mass to 100% by mass) with respect to the entire monofilament yarn (multifilament yarn), and the entire monofilament yarn is usually formed of the aramid fiber. The untreated yarn can be usually used in a form of a twisted cord or a twisted yarn cord (untreated twisted yarn cord) obtained by twisting a multifilament yarn (raw yarn) in order to increase tensile strength. That is, in many cases, the cord is used as a cord, for example, a twisted cord (twisted yarn cord) in which these multifilament yarns are used as a core yarn (untwisted yarn, preferably a primary twisted yarn) and finally twisted in a predetermined direction (e.g., the same direction as or a direction opposite to the primary twisted yarn). The average diameter (average wire diameter) of the core yarn may be, for example, about 0.1 mm to 1.2 mm (e.g., 0.2 mm to 1 mm), preferably about 0.3 mm to 0.8 mm (e.g., 0.4 mm to 0.7 mm), and the average diameter (average wire diameter) of the twisted yarn cord (or the cord) may be, for example, about 0.1 mm to 3.5 mm (e.g., 0.2 mm to 3 mm), preferably about 0.3 mm to 2.5 mm (e.g., 0.35 mm to 2 mm), more preferably about 0.4 mm to 2 mm (e.g., 0.5 mm to 1.5 mm), particularly preferably about 0.5 mm to 1 mm. The twisted yarn cord (untreated twisted yarn cord) may be a twisted yarn cord (single twisted yarn) formed by twisting at least one raw yarn right (S-twist) or left (Z-twist), but from the viewpoint of strength, a twisted yarn cord obtained by twisting a plurality of raw yarns is preferable. The twisted yarn cord formed by twisting a plurality of raw yarns may be a yarn (e.g., plied yarn, Koma twist yarn or Lang lay yarn) obtained by secondary twisting a plurality of single twisted yarns as a primary twisted yarn, or may be a yarn (e.g., corkscrew yarn) formed by aligning and twisting a single twisted yarn and a raw yarn (untwisted yarn). A single twist direction (primary twist direction) and a secondary twist direction may be either the same direction (Lang twist) or an opposite direction (plied twist). Among these twisted yarn cords, a twisted yarn cord (plied yarn or Lang lay yarn) in which a plurality of single twisted yarns are used as a primary twisted yarn and secondarily twisted in two stages is preferable in terms of prevention of untwisting and excellent bending fatigue resistance. The number of the primary twisted yarns constituting the twisted yarn may be, for example, about 2 to 5, preferably about 2 to 4, and more preferably about 2 to 3. The number of twists of the primary twist may be, for example, about 20 times/m to 300 times/m, preferably about 30 times/m to 200 times/m, and more preferably about 50 times/m to 180 times/m (particularly about 100 times/m to 150 times/m). In the primary twist, a twist factor (T.F.) represented by the following formula (1) can be selected from a range of, for example, about 0.01 to 10, and is preferably about 1 to 6 in the case of a plied yarn, and about 0.2 to 2 in the case of a Lang lay yarn. Twist factor(T.F.)=[Number of twists(times/m)×√Total fineness(tex)]/960 (1) The number of twists of the secondary twist is not particularly limited, and may be, for example, about 30 times/m to 200 times/m, preferably about 40 times/m to 180 times/m, and more preferably about 50 times/m to 150 times/m (e.g., about 60 times/m to 100 times/m). In the secondary twist, the twist factor (T.F.) represented by the formula (1) can be selected from a range of, for example, about 0.01 to 10, and is preferably about 1 to 6 in the case of a piled yarn and about 2 to 5 in the case of a Lang lay yarn. In a case where a twisted configuration of a twisted yarn cord formed by twisting a plurality of raw yarns is represented by (the number of raw yarns aligned in primary twist)×(the number of primary twisted yarns aligned in secondary twist), the twisted yarn cord may be a twisted yarn cord having a configuration of 1×2, 1×3, 1×5, 2×3, 2×5, 3×5, etc. In the present invention, the reinforcing fiber may be treated with a first treatment agent to form a raw yarn (multifilament yarn or twisted yarn), but in many cases, a raw yarn and/or a cord (hereinafter, may be simply referred to as an untreated yarn) is treated. The untreated yarn may be in a state of an untwisted raw yarn (untreated raw yarn), or may be in a state of a twisted yarn formed by twisting a raw yarn (untreated twisted yarn cord). Since the untreated twisted yarn cord is formed by twisting the raw yarn and the fiber, the untreated twisted yarn cord has a property that the first treatment agent is less likely to penetrate between the filaments (fibers) inside the untreated twisted yarn cord. Therefore, in the untreated twisted yarn cord, usually, the treatment agent cannot firmly adhere to the filaments inside the cord, and the adhesion to the rubber tends to be lowered. On the other hand, it is also conceivable to treat a raw yarn with a treatment agent and twist the raw yarn to produce a twisted yarn cord, which is further treated with a treatment agent. In this method, although fraying and adhesiveness are improved, the flexibility of the fiber is impaired and the bending fatigue resistance tends to be lowered. Since a treatment process is provided before and after the twisted yarn, the process becomes complicated, and when the treatment agent adheres to the raw yarn, the adhesiveness is increased and the twisted yarn workability is also decreased. In the present invention, since the permeability of the first treatment agent is excellent, even when the twisted yarn cord is used, the permeability or impregnability into the cord (between the monofilaments (or between the fibers) and/or between the multifilaments (or between the raw yarns)) can be improved, and the adhesion to the rubber can also be improved. Therefore, in the present invention, the untreated yarn of the cord for a power transmission belt exhibits an excellent effect regardless of whether the untreated yarn is a raw yarn or a twisted yarn cord, and it is particularly effective in treating the twisted yarn cord. In the present invention, even in the case of the twisted yarn cord, it is possible to improve the fraying resistance, the bending fatigue resistance, and the adhesiveness to a rubber since the specific first treatment agent having excellent impregnability or permeability into the cord (between the monofilaments and/or between the multifilament yarns of the reinforcing fiber) is used. In particular, even when the twisted yarn cord is formed of a multifilament yarn containing a para-aramid fiber (in particular, a multifilament yarn composed of a monofilament yarn of a para-aramid fiber), it is possible to effectively prevent the fibers from fraying on a side surface of the power transmission belt, and to improve the bending fatigue resistance of the power transmission belt. The reinforcing fiber, the raw yarn (multifilament yarn), and/or the cord may be pretreated with a silane coupling agent, an RFL treatment liquid, or the like, if necessary. [First Treatment Agent] The treated yarn (twisted cord, etc.) is treated with at least the first treatment agent (first aqueous treatment agent), and the first treatment agent contains an epoxy resin (A), a polycarbonate polyol (B), and a blocked polyisocyanate (C) to form an aqueous treatment agent. The first treatment agent permeates or impregnates between the monofilaments of the reinforcing fibers and/or the multifilament yarn (raw yarn) to firmly bond the reinforcing fibers and the raw yarn, fiber fraying resistance on a power transmission surface (e.g., a frictional power transmission surface of a frictional power transmission belt such as a V-ribbed belt) is high, and the bending fatigue resistance can be greatly improved. In the following description, an epoxy equivalent can be measured in accordance with a method defined in JIS K7236 (2009). A hydroxyl group value and an acid value can be measured in accordance with a method defined in JIS K0070 (1992). The molecular weight can be measured by using gel permeation chromatography (GPC) as a molecular weight in terms of polystyrene. The hydroxyl group equivalent can be converted from the hydroxyl group value. [Epoxy Resin (A)] The epoxy resin (epoxy compound) (A) usually has a plurality of epoxy groups (oxirane rings or glycidyl groups) in one molecule in many cases, and may be a glycidyl ether type epoxy resin, a glycidyl ester type epoxy resin (glycidyl ester of a polyvalent carboxylic acid (e.g., aromatic polyvalent carboxylic acid) such as diglycidyl phthalate or diglycidyl hexahydrophthalate), a glycidyl amine type epoxy resin (a resin obtained by glycidylating an amino group of an amino compound (e.g., aromatic amine) such as tetraglycidyl aminodiphenylmethane or triglycidyl-p-aminophenol), an alicyclic epoxy resin (an epoxy resin obtained by epoxidizing a double bond of a cycloalkene ring such as a cycloalkenene oxide type epoxy resin such as cyclohexene oxide or a dicyclopentadiene type epoxy resin), a heterocyclic epoxy resin (triglycidyl isocyanurate, etc.), or the like. Examples of the glycidyl ether type epoxy resin include aliphatic epoxy resins (e.g., polyglycidyl ethers of polyols such as diols such as ethylene glycol, propylene glycol, neopentyl glycol, polyethylene glycol, and polypropylene glycol, and triols such as glycerin), alicyclic epoxy resins (e.g., cycloalkanediol diglycidyl ethers such as cyclohexanedimethanol diglycidyl ether, and crosslinked cyclic epoxy resins such as dicyclopentadiene diol diglycidyl ether), and aromatic epoxy resins. The epoxy resin preferably contains an aromatic epoxy resin. Examples of the aromatic epoxy resin include arene type epoxy resins (benzene type epoxy resins such as diglycidyloxybenzene, naphthalene type epoxy resins such as diglycidyloxynaphthalene and bis(2,7-dihydroxynaphthalene)methyltetraglycidyl ether, etc.), novolak type epoxy resins (phenol novolak type epoxy resins, cresol novolak type epoxy resins, etc.), and bisphenol type epoxy resins. The epoxy resin preferably contains a bisphenol type epoxy resin (A1) which has a rigid skeleton and thus has excellent mechanical strength of a cured product, and has a hydroxyl group in a molecule and thus can be crosslinked by a polyisocyanate. In the bisphenol type epoxy resin (A1), examples of the bisphenols include bisphenols (4,4′-dihydroxybiphenyl, etc.), bis(hydroxyphenyl)alkanes [e.g., bis(hydroxyphenyl) C1-10alkanes such as bis(4-hydroxyphenyl)methane (bisphenol F), 1,1-bis(4-hydroxyphenyl)ethane (bisphenol AD), 2,2-bis(4-hydroxyphenyl)propane (bisphenol A), 2,2-bis(4-hydroxy-3-methylphenyl)propane, 2,2-bis(4-hydroxyphenyl)butane, 2,2-bis(4-hydroxyphenyl)octane, 2,2-bis(3,5-dimethyl-4-hydroxyphenyl)propane, 2,2-bis(3-cyclohexyl-4-hydroxyphenyl)propane, bis(4-hydroxyphenyl)diphenylmethane, 2,2-bis(3,5-dichloro-4-hydroxyphenyl)propane, 2,2-bis(3,5-dibromo-4-hydroxyphenyl)propane], bis(hydroxyphenyl)cycloalkanes (e.g., 1,1-bis(4-hydroxyphenyl)cyclohexane, 1,1-bis(4-hydroxyphenyl)-3,3,5-trimethylcyclohexane), bis(hydroxyphenyl) ethers (e.g., 4,4′-dihydroxydiphenyl ether), and bis(hydroxyphenyl) sulfones (e.g., 4,4′-dihydroxydiphenylsulfone), bis(hydroxyphenyl) sulfoxides (e.g., 4,4′-dihydroxydiphenyl sulfoxide), and bis(hydroxyphenyl) sulfides (e.g., 4,4′-dihydroxydiphenyl sulfide). Furthermore, the epoxy resin also includes a phenoxy resin which is a high molecular weight bisphenol type epoxy resin (such as a bisphenol A type epoxy resin). These epoxy resins may be used alone or in combination of two or more. Among these epoxy resins, an aliphatic epoxy resin is likely to have a decrease in durability, and thus an aromatic epoxy resin (a glycidyl ether type epoxy resin, etc.) is usually used in many cases, and a bisphenol type epoxy resin (a bisphenol F type epoxy resin and/or a bisphenol A type epoxy resin) (A1) is preferable from the viewpoint of excellent adhesiveness to a reinforcing fiber (para-aramid fiber, etc.). The epoxy resin may usually have two or more (e.g., 2 to 10, preferably 2 to 6, more preferably 2 to 4, and particularly about 2 or 3) epoxy groups in the molecule. The epoxy equivalent of such an epoxy resin can be selected from a range of, for example, about 100 g/eq to 15000 g/eq (e.g., 150 g/eq to 10000 g/eq) depending on the type of the epoxy resin, and may be usually about 200 g/eq to 5000 g/eq (e.g., 250 g/eq to 3000 g/eq or 450 g/eq to 5000 g/eq), preferably about 300 g/eq to 2500 g/eq (e.g., 400 g/eq to 2000 g/eq), more preferably about 450 g/eq to 1500 g/eq (e.g., 500 g/eq to 1000 g/eq), or about 400 g/eq to 4500 g/eq (e.g., 500 g/eq to 3500 g/eq). In particular, the epoxy equivalent of the bisphenol type epoxy resin (typically bisphenol A type epoxy resin) may be about 450 g/eq to 5000 g/eq (e.g., about 600 g/eq to 4500 g/eq), preferably about 700 g/eq to 4000 g/eq (e.g., about 750 g/eq to 3500 g/eq), more preferably about 800 g/eq to 3300 g/eq (e.g., about 900 g/eq to 3000 g/eq), and particularly about 1200 g/eq to 2700 g/eq (e.g., about 1500 g/eq to 2500 g/eq). When the epoxy equivalent is too large, the reactivity may be decreased and the force for bonding the fibers may be decreased, and when the epoxy equivalent is too small, the heat resistance and bending fatigue resistance of the belt may be decreased. Furthermore, the epoxy resin may be a monomer or a multimer (e.g., a 2 to 15-mers, preferably a 2 to 10-mers), and may be a mixture of the monomer and the multimer. The epoxy resin preferably has a hydroxyl group (secondary hydroxyl group). That is, the epoxy resin preferably contains at least a multimer. The weight average molecular weight of the epoxy resin can be selected from a range of, for example, about 300 to 60000 (e.g., 400 to 30000), and may be about 500 to 10000 (e.g., 750 to 7500), preferably about 900 to 5500 (e.g., 100 to 5000), more preferably about 1500 to 4000 (e.g., 1600 to 4000), and particularly about 2000 to 3700 (e.g., 2500 to 3500). The epoxy resin may be liquid or viscous at a room temperature (20° C.), or may be solid. A softening temperature of the solid epoxy resin may be about 60° C. to 170° C. (e.g., 70° C. to 160° C.), preferably about 80° C. to 155° C. (e.g., 90° C. to 150° C.), and more preferably about 100° C. to 145° C. (e.g., 110° C. to 140° C.) in a ring and ball method. The epoxy resin may be in the form of an aqueous solution dissolved in an aqueous medium, and is usually an aqueous dispersion dispersed in an aqueous medium in many cases. Examples of the epoxy resin (e.g., bisphenol A type epoxy resin which is solid at a room temperature) include “jER (registered trademark) 1007”, “jER (registered trademark) 1009F”, and “jER (registered trademark) 1007F” manufactured by Mitsubishi Chemical Corporation. Examples of the aqueous dispersion of such an epoxy resin include “Yuka Resin (registered trademark) KE-307E” and “Yuka Resin (registered trademark) NE-307H” manufactured by Yoshimura Oil Chemical Co., Ltd. The epoxy resin is effective for improving the adhesiveness of the cord (the adhesiveness between the reinforcing fibers, the adhesiveness between the raw yarns, and the adhesiveness to elastomer (or rubber)), but a cured coating film of the epoxy resin represented by the aromatic epoxy resin has relatively high brittleness (brittleness), and there is a concern that the bending fatigue resistance may be reduced. In the present invention, even when an epoxy resin (e.g., bisphenol type epoxy resin) that forms a brittle cured coating film is used, the adhesiveness of the reinforcing fiber and the raw yarn that form the cord can be improved and the toughness can also be improved by combining with a polycarbonate polyol and a polyisocyanate. For this reason, in the present invention, both the fraying resistance and the bending fatigue resistance can be achieved, and the adhesiveness of the power transmission belt to the matrix elastomer (or rubber) can also be improved. [Polycarbonate Polyol (B)] The polycarbonate polyol (B) is obtained by a reaction between polyol and carbonates (dialkyl carbonates, alkylene carbonates, etc.). The polyol contains at least diol, and a branched structure may be introduced by using polyol (alkanepolyol such as glycerin, trimethylolpropane, pentaerythritol, etc.) in combination. Examples of the diol include C2-10alkanediols such as ethylene glycol, propylene glycol, trimethylene glycol, 1,4-butanediol, 1,6-hexanediol, neopentyl glycol, 3-methyl-1,5-pentanediol, and octanediol, and polyalkylene glycols such as diethylene glycol and dipropylene glycol. As the diol, aromatic diol (e.g., bisphenol such as bisphenol A or an ethylene oxide adduct thereof), alicyclic diol (e.g., cyclohexanedimethanol), or the like may be used in combination. The diol contains, for example, C2-10alkanediol such as 1,6-hexanediol in many cases. Therefore, the polycarbonate polyol (B) preferably contains a polycarbonate diol (B1). An average number of functional groups (the number of hydroxyl groups) of the polycarbonate polyol may be at least about 2, preferably about 2 to 6, and more preferably about 2 to 4 (e.g., about 2 to 3). The hydroxyl group value of the polycarbonate polyol can be selected from a range of about 30 mgKOH/g to 500 mgKOH/g (e.g., 50 mgKOH/g to 400 mgKOH/g), and may be, for example, about 80 mgKOH/g to 500 mgKOH/g (e.g., 100 mgKOH/g to 400 mgKOH/g), preferably about 120 mgKOH/g to 350 mgKOH/g (e.g., 150 mgKOH/g to 300 mgKOH/g), and more preferably about 170 mgKOH/g to 270 mgKOH/g (e.g., 200 mgKOH/g to 250 mgKOH/g). The hydroxyl group equivalent of the polycarbonate polyol can be selected from a range of, for example, about 200 g/eq to 2000 g/eq (e.g., 230 g/eq to 1700 g/eq), and may be about 200 g/eq to 1000 g/eq (e.g., 220 g/eq to 800 g/eq), preferably about 200 g/eq to 600 g/eq (e.g., 230 g/eq to 550 g/eq), and more preferably about 200 g/eq to 300 g/eq (e.g., 230 g/eq to 275 g/eq). When the hydroxyl group equivalent is too small, the toughness of the adhesive coating film due to the epoxy resin becomes insufficient, and when the hydroxyl group equivalent is too large, the adhesion strength may be decreased. The hydroxyl group equivalent can be calculated based on the hydroxyl value and based on the formula: hydroxyl group equivalent=1/[(hydroxyl group value mgKOH/g)/1000/56.1]. The number average molecular weight of the polycarbonate polyol can be selected from a range of, for example, about 150 to 5000 (e.g., 175 to 4000), and may be about 200 to 3000 (e.g., 225 to 2500), preferably about 250 to 2000 (e.g., 300 to 1500), and more preferably about 400 to 1000 (e.g., 300 to 700). These polycarbonate polyols (B) may be used alone or in combination of two or more thereof. The polycarbonate polyol (B) can form an adhesive coating film having high toughness, and can effectively improve the fraying resistance and the bending fatigue resistance. The polycarbonate polyol (B) may be in the form of an aqueous solution dissolved in an aqueous medium, or may be an aqueous dispersion dispersed in an aqueous medium. As the polycarbonate polyol (B), for example. “UH-50” and “UH-100” of the “ETERNACOLL (registered trademark)” series manufactured by Ube Industries, Ltd., and “976” and “965” of the “Nippolan (registered trademark)” series manufactured by Tosoh Corporation can be used. Examples of the aqueous dispersion of such a polycarbonate polyol include “Yuka Resin (registered trademark) PEP-50” and “Yuka Resin (registered trademark) PEP-90” manufactured by Yoshimura Oil Chemical Co., Ltd. An amount of the polycarbonate polyol to be used is not particularly limited, and a molar ratio of the hydroxyl group of the epoxy resin (A) to the hydroxyl group of the polycarbonate polyol (B) may be selected from a range of, for example, the former/the latter of about 30/70 to 97/3 (e.g., about 35/65 to 95/5), about 45/55 to 95/5 (e.g., about 50/50 to 92/8), preferably about 45/55 to 93/7 (e.g., about 45/55 to 85/15), more preferably about 55/45 to 90/10 (e.g., about 60/40 to 87/13), still more preferably about 65/35 to 85/15 (e.g., about 70/30 to 80/20), and may be about 50/50 to 85/15 (e.g., about 60/40 to 80/20), particularly about 70/30 to 80/20. When the amount of the epoxy resin (A) is too small, the adhesiveness between the fibers of the cord and between the raw yarns, and the adhesiveness between the elastomers (or rubbers) may be reduced, and when the amount of the epoxy resin (A) is too large, the toughness of the adhesive coating film may be reduced, the fraying resistance and the bending fatigue resistance may be reduced, and the strength retention rate may be reduced. A ratio of the polycarbonate polyol (B) to the epoxy resin (A) may be about 2.5 parts by mass to 50 parts by mass (e.g., 3 parts by mass to 45 parts by mass), preferably about 5 parts by mass to 40 parts by mass (e.g., 7 parts by mass to 35 parts by mass), more preferably about 8 parts by mass to 30 parts by mass (e.g., 10 parts by mass to 25 parts by mass), or about 7.5 parts by mass to 20 parts by mass (e.g., 10 parts by mass to 15 parts by mass), in terms of solid content, with respect to 100 parts by mass of the epoxy resin (A). [Blocked Polyisocyanate (C)] When the blocked polyisocyanate which is protected by a blocking agent is used, the blocked polyisocyanate does not react in the process of dipping or impregnating the cord (reinforcing fiber or raw yarn) in the first treatment agent, and the curing reaction proceeds by heating in the process of vulcanizing the elastomer (or rubber) such as the power transmission belt, so that the operability can be improved. The polyisocyanate protected by a blocking agent may be any polyisocyanate as long as the polyisocyanate can generate a free (or reactive) isocyanate group by heating, and a polyisocyanate having a plurality of isocyanate groups in one molecule (e.g., diisocyanate) is usually used in many cases. Examples of the polyisocyanate include aliphatic polyisocyanates [diisocyanates such as propylene diisocyanate, trimethylene diisocyanate, tetramethylene diisocyanate, hexamethylene diisocyanate (HDI), trimethylhexamethylene diisocyanate (TMDI), and lysine diisocyanate (LDI); triisocyanates or polyisocyanates such as 1,6,11-undecanetriisocyanatemethyloctane and 1,3,6-hexamethylene triisocyanate]; alicyclic polyisocyanates [diisocyanates such as cyclohexane 1,4-diisocyanate, isophorone diisocyanate (IPDI), hydrogenated xylylene diisocyanate, and hydrogenated bis(isocyanatophenyl)methane; triisocyanates or polyisocyanates such as bicycloheptane triisocyanate]; aromatic polyisocyanates [diisocyanates such as phenylene diisocyanate, tolylene diisocyanate (TDI), xylylene diisocyanate (XDI), tetramethylxylylene diisocyanate (TMXDI), naphthalene diisocyanate (NDI), bis(isocyanatophenyl)methane (MDI), toluidine diisocyanate (TODI), and 1,3-bis(isocyanatophenyl)propane; and triisocyanates or polyisocyanates]. The polyisocyanate may be a derivative such as a multimer (dimer, trimer, tetramer, etc.), an adduct, a modified product (biuret modified product, allophanate modified product, urea modified product, etc.), a urethane oligomer having a plurality of isocyanate groups, or the like. Examples of the modified product or derivative of polyisocyanate include adducts of polyisocyanate (aliphatic polyisocyanate such as hexamethylene diisocyanate) and polyhydric alcohol (trimethylolpropane or pentaerythritol), biuret of the polyisocyanate, and multimers of the polyisocyanate (e.g., aliphatic polyisocyanate) (e.g., polyisocyanate having an isocyanurate ring such as trimer of hexamethylene diisocyanate). These polyisocyanates may be used alone or in combination of two or more. Among these polyisocyanates, aliphatic polyisocyanates or derivatives thereof (e.g., HDI or trimers thereof), aromatic polyisocyanates (TDI, MDI, etc.), and the like are widely used. Furthermore, the isocyanate group of the polyisocyanate is protected by a blocking agent to form a blocked polyisocyanate (or thermal reaction type polyisocyanate). When the blocked polyisocyanate is used, the stability in the aqueous medium can be improved since the isocyanate group is protected by the blocking agent, and the treatment efficiency of the cord (the reinforcing fiber and the raw yarn) can be improved since the blocked polyisocyanate is inactive and is not involved in curing in the process of forming the cord. The blocking agent is dissociated by heating, the isocyanate group is activated, and the isocyanate group reacts with the reactive group of the epoxy resin, the polycarbonate polyol and/or the reinforcing fiber (and the raw yarn) to be cured, whereby a cord can be formed with high adhesiveness, and the fraying resistance and the bending fatigue resistance can be improved. Therefore, the adhesiveness of the belt to the elastomer (or rubber) and the abrasion resistance and durability of the belt can be improved. Furthermore, since the treatment agent is an aqueous treatment agent containing polyisocyanate, the preparation of the solution is simple, and the environmental load is small. As the blocked polyisocyanate, common heat-reactive polyisocyanates can be used. Examples of the blocking agent (protective agent) include C1-24monoalcohols such as methanol, ethanol, and isopropanol or alkylene oxide adducts thereof (e.g., C2-4alkylene oxide adducts such as ethylene oxide); phenols such as phenol, cresol, and resorcin; oximes such as acetoxyme, methyl ethyl ketoxime, and cyclohexane oxime; lactams such as ε-caprolactam and valerolactam; and secondary amines such as dibutylamine and ethyleneimine. These blocking agents may be used alone or in combination of two or more. Among these, oximes, lactams, and the like are widely used. The blocked polyisocyanate (or heat-reactive polyisocyanate) may be in the form of an aqueous solution, but is often in the form of an aqueous dispersion. The blocked polyisocyanate (heat-reactive isocyanate) may be, for example, a water-soluble blocked polyisocyanate or a water-dispersible blocked polyisocyanate into which a hydrophilic group (an acidic group such as a carboxyl group or a sulfonic acid group or a salt thereof, an oxyethylene group, etc.) is introduced. The content of the isocyanate group in the blocked polyisocyanate (heat-reactive isocyanate) is not particularly limited, and may be, for example, about 1% by mass to 50% by mass, preferably about 3% by mass to 40% by mass, and more preferably about 5% by mass to 30% by mass. The isocyanate group equivalent of the blocked polyisocyanate may be about 200 g/eq to 600 g/eq (e.g., 220 g/eq to 570 g/eq), preferably about 250 g/eq to 500 g/eq (e.g., 270 g/eq to 450 g/eq), and more preferably about 300 g/eq to 400 g/eq (e.g., 330 g/eq to 380 g/eq). The isocyanate group equivalent can be calculated based on the number of isocyanate groups and the molecular weight, and can also be calculated from the isocyanate group content I (% by mass) according to the calculation formula: isocyanate group equivalent=42/(I/100). A dissociation temperature of the blocked polyisocyanate (temperature at which the blocking agent is dissociated and the active isocyanate group is regenerated) is usually equal to or higher than a drying temperature of the cord impregnated with the treatment agent. When the dissociation temperature is high, the drying temperature can be increased, and thus the productivity of the cord can be improved. The dissociation temperature may be, for example, 120° C. or higher (e.g., 140° C. or higher), preferably 150° C. or higher (e.g., 160° C. or higher), or more preferably 170° C. or higher (e.g., 180° C. or higher), and may be, for example, about 120° C. to 250° C. (e.g., 150° C. to 240° C.), preferably 160° C. to 230° C. (e.g., 170° C. to 220° C.), or more preferably 175° C. to 210° C. (e.g., 180° C. to 200° C.). When the dissociation temperature is too low, the drying temperature cannot be increased, so that the drying efficiency may be reduced and the productivity of the cord may be reduced. The blocked polyisocyanate may be in the form of an aqueous solution dissolved in an aqueous medium, or may be an aqueous dispersion dispersed in an aqueous medium. Examples of the blocked polyisocyanate (C) include blocked polyisocyanates of a general-purpose diisocyanate (a diisocyanate based polyisocyanate such as hexamethylene diisocyanate (HDI) or a derivative thereof (polyisocyanate having an isocyanurate ring, etc.), diphenylmethane diisocyanate (MDI), etc.) blocked with a general-purpose blocking agent (oximes such as methyl ethyl ketoxime, lactams such as ε-caprolactam, etc.). As the aqueous dispersion of such blocked polyisocyanate, for example, “Yuka Resin (registered trademark) AK-81” manufactured by Yoshimura Oil Chemical Co., Ltd., “BN-27” and “BN-69” of the “Elastron (registered trademark)” series manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., and the like can be used. An amount of the blocked polyisocyanate (C) to be used can be selected from a range of about 0.1 mole to 2 mole (e.g., 0.2 mole to 1.8 mole) of the isocyanate group (blocked isocyanate group) of the blocked polyisocyanate with respect to 1 mole of the total amount of the hydroxyl groups of the epoxy resin (A) and the polycarbonate polyol (B), and may be about 0.3 mole to 1.7 mole (e.g., 0.5 mole to 1.5 mole), preferably about 0.7 mole to 1.3 mole (e.g., 0.8 mole to 1.2 mole), more preferably about 0.7 mole to 1.15 mole (e.g., 0.75 mole to 1.1 mole), or about 0.75 mole to 1.25 mole (e.g., 0.9 mole to 1.1 mole). [Curing Agent] The first treatment agent according to the present invention may contain a curing agent in addition to the above components. As the curing agent, various curing agents for the epoxy resin can be used, and the curing agent may function as a curing agent for the blocked polyisocyanate. Examples of the curing agent include amines (amines having at least a primary amino group, amines having a tertiary amino group, etc.), polyamide resins (polyaminoamides having a plurality of amino groups and at least one amide group, polyaminoamides having an imidazole ring, etc.), acid anhydride curing agents (aliphatic acid anhydrides such as dodecenyl succinic anhydride and polyadipic anhydride, alicyclic acid anhydrides such as hexahydrophthalic anhydride, aromatic acid anhydrides such as pyromellitic anhydride and benzophenonetetracarboxylic anhydride, etc.), mercaptan curing agents (mercaptopropionic acid ester, etc.), imidazole-based curing agents, resol phenol resins, amino resins (urea resin, melamine resin, etc.), latent curing agents (dicyandiamides such as dicyandiamide and dicyandiamide-modified polyamine, organic acid hydrazides, Lewis acids (boron trifluoride-amine complexes, etc.), etc.). Examples of the amines include aliphatic amines such as diethylenetriamine, triethylenetetramine, dipropylenediamine, hexamethylenediamine, trimethylhexamethylenediamine, and triethanolamine; alicyclic amines such as menthenediamine, isophoronediamine, bis(aminomethyl)cyclohexane, and bis(aminocyclohexyl)methane; aromatic amines such as phenylenediamine, bis(aminophenyl)methane, bis(4-amino-3-methylphenyl)methane, bis(aminophenyl)sulfone, benzyldimethylamine, 2-(dimethylaminomethyl)phenol, and 2,4,6-tris(dimethylaminomethyl)phenol; aromatic aliphatic amines such as xylylenediamine; heterocyclic amines such as N,N′-dimethylpiperazine, 1,8-diazabicyclo[5.4.0]undecene-7 (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO), 3,9-bis(3-aminopropyl)-2,4,8,10-tetraoxaspiro[5.5]undecane (ATU), and phthalocyanine tetramine; modified polyamines using these polyamines (adducts to epoxy resin, Michael adduct to unsaturated compound, reaction product with methylol compound). These curing agents may be used alone or in combination of two or more. The curing agent may be a curing agent that can impart high heat resistance, for example, a curing agent containing at least an imidazole-based curing agent. The imidazole-based curing agent is useful for improving the heat resistance of the adhesive coating film of the cord. In the imidazole-based curing agent, a 2-position of an imidazole ring may be substituted with a substituent such as a linear or branched C1-24alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, a hexyl group, an octyl group, a decyl group, an undecyl group, a dodecyl group, a tetradecyl group, a hexadecyl group, and an octadecyl group, a C3-10cycloalkyl group such as a cyclohexyl group, and a C6-12aryl group such as a phenyl group and a naphthyl group. The imidazole ring may have a hydrogen atom at a 4-position, and the 4-position may be substituted with a linear or branched C1-6alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, or a butyl group. Furthermore, the imidazole-based curing agent may be various modified products, and may be, for example, a cyanoethylated product in which an active hydrogen atom of an imino group (NH group) at a 1-position of an imidazole ring is cyanoethylated, a triazine product in which a cyanoethyl group of a cyanoethylated product is triazine, a modified product in which a hydroxymethyl group, an alkoxymethyl group, or the like is introduced at a 4-position and/or 5-position, or a salt thereof (a polyvalent carboxylic acid salt such as trimellitic acid, an isocyanuric acid salt, etc.). A ratio of the curing agent with respect to 100 parts by mass of the epoxy resin may be, for example, about 1 part by mass to 10 parts by mass (e.g., about 2 parts by mass to 8 parts by mass), preferably about 2.5 parts by mass to 7.5 parts by mass (e.g., about 3 parts by mass to 7 parts by mass), and more preferably about 3.5 parts by mass to 6.5 parts by mass (e.g., about 4 parts by mass to 6 parts by mass). [Curing Accelerator] The first treatment agent may further contain a curing accelerator in order to accelerate the curing reaction by the curing agent. As the curing accelerator, a curing agent or a curing accelerator commonly used as a curing accelerator of an epoxy resin can be used, and a tertiary amine is preferable from the viewpoint that the epoxy resin can be cured under appropriate conditions. Examples of the tertiary amines include aliphatic amines such as triethylamine, triethanolamine, and dimethylaminoethanol, aromatic amines such as benzyldimethylamine and 2,4,6-tris(dimethylaminomethyl)phenol, and heterocyclic amines such as DBU, DABCO, and ATU. These tertiary amines may be used alone or in combination of two or more. A ratio of the curing accelerator may be 10 parts by mass or less (e.g., about 1 part by mass to 10 parts by mass, preferably about 2 parts by mass to 6 parts by mass, and more preferably about 3 parts by mass to 5 parts by mass) with respect to 100 parts by mass of the epoxy resin. [Aqueous Medium (Aqueous Solvent)] The first treatment agent containing the epoxy resin (A), the polycarbonate polyol (B), and the blocked polyisocyanate (C) forms an aqueous treatment agent containing an aqueous medium (aqueous solvent). The aqueous medium (aqueous solvent) may be water alone or a mixed solvent of water and a water-soluble organic solvent. Examples of the water-soluble organic solvent include alcohols such as methanol, ethanol, propanol, and isopropanol; ketones such as acetone; ethers such as dioxane; amides such as N,N-dimethylformamide and N,N-dimethylacetamide; N-methylpyrrolidone; sulfoxides such as dimethyl sulfoxide; glycol ethers (e.g., cellosolves such as methyl cellosolve, ethyl cellosolve, and butyl cellosolve; carbitols such as methyl carbitol and ethyl carbitol; propylene glycol monomethyl ether); glycol ether esters (e.g., cellosolve acetate such as methyl cellosolve acetate, carbitol acetate, propylene glycol monomethyl ether acetate); glyme and diglyme. These water-soluble organic solvents may be used alone or in combination of two or more thereof. The aqueous medium (aqueous solvent) is a solvent containing water as a main component, for example, a solvent having a water content of 50% by mass or more (preferably 75% by mass or more, and more preferably 90)% by mass or more) in many cases, and water may be used alone from the viewpoint of reducing the environmental load. When a hydrophobic (or immiscible) organic solvent is used to hydrate at least one component selected from the epoxy resin (A), the polycarbonate polyol (B) and the blocked polyisocyanate (C) (in the form of an aqueous solution or an aqueous dispersion), some or unavoidable organic solvent may remain. [Other Components (Additives)] The polycarbonate polyol (B) may be used in combination with polymer polyol, for example, polyacryl polyol, polyester polyol, polyether polyol, if necessary. Furthermore, the first treatment agent or the aqueous solvent may contain a surfactant or a dispersant, and such a component may be derived from a surfactant or a dispersant used in hydration of at least one component selected from the components (A), (B), and (C). If necessary, the first treatment agent may contain various additives, for example, additives such as stabilizers (antioxidants, ultraviolet absorbers, heat stabilizers, etc.), antioxidants, reactive diluents (low viscosity glycidyl ethers, e.g., monoglycidyl ethers), surface treatment agents, adhesiveness improvers, tackifiers, coupling agents, plasticizers, leveling agents, viscosity modifiers or rheology modifiers, preservatives, bactericides, fillers or fillers, colorants, and lubricants. A total amount of these additives may be 30% by mass or less (e.g., 0.01% by mass to 30% by mass), preferably about 0.05% by mass to 20% by mass (e.g., 0.1% by mass to 10% by mass) with respect to the total solid content of the first treatment agent. The first treatment agent contains the epoxy resin (A), the polycarbonate polyol (B), and the blocked polyisocyanate (C), and may contain an aqueous urethane resin in which a reaction product of polyol such as polyester polyol or polyether polyol and polyisocyanate (such as aromatic polyisocyanate) is dissolved or dispersed in an aqueous medium, and/or a rubber latex. In the present invention, even when such components are not contained, it is possible to enhance the fraying resistance, the bending fatigue resistance, and the adhesiveness to the elastomer (or rubber). A solid content concentration of the first treatment agent (first treatment liquid) can be selected from a range of, for example, about 1% by mass to 70% by mass (e.g., about 3% by mass to 50% by mass), and may be usually about 5% by mass to 40% by mass (e.g., about 7% by mass to 30% by mass), and preferably about 10% by mass to 25% by mass (e.g., about 10% by mass to 20% by mass). When the solid content concentration is too high, the impregnability and permeability of the treatment agent into the fibers may be reduced, and the bending fatigue resistance of the belt may be reduced. (Treatment with First Treatment Agent) The first treatment agent (first treatment liquid) may be prepared by dissolving or dispersing each component in an aqueous medium, or may be prepared by mixing solutions or dispersions of each component. If necessary, a water-insoluble component (e.g., a curing agent) may be dissolved and mixed in a water-soluble organic solvent. The treatment of the untreated yarn (reinforcing fiber, raw yarn (multifilament yarn), or twisted yarn cord) with the first treatment agent (first treatment liquid) can be performed by a common method, for example, a method such as spraying, coating, or dipping, and a dipping method is often used. An immersion time is not particularly limited, and may be selected, for example, from a range of about 1 second to 10 minutes (e.g., 2 seconds to 1 minute), or may be about 1 second to 30 seconds (e.g., 2 seconds to 25 seconds), and preferably about 2 seconds to 20 seconds (e.g., 5 seconds to 15 seconds). After the twisted yarn cord is treated with the first treatment agent, the twisted yarn cord may be dried as necessary. A drying temperature may be, for example, about 80° C. to 200° C. (e.g., about 100° C. to 190° C.), or preferably about 120° C. to 180° C. (e.g., about 130° C. to 170° C.). A drying time may be, for example, about 10 seconds to 30 minutes, preferably about 30 seconds to 10 minutes, and more preferably about 1 to 5 minutes, depending on the drying temperature. Furthermore, the drying may be performed by applying tension to the twisted yarn cord. The tension may be, for example, about 5 N to 20 N (e.g., about 7 N to 17 N), and preferably about 10 N to 15 N. When the twisted yarn cord is dried under the action of tension, the treatment agent is easily fitted to the twisted yarn cord, unevenness in twisting can be reduced, and variation in the diameter of the twisted yarn caused by unevenness in twisting can be reduced. An adhesion ratio of the first treatment agent to the untreated yarn (twisted yarn cord, etc.) [(mass after treatment with first treatment agent−mass before treatment with first treatment agent)/mass after treatment with first treatment agent×100] can be selected from a range of, for example, about 0.1% by mass to 10% by mass (e.g., about 0.2% by mass to 7% by mass) in terms of solid content, and may be about 0.5% by mass to 5% by mass, preferably about 0.7% by mass to 4 mass %, and more preferably about 1% by mass to 3 mass %. An average thickness of the coating film formed by the first treatment agent can be selected from a range of, for example, about 0.001 μm to 20 μm (e.g., 0.005 μm to 15 μm), and may be about 0.01 μm to 10 μm (e.g.e, 0.05 μm to 5 μm), and preferably about 0.1 μm to 3 μm (e.g., 0.5 μm to 2 μm). [Second Treatment Agent] In the present invention, the cord (untreated yarn) is treated with at least the first aqueous treatment agent, and may be further treated with a second aqueous treatment agent. Therefore, the aqueous treatment agent according to the present invention may contain the first aqueous treatment agent (first aqueous treatment liquid) and the second aqueous treatment agent (second aqueous treatment liquid), and the present invention also includes a set of the first aqueous treatment agent and the second aqueous treatment agent. The second treatment agent (second aqueous treatment agent) contains at least resorcin (R), formaldehyde (F), and rubber or latex (L), and forms a resorcin-formaldehyde-latex (RFL) treatment liquid. When the treatment is performed with such a second treatment agent, the adhesiveness between the cord and the power transmission belt main body can be further improved even when the second treatment agent is aqueous. The resorcin (R) and the formaldehyde (F) may be contained in the form of a mixture of the resorcin (R) and the formaldehyde (F), or may be contained in the form of a condensate thereof (an RF condensate, a condensate having a methylol group, etc.). The RF condensate (such as condensate having a methylol group, etc.) and the epoxy resin (A) and/or the blocked polyisocyanate (C) of the first treatment agent may be reacted or co-cured by the treatment with the second treatment agent in combination with the curing agent of the first treatment agent. By such a reaction or co-curing, a tough and strong coating film may be formed on the surface of the cord, and the fraying of the cord may be prevented to a high degree. Depending on the type of the latex (L), the epoxy resin (A), the blocked polyisocyanate (C), and/or the RF condensate (such as condensate having a methylol group, etc.) may react with the rubber of the latex (L). The RF condensate is not particularly limited, and examples thereof include a novolac type, a resol type, and a combination (or a mixture) thereof. The RF condensate is preferably a combination (mixture) of the novolac type and the resol type since the RF condensate reacts with the epoxy resin of the first treatment agent and can form a coating film of the condensate on a surface of a treatment layer of the first treatment agent. When the novolac type and the resol type are combined, the epoxy resin cured between the fibers in the cord (untreated yarn such as twisted yarn cord) by the first treatment agent reacts with the novolac type RF condensate, and a larger amount of resol type RF condensates can form a strong RFL coating film having a high crosslinking density in a surface layer portion (or an outer peripheral portion) of the cord (twisted yarn cord, etc.). Therefore, it is possible to form a cured product in which the epoxy resin cured product for bonding the fibers to each other and the RFL coating film on the outer peripheral portion of the cord (untreated yarn such as twisted yarn cord) are integrated by the reaction between the novolac type RF condensate and the epoxy resin, and it is possible to improve the adhesiveness and the fraying resistance between the cord (untreated yarn such as twisted yarn cord) and the rubber. The RF condensate may be, for example, a reaction product (e.g., initial condensate or prepolymer) produced by a reaction between resorcin and formaldehyde in the presence of water and a base catalyst (alkali metal salt such as sodium hydroxide; an alkaline earth metal salt; ammonia, etc.). As long as the effects of the present invention are not inhibited, aromatic phenols (monohydroxyarene such as phenol and cresol) may be used in combination with resorcin, and di- or polyhydroxyarene such as catechol and hydroquinone may be used in combination with resorcin. As the formaldehyde, a condensate of formaldehyde (e.g., trioxane, paraformaldehyde) may be used, or an aqueous solution of formaldehyde (formalin, etc.) may be used. A ratio (usage ratio) of the formaldehyde can be selected from a range of about 0.1 mole to 5 mole (e.g., 0.2 mole to 2 mole) with respect to 1 mole of resorcin, and in the case of producing a mixture of the resol type and the novolac type, the ratio may be, for example, about 0.3 mole to 1 mole (e.g., 0.4 mole to 0.95 mole), preferably about 0.5 mole to 0.9 mole (e.g., 0.6 mole to 0.85 mole), and more preferably about 0.65 mole to 0.8 mole (e.g., 0.7 mole to 0.8 mole). When the ratio of the formaldehyde is too large, the ratio of the resol type RF condensate is too large, and thus there is a risk that the reaction with the epoxy resin may be decreased. Conversely, when the ratio of the formaldehyde is too small, the ratio of novolak type RF condensate is too small, and thus the strength of the coating film formed by the second treatment agent may be decreased. The rubber or elastomer constituting the latex is not particularly limited as long as flexibility can be imparted to the cord, and examples thereof include diene rubbers [e.g., natural rubber, isoprene rubber, butadiene rubber, chloroprene rubber, styrene-butadiene rubber, vinylpyridine-styrene-butadiene copolymer rubber, acrylonitrile-butadiene rubber (nitrile rubber), hydrogenated products of these diene rubbers], olefin rubbers [e.g., ethylene-α-olefin rubber (ethylene-α-olefin elastomer), polyoctenylene rubber, ethylene-vinyl acetate copolymer rubber, chloroprene rubber, chlorosulfonated polyethylene rubber, alkylated chlorosulfonated polyethylene rubber], acrylic rubbers, silicone rubbers, urethane rubbers, epichlorohydrin rubbers, fluororubbers, and combinations thereof. These rubbers may be used alone or in combination of two or more. As the latex, a latex of a vinylpyridine-styrene-butadiene copolymer rubber is often used. As the rubber, the same or similar rubber or elastomer as the rubber or elastomer into which the cord is embedded may be preferably used. A ratio of the latex can be selected from a range of about 30 parts by mass to 700 parts by mass (e.g., 40 parts by mass to 650 parts by mass) with respect to 100 parts by mass of the solid content of the RF condensate in terms of the solid content, and may be, for example, about 50 parts by mass to 600 parts by mass (e.g., 75 parts by mass to 500 parts by mass), preferably about 100 parts by mass to 400 parts by mass (e.g., 125 parts by mass to 300 parts by mass), and more preferably about 150 parts by mass to 250 parts by mass (e.g., 200 parts by mass to 250 parts by mass). The second treatment agent usually contains the same aqueous medium (water, etc.) as the first treatment agent. If necessary, the second treatment agent may contain an additive (e.g., a vulcanizing agent, a vulcanization accelerator, or a co-vulcanizing agent) involved in vulcanization or crosslinking of the rubber or the elastomer, in addition to the same additive (e.g., an additive such as a surfactant, a dispersant, or a stabilizer) as the first treatment agent. A solid content concentration of the second treatment agent may be, for example, about 0.1% by mass to 30% by mass (e.g., about 0.5% by mass to 27% by mass), preferably about 1% by mass to 25% by mass (e.g., about 2% by mass to 20% by mass), and more preferably about 3% by mass to 17% by mass (e.g., about 5% by mass to 15% by mass). The second treatment agent having such a solid content concentration can adjust an adhesion amount to the untreated yarn (twisted yarn cord, etc.) treated with the first treatment agent to an appropriate range, and can efficiently improve the properties of the cord. The treatment method using the second treatment agent is the same as the treatment method using the first treatment agent. A drying temperature may be the same as the drying temperature in the treatment with the first treatment agent, and may be about 150° C. to 250° C. (e.g., 170° C. to 220° C.). An adhesion ratio of the second treatment agent [(mass after treatment with second treatment agent−mass before treatment with second treatment agent)/mass after treatment with second treatment agent×100] can be selected from a range of, for example, about 0.1% by mass to 25% by mass (e.g., 0.5% by mass to 22% by mass) in terms of solid content, and may be about 1% by mass to 20% by mass (e.g., 1.5% by mass to 15% by mass), and preferably about 2% by mass to 10% by mass (e.g., 3% by mass to 8% by mass). An average thickness of the coating film formed by the second treatment agent may be, for example, about 1 μm to 30 μm, preferably about 2 μm to 25 μm, and more preferably about 5 μm to 20 μm. As described above, although the second treatment agent is not necessarily required, the ratio (mass ratio) of the adhesion amount of the first treatment agent to the adhesion amount of the second treatment agent can be selected from a range of, for example, the former/latter of about 0.1/1 to 20/1 (e.g., 0.3/1 to 15/1) in terms of solid content, and may be about 0.5/1 to 12/1, preferably about 1/1 to 10/1, and more preferably about 2/1 to 8/1 (e.g., 3/1 to 7/1). In the present invention, after the treatment with the second treatment agent, if necessary, the treatment may be performed with a third treatment agent containing rubber or elastomer (e.g., aqueous rubber latex, organic solvent solution of rubber, or rubber glue), but from the viewpoint of simplifying the process and improving the working environment and environmental load, it is preferable not to use the third treatment agent (rubber glue) containing at least an organic solvent. Furthermore, in the present invention, even when the third treatment agent is not used, a cord having high fraying resistance and bending fatigue resistance can be produced, and the adhesiveness to the matrix rubber can be improved. An average diameter of the cord may be, for example, about 0.3 mm to 3.6 mm (e.g., about 0.4 mm to 3.5 mm), preferably about 0.5 mm to 3.3 mm (e.g., about 0.55 mm to 3.1 mm), and more preferably about 0.6 mm to 2.7 mm (e.g., about 0.7 mm to 2.5 mm). [Power Transmission Belt and Method for Producing Same] The cord is prepared by treating an untreated yarn with the first aqueous treatment agent. The cord may be prepared by treating the untreated yarn with the first aqueous treatment agent and then treating the yarn with the second aqueous treatment agent. The cord includes a cured product obtained by the first treatment agent penetrating into the surface and between the fibers (or between the raw yarns) and being cured. When the cord is further treated with the second treatment agent, the cord includes a cured product obtained by an RFL liquid reacting with the epoxy resin and being cured. Such a cord is suitable as a cord for rubber reinforcement, for example, a cord for a power transmission belt. The cord is usually used by being embedded in a rubber layer (crosslinked rubber layer) of a power transmission belt. The power transmission belt includes the cord, and usually includes a rubber layer in which the cord (usually a plurality of cords) is embedded along a longitudinal direction (or a circumferential direction) of a belt main body in many cases, and the cord may be embedded in parallel at a predetermined pitch in parallel with the longitudinal direction of the belt main body. An interval (spinning pitch) between adjacent cords may be, for example, about 0.5 mm to 4 mm (e.g., about 0.6 mm to 2.5 mm), preferably about 0.7 mm to 2.3 mm (e.g., about 0.8 mm to 2 mm), or about 0.5 mm to 2 mm (e.g., about 0.6 mm to 1.8 mm), more preferably about 0.7 mm to 1.7 mm (e.g., about 0.8 mm to 1.5 mm), depending on the cord diameter. The rubber layer of the power transmission belt can be formed of a composition containing an unvulcanized rubber, and examples of the unvulcanized rubber include diene rubbers (natural rubber, isoprene rubber, butadiene rubber, chloroprene rubber, butyl rubber, styrene-butadiene rubber (SBR), vinylpyridine-styrene-butadiene rubber, acrylonitrile-butadiene rubber (nitrile rubber: NBR), acrylonitrile-chloroprene rubber, hydride nitrile rubber (HNBR), and the like), ethylene-α-olefin elastomers, chlorosulfonated polyethylene rubbers (CSM), alkylated chlorosulfonated polyethylene rubbers (ACSM), epichlorohydrin rubbers, acrylic rubbers, silicone rubbers, urethane rubbers, and fluororubbers. These rubber components may be carboxylated, such as carboxylated SBR and carboxylated NBR. These rubber components may be used alone or in combination of two or more. The rubber component preferably contains at least ethylene-α-olefin elastomer (or ethylene-α-olefin-rubber) such as an ethylene-propylene copolymer (EPM) or an ethylene-propylene-diene terpolymer (EPDM) as a main component. An unvulcanized rubber composition may contain common components, and examples of such components include reinforcing agents such as carbon black and/or silica, short fibers, polyvalent metal salts of (meth)acrylic acid such as zinc(meth)acrylate and magnesium(meth)acrylate, vulcanizing agents and/or crosslinking agents (organic peroxides, etc.), co-crosslinking agents (alkane polyol poly(meth)acrylates such as ethylene glycol dimethacrylate, butanediol dimethacrylate, and trimethylolpropane trimethacrylate; triallyl(iso)cyanurate; bismaleimide such as N,N′-m-phenylene bismaleimide and N,N′-(4,4′-diphenylmethane bismaleimide), vulcanization aids, vulcanization accelerators (thiuram based accelerators, etc.), vulcanization accelerator aids (stearic acid, etc.), vulcanization retarders, metal oxides (e.g., zinc oxide, magnesium oxide, calcium oxide, barium oxide, iron oxide, copper oxide, titanium oxide, and aluminum oxide), fillers (clay, calcium carbonate, talc, mica, etc.), plasticizers, surfactants, softeners (paraffin oil, naphthenic oil, etc.), processing agents or processing aids (stearic acid metal salt, wax, paraffin, etc.), anti-aging agents (aromatic amine based anti-aging agents, benzimidazole based anti-aging agents, etc.), adhesiveness improvers [resorcin-formaldehyde co-condensate, melamine resin such as hexamethoxymethylmelamine, co-condensate thereof (such as resorcin-melamine-formaldehyde co-condensate), etc], colorant, tackifier, coupling agent (silane coupling agent, etc.), stabilizer (antioxidant, ultraviolet absorber, heat stabilizer, etc.), lubricant, flame retardant, and antistatic agents. These additives may be used alone or in combination. The power transmission belt may include a cover fabric (or a reinforcing fabric) such as canvas. Examples of such a power transmission belt include a V-ribbed belt, a flat belt, a toothed belt and a V-belt such as a wrapped V-belt and a raw edge V-belt. Typically, the power transmission belt may include an adhesive rubber layer and a compression rubber layer on one surface of the adhesive rubber layer, and the cord may be embedded in the adhesive rubber layer. A tension rubber layer may be provided on the other surface of the adhesive rubber layer. In the power transmission belt, a part of the belt main body formed of the rubber layer (e.g., the surface of the tension rubber layer and/or the compression rubber layer) or the whole thereof may be covered (or laminated) with a reinforcing fabric. FIG.1is a schematic cross-sectional view showing a V-ribbed belt as an example of a power transmission belt. In this example, the belt includes an adhesive rubber layer2in which cords1are embedded in a longitudinal direction of the belt, a compression rubber layer3formed on one surface (inner peripheral surface) of the adhesive rubber layer, and a tension rubber layer4formed on the other surface (outer peripheral surface or back surface) of the adhesive rubber layer, and V-shaped groove ribs5are formed on the compression rubber layer3. The compression rubber layer3contains polyamide short fibers6in order to improve lateral pressure resistance of the power transmission belt. In many cases, the adhesive rubber layer2, the compression rubber layer3, and the tension rubber layer4are each formed of a rubber composition. Further, a reinforcing fabric formed of a woven fabric, a nonwoven fabric, a knitted fabric, and the like may be laminated on the back surface of the tension rubber layer4. FIG.2is a schematic cross-sectional view showing a raw edge V-belt that is another example of the power transmission belt. The belt shown inFIG.2is configured in the same manner as inFIG.1except that the ribs5are not formed on the compression rubber layer3and that the belt has a trapezoidal shape in which a belt width is decreased from an outer peripheral surface toward an inner peripheral surface. In the compression rubber layer3, a plurality of cogs (convex portions) may be formed at predetermined intervals along the longitudinal direction of the belt. The reinforcing fabric formed of a woven fabric, a nonwoven fabric, a knitted fabric, and the like may be laminated on the surface (inner peripheral surface) of the compression rubber layer3and the surface (outer peripheral surface) of the tension rubber layer4. These power transmission belts are formed by sequentially winding an unvulcanized rubber sheet for a compression rubber layer and an unvulcanized rubber sheet for a first adhesion rubber layer around a cylindrical forming drum, spirally spinning a cord thereon, further sequentially winding an unvulcanized rubber sheet for a second adhesion rubber layer and an unvulcanized rubber sheet for a tension rubber layer to form a laminate, vulcanizing the laminate to produce a vulcanization belt sleeve, and cutting the cylindrical vulcanization belt sleeve in a circumferential direction. At the time of this cutting, the cords arranged or oriented in the circumferential direction are also cut, and the cords are exposed to a side surface (cut surface) of the power transmission belt. When the cord is exposed to the side surface of the power transmission belt, the cord is easily loosened, and pop-out occurs in which the cord protrudes from the side surface of the power transmission belt with the cord loosened from the side surface of the power transmission belt as a starting point. The pop-out cord may be wound around a shaft of a rotating pulley to break the power transmission belt. However, since the cord treated with the specific treatment agent is embedded in the rubber layer (the adhesive rubber layer in the power transmission belt shown inFIG.1andFIG.2) and the binding property between the filaments of the cord is high, the cord is not loosened on the side surface of the power transmission belt, popping out of the cord can be effectively prevented, and the durability of the power transmission belt can be significantly improved. The power transmission belt can be manufactured by a common method including an embedding step of embedding a cord in a rubber layer along a longitudinal direction of a belt depending on a type of the belt. For example, the power transmission belt can be manufactured by a method in which a cylindrical laminate in which a cord treated with a specific treatment agent is sandwiched or embedded between a pair of unvulcanized rubber sheets (including unvulcanized laminated rubber sheets) may be vulcanized to produce a power transmission belt precursor (vulcanized belt sleeve), and the cylindrical power transmission belt precursor may be cut in the circumferential direction. In the present invention, even when the cutting is performed in this manner, fluffing or fraying of the cord is not generated on the side surface (transmission surface) of the power transmission belt. In many cases, the pair of unvulcanized rubber sheets are formed of the same or different rubber compositions. EXAMPLE The present invention is described below in detail based on examples. However, the present invention is not limited by the examples. [Preparation of Cord] Aramid twisted yarn cord: a plied twisted cord having a total fineness of 3300 dtex (the number of filaments: 3000) formed by twisting a bundle of aramid fibers having a fineness of 1100 dtex (the number of filaments: 1000) in a twisted configuration of 1×3 with a primary twist factor of 4.0 and a secondary twist factor of 2.8. [First Treatment Agent] A first treatment agent containing the following components was prepared. That is, components of the first treatment agent shown in Table 1 were mixed and stirred at a room temperature for 10 minutes to prepare various first treatment agents (treatment liquids). Aqueous dispersion of bisphenol A type epoxy resin: “Yuka Resin NE-307H” (aqueous dispersion of a bisphenol A type epoxy resin (jER #1007, manufactured by Mitsubishi Chemical Corporation) having a molecular weight of about 2900, an epoxy equivalent of 1750 g/eq to 2200 g/eq, and a softening point (ring and ball method) of 128° C.) manufactured by Yoshimura Oil Chemical Co., Ltd., solid content concentration: 50% by mass Aqueous dispersion of polycarbonate polyol: “Yuka Resin PEP-50” manufactured by Yoshimura Oil Chemical Co., Ltd., solid content concentration: 60% by mass, hydroxyl group equivalent (solid content): 267 g/eq As described above, the hydroxyl group equivalent can be calculated based on a hydroxyl group value and the formula: hydroxyl group equivalent=1/[(hydroxyl group value mgKOH/g)/1000/56.1]. Aqueous dispersion of blocked polyisocyanate: “Yuka Resin AK-81” (aqueous dispersion of blocked polyisocyanate based on hexamethylene diisocyanate and having a dissociation temperature of about 140° C.) manufactured by Yoshimura Oil Chemical Co., Ltd., solid content concentration of 45% by mass, isocyanate group equivalent (solid content) of 360 g/eq Imidazoles: “Curezol 2E4MZ-CN” manufactured by Shikoku Chemicals Corporation TABLE 1First Treatment AgentABCDEFGHIJCompositionAqueous dispersion of bisphenol200200200200200200200200200200(parts by mass)A type epoxy resinAqueous dispersion of12040150180740404040polycarbonate polyolAqueous dispersion of blocked440300250220550230230350180410polyisocyanateImidazoles5555555555Water17001200100012002100950105013009501400Total2465174514701625303513921525189513752055Solid content concentration (% by mass)15%15%15%12%15%15%15%15%15%15%Ratio of functional groups *1:1:23:1:49:1:101:0:10.67:1:1.6719:1:2031.:3.23:1:4.83:1:2.43:1:5.6Mole ratio of OH groups * *50/5075/2590/10100/040/6095/575/2575/2575/2575/25Mole ratio of NCO groups * * *1111110.81.20.61.4In the table, * “ratio of the number of functional groups” indicates a ratio of the number of OH groups of the bisphenol A type epoxy resin:the number of OH groups of the polycarbonate polyol:the number of NCO groups of the blocked polyisocyanate.* * “molar ratio of OH groups” indicates the ratio of the number of OH groups of the bisphenol A type epoxy resin to the number of OH groups of the polycarbonate polyol.* * * “molar ratio of NCO group” indicates a ratio of the number of moles of NCO group of blocked polyisocyanate to 1 mole of the total amount of OH group of bisphenol A type epoxy resin and polycarbonate polyol. [Second Treatment Agent] Constituent components of the second treatment agent (RFL liquid) are shown in Table 2. First, resorcin was added to water and dissolved by stirring at a room temperature for 30 minutes, then 37% by mass of formalin was added, and the mixture was further stirred at a room temperature for 30 minutes to react to obtain a liquid X. Next, latex was diluted with water to prepare a liquid Y, and the liquid X was added thereto and mixed with stirring. The mixed solution was aged at 25° C. for 6 days and then diluted with water (liquid Z) so as to have a solid content concentration of 10% by mass to prepare a second treatment agent (RFL solution). As the latex, a styrene-butadiene-vinylpyridine copolymer (manufactured by Zeon Corporation) was used. TABLE 2Second Treatment AgentConstituent componentParts by massLiquid XResorcin12237% by mass of formalin68Water400Liquid YVinvlpyridine-styrene-butadiene copolymer latex800(solid content concentration: 41.5% by mass)Water510Liquid ZWater2900Total (X + Y + Z)4800Solid content concentration10% by mass [Preparation of Cord] The untreated aramid twisted yarn cord was immersed in a first treatment agent (25° C.) for 10 seconds and then dried at 150° C. for 2 minutes, and subsequently immersed in a second treatment agent (25° C.) for 10 seconds and then dried at 200° C. for 2 minutes to obtain a treated cord to which an adhesive component was attached. [Preparation of V-Ribbed Belt] The following components were used as an unvulcanized rubber composition of a belt. EPDM: “IP3640” manufactured by DuPont Dow Elastomer Japan Co., Ltd., Mooney viscosity: 40 (100° C.) Zinc oxide: “Zinc Oxide third grade” manufactured by Seido Chemical Industry Co., Ltd. Carbon black HAF: “SEAST 3” manufactured by Tokai Carbon Co., Ltd. Stearic acid: “Stearic acid Tsubaki” manufactured by NOF Corporation Hydrous silica: “Nipsil VN3” manufactured by Tosoh Silica Corporation, BET specific surface area: 240 m2/g Resorcin-formaldehyde condensate: less than 20% of resorcinol and less than 0.1% of formalin Antioxidant: “Nonflex OD3” manufactured by Seiko Chemical Co., Ltd. Vulcanization accelerator DM: di-2-benzothiazolyl disulfide Hexamethoxymethylol melamine: “PP-1890S” manufactured by Power Plast Polyamide short fiber: “66 Nylon” manufactured by Asahi Kasei Corporation Cotton short fiber: average fiber diameter 13 μm, average fiber length 6 mm Mercaptobenzimidazole: “Nocrac MB” manufactured by Ouchi Shinko Chemical Industrial Co., Ltd. Paraffinic softener: “Diana Process Oil” manufactured by Idemitsu Kosan Co., Ltd. Dibenzoyl-quinone dioxime: “Vulnoc DGM” manufactured by Ouchi Shinko Chemical Industrial Co., Ltd. Organic peroxide; “Percadox 14RP” manufactured by Kayaku Akzo Co., Ltd. First, a rubber-attached cotton canvas of one ply (one stack) was wound around an outer periphery of a cylindrical molding mold having a smooth surface, and an unvulcanized adhesive rubber layer sheet formed of a rubber composition shown in Table 3 was wound around an outside of the cotton canvas. Next, the treatment cord was spirally spun and wound on the adhesive rubber layer sheet in a state of being arranged in parallel at a pitch of 0.95 mm, and further, the unvulcanized adhesive rubber layer sheet formed of the rubber composition and the unvulcanized compression rubber layer sheet formed of the rubber composition shown in Table 4 were wound thereon in order. In a state in which a vulcanization jacket was disposed outside the compression rubber layer sheet, a molding mold was placed in a vulcanization can and vulcanized. A cylindrical vulcanized rubber sleeve formed by vulcanization was taken out from the molding mold, a plurality of V-shaped grooves were simultaneously ground in the compression rubber layer of the vulcanized rubber sleeve by a grinder, and then the vulcanized rubber sleeve was cut in a circumferential direction by a cutter so as to be sliced, thereby obtaining a V-ribbed belt having three ribs and a circumferential length of 1100 mm. The belt does not include a tension rubber layer. TABLE 3Composition for adhesive rubber layerConstituent componentParts by massEPDM100Stearic acid1Zinc oxide5Carbon black HAF35Hydrous silica20Resorcin-formaldehyde condensate2Antioxidant2Vulcanization accelerator DM2Hexamethoxymethylol melamine2Sulfur1Total170 TABLE 4Composition for compression rubber layerConstituent componentParts by massEPDM100Polyamide short fiber15Cotton short fiber25Zinc oxide5Stearic acid1Mercaptobenzoimidazole1Carbon black HAF60Paraffin softener10Organic peroxide4Dibenzoyl-quinone dioxime2Total223 Then, the characteristics of the obtained V-ribbed belt and cord were evaluated as follows. [Evaluation of Fraying Resistance] The side surface of the belt was visually observed, and the presence or absence of the fraying of the cord was confirmed. [Evaluation of Adhesiveness Between Cord and Rubber] As shown inFIG.3, in the obtained V-ribbed belt, after the canvas on the back surface of the belt was separated, two adjacent cords1aamong the cords1adhered to the rubber layer were pulled from the adhesive rubber layer2on the compression rubber layer side. In a state in which one end of the V-ribbed belt was gripped by a gripping tool21a, cords1awere gripped by a gripping tool21bof an autograph (“AGS-J10 kN” manufactured by Shimadzu Corporation), and a separating test was performed at a tensile speed of 50 mm/min to measure the adhesive force between the cords1and the adhesive rubber layer2at an atmospheric temperature of 23° C. (two-cord separating force). [Traveling Durability Test] As shown inFIG.4, the obtained V-ribbed belt was wound around a driving pulley11(diameter: 120 mm, number of revolutions: 4900 rpm), a driven pulley12(diameter; 120 mm, load: 8.8 kW) an idler pulley13(diameter: 85 mm), and a tension pulley14(diameter: 45 mm, axial load: 60 kgf (constant)), and allowed to run at an atmospheric temperature of 120° C. to the end of life. The bending fatigue resistance was evaluated by terminating a running time at 200 hours and measuring the tensile strength before and after the traveling durability test. Specifically, the tensile strength of the belt before and after running for 200 hours was measured by using a universal testing machine (“UH-200kNX” manufactured by Shimadzu Corporation) under the condition of a tensile speed of 50 mm/min, and the strength at the time of breaking of the belt was measured. The strength retention rate was calculated by the following formula. Strength retention rate (%)=(Strength after traveling durability test/Strength before traveling durability test)×100 The results are shown in Table 5 below. TABLE 5ComparativeExamplesExample1234567891First treatment agentABCEFGHIJDFraying resistanceNoNoNoNoNoNoNoNoNoFrayingfrayingfrayingfrayingfrayingfrayingfrayingfrayingfrayingfrayingTwo-cord separating force (N)13.514.212.912.012.413.213.412.312.711.6TravelingLife (h)330350300300300320300300300220DurabilityFailure modeRibRibRibRibRibRibRibRibRibBreakcrackcrackcrackcrackcrackcrackcrackcrackcrackStrong72%75%67%72%61%70%67%67%60%51%retention rate Examples 1 to 9 were excellent in fraying resistance, adhesiveness, and bending fatigue resistance. In Comparative Example 1 in which polycarbonate polyol was not contained, the toughness of the adhesive coating film was low, and therefore the fraying resistance and the bending fatigue resistance were deteriorated. From the comparison of Examples 1 to 5, it can be seen that when the ratio of the polycarbonate polyol is too low, the strength retention rate (bending fatigue resistance) was decreased. That is, in Examples 3 and 5, the amount of epoxy is relatively large with respect to the polyol, and the toughness of the adhesive coating film is not so high. On the other hand, it can be seen that when the ratio of the polycarbonate polyol is too high, the two-cord separating force (adhesiveness) was decreased. Further, from the comparison between Example 2 and Examples 6 to 9, it can be seen that when the ratio of the blocked polyisocyanate is too low, the adhesiveness is lowered. On the other hand, it can be seen that when the ratio of the blocked polyisocyanate is too high, the bending fatigue resistance was decreased. Although the present invention has been described in detail with reference to a specific embodiment, it is obvious to those skilled in the art that various changes and modifications may be made without departing from the gist and the scope of the invention. This application is based on Japanese Patent Application 2019-051513 filed on Mar. 19, 2019, and Japanese Patent Application 2020-33980 filed on Feb. 28, 2020, contents of which are incorporated by reference herein. INDUSTRIAL APPLICABILITY An aqueous treatment agent according to the present invention is useful for preparing a cord of a power transmission belt. When such a cord is used, the fraying resistance and the bending fatigue resistance of the power transmission belt can be greatly improved, the adhesiveness to rubber can be improved, and the durability can be greatly improved. Therefore, it can be effectively used for a power transmission belt for increasing tensile strength by being bonded to a rubber layer such as a V-belt. REFERENCE SIGNS LIST 1Cord2Adhesive rubber layer3Compression rubber layer4Tension rubber layer5Rib | 78,822 |
11859079 | DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention will now be described by way of several worked examples of embodiments in accordance with the present invention. The following description is not to limit the generality of the above description. Example 1 In this example, an epoxy component in the form of glycerol triglycidyl ether (GTE) was used to form a fully crossed linked and cured epoxy resin. Isophorone diamine (IPD) was used as both the reactive component and the curing component as it advantageously reacts slower and therefore was found to be more suited to larger scale industrial applications. 100 g of 0.59 mol isophorone diamine (IPD) was added carefully to 2000 g of 7.69 mol glycerol triglycidyl ether (GTE) and stirred with a mechanical stirrer on medium for 1 hour followed by stirring overnight on a low setting. The precursor composition produced by this reaction was viscous and clear with a light-yellow tinge in appearance. The precursor composition was placed in an appropriate dry container ready for further blending with additives. In this respect, the stored precursor composition is able to be formulated as required depending on the final application. Various additives can be added to impart different properties. A portion of the precursor composition was then modified by being blended with additives. Pigment (10 g, 7.2 parts) was added portion wise into a stirred mixture of 100 g (71.9 parts) of the precursor composition and stirred for 5 minutes. Talc (9 g, 6.5 parts), silica flour (16.0 g, 11.5 parts) and Aerosil 200 [hydrophilic fumed silica] (4.03 g, 2.9 parts) were added in a similar fashion with 10 minute breaks between each additive. The resulting blended precursor composition was stirred for 5 hours at 1200 to 1000 revolutions per minute. Stirring was deemed complete after spreading a thin layer over glass, and checking for uniformity of the additives. The blended precursor composition was then stored until ready for use. In this example, further isophorone diamine (1.9g) was used as a curing component to form a cured aliphatic epoxy resin and was rapidly and thoroughly mixed into 10.25 g of the blended precursor composition, and rapidly mixed for 3 to 4 minutes. The resultant epoxy resin was applied to a timber surface using a paint brush and allowed to cure. The coating was tacky within 3 hours and cured within 6 hours, with optimum curing and strength achieved after 24 hours. Curing time can be fast tracked by curing in an oven. The amount of the IPD curing component required to achieve a fully crosslinked molecular structure in the cured epoxy resin would usually be based on an epoxy equivalent weight (EEW) calculation. However, in the present example, NMR was used instead of a traditional titration analysis to determine the amount of curing component required. Example 2—Formation of Cured Disks for Testing Cured disks were prepared using Araldite™ and Megapoxy™ for purposes of comparison with the cured epoxy resin of the present invention. Araldite™ liquids were dispensed from two tubes into a plastic cup, mixed and allowed to cure, while Megapoxy™ liquids were mixed in a 2:1 (w/w) ratio and also allowed to cure in a cup. For comparison, 10 g of the precursor composition (pre-blending) prepared in Example 1 had 3.13 g of IPD added to it as a curing component, and the subsequent epoxy resin of the present invention was cured in a plastic cup to form a clear, cured epoxy resin. After further curing in an oven at 80° C. for 4 hours, strips of each of the Araldite™, Megapoxy™ and GTE/IPD resin were prepared for testing purposes. Example 3—Formation of Cured Cylinders for Testing Clear polymer cylinders were made in the same manner as described in paragraph [0035] above for hardness testing and for comparison to a commercial BPA epoxy resin product. The hardness results showed that the BPA epoxy resin product was 31 MPa compared to epoxy resin blocks in accordance with the present invention of 68 to 77 MPa. Example 4—Coating With Clear Epoxy Resin for Testing A clear precursor composition was made by mixing 50 g of IPD with 1000 g of GTE and stored for two weeks. Prior to application as a coating to flooring, 250 g of IPD was added to the precursor composition and thoroughly stirred. The resulting mixture was applied to an exposed aggregate concrete floor with a squeegee and allowed to harden. A clear glossy finish was achieved. Two weeks later, and without pre-preparation, a second coating was applied adjacent to the hardened first application. Again, a clear glossy finish was achieved with no noticeable join between the first and second applications. Example 5—Application of the Epoxy Resin (Tinted) for Flooring Pigment (10 g, 7.2 parts) was added portion wise into a stirred mixture of precursor composition (100 g, 71.9 parts) and stirred for 5 minutes. Talc (9 g, 6.5 parts), Silica flour (16.0 g, 11.5 parts) and Aerosil 200 (4.03 g, 2.9 parts) were added in a similar fashion with 10 minute breaks between each additive. The resulting mixture was stirred for 5 hours at 1200-1000 revolutions per minute. Stirring was deemed complete after spreading a thin layer over glass, checking for uniformity of the additives. The precursor composition was then stored until ready for use. When application was eventually required, IPD (30 g) was applied to the formulation described in Example 1 and rapidly mixed. The mixture was then applied with a paint brush to various materials. The applications tested four different dyes white, ochre, black and grey (a combination of white and black dyes), which were applied to timber resulting in a very hard, high gloss finish. A UV lamp test was conducted for 48 hours on the white dyed epoxy applied as a coating on timber. This test showed initial darkening and returned to standard white colour within a few hours, which was determined to be not due to resin but the titanium oxide dye. Finally, other modifications and improvements may also be made to the compositions and resins described above without departing from the scope of the present invention. | 6,153 |
11859080 | It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. DETAILED DESCRIPTION OF THE PRESENT INVENTION In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention. In some embodiments, this invention is directed to a composition comprising hydroxyapatite [Ca10(PO4)6(OH)2)] and doped inorganic fullerene-like nanoparticles (IF-NPs) or doped inorganic nanotubes (INT). In some embodiments, this invention is directed to a film comprising hydroxyapatite [Ca10(PO4)6(OH)2)] and doped inorganic fullerene-like nanoparticles (IF-NPs) or doped inorganic nanotubes (INTs). In some embodiments, this invention provides a composite comprising PLLA (Poly(L-lactic acid), hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B(dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te. In some embodiments, this invention provides a composite comprising a biodegradable polymer, hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, a biocompatible surfactant and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBr-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te. In other embodiments, the biodegradable polymer is poly(lactic acid) (PLA), Poly(L-lactide) (PLLA) or poly-D-lactide (PDLA). In other embodiment the biocompatible surfactant is a fatty acid having between 12-24 carbons. In other embodiments, the biocompatible surfactant is oleic acid (C18H34O2). In some embodiments, the biocompatible surfactants refer to surface active group of amphiphilic molecules which are manufactured by chemical processes or purified from natural sources or processes. These can be anionic, cationic, nonionic, and zwitterionic. A variety of biocompatible surfactants include a fatty acid, arabic gum, poloxamer, poloxamines, pluronic acid, PEG, Tween-80, gelatin, dextran, pluronic L-63, PVA, methylcellulose, lecithin and DMAB, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate), phospholipid compounds or phospholipid mixtures (phospholipid choline (lecithin), such as lecithin of soy or egg), sorbitan, such as fatty acid-substituted sorbus, a sugar alcohol surfactant (available commercially under the name of SPAN® or ARLACEL®), a fatty acid ester of polyethoxylated sorbitol (TWEEN®), a polyethylene glycol derived from fatty acids such as castor oil Ester (EMULFOR): polyethoxylated fatty acids (for example, stearic acid available under SIMULSOL M-53), polyethoxylated isooctylphenol/formaldehyde polymer (TYLOXAPOL), polyoxyethylene fat Alcohol ethers (BRIJ®); polyoxyethylene phenyl ether (TRITON®); polyoxyethylene isooctyl phenyl ether (TRITON® X). In some embodiments, the composite comprises PLLA (Poly(L-lactic acid), hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, oleic acid, and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te. Incorporated hydroxyapatite in the PLLA matrix improves the flexibility of the bioceramic HA and consequently, produce biodegradable ceramic-polymer composites, which is an alternative to the traditional materials used for implants or bone repair and for tissue engineering. However, both PLLA and HA and their composites, suffer from low toughness, which limit their application in the human body. Reinforcing the PLLA/HA composite with inorganic fullerene-like nanoparticles (IF-NPs) or inorganic nanotubes (INT) (such as INT-WS2) can remedy this disadvantage. The INT-WS2are multiwall nanostructures 1-20 μm long with diameter of 30-150 nm (aspect ratio of 50-100 and even larger). They are nontoxic with very good mechanical properties (Young's modulus 150-170 GPa, bending modulus of 217 GPa, tensile strength between 10-22 GPa, and strain ε>10%). HA does not disperse well in the PLLA matrix and tend to agglomerate as secondary particles a few micrometers in size. This is because HA is hydrophilic, while the organic solvents used to dissolve the polymers are mostly hydrophobic. However, a biocompatible surfactant such as oleic acid (OA), which is an amphiphilic surfactant, used to mediate the interaction between the HA (hydrophilic ceramic) and hydrophobic polymer, like PLLA. Therefore, oleic acid induces a homogeneous dispersion of the HA in the PLLA matrix. In some embodiments, this invention is directed to a film comprising the composition/composite of this invention. In some embodiments, this invention is directed to a film comprising hydroxyapatite [Ca10(PO4)6(OH)2)] and doped inorganic fullerene-like nanoparticles (IF-NPs) or doped inorganic nanotubes (INT), wherein the film is coated on a solid substrate. In some embodiments, this invention is directed to a film comprising a biodegradable polymer, hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, a biocompatible surfactant, and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te. In some embodiments, this invention is directed to a film comprising PLLA (Poly(L-lactic acid), hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, oleic acid, and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te. In other embodiments, the inorganic nanotubes (INT) are WS2. In other embodiments, the inorganic fullerene-like nanoparticles (IF-NPs) or the inorganic nanotubes (INT) are doped by rhenium and niobium. Inorganic Fullerene-like (IF) nanoparticles and/or inorganic nanotubes (INT) of this invention each having the formula A1-xBx-chalcogenide wherein A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; and x is below or equal 0.003, and the chalcogenide is selected from the S, Se, Te. In other embodiments, x is below or equal 0.001. For example, doped IF-NP or doped INT of the invention may be IF-Mo1-xNbxS2, IF-Mo1-xRexS2, INT-Mo1-xNbxS2, INT-Mo1-xRexS2, IF-W1-xNbxS2, IF-W1-xRexS2, INT-W1-xNbxS2, INT-W1-xRexS2or the alloys of WMoS2, WMoSe2, TiWS2, TiWSe2, where Nb or Re are doped therein. Within the alloys of the invention, taking WMo, TiW for example, the ratio between W and Mo or Ti and W may be 0.65-0.75 of one metal or transition metal and 0.25-0.35 of the other metal or transition metal, e.g. W0.7Mo0.29Nb0.01S2(given with the percentage of the Nb dopant). In one embodiment, the rhenium or niobium atoms serve as dopants in the lattice of the IF-NPs/INTs. The dopants substitute for the molybdenum or tungsten atoms, which lead to an excess of negative charge carriers being trapped on the IF-NPs/NT surfaces. In other embodiments, the concentration of the dopants is below or equal to 0.3 at %. In other embodiments, the concentration of the dopants is between 0.01 to 0.1 at %. In other embodiments, the concentration of the dopants is between 0.01 to 0.07 at/o. In other embodiments, the concentration of the dopants is between 0.01 to 0.05 at %. The doped IF-nanoparticles/inorganic nanotubes behave like charged colloids, which do not agglomerate and form stable suspensions in oils and various fluids. This is in contrast to the undoped IF-NPs/INTs, as their structure allows rolling. Additionally, the doped IF-NPs and doped INTs have higher conductivity, higher carrier density, lower activation energy, and lower resistance than the undoped ones. In some embodiments, this invention is directed to a composition/composite or a film comprising hydroxyapatite [Ca10(PO4)6(OH)2), HA] and doped IF-NPs/doped INTs. In other embodiment, the composition of the film further comprises brushite, portlandite, other HA minerals or combination thereof. In some embodiments, this invention is directed to a composition/composite and/or a film comprising hydroxyapatite [Ca10(PO4)6(OH)2), HA] and doped IF-NPs or doped INTs. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 0.2 wt % to 5 wt % of the composition and/or film. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 0.2 wt % to 2 wt % of the composition and/or film. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 0.2 wt % to 1 wt %. In other embodiment, the concentration of doped IF-NPs or doped INTs is between 0.2 wt % to 1.5 wt %. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 0.5 wt % to 1.5 wt %. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 0.5 wt % to 2 wt %. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 1 wt % to 5 wt %. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 0.5 wt % to 3 wt %. In other embodiment, the concentration of the doped IF-NPs or doped INTs is between 1.5 wt % to 5 wt %. In some embodiments, this invention is directed to a composition/composite and/or a film comprising PLLA (Poly(L-lactic acid), hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te; wherein the concentration of inorganic fullerene-like nanoparticles or inorganic nanotubes is between 0.1 wt % to 5 wt % of the composition. In other embodiments, the concentration of HA is between 20 wt % to 60 wt % of the composition. In some embodiments, this invention is directed to a composition/composite and/or a film comprising PLLA (Poly(L-lactic acid), hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, oleic acid and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te; wherein the concentration of inorganic fullerene-like nanoparticles or inorganic nanotubes is between 0.1 wt % to 5 wt % of the composition. In other embodiments, the concentration of HA is between 20 wt % to 60 wt % of the composition. In some embodiments, this invention is directed to a composite and/or a film comprising a biodegradable polymer, a biocompatible surfactant, hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW, and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te; wherein the concentration of inorganic fullerene-like nanoparticles or inorganic nanotubes is between 0.1 wt % to 5 wt % of the composition. In another embodiment between 0.1 wt % to 1 wt %. In another embodiment between 0.2 wt % to 1 wt %. In another embodiment between 0.1 wt % to 2 wt %. In another embodiment between 0.1 wt % to 3 wt %. In another embodiment between 0.5 wt % to 3 wt %. In another embodiment between 0.5 wt % to 2 wt %. In another embodiment between 1 wt % to 5 wt %. In another embodiment, the biodegradable polymer is PLLA. In another embodiment, the biocompatible surfactant is oleic acid. In some embodiments, this invention is directed to a composition and/or a film comprising a biodegradable polymer, a biocompatible surfactant, hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te; wherein the concentration of HA is between 20 wt % to 60 wt % of the composition. In another embodiment between 20 wt % to 30 wt %. In another embodiment between 20 wt % to 40 wt %. In another embodiment between 20 wt % to 50 wt %. In another embodiment between 30 wt % to 40 wt %. In another embodiment between 30 wt % to 50 wt %/o. In another embodiment between 30 wt % to 60 wt %. In another embodiment, the biodegradable polymer is PLLA. In another embodiment, the biocompatible surfactant is oleic acid. The term “composite” or “composition” is used herein interchangeably referring to hydroxyapatite is embedded in different matrices (biodegradable polymers), such as the PLLA. The composite/composition of this invention further comprises inorganic particles such as inorganic fullerene-like nanoparticles or inorganic nanotubes and a biocompatible surfactant (i.e. oleic acid) In some embodiments, this invention is directed to a film comprising PLLA (Poly(L-lactic acid), hydroxyapatite [Ca10(PO4)6(OH)2)] nanoparticles, oleic acid and inorganic fullerene-like nanoparticles or inorganic nanotubes; wherein the inorganic fullerene-like nanoparticles or inorganic nanotubes is A1-xBx-chalcogenide where A is a metal or transition metal or an alloy of one metals or transition metals including at least one of the following: Mo, W, Re, Ti, Zr, Hf, Nb, Ta, Pt, Ru, Rh, In, Ga, InS, InSe, GaS, GaSe, WMo, TiW; and B (dopant) is a metal transition metal selected from the following: Si, Nb, Ta, W, Mo, Sc, Y, La, Hf, Ir, Mn, Ru, Re, Os, V, Au, Rh, Pd, Cr, Co, Fe, Ni; x is between 0 to 0.003; and the chalcogenide is selected from the S, Se, Te; wherein the film is prepared by solvent casting on a solid surface, wherein the film HA nanoparticles and the organic fullerene-like nanoparticles or inorganic nanotubes within the film are dispersed in the PLLA. In other embodiments, the film provides Young's modulus being 1.5 to 3 times higher compared to a film comprising PLLA and HA. In other embodiments, the film provides a toughness being 2 to 10 times higher compared to a film comprising PLLA and HA. In other embodiments, the film provides a hardness being 1.5 to 3 times higher compared to PLLA film. In other embodiment, the film provides higher thermal stability compared to a PLLA film or a film comprising PLLA and HA. In other embodiment, there is no chemical bonding between each of the PLLA, HA and the inorganic fullerene-like nanoparticles or inorganic nanotubes. In some embodiments the composite described herein The composite of claim6, wherein the film provides an improved hardness by 1.2 to 3, Young Modules by 1.5 to 3, Toughness by 2 to 5, Yield Strength by 1.2 to 3 and Strain at failure by 1.1 to 3 compared to PLLA film. In other embodiments, the film is formed by mixing each of PPLA, HA and INT or IF in an organic solvent followed solvent casting on a solid substrate and drying the formed film. In another embodiment, the organic solvent is chloroform, THF or mixtures thereof. In other embodiment, the oleic acid is used to homogenize the mixture between the HA phase and the other two components (PLLA and INT/IF). In other embodiments, minute amounts of a solvent remain in the film. In other embodiments, no solvent remains in the film. In other embodiments, other means of film formation include hot-pressing, or extrusion and subsequent 3D printing. The control of the interfacial interaction between the two majority phases, i.e. PLLA and HA and the minority phase—INT-WS2has major implications on the mechanical stability of the nanocomposite subdued to different stress and environmental conditions. The oleic acid, which is used for compatelizing HA in different polymer phases, was found to be indispensable. First, both oleic acid and HA nanoparticles are non-toxic and biocompatible phases. The FTIR, XRD and Raman measurements as presented in Example 6 do not reveal any specific chemical bonding between each of the four components (PLLA, HA, OA and INT) or a new phase forming during the preparation of the film. It remains to be seen if other specific functionalization processes of the nanotubes surface could further improve their interfacial interaction with the matrix and influence the mechanical behavior of the nanocomposite without sacrificing its biocompatibility. The INT and IF within the compositions described herein have no specific interaction with the polymer-HA, their large surface area and aspect ratio (50-100) as well as their mechanical strength (10-22 GPa), large strain (10%), non-toxic nature and their facile dispersion make the INT/IF suited for reinforcing biodegradable polymer, even if added in minute amounts (˜0.1 wt %˜5 wt %). In some embodiment, the film of this invention is coated on a solid substrate. In other embodiments, the film described herein is formed by solvent casting. In other embodiment, the solid substrate is biocompatible. In other embodiments, the solid substrate is metallic biocompatible. In other embodiments the solid and biocompatible substrate is titanium, alloys of titanium, Ti6Al4V, Co—Cr alloys, magnesium, stainless steel, shape memory alloys of nickel-titanium, silver, tantalum, zirconium, novel ceramics such as alumina or zirconia or any other electrical-conductive substrate. In other embodiment, the titanium is porous. In other embodiment, to improve the coating of the film onto the solid substrate the composition and/or film further comprises a cationic surfactant. In other embodiment a cationic surfactant comprises an ammonium group. Non limiting examples of cationic surfactant include: alkyltrimethylammonium salts: cetyl trimethylammonium bromide (CTAB) and cetyl trimethylammonium chloride (CTAC); benzalkonium chloride (BAC); cetylpyridinium chloride (CPC) or benzethonium chloride (BZT). In other embodiment, to improve the coating of the film to the solid substrate the composition and/or film further comprises a polymeric binder. In other embodiments a non-limiting example of a polymer binder include a poly(lactic acid) (PLAs) based polymer. In some embodiments this invention provides methods for coating a solid substrate with the composition of this invention to form a film on a solid substrate. In other embodiments, the methods of coating include; (i) electrophoretic deposition (solution); (ii) plasma spray (in vacuum); (iii) ion beam coating (in vacuum); (iv) e-beam evaporation [Cen Chen et al. Biomimetic apatite formation on calcium phosphate-coated titanium in Dulbecco's phosphate-buffered saline solution containing CaCl2) with and without fibronectin,Acta Biomaterialia, (2010) 6, 2274-2281]; (v) thermal deposition; vacuum deposition [D. Predoi et al. Characteristics of hydroxyapatite thin films,J. Optoelect and Adv. Mat., (2007), 9(12), 3827-3831]; (vi) physical vapor deposition (PVD) [Ohad Goldbart et al. New Deposition Technique for Metal Films Containing Inorganic Fullerene-Like (IF) Nanoparticles,Chem Phys Chem, (2013), 14, 2125-2131; Olga Elianov MSc thesis submitted to the Faculty of Dental Medicine, Hadassah-Hebrew University, Jerusalem 91120, Israel (March 2018); (vii) aerosol deposition [C. Piccirillo, et al. Aerosol assisted chemical vapour deposition of hydroxyapatite-embedded titanium dioxide composite thin films,J. of photochem. And photobiol. A: Chemistry(2017), 332, 45-53]; (viii) sol gel deposition (ix) dip coating; or (x) solvent casting. Each represents a separate embodiment of this invention. Electrophoretic Deposition: The electrophoresis coating technique is an inexpensive process capable of a high deposition rate while maintaining control of the coating thickness and morphology on the metal. In addition, this technique has a wide range of materials permitting coating of variety of shapes and sizes, all resulting in a quality surface with uniform thickness. The electrophoresis coating technique also has high material efficiency and can perform at low temperatures. The electrophoresis coating technique requires several steps, including surface treatments, which are used to clean the electrode from contaminants, improve the mechanical properties to create a uniform coating, and achieve better adhesion deposition. Electrophoresis coating is performed by dipping two electrodes into a container of electrolyte solution. A constant power supply creates an electrical field in the solution, which moves the charging colloid toward the opposite electrode. The deposition is obtained by chemical oxidation and reduction. The final step is an annealing process, and is done to achieve a smooth and continuous coating characterized by good adhesion to the surface. The electrophoretic deposition for coating the composition of this invention on a porous solid substrate is conducted at a relatively low temperature using an aqueous electrolyte containing calcium and phosphate salts. In this method, the calcium phosphate is deposited on the cathode as a result of a pH increase in the vicinity of the cathode and by the reduction of the H+ion accompanying the generation of H2gas and OH·ions. The production of H2on the cathode's surface inhibits the nucleation or absorption of calcium phosphate on the cathode. Adding an alcohol such as ethanol to the electrolyte solution resolves this problem. In some embodiment, this invention provides a method of coating a metal substrate with the composition of this invention, wherein the method comprises electrophoretic deposition having an electrolyte comprising a calcium salt, a phosphate salt and doped inorganic fullerene like nanoparticles, and thereby forming a film of desirable composition on the substrate. The coating process of the film of this invention depends on achieving the proper pH solution that allows quality coatings, which in turn, relies on the nanoparticles' zeta potential measurement. In other embodiment, the composition has a positive zeta potential at pH below 6.5. In neutral pH (7) the nanoparticles are negatively charged, which reflects the extra negative charge induced by native defects in the lattice and chemisorbed negatively charged moieties, like OH— groups. This extra negative charge is neutralized in very low pH (up to pH=2) by positively charged chemical moieties, like protons, etc. In either very low and very high pH, the Debye screening radius is very small (few nm) leading to agglomeration of the nanoparticles and their precipitation. Thus, the electrophoresis coating process of the composition of this invention is performed at pH 6-7 to: 1) avoid damaging the surface of the nanoparticles; 2) provide a stable working solution; and 3) achieve a uniform coating of the substrate. Within this pH range, the nanoparticles gained a negative charge and the deposition was performed on the anode. In some embodiments the methods for coating a metal substrate with the composition of this invention is performed by electrophoretic deposition. In another embodiment, the metal substrate is pretreated for example with carbon paper to obtain a smooth surface and then the metal substrate is anodized prior to the electrophoretic deposition. In other embodiment, the metal substrate is anodized in electrolyte solution containing a fluoride ion. In other embodiment, the electrophoretic deposition is conducted as presented in Example 1. Anodization is an electrochemical method for producing a protective layer on metal by forming a metal oxide layer which makes the metal substrate biocompatible. The metal oxide layer is a few tens of microns thick with micro pores to maintain homogeneity. The anodization process creates a porous surface, which improves and increases osseointegration (the functional connection between the human bone and the implant), and thereby increase the osteoblast adhesion (bone cell). In another embodiment the electrophoretic deposition (EPD) is conducted between 2 to 5 hours. In another embodiment the electrophoretic deposition is conducted for 2, 3, 4 or 5 hours. Each represents a separate embodiment of this invention. In some embodiments, this invention provides HA coatings containing up to 5 wt % doped IF-NPs or doped INTs deposited on a porous metallic biocompatible substrate by electrophoretic deposition using DC bias. The major phase in each coating is hydroxyapatite which incorporates small amounts of doped IF-NPs or doped INTs. In other embodiments, the metal substrate was a titanium substrate. In other embodiments, the doped inorganic fullerene-like nanoparticle is Re:IF-MoS2. In other embodiments, the doped inorganic fullerene-like nanoparticle is Re:IF-W S2. In other embodiments, the doped inorganic fullerene-like nanoparticle is Nb:IF-MoS2. In other embodiments, the doped inorganic fullerene-like nanoparticle is Nb:IF-WS2. In other embodiments, the doped inorganic nanotube is Re:INT-MoS2. In other embodiments, the doped inorganic nanotube is Re:INT-WS2. In other embodiments, the doped inorganic nanotube is Nb:INT-MoS2. In other embodiments, the doped inorganic nanotube is Nb:INT-WS2. In some embodiments, the film formed on the metal substrate has low friction coefficient of between 0.05 to 0.15. In another embodiment, the film formed by EPD on the metal substrate has low friction coefficient of between 0.05 to 0.1. In another embodiment, the low friction is maintained after annealing. In another embodiment, the film maintains its mechanical robustness. Uses Thereof Artificial bone implants became a major health concern. Hydroxyapatite (Ca10(PO4)6(OH)2; HA) is the main constituency of the bone. Hydroxyapatite, is chemically similar to the calcium phosphate mineral present in bone and biological hard tissue. The composition/composite and film of this invention are bioceramic suitable for implants and bone repair, and for tissue engineering. In some embodiments, this invention provides an implant, a bone repair or a tissue engineering comprising the composition/composite described herein. The composition/composite and film of this invention are for use in dental and orthopedic implants having very low friction, good adhesion to the underlying rough substrate even under very high load (600 MPa). The composition and film of this invention have high biocompatibility, specifically as a bone substitute. The composition/film prepared by the methods of this invention form a homogeneous structure, having slow degradability rate and both osseointegration and osteoconductive characteristics, which improve bone growth. In some embodiments, this invention provides a dental or orthopedic implant comprising the composition of this invention. In other embodiments, this invention provides a dental or orthopedic implant comprising a film on a biocompatible substrate, wherein the film comprises the composition of this invention. In some embodiments, this invention provides a bone regeneration therapy comprising administering an artificial bone implant comprising the composition of this invention. In some embodiments, this invention provides a method of osseointegration comprising contacting an artificial bone implant comprising the composition of this invention in a bone needs to be improved. In other embodiments, the artificial bone implant comprises a biocompatible substrate coated by a film, wherein the film comprises the composition of this invention. The methods of this invention for osseointegration or for bone regeneration provide fast fixation and spontaneous binding of the HA to neighboring bone, having osteoconductive properties, resulting in deposition of biological apatite on the surface of the implant and thereby bone healing around the implant. The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention. EXAMPLES Example 1 Preparation of a Film of Hydroxyapatite (HA) and Rhenium Doped Fullerene Like MoS2(Re:IF-MoS2) on Titanium Substrate A titanium electrode (30×5×0.3 mm, 97 wt % purity) was polished with silicon carbide paper to a mirror finish. It was subsequently cleaned by sonicating in a series of solvents, i.e., acetone, ethanol, methanol, isopropanol and finally distilled water, then dried under a nitrogen stream. The surface morphology of the titanium before the pretreatment preceding the anodization is presented inFIGS.11A-11B. Visibly, the fresh surface was heavily contaminated with a dense network of scratches. After treatment of the titanium with different solvents, a smooth surface with low density of scratches and clean from contaminants was obtained (FIGS.11C-11D). The smooth surface was imperative for achieving reproducible tribological measurements. Titanium Anodization An electrochemical cell containing two-electrodes, i.e., platinum (cathode) and titanium (anode) was used. The electrolyte solution contained 1 M (NH4)2SO4and 0.5 wt % NH4F. All electrolytes were prepared from reagent grade chemicals and deionized water. The electrochemical treatment was conducted with a DC power source operated at 2.5 V and 1.5 A, at room temperature for 2.5 h. After the electrochemical treatment, the samples were rinsed with deionized water and dried under nitrogen stream. The surface of the titanium after anodization is displayed inFIGS.12A-12C. Visibly the anodized titanium surface consists of a dense array of (TiO2) nanotubes with the range of pore diameters between 50-130 nm, which form a highly organized, roughly hexagonal, pattern on the Ti surface. Electrophoretic Deposition (EPD) The detailed synthesis of the Re:IF-MoS2nanoparticles (Re content <0.1 at %), which were added to the coating processes, was reported before [Yadgarov, L.; et al. Investigation of Rhenium-Doped MoS2Nanoparticles with Fullerene-Like Structure.Z. Anorg. Allg. Chem.2012, 638, 2610-2616]. Three different chemical baths were used for electrophoretic deposition of HA+IF NP on the porous titanium substrate. Titanium samples were used as the working electrode (cathode), while a platinum plate served as the anode. The final volume of all three electrolyte solutions containing 1 mg of the IF NP was 50 mL. Solution A: The electrolyte solution consisted of 42 mM Ca(NO3)2and 25 mM NH4H2PO4, 1 mg Re:IF-MoS2sonicated in 3 mM cetyl trimethylammonium bromide (CTAB). Ethyl alcohol was added into the above solution in a 1:1 ratio in order to reduce the hydrogen evolution on the titanium electrode. The initial pH of the electrolyte solution was 4.5. The coating process was carried out at 40° C. with a DC power supply at 20 V bias and 0.11 A for 3 h. The samples were washed with deionized water and dried for 24 h at 100° C. Solution B: The electrolyte solution consisted of 5.25 mM Ca(NO3)2, 10.5 mM NH4H2PO4, and 150 mM NaCl. The initial pH of the solution was adjusted to 5.30 by adding NaOH. 1 mg Re:IF-MoS2was sonicated in distilled water for 15 min and added to the electrolyte solution. The coating process was conducted with a DC power source operated at 2.5 V and 0.11 A at room temperature for 3 h. Solution C: The electrolyte solution consisted of 3 mM Ca(NO3)2and 1.8 mM KH2PO4, 1 mg Re:IF-MoS2sonicated in 3 mM CTAB. The initial pH of the electrolyte solution was 5. The coating process was conducted with a DC power source operated at 6 V and 1 A at room temperature for 1 h. The resulting samples, after coating, were washed with deionized water and dried in room temperature. The formal molar Ca/P ratio in HA is 5:3 (1.67). The Ca/P ratio in each coating was calculated based on semi-quantitative. Energy dispersive spectroscopy (EDS) analysis. For solution A, the ratio was found to be 2.6. The higher abundance of calcium in this coating could be attributed to the presence of portlandite (Ca(OH)2). The Ca/P ratio of the coating obtained from solution B, which was highly crystalline and discontinuous was 1.5, which agrees well with the HA formula (1.66). The ratio is 1 for the coating obtained from solution C, which can be ascribed to the presence of calcium pyrophosphate phase (Ca2(P2O7)) in the coating—see XRD analysis (Example 3). The bath showing the most uniform coating and good adhesion (solution A) was then further studied by changing the deposition time to 2, 3 and 4 hours and subsequent annealing at 700° C. for 1 h. Characterization High-Resolution Scanning Electron Microscopy (HRSEM) and High-Resolution Transmission Electron Microcopy (HRTEM): The surface morphology of the titanium samples was analyzed by (HRSEM) (Zeiss Ultra 55) after each step. For topographical information, the secondary electrons were recorded using the SE2 and In-lens detectors. For atomic number contrast the backscattering electron (BSE) detector was used. In order to avoid the sample charging during the analysis, the imaging was done under relatively low accelerating voltage (2-5 kV) and low current. Energy dispersive spectroscopy (EDS) analysis (EDS Bruker XFlash/60 mm) of the samples was undertaken as well. The reported results of the EDS were based on standard-less analysis and hence is semi-quantitative in nature. TEM was performed with a JEOL 2100 microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV, equipped with a Thermo Fisher EDS analyzer. High-resolution TEM (HRTEM) images were recorded with a Tecnai F30 UT (FEI) microscope (FEI, Eindhoven, the Netherlands) operating a 300 kV. The TEM grids were prepared by dripping an ethanolic solution of the nanoparticles onto a collodion-coated Cu grids. The surface morphology of the HA film prepared via solution A (FIGS.2A-2B) and solution C was more homogeneous and could be successfully combined with the Re:IF-MoS2NP in the films as opposed to the film obtained from solution B, which was highly crystalline but non-uniform. The surface morphology of the film obtained from solutions B and C are shown inFIGS.9A-9B and10A-10B, respectively The SEM images of the surface of the HA films with Re:IF-MoS2nanoparticles obtained from solution A for different deposition periods are shown inFIGS.3A-3D. The surface of the coated film shows defects, including the presence of cracks and pores with circular shape. Such pores can be probably attributed to the formation of H2(g) bubbles during the coating process. Interestingly, the bias applied during EPD for solution B (and C) was appreciably smaller (2.5 V) compared to solution A (20 V). On the other hand, the film obtained by EPD from solution A was quasi-continuous. It was highly crystalline but less uniform in the case of solution B, i.e., the apparent current density was higher than that calculated on the basis of the formal electrode surface. The higher voltage used for the EPD from solution A implied a much higher rate of hydrogen production, which could explain the porous structure of this film. The density of the pores and their sizes could be possibly tuned by the bias applied on the cathode during the electrophoretic deposition. Furthermore, addition of surface active agents, like CTAB and others, could reduce the size of the pores. The large cracks are diminished, and the pore-size decreased as the coating time was prolonged. The thickness of the coating was a few microns, therefore the nanoparticles could have been buried under the film surface and even be closer to the titanium substrate. Using low energy beam (2 keV) in the BSE mode, the IF NP could be nevertheless observed (FIG.3D). Example 2 Zeta Potential Results of Hydroxyapatite (HA) and Rhenium Doped Fullerene Like MoS2(Re:IF-MoS2) Film on Titanium Substrate The surface charge of the HA suspension with and without the nanoparticles was determined by zeta potential (ZP) measurements using ZetaSizer Nano ZS (Malvern Instruments Inc., Malvern, UK) with a He—Ne light source (632 nm). To prepare the samples for these measurements, IF (0.6 mg) NP were deagglomerated in 20 mL purified water by sonicating for 5-10 minutes using an ultrasonic bath (seeFIG.1Cfor a SEM image of such an agglomerate). Subsequently, 0.2 mL of the IF suspension was added to 1.5 mL aqueous solutions with pH varying from 1 to 12 and sonicated for an extra 5 min. Before the addition of the IF NP, the pH of each solution was adjusted using concentrated NaOH or HCl. The final concentration of the IF NP was 0.004 mg/mL. The ZP of the solutions was measured in a folded capillary cell (DTS1060) made from polycarbonate with gold plated beryllium/copper electrodes. FIGS.4A-4Bshow the results of the Zeta potential (ZP) measurements performed with the three solutions containing Re:IF-MoS2nanoparticles as a function of pH—up to pH=7. The ZP of all the solutions containing the nanoparticles was positive for pH below 6.5. At higher pH the ZP of solution B became negative, while that of solutions A and C remain positive. This difference can be attributed to the addition of the CTAB, which is a cationic surfactant, to solutions A and C. The (positive) ZP of the natural solutions used for EPD is marked onFIG.4Afor all three solutions. The ZP measurements showed that the species in the HA solution containing the IF NP were positively charged and consequently, the HA film could be deposited on the negative electrode (Ti) during the EPD process. The ZP of the IF NP in pure water, ethanol solution, CTAB in water, and the three solutions used for the EPD (included also inFIG.4A) are summarized inFIG.4B, the errors of the ZP measurements were about 2%. Example 3 X-Ray Diffraction (XRD) of Hydroxyapatite (HA) and Rhenium Doped Fullerene Like MoS2(Re:IF-MoS2) Film The film was removed from the Ti substrate and carefully crushed into a powder. The powder was analyzed by X-ray powder diffraction (XRD) using TTRAX III (Rigaku, Tokyo, Japan) theta-theta diffractometer equipped with a rotating copper anode X-ray tube operating at 50 kV/200 mA. A scintillation detector aligned at the diffracted beam was used after a bent Graphite monochromator. The samples were scanned in specular diffraction mode (θ/2θ scans) from 10 to 80 degrees (2θ) with step size of 0.025 degrees and scan rate of 0.5 degree per minute. Phase identification and quantitative analysis were performed using the Jade 2010 software (MDI) and PDF-4+ (2016) database. The results of the XRD analyses are summarized inFIGS.5A-5Band in Table 1. The XRD patterns of the different coatings obtained from solutions A, B and Care shown inFIG.5A. The major phase obtained by EPD of these solutions is HA. Nonetheless, the coating obtained from solution A contained appreciable amounts (25 wt %) of portlandite (Ca(OH)2). Solution B, on the other hand, contained, in addition to the HA, also significant amounts of brushite-(CaH(PO4)·2H2O). The film obtained from solution C contained calcium pyrophosphate-(Ca2(P2O7)). The presence of the Re:IF-MoS2nanoparticles in the coatings is confirmed by the tiny peak at 14.3°. The content of the IF NP is calculated as 0.2 wt % for solution A, 1.5 wt % for solution B and 1.4 wt % for solution C. This amount is rather small but could nevertheless lead to major improvements of the tribological properties of the film without compromising its mechanical robustness. Following the annealing of the film obtained from solution A (FIG.5B), the HA became biphasic calcium phosphate (BCP), i.e., intimate mixture of two phases: HA (73.6 wt %) and β-TCP (5.9 wt %), and 0.1 wt % Re:IF-MoS2NP. TABLE 1Composition of the films deposited from different solutions determinedfrom the XRD analysis.CalciumEPD filmsHAPortlanditeBrushitePyrophosphateβ-TCPRe:IF-MoS2Film obtained74.8 wt %25 wt %0.2 wt %from solution AFilm obtained17.2 wt %81.3 wt %1.5 wt %from solution BFilm obtained81.1 wt %17.5 wt %1.4 wt %from solution CFilm obtained73.6 wt %20.4 wt %5.9 wt %0.1 wt %from solution Aafter annealing The XRD patterns of the films obtained from solution A without the NP (a) and with the IF NP for different deposition times (b-c) is shown inFIG.6. The percentages of the compounds in each film is presented in Table 2. The major phase in the films was hydroxyapatite. The relative amount of the portlandite in the film increased with extending deposition times (FIG.6). The relative amount of the calcium oxide didn't vary with the deposition time which was also true for the relative content of the IF NP. Although the signal of the IF NP was non-visible inFIG.6, their presence is confirmed through both electron microscopy (FIGS.3A-3D) and the Raman measurements (FIG.7). TABLE 2Composition of the film determined via XRD analysisfor different deposition times (from solution A).CalciumEPD filmsHAPortlanditeOxideRe:IF-MoS2Film obtained from87.8 wt %4.6 wt %7.6 wt %solution A withoutRe:IF-MoS2(3 h)Film obtained from82.6 wt %7.4 wt %9.1 wt %0.3 wt %solution A (2 h)Film obtained from80.4 wt %11.3 wt %8.0 wt %0.3 wt %solution A (3 h)Film obtained from77.8 wt %13.6 wt %8.3 wt %0.3 wt %solution A (4 h) Example 4 Raman Spectroscopy of Hydroxyapatite (HA) and Rhenium Doped Fullerene Like MoS2(Re:IF-MoS2) Film Raman spectra of the powders ground from the films were obtained with Horiba-Jobin Yivon (Lille, France) LabRAM HR Evolution set-up using solid state laser with a wavelength of 532 nm. The instrument was equipped with Olympus objectives MPlan N 100×NA 0.9. The measurements were conducted using a 600 grooves/mm grating. Each spectrum was acquired for 20 s and the spectra were averaged 100 times, which enabled using low excitation power thereby preserving the sample integrity. The spectral ranges collected were from 100 to 1800 cm−1. The Raman spectra of HA+IF films prepared from solution A for different deposition times (2, 3 and 4 hours) are shown inFIG.7. The spectra showed the characteristic vibration bands of calcium hydroxide (wide peak at 1600 cm−1) and poorly crystalline phosphoric moieties, especially phosphate PO4−3bands at 469 (v2), 562-603 (v4), 962 (v1) and 1000-1104 cm−1(v3). These bands are typical of HA. The Raman spectra showed also the typical MoS2modes at 383 (E2g) and 408 cm−1(A1g). Interestingly, in contrast to the XRD pattern (FIG.6), the Raman bands of the IF NP in the HA film are easily discerned here. Example 5 Tribological Results of Hydroxyapatite (HA) and Rhenium Doped Fullerene Like MoS2(Re:IF-MoS2) Film A home-made ball-on-flat rig was used for the tribological tests. The tests were carried-out at room temperature and humidity of ˜40%. Each test was repeated 5-times. Tribological tests were performed on the titanium samples at every step of the experimental procedure. The tribological testing was done under dry friction conditions. This testing method utilizes flat lower samples and a ball-shaped upper specimen, which slides against the flat specimen. The two surfaces move relative to each other in a linear, back and forth sliding motion, under a prescribed set of conditions. In this testing method, the load is applied vertically downwards through the ball against the horizontally mounted flat specimen. Two measurements procedures were used in these series of tests. Sliding speed of 0.3 mm/s was common to both series. In one series of measurements the load was 10 g; the diameter of the ball (hard steel—AISI 301) was 10 mm and consequently a Hertzian pressure of 150 MPa was applied on the film (20 cycles). In another series, the load was 20 g, the diameter of the ball 2 mm, i.e., a Hertzian pressure of 600 MPa was applied, and the number of cycles was 100. Table 3 summarizes the data for the friction coefficient and surface roughness of the different samples under dry conditions. In general, the friction coefficient was found to go down along with the stages of the experimental procedure of preparing the film. The low friction coefficient of the HA film obtained from solution A can be attributed to the IF nanoparticle structure. The nanoparticles exhibited facile rolling when released from the film surface. In addition, gradual peeling/crushing of the NP and material transfer from the film surface to the counter surface of the ball contributed to the facile shearing of the mating surfaces and low friction coefficients. Interestingly, the friction coefficient of the HA film obtained from solution A was maintained also after 700° C. annealing. TABLE 3Summary of the initial and final friction coefficients and the initialroughness for different stages of preparation of the compositeHA + IF film. Measurement conditions: diameter of the testball 10 mm; load = 10 g (Hertzian pressure—P = 150 MPa).InitialFinal CoefficientInitialCoefficientof FrictionRoughnessTested filmof Friction(after 20 Cycles)(μm)Titanium after0.50 ± 0.010.60 ± 0.020.23 ± 0.03surface treatmentTitanium after0.15 ± 0.010.23 ± 0.030.50 ± 0.05anodizationFilm of HA with0.11 ± 0.010.13 ± 0.010.45 ± 0.4Re:IF-MoS2NPobtained fromsolution A onanodized titaniumFilm of HA with0.21 ± 0.020.43 ± 0.080.37 ± 0.03Re:IF-MoS2NPobtained fromsolution B onanodized titaniumFilm of HA with0.37 ± 0.230.30 ± 0.180.57 ± 0.02Re:IF-MoS2NPobtained fromsolution C onanodized titaniumFilm of HA with0.12 ± 0.010.11 ± 0.020.49 ± 0.7Re:IF-MoS2NPobtained fromsolution A onanodized titaniumafter annealing Table 4 shows the dry friction coefficient of the coatings obtained from solutions A without (3 h) and with the NP after 2, 3 and 4 h of deposition time on the anodized titanium substrate. A higher Hertzian pressure (600 MPa) was used for the tribological test. The dry friction coefficient was reduced with increasing coating-time of the film. Following the 4 h deposition time the friction coefficient was very low (0.12) attesting to the quality of the composite film. TABLE 4The initial and final friction coefficients and the initialroughness of the coating on titanium substrate obtainedfrom solution A for different periods of deposition.Measurement conditions: diameter of the test ball 2 mm;load 20 g and Hertzian pressure of P = 600 MPa.InitialFinal CoefficientInitialCoefficientof FrictionRoughnessTested filmof Friction(after 100 Cycles)(μm)Pure HA film0.66 ± 0.080.78 ± 0.041.59 ± 0.28obtained fromsolution Awithout NP after3 h depositionHA film with0.75 ± 0.050.63 ± 0.030.49 ± 0.05Re:IF-MoS2NP obtainedfrom solutionA after 2 hHA film with0.53 ± 0.030.55 ± 0.040.57 ± 0.17Re:IF-MoS2NP obtainedfrom solutionA after 3 hHA film with0.13 ± 0.010.12 ± 0.020.48 ± 0.02Re:IF-MoS2NP obtainedfrom solutionA after 4 h Therefore, it is clear that the extended deposition of the composite film resulted in lower friction under very high load. However, the mechanical stability of the film might have been partially compromised. The surface roughness of the films was in the sub-micron range for all the films containing the NP. FIGS.8A-8Dshow optical micrographs of the wear of the ball and the wear track on the film (inset) after different periods of EPD (600 MPa) and 100 cycles. In analogy to the friction coefficient, the visible wear scar on the ball and the wear track on the film were markedly reduced with the deposition time of the HA+IF NP film. Example 6 Methods for Film Formation Sol-Gel Deposition: A solution of 3M (C2H5O)3PO was hydrolyzed for 24 h in a sealed container under vigorous stirring, 3 M Ca(NO3)24H2O was added dropwise with 1 mgr Re:IF-MoS2nanoparticles in anhydrous ethanol. The mixed sol solution agitated for additional 30 min and kept static at ambient duration time for 24 h. Ti6Al4V substrate was dip coated in the sol solution, then dried at 80° C. and followed by annealing in vacuum at 900° C. for 5 h. Dip Coating: 3 mM Ca(NO3)2, 1.8 mM KH2PO4were dissolved in distilled water, then adding 1 mgr Re:IF-MoS2nanoparticles after dispersion. Immersing titanium substrate in the solution at 37° C. and sealed the container for 24 h, finally the substrate was drying at room temperature, followed by annealing at 700° C. for 3 h. Casting Molding PLLA was dissolved in dichloromethane and adding hydroxyapatite powder with Re:IF-MoS2nanoparticles to the polymer solution, split the solution to Teflon mold and drying at room temperature. Example 7 Film Formation Including PLLA, HA and INT-WS2 Materials Poly L-lactic acid (PLLA) with an inherent viscosity midpoint of 2.4 dl/g was purchased from Corbion (Gorinchem, The Netherlands). Oleic acid (OA, ≥99%) and Hydroxyapatite (HA, nanopowder, <200 nm particle size (BET), ≥97%, synthetic) were purchased from Sigma Aldrich Chemical Company. INT-WS2with diameter between 30-150 nm and length between 1-20 micrometer were synthesized using a published procedure [Chithaiah, P.; Ghosh, S.; Idelevich, A.; Rovinsky, L.; Livneh, T.; Zak, A. Solving the “MoS2nanotubes” synthetic enigma and elucidating the route for their catalyst-free and scalable production. ACS Nano 2020, 14, 3004-3016]. Briefly, the precursor nanoparticles of tungsten trioxide, grew into high aspect ratio tungsten suboxide nanowhiskers under mild reducing atmosphere at 840° C. Subsequently, sulfurization of the nanowhiskers result in hollow WS2nanotubes. The PLLA/HA/INT films were prepared by the solvent casting method according to the following procedure. PLLA neat films: 0.75 g of PLLA pellets was dissolved in 20 ml chloroform and mechanically mixed. Subsequently, the solution was poured onto a Teflon plate for drying in the hood with an aluminum foil cover punctuated with 10 holes. PLLA films with 40 wt % hydroxyapatite: 0.75 g of PLLA pellets was dissolved in 15 ml chloroform; 300 mg of hydroxyapatite nanoparticles were mixed with 5 ml chloroform and 150 μl oleic acid for 30 min. The two solutions were mixed together using a magnetic stirrer for 5 min before pouring onto a Teflon plate and were then dried in the hood using aluminum foil cover punctuated with 10 holes. PLLA fin with 0.5 wt % INT-WS2: First, 3.8 mg INT-WS2powder was dispersed in 5 ml ethanol for 3 min and vacuum annealed for 1.5 h at 80° C. Next, 0.75 g of PLLA pellets was dissolved in 15 ml chloroform and mechanically mixed for 5 hours; then the annealed INT-WS2were dispersed in 5 ml chloroform for 3 min. Finally, the two solutions were mixed together using a magnetic stirrer for 5 min before pouring onto a Teflon plate for drying in the hood with an aluminum foil cover punctuated with 10 holes. PLLA films with 40 wt % hydroxyapatite and 0.25, 0.5 and 0.75 wt % INT-WS2: First, 1.9, 3.8 or 5.6 mg of INT-WS2powder were dispersed in 5 ml ethanol for 3 min and vacuum annealed for 1.5 h at 80° C. Next, 0.75 g PLLA pellets was dissolved in 10 ml chloroform, while 300 mg of hydroxyapatite nanoparticles were dispersed in 5 ml chloroform and 150 μl oleic acid for 30 min. Afterwards, the annealed INT-WS2were dispersed in 5 ml chloroform for 3 min. Finally, the three solutions were mixed together using a magnetic stirrer for 5 min before pouring onto a Teflon plate for drying in the hood with aluminum foil cover punctuated with 10 holes. The dried samples were vacuum annealed for 5 days at 45° C. The thickness of the films was determined by caliper and was on the average 80 μm for pure PLLA; 113 μm for PLLA+HA; and 93 μm for PLLA+HA+INT. The densification of the films upon the addition of the nanotubes was attributed to alignment of the polymer molecules along the nanotubes surface. The texturing of the polymer molecules was an indirect evidence to the non-specific but nevertheless strong nanotube-polymer interfacial interaction and explained the mechanical reinforcement of the PLLA by the INT. Example 8 Characterization of Films of this Invention Comprising PLLA, HA and INT-WS2 X-Ray Diffraction Comparison between the XRD patterns of the nano composite films and HA (hydroxyapatite) powder is shown inFIG.13. The XRD pattern of the composite PLLA and HA and INT-WS2film contains peaks of the different components, which indicates that the composition and structure of PLLA and HA and nanotubes were not affected by the fabrication process of the film. Using the Pawley-based WPF analysis, the degree of crystallinity of the samples was calculated by comparing the total area under all the crystal peaks and the area under the amorphous halo. The results are presented in Table 1. The degree of crystallinity of the PLLA film was calculated to be 32.8%. After adding 0.5 wt % of INT-WS2the degree of crystallinity is slightly increased to 33.2%. However, after adding HA nanoparticles to the PLLA film, the degree of crystallinity increased significantly to 37.1% and remained essentially the same even after adding 0.25-0.75 wt % of INT-WS2. The average crystallite size of the different compositions was estimated using the Williamson-Hall approach from the XRD peak widths fitted in the Pawley-based WPF analysis and the Scherrer equation using the main peak of the PLLA at 16.5° and is reported also in Table 5. The largest crystallite size (171 Å) occurred for the neat PLLA film. As expected, the foreign ingredients (HA and INT) served as crystallization nuclei for the PLLA and reduce its average crystallite size. TABLE 5The degree of crystallinity of PLLA film andHA/INT-WS2/PLLA nanocomposites.Degree ofAverage crystallite sizecrystallinity[Å][%]WPFSample typeWPFScherrerPLLAHAPLLA film32.8 ± 0.9170171 ± 2—PLLA film with37.1 ± 3.0130128 ± 5291 ± 1840 wt % HAPLLA film with33.2 ± 1.6165162 ± 2—0.5 wt % INT-WS2PLLA film with38.2 ± 2.5145145 ± 5331 ± 2040 wt % HA and0.25 wt % INT-WS2PLLA film with42.7 ± 2.7140144 ± 4353 ± 1940 wt % HA and0.5 wt % INT-WS2PLLA film with42.1 ± 2 s · 2150145 ± 4342 ± 1640 wt % HA and0.75 wt % INT-WS2 High-Resolution Scanning Electron Microscopy (HR-SEM) FIGS.14A-14Cshow the SEM images of HA powder (FIG.14A), and a cross-section of PLLA with 40 wt % HA film in secondary electrons (SE) mode (FIGS.14B and14C). Visibly (FIG.14A), the HA particles constitute a bimodal size-distribution made of micron-size agglomerates and a majority phase of a well-dispersed HA nanoparticles (<50 nm). The surface of the large agglomerates is decorated with the small HA NPs. However, the same agglomerates appeared to have a smooth and uniform surface, i.e. free of the decorating HA NPs after being incorporated into the polymer (FIGS.14B and14C—marked with green arrows). To further understand this effect, the HA phase was washed in ultrasonic bath with the polymer-free solvent (chloroform) containing the oleic acid. It was found that the surface of the large spherical HA agglomerates became smooth and free of the HA NPs decoration after this washing procedure. Therefore, the solvent treatment seem to be responsible for the “cleaning” of the HA spherical agglomerates. These smooth spherical agglomerates of HA, are likely to impair the mechanical properties of the film. FIGS.14B,14Cshow that the HA nanoparticles (NPs) were well dispersed in the polymer matrix, i.e. no phase separation or excessive additional agglomeration was observed, which was not the case in the absence of oleic acid. Furthermore,FIG.14Cshows that the HA agglomerates (>0.5 μm) were not damaged during the film breaking process, but were uprooted as a whole from the polymer matrix surface. Furthermore, the hemispherical depressions inFIG.14C(red arrows) are indicative of entire HA agglomerates, which were uprooted from the polymer matrix during fracture, possibly being stuck to the other surface of the broken contact. Consequently, one can conclude that the strain was not well transferred to these agglomerates during fracture and hence they adversely affected the mechanical strength of the film. HR-SEM images of the cross section of PLLA reinforced with 40 wt % HA and 0.75 wt % INT-WS2are presented inFIGS.15A-15B. It can be seen inFIG.15Athat there was no phase separation and consequently a very good compatibility between the nanotubes and the PLLA matrix and between the HA NPs and nanotubes. The nanotubes protrude from the broken surface, which indicates that they carry some of the applied stress transferred to them from the matrix. In addition, other similar scans show that the INT-WS2, are fully dispersed in the PLLA matrix. Visibly, the nanotubes protrude from the PLLA matrix, suggesting that they reinforce the polymer via a bridging and pullout mechanisms. EDS elemental mappings of the PLLA film with 40 wt % HA and 0.75 wt % INT-WS2are presented inFIGS.16A-16D. The carbon mapping, displayed inFIG.16B, shows that the strong carbon signal was evenly distributed throughout the film. This observation reflects the fact that the matrix of the material was PLLA whose chemical composition was mostly carbon.FIG.16Cpresents the phosphorus mapping, which was a major component of HA. It can be seen that the HA NPs were well dispersed throughout the film. However, the bimodal distribution with distinct micron and submicron-sized spherical agglomerated and evenly distributed HA nanoparticles was clearly discernable. The INT-WS2distribution are represented by the tungsten mapping inFIG.16D, which showed that the nanotubes were well dispersed in the PLLA matrix. Tensile Test The mechanical properties derived from the stress-strain curves of the films are displayed inFIG.17and presented in Table 6. The Young's modulus of PLLA film with 40 wt % HA (2.4 GPa) increased 1.5 times compared to the neat PLLA film (1.55 GPa), while the yield strength (26.7 MPa) and strain at failure (2.1%) of PLLA film with 40 wt % HA decreased to 0.85 and 0.75 of their values, respectively. Therefore, the toughness of PLLA film with 40 wt % HA (0.3 MPa) was reduced by half compared to that of neat PLLA film (0.6 MPa). This is not surprising, since the HA is an oxide with a small strain to failure. Also, the binding of the HA to the PLLA is not chemical in nature and is rather weak (mostly van der Waals and polar interactions). These two factors adversely affected the fracture toughness of the composite. However, the indentation hardness and modulus of the PLLA+HA composite was appreciably higher than that of pure PLLA (see below). Obviously, the most rational way to mediate between the HA and the PLLA phases and increase the mechanical properties of the nanocomposite would be through surface functionalization. The surface functionalization showed chemically versatility and biocompatibility in order to permit the three constituents (PLLA, HA and INT-WS2) to optimally interact with each other and exhibit no biotoxicity effects. The Young's modulus and yield strength of PLLA film with 0.5 wt %/o INT-WS2(2.25 GPa and 44.6 MPa, respectively) increased 1.45 times compared to the neat PLLA film, while the strain at failure of the film with 0.5 wt % INT-WS2(6.8%) increased 2.5 times. Therefore, the toughness of the PLLA film with 0.5 wt % INT-WS2(2.4 MPa) increased significantly by 4 times compared to the toughness of the neat PLLA film. The Young's modulus of the PLLA film with 40 wt % HA and 0.5 wt % of INT-WS2(3.8 GPa) increased up to 1.7 times compared to the PLLA film with 40 wt % HA and to the PLLA film with 0.5 wt % INT-WS2. The yield strength of the PLLA film with 40 wt % HA and 0.5 wt % of INT-WS2(62.7 MPa) increased by 2.35 and 1.4 times compared to the PLLA film with 40 wt % HA and to the PLLA film with 0.5 wt % INT-WS2. The strain at failure of the PLLA film with 40 wt % HA and 0.5 wt % of INT-WS2(3.2%) increased 1.5 times compared to the PLLA film with 40 wt % HA. However, the PLLA film with 0.5 wt % INT-WS2, had strain at failure only half the value of the PLLA film with 40 wt % HA and 0.5 wt % of INT-WS2. Therefore, the toughness of the PLLA film with 40 wt % HA and 0.5 wt % INT-WS2(1.4 MPa) increased significantly by 4.7 times compared to the PLLA film with 40 wt % HA and decreased to 0.6 times the value of the PLLA film with 0.5 wt % INT-WS2. TABLE 6The mechanical properties of PLLA film andHA/INT-WS2/PLLA nanocomposites from tensile testing.Young'sYieldStrain atModulusStrengthfailureToughnessSample type(GPa)(MPa)(%)(MPa)PLLA film1.55 ± 0.1531.0 ± 2.42.7 ± 1.30.6 ± 0.2PLLA film with2.4 ± 0.126.7 ± 1.12.1 ± 0.10.3 ± 0.140 wt % HAPLLA film with2.25 ± 0.244.6 ± 4.656.8 ± 1.02.4 ± 0.50.5 wt % INT-WS2PLLA film with2.7 ± 0.442.5 ± 5.87.3 ± 1.02.6 ± 0.340 wt % HA and0.25 wt % INT-WS2PLLA film with3.8 ± 0.562.7 ± 1.23.2 ± 1.61.4 ± 0.740 wt % HA and0.5 wt % INT-WS2PLLA film with2.7 ± 0.3539.6 ± 4.95.8 ± 0.71.8 ± 0.2540 wt % HA and0.75 wt % INT-WS2 Micro Hardness Test FIG.18shows the results of the micro-hardness test of PLLA film and the PLLA/HA/INT-WS2nanocomposites. The addition of HA nanoparticles to the PLLA film increased the hardness value (26.8 HV) by 1.4 times compared to the hardness value of the neat PLLA film (18.9 HV). In addition, the hardness of the PLLA film with 0.5 wt % INT-WS2(23 HV) increased 1.2 times compared to the hardness of the neat PLLA film. Amore significant increase in the hardness was achieved with the combination of HA and INT-WS2in PLLA. The optimum hardness value was obtained for the films containing PLLA with 40 wt % HA NPs and 0.5 wt % INT-WS2with 38.5 HV, a value that is two-times higher than the hardness of the pure PLLA film. It can be deduced that a small amount of nanotubes added to the matrix bridges the gap between the HA nanoparticles creating a uniform network of hardening material. Beyond the optimal concentration of 0.5 wt %, the nanotubes have a deleterious effect on the hardness of the nanocomposite, likely due to agglomeration. Nanomechanical Testing (Nanoindentation Tests) While microhardness measurements provided an average hardness value, the domain size in the present nanocomposite call for a more local measurements, which will report on possible inhomogeneities in the film. The results from the nanoindentation analysis are presented in Table 7. The results of the nanoindentation experiments are consistent with the Vickers micro-hardness tests discussed above. The addition of 0.5 wt % INT-WS2to the PLLA film caused almost no change in the Young's modulus (3.4 GPa) and hardness (0.18 GPa) values compared to the parameters of the neat PLLA film with Young's modulus of 3.3 GPa and harness of 0.16 GPa. However, the Young's modulus and hardness of the PLLA film with 40 wt % HA (4.9 GPa, 0.24 GPa) increased by 1.5 times each compared to the Young's modulus and hardness of the neat PLLA film. The addition of a small amount of nanotubes to the PLLA film with HA increased the Young's modulus and hardness significantly with the optimum being the addition of 0.25 wt % INT-WS2. The Young's modulus and hardness of the PLLA film with 40 wt % HA and 0.25 wt % INT-WS2(5.6 GPa, 0.36 GPa) increased significantly by 1.7 and 2.25 times, respectively, compared with the values of the neat PLLA film and even the PLLA+HA. Notwithstanding the large fraction of the HA in the film (40 wt %), the hardness values measured here are more than an order of magnitude lower than those reported for a pure HA single crystal. It can be assumed that the difference between the value of hardness measured here and the value observed in the literature, originates from the presence of HA NP agglomerates, which degrade the mechanical properties of the material. Obviously also, the mechanical properties of the nanocomposite are compromised due to the weak links between the HA and the PLLA. Larger statistical variations for some of the composite samples were consistent with local inhomogeneities in the nanoparticle distribution, as is supported by the EDS measurements and mapping, and the Raman studies (below). Nanoindentation results show relative uncertainties an order of magnitude higher in comparison with the microhardness data. This can be attributed to the scale of the inhomogeneities within the sample: EDS mappings inFIG.16show HA “pockets” of several μm extent, and WS2inhomogeneities on a smaller scale. The area of the microindentation imprint varies between 1000-2500 μm2(axial length of 30-50 μm) whereas for the nanoindentations the relevant indentation size is on the scale of the HA pockets, and INT length, 4-5 μm. TABLE 7Parameters determined from the nanoindentation ofPLLA film and HA/INT-WS2/PLLA nanocomposites.Young'sModulusHardnessSample type(GPa)(GPa)PLLA film3.3 ± 0.40.16 ± 0.05PLLA film with4.9 ± 0.70.24 ± 0.0640 wt % HAPLLA film with3.4 ± 0.70.18 ± 0.080.5 wt % INT-WS2PLLA film with5.6 ± 1.20.36 ± 0.1540 wt % HA and0.25 wt % INT-WS2PLLA film with4.6 ± 0.80.25 ± 0.0840 wt % HA and0.5 wt % INT-WS2PLLA film with4.3 ± 0.60.22 ± 0.0740 wt % HA and0.75 wt % INT-WS2 Thermal Properties of PLLA Film and HA/NT-WS2/PLLA nanocomposites by DSC The thermal behavior of the different PLLA films and PLLA/HA/INT-WS2nanocomposites films were measured using DSC. The results are summarized in Table 8, and the heating and cooling curves are presented inFIG.19. The addition of 40 wt % HA nanoparticles to the PLLA film increased the glass transition temperature-Tg(62.7° C.) by merely 1.9% compared to the neat PLLA film (61.5° C.). The addition of 0.5 wt % INT-WS2to the PLLA film increased Tg(66.7° C.) significantly by 8.5% compared to the Tgof the neat PLLA film. Therefore, the PLLA film with 0.5 wt % INT-WS2has the highest thermal deformation resistance of the films tested. The cold crystallization temperature—Tccof the PLLA film with 0.5 wt % INT-WS2(107.9° C.) is lower than the Tccof the neat PLLA film (114.1° C.). In addition, the PLLA film with 40 wt % HA has lower Tcc(93.6° C.) than both the neat PLLA film and the PLLA film with 0.5 wt % INT-WS2, which indicates that the PLLA with 40 wt % HA NPs film consists of smaller crystallites, compared to the neat PLLA film. The lower ΔHccof PLLA film with 40 wt % HA and PLLA film with 0.5 wt % INT-WS2compared to the neat PLLA film also indicates the presence of bigger PLLA crystallites in the neat PLLA film, which is consistent with the findings from XRD (see discussion of Table 5 data, above). The addition of 40 wt % HA nanoparticles to the PLLA reduced Tm(177.6° C.) compared to the neat PLLA film (179.6° C.), while the addition of 0.5 wt % INT-WS2resulted in increased Tm(181.7° C.). Therefore, the PLLA film with 0.5 wt % INT-WS2has the highest thermal stability. The ΔHmvalues of the PLLA films with 40 wt % HA (33.2 J/g) and PLLA film with 0.5 wt % INT-WS2(34.3 J/g) are lower compared to the PLLA film (39.1 J/g). Therefore, the HA nanoparticles and the INT-WS2each, independently lower the energy required for breaking the polymer chain-chain interactions. The lower T, and higher ΔHcof PLLA film with 40 wt % HA (96.9° C., 5.6 J/g) compared to the PLLA film (101.6° C., 2.0 J/g), shows that the PLLA with 40 wt % HA film has a higher cooling rate and smaller crystal nuclei. The higher T, and higher ΔHcof PLLA film with 0.5 wt % INT-WS2(116.9° C., 34.9 J/g) compared to the PLLA film, indicate that the PLLA film with 0.5 wt % INT-WS2had lower cooling rate and even smaller crystal nuclei. The degree of crystallinity—Xcand (1−λ)cof the PLLA film with 40 wt % HA (32.2%, 6.0%) was higher compared to the neat PLLA film (7.5%, 2.2%), which indicated that the PLLA film with 40 wt % HA was harder and denser than the neat PLLA film. However, the Xcand (1-λ)cof PLLA film with 0.5 wt % INT-WS2(33.5%, 36.7%) was even higher compared to the PLLA film with 40 wt % HA, therefore, the PLLA film with 0.5 wt % INT-WS2was the hardest and the densest film among the three. However, the results of micro-hardness test and the nanoindentation tests, show that the hardest film among the three was not the PLLA film with 0.5 wt % INT-WS2, but the PLLA film with 40 wt % HA. The reason for the difference between the estimated hardness trend and the mechanical measurements could possibly be linked to the nuclei size. The crystallites of the PLLA film with 0.5 wt % INT-WS2were larger than the crystallites of the PLLA film with 40 wt % HA. Although not directly relevant, these results are consistent with the Hall-Petch effect, usually associated with polycrystalline metallic films. According to this law, as the size of the crystallites is reduced, the area of their grain boundaries increase, thereby increasing the hardness of the material. PLLA films with 40 wt % HA and 0.25-0.75 wt % INT-WS2have thermal properties (Tg, Tcc, Tmand Tc) similar to the PLLA film with 40 wt % HA. Consequently, the PLLA film with 40 wt % HA and 0.25-0.75 wt % INT-WS2have smaller thermal deformation resistance, crystallite size, thermal stability and lower cooling rate compared with the PLLA film with 0.5 wt % INT-WS2. However, the PLLA film with 40 wt % HA and 0.25-0.75 wt % INT-WS2had better thermal deformation resistance, smaller crystallites, smaller thermal stability and lower cooling rate compared to the neat PLLA film. The ΔHccof PLLA film with 40 wt % HA and 0.5-0.75 wt % INT-WS2(2.5-2.4 J/g) was lower compared to the other samples, which was attributed to the smaller crystallites in the nanocomposite films, due to the combined addition of HA nanoparticles and INT-WS2to the PLLA film. The PLLA film with 40 wt % HA and 0.25 wt % INT-WS2had lower ΔHcc(4.8 J/g) compared to the neat PLLA film and higher ΔHcccompared to the rest of the samples. This data demonstrates that addition of a small amount of INT-WS2combined with 40 wt % HA produced smaller crystallites compared to the neat PLLA film. The ΔHmof the PLLA film with 40 wt % HA and 0.25-0.75 wt % INT-WS2was lower compared to the other samples, thus the combined addition of HA nanoparticles and INT-WS2to the PLLA film decreased the flexibility of the polymer chains and the energy required to break the interaction between the polymer chains. PLLA films with 40 wt % HA and 0.25-0.75 wt % INT-WS2had similar Xcand (1-λ)cto PLLA film with 40 wt % HA, but lower Xcand (1-λ)ccompared to the PLLA film with 0.5 wt % INT-WS2. The Xcand (1-λ)cof PLLA films with 40 wt % HA and 0.5 wt % and 0.75% INT-WS2were very similar, thus they are equally hard. However, the PLLA film with 40 wt % HA and 0.25 wt % INT-WS2had lower Xcbut higher (1-λ)ccompared to the PLLA film with 40 wt % HA and 0.5-0.75 wt % INT-WS2. Therefore, the PLLA film with 40 wt % HA and 0.25 wt % INT-WS2was more elastic but not as hard as the PLLA films with 40 wt % HA and 0.5-0.75 wt % INT-WS2. This is in agreement with the results of the mechanical properties. TABLE 8Thermal properties of PLLA film and HA/INT-WS2/PLLA nanocomposites.TgTccΔHccTmΔHmTcΔHcXc(1-λ)cSample type[° C.][° C.][J/g][° C.][J/g][° C.][J/g][%][%]PLLA film61.5114.132.1179.639.1101.62.07.52.2PLLA film with 40 wt % HA62.793.63.2177.633.296.95.632.26.0PLLA film with 0.5 wt % INT-WS266.7107.93.1181.734.3116.934.233.536.7PLLA film with 40 wt % HA and62.993.54.8177.331.197.28.528.39.20.25 wt % INT-WS2PLLA film with 40 wt % HA and 0.562.992.62.5177.232.698.35.532.35.9wt % INT-WS2PLLA film with 40 wt % HA and62.695.22.4177.032.399.35.032.15.40.75 wt % INT-WS2 Micro-Raman Spectroscopy The Raman spectra of the different PLLA films and PLLA/HA/INT-WS2nanocomposite films are presented inFIG.20. The PLLA film with 40 wt % HA and the neat PLLA film had exactly the same pattern of peaks and at the same energy (873 cm−1, 1452 cm−1), except the peak of the HA at 960 cm−1. In addition, comparing the PLLA film with 0.5 wt % INT-WS2to the neat PLLA film also showed the same pattern of peaks and intensity, except the peaks of the INT-WS2at 350 cm−1and 418 cm−1. The match between the different spectra patterns was excellent, indicating that no chemical reaction took place between the different ingredients of the nanocomposite, as all the identified peaks belong to the pure reagents, with no missing peaks. Hence the chemical composition of the PLLA was not affected by the addition of the HA NPs and INT-WS2, or from the production process of the film as suggested above. The band of 1379 cm−1was associated with chloroform. That band was seen in the spectra of all the different PLLA films and PLLA/HA/INT-WS2nanocomposites films. The existence of this peak indicated that residual amounts of the solvent remained in the films. The film of PLLA with 0.5 wt % INT-WS2and the films of PLLA with 40 wt % HA and 0.25-0.75 wt % INT-WS2, present peaks at 350 cm−1and 418 cm−1, which are associated with the E2gand A1gmodes of the INT-WS2. Oleic acid was a component which was incorporated only into the films of PLLA with 40 wt % HA and 0.25-0.75 wt % INT-WS2. However, the main peak associated with the oleic acid at 1655 cm−1is rather small and can be observed by focusing on the portion of the spectrum near this peak (dashed square) and magnifying the scale. The low intensity of the peak reflects the fact that the oleic acid concentration is very low in the films (150 μl). Raman intensity mapping of the PLLA with 40 wt % HA and 0.5 wt % INT-WS2films were carried out (not shown). Intensity mapping of the PLLA peak at 873 cm−1showed a relatively uniform Raman light scattering intensity on the entire scanned area, with minimum value of 60% with respect to the maximum (normalized) intensity. This indicates, as suggested above, that the PLLA film was uniform and that it was not affected by the addition of the solvent, HA, or and INT-WS2, nor from the fabrication process of the film. The result showed also that no chemical reactions occurred between the four main components during their mixing and processing of the film. Furthermore, the intensity mapping of HA NPs at 960 cm−1showed a good dispersion of the HA nanoparticles in the film, which confirms the observation of a uniform HA distribution obtained via SEM imaging. Notwithstanding the limited resolution of the technique (>1 μm), the INT-WS2were clearly seen as elongated shapes throughout the film in the Raman mapping. Obviously, the asymmetric shape of the nanotube does not reflect its genuine shape, since the coarse size of the focused laser beam (1-2 μm) is at least 10-times larger than the tube diameter (˜100 nm). Moreover, the nanotubes are fully dispersed in the film. Their long axis seem to be within the film plan and oriented roughly in the x-direction. The preferred directionality of the tubes could be related to the mode of evaporation of the solvent from the casted film. Raman mapping of oleic acid at 1655 cm−1presents relatively strong and uniform intensity throughout the film area, with minimum value of (normalized) intensity around 40%. Thus, it can be concluded, that the oleic acid was uniformly dispersed throughout the nanocomposite film during its preparation. FTIR Spectroscopy FTIR of different PLLA/HA/INT-WS2nanocomposite films was conducted and the results of the spectra are displayed inFIG.21. Visibly, PLLA peaks were observed for all different films at the same position, except for the two peaks in 1044 cm−1and 1086 cm−1which overlap with the two IR peaks of HA in 1033 cm−1and 1093 cm−1. No extra peaks occur due to the addition of HA and nanotubes to the PLLA. The nanotubes peaks (<500 cm−1) were not observed due to the predominant PLLA peak in this region. Thus, consistently with the previous measurements, the FTIR indicates that the four components (PLLA, HA, OA(=oleic acid) and INT) are mixed together uniformly and they are compatible with each. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. | 79,979 |
11859081 | DETAILED DESCRIPTION OF EMBODIMENTS Definitions As used herein, the term “PLA” refers to polylactide or poly(lactic acid). As used herein, the term “PHA” refers to poly-3-(hydroxyalkanoate). As used herein, the term “PHB” refers to polyhydroxybutyrate As used herein, the term “PCL” refers to polycaprolactone. As used herein, the term “PBS” refers to polybutylene succinate. As used herein, the term “PBAT” refers to poly(butylene adipate-co-terephthalate). As used herein, the term “PET” refers to polyethylene terephthalate. As used herein, the term “DCP” refers to dicumyl peroxide, an example of a free radical initiator. As used herein, the term “TAM” refers to triallyl trimesate, an example of an allylic coagent. As used herein, the term “TAIC” refers to triallyl isocyanurate, an example of an allylic coagent. As used herein, the term “PETA” refers to pentaerythritol triacrylate, an example of an acrylate based coagent. As used herein, the term “TMPTMA” refers to trimethylolpropane trimethacrylate, an example of an acrylate based coagent. As used herein, the term “BF” refers to an additive that is a nucleating and reinforcing agent that is useful for improving the thermo-mechanical properties of biopolyesters. As described herein, the BF additive is a particulate of thermoset biopolyester. As used herein, the term “a-BF” refers to an acrylate based additive. As used herein, the term “nucleating agent” refers to a material that induces nucleation, which refers to a first step in formation of either a new thermodynamic phase or a new structure. As used herein, the term “BF-reinforced composite” refers to a composite material product prepared by mixing polyester and BF. In some instances, the polyester base is specified in the term, for example “BF-reinforced PLA composite”. As used herein, the term “thermoplastic”, or “thermosoftening plastic”, is a plastic material (e.g., polymer) that becomes pliable or moldable above a specific temperature and solidifies upon cooling. As used herein, the term “fine” in regard to particle size of BF refers to a particle size that is sufficiently small to allow the BF to reinforce and nucleate a thermoplastic polyester; for example, a particle diameter of about 10 to about 150 micrometers. As used herein, the term “thermoset” refers to a crosslinked material that has a substantial (e.g., 20% or higher) gel content. A primary physical difference between thermoset plastics and thermoplastics is that thermoplastics can be melted and remelted back into a liquid with the application of heat, whereas thermoset plastics remain in a permanent solid state. That is, thermoset materials are non-reversibly crosslinked to an extent that they do not melt to form a liquid, thus a thermoset material would not be suitable for melt processing (e.g., not suitable to be pelletized via an extruder) when undiluted by a thermoplastic. As used herein, the term “biobased” or “bioderived” means derived, in whole or in part, from plant, animal, marine, or forestry products. As used herein, the term “biodegradable” means compostable or degradable under certain conditions. As used herein, the term “biopolyester” means a polyester that is biobased, bioderived, and/or biodegradable. As used herein, the term “conditioned” refers to a material which has been subjected to controlled crystallization conditions. EMBODIMENTS Described herein are composite materials comprising thermoplastic polyester and a particulate material that acts to reinforce the thermoplastic polyester. The particulate material comprises thermoset biopolyester. In some embodiments, the thermoplastic polyester that is reinforced is a polyester derived from non-biobased sources (e.g., petroleum based). In one embodiment, a biobased composite material comprises a biopolyester and a bioderived additive. The presence of the bioderived additive provides improved thermo-mechanical properties to the biopolyester, without affecting its status as a fully bioderived polyester. As described in the figures and examples herein, the biobased additive is added to a biopolyester to form a composite material. The composite material, which is referred to herein as BF-reinforced biopolyester, is biobased and has improved properties, including melt strength, impact resistance, and resistance to heat distortion relative to the biopolyester in the absence of the biobased additive. In another embodiment, a biobased additive is provided that acts as both a nucleating and a reinforcement agent when present in polyesters (e.g., biopolyesters). Other embodiments are provided such as the biobased additive. Also described herein are methods of making the composite material and the biobased additive, and articles of manufacture prepared using the composite material. Although the biobased additive of embodiments of the present invention can be used for any thermoplastic polyester (e.g., conventional thermoplastic polyesters such as, for example, polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyethylene succinate (PES), polybutylene succinate (PBS), and poly(butylene adipate-co-terephthalate) (PBAT) and biodegradable polyesters (e.g., PCL, PBAT, PES, PBS)), the improved properties are particularly advantageous for bioderived polyesters. Examples of thermoplastic bioderived polyesters include polylactide (PLA), poly-3-(hydroxyalkanoates) (PHAs) including polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). Embodiments of the invention provide a biobased additive (BF) that acts as a nucleating and reinforcing agent when present in thermoplastics such as biopolyesters (e.g., biobased and/or biodegradable polyesters). BF is particularly suitable for bioplastics since a primary component of BF is biobased (e.g., PLA, PHA) or biodegradable (e.g., PCL, PBS, PBAT). Presence of BF improves thereto-mechanical properties of polyesters such as melt strength, impact resistance, and resistance to heat distortion. It also reduces or even eliminates the need for other additives such as plasticizers and traditional nucleating agents. Further, it eliminates the need for an annealing step to induce crystallization that is required for adequate processability in biopolyesters or composites thereof with traditional nucleating agents/processing additives. Advantageously, such an annealing step is not necessary for a BF-reinforced composite. In one embodiment, a method of making BF is provided. BF has three components, its main component is a biopolyester, and there are two other components: a free-radical initiator (e.g., DCP), and a crosslinking agent (e.g., an allylic based coagent such as TAM or an acrylate based coagent such as PETA). In one embodiment, BF is prepared by mixing and heating a biopolyester. The temperature to which it should be heated should melt the biopolyester and be at or above the decomposition temperature of the initiator. Once heated, and while mixing, about 0.5 wt % to about 2.5 wt % free-radical initiator and about 0.5 wt % to about 2.5 wt % of crosslinking agent are added. Mixing continues while the crosslinking reactions occur, until a thermoset product is obtained. Although mixing, for example mechanical mixing, affects the particle size of the BF, optionally, a grinder is used to make the BF a particular particle size. Suggested BF particle size is a diameter of 10-150 micrometers. (The smaller the particle size, the more impact BF will have on reinforcing and nucleating a biopolymer.) In one embodiment, BF is used as an additive to a biopolyester that is subsequently processed using thermoforming, injection molding, or extrusion. One example of an extrusion process is 3D printing. In one embodiment, BF is prepared from dried PLA that has been compounded (i.e., melt mixed) with DCP and TAM. Results provided herein show that the presence of 10 wt % BF in a biopolyester composite decreased the crystallization half-time of the biopolyester composite by ten-fold. A suggested range of the amount of BF in the biopolyester composite is from about 1 wt % to about 50 wt %. In one embodiment, a BF-reinforced composite is provided that is prepared from a biopolyester and a thermoset additive prepared from: a biopolyester; a crosslinking initiator; and an allylic based coagent. In one embodiment, a BF-reinforced composite is provided that is prepared from a polyester, a crosslinking initiator and an acrylate based coagent. Examples of allylic based coagents include TAM, or TAIC, An example of an acrylate based coagent is PETA. An example of a crosslinking initiator is DCP. Other examples include Luperox® L101, Luperox® L130. PLA can be processed using conventional thermoplastics processing equipment including injection molding, blow molding, film casting, etc. However, PLA has a narrow processing window because of its sensitivity to processing temperatures that are too high above its melting point. Additionally, PLA has a lack of melt strength, a slow crystallization rate, and is considered to have poor engineering properties such as impact strength and heat distortion resistance. Such drawbacks have limited the applicability of PLA to low-cost commodity applications (e.g., food packaging) and biomedical applications (e.g., drug delivery) where biocompatibility and biodegradability are desired. Properties of PLA depend significantly on its molecular weight and the stereochemical makeup of its polymer backbone which is controlled by polymerization with D-lactide, L-lactide, or D,L-lactide, to form random or block stereocopolymers. The rheological properties of PLA depend on the molecular weight and molecular weight distributions (MWD), presence of branching, and the stereochemical makeup. Commercially available linear PLA is reported to lack the level of strain hardening, and therefore melt strength, needed for normal processing operations, limiting the success of PLA in operations involving high stretch rates such as film blowing, thermoforming, and foaming. Nucleating agents are commonly added to impart crystallinity, or to enhance crystallization kinetics (Li, H., et al.,Polymer,48(23), 6855-6866 (2007), Withey R. E., et al.,Polymer,40(18), 5147-5152 (1999).). Nucleating agents work by providing sites around which polymer chains can crystallize. They can alter the crystallization temperature and thus the rate of crystallization. For neat PLA, even with the addition of nucleating agents, an annealing step is still required to induce crystallization, and to obtain satisfactory heat distortion resistance. Generally in PLA the maximum achievable crystallinity is dictated by the amount of D-lactide. Mixtures of D-lactide and L-lactide based PLA can crystallize in the form of a stereocomplex, which improves the temperature resistance of the material, while also acting as a nucleating agent for the crystallization of PLA. Various nucleating agents have been tested in PLA and PHB formulations to promote crystallization including talc, sodium stearate, calcium lactate etc., often together with a plasticizer. Various nanofillers have also been tested in PLA, and PHAs. More recently, biobased cellulosic additives, such as cellulose nanowhiskers, nanofibers, and nanocrystals have been used to achieve reinforcement and to increase the crystallization rate of biopolymers, while maintaining the completely biobased nature of the formulations. This approach comes with many challenges, given the incompatibility with polymer matrices, which necessitates the use of various functionalization methods, such as silylation of the cellulose nanocrystals. Advantageously, the reinforced biopolyester is substantially all biobased. Assuming 10% for an amount of reinforcing additive and 90% as the amount of matrix biopolymer, with the additive being 2% DCP and TAM, the resulting amount is 0.2% non-biobased material and 99.8% biobased material. Assuming 2% for an amount of reinforcing additive results in an even higher amount of biobased material. In one embodiment, an article of manufacture is provided that is made from BF-reinforced composite materials, prepared from mixing BF and polyesters (e.g., PLA, PHA and/or PCL). Such articles include a wide range of articles from clothing, packaging (e.g., clam shell type food packaging), bottles, office stationary, food containers, disposable cutlery, disposable cups, disposable plates, toys, an article of manufacture made from plastic, injection molded articles, consumer products, building materials, products of 3D printing/additive manufacturing, packaging, foams, and automotive applications. PLA, as well as PHAs and PCL are used in biomedical applications. Accordingly, BF-reinforced PLA, BF-reinforced PHA, and BF-reinforced PCL offer improved properties and are also suitable for use to prepare biomedical articles, such as sutures, stents, dialysis media, and drug delivery devices. As described in the Working Examples and shown in the figures, tests were conducted to quantify the effect of the presence of various amounts of BF and a-BF in BF-reinforced PLA composite material samples versus neat PLA. Referring toFIG.1A, a graph is presented that shows complex viscosity as a function of angular frequency of PLA compared to composites of PLA and specified amounts of BF. These rheological measurements were conducted from low to high frequencies at 190° C. Referring toFIG.1B, a graph is presented that shows storage modulus as a function of angular frequency of PLA compared to composites of PLA and specified amounts of BF. These rheological measurements were conducted from low to high frequencies at 190° C. Referring toFIG.2A, a non-isothermal melt crystallization thermogram is shown of processed PLA and BF-reinforced PLA composites with various BF loadings, at a cooling rate of ° C./min. Referring toFIG.2B, a graph is shown that presents degree of crystallinity of processed PLA and BF-reinforced PLA composites with various BF loadings at a cooling rate of 5° C./min. Non-isothermal crystallization behavior from the melt state is of increasing technological importance since these conditions are the closest to industrial processing situations. The degree of crystallinity (Xc) was calculated according to Eq. 1. Xc(%)=(ΔHc-ΔHCCΔHmo(ϕPLA100))×100Eq.1 where ΔHc, ΔHccand ΔHomare the enthalpy of fusion, and enthalpy of cold crystallization of the component and the melting enthalpy of perfectly crystalline PLA (93.6 J/g), respectively, and ϕPLAis the matrix weight percent in the samples.FIG.2Ashows that neat PLA exhibited no exothermic peak on the cooling curve, indicating that it does not crystallize during the cooling process. As expected, the inclusion of BF in the linear PLA significantly affected the degree of crystallization (Xc) and crystallization temperature (Tc). The degree of crystallinity, Xcincreases from 3 to 50 and 55% in the BF-reinforced PLA composite with 5 and 10 wt % of BF, respectively, as shown inFIG.2B. The Xcvalue, however, drops to 27% as the BF concentration in the BF-reinforced composite was further raised to 20 and 50 wt %. This phenomenon indicated that dispersed particles played a dual role during non-isothermal melt crystallization: increase of crystal nucleation induced by the heterogeneous mechanism, leading to enhanced degree of crystallinity, in competition with the restriction of chain mobility that makes chain packing in the crystal structure more difficult. Due to strong interactions between PLA polymer chains and BF, the PLA molecules at the surface of the dispersed particles were partially immobilized. Referring toFIG.2C, a non-isothermal melt crystallization (first heating) thermogram is shown of unconditioned PLA and BF-reinforced PLA composites with various BF loadings at a heating rate of 5° C./min. Both unconditioned BF-reinforced PLA composites and PLA showed similar thermal properties. Referring toFIG.2D, a non-isothermal melt crystallization (first heating) thermograph obtained at a heating rate of 5° C./min is shown of PLA and BF-reinforced PLA composites with various BF loadings that were conditioned at 100° C. prior to testing. The BF-reinforced PLA composites do not exhibit the cold crystallization peaks that are characteristic of the neat PLA. This shows that the BF samples have attained substantial amounts of crystallization upon conditioning. Contrarily, a cold crystallization peak is present in the a-BE sample, consistent with PLA. Referring toFIG.2E, a graph is shown that presents the crystallinity of conditioned PLA and BF-reinforced PLA composites with various BF loadings, obtained from the first heating scan. The degree of crystallinity was calculated as previously described. As expected, exposure to controlled crystallization conditions results in higher crystallinities in the BF samples compared to PLA alone. Referring toFIG.3, a plot is shown to display isothermal crystallization behavior of BF-reinforced composites at Tcof 130° C. As shown, incorporation of BF into PLA significantly promoted the onset of crystallization and promoted the crystallization rate. It is believed that the inclusion of BF particles led to an enhancement of the density of nuclei, increasing the crystallization rate at the early stage of crystallization. Referring toFIG.4, crystallization half times were calculated and plotted inFIG.4. At low-crystallization temperatures, the decreased chain mobility significantly retarded the crystallization rate, whereas at high-crystallization temperatures, a considerable decrease in nucleation density hindered the crystallization growth although the chain mobility was high. Notably, the crystallization half time of PLA was above 10 min at 110° C. while it dropped to 1 min after the inclusion of 10 wt % BF. This result confirms the marked nucleating effect of BF in BF-reinforced PLA. Referring toFIG.5A, a polarized optical micrograph is shown of neat PLA isothermally crystallized at 130° C. Referring toFIG.5B, a polarized optical micrograph is shown of a BF-reinforced PLA composite (10 wt % BF) isothermally crystallized at 130° C. The nucleating effect of the biofiller is evident by the change in the spherulitic morphology and spherulite density of crystallized PLA. In the presence of the nucleating agent, more spherulites nucleate and grow, thus increasing their density, while limiting their size. Referring toFIG.6, a plot is shown that graphically shows tensile mechanical properties of PLA and BF-reinforced PLA composites with various BF loadings. Referring toFIG.7A, a plot is shown that graphically shows flexural behavior of PLA and BF-reinforced PLA composites with specified BF loadings. Referring toFIG.7B, a graph is shown of the flexural moduli for the conditioned at 100° C. BF-reinforced PLA composites at specified BF loadings. All BF-reinforced PLA composites have higher flexural moduli compared to PLA alone. Referring toFIG.8A, a graph is shown of notched-Izod impact strength tests of PLA and BF-reinforced PLA composites with specified BF loadings. Referring toFIG.8B, a graph is shown of notched-Izod impact strength results for conditioned at 100° C. PLA and BF-reinforced PLA composites at specified BF loadings. All BF-reinforced PLA composites have higher impact strength compared to PLA alone. Referring toFIG.9A, a graph is shown of the particle size distribution in one embodiment. Particle size is shown to range from 20 μm to 100 μm. Referring toFIG.9B, an optical microscopic image is shown of the particles dispersed evenly in one embodiment. Referring toFIG.10, a table is shown of the heat distortion temperatures (HDT) of conditioned PLA and BF-reinforced PLA composites at specified BF loadings. All BF-reinforced PLA composites were shown to have higher HDT than PLA. Referring toFIG.11an image is shown where the visual comparison between PLA and PLA-BF show similar hydrolytic degradation after week5. Notably, the composite materials described herein have been shown to possess improved impact properties. As described in the Working Examples and shown in the figures, a variety of improved properties of BF-reinforced biopolyester relative to neat biopolyester have been observed and quantified. Samples of BF were tested for gel content as described in Example 9. A gel content of about 90% or higher was determined for a representative example of BF. Gel content of about 20% or higher is expected to have the reinforcing and nucleating effect on biopolyester that was shown herein. A gel content of about 20% of higher is not significantly deformed in the presence of heat and remains solid (i.e, does not melt) in the presence of heat and therefore is not suitable for melt processing. The following working examples further illustrate embodiments of the invention and are not intended to be limiting in any respect. WORKING EXAMPLES Polylactide or PLA used in experiments described herein was PLA 3251D and was purchased from NatureWorks LLC Co. (USA). The selected grade is a semi-crystalline linear polymer with melt flow rate (MFR) of 35 g/10 min under a load of 2.16 kg at 190° C., and 80 g/10 min at 210° C. (ASTM D1238), as reported by the manufacturer. Dicumyl peroxide (DCP) and triallyl-trimesate (TAM), supplied by Sigma Aldrich and Monomer Polymer & Dajac Labs, respectively, were used as initiator and crosslinking agent in this work, respectively. Example 1. Melt Compounding of PLA with DCP and TAM Melt compounding of PLA with initiator and crosslinking agent was performed using a Haake Polylab R600 internal batch mixer. Before compounding, the PLA was dried at 60° C. in a vacuum oven for 24 h. The dried PLA was then directly mixed in the molten state with 1 wt % of dicumyl peroxide (DCP) and 1 wt % of triallyl trimesate (TAM), trimethylolpropane trimethacrylate (TMPTMA) at a set temperature of 185° C. The mixing was conducted at a rotation speed of 120 rpm for 10 min. The PLA was chemically reacted with the cross-linking agents during the melt compounding leading to the production of an agglomeration of thermoset (i.e., cross-linked) polymer. The thermoset polymer was then ground using a grinder to smaller size detached particles. A particulate of thermoset PLA that has a particulate diameter of about 10 to about 150 micrometers is referred to herein as BF. The melt mixing of PLA with various quantities of BF (1, 5, 7, 10, 20 and 50 wt %) and a-BF (5 wt %) was carried out using the laboratory internal mixer (Haake) for 5 min under the aforementioned conditions. The melt mixing of PHB with 5 wt % of BF was carried out using the Haake mixer under the aforementioned conditions. Based on preliminary results, the use of PHB does not offer any improvements compared with the PLA based materials. To further investigate the effects of scale-up on the properties of the BF samples, melt mixing of PLA with 5 wt % BF was performed using a twin screw corotating extruder (Coperion ZSK 18 ML). The temperature profile was 170/190/190/190/190/190/190° C. (hopper to die). The extrusion was performed at a feeder speed of 30 min−1with a screw speed of 120 min−1and an average residence time of 2.5 min. No significant differences in thermal properties were detected between the Haake and Coperion processed samples. Samples were subjected to controlled crystallization conditions using a Carver Hydraulic Press. The conditioned samples were molded at 190° C. for 5 minutes and were transferred to a compression molding machine at 100° C. After 5 minutes the mold was removed and the samples were extracted. Example 2. Rheological Properties The prepared disk-shaped samples were used to measure dynamic rheological properties. Small amplitude oscillatory shear (SAGS) experiments were conducted using a rotational rheometer with parallel plate flow geometry of 25 mm diameter and 1.0 mm gap size. Example 3. Non-Isothermal and Isothermal Melt-Crystallization Behavior The non-isothermal and isothermal melt-crystallization behavior of the samples were investigated using a differential scanning calorimeter, DSC-Q 1000 (TA Instruments, USA) under a N2atmosphere. In initial tests, samples (˜10 mg) encapsulated in aluminum standard pans, heated at a scanning rate of 10° C./min from 30 to 190° C. and held for 5 min at this temperature. Samples were then cooled down to 30° C. at a rate of 5° C./min to determine the crystallization enthalpy (ΔHc) and degree of crystallinity (Xc). In subsequent tests, samples were heated from 30 to 210° C. at a rate of 5° C./min and held for 5 min at this temperature. Samples were cooled down to −30° C. at a rate of 5° C./min. Example 4. Isothermal Melt-Crystallization Behavior The isothermal melt-crystallization behavior was studied at 90 to 130° C. All the specimens were first heated at 60° C./min to 200° C. and held there for 5 min. The molten samples were then cooled down to crystallization temperature (Tc) at 50° C./min. The samples were kept at Tc until the crystallization was complete. Example 5. Tensile and Flexural Properties Tensile and flexural properties were determined from the compression molded dog-bone and rectangular specimens, respectively. The samples were molded at 190° C. and were subsequently conditioned at 100° C. in temperature controlled compression molding platens. Tensile testing was performed according to ASTM D 638 on an Instron tensile machine equipped with a 5 kN load cell and a cross head speed of 5 mm min−1. Flexural tests were performed according to ISO 178 with a cross head speed of 2 mm min−1. All the mechanical tests were performed 48 h after molding and the reported values were obtained by averaging over eight specimens for each composition. Example 6. Impact Strength The rectangular compression molded bars that were conditioned at 100° C. were used to obtain the impact strength of composites of PLA and various amounts of BF particles. The Izod impact test was performed on notched samples using a pendulum impact tester (available from Satec System Inc.), according to ASTM D256. The values reported here are the averages of five specimens for each composition. Example 7. Heat Deflection Temperature (HDT) Specimens (127 mm×13 mm×3 mm) were prepared by compression molding using a Carver Hydraulic Press at 190° C. with a residence time of 5 minutes. The mold was removed and cooled at 100° C. for 5 minutes. Specimens were lowered in a silicon oil bath, and the temperature was increased from 23° C. at a heating rate of 120° C./h until 0.25 mm deflection occurred under a load of 1.82 MPa, in accordance with ASTM D648. At least three specimens were tested, and the average value was reported. Example 8. Complex Viscosity (q*) and Elastic Modulus (G′) of Processed PLA (“PPLA”) and BF-Reinforced PLA The complex viscosity (η*) and elastic modulus (G′) of the processed PLA (PPLA) and BF-reinforced PLA with specified BF amounts were plotted as functions of frequency (w) at 190° C. inFIGS.1A and18. The incorporation of BF into the neat PLA changed its rheological response. The magnitude of the complex viscosity and elastic modulus were respectively increased by seven times and four order of magnitudes at low frequencies, depending on the BF content. Meanwhile, the transition from a Newtonian plateau to the so-called shear-thinning regime shifted to a lower frequency as BF content was increased. The observed change in the viscosity behavior was attributed to the BF-polymer and BF-BF interactions. Example 9. Isothermal Crystallization Behavior The isothermal crystallization behaviors of PLA and BF-reinforced PLA composites was investigated by DSC. Results in the form of thermograms for all systems at Tcof 130° C. are presented inFIG.3. The incorporation of BF into the PLA significantly promoted the onset of crystallization and increased the crystallization rate. The crystallization rate is, first, controlled by nucleation and then crystal growth and packing. It is believed that the inclusion of BF particles leads to an enhancement of the density of nuclei, increasing the crystallization rate at the early stage of crystallization. To further analyze the effect of BF on the crystallization behavior of PLA, the isothermal crystallization behavior of BF-reinforced PLA with 10 wt % BF was investigated in a temperature ranging from 90 to 130° C. The crystallization half times (t1/2) were calculated and plotted inFIG.4. The t1/2value initially decreases with increasing the crystallization temperature, followed by an increase above 110° C. Such a behavior is expected in polymer due to a balance between two opposing effects. At low-crystallization temperatures, close to the glass transition temperature, the decreased chain mobility significantly retards the crystallization rate, whereas at high-crystallization temperatures, close to equilibrium melting temperature, a considerable decrease in nucleation density hinders the crystallization growth although the chain mobility is high. Notably, the t1/2of PLA is above 10 min at 110° C. while it, remarkably, drops to 1 min after the inclusion of 10 wt % BF. This result, indeed, confirms the marked nucleating effect of BF in BF-reinforced PLA. Example 10. Particle Size Distribution An optical microscope (Olympus BX51, Toronto, Canada) was used to observe compression molded specimens, BF was found to be evenly dispersed in one of the embodiments (10 wt %). Particle size ranged from 20 μm to 100 μm. Example 11. Gel Content Study Gel content analysis of BF was conducted by extraction into boiling chloroform from a 120 mesh stainless steel sieve for 6 hours, according to ASTM D 2765 (Takamura, M., et al.,Polym. Degrad. Stabil.,93 (2008) 1909-1916, and Sen-lin Yang, et al.,Polymer Testing27 (2008) 957-963). Residual polymer was dried to constant weight, with gel content reported as a weight percentage of unextracted material. Gel contents of PLA samples have been quantified as low as 3 wt %, which is considered the detection limit, and as high as fully crosslinked. Gel content amounts between these low and high amounts are possible depending on the amounts of crosslinking agent(s). Samples of BF were tested and found to be insoluble in chloroform. This result was interpreted as indicative of a gel content of about 90% or higher. Example 12. Hydrolytic Degradation Study Hydrolytic degradation tests were conducted at 60° C. in a Thermo Scientific Forma 3911 environmental chamber. Individual films with 1×3 cm2dimensions and a thickness of 200 μm were placed in tared scintillation vials containing 20 ml of phosphate buffer solution. On a weekly basis, two samples per formulation were extracted for further characterization and the PBS was replaced for all remaining vials. Gel permeation chromatography (GPC) was performed using a Viscotek 270 max separation module equipped with triple detectors: differential refractive index (DRI), viscosity (IV), and light scattering (low angle, LALS and right angle, RALS). The separation module was maintained at 40° C. and contained two porous PolyAnalytik columns in series with an exclusion molecular weight limit of 20×106g·mol-1. HPLC grade THF was used as the eluent at a flow rate of 1 mL·min−1. Samples were prepared for GPC analysis by dissolving film cross-sections in THF to achieve solutions with concentrations of 2.5 mg ml−1. In preliminary tests, BF did not significantly impact the degradation of PLA. After 5 weeks of hydrolytic exposure, both non-BF reinforced PLA and PLA with 1 wt % BF were observed to undergo 94% loss in molar mass and 17% loss in mass. Also shown inFIG.11, the samples showed similar breakdown of structural integrity. All publications listed and cited herein are incorporated herein by reference in their entirety. It will be understood by those skilled in the art that this description is made with reference to certain preferred embodiments and that it is possible to make other embodiments employing the principles which fall within its spirit and scope as defined by the claims. | 32,184 |
11859082 | DETAILED DESCRIPTION As used herein, the terms “a,” “an,” and “the” do not denote a limitation of quantity and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. “Or” means “and/or” unless clearly indicated otherwise. As used herein, an “acid-labile group” refers to a group in which a bond is cleaved by the catalytic action of an acid, optionally and typically with thermal treatment, resulting in a polar group, such as a carboxylic acid or alcohol group, being formed on the polymer, and optionally and typically with a moiety connected to the cleaved bond becoming disconnected from the polymer. Such acid is typically a photo-generated acid with bond cleavage occurring during post-exposure baking. Suitable acid-labile groups include, for example: tertiary alkyl ester groups, secondary or tertiary aryl ester groups, secondary or tertiary ester groups having a combination of alkyl and aryl groups, tertiary alkoxy groups, acetal groups, or ketal groups. Acid-labile groups are also commonly referred to in the art as “acid-cleavable groups,” “acid-cleavable protecting groups,” “acid-labile protecting groups,” “acid-leaving groups,” “acid-labile groups,” and “acid-sensitive groups.” “Substituted” means that at least one hydrogen atom on the group is replaced with another atom or group, provided that the designated atom's normal valence is not exceeded. When the substituent is oxo (i.e., ═O), then two hydrogens on the carbon atom are replaced. Combinations of substituents or variables are permissible. Exemplary groups that may be present on a “substituted” position include, but are not limited to, nitro (—NO2), cyano (—CN), hydroxy (—OH), oxo (═O), amino (—NH2), mono- or di-(C1-6)alkylamino, alkanoyl (such as a C2-6alkanoyl group such as acyl), formyl (—C(═O)H), carboxylic acid or an alkali metal or ammonium salt thereof; esters (including acrylates, methacrylates, and lactones) such as C2-6alkyl esters (—C(═O)O-alkyl or —OC(═O)-alkyl) and C7-13aryl esters (—C(═O)O-aryl or —OC(═O)-aryl); amido (—C(═O)NR2wherein R is hydrogen or C1-6alkyl), carboxamido (—CH2C(═O)NR2wherein R is hydrogen or C1-6alkyl), halogen, thiol (—SH), C1-6alkylthio (—S-alkyl), thiocyano (—SCN), C1-6alkyl, C2-6alkenyl, C2-6alkynyl, C1-6haloalkyl, C1-9alkoxy, C1-6haloalkoxy, C3-12cycloalkyl, C5-18cycloalkenyl, C6-12aryl having at least one aromatic ring (e.g., phenyl, biphenyl, naphthyl, or the like, each ring either substituted or unsubstituted aromatic), C7-19arylalkyl having 1 to 3 separate or fused rings and from 6 to 18 ring carbon atoms, arylalkoxy having 1 to 3 separate or fused rings and from 6 to 18 ring carbon atoms, C7-12alkylaryl, C4-12heterocycloalkyl, C3-12heteroaryl, C1-6alkyl sulfonyl (—S(═O)2-alkyl), C6-12arylsulfonyl (—S(═O)2-aryl), or tosyl (CH3C6H4SO2—). When a group is substituted, the indicated number of carbon atoms is the total number of carbon atoms in the group, excluding those of any substituents. For example, the group —CH2CH2CN is a C2alkyl group substituted with a cyano group. In the present specification, “(meth)acrylate” represents “at least one of acrylate and methacrylate.” In addition, “(meth)acrylic acid” means “at least one of acrylic acid and methacrylic acid”. The term “alkyl”, as used herein, means a branched or straight chain saturated aliphatic hydrocarbon group having the specified number of carbon atoms, generally from 1 to about 12 carbon atoms. The term C1-C6alkyl as used herein indicates an alkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Other embodiments include alkyl groups having from 1 to 8 carbon atoms, 1 to 4 carbon atoms or 1 or 2 carbon atoms, e.g. C1-C6alkyl, C1-C4alkyl, and C1-C2alkyl. When C0-C4alkyl is used herein in conjunction with another group, for example, (cycloalkyl)C0-C4alkyl, the indicated group, in this case cycloalkyl, is either directly bound by a single covalent bond (C0), or attached by an alkyl chain having the specified number of carbon atoms, in this case 1, 2, 3, or 4 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl. The term “cycloalkyl”, as used herein, indicates a saturated hydrocarbon ring group, having only carbon ring atoms and having the specified number of carbon atoms, usually from 3 to about 8 ring carbon atoms, or from 3 to about 7 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl as well as bridged or caged saturated ring groups such as norborane or adamantane. The term “heterocycloalkyl”, as used herein, indicates a saturated cyclic group containing from 1 to about 3 heteroatoms chosen from N, O, and S, with remaining ring atoms being carbon. Heterocycloalkyl groups have from 3 to about 8 ring atoms, and more typically have from 5 to 7 ring atoms. Examples of heterocycloalkyl groups include morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl groups. A nitrogen in a heterocycloalkyl group may optionally be quaternized. In citations for a group and an atomic group in the present specification, in a case where the group is denoted without specifying whether it is substituted or unsubstituted, the group includes both a group and an atomic group not having a substituent, and a group and an atomic group having a substituent. For example, an “alkyl group” which is not denoted about whether it is substituted or unsubstituted includes not only an alkyl group not having a substituent (unsubstituted alkyl group), but also an alkyl group having a substituent (substituted alkyl group). Disclosed herein is a surface leveling agent that is used in surface-active chemical applications, in particular lithographical compositions for manufacturing spin-on thin films. The surface leveling agent comprises a copolymer of two or more polymeric units that have ether linkages in the chain backbone. The two or more polymeric units are free of fluorine. In an optional embodiment, the polymers are free of surface energy reducing moieties that comprise silicon. While the aforementioned ether linkages are a part of the polymer backbone, there may be additional functional linkages present in pendent groups (side chains) that are covalently bonded to the copolymer backbone. In an embodiment, these side chains may comprise only carbon-carbon linkages. In another embodiment, these side chains may comprise carbon-carbon linkages in addition to other functional linkages or functional groups, including but not limited to, ether, ester, amide, sulfonate, hydroxy, thiol, cyano, amine, thiol, aldehyde, carboxyl, alkyl halide, ketone, allyl, allenyl, norbornyl, ethynyl, acrylates, methacrylates, itaconates, maleimides, maleic anhydrides, and the like. In other words, the side chain may contain heteroatoms such as nitrogen, sulfur, oxygen, and the like. The surface leveling agent may be used not only in compositions for lithographic applications, but may also in other applications such as, for examples, coatings, paints, inks, plating solutions, medical, anticorrosion, and lubrication technologies, where good surface leveling and glossy appearance are desirable. In an exemplary embodiment, at least one of the polymeric unit of the copolymer is a polyoxetane having at least one alkyl substituent along the chain backbone (hereinafter polyalkyloxetane). The polyoxetane backbone provides the surface leveling agent copolymer with hydrophilicity that facilitates miscibility with bases and with water, while the alkyl side chain provides hydrophobicity for the migration of the surface leveling agent to an interface in a thin film or a coating. The interface may lie between the spin-on layer and air, or alternatively, between two layers in a multilayer film. In an embodiment, the polyalkyloxetane is present in the copolymer in an amount of greater than 40 mole percent (mol %). In an embodiment, the polyoxetane copolymer may contain one or more end functional groups. The end functional group may comprise a hydroxyl, a thiol, a cyano, an amine or a sulfonate. The aforementioned alkyl substituent may be a straight chain, a branched chain or may optionally contain other atoms such as O, S, N, P, or other functional linkages, which include an ether, an ester, an amide, an imide, a urethane or a urea. The alkyl substituent may also optionally contain an end functional group such as for example, a hydroxyl, a thiol, a cyano, an amine or a sulfonate. The surface leveling agent may be hydrophobic for certain applications, while it may be hydrophilic enough so as to be miscible with water or an aqueous developer in other applications. The lack of fluorine or silicon containing moieties in the surface leveling agent minimizes defect formation. The lack of fluorine containing moieties complies with environmental regulations. The surface leveling agent is preferably a copolymer comprising two or more different repeat units. In an embodiment, the surface leveling agent may be a copolymer that comprises three of more different repeat units. The copolymer may be a random copolymer, alternating copolymer, a block copolymer, a star block copolymer, a hyperbranched polymer, a comb copolymer, a dendrimer, a gradient copolymer, or the like, with a random copolymer or a block copolymer being preferred. Combinations of random copolymers and block copolymers may also be used in a surface leveling agent. In an embodiment, the copolymer comprises first polymerized units of the formula (1): wherein R1is H or a substituted or unsubstituted C1-C6alkyl group; and R2is a substituted or unsubstituted C3-C20alkyl group that optionally includes one or more of —O—, —S—, —N—, —C(O)—, or —C(O)O—, —N—C(O)—, —C(O)—NR—; wherein R is H or a substituted or unsubstituted C1-C6alkyl group; and a second polymerized unit of the formula (2): wherein R3is a substituted or unsubstituted C1-C6alkyl group that optionally includes one or more of —O—, —N—, —S—, —C(O)—, or —C(O)O—; wherein the first polymerized units and the second polymerized units are chemically different, and the copolymer is free of fluorine. In a preferred embodiment, R3is an unsubstituted C1-C5alkyl. When R3is substituted, the substituent chain length has two or less carbon atoms. A hydrophilic polymeric unit of formula (2) can provide benefits of miscibility of the surfactant leveling agent with water or aqueous base developer. In an embodiment, R2has a branched structure. In an embodiment, R2has two or more branches. In an embodiment, the surface leveling agent comprises a polymerized unit of formula (1), a polymerized unit of formula (2) and comprises an additional polymerized unit of formula (3) wherein R4is a substituted or unsubstituted C2to C4alkyl group that optionally includes one or more of —O—, —S—, —N—, —C(O)—, or —C(O)O—, —N—C(O)—, —C(O)—NR—; wherein R is H or a substituted or unsubstituted C1-C6alkyl group. As a specific example of the above-mentioned embodiment, the first polymerized units are of the formula (1-1), the second polymerized units are of the formula (2-1), and the third polymerized units are of the formula (4): wherein R4is a substituted or unsubstituted C3-C20alkyl group that optionally includes one or more of —O—, —S—, —N—, —C(O)—, or —C(O)O—; Examples of the precursor to the polymerized units of formula (1) include 3-methyloxelane, 3-ethyloxetane, 3-propyloxetane, 3-butyloxetane, 3-neupentyloxetane, 3-pentyloxetane, 3-hexyloxetane, 3-(2,2-dimethylbutyl)oxetane, 3-methxymethyloxetane, 3-ethoxymethyloxetane, 3-propoxymethyloxetane, 3-butoxymethyloxetane, 3-neupentoxymethyloxetane, 3-pentoxymethyoxetane, 3-hexoxymethyloxetane, 3-(2,2-dimethylbutoxy)oxetane, 3-methxyethyloxetane, 3-ethoxyethyloxetane, 3-propoxyethyloxetane, 3-butoxyethyloxetane, 3-neupentoxyethyloxetane, 3-pentoxyethyloxetane, 3-hexoxyethyloxetane, 3-butyloxymethyl-3-methyloxetane, 3-butyloxymethyl-3-ethyloxetane, 3-butyloxymethyl-3-propyloxetane, 3-butyloxymethyl-3-butyloxetane, 3-butyloxymethyl-3-pentyloxetane, 3-butyloxymethyl-3-neupentyloxetane, 3-butyloxymethyl-3-hexyloxetane, 3,3-dimethyloxetane, 3-ethyl-3-[(2-ethylhexyloxy)methyl]oxetane, 3-ethyl-3-hydroxymethyl oxetane, 3-ethyl-3-hydroxymethyl oxetane, 3-methyl-3-oxetanemethanol, 3-(1-methylethyl)-oxetane, or a combination thereof. Examples of the precursor to the polymerized units of formula (2) include formaldehyde, ethylene oxide, propylene oxide, tetrahydrofuran, 1,4-dioxane, or a combination thereof. Examples of the precursors to the polymerized unit of formula (3) includes 1,3-propane diol, 2,2-dimethyl-1,3-propane diol, 1,4-butane diol, 1,5-pentane diol, 1,6-hexane diol, or a combination thereof. A preferred precursor to the polymerized units of formula (1) is 3-butyloxymethyl-3-methyloxetane, while a preferred precursor to the polymerized units of formula (2) is tetrahydrofuran. A preferred precursor to the polymerized units of formula (3) is 2,2-dimethyl-1,3-propane diol. The surface leveling agent may have a weight average molecular weight of 500 to 30,000 grams per mole (g/mole), preferably 800 to 12,000 g/mole and more preferably 1000 to 10,000 g/mole. The polymerized units of formula (1) are typically present in the copolymer in an amount of greater than 40 mol %, preferably greater than 50 mol % and more preferably greater than 60 mol %, based on the total number of moles in the copolymer. The polymerized units of formula (1) are present in the copolymer in an amount of less than 80 mol %, preferably less than 75 mol % and more preferably greater than 70 mol %, based on the total number of moles in the copolymer. In an embodiment, the polymerized units of formula (2) are present in the copolymer in an amount of greater than 20 mol %, preferably greater than 25 mol % and more preferably greater than 70 mol %, based on the total number of moles in the copolymer. In an embodiment, the polymerized units of formula (2) are present in the copolymer in an amount of less than 60 mol %, preferably less than 50 mol % and more preferably less than 40 mol %, based on the total number of moles in the copolymer. In an embodiment, the polymerized units of formula (3) are present in the copolymer in an amount of greater than or equal to 0.01 mol %, preferably greater than or equal to 1 mol % and more preferably greater than or equal to 2 mol %, based on the total number of moles in the copolymer. In an embodiment, the polymerized units of formula (3) are present in the copolymer in an amount of less than or equal to 20 mol %, preferably less than or equal to 15 mol % and more preferably less than or equal to 10 mol %, based on the total number of moles in the copolymer. The surface leveling agent may be used in a variety of different compositions. In an embodiment, the surface leveling agent may be used in a solvent or solvent mixture only, acting as a rinse formulation. In another embodiment, the surface leveling agent may be used in a composition that comprises a matrix polymer, a solvent, and other optional additives. In an embodiment, the surface leveling agent may be used in a photoresist composition that contains a polymeric matrix resin, an optional quencher, one or more photoacid generators, optional additives and a solvent. The matrix polymer is preferably a copolymer that contains at least one or more repeating units that contain an acid-labile group and/or contains crosslinkable functionalities. If used, the surface leveling agent is present in an amount of from 0.001 to 100 wt %, more preferably 0.001 to 1 wt %, based on total solids of the composition. In another embodiment, the surface leveling agent may be used in a photoresist composition that in addition to the surface leveling agent, contains a matrix polymer, a photoacid generator, an optional quencher, a solvent, and other optional additives. In another embodiment, the surface leveling agent may be used in a composition that contains in addition to the surface leveling agent, a matrix polymer, at least one thermally activated acid generator, and a solvent. Other optional additives may be added to the composition, including but not limited to crosslinking agents. Examples of compositions that contain thermal activated acid generators can be antireflective coating compositions, top coat, photoresist trimming or pattern enhancement composition, and other underlayer compositions. The polymer matrix that is used in the foregoing compositions may include a thermoplastic polymer, a blend of thermoplastic polymer, a thermosetting polymer, or a blend of a thermoplastic polymer with a thermosetting polymer. The polymer matrix may also include a blend of polymers, copolymers, terpolymers, or combinations comprising at least one of the foregoing polymers. The polymer matrix may also include an oligomer, a homopolymer, a copolymer, a block copolymer, an alternating copolymer, a random polymer, a random copolymer, a random block copolymer, a gradient copolymer, a graft copolymer, a star block copolymer, a dendrimer, a polyelectrolyte (polymers that have some repeat groups that contain electrolytes), a polyampholyte (a polyelectrolyte having both cationic and anionic repeat groups), an ionomer, or the like, or a combination thereof. The copolymer may be available as a dissolved molecule, in the form of a microparticle, or in the form of a nanoparticle dispersion or suspension in the solvent or solvent mixtures. Examples of thermoplastic polymers include polyacetals, polydienes, polyacrylics, polycarbonates, polyalkyds, polystyrenes, polyolefins, polyesters, polyamides, polyaramides, polyamideimides, polyarylates, polyurethanes, epoxies, phenolics, silicones, polyarylsulfones, polyethersulfones, polyphenylene sulfides, polysulfones, polyimides, polyetherimides, polytetrafluoroethylenes, polyetherketones, polyether ether ketones, polyether ketone ketones, polybenzoxazoles, polyoxadiazoles, polybenzothiazinophenothiazines, polybenzothiazoles, polypyrazinoquinoxalines, polypyromellitimides, polyguinoxalines, polybenzimidazoles, polyoxindoles, polyoxoisoindolines, polydioxoisoindolines, polytriazines, polypyridazines, polypiperazines, polypyridines, polypiperidines, polytriazoles, polypyrazoles, polycarboranes, polyoxabicyclononanes, polydibenzofurans, polyphthalides, polyacetals, polyanhydrides, polyvinyl ethers, polyvinyl thioethers, polyvinyl alcohols, polyvinyl ketones, polyvinyl halides, polyvinyl nitriles, polyvinyl esters, polysulfonates, polysulfides, polythioesters, polysulfones, polysulfonamides, polyureas, polyphosphazenes, polysilazanes, polysiloxanes, or the like, or a combination thereof. Examples of thermosetting polymers include epoxy polymers, unsaturated polyester polymers, polyimide polymers, bismaleimide polymers, bismaleimide triazine polymers, cyanate ester polymers, vinyl polymers, benzoxazine polymers, benzocyclobutene polymers, acrylics, alkyds, phenol-formaldehyde polymers, novolacs, resoles, melamine-formaldehyde polymers, urea-formaldehyde polymers, hydroxymethylfurans, isocyanates, diallyl phthalate, triallyl cyanurate, triallyl isocyanurate, unsaturated polyesterimides, or the like, or a combination thereof. The composition may comprise a crosslinking agent to facilitate the formation of the thermosetting polymer. The crosslinking agent may be self-crosslinking, an acid or base activated crosslinking agent, a free-radical crosslinking agent, or a combination thereof. Suitable solvents include, for example: aliphatic hydrocarbons such as hexane and heptane; aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as dichloromethane, 1,2-dichloroethane and 1-chlorohexane, alcohols such as methanol, ethanol, 1-propanol, iso-propanol, tert-butanol, 2-methyl-2-butanol and 4-methyl-2-pentanol, propylene glycol monomethyl ether (PGME), ethers such as diethyl ether, tetrahydrofuran, 1,4-dioxane and anisole; ketones such as acetone, methyl ethyl ketone, methyl iso-butyl ketone, 2-heptanone and cyclohexanone (CHO); esters such as ethyl acetate, n-butyl acetate, propylene glycol monomethyl ether acetate (PGMEA), ethyl lactate (EL), hydroxyisobutyrate methyl ester (HBM) and ethyl acetoacetate; lactones such as gamma-butyrolactone (GBL) and epsilon-caprolactone; lactams such as N-methyl pyrrolidone; nitriles such as acetonitrile and propionitrile; cyclic or non-cyclic carbonate esters such as propylene carbonate, dimethyl carbonate, ethylene carbonate, propylene carbonate, diphenyl carbonate, and propylene carbonate; polar aprotic solvents such as dimethyl sulfoxide and dimethyl formamide; water; and combinations thereof. Of these, preferred solvents are PGME, PGMEA, EL, GBL, HBM, CHO, and combinations thereof. The total solvent content (i.e., cumulative solvent content for all solvents) in the composition is typically from 40 to 99 wt %, for example, from 70 to 99 wt %, or from 85 to 99 wt %, based on total weight of the photoresist composition. The desired solvent content will depend, for example, on the desired thickness of the coated photoresist layer and coating conditions. The composition is first manufactured by mixing together the matrix polymer, the surface leveling agent and any other solid components and optional additives, with solvent. The composition may be subjected to additional processes such as filtration, ion exchange, and the like, before being used. The composition can be applied to a substrate by spin-coating, dipping, roller-coating or other coating methods. The substrate may include electronic device substrates, metal, wood, paper, polymeric substrates or underlayers. The solids content of the coating solution may be varied to provide films of variable thicknesses. The solids content may also be based upon the specific coating equipment utilized, the viscosity of the solution, the speed of the coating tool and the amount of time used for the spinning. In an embodiment, the layer of the composition may be applied in a single application. In another embodiment, the layer of composition may be applied in multiple applications. Depending on the particular coating composition, it may be beneficial to soft-bake the composition layer (disposed on the substrate) to minimize the solvent content in the film. The soft-bake facilitates the formation of a tack-free coating and improves adhesion of the composition layer to the substrate. The soft-bake can be conducted on a hotplate or in an oven, or alternatively may be conducted using ultraviolet light or using lasers. In the case of the photoresist composition, the layer may then be exposed patternwise to permit irradiation through a photomask, via direct write or contact or via a pattern produced using light interference to create a difference in solubility between exposed and unexposed regions. This irradiation forms a latent image in the layer. The photomask has optically transparent and optically opaque regions that correspond to regions of the composition layer that are to be exposed or unexposed by the activating radiation. The composition layer may optionally undergo a postexposure bake process after which it is developed to provide a resist relief image. In an embodiment, an alkaline developing solution is used to remove exposed portions of the composition layer. Examples of the alkaline developing solution include aqueous solutions of tetramethyl ammonium hydroxide, sodium hydroxide and potassium hydroxide. The exposed portions can form a pattern such as a hole (e.g., contact, via or bump pattern) or trench (e.g., line-space) pattern. In one embodiment, the surface leveling agent is advantageous because it is miscible with water and aqueous developers. In another embodiment, the surface leveling agent displays an appropriate level of hydrophobicity and is not miscible with water and aqueous developers, which renders it miscible with an organic solvent developer. It does not contain fluorine and is therefore environmentally friendly. The surface leveling agent may be used as a wetting agent for improving flow control. These properties (surface leveling and wetting capability) provide good optical properties (e.g., high gloss and good distinctness of image) to coatings. The surface leveling agent can therefore be blended with a wide variety of solutions, waxes, polishes, coatings, blends, and the like. In an embodiment, the surface leveling agent may be used in floor polish formulations, painting, powder coating compositions, or the like. The invention will now be exemplified by the following non-limiting examples. Example Example 1 This prophetic example is conducted to demonstrate the synthesis of the monomeric repeat unit that is used in the copolymer (the surface leveling agent). The copolymer is manufactured using 3-butyloxymethyl-3-methyloxetane as one of the monomers and tetrahydrofuran and the other monomer. In other words, 3-butyloxymethyl-3-methyloxetane is used to manufacture the first polymeric unit of the copolymer and tetrahydrofuran is used to manufacture the second polymeric unit of the copolymer. The synthesis of the 3-butyloxymethyl-3-methyloxetane monomer is performed as follows. A dispersion of 50 weight percent (2.8 grams, 58.3 mmol) sodium hydride in mineral oil, is washed twice with hexane and suspended in 35 milliliters of dimethyl formamide. 3.9 grams (52.6 mmol) of butanol is then added to the dispersion and the mixture stirred for 45 minutes, A solution of 10.0 grams (39 mmol) of 3-hydroxymethyl-3-methyloxetane p-toluenesulfonate in 15 milliliters of dimethyl formamide is added and the mixture is heated at 80° C. for 20 hours, when1H-NMR analysis of an aliquot sample shows that the starting sulfonate is completely consumed. The mixture is then poured into 100 milliliters of ice water and extracted with 2 volumes of methylene chloride. The combined organic extracts are washed twice with water, twice with 2 weight percent aqueous hydrochloric acid, brine, dried over magnesium sulfate, and evaporated to give 3-(2,2,2-trifluoroethoxymethyl)-3-methyloxetane as an oil containing less than 1 weight percent dimethyl formamide. The oil is distilled under reduced pressure to give analytically pure 3-butyloxymethyl-3-methyloxetane monomer. The synthesis of the (3-methyloxetan-3-yl) methyl 3,3-dimethylbutanoate monomer is performed as follows. Into a 100 mL four-necked flask, 10.0 g of 3,3-dimethylbutanoic anhydride (46.7 mmol), 0.6 g of dimethylaminopyridine (4.7 mmol), are dissolved in 250 ml of dichloromethane. 6.0 g of 3-ethyl-3-hydroxymethyloxetane (51.4 mmol) is added slowly under ice water bath, and the reaction is left stirring at room temperature for 24 hours. The reaction mixture is then washed with saturated sodium bicarbonate aqueous solution, water and brine, and then dried over magnesium sulfate overnight. The solvent is removed and the oil is distilled under reduced pressure to give analytically pure (3-ethyloxetan-3-yl)methyl 3,3-dimethylbutanoate. Example 2 This prophetic example is conducted to demonstrate the synthesis of the polymer that is used as the surface leveling agent. The method of preparing a copolymer using a functional oxetane and tetrahydrofuran is detailed below. A 400 ml flask (fitted with a condenser, a thermocouple temperature probe and a mechanical stirrer) is charged with anhydrous methylene chloride (100 ml) and 1,4-butanediol (2.03 g, 22.6 mmoles). BF3THF (29.6 g, 211.7 moles), 2,2-dimethyl-1,3-propanediol (22.1 g, 211.7 mmol) is then added, and the mixture is stirred for 10 minutes. A solution of 3-butyloxymethyl-3-methyloxetane (67 g. 423.4 moles) in anhydrous methylene chloride (30 ml) is then pumped into the vessel over 5 hours. The reaction temperature is maintained between 38 and 42° C. throughout the addition. The mixture is then refluxed for an additional 2 hours (while simultaneously being stirred), after which1H-NMR indicated ≤98% conversion. The reaction is quenched with 10% aqueous sodium bicarbonate (200 ml) and the organic phase is washed with 3% aq. HCl (200 ml) and with water (200 ml). The organic phase is dried over sodium sulfate, filtered, and stripped of solvent under reduced pressure to give of (3-methyloxetan-3-yl) methyl 3,3-dimethylbutanoate monomer as a clear oil. Example 3 This prophetic example is conducted to determine the resist coating and development properties of the resist composition. The formulations R1-R4 (resist compositions) and CR1-CR2 (comparative resist compositions) are prepared with components and in amounts shown in Table 1. In Table 1, the number in the parenthesis indicates the weight ratio of each component. The structures represented by C1-2, D1-2, and S1-2 are depicted below the Table 2. It is to be noted that all of the polymers in Table 1 are prepared according to this general synthesis protocol. TABLE 1(In this Table 1, all the numbers in the parenthesis represent themole percent of the monomer in the copolymer). Monomer numbers (e.g.,Monomer 1) relate to the structures shown immediately above.Matrix ResinPolymerMonomer 1Monomer 2Monomer 3Monomer 4A11 (40%)2 (30%)3 (20%)4 (10%)A25 (40%)2 (40%)6 (20%) TABLE 2SurfaceResistlevelingCompo-PolymerAdditiveAdditiveagentSolventsition1 (grams)1 (grams)2 (grams)(grams)(grams)R1A1 [3.78]C1 [0.88]D1 [0.18]E1 [0.01]S1/S2[33.95/63.05]R2A2 [3.78]C1 [0.88]D1 [0.18]E1 [0.01]S1/S2[33.95/63.05]CR1A1 [3.78]C1 [0.88]D2 [0.18]E2 [0.01]S1/S2[33.95/63.05]CR2A2 [3.78]C2 [0.88]D2 [0.18]E2 [0.01]S1/S2[33.95/63.05] The structures for the additives (C1, C2, D1 and D2) and the solvents (S1 and S2) are shown immediately below. Polymer A1 and A2 have a weight average molecular weight of 8000 g/mole. E1 is the poly-3-(2,2,2-trifluoroethoxymethyl)-3-methyloxetane as described in the synthesis Example 2. E2 is PolyFox PF-656, which is a commercial material from Omnova Solution Incorporation Each formulation is made with the components shown in Table 2 and mixed together overnight (via stirring), then passed through 0.2 micrometer filter, spun coat on a wafer and then exposed to patterns of 65 nm/130 nm pitch line/space under an ASML 1100 scanner at increasing focus with an increasing dose and then post exposed baked (PEB) at 100° C. for 60 seconds. Following PEB, the wafers are developed in 0.26 N aqueous TMAH developer for 12 seconds, rinsed with distilled water and spun dry. Coating defect evaluation is performed on a spin-coated wafer of the compositions described above. After spin-coating the composition onto a hexamethyidisilazane (HDMS) primed silicon wafer, metrology is carried out on a Hitachi CG4000 CD-SEM and SP2 tool evaluation. Defect counts and haze value are measured for comparison. To evaluate patterning defects, immersion lithography is carried out with a TEL Lithius 300 mm wafer track and ASML 1900i immersion scanner at 1.3 NA (numerical aperture), 0.86/0.61 inner/outer sigma, and dipole illumination with 35Y polarization. Wafers for photolithographic testing are coated with 800 Å AR40A bottom antireflective coating (BARC) using a cure of 205° C./60 seconds. Over the AR40A layer is coated 400 Å of AR104 BARC using a cure of 175° C./60 sec. Over the BARC stack is coated 900 Å of photoresist using a 90° C./60 second soft bake. Wafers are exposed to patterns of 55 nm/110 nm pitch line/space at increasing focus and increasing dose and then post exposure baked (PEB) at 100° C./60 seconds. Following PEB, wafers are developed in 0.26 N aqueous TMAH developer for 12 sec, rinsed with distilled water, and spun dry. Metrology is then carried out on a Hitachi CG4000 CD-SEM and defect numbers are counted for comparison. It is expected that the surface leveling agent described in this invention can be advantageously used to produce photoresist compositions that do not contain a large number of coating and patterning defects. | 32,028 |
11859083 | DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS The present disclosure is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Like numbers in the drawings indicate like components throughout the views. As used in the description herein and throughout the claims that follow, unless the context clearly dictates otherwise, the meaning of “a”, “an”, and “the” includes plural reference, and the meaning of “in” includes “in” and “on”. Titles or subtitles can be used herein for the convenience of a reader, which shall have no influence on the scope of the present disclosure. The terms used herein generally have their ordinary meanings in the art. In the case of conflict, the present document, including any definitions given herein, will prevail. The same thing can be expressed in more than one way. Alternative language and synonyms can be used for any term(s) discussed herein, and no special significance is to be placed upon whether a term is elaborated or discussed herein. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms is illustrative only, and in no way limits the scope and meaning of the present disclosure or of any exemplified term. Likewise, the present disclosure is not limited to various embodiments given herein. Numbering terms such as “first”, “second” or “third” can be used to describe various components, signals or the like, which are for distinguishing one component/signal from another one only, and are not intended to, nor should be construed to impose any substantive limitations on the components, signals or the like. [Low-Dielectric Rubber Resin Material] A low-dielectric rubber resin material (or a rubber resin material for short) of the present disclosure contains a liquid rubber. By controlling properties of the liquid rubber, an amount of the liquid rubber added in the rubber resin material can be higher than an upper limit of the amount of the liquid rubber allowable in the conventional technology. Therefore, the rubber resin material of the present disclosure is more suitable to be used as a high frequency substrate material. Specifically, the rubber resin material of the present disclosure includes a low-dielectric rubber resin composition (or a rubber resin composition for short) and inorganic fillers. The inorganic fillers are uniformly dispersed in the rubber resin composition. Specific properties of the rubber resin composition and the inorganic fillers will be illustrated below. [Low-Dielectric Rubber Resin Composition] The rubber resin composition of the present disclosure includes: 5 wt % to 40 wt % of the liquid rubber, 20 wt % to 70 wt % of a polyphenylene ether resin, 5 wt % to 30 wt % of a bismaleimide resin, and 20 wt % to 45 wt % of a crosslinker. Through the aforesaid components and contents, the rubber resin composition of the present disclosure can be used to manufacture a low-dielectric metal substrate (or a metal substrate for short) that has good dielectric properties and a good thermal resistance. In addition, the metal substrate can have a strong adhesive force with a metal layer (i.e., having an appropriate peeling strength). Property tests for the metal substrate will be illustrated below. When the liquid rubber has a molecular weight ranging from 800 g/mol to 6000 g/mol, flowability of the rubber resin composition can be enhanced. Accordingly, a glue filling property of the rubber resin composition can also be enhanced. Preferably, the molecular weight of the liquid rubber ranges from 800 g/mol to 5500 g/mol. It is worth mentioning that in the present disclosure, an amount of the liquid rubber in the rubber resin composition can be increased due to control of the molecular weight and the iodine value of the liquid rubber. Specifically, based on a total weight of the rubber resin composition being 100 wt %, the amount of the liquid rubber is higher than 25 wt %. In an exemplary embodiment, the rubber resin composition contains 25 wt % to 40 wt % of the liquid rubber. In certain embodiments, the liquid rubber includes a liquid diene rubber. Specifically, the liquid diene rubber includes a polybutadiene resin. The polybutadiene resin is a polymer polymerized from butadiene monomers, such as a butadiene homopolymer or a copolymer formed from butadiene and other monomers. In an exemplary embodiment, the liquid diene rubber is a copolymer formed from butadiene and styrene. In other words, monomers forming the liquid rubber include styrene and butadiene. A styrene monomer and a butadiene monomer can be randomly arranged forming a random copolymer, or can be regularly arranged forming an alternating copolymer or a block copolymer. Based on a total weight of the liquid rubber being 100 wt %, an amount of the styrene monomer ranges from 10 wt % to 50 wt %. When the liquid rubber contains 10 wt % to 50 wt % of the styrene monomer, the adhesive force between the metal substrate and the metal layer (i.e., the peeling strength of the metal substrate) can be enhanced. Preferably, the liquid rubber contains 15 wt % to 50 wt % of the styrene monomer. When the amount of the styrene monomer is higher than 50 wt %, since the styrene monomer has no reactive groups to be crosslinked, a crosslinking degree of the rubber resin material will be decreased, thereby negatively influencing the thermal resistance of the metal substrate. Specifically, the butadiene monomer has two double bonds. Hence, different ways of polymerizing the butadiene monomer can result in different structures of the polybutadiene resin. In other words, the polybutadiene resin can include one or more structures of: cis-1, 4-polybutadiene, trans-1, 4-polybutadiene, and 1, 2-polybutadiene. When the butadiene is polymerized through a 1, 4-addition reaction, the structure of cis-1, 4-polybutadiene or trans-1, 4-polybutadiene can be formed. In the structure of cis-1, 4-polybutadiene or trans-1, 4-polybutadiene, neither cis-1, 4-polybutadiene nor trans-1, 4-polybutadiene has an unsaturated side chain. When the butadiene is polymerized through a 1, 2-addition reaction, the structure of 1, 2-polybutadiene can be formed. In the structure of 1, 2-polybutadiene, 1, 2-polybutadiene has an unsaturated side chain (such as an ethylene group). In an exemplary embodiment, based on a total weight of the butadiene monomers being 100 wt %, 30 wt % to 90 wt % of the butadiene monomers (after being polymerized) have a side chain containing an ethylene group. Preferably, based on the total weight of the butadiene monomers being 100 wt %, 30 wt % to 80 wt % of the butadiene monomers (after being polymerized) have the side chain containing an ethylene group. When the liquid rubber has at least one unsaturated side chain containing an ethylene group (or an ethylene side chain), a crosslinking density and a thermal resistance of the rubber resin composition after being crosslinked can be enhanced. In the present disclosure, an amount of the unsaturated side chain containing an ethylene group (or an ethylene side chain) in the liquid rubber can be quantified by an iodine value through a chemical analysis. The higher the amount of the unsaturated side chain containing an ethylene group (or an ethylene side chain) is, the higher the iodine value of the liquid rubber is. Physical properties of the rubber resin composition after being crosslinked can be enhanced by the unsaturated side chain containing an ethylene group (or an ethylene side chain). Specific measurements of the iodine value of the liquid rubber will be illustrated below. In the present disclosure, a molecular weight of the polyphenylene ether resin ranges from 1000 g/mol to 20000 g/mol. Preferably, the molecular weight of the polyphenylene ether resin ranges from 2000 g/mol to 10000 g/mol. More preferably, the molecular weight of the polyphenylene ether resin ranges from 2000 g/mol to 2200 g/mol. When the molecular weight of the polyphenylene ether resin is lower than 20000 g/mol, a solubility of the polyphenylene ether resin in a solvent can be enhanced, which is advantageous for preparing the rubber resin composition. In an exemplary embodiment, the polyphenylene ether resin can have at least one modified group. The modified group can be selected from the group consisting of: a hydroxyl group, an amino group, an ethylene group, a styrene group, a methacryl group, and an epoxy group. The modified group of the polyphenylene ether resin can provide an unsaturated bond, so as to facilitate a crosslinking reaction. In this way, a material that has a high glass transition temperature and a good thermal resistance can be obtained. In the present embodiment, two molecular ends of the polyphenylene ether resin each have the modified group, and the two modified groups are the same. In an exemplary embodiment, the polyphenylene ether resin can include one kind of polyphenylene ether or various kinds of polyphenylene ether. For example, the polyethylene ether can be a polyphenylene ether that has two hydroxyl modified groups at molecular ends thereof, a polyphenylene ether that has two methacryl modified groups at molecular ends thereof, a polyphenylene ether that has two styrene modified groups at molecular ends thereof, or a polyphenylene ether that has two epoxy modified groups at molecular ends thereof. However, the present disclosure is not limited thereto. In certain embodiments, the polyphenylene ether resin includes a first polyphenylene ether and a second polyphenylene ether. Molecular ends of both the first polyphenylene ether and the second polyphenylene ether each have at least one modified group. The modified group can be selected from the group consisting of: a hydroxyl group, an amino group, an ethylene group, a styrene group, a methacryl group, and an epoxy group. In addition, the modified group of the first polyphenylene ether and the modified group of the second polyphenylene ether can be different from each other. Specifically, a weight ratio of the first polyphenylene ether to the second polyphenylene ether ranges from 0.5 to 1.5. Preferably, the weight ratio of the first polyphenylene ether to the second polyphenylene ether ranges from 0.75 to 1.25. More preferably, the weight ratio of the first polyphenylene ether to the second polyphenylene ether is 1. For example, the first polyphenylene ether and the second polyphenylene ether can each be one of the polyphenylene ether having two hydroxyl modified groups at the molecular ends thereof, the polyphenylene ether having two methacryl modified groups at the molecular ends thereof, the polyphenylene ether having two styrene modified groups at the molecular ends thereof, and the polyphenylene ether having two epoxy modified groups at the molecular ends thereof. However, the present disclosure is not limited thereto. In the present disclosure, an average molecular weight of the bismaleimide resin ranges from 500 g/mol to 4500 g/mol. Preferably, the average molecular weight of the bismaleimide resin ranges from 500 g/mol to 3500 g/mol. More preferably, the average molecular weight of the bismaleimide resin ranges from 500 g/mol to 3000 g/mol. The bismaleimide resin has at least two functional groups, such that the peeling strength of the metal substrate can be enhanced. For example, the bismaleimide resin can be bis(3-ethyl-5-methyl-4- maleimidophenyl)methane (e.g., the model KI-70 produced by KI Chemical Industry Co., LTD. and the model BMI-5100 produced by Daiwakasei Industry Co., LTD.), (4,4′-methylene diphenyl)bismaleimide (e.g., the model BMI-1000, BMI-1000H, BMI-1000S, BMI-1100, or BMI-1100H produced by Daiwakasei Industry Co., LTD.), phenylmaleimide oligomers (e.g., the model BMI-2000 or BMI-2300 produced by Daiwakasei Industry Co., LTD.), m-phenylene bismaleimide (e.g., the model BMI-3000 or BMI-3000H produced by Daiwakasei Industry Co., LTD.), bisphenol A diphenyl ether bismaleimide (e.g., the model BMI-4000 produced by Daiwakasei Industry Co., LTD.), 3,3′-dimethyl-5,5′-diethyl-4,4′-diphenylethane bismaleimide (e.g., the model BMI-5100 produced by Daiwakasei Industry Co., LTD.), (4-methyl-1, 3-phenylene) bismaleimide (e.g., the model BMI-7000 or BMI-7000H produced by Daiwakasei Industry Co., LTD), or 1,6-bismaleimide-(2,2,4-trimethyl)hexane (e.g., the model BMI-TMH produced by Daiwakasei Industry Co., LTD.). However, the present disclosure is not limited thereto. The crosslinker of the present disclosure can enhance a crosslinking degree of the polyphenylene ether resin and the liquid rubber. In the present embodiment, the crosslinker can include an allyl group. For example, the crosslinker can be triallyl cyanurate (TAC), triallyl isocyanurate (TRIC), diallyl phthalate, divinylbenzene, triallyl trimellitate, or any combination thereof. Preferably, the crosslinker can be triallyl isocyanurate. However, the present disclosure is not limited thereto. [Inorganic Fillers] An addition of the inorganic fillers can help decrease a viscosity of the rubber resin material and decrease a dielectric constant of the rubber resin material. For example, the inorganic fillers can be silicon dioxide, titanium dioxide, aluminum hydroxide, aluminum oxide, magnesium hydroxide, magnesium oxide, calcium carbonate, boron oxide, calcium oxide, strontium titanate, barium titanate, calcium titanate, magnesium titanate, boron nitride, aluminum nitride, silicon carbide, cerium oxide, or any combination thereof. However, the present disclosure is not limited thereto. In an exemplary embodiment, the inorganic fillers include silicon dioxide. The silicon dioxide can be fused silica or crystalline silica. Preferably, the silicon dioxide is fused silica. An appearance of the inorganic fillers can be spherical. An average particle size of the inorganic fillers ranges from 0.3 μm to 30 μm. The particle size of the inorganic fillers is within a range between 0.3 μm and 30 μm, such that the inorganic fillers can be uniformly dispersed in the rubber resin composition. In an exemplary embodiment, a purity of the inorganic fillers is higher than or equal to 99.95%. In other words, an amount of metal impurities in the inorganic fillers is lower than or equal to 500 ppm. Specifically, an amount of calcium element in the inorganic fillers is lower than or equal to 200 ppm, an amount of aluminum element in the inorganic fillers is lower than or equal to 200 ppm, and an amount of iron element in the inorganic fillers is lower than or equal to 100 ppm. When the purity of the inorganic fillers is higher than or equal to 99.95%, a dielectric dissipation factor of the metal substrate can be maintained to be lower than or equal to 0.002 (10 GHz). Preferably, the dielectric dissipation factor of the metal substrate is lower than or equal to 0.0018. An amount of the inorganic fillers can be adjusted according to product requirements. In certain embodiments, based on the total weight of the rubber resin composition being 100 phr, the amount of the inorganic fillers ranges from 30 phr to 250 phr. Preferably, based on the total weight of the rubber resin composition being 100 phr, the amount of the inorganic fillers ranges from 30 phr to 200 phr. More preferably, based on the total weight of the rubber resin composition being 100 phr, the amount of the inorganic fillers ranges from 30 phr to 100 phr. However, the present disclosure is not limited thereto. [Property Test] In order to prove that the rubber resin material can be used as the high frequency substrate material, 5 wt % to 40 wt % of the liquid rubber, 20 wt % to 70 wt % of the polyphenylene ether resin, 5 wt % to 30 wt % of the bismaleimide resin, and 20 wt % to 45 wt % of the crosslinker are mixed to form the rubber resin composition. In addition, the inorganic fillers are further added into the rubber resin composition, so as to form the rubber resin material of Examples 1 to 6 and Comparative Examples 1 to 3. Specific contents of the rubber resin material of Examples 1 to 6 and Comparative Examples 1 to 3 are listed in Table 1. In Table 1, the liquid rubber can be a butadiene/styrene copolymer A, a butadiene/styrene copolymer B, a butadiene/styrene copolymer C, or a butadiene homopolymer. Specific properties of the butadiene/styrene copolymer A, the butadiene/styrene copolymer B, the butadiene/styrene copolymer C, and the butadiene homopolymer are listed in Table 2. The iodine value of the liquid rubber in each of Examples 1 to 6 and Comparative Examples 1 to 3 is listed in Table 1. In order to measure the iodine value of the liquid rubber, 0.3 mg to 1 mg of the liquid rubber is completely dissolved in chloroform, and is placed in the dark for 30 minutes after a Wijs solution is added thereinto. Next, 20 ml of a potassium iodide solution (100 g/L) and 100 ml of water are added to form an analyte. Subsequently, the analyte is titrated by a sodium thiosulfate solution (0.1 mol/L) which is used as a titrant. When a color of the analyte becomes light yellow, a few drops of a starch solution are dripped into the analyte. Then, the analyte is further titrated until a blue color of the analyte disappears. In Table 1, the polyphenylene ether resin refers to a polyphenylene ether resin produced by Saudi Basic Industries Corporation (SABIC) as the model SA9000, and the SA9000 polyphenylene ether resin has methacryl groups at two molecular ends thereof. The bismaleimide resin is bis(3-ethyl-5-methyl-4-maleimidphenyl)methane, such as the model KI-70 produced by KI Chemical Industry Co., LTD. or the model BMI-5100 produced by Daiwakasei Industry Co., LTD. The crosslinker is triallyl isocyanurate (TRIC). In Table 1, three kinds of the inorganic fillers that have different purities are used. The three kinds of the inorganic fillers have 85 ppm, 168 ppm, and 868 ppm of metal impurities, respectively. The metal impurities include the aluminum element, the calcium element, and the iron element. Subsequently, a glass fiber cloth produced by Nan Ya Plastics Corporation as the model 1078 is immersed into the rubber resin material in each of Examples 1 to 6 and Comparative Examples 1 to 3. After immersion, drying, and molding, a prepreg is obtained. After the prepreg is processed, a metal layer is disposed on the prepreg, so as to form the metal substrate of Examples 1 to 6 and Comparative Examples 1 to 3. Properties of the metal substrate of Examples 1 to 6 and Comparative Examples 1 to 3 are listed in Table 3. In Table 3, the properties of the metal substrate are measured by methods below.(1) Dielectric constant (10 GHz): detecting a dielectric constant of the metal substrate at 10 GHz by a dielectric analyzer (model: HP Agilent E5071C).(2) Dielectric dissipation factor (10 GHz): detecting the dielectric dissipation factor of the metal substrate at 10 GHz by the dielectric analyzer (model: HP Agilent E5071C).(3) Peeling strength: measuring the peeling strength of the metal substrate according to the IPC-TM-650-2.4.8 test method.(4) Thermal resistance: heating the metal substrate in an autoclave at a temperature of 120° C. and a pressure of 2 atm for 120 minutes, and then putting the said metal substrate into a soldering furnace of 288° C., so as to record the time needed for delamination. TABLE 1ExampleComparative ExampleUnit: phr123456123Rubber resinLiquidButadiene/styrene303000101530030compositionrubbercopolymer AButadiene/styrene0030000000copolymer BButadiene/styrene000302015000copolymer CButadiene0000000300homopolymerIodine value383934363537283536(g/100 g)Polyphenylene ether404040404040454040resin (SA9000)Crosslinker (TAIC)202020202020252020BismaleimideKI-701001010101001010resinBMI-51000100000000InorganicSilicon dioxide40404040204040400fillers(impurity: 168 ppm)Silicon dioxide0000200000(impurity: 85 ppm)Silicon dioxide0000000040(impurity: 868 ppm) TABLE 2Amount of the butadienemonomers (after beingAmount ofpolymerized) having aMolecularstyreneside chain containingLiquid rubberweightmonomeran ethylene groupButadiene/styrene5300 g/mol35 wt %70 wt %copolymer AButadiene/styrene4500 g/mol20 wt %70 wt %copolymer BButadiene/styrene3200 g/mol28 wt %30 wt %copolymer CButadiene3900 g/mol0 wt %70 wt %homopolymer TABLE 3ExampleComparative Example123456123MetalDielectric constant3.393.283.283.313.303.223.283.313.48substrate(10 GHz)Dielectric dissipation1.61.71.71.71.61.61.81.72.5factor (10 GHz) × 103Peeling strength (lb/in)6.26.16.36.56.66.54.34.56.2Thermal resistanceOKOKOKOKOKOKOKOKOK According to results in Table 1 and Table 3, by controlling contents of the liquid rubber, the polyphenylene ether resin, the bismaleimide resin, and the crosslinker, the metal substrate of Examples 1 to 6 can have good dielectric properties, a good peeling strength, and a good thermal resistance. Even when the rubber resin composition contains a high content (higher than 25 wt %) of the liquid rubber, the metal substrate of the present disclosure can still have a good peeling strength. Specifically, in the present disclosure, the iodine value of the liquid rubber ranges from 30 g/100 g to 60 g/100 g. Preferably, the iodine value of the liquid rubber ranges from 30 g/100 g to 50 g/100 g. More preferably, the iodine value of the liquid rubber ranges from 30 g/100 g to 40 g/100 g. Specifically, in the present disclosure, the dielectric constant (10 GHz) of the metal substrate is lower than or equal to 3.5. Preferably, the dielectric constant (10 GHz) of the metal substrate ranges from 2.5 to 3.5. More preferably, the dielectric constant (10 GHz) of the metal substrate ranges from 3.0 to 3.5. The dielectric dissipation factor (10 GHz) of the metal substrate is lower than or equal to 0.002. Preferably, the dielectric dissipation factor (10 GHz) of the metal substrate is lower than or equal to 0.0018. More preferably, the dielectric dissipation factor (10 GHz) of the metal substrate is lower than or equal to 0.0017. The peeling strength of the metal substrate ranges from 5.5 lb/in to 7 lb/in. Preferably, the peeling strength of the metal substrate ranges from 6 lb/in to 7 lb/in. According to Comparative Examples 1 and 2, when the bismaleimide resin is absent from the rubber resin composition (Comparative Example 1) or the styrene monomer is absent from the liquid rubber (Comparative Example 2), the reactivity of the rubber resin composition is decreased, thereby negatively influencing the peeling strength of the metal substrate. In Comparative Example 1, the rubber resin composition does not contain the bismaleimide resin, which causes the metal substrate to have a low peeling strength. In Comparative Example 2, the liquid rubber only contains the butadiene homopolymer (RICON® 150) and does not contain the styrene monomer, thereby causing the peeling strength of the metal substrate to be low. An addition of the silicon dioxide can not only enhance the dielectric constant of the metal substrate but can also influence the dielectric dissipation factor of the metal substrate. According to Comparative Example 3, when the amount of the metal impurities in the inorganic fillers is higher than 500 ppm, the dielectric dissipation factor of the metal substrate is increased due to the metal impurities. Accordingly, the rubber resin material is not suitable to be used as a high frequency transmission material. [Beneficial Effects of the Embodiments] In conclusion, in the low-dielectric rubber resin material and the low-dielectric metal substrate provided by the present disclosure, by virtue of “a molecular weight of the liquid rubber ranging from 800 g/mol to 6000 g/mol” and “an iodine value of the liquid rubber ranging from 30 g/100 g to 60 g/100 g”, the rubber resin material can be used as the high frequency transmission material. Further, by virtue of “adding the bismaleimide resin” and “monomers forming the liquid rubber including a styrene monomer”, the peeling strength of the metal substrate can be enhanced. Moreover, by virtue of “a purity of the inorganic fillers being higher than or equal to 99.95%” and “an amount of metal impurities in the inorganic fillers being lower than or equal to 500 ppm”, the dielectric dissipation factor of the metal substrate can be decreased. The foregoing description of the exemplary embodiments of the disclosure has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to explain the principles of the disclosure and their practical application so as to enable others skilled in the art to utilize the disclosure and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present disclosure pertains without departing from its spirit and scope. | 25,266 |
11859084 | EXAMPLES 1. Components used Commercial polymers (pellets): Polyamides (component A): Nylon-6,6 (PA 6,6-GR): Ultramid® A27 (from BASF AG, Germany) Further polymers (component C): Polyester elastomer (TPE-E): Hytrel® 4056 (from DuPont, USA) Polyethylene terephthalate: Polyclear® 1101 (from Invista, Germany) Polybutylene terephthalate: Ultradur® B4500 (from BASF, Germany) Polystyrene: Styrolution PS 454 N natural (from Ineos, Germany) ABS: Novodur P2-H (from Styrolution, Germany) Polyglycol: Polyglycol 8000 P (from Clariant, Germany) Polycarbonate: Makrolon 3208 (from Covestro, Germany) Polycaprolactone: PC Resin 2-2 (from Changxing, China) Component F: PPG HP 3610 EC 10 4.5 mm glass fibers (from PPG Ind. Fiber Glass, the Netherlands) Flame retardant (component B): Aluminum salt of diethylphosphinic acid, referred to hereinafter as Depal Further flame retardants: Component D: Aluminum salt of phosphorous acid, referred to hereinafter as Phopal Component E: Melamine polyphosphate (Melapur® 200/70, (from BASF, Germany), referred to as MPP Compatibilizer (component G): Zemac® E60, polyethylene with maleic anhydride (Vertellus, USA) Nexamitee® 56 (Nexam Chemicals, Sweden) Nexamite® A99 (Nexam Chemicals, Sweden) Licocene® PE MA 4351 (Clariant, Germany) Lotader® AX 8700 (Arkema, France) Trimellitic anhydride Phosphonites (component H): Sandostab® P-EPQ, from Clariant GmbH, Germany Wax components (component F)): Licowax® E, from Clariant Produkte (Deutschland) GmbH, Germany (esters of montan wax acid), component I) 2. Production, processing, and testing of flame-retardant polymer molding compounds The flame retardant components were mixed with the phosphonite, lubricants, and stabilizers in the ratio specified in the table and incorporated via the side intake of a twin-screw extruder (Leistritz ZSE 27/44D) into PA 6,6 at temperatures of 260 to 310° C., and into PPA at 300-340° C. The glass fibers were added via a second side intake. The homogenized polymer strand was drawn off, cooled in a water bath, and then pelletized. After sufficient drying, the molding compounds were processed into test specimens on an injection-molding machine (Arburg 320 C Allrounder) at melt temperatures of 250 to 340° C., and tested and classified for flame retardancy using the UL 94 test (Underwriter Laboratories). The UL 94 fire classifications are as follows:V-0: Afterflame time never longer than 10 sec, total of afterflame times for 10 flame applications not more than 50 sec, no flaming drops, no complete consumption of the specimen, afterglow time for specimens never longer than 30 sec after end of flame application.V-1: Afterflame time never longer than 30 sec after end of flame application, total of afterflame times for 10 flame applications not more than 250 sec, afterglow time for specimens never longer than 60 sec after end of flame application, remaining criteria as for V-0.V-2 Cotton indicator ignited by flaming drops, remaining criteria as for V-1 Not classifiable (ncl): Does not conform to fire classification V-2. The flowability of the molding compounds was determined by establishing the melt volume flow rate (MVR) at 275° C./2.16 kg. A sharp rise in the MVR value indicates polymer degradation. Fillers also have an influence on the MVR. Tensile strength (N/mm2), elongation at break, and tear strength were measured according to DIN EN ISO 527 (%); impact resistance [kJ/m2], and notched impact strength [kJ/m2] were measured according to DIN EN ISO 179. The change in color after water storage was determined by storing 1 and 3 mm thick plates semi-immersed in water for seven days. The color (Lab values) was then measured in accordance with DIN 6174 using a CM 3600d spectrophotometer with white and black standards and measuring apertures, and the results were evaluated using SpectraMagic NX software (from Minolta Europe GmbH, Germany). For comparability, all tests in the respective series, unless stated otherwise, were performed under identical conditions (temperature programs, screw geometry, injection molding parameters, etc.). All amounts are reported as % by weight and are based on the polymer molding compound including the flame retardant combination and additives. Table 1 shows polyamide molding compounds that contain component A) and component B) as a flame retardant mixture. These show clearly measurable corrosion. All amounts are reported as % by weight and are based on the polymer molding compound including the flame retardant combination and additives. TABLE 1PA 66 GF30 V-0 with phosphinates. C1 and C2 are comparative exampleswithout addition of PET; I1 and I2 are inventive examplesExampleC1I1C2I2Nylon 6647.637.647.637.6Glass fibers30303030Depal13.313.316.416.4MPP6.76.7Zinc borate11Phopal3.63.6Polyethylene terephthalate1010Carbon black masterbatch 30% in PA 62222Licowax E0.30.30.30.3PEP-Q0.10.10.10.1Color before storage in waterblackblackblackblackL value26.926.525.726.4Color after storage in watergrayblackgrayblackL value33.327.234.227.2Delta Eab (D65) after 7 days in water6.30.28.50.8UL 94 0.4 mmV-0V-0V-0V-0UL 94 0.8 mmV-0V-0V-0V-0UL 94 1.6 mmV-0V-0V-0V-0E modulus/N/mm210901.5511280.2510858.9111358.64Tensile strength/N/mm2143.02133.72144.11138.33Elongation at break/%2.532.163.092.21 Comparative examples C1and C2show that flame-retardant polyamide 66 GF30 compounds undergo a change in color after storage in water for seven days at room temperature. This is observed both when using Depal together with melamine polyphosphate and when using Depal with Phopal. Examples I1 and I2 show that the addition of 10% polyethylene terephthalate almost completely eliminates the change in color after storage in water. At the same time, the UL 94 V-0 fire classification is maintained and the mechanical values are at a high level. Shaped bodies according to the invention have high surface quality, are easy to process, and are resistant to thermal aging. Example 3 shows that addition of PET achieves good color stability, even when using a compatibilizer. This also improves the phase compatibility of PA 66 and PET, which leads to higher mechanical values. TABLE 2PA66/PET GF 30 V-0 with and without compatibilizerExampleC3I3Nylon 665644Depal1212ZeMac E60-P EMA copolymer2Glass fibers3030Carbon black masterbatch (30% carbon22black in PA 6)Polyethylene terephthalate10dEab (D65) 7 days in water3.390.21 TABLE 3PA66/PET GF 30 V-0, I4-6: Variation of the PETcontent, C4 with polystyrene instead of PETExampleC4I4I5I6PA 6646525046Depal12121212Glass fibers30303030Carbon black masterbatch (30% in PA 6)2222PET4610PS10dE*ab (D65) 7 days in water3.650.160.160.15 Examples I4 and I5 show that addition of even a relatively small amount of PET results in there being no change in color after storage in water. Example 7 shows that addition of PBT and of a polyester elastomer likewise results in less change in color after storage in water. On the other hand, comparative examples C4and C5show that the addition of polystyrene or polyethylene glycol has no influence on the change in color after storage in water. TABLE 4Addition of polyglycol, polyester elastomer, and PBTExampleC5I7I8C6PA 6656464646Depal12121212Glass fibers30303030Carbon black masterbatch (30% in PA 6)2222Polyglycol10PBT10Polyester elastomer10dE*ab (D65)4.150.320.154.81 TABLE 5Addition of PC, ABS, SEBS, and polycaprolactoneExampleC7C8C9C10I9PA 664646464646Depal1212121212Glass fibers3030303030Carbon black masterbatch22222(30% in PA 6)ABS Novodur P2H-AT10PC Makrolon 320810Polycaprolactone10Lotader AX 87005SEBS Kraton FG 1901 GT10PET Polyclear 33005dE*ab (D65)3.251.281.61.920.18Surface qualitygoodmoderatemoderategoodgood Example I9 shows that no change in color is observed after storage in water even when PET and a compatibilizer (Lotader®) are added. With the addition of the other polymers, on the other hand, a discoloration after storage in water is observed as well as a deterioration in surface quality, recognizable by glass fibers at the surfaces (“cloudy” surfaces). | 8,076 |
11859085 | DESCRIPTION There are many examples of synthetic based polymer plastics and variations thereof that we rely for their various useful mechanical properties and in many cases, these compositions have an adverse effect on the environment not only in the production processing but at their end of life usefulness. The Nation's ability to dispose of these plastics is increasingly costly and difficult. PP (Polypropylene) is stiffer than other polymers and is more resistant to heat and widely used in hot food containers, car parts, disposable diapers and thermal vests to name a few. PET (Polyethylene Terephthalate) is primarily used for food and drink packaging due to its ability to prevent oxygen from permeating the material to spoil the product inside and its malleability. Although PET is most likely to be picked up by recycling programs this type of plastic contains antimony trioxide—a matter that is considered as a carcinogen—capable of causing cancer in a living tissue. In addition as noted further, while PET is highly recyclable, global changes in exporting plastic waste poses a National threat given machinery and processing resources to recycle it. Consequently much of this and other polymer plastic materials end up in landfills taking up to hundreds or more years to decompose while emitting harmful methane (CH4) and Carbon Dioxide (CO2) gases. According to the Environmental Protection Agency (EPA) landfill gas is comprised of approximately 50% methane and 50% Carbon Dioxide. Methane is a greenhouse gas up to 35 times as potent as carbon dioxide as a driver of climate-change over the span of a century. Landfills are the United States' third largest source of methane emissions. HDPE (High Density Polyethylene) has long, virtually unbranched polymer chains which makes it very dense and thus, stronger and thicker than PET. HDPE is commonly used as grocery bags, juice containers, medicine bottles and garden planters. It is known the aforementioned items in their category commonly have a limited product-lifecycle or are simply intended for single use such as food and pet packaging, utensils, straws and others which constitute about 40% of all plastic waste. While consumer goods companies and retailers commit to increasing recycled content in their packaging to an average of 25% by 2025 (compared with the current global average of just 2%), less than 5% of US plastics are recycled with the remainder landfilled, burned or exported. According to a report published by Science Advances, only 9% of all plastic ever produced has been recycled with the majority ending up in landfills or the natural environment. Plastics are made with oil so when oil prices fall, it is inherently cheaper to make fresh plastic. Also in this case using recycled plastic can be more expensive because it has to be sorted and cleaned. In January 2018 China implemented The National Sword Policy resulting in an estimated 111 million metric tons of plastic waste that will be displaced by 2030. Other countries including India, Indonesia, and Hong Kong have since followed China's lead in limiting the export of plastic waste to their countries. 89% of historical exports consist of polymer groups often used in single-use plastic food packaging (polyethylene, polypropylene, and polyethylene terephthalate). The U.S. alone produced 26 million metric tons of plastic waste in 2016 alone. Plastic recycle and processing backups have been reported in multiple international countries further contributing to the importance of the invention and attributes disclosed herein. Plastic PET bottles are the primary source for recyclers. An investment of at least $3 billion in U.S. processing plants alone will be needed over the next decade to achieve companies' goals to increase recycled material content according to a report by research and analysis firm Wood Mackenzie, which does not include additional investments in collection or improved material recovery facilities to manage the magnitude of plastic waste otherwise destined to the landfill. The price of virgin PET is cheaper than recycled PET for the first time in decades. It is also worth noting the cost of producing virgin plastic relies on the cost of petroleum and natural gas which continually fluctuate (in addition to cost of disposal based on supply and demand), therefore creating fluid economic scale of plastic product production when their product wholesale prices remain generally the same as does overhead manufacturing production costs. This potentially also leads to a detrimental imbalance of industry which can be minimized or avoided through the adoption of the hemp and PBAT biopolymer invention. According to The National Geographic Society in their first study in 2018, 91% of all plastic waste isn't recycled and only 12% has been incinerated. Of the 8.3 billion metric tons that has been produced, 6.3 billion metric tons has become plastic waste. Up to six times more plastic was burned in the U.S. in 2018 than was recycled. Burning plastic produces CO2 and can release toxins. The U.S. has seen only one new incinerator since 1997. In 2016, U.S. waste incinerators released the equivalent of 12 million tons of carbon dioxide, more than half of which came from plastics. According to a 2019 report, Plastic & Climate: The Hidden Costs of a Plastic Planet, globally burning plastic packaging adds 16 million metric tons of greenhouse gases into the air, which is equivalent to more than 2.7 million homes' electricity use for one year, and clearly shows incinerating plastic creates the most CO2 emissions among any plastic waste management method. Carbon dioxide emissions mainly come from burning organic materials such as coal, oil, gas, wood, and solid waste. Since most packaging includes some level of polypropylene which uses fossil fuels such as crude oil, plastic is the largest contributing offender to emitting carbon dioxide or methane gases both when incinerated and left to degrade under their natural cycle taking tens if not hundreds or thousands of years. Due to synthetic polymer mechanical attributes or other additives that make up the many products we rely on today (PP, PET, HDPE and others) several additives have been designed to break down synthetically formulated compounds including the polypropylene polymers. This is commonly known as “OXO” biodegradable or “degradable” as micro plastics that cannot be seen by the human eye are created and still exist upon degradation and contribute to polluting our environment and oceans. Micro plastics can come from a variety of sources including larger plastic pieces that have broken apart, resin pellets used for plastic manufacturing, or in the form of microbeads. Of the 260 million tons of plastic the world produces each year, about 10 percent ends up in the Ocean, according to a Greenpeace report. Seventy percent of the mass eventually sinks, damaging life on the seabed. As of 2020, scientists calculate that the top 200 meters of ocean alone contains up to 21 million metric tons of micro plastic which does not include micro fibers. While the present invention presents many advantages over other polymer and biopolymer plastics and alternatives, one of the more prominent advantages in the unique invention described herein is the use of natural and sustainable composition as a plastic alternative resulting in zero emissions of harmful gases currently created from landfill plastic waste and requires no modifications to standard manufacturing or waste management disposal processing nor facility equipment. An additional prominent feature is the biodegradability and compostability of the composition that creates no micro plastic residue and the end of a products useful life. In addition, the advantages of a composition that contains zero petroleum polymers while still meeting the needs of various plastic product attributes for rigidity, form, and weight, is also naturally UV and fire resistant. There are many other unique advantages of the invention described herein such as the abundance of limestone and hemp in the United. States and other countries. Hemp, for example, is grown in mixed crop farming to benefit other crops, matures within approximately four months when it is then harvested, and requires no special fertilizers or pesticides to replant immediately for the next sustainable crop. It can grow several years in the same location with no special requirements, requires less water due to its 3 foot root depth and can be planted densely with no adverse planting effects. Recycling is broken, commercial composting facilities limited and municipalities are cancelling select plastic recycling due to lack of equipment, capabilities and steep capital investments not previously anticipated with the drastic changes regarding overseas export of waste. Therefore, a sustainable biodegradable and compostable plastic alternative such as the composition disclosed herein using widely available natural and renewable materials is an important advancement to reducing both the current and future dilemma of plastic waste. The present invention addresses those issues with a substrate composition that is capable of exhibiting mechanical properties of other polymers used to produce various plastic products without the harmful effects of current polymer and mixed biopolymer resins to the environment. A hemp and biopolymer substrate as disclosed herein is comprised of hemp. When Hemp fibers are extracted from stems what remains is 70-77% cellulose. Cellulose is a homogeneous linear polymer constructed of repeating glucose units: the building blocks of trees and plants. Industrial hemp has been scientifically proven to absorb more CO2 per hectare than any forest or commercial crop. The qualities and environmental benefits such as the fully sustainable use of all hemp plant parts and wherein the hemp stalk traps Carbon Dioxide out of the atmosphere indefinitely and releases oxygen as a byproduct, combined with the current global plastic waste, recycling and greenhouse gas emissions problems, the use of and adoption of the described biopolymer invention presents an important long term improved solution to plastics production and environmental issues resulting from it. A hemp and biopolymer substrate also comprises of PLA (Polyactic Acid or other variations such as PCL's, thermal plastic aliphatic polyester). PLA is a type of resin from plants, with high strength, outstanding plasticity and easy to be machine-shaping ability. PLA is a bio based compound derived from natural resources and offers a significant reduction in carbon footprint compared to oil-based plastics. Limestone (CaCO3), another compound comprised herein is non-toxic, harmless to human beings or the environment. Limestone makes up about 10% of the total volume of all sedimentary rocks and in processed powdered form found to be inexpensive, simple to process and widely abundant. PBAT or aliphatic-aromatic co polyester's such as that known under one brand name of Ecoflex® has properties similar to PE-LD due to its high molecular weight and its long chain branched molecular structure. This serves as an elongation, malleable or flexible component with physical properties that are suitable to blown film, cast film or any number of other manufactured processing systems. Additionally PBAT's are biodegradable and compostable, and include uses such as the production of bags, packaging, and other such useful products in similar categories. It is widely understood that many of these noted compounds have shortfalls in becoming synthetic polymer plastic replacements of their own accord due to various limitations including but not limited to melt temperature, lack of elasticity, brittleness and other such attributes required of useful plastic product replacements, thereby further supporting the need for a unique hemp and PBAT biopolymer that can address and overcome known issues. An exemplary embodiment of a hemp and PBAT biopolymer substrate of the present invention comprises approximately ten to thirty percent (10-30%) of Calcium Carbonate (CaCO3) by weight in a one to three (1-3) powdered micron form, industrial Hemp from approximately fifteen to thirty five percent (15% to 35%) by weight, approximately three to eighteen percent (3-30%) PLA (Polylactic Acid) or PCL (Polycaprolactone) by weight which can include any one of a series of compounds such as those sold under the brand names Luminy®, Capa6500® or any similar such product known in the art. PCL (Polycaprolactone) is a biodegradable polyester. Both PLA and PCL's are thermoplastics. In another exemplary embodiment, the substrate comprises of approximately ten to forty percent (10-40%) biodegradable aliphatic-aromatic polyester (PBAT) by weight such as that sold under the trademark of Ecoflex®, or any one of a series of PBAT's formulated for biodegradability. Though the product Exoflex® is not limited to the invention as described herein, Ecoflex® biodegrades to the basic monomers 1,4-butanediol, adipic acid and terephthalic acid and eventually to carbon dioxide, water and biomass when metabolized in the soil or compost under standard conditions. In another exemplary embodiment, the substrate comprises approximately twenty to thirty five percent (20-35%) hemp by weight or reinforced polymer granules such as those sold under the brand names GreenGran®, Trifilon® or others (which can include any one of a series of compounds that are bio-based composites containing hemp). Both are resins derived from starch (and cellulose), which are plant-based and often reinforced with industrial hemp fibers. In another exemplary embodiment, the substrate may include a biodegradation additive from approximately three fourths of a percent to two percent (0.75-2.0%) by weight. The biodegradation additive may include a chemo-attractant such as Ecopure® (which can include any one of a series of formulations of organic molecules or polymer chains that are tailored to non-biodegradable polymers) or any similar such biodegradable additive known in the art. These products allow microbes to create a biofilm that coats the plastic waste. The biofilm forms on treated plastics once it comes in contact with certain enzymes and microbes found in landfills, compost piles or the ocean. These microbes send out chemical signals, a part of the quorum sensing process, that attract other microbes. Through this process, the microbes break down the chemical bonds of polymer chains to biodegrade them at an accelerated rate. Although several embodiments have been described in detail for purposes of illustration, various modifications may be made to each of the formulations compound inclusion percentages without departing from the scope and spirit of the invention. Accordingly, the invention is not to be limited in scope to any particular product or manufacturing process. All references to percentages herein are references to percentage by weight, unless specifically noted otherwise. While the present invention and corresponding methods have been described in connection with various illustrative embodiments, it is to be understood that other similar embodiments may be used or modifications and additions may be made to the described embodiments for performing the same function disclosed herein without deviating therefrom. Further, all embodiments disclosed are not necessarily in the alternative, as various embodiments may be combined or subtracted to provide the desired characteristics. Variations can be made by one having ordinary skill in the art without departing from the spirit and scope and process hereof. Therefore, the biopolymer plastic alternative composition and corresponding methods disclosed herein should not be limited to any single embodiment, but rather construed in breadth and scope in accordance with the recitations of the subject matter. In certain embodiments, the percentages of each compound for a hemp and PBAT biopolymer substrate falls within the ranges disclosed herein. However, specific percentages of each component or compound's composition are dependent upon the desired end product and its desired mechanical properties. Depending on the particular composition used within these ranges and the desired end product application, the biopolymer substrate's properties will vary and make it suitable for various plastic applications such as, but not limited to, consumer goods, plastic auto-parts, single use packaging, toys and games, food trays, medical trays, disposable cups, furniture, water bottles, medical packaging, pet toys, office supplies, sporting goods, electrical and household goods and any other variety of materials manufactured from the same, for various industries. In the spirit of various embodiments a hemp and PBAT composition wherein the compound loads can be modified within the scope identified herein can create a diverse set of varying products including but not limited to those for aerospace, food and product packaging (included but not limited to plastic bags and containers whether malleable or rigid), kitchen utensils (both single use and intended longer life product uses such as straws, plates or containers respectively) or a portion incorporated thereof, household and business items (such as folio sleeves or a writing apparatus, garbage can, plastic bag, cling film and picture frames), marine and boating parts, floating devices of various forms, outdoor furniture or gardening materials, toys and games, beverage apparatuses including but not limited to cups and containers, baby supplies (including but not limited to diapers, cribs, safety devices, toys or other products in this category), auto parts, medical devices or medical parts, cosmetic containers, gaming supplies, pet toys, novelty items both seasonal and non-seasonal such as holiday ornaments and tchotchke's, and apparel including but not limited to shoes or laces, clothes hangers, jewelry or other such items as known to the apparel and apparel accessory industry. Description of Preferred Embodiments In one exemplary embodiment which has properties suitable for malleable plastic parts such as a light switch plate cover comprises of approximately: (A) 25-35% Calcium Carbonate; (B) 15-22.5% Hemp from seeds (oil, powder or alternative aggregated form representing a biodegradable hemp resin), (C) 1%-2% biodegradable Eco-Pure® additive, (D) 15-25% EcoFlex® PBAT, (E) 20-30% PLA as a Bio-Polymer (F) 20-25% hemp. In another exemplary embodiment which has properties suitable for rigid plastic parts, in this example specifically compounding the substrate for a Yoyo, the embodiment is comprised of the following approximate compounds: (A) 30-40% hemp from seeds, (B) 15-25% Limestone (CaCO3), (C) 20-30% PLA as a Biopolymer (D) 15-40% PBAT, and (E) 1-2% biodegradable additive. And, in an exemplary embodiment as a sheet for thermoplastic forming such as is used to manufacture food service trays, the substrate comprises of approximately: (A) 25-40% PBAT, (B) 25-30% Limestone, (C) 0.05-1% Ecopure®, and (D) 15-30% PLA. Process for Formulation Akin to creating a relevant mixture for a 2 foot concrete retaining wall versus a 10 foot concrete retaining wall wherein formulation attributes are all critical to the function, longevity and structural strength of any particular item, each has their own mechanical density, strength, and elasticity end product requirements to fulfill their role as a useful item. The ultimate composition and properties of the disclosed invention is dependent on its end use is related to the process of mixing dry compounds so as to be cohesively mixed first, then any remaining liquid compounds separately mixed also in cohesion with each other which is the first key to a successful compound. Following, powder and liquid compounded materials are then mixed in the process below as to create ultimate even dispersion and cohesion of a final compound ready for manufacturing. First, the composition of the substrate requires the application of CaCO3 to be evenly dispersed throughout the resin blend in various amounts ranging from 30% to 50% by weight and with other powdered compounds. Whether the CaCO3 is treated or untreated but in a powered state and 90%+ pure in sizes ranging from 1-3 microns, it is then sifted into other powdered compounds to achieve an evenly distributed powder mix. The amount of other powders is in the ratio of 2-20% by weight. Powdered compounds must be blended in a dry and powdered state. Any additional dry compounds are mixed in the percentage ranges identified herein including those such as PBAT, PLA, PCL or hemp granules. Second, the compounding process then requires all liquids such as hemp oil from seeds, Biopolymers or other additive liquids to be consistently blended as a separate batch, independent of the first, and heated to a range of between 150 and 300 degrees Fahrenheit, processed and mixed in a heated agitation for a period of time between 2-4 hours for a consistently dispersed and blended integration of the inclusive liquid compounds prior to any powdered compound batch introductions. Processing temperature ranges may vary depending on formulation, manufacturing process and properties desired of a final product. In the final stage of compounding, the process requires a merging meld of the heated processed and agitated liquid batch, with the processed agitated powdered batch to create a slurry or roux wherein a mixture is created in a heated state where they can thermodynamically activate at temperatures in the 150-330 Fahrenheit range. It is in this range which there is cohesion between the two within this specific temperature value. The blend of Limestone or other dry compounds must be added in an even fill to the melted batch compounds mass ratio whereas by weight, the liquid batch is between 20-50% of the dry batch formulation. The two batches of compounds are added together and must be continuously agitated for a period of time between 2-hours to its desired consistency for any given manufacturing process requirement. The resulting biopolymer composition is now ready and compatible for any given manufacturing process such as thermoforming, extrusion blow molding, molding or other methods and processing as known in the art. There must be a short thermal process in correlation to the slurry agitation within specific time parameters prior to either the vacuum forming or an injection molding processes depending upon desired end product formation. There is an injection of air in the majority of end product processes that is directly relevant to the substrates native characteristic as applied to the end product and must be observed as a product specific time value. The process as described produces the substrate in a balanced and even dispersion suitable for standard manufacturing production equipment and processes as described, but not necessarily limited to those as provided as examples herein. During the early stages of production and development in identifying a successful composition as a replacement for synthetic polymer products, several challenges ensued including notably, working with non-synthetic compounds in order to achieve various attributes of various useful plastic replacement items. By identifying specialized and varying non synthetic polymers in various selective loads by weight and combining the compounds in the disclosed process, the hemp and PBAT biopolymer composition formed a strong cohesion to retain not only the best of the initial compounds' various unique structure and attributes, but that with a uniform compounded master batch. This technique led to a successful composition, regardless of varying thermal and other properties of each compound noted herein to create a highly efficient and durable biopolymer suited to traditional manufacturing processing as a replacement to traditional synthetic or mixed synthetic plastic products as a biodegradable and compostable replacement. It should be noted that in of themselves, each of the herein discussed individual compounds are challenged to create a meaningful use as a plastic replacement product for several reasons. For example tensile strength (enough durability or flexibility to be useful), manufacturing challenges (elongation or lack thereof wherein reaction to processing heat may compromise a material), and expected everyday functional uses serving a purposeful life for that individual product. This also includes microwavable and freezable products. Additionally, attributes such as elongation, stretch and flexibility to achieve certain product requirements or alternately stiffness, bending stiffness, bend modulus or rigidity are also generally challenged to produce meaningful products as intended for their useful life. A hemp and PBAT composition that in certain embodiments can be customized for the use in blow film extrusion, extrusion blow molding, extrusion, thermoforming, rotational and vacuum forming, injection molding, blow molding, CNC machining, 3D printing or other such plastic processing methods as known in the art for the production of various materials as stated herein. BACKGROUND The various embodiments and aspects described herein relate to a composition that can be used as an alternative to producing various plastic products and objects. Various issues relating to synthetic polymer based plastics with harmful impact to the environment and end of life disposal challenges are commonly known. Accordingly, there is a need for an improved plastic alternative compound without reliance on fossil fuels, greenhouse gas contributions, manufacturing changes or specialized knowledge as practiced, and understood in the art. | 25,858 |
11859086 | DETAILED DESCRIPTION A composition is useful for preparing a wood plastic composite article. The composition comprises: 15 weight % to 70 weight % of (a) a lignocellulosic-based filler; 29.5 weight % to 84.5 weight % of (b) an ethylene-based polymer; 0.5 weight % to 6 weight % of (c) a polydiorganosiloxane of formula where each R is an independently selected monovalent hydrocarbon group of 1 to 18 carbon atoms that is free of aliphatic unsaturation, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of >350 mPa·s to 100,000 mPa·s measured at 25° C. at 0.1 RPM to 50 RPM on a Brookfield DV-III cone & plate viscometer with #CP-52 spindle; and 0 to 4 weight % of (d) a maleated ethylene-based polymer; each based on combined weights of starting materials (a), (b), (c), and (d) in said composition. (a) Lignocellulosic-Based Filler The composition described above comprises starting material (a) a lignocellulosic-based filler. The lignocellulosic-based filler comprises, alternatively consists essentially of, alternatively consists of, a lignocellulosic material. Typically, the lignocellulosic-based filler consists of the lignocellulosic material. The lignocellulosic-based filler, as well as the lignocellulosic material, may comprise any matter derived from any plant source. When the lignocellulosic-based filler consists essentially of or consists of lignocellulosic material, the lignocellulosic material may also include some water or moisture content, although the lignocellulosic material, as well as the lignocellulosic-based filler, is typically dry, i.e., does not contain any free moisture content but for that which may be associated with the relative humidity in an environment in which the lignocellulosic-based filler is prepared, derived, formed, and/or stored. The same is typically true for other species of (a) the lignocellulosic-based filler, but is noted in regards to lignocellulosic-based fillers as lignocellulosic materials generally include some water content as harvested/prepared before any drying or end use. The lignocellulosic-based filler typically comprises carbohydrate polymers (e.g., cellulose and/or hemicellulose), and may further comprise an aromatic polymer (e.g., lignin). The lignocellulosic-based filler is typically a natural lignocellulosic material, i.e., is not synthetically derived. For example, the lignocellulosic-based filler is typically derived from wood (hardwood, softwood, and/or plywood). Alternatively or in addition, the lignocellulosic-based filler may comprise lignocellulosic material from other non-wood sources, such as lignocellulosic material from plants, or other plant-derived polymers, for example agricultural by-products, chaff, sisal, bagasse, wheat straw, kapok, ramie, henequen, corn fiber or coir, nut shells, flax, jute, hemp, kenaf, rice hulls, abaca, peanut hull, bamboo, straw, lignin, starch, or cellulose and cellulose-containing products, and combinations thereof. The lignocellulosic-based filler may be virgin, recycled, or a combination thereof. Alternatively, the lignocellulosic-based filler may comprise a wood filler. “Wood” is as described in The Chemical Composition of Wood by Pettersen, Roger C., U.S. Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, Wis., Chapter 2. Wood may comprise lignin in an amount of 18% to 35% and carbohydrate in an amount of 65% to 75%, and optionally inorganic minerals in an amount up to 10%. The carbohydrate portion of wood comprises cellulose and hemicellulose. Cellulose content may range from 40% to 50% of the dry wood weight and hemicellulose may range from 25% to 35%. Alpha-cellulose content may be 29% to 57%, alternatively 40% to 50%, based on dry weight of the wood filler. The wood filler is derived from wood, e.g., hardwood and/or softwood. Specific examples of suitable hardwoods from which the wood filler may be derived include, but are not limited to, ash, aspen, cottonwood, basswood, birch, beech, chestnut, gum, elm eucalyptus, maple, oak, poplar, sycamore, and combinations thereof. Specific examples of suitable softwoods from which the wood filler may be derived include, but are not limited to, spruce, fir, hemlock, tamarack, larch, pine, cypress, redwood, and combinations thereof. Fillers derived from combinations of different hardwoods, combinations of different softwoods, or combinations of hardwood(s) and softwood(s) may be used together as the wood filler. Alternatively, the lignocellulosic-based filler may consist essentially of a wood filler. Alternatively, the lignocellulosic-based filler may consist of a wood filler. The lignocellulosic-based filler may have any form and size, e.g., from nanometer to millimeter particle size. For example, the lignocellulosic-based filler may comprise a powder, a pulp, a flour, sawdust, a fiber, a flake, a chip, a shaving, a strand, a scrim, a wafer, a wool, a straw, a particle, or any combination thereof. The lignocellulosic-based filler may be formed via a variety of techniques known to one of skill in the art, typically as a function of the form thereof. For example, the lignocellulosic-based filler can be prepared by comminuting logs, branches, industrial wood residue, or rough pulpwood. The lignocellulosic-based filler may be comminuted to a desired particle size. For example, the lignocellulosic-based filler may be comminuted with any convenient equipment, such as a hammer mill, which results in the lignocellulosic-based filler having a particle size suitable for use in mixing processes. The desired particle size is typically selected by one of skill in the art based on the particular mixing process utilized and desired properties of the wood plastic composite article. By particle size, it is meant the dimensions of the lignocellulosic-based filler, regardless of shape, and includes, for example, dimensions associated with the lignocellulosic-based filler when in the form of fibers. As known in the art, lignocellulosic-based fillers may be pelletized, or otherwise in the form of pellets, which may substantially maintain shape and dimension when incorporated into the composition or which may form smaller particles in the composition. The shape and dimensions of the lignocellulosic-based filler is also not specifically restricted. For example, the lignocellulosic-based filler may be spherical, rectangular, ovoid, irregular, and may be in the form of, for example, a powder, a flour, a fiber, a flake, a chip, a shaving, a strand, a scrim, a wafer, a wool, a straw, a particle, and combinations thereof. Dimensions and shape are typically selected based on the type of the lignocellulosic-based filler utilized, the selection of other starting materials included within the WPC composition, and the end use application of the WPC article formed therewith. Starting material (a) may be one lignocellulosic-based filler or may be a combination of two or more lignocellulosic-based polymers that differ from one another by at least one property such as plant source from which the lignocellulosic-based filler was derived, lignin content, alpha-cellulose content, method of preparation, filler shape, filler surface area, average particle size, and/or particle size distribution. Starting material (a) may be present in the composition in an amount of 15% to 70%, alternatively 45% to 65%, based on combined weights of starting materials (a), (b), (c) and (d). (b) Ethylene-Based Polymer The composition described above further comprises starting material (b) an ethylene-based polymer. As used herein, “ethylene-based” polymers are polymers prepared from ethylene monomers as the primary (i.e., greater than 50%) monomer component, though other co-monomers may also be employed. “Polymer” means a macromolecular compound prepared by reacting (i.e., polymerizing) monomers of the same or different type, and includes homopolymers and interpolymers. “Interpolymer” means a polymer prepared by the polymerization of at least two different monomer types. This generic term includes copolymers (usually employed to refer to polymers prepared from two different monomer types), and polymers prepared from more than two different monomer types (e.g., terpolymers (three different monomer types) and tetrapolymers (four different monomer types)). The ethylene-based polymer can be an ethylene homopolymer. As used herein, “homopolymer” denotes a polymer comprising repeating units derived from a single monomer type, but does not exclude residual amounts of other components used in preparing the homopolymer, such as catalysts, initiators, solvents, and chain transfer agents. Alternatively, the ethylene-based polymer can be an ethylene/alpha-olefin (“α-olefin”) interpolymer having an α-olefin content of at least 1%, alternatively at least 5%, alternatively at least 10%, alternatively at least 15%, alternatively at least 20%, or alternatively at least 25 wt % based on the entire interpolymer weight. These interpolymers can have an α-olefin content of less than 50%, alternatively less than 45%, alternatively less than 40%, or alternatively less than 35% based on the entire interpolymer weight. When an α-olefin is employed, the α-olefin can have 3 to 20 carbon atoms (C3-C20) and be a linear, branched or cyclic α-olefin. Examples of C3-20 α-olefins include propene, 1-butene, 4-methyl-1-pentene, 1-hexene, 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, and 1-octadecene. The α-olefins can also have a cyclic structure such as cyclohexane or cyclopentane, resulting in an α-olefin such as 3-cyclohexyl-1-propene (allyl cyclohexane) and vinyl cyclohexane. Illustrative ethylene/α-olefin interpolymers include ethylene/propylene, ethylene/1-butene, ethylene/1-hexene, ethylene/1-octene, ethylene/propylene/1-octene, ethylene/propylene/1-butene, and ethylene/1-butene/1-octene. Starting material (b) can be one ethylene-based polymer or a combination of two or more ethylene-based polymers (e.g., a blend of two or more ethylene-based polymers that differ from one another by at least one property such as monomer composition, monomer content, catalytic method of preparation, molecular weight, molecular weight distributions, and/or densities). If a blend of ethylene-based polymers is employed, the polymers can be blended by any in-reactor or post-reactor process. The ethylene-based polymer for starting material (b) may be selected from the group consisting of High Density Polyethylene (HDPE), Medium Density Polyethylene (MDPE), Low Density Polyethylene (LDPE), Linear Low Density Polyethylene (LLDPE), Low Density Low Molecular Weight Polyethylene (LDLMWPE), and a combination thereof. Alternatively, the ethylene-based polymer can be a LLDPE. LLDPEs are generally ethylene-based polymers having a heterogeneous distribution of comonomer (e.g., α-olefin monomer), and are characterized by short-chain branching. For example, LLDPEs can be copolymers of ethylene and α-olefin monomers, such as those described above. LLDPEs may have densities ranging from 0.91 g/cm3to 0.94 g/cm3. Densities for the LLDPEs and other ethylene-based polymers described herein are determined by ASTM D792-13. LLDPEs suitable for use herein can have a melt index (I2) of 1 g/10 min to 20 g/10 min, alternatively >2 g/10 min, alternatively 2.3 g/10 min to 20 g/10 min, alternatively 2.3 g/10 min to 12 g/10 min, alternatively 2.3 g/10 min to 6 g/10 min. Values for I2for LLDPEs and other ethylene-based polymers are determined at 190° C. and 2.16 Kg according to ASTM D1238-13. The LLDPE can have a melting temperature of at least 124° C., alternatively 124° C. to 135° C., and alternatively 124° C. to 132° C. Melting temperatures for LLDPEs and other polyethylene-based polymers are determined by DSC according to ASTM D3418-15. LLDPE's are known in the art and may be produced by known methods. For example, LLDPE may be made using Ziegler-Natta catalyst systems as well as single-site catalysts such as bis-metallocenes (sometimes referred to as “m-LLDPE”), post-metallocene catalysts, and constrained geometry catalysts. LLDPEs include linear, substantially linear or heterogeneous polyethylene copolymers or homopolymers. LLDPEs may contain less long chain branching than LDPEs, and LLDPEs include: substantially linear ethylene polymers which are further defined in U.S. Pat. Nos. 5,272,236, 5,278,272, and 5,582,923; homogeneously branched linear ethylene polymer compositions such as those in U.S. Pat. No. 3,645,992; and/or heterogeneously branched ethylene polymers such as those prepared according to the process disclosed in U.S. Pat. No. 4,076,698. The LLDPEs can be made via gas-phase, solution-phase or slurry polymerization or any combination thereof, using any type of reactor or reactor configuration known in the art. Alternatively, the ethylene-based polymer can be a MDPE. MDPEs are ethylene-based polymers having densities generally ranging from 0.926 g/cm3to 0.940 g/cm3. Alternatively, the MDPE can have a density ranging from 0.930 g/cm3to 0.939 g/cm3. The MDPE can have I2of 0.1 g/10 min to 20 g/10 min, alternatively >2 g/10 min, alternatively 2.3 g/10 min to 20 g/10 min, alternatively 2.3 g/10 min to 12 g/10 min, and alternatively 2.3 g/10 min to 6 g/10 min. The MDPE can have a melting temperature of at least 124° C., alternatively 124° C. to 135° C., and alternatively 124° C. to 132° C. MDPE may be made using chromium or Ziegler-Natta catalysts or using metallocene, constrained geometry, or single site catalysts, and typically have MWD greater than 2.5. Alternatively, the ethylene-based polymer can be a HDPE. HDPEs are ethylene-based polymers having densities of at least 0.940 g/cm3. Alternatively, the HDPE can have a density of >0.940 g/cm3to 0.970 g/cm3, alternatively >0.940 g/cm3to 0.965 g/cm3, alternatively >0.940 to 0.952 g/cm3. The HDPE can have a melting temperature of at least 124° C., alternatively 124° C. to 135° C., alternatively 124° C. to 132° C., and alternatively 131° C. to 132° C. The HDPE can have I2of 0.1 g/10 min to 66 g/10 min, alternatively 0.2 g/10 min to 20 g/10 min, alternatively >2 g/10 min, alternatively 2.3 g/10 min to 20 g/10 min, alternatively 3 g/10 min to 12 g/10 min, alternatively 4 g/10 min to 7 g/10 min. The HDPE can have a PDI of 1.0 to 30.0, alternatively 2.0 to 15.0, as determined by GPC. The HDPE suitable for use herein can be unimodal. As used herein, “unimodal” denotes an HDPE having a MWD such that its GPC curve exhibits only a single peak with no discernible second peak, or even a shoulder or hump, relative to such single peak. In contrast, “bi-modal” means that the MWD in a GPC curve exhibits the presence of two component polymers, such as by having two peaks or where one component may be indicated by a hump, shoulder, or tail relative to the peak of the other component polymer. The HDPE used herein may be unimodal. HDPEs are known in the art and may be made by known methods. For example, HDPEs may be prepared with Ziegler-Natta catalysts, chrome catalysts or even metallocene catalysts. Alternatively, the ethylene-based polymer for starting material (b) may be selected from the group consisting of HDPE, MDPE, LLDPE, and a combination thereof. Alternatively, the ethylene-based polymer for starting material (b) may be selected from the group consisting of HDPE, LLDPE, and a combination thereof. Alternatively, the ethylene-based polymer for starting material (b) may be selected from the group consisting of HDPE and LLDPE. Alternatively, the ethylene-based polymer for starting material (b) may be HDPE. Preparation methods for ethylene-based polymers are well known in the art. Any methods known or hereafter discovered for preparing an ethylene-based polymer having the desired properties may be employed for making the ethylene-based polymer. Suitable LLDPEs, MDPEs, and HDPEs may be prepared by methods described above or those disclosed in PCT Publication No. WO2018/049555 and U.S. Patent Application Publication No. 2019/0023895, and the references cited therein. Suitable ethylene-based polymers are commercially available from The Dow Chemical Company of Midland, Mich., USA. Examples of suitable ethylene-based polymers are shown below in Table 1. TABLE 1Ethylene-Based PolymersMeltingDensityI2(g/TemperatureType(g/cm3)10 min)(° C.)high density polyethylene0.95012132narrow molecular weight0.9526.8131distributionhigh density polyethylenehomopolymerhigh density polyethylene0.9524.4131high density polyethylene0.95210130high density polyethylene0.95420130high density polyethylene0.9610.80133homopolymerhigh density polyethylene0.9658.3133homopolymer with a narrowmolecular weight distributionethylene/1-octene linear-low-0.9172.3123density polyethylene copolymerethylene/1-octene linear-low-0.9196.0124density polyethylene copolymerpolyethylene resin, which is a0.91725124narrow molecular weightdistribution copolymer The ethylene-based polymer for use in the composition may comprise virgin polymer and/or recycled polymer. Without wishing to be bound by theory, it is thought that the ethylene-based polymer may comprise ≥50% recycled polyethylene. The recycled ethylene-based polymer, if utilized, may be sourced from industrial production streams, as well as from post-industrial and/or post-consumer sources. The selection of the specific ethylene-based polymer, as well as any ratio of virgin polymer to recycled polymer, if utilized in concert, is typically a function of cost and desired properties of the WPC article formed therewith. Starting material (b) may be present in the composition in an amount of 29.5% to 84.5%, alternatively 30% to 60%, alternatively 35% to 55%, and alternatively 40% to 50%, based on combined weights of starting materials (a), (b), (c) and (d). (c) Polydiorganosiloxane The composition described above further comprises starting material (c) a polydiorganosiloxane. The polydiorganosiloxane comprises formula where each R is an independently selected monovalent hydrocarbon group of 1 to 18 carbon atoms that is free of aliphatic unsaturation, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of >350 mPa·s to 100,000 mPa·s measured at 25° C. at 0.1 RPM to 50 RPM on a Brookfield DV-III cone & plate viscometer with #CP-52 spindle. One skilled in the art would recognize that rotation rate decreases as viscosity increases and would be able to select the appropriate rotation rate when using this test method to measure viscosity. Alternatively, viscosity may be 1,000 mPa·s to 50,000 mPa·s; alternatively 1,000 mPa·s to 20,000 mPa·s, and alternatively 5,000 mPa·s to 50,000 mPa·s measured as described above. Alternatively, viscosity may be 5,000 mPa·s to 20,000 mPa·s, alternatively 5,000 mPa·s to 15,000 mPa·s, and alternatively 5,000 mPa·s to 12,500 mPa·s, measured according to the test method described above at 5 RPM. Alternatively, the polydiorganosiloxane may be a trialkyl-siloxy terminated polydialkylsiloxane. Alternatively, each R may be an alkyl group of 1 to 18 carbon atoms, alternatively 1 to 12 carbon atoms, alternatively 1 to 6 carbon atoms, and alternatively 1 to 4 carbon atoms. Suitable alkyl groups include methyl, ethyl, propyl (including n-propyl and iso-propyl), and butyl (including n-butyl, tert-butyl, sec-butyl, and iso-butyl). Alternatively, each R may be methyl. Suitable polydiorganosiloxanes may be prepared by methods known in the art such as hydrolysis and condensation of appropriate organohalosilane monomers and/or equilibration of linear and cyclic polyorganosiloxanes optionally with endcapping. The polydiorganosiloxane may be a trimethylsiloxy-terminated polydimethylsiloxane, which is commercially available. Trimethylsiloxy-terminated polydimethylsiloxanes with viscosities of >350 mPa·s to 100,000 mPa·s are commercially available from Dow Silicones Corporation of Midland, Mich., USA. Starting material (c) may be one polydiorganosiloxane or may be a combination of two or more polydiorganosiloxanes that differ from one another by at least one property such as selection of R groups and viscosity. Starting material (c) may be present in the composition in an amount of 0.5% to 6%, alternatively 1% to 4%, alternatively 0.5% to 3%, alternatively 1% to 2%, and alternatively 2% to 4%, based on combined weights of starting materials (a), (b), (c) and (d). (d) Maleated Ethylene-Based Polymer The composition described above may optionally further comprise starting material (d) a maleated ethylene-based polymer. As used herein, the term “maleated” indicates a polymer (e.g., an ethylene-based polymer) that has been modified to incorporate a maleic anhydride monomer. Maleic anhydride can be incorporated into the ethylene-based polymer by any methods known or hereafter discovered in the art. For instance, the maleic anhydride can be copolymerized with ethylene and other monomers (if present) to prepare an interpolymer having maleic anhydride residues incorporated into the polymer backbone. Alternatively, the maleic anhydride can be graft-polymerized to the ethylene-based polymer. Techniques for copolymerizing and graft polymerizing are known in the art. The maleated ethylene-based polymer may be an ethylene-based polymer having maleic anhydride grafted thereon. The ethylene-based polymer prior to being maleated can be any of the ethylene-based polymers described above, alternatively, the ethylene-based polymer used for maleating may have a melt index lower than that melt index of the ethylene-based polymer described above. The starting ethylene-based polymer can be selected from a linear-low density polyethylene, a medium-density polyethylene, and a high-density polyethylene. Alternatively, the starting ethylene-based polymer can be a high-density polyethylene. The maleated ethylene-based polymer may have a density of at least 0.923 g/cm3. Alternatively, the maleated ethylene-based polymer can have a density of 0.923 g/cm3to 0.962 g/cm3, alternatively 0.940 g/cm3to 0.962 g/cm3, and alternatively 0.923 g/cm3to 0.940 g/cm3. Density of the maleated ethylene-based polymer may be determined by ASTM D792-13. The maleated ethylene-based polymer may have I2of 0.1 g/10 min to 25 g/10 min, alternatively 1 g/10 min to 2 g/10 min, alternatively 2 g/10 min to 25 g/10 min, alternatively 2 g/10 min to 12 g/10 min, alternatively 3 g/10 min to 25 g/10 min, and alternatively 3 g/10 min to 12 g/10 min. Values for I2for maleated ethylene-based polymers are determined at 190° C. and 2.16 Kg according to ASTM D1238-13. The maleated ethylene-based polymer can have a maleic anhydride content of at least 0.25%, alternatively an amount of 0.25% to 2.5%, and alternatively 0.5% to 1.5%, each based on the total weight of the maleated ethylene-based polymer. Maleic anhydride concentrations may be determined by a titration method, which takes dried resin and titrates with 0.02N KOH to determine the amount of maleic anhydride. The dried polymers are titrated by dissolving 0.3 to 0.5 grams of maleated ethylene-based polymer in 150 mL of refluxing xylene. Upon complete dissolution, deionized water (four drops) is added to the solution and the solution is refluxed for 1 hour. Next, 1% thymol blue (a few drops) is added to the solution and the solution is over titrated with 0.02N KOH in ethanol as indicated by the formation of a purple color. The solution is then back-titrated to a yellow endpoint with 0.05N HCl in isopropanol. Suitable maleated ethylene-based polymers for starting material (d) may be prepared by known methods, such as those disclosed in PCT Publication No. WO2018/049555 and the references cited therein. Alternatively, maleated ethylene-based polymers may be prepared by a process for grafting maleic anhydride on an ethylene-based polymer, which can be initiated by decomposing initiators to form free radicals, including azo-containing compounds, carboxylic peroxyacids and peroxyesters, alkyl hydroperoxides, and dialkyl and diacyl peroxides, among others. Many of these compounds and their properties have been described (Reference: J. Branderup, E. Immergut, E. Grulke, eds. “Polymer Handbook,” 4th ed., Wiley, New York, 1999, Section II, pp. 1-76.). Alternatively, the species that is formed by the decomposition of the initiator may be an oxygen-based free radical. Alternatively, the initiator may be selected from the group consisting of carboxylic peroxyesters, peroxyketals, dialkyl peroxides, and diacyl peroxides. Exemplary initiators, commonly used to modify the structure of polymers, are listed in U.S. Pat. No. 7,897,689, in the table spanning col. 48 line 13-col. 49 line 29. Alternatively, the grafting process for making maleated ethylene-based polymers can be initiated by free radicals generated by thermal oxidative processes. Suitable maleated ethylene-based polymers are commercially available from The Dow Chemical Company, of Midland, Mich., USA, such as those described below in Table 2. TABLE 2Examples of Maleated Ethylene-Based Polymersa random ethylenehigh densitycopolymerpolyethylene graftedincorporating awith very highmonomer which ismaleic anhydrideclassified as being a maleiccopolymerTypeanhydride equivalentgraft levelDensity (g/cm3)0.9400.962I2(g/10 min)252.0Melting Temperature108130(° C.) In Table 2, melting temperature of the random ethylene copolymer incorporating a monomer which is classified as being a maleic anhydride equivalent was measured by DSC according to ASTM D3418-15, and melting temperature of the high density polyethylene grafted with very high maleic anhydride copolymer graft level was measured by DSC wherein a film was conditioned at 230° C. for 3 minutes before cooling at a rate of 10° C. per minute to a temperature of −40° C. After the film was kept at −40° C. for 3 minutes, the film was heated to 200° C. at a rate of 10° C. per minute. Starting material (d) can be one maleated ethylene-based polymer or a combination of two or more maleated ethylene-based polymers (e.g., a blend of two or more maleated ethylene-based polymers that differ from one another by at least one property such as monomer composition, monomer content, catalytic method of preparation, molecular weight, molecular weight distributions, and/or densities). The maleated ethylene-based polymer may be present in the composition in an amount of 0 to 4%. Alternatively, the maleated ethylene-based polymer may be present in an amount of 0 to 2%, alternatively >0% to 2%, alternatively 1% to 3%, and alternatively 1% to 2%, based on combined weights of starting materials (a), (b), (c), and (d). Additional Starting Materials The composition described above may optionally further comprise one or more additional starting materials. For example, one or more additional starting materials may be selected from the group consisting of (e) an additional filler which is distinct from the lignocellulosic-based filler of starting material (a), (f) a colorant, (g) a blowing agent, (h) a UV stabilizer, (i) an antioxidant, (j) a process aid, (k) a preservative, (l) a biocide, (m) a flame retardant, (n) an impact modifier, and (o) a combination of two or more of starting materials (e) to (n). Each additional starting material, if utilized, may be present in the composition in an amount of greater than 0 to 30% based on combined weights of all starting materials in the composition. The composition may also include other optional additives, as known in the art. Such additives are described, for example, in Walker, Benjamin M., and Charles P. Rader, eds. Handbook of thermoplastic elastomers. New York: Van Nostrand Reinhold, 1979; Murphy, John, ed. Additives for plastics handbook. Elsevier, 2001. (e) Additional Filler The composition may optionally further comprise starting material (e) a filler distinct from the lignocellulosic-filler described above as starting material (a). Specific examples of suitable fillers include, but are not limited to, calcium carbonate, silica, quartz, fused quartz, talc, mica, clay, kaolin, wollastonite, feldspar, aluminum hydroxide, carbon black, and graphite. Alternatively, this filler may be a mineral filler. Alternatively, this filler may be selected from the group consisting of calcium carbonate, talc, and combinations thereof. Suitable fillers are known in the art and are commercially available, e.g., ground silica is sold under the name MIN-U-SIL by U.S. Silica of Berkeley Springs, W. Va., USA. Suitable precipitated calcium carbonates include Winnofil™ SPM from Solvay and Ultra-pflex™ and Ultra-pflex™ 100 from Specialty Minerals, Inc. of Quinnesec, Mich., USA. The shape and dimensions of the filler is not specifically restricted. For example, the filler may be spherical, rectangular, ovoid, irregular, and may be in the form of, for example, a powder, a flour, a fiber, a flake, a chip, a shaving, a strand, a scrim, a wafer, a wool, a straw, a particle, and combinations thereof. Dimensions and shape are typically selected based on the type of the filler utilized, the selection of other starting materials included within the solid carrier component. Regardless of the selection of the filler, the filler may be untreated, pretreated, or added in conjunction with an optional filler treating agent, described below, which when so added may treat the filler in situ or before incorporation of the filler in the composition described above. Alternatively, the filler may be surface treated to facilitate wetting or dispersion in the composition, which when so added may treat the filler in situ in the composition. The filler treating agent may comprise a silane such as an alkoxysilane, an alkoxy-functional oligosiloxane, a cyclic polyorganosiloxane, a hydroxyl-functional oligosiloxane such as a dimethyl siloxane or methyl phenyl siloxane, an organosilicon compound, a stearate, or a fatty acid. The filler treating agent may comprise a single filler treating agent, or a combination of two or more filler treating agents selected from similar or different types of molecules. The filler treating agent may comprise an alkoxysilane, which may be a mono-alkoxysilane, a di-alkoxysilane, a tri-alkoxysilane, or a tetra-alkoxysilane. Alkoxysilane filler treating agents are exemplified by hexyltrimethoxysilane, octyltriethoxysilane, decyltrimethoxysilane, dodecyltrimethoxysilane, tetradecyltrimethoxysilane, phenyltrimethoxysilane, phenylethyltrimethoxysilane, octadecyltrimethoxysilane, octadecyltriethoxysilane, and a combination thereof. In certain aspects the alkoxysilane(s) may be used in combination with silazanes, which catalyze the less reactive alkoxysilane reaction with surface hydroxyls. Such reactions are typically performed above 100° C. with high shear with the removal of volatile by-products such as ammonia, methanol and water. Suitable filler treating agents also include alkoxysilyl functional alkylmethyl polysiloxanes, or similar materials where the hydrolyzable group may comprise, for example, silazane, acyloxy or oximo. Alkoxy-functional oligosiloxanes can also be used as filler treating agents. Alkoxy-functional oligosiloxanes and methods for their preparation are generally known in the art. Other filler treating agents include mono-endcapped alkoxy functional polydiorganosiloxanes, i.e., polyorganosiloxanes having alkoxy functionality at one end. Alternatively, the filler treating agent can be any of the organosilicon compounds typically used to treat silica fillers. Examples of organosilicon compounds include organochlorosilanes such as methyltrichlorosilane, dimethyldichlorosilane, and trimethyl monochlorosilane; organosiloxanes such as hydroxy-endblocked dimethylsiloxane oligomer, silicon hydride functional siloxanes, hexamethyldisiloxane, and tetramethyldivinyldisiloxane; organosilazanes such as hexamethyldisilazane and hexamethylcyclotrisilazane; and organoalkoxysilanes such as alkylalkoxysilanes with methyl, propyl, n-butyl, i-butyl, n-hexyl, n-octyl, i-octyl, n-decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl substituents. Organoreactive alkoxysilanes can include amino, methacryloxy, vinyl, glycidoxy, epoxycyclohexyl, isocyanurato, isocyanato, mercapto, sulfido, vinyl-benzyl-amino, benzyl-amino, or phenyl-amino substituents. Alternatively, the filler treating agent may comprise an organopolysiloxane. Alternatively, certain filler treating agents, such as chlorosilanes, may be hydrolyzed at the filler surface. Alternatively, the filler treating agent may take advantage of multiple hydrogen bonds, either clustered or dispersed or both, as the method to bond the organosiloxane to the surface of the filler. The organosiloxane capable of hydrogen bonding has an average, per molecule, of at least one silicon-bonded group capable of hydrogen bonding. The group may be selected from: a monovalent organic group having multiple hydroxyl functionalities or a monovalent organic group having at least one amino functional group. Hydrogen bonding may be a primary mode of bonding of the organosiloxane to the filler. The organosiloxane may be incapable of forming covalent bonds with the filler. The organosiloxane capable of hydrogen bonding may be selected from the group consisting of a saccharide-siloxane polymer, an amino-functional organosiloxane, and a combination thereof. Alternatively, the polyorganosiloxane capable of hydrogen bonding may be a saccharide-siloxane polymer. Alternatively, the filler treating agent may comprise alkylthiols such as octadecyl mercaptan and others, and fatty acids such as oleic acid, stearic acid, titanates, titanate coupling agents, zirconate coupling agents, and a combination thereof. One skilled in the art could optimize a filler treating agent to aid dispersion of the filler without undue experimentation. Starting material (e) may be one additional filler or a combination of two or more additional fillers that differ from one another by at least one property such as type of filler, method of preparation, treatment or surface chemistry, filler composition, filler shape, filler surface area, average particle size, and/or particle size distribution. The additional filler, when present, may be added to the composition in an amount of >0% to 30%, alternatively 5% to 15%, and alternatively 10% to 15%, based on combined weights of all starting materials in the composition. When selecting starting materials to include in the composition, there may be overlap between types of starting materials because certain starting materials described herein may have more than one function. For example, (e) the additional filler may be useful as an additional filler and as a colorant, and even as a flame retardant, e.g., carbon black. When selecting starting materials for the composition, the components selected are distinct from one another. Method of Making This invention further relates to a method for preparing a wood plastic composite (WPC) article. The method comprises: (1) combining starting materials comprising 15 weight % to 70 weight % of (a) a lignocellulosic-based filler; 29.5 weight % to 84.5 weight % of (b) an ethylene-based polymer; 0.5 weight % to 6 weight % of (c) a polydiorganosiloxane of formula where each R is an independently selected monovalent hydrocarbon group of 1 to 18 carbon atoms that is free of aliphatic unsaturation, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of >350 mPa·s to 100,000 mPa·s measured at 25° C. at 0.1 RPM to 50 RPM on a Brookfield DV-III cone & plate viscometer with #CP-52 spindle; and 0 to 4 weight % of (d) a maleated ethylene-based polymer; each based on combined weights of starting materials (a), (b), (c), and (d); thereby preparing a composition; and (2) preparing the WPC article from the composition. In step (1), the composition is formed by combining at least (a) the lignocellulosic-based filler, (b) the ethylene-based polymer, and (c) the polydiorganosiloxane along with any optional starting materials present in the composition. When (c) the polydiorganosiloxane is in the form of a solid carrier component, the method may comprise combining (a) the lignocellulosic-based filler, (b) the ethylene-based polymer, and the solid carrier component comprising (c) the polydiorganosiloxane. The starting materials of the composition may be combined in any order and via any suitable manner. For example, (b) the ethylene-based polymer may be melted before, during, and/or after formation of the composition. For example, (b) the ethylene-based polymer may be heated before and/or during combining the starting materials such that (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane are combined with a melted form of (b) the ethylene-based polymer. Starting materials (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane be combined with the melted form of (b) the ethylene-based polymer in any order, e.g., individually, sequentially, together, or simultaneously. Alternatively, however, (b) the ethylene-based polymer may be combined with (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane before heating or melting (b) the ethylene-based polymer such that (b) the ethylene-based polymer is in solid and unmelted or unsoftened form when preparing the composition. Alternatively, (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane may be combined and heated, then added to (b) the ethylene-based polymer in solid or liquid form when preparing the composition. Starting material (b) the ethylene-based polymer is heated before, during, and/or after formation of the composition to a temperature that is greater than the melting temperature of (b) the ethylene-based polymer, e.g., 10° C. to 90° C., alternatively 10° C. to 40° C., higher than the melting temperature of (b) the ethylene-based polymer. This ensures melting rather than mere softening of (b) the ethylene-based polymer. Alternatively, lower temperatures may be utilized in combination with shear or mixing to ensure softening and/or melting of (b) the ethylene-based polymer. Starting material (c) the polydiorganosiloxane may be in liquid form or delivered in the form of solid carrier component. The solid carrier component is a solid at room temperature and is a combination comprising (i) the polydiorganosiloxane described above as starting material (c) and (ii) a polymer component selected from the group consisting of an ethylene-based polymer (as described above for starting material (b)), a maleated ethylene-based polymer (as described above for starting material (d)), or a combination of both the ethylene-based polymer and the maleated-ethylene based polymer. The solid carrier component may optionally further comprise a filler, as described below. Alternatively, (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane and at least one other starting material (e.g., one or more of the additional starting materials (e) to (n) described above) may be combined to give a mixture, and the mixture may be combined with (b) the ethylene-based polymer (and any other additional starting materials) to give the composition. Combining (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane may be referred to as surface treating, wetting, or pre-treating (a) the lignocellulosic-based filler, which may be further to or alternatively to surface treating (a) the lignocellulosic-based filler as set forth herein. Alternatively, (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane may be combined by spraying, impregnation, blending or mixing. Combining (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane may further comprise heating, e.g., to combine (c) the polydiorganosiloxane with (a) the lignocellulosic-based filler. Optionally, the resulting combination of (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane may be compacted before being pelletized to form the pellet if a pellet is utilized. Combining (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane may be performed in a separate process or may be integrated into an existing (e.g., extrusion) process for making a WPC article in a pre-mixing step. In the pre-mixing step, the starting materials may be blended together before feeding into an extruder, e.g., all or a portion of (a) the lignocellulosic-based filler, (c) the polydiorganosiloxane and (b) the ethylene-based polymer and one or more optional starting materials, may be mixed in the pre-mixing step and thereafter fed to an extruder. Alternatively, (c) the polydiorganosiloxane may be present in a solid carrier component which comprises, alternatively consists essentially of, alternatively consists of: (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane; and the solid carrier component may be heated. Alternatively, this solid carrier component may be heated in a vacuum. This can be performed for multiple reasons, such as to evaporate the carrier vehicle (if any), to evaporate other components present in the mixture used to form the solid carrier component or to improve the mechanical properties of the solid carrier component before using in the method. The composition may be formed under mixing or shear, e.g., with suitable mixing equipment. For example, the composition may be formed in a vessel equipped with an agitator and/or mixing blades. The vessel may be, for example, an internal mixer, such as a Banbury, Sigma (Z) Blade, or Cavity Transfer style mixer. Alternatively or in addition, the composition may be formed in or processed by an extruder, which may be any extruder, e.g., a single screw extruder with rotational and/or reciprocating (co-kneader) screws, as well as multi-screw devices comprising two or more screws, which may be aligned tangentially or partially/fully intermeshing, revolving in either a co- or counter-rotational direction. Alternatively, a conical extruder may be used for forming the WPC composition described herein. In the method for preparing the WPC article as described above, the method further comprises forming the WPC article from the composition in step 2). The composition may be prepared, e.g., in the vessel, and subsequently removed from the vessel to form the article with separate equipment. Alternatively, the same equipment may be utilized to prepare the composition and subsequently form the WPC article. For example, the composition may be prepared and/or mixed in an extruder, and the extruder may be utilized to form the WPC article with the composition. Alternatively, the WPC article may be formed via molding, e.g., with an injection, compression, or transfer molding process. The composition may be formed independently and disposed in the mold once formed. The method described above comprises forming the WPC article from the composition, which may comprise forming the composition into a desired shape. The desired shape depends on end use applications of the WPC article. One of skill in the art understands how dies for extrusion and molds for molding may be selected and created based on the desired shape of the WPC article. The method may be performed continuously or semi-continuously in an extruder, such as a twin screw extruder (in which the screws are concurrently rotated, partially or fully intermeshing, alternatively counter rotated aligned either tangentially or partially or fully intermeshing). Starting material (c) the polydiorganosiloxane (in liquid state or as part of a solid carrier component) may be disposed in the extruder concurrently with (a) the lignocellulosic-based filler and (b) the ethylene-based polymer. Alternatively, the polydiorganosiloxane may be disposed in the extruder after melting (b) the ethylene-based polymer and before adding (a) the lignocellulosic-based filler. Alternatively, the polydiorganosiloxane may be disposed in the extruder after (a) the lignocellulosic-based filler and (b) the ethylene-based polymer and before the WPC article exits the extruder. Alternatively, (a) the lignocellulosic-based filler may be disposed in the extruder concurrently with the polydiorganosiloxane, where they are heated to effect surface treatment of (a) the lignocellulosic-based filler with (c) the polydiorganosiloxane, then (b) the ethylene-based polymer may be disposed in the extruder to give a mixture and the temperature increased to a temperature suitable for compounding the mixture and forming the WPC article. The extruder may have one or more zones, such as 1 to 3, or 3 to 8, or 1 to 12, zones, where starting materials can be added. The zones may be heated at different temperatures. Alternatively, (b) the ethylene-based polymer may be disposed in a first zone of the extruder, which is heated at +/−30° C. within the melting temperature of (b) the ethylene-based polymer. Starting material (c) the polydiorganosiloxane, which may be delivered in a solid carrier component, may be disposed in a second or later zone of the extruder, which may be heated at 10° C. to 90° C. above the melting temperature of (b) the ethylene-based polymer. As noted above, the temperature utilized is typically less than a degradation temperature of the starting materials of the composition. Alternatively, the die of the extruder may also be heated, and the temperatures utilized by the extruder, including the temperature of any zone and the die, may be selected such that the temperatures do not exceed a degradation temperature of (a) the lignocellulosic-based filler. The degradation temperature of (a) the lignocellulosic-based filler is contingent on the selection thereof, as understood by one of skill in the art. The method described above may be used to produce various WPC articles, such as building materials. Such WPC building materials include residential and/or commercial building and construction products and applications, e.g., decking, railing, siding, fencing, window framing, trim, skirts, and flooring. When the building material is decking, the method may optionally further comprise step 3), adding a cap stock layer after step 2). Solid Carrier Component Composition As described above, (c) the polydiorganosiloxane may be added to the composition for preparing the WPC article in the form of a solid carrier component. The solid carrier component may comprise, alternatively may consist essentially of, and alternatively may consist of: 5 weight % to 35 weight % of (i) the polydiorganosiloxane described above as starting material (c); 65 weight % to 95 weight % of (ii) a polymer component selected from the group consisting of:an ethylene-based polymer as described above for starting material (b),a maleated ethylene-based polymer as described above for starting material (d), anda combination of both the ethylene-based polymer and the maleated ethylene-based polymer; and 0 to 10% of (iii) a filler. Starting material (i) the polydiorganosiloxane in the solid carrier component is as described above for starting material (c). Starting material (ii) the polymer component may comprise the ethylene-based polymer and may be free of maleated ethylene-based polymer. The ethylene-based polymer in the solid carrier component is as described above for starting material (b). Alternatively, in the solid carrier component the ethylene-based polymer may be selected from the group consisting of LLDPE, HDPE and a combination thereof, alternatively the ethylene-based polymer in the solid carrier component may be HDPE. The HDPE used in the solid carrier component may have a melt index >2 g/10 min, alternatively 2.3 g/10 min to 20 g/10 min, alternatively 2.3 g/10 min to 12 g/10 min, alternatively 2.3 g/10 min to 6 g/10 min, alternatively 4.4 g/10 min to 20 g/10 min, and alternatively 4.4 g/10 min to 12 g/10 min. Alternatively, (ii) the polymer component may be a maleated-ethylene based polymer, and the solid carrier component may be free of ethylene-based polymer. The maleated ethylene-based polymer for use in the solid carrier component may be as described above for starting material (d). Alternatively, (ii) the polymer component may include both an ethylene-based polymer and a maleated ethylene-based polymer. The filler in the solid carrier component is optional. When present, the filler may comprise a lignocellulosic-based filler as described above for starting material (a), an additional filler, such as a mineral filler, as described above as starting material (e), or a combination of both the lignocellulosic-based filler and the additional filler. Alternatively, the filler in the solid carrier component may be a mineral filler, and alternatively the mineral filler may be selected from the group consisting of talc, calcium carbonate, and a combination thereof. Alternatively, the filler in the solid carrier component may be talc. The solid carrier component may alternatively comprise 10% to 30% of (i) the polydiorganosiloxane, 70% to 90% of (ii) the polymer component, and 0 to 10% of (iii) the filler. Alternatively, the solid carrier component may comprise 10% to <25% of (i) the polydiorganosiloxane, alternatively 10% to 20% of the polydiorganosiloxane. Alternatively, the solid carrier component may contain 0% filler. Alternatively, the solid carrier component may comprise >75% to 90% of (ii) the polymer component, alternatively 80% to 90% of (ii) the polymer component. The solid carrier component is a solid at ambient temperature and pressure (e.g., 25° C. and 1 atmosphere). The solid carrier component may be formed by combining the starting materials in any order. The solid carrier component may be prepared by forming a mixed composition from (ii) the polymer component and (i) the polydiorganosiloxane, and when present (iii), the filler, by dispersing under mixing or shear, e.g., with suitable mixing equipment. For example, the mixed composition may be dispersed in a vessel equipped with an agitator and/or mixing blades. The vessel may be, for example, an internal mixer, such as a Banbury, Sigma (Z) Blade, or Cavity Transfer style mixer. Alternatively or in addition, the mixed composition may be dispersed in or processed by an extruder, which may be any extruder, e.g., a single screw extruder with rotational and/or reciprocating (co-kneader) screws, as well as multi-screw devices comprising two or more screws, which may be aligned tangentially or partially/fully intermeshing, revolving in either a co- or counter-rotational direction. Alternatively, a conical extruder may be used to disperse the mixed composition described herein. The solid carrier components prepared as described above are re-processable and may be prepared for feeding in subsequent processes. The mixed composition prepared as described above may be, for example, substantially continuous ribbons or discontinuous pellets or particles or powders. Substantially continuous ribbons can be formed by pressurizing the mixed composition and passing it through a die to create continuous strands or tapes that are subsequently cooled before being suitably packaged. Alternatively, the strand or tape may be comminuted to form pellets or powders. The mixing device may also produce the pressure and/or heat needed to process the mixed composition through the die when the mixing device is an extruder, which may be any extruder, e.g., BUSS kneader, or a single screw extruder with rotational and/or reciprocating (co-kneader) screws, as well as multi-screw devices comprising two or more screws, which may be aligned tangentially or partially/fully intermeshing, revolving in either a co- or counter-rotational direction. A conical extruder may be used for mixing and pressurizing the mixed composition. Alternately, a gear pump may be used to generate the pressure needed for extrusion after the starting materials have been mixed to form the mixed composition. Discontinuous forms of the mixed composition may be created by chopping continuous ribbons of mixed composition into shorter lengths. Alternatively, large pieces of mixed composition may be reduced to usable sizes by use of a grinder or shredder. The solid carrier component may be formed by a method performed continuously or semi-continuously in an extruder, such as a twin screw extruder (in which the screws are concurrently rotated, partially or fully intermeshing, alternatively counter rotated aligned either tangentially or partially or fully intermeshing). Alternatively, (i) the polydiorganosiloxane may be disposed in the extruder concurrently with the polymer component and optionally (iii) the filler. Alternatively, (i) the polydiorganosiloxane may be disposed in the extruder after melting (ii) the polymer component (and before adding (iii) the filler, if any will be added to the mixed composition). Alternatively, (i) the polydiorganosiloxane may be disposed in the extruder after (iii) the filler, when present, and before (ii) the polymer component, and before the mixed composition exits the extruder. Alternatively, (iii) the filler may be disposed in the extruder concurrently with (i) the polydiorganosiloxane, then the polymer component may be disposed in the extruder to give a mixture and the temperature increased to a temperature suitable for compounding the mixture. The extruder may have one or more zones, such as 1 to 3, alternatively 1 to 12, alternatively 3 to 12, or alternatively 3 to 10 zones, where starting materials can be added. The zones may be heated at different temperatures and incorporate various functional stages including conveying, melting, mixing, deaeration, vacuum, pressurization, and forming. Alternatively, (ii) the polymer component may be disposed in a first zone of the extruder, which is heated at +/−30° C. within the melting temperature of the polymer component. The (i) polydiorganosiloxane may be disposed in a second zone of the extruder, which is heated at 10° C. to 90° C. above the melting temperature of (ii) the polymer component. Starting material (iii), the filler, when present, is disposed in one or more of the first, second, or subsequent zones of the extruder. As noted above, the temperature utilized is typically less than a degradation temperature of the starting materials of the solid carrier component. The mixture may be stripped to remove any air, moisture or byproducts prior to pressurization and forming in the die of the extruder. The vacuum, pressurization, and forming zones may also be heated, and the temperatures utilized by the extruder, including the temperature of any zone and the die, does not exceed a degradation temperature of starting materials (i), (ii), and, when present (iii). The degradation temperature of starting materials (i), (ii), and (iii) is contingent on the selection thereof, as understood by one of skill in the art. The resulting extruded strand may be comminuted by any convenient means to form the solid carrier component. The solid carrier component is typically in particulate form, and may be, for example, in the form of particles, pellets, or powders. An average particle size of the solid carrier component is a function of desired properties and end use thereof. The solid carrier component may be a powder. Alternatively, the solid carrier component may be a pellet. Pellets typically have greater average particle sizes than powders. EXAMPLES These examples are intended to illustrate the invention to one skilled in the art and are not to be interpreted as limiting the scope of the invention set forth in the claims. The starting materials in Table 3 were used in these examples. TABLE 3Starting MaterialsMaterialDescriptionLLDPEPolyethylene with I2= 2.3 g/10 min and a density of 0.92 g/cm3LLDPE 2Polyethylene with I2= 6 g/10 min and a density of 0.919 g/cm3, and a meltingtemperature of 124° C.HDPEPolyethylene with I2= 6.8 g/10 min and a density of 0.952 g/cm3HDPE 2high density polyethylene homopolymer with I2= 0.8 g/10 min, a density of0.961 g/cm3, and a melting temperature of 133° C.HDPE 3high density polyethylene homopolymer with a narrow molecular weightdistribution with I2= 8.3 g/10 min, a density of 0.965 g/cm3, and meltingtemperature of 133° C.HDPE 4high density polyethylene homopolymer with I2= 1.5 g/10 min, a density of0.955 g/cm3, and a melting temperature of 130° C.HDPE 5high density polyethylene homopolymer with I2= 20 g/10 min @ 190/21.6 kg, adensity of 0.954 g/cm3, and a melting temperature of 130° C.HDPE 6high density polyethylene homopolymer with I2= 4.4 g/10 min @ 190/2.16 kg, adensity of 0.952 g/cm3, and a melting temperature of 131° C.MRFTalco 0130WNAPC Multicolor flake (high density polyethylene)Si-350trimethylsiloxy-terminated polydimethylsiloxane with a viscosity of 350 mPa · sSi-1000trimethylsiloxy-terminated polydimethylsiloxane with a viscosity of 1000 mPa · sSi-5000trimethylsiloxy-terminated polydimethylsiloxane with a viscosity of 5000 mPa · sSi-12500trimethylsiloxy-terminated polydimethylsiloxane with a viscosity of 12500 mPa · sSi-16500Blend of 15% trimethylsiloxy-terminated polydimethylsiloxane and 85% bis-hydroxyl-terminated polydimethylsiloxane with a viscosity of 16500mPa · sSi-60000trimethylsiloxy-terminated polydimethylsiloxane with a viscosity of 60000 mPa · sSi-100000trimethylsiloxy-terminated polydimethylsiloxane with a viscosity of 100,000mPa · sMAPEhigh density polyethylene grafted with very high maleic anhydride copolymergraft level with density 0.962 g/cm3and I2= 2.0 g/10 minFiller40M1 Sixty mesh wood flour purchased from American Wood Fibers composedof primarily hardwoods such as maple, poplar, ash and beech. The hydroscopicnature of wood results in moisture contents of up to 10% despite being dried atthe time of milling. To compensate for these variations, the wood content wasadjusted in the final formulation for moisture content to result in consistentlevels of dried wood for all samples. Moisture was removed from the wood byuse of a vacuum vent on the extruder shortly after the introduction of the woodto the polymer system. Using this system the water was removed for uniformlydry pellets at the time of processing. The wood flour consisted of the followingparticle size distribution:>850 μm: 0-1%425-850 μm: 15-35%250-425 μm: 30-60%180-250 μm: 10-25%150-180 μm: 0-15%Balance Pan 0-23% The ethylene-based polymers (PE) and maleated ethylene-based polymer (MAPE) in Table 3 are each commercially available from The Dow Chemical Company of Midland, Mich., USA. In Table 3, densities were measured by ASTM D792-13; I2values were measured by ASTM D1238-13 at 190° C. and 2.16 Kg load; and Melting Temperatures were measured by DSC, where a film was conditioned at 230° C. for 3 minutes before cooling at a rate of 10° C. per minute to a temperature of −40° C. After the film was kept at −40° C. for 3 minutes, the film was heated to 200° C. at a rate of 10° C. per minute. The polydiorganosiloxanes are each commercially available from Dow Silicones Corporation of Midland, Mich., USA, and their viscosities were measured at 25° C. at 0.1 to 50 RPM on a Brookfield DV III cone & plate viscometer with #CP-52 spindle. Reference Example 1—Procedure for Preparing WPC Samples Compositions for these examples were produced using a twin screw extruder. The composition was processed in the twin screw extruder and made into a granular format by chopping extruded strands. The granular pellets could then be used in subsequent processes. Starting material (a), the lignocellulosic-based filler, was added independent of (b) the ethylene-based polymer, and (c) the polydiorganosiloxane through a secondary feed system located at a downstream position on the extruder barrel. By mixing of the solids into the blend of fully melted ethylene-based polymer and polydiorganosiloxane, higher filler content samples could be produced than would have been possible with all materials being fed at the same location. Injection molding was utilized for producing test specimens. Tensile bars were produced and tested in accordance with ASTM D638-14. Each composition was processed with the same conditions for both compounding in the twin screw extruder and injection molding equipment for consistency. For each example, total feed rates, RPM, temperatures, and equipment configurations remained constant for each composition for both the compounding extruders and injection molding equipment. The parameters associated with extrusion, as well as the average break strength of the wood plastic composite article formed by each example, the strand quality, and color of the final injection molded tensile bars is set forth below in the tables below. Melt temperature was obtained with a thermocouple hand probe. As this measurement required a level of technique due to the manual method, it was subject to a high level of variation. Experience showed that results could differ by up to 10° C. depending on operator and technique. In the case of these tests, care was taken to use the same operator and technique per system of (a) lignocellulosic-based filler and (b) ethylene-based polymer to minimize this error. Extruder torque was noted as a relative percent of the extruder maximum torque. Break strength was measured by producing five samples which were averaged. Testing was performed in accordance with ASTM D638-14. Color (Y) was also measured to quantify the level of thermal decomposition occurring in the wood filler. The Y-value or the luminance was measure as a gauge of the darkening of the wood plastic composite during processing. Higher values of Y correspond to a lighter brown color of the wood. The Y value was measured using an average of 2 measurements on 5 separate injection molded tensile dog bone samples (average of 10 measurements) using a BYK spectro-guide 45/0 gloss meter with D65 illuminant and 10 observer. Strand quality was assigned by visually evaluating for melt fracture, ability to maintain strength for pelletization, and roughness. Comparative Example Compositions are shown in Table 4. Amounts of each starting material are in weight %. TABLE 4(b)(c)Com-Ethylene-Polydimethyl-(d)(a)parativeBasedsiloxanePDMSCompatibilizerFillerExamplePolymer(PDMS)AmountAmountAmount1LLDPENone00552LLDPENone02553LLDPESi-35012554LLDPESi-3502255 In Table 4, the balance of each composition was (b) Ethylene-Based Polymer. Comparative Examples 1 and 2 show controls where no polydiorganosiloxane was added. Comparative Examples 3 and 4 show controls where the polydiorganosiloxane selected had a viscosity too low for this application under the conditions tested. Table 5 shows performance of the samples prepared as shown in Table 4. TABLE 5Com-para-Ex-MeltAvg.tivetruderTemper-BreakExam-TorqueatureStrengthColorple(%)(° C.)(MPa)(Y)Observations1822458.26.7die drool, cannot pelletize28324927.44.5uneven blending, diesurge, smooth to roughstrand351NA*25.510.8uneven die flow - couldnot measure melt tempdue to surging and solidsin stream444NA*24.010.5strand surging at die head/uneven die flow - couldnot measure temperatureNA* means not applicable. Working Example Compositions are shown in Table 6. Amounts of each starting material are in weight %. TABLE 6(c)(d)(a)Working(b)(c)PDMSCompatibilizerFillerExamplePolymerPDMSAmountAmountAmount1HDPESi-500020552HDPESi-500022553LLDPESi-500020554LLDPESi-500022555LLDPESi-500022656LLDPESi-500022457LLDPESi-500012558LLDPESi-500042559LLDPESi-1000125510LLDPESi-1000225511LLDPESi-1000425512LLDPESi-60000125513LLDPESi-60000225514LLDPESi-60000425515LLDPESi-50000.525516LLDPESi-5000625517LLDPESi-12500125518LLDPESi-12500225519LLDPESi-12500425520LLDPESi-100000125521LLDPESi-100000225522LLDPESi-100000425523MRFSi-60000605524MRFSi-20000.5055 Starting Material (b) Polymer was the balance of each sample shown in Table 6. Table 7 shows performance of the samples prepared as shown in Table 6. TABLE 7MeltAvg.ExtruderTemper-BreakWorkingTorqueatureStrengthColorExample(%)(° C.)(MPa)(Y)Observations15621613.927.4good strands25521441.116.8good strands,minimal unevenflow3572146.720.4good strands45221224.713.6good strands,uneven flow55421226.716.3good strands,uneven flow65021522.09.4good strands,slightly shiny76322526.18.7good strands84220421.019.2good strands,minimal unevenflow95622126.811.5good strands104520924.310.0good strands113920122.112.6good strands,slightly shiny126924525.19.6good strands136023124.514.3good strands,minimal unevenflow144921221.615.4good strands,minimal unevenflow157224227.27.7good strands,minimal unevenflow163719620.114.9good strands,uneven flow176824024.814.2good strands185522423.39.4good strands194420020.815.2good strands,uneven flow,some curlingand potentialunmelts207224925.16.9good strands216324024.09.8good strands225221921.513.1good strands234921411.9NDgood strands246422815.9NDrough strandsND = not determined In this Reference Example A, a solid carrier component in pellet form was produced using a 26 mm twin screw extruder. Starting material (ii) the ethylene-based polymer, and when used, (ii) the maleated ethylene-based polymer, were fed in via the feed throat in the first barrel section. When used, (ii) the filler CaCO3 (Calcium carbonate which was untreated and had an average particle size of 3 μm) was also fed in via the feed throat in the first barrel section. Starting material (i) the polydiorganosiloxane was injected into the fourth of eleven barrel sections onto a screw section with mixing. The resulting composition was pelletized using a Gala underwater pelletizer for consistency and collected for testing. All samples were cooled to room temperature and aged a minimum of 48 hours before any testing. In this Reference Example B, A solid carrier component in pellet form was produced using a 25 mm twin screw extruder. Starting material (b) the ethylene-based polymer, and when used, (d) the maleated ethylene-based polymer, were fed in via the feed throat in the first barrel section. Starting material (c) the polydiorganosiloxane was injected into the fourth of twelve barrel sections onto a screw section with mixing. The resulting composition was cooled via full immersion water bath and pelletized using a strand pelletizer. In this Reference Example C, bleed of the polydiorganosiloxane from the pellets prepared in Reference Example A and Reference Example B as described above was evaluated, as follows. Each sample (4 g) was placed into pre-weighed aluminum pans lined with Whatman™ #1 filter paper (5.5 cm diameter) such that the surface of the aluminum pan was covered fully by the filter paper, but the filter paper was not bent. The pellets were evenly spread out across the filter paper in a semi-uniform layer. The samples were left standing at room temperature on the bench or at the said temperature in a convection oven for the Aging Time. After aging, the pellets were left to stand at room temperature for at least 4 hours, and the pellets were placed in a 20 mL scintillation vial. The filter paper was weighed to determine aged filter paper weight. Bleed was determined according to the formula below: Bleed(%)=100×AgedFilterPaperWeight-StartingFilterPaperWeightTotalPelletWeight×FractionSiloxaneinPellet Compositions, aging conditions and polydiorganosiloxane bleed for the pellets prepared according to Reference Example A (25 through 27 & 35) and Reference Example B (28 through 34) and tested according to Reference Example B are reported below in Table 9. TABLE 9(ii)(iii)AgingAging(ii)PE(i)PDMSMAPECaCO3TimeTemp%SamplePE(%)PDMS(%)(%)(%)(weeks)(° C.)Bleed25 (working)HDPE 360Si-12500202002700.3626 (working)HDPE 380Si-1250020002700.3127 (working)HDPE 640Si-165002525102700.828 (working)HDPE 560Si-1250020200270029 (working)LLDPE 295Si-3505200270030 (working)HDPE70Si-6000030002700.0131 (working)HDPE75Si-600002500270032 (working)HDPE80Si-600002000270033 (working)none0Si-125005950270034 (comparative)HDPE 460Si-12500202002704.0435 (comparative)HDPE 260Si-125002020027013.2 Problem to be Solved WPC articles are commonly produced by high shear methods such as extrusion or injection molding. Lignocellulosic-based fillers are used to alter mechanical properties, decrease cost (because these are typically less expensive than the ethylene-based polymers), decrease density, and/or meet end use requirements for various applications. Adding fillers can make the starting materials difficult to process because the filler generally increases the viscosity of the melted ethylene-based polymer. When the starting materials are processed with a high shear method, these fillers can require more work to process resulting in higher temperatures and limited extrusion rates. This increase in temperature and stress can result in thermal or mechanical decomposition of the lignocellulosic-based filler. Similarly, some ethylene-based polymers can suffer from decomposition under the mechanical or thermal stress from processing. This decomposition translates in poor mechanical properties, discoloration, poor aesthetics, and/or other undesirable defects in the WPC article produced. Similarly, such processing difficulties translate in the need for a higher energy input for processing, increased torque, and reduced processing speed. Combined these effects can result in lower output for compounders and/or poor product quality. INDUSTRIAL APPLICABILITY The EXAMPLES above show that by adding a polydiorganosiloxane during processing, torque can be substantially reduced. Reducing torque also reduces energy requirements and reduces the melt temperature of the composition. This temperature reduction can enable higher throughputs, improved material properties, higher filler loadings, improve properties of the WPC article, and/or decrease costs associated with producing the WPC article. This reduction in torque, pressure, work, and temperature can also minimize or eliminate process related decomposition of the ethylene-based polymer and/or filler. It has been surprisingly found that this melt temperature reduction (on the order of 5° C. to 30° C., alternatively 10° C. to 20° C.) can be obtained by using a polydiorganosiloxane without silicon bonded groups other than monovalent hydrocarbon groups free of aliphatic unsaturation, e.g., trimethylsiloxy-terminated polydimethylsiloxane. It has also been found that polydiorganosiloxanes with viscosity greater than 350 mPa·s but less than or equal to 100,000 mPa·s provide one or more of the benefits described above. Working examples 1-22 show that using 0.5% to 6% of polydiorganosiloxane in the composition can significantly reduce torque to values of 39% to 72% with polydiorganosiloxane, as compared to 81% to 82% without polydiorganosiloxane as observed in comparative examples 1 and 2. Additionally, working examples 1 to 11, 13 to 19 and 21 to 22 show melt temperatures below the melt temperatures in comparative examples 1 and 2. Alternatively, the viscosity of the polydiorganosiloxane may be 5,000 mPa·s to 20,000 mPa·s. It was found that for high viscosity siloxanes (i.e., >100,000 mPa·s, alternatively 60,000 mPa·s), the change in melt temperature during extrusion was less significant under the conditions tested in the examples and comparative examples above, making certain high viscosity polydimethylsiloxanes less useful than polydimethylsiloxanes with lower viscosities. Working examples 12 and 20 have lower levels (1%) of higher viscosity 60,000 mPa·s and 100,000 mPa·s, respectively. For lower viscosity polydimethylsiloxanes (e.g., 350 mPa·s) under the conditions of the comparative examples above, the low viscosity polydimethylsiloxane may have not adequately distributed through the ethylene-based polymer resulting in the observed surging at the extrusion die in comparative examples 3 and 4. Additionally, it has also been found that using polydiorganosiloxanes with viscosity greater than 350 mPa·s but less than or equal to 100,000 mPa·s in combination with a wood filler that is a combination of hardwoods such as maple, poplar, ash and beech with typical alpha-cellulose levels of 42-47%, 45%, 40-41%, and 49% and lignin levels of 21-22%, 16%, 26% and 22%, respectively according to results reported by R. C. Pettersen in the book chapter entitled “The Chemical Composition of Wood,” enable composites to be produced with reduced darkening compared to samples that did not have the additive. The level of lignin in the wood flour is much higher than defined in U.S. Pat. No. 6,743,507; where it was outlined that cellulose pulp fibers were required to contain greater than 80% alpha-cellulose and less than 2% lignin in order to achieve a reduction is discoloration. The color was measured using the Luminance (Y) value of the XYZ scale, which represents a scaled of light to dark (100 being white and 0 being black/no reflected light). The results show that in the absence of the polydiorganosiloxane, the Y value is low (4.5 to 6.7) in comparative examples 1 and 2. However, Y is 6.9 to 27.4 when polydiorganosiloxane is added, with the lowest Y values reflecting lower levels of polydiorganosiloxanes. The Examples 25 to 27 showed that a solid carrier component could be prepared including a polydiorganosiloxane as described herein. Examples 25 to 27 showed that a solid carrier component with low bleed of the polydiorganosiloxane can be prepared. “Low bleed” means that siloxane migrating out of the solid carrier component is <1.5% after aging at 70° C. for at least 2 weeks, as measured by the test method in Reference Example B. Working Examples 25 and 26 showed that a low bleed solid carrier component could be prepared using 60 weight % to 80 weight % of HDPE, 0 to 20 weight % of a maleated ethylene-based polymer, and up to 20 weight % of a bis-trimethylsiloxy-terminated polydimethylsiloxane to prepare low bleed pellets. DEFINITIONS AND USAGE OF TERMS Unless otherwise indicated by the context of the specification: all amounts, ratios, and percentages herein are by weight; the articles ‘a’, ‘an’, and ‘the’ each refer to one or more; and the singular includes the plural. The SUMMARY and ABSTRACT are hereby incorporated by reference. The transitional phrases “comprising”, “consisting essentially of”, and “consisting of” are used as described in the Manual of Patent Examining Procedure Ninth Edition, Revision 08.2017, Last Revised January 2018 at section § 2111.03 I., II., and III. The use of “for example,” “e.g.,” “such as,” and “including” to list illustrative examples does not limit to only the listed examples. Thus, “for example” or “such as” means “for example, but not limited to” or “such as, but not limited to” and encompasses other similar or equivalent examples. The abbreviations used herein have the definitions in Table 10. TABLE 10AbbreviationsAbbreviationDefinition° C.degrees CelsiuscmcentimetersDSCdifferential scanning calorimetryggramsGPCgel permeation chromatographyHDPEhigh-density polyethyleneKgkilogramsLLDPElinear-low-density polyethyleneMAPEmaleated ethylene-based polymerMDPEmedium-density polyethylenemgmilligramsminminutesmLmillilitersmmmillimetersmPa · smilliPascal · secondsMWDmolecular weight distributionNnormalPDIpolydispersity indexPDMStrimethylsiloxy-terminated polydimethylsiloxanePEethylene-based polymerPTFEpolytetrafluoroethyleneRPMrevolutions per minuteULDPEultra low density polyethylene, which has a density of0.880 to 0.912 g/cm3, and which may be preparedwith Ziegler-Natta catalysts, chrome catalysts, orsingle-site catalysts including, but not limited to,bis-metallocene catalysts and constrained geometrycatalystsμLmicrolitersμmmicrometersWPCwood plastic composite The following test methods were used to measure properties of the starting materials herein. Melt indices of ethylene-based polymers and maleated ethylene-based polymers, abbreviated I2or 12, were measured according to ASTM D1238-13 at 190° C. and at 2.16 Kg loading. Melt index values are reported in g/10 min. Samples of ethylene-based polymers and maleated ethylene-based polymers were prepared for density measurement according to ASTM D4703. Measurements were made, according to ASTM D792, Method B, within one hour of sample pressing. Peak melting point (Melting Temperature) of ethylene-based polymers and maleated ethylene-based polymers was determined by DSC, where a film was conditioned at 230° C. for 3 minutes before cooling at a rate of 10° C. per minute to a temperature of −40° C. After the film was kept at −40° C. for 3 minutes, the film was heated to 200° C. at a rate of 10° C. per minute. “MWD” is defined as the ratio of weight average molecular weight to number average molecular weight (Mw/Mn). Mwand Mnare determined according to conventional GPC methods. Viscosity of each polydiorganosiloxane was measured at 0.1 to 50 RPM on a Brookfield DV-III cone & plate viscometer with #CP-52 spindle. One skilled in the art would recognize that rotation rate decreases as viscosity increases and would be able to select the appropriate rotation rate when using this test method to measure viscosity. The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. With respect to any Markush groups relied upon herein for describing particular features or aspects, different, special, and/or unexpected results may be obtained from each member of the respective Markush group independent from all other Markush members. Each member of a Markush group may be relied upon individually and or in combination and provides adequate support for specific embodiments within the scope of the appended claims. Furthermore, any ranges and subranges relied upon in describing the present invention independently and collectively fall within the scope of the appended claims, and are understood to describe and contemplate all ranges including whole and/or fractional values therein, even if such values are not expressly written herein. One of skill in the art readily recognizes that the enumerated ranges and subranges sufficiently describe and enable various embodiments of the present invention, and such ranges and subranges may be further delineated into relevant halves, thirds, quarters, fifths, and so on. As just one example, a range of “1 to 18” may be further delineated into a lower third, i.e., 1 to 6, a middle third, i.e., 7 to 12, and an upper third, i.e., from 13 to 18, which individually and collectively are within the scope of the appended claims, and may be relied upon individually and/or collectively and provide adequate support for specific embodiments within the scope of the appended claims. In addition, with respect to the language which defines or modifies a range, such as “at least,” “greater than,” “less than,” “no more than,” and the like, it is to be understood that such language includes subranges and/or an upper or lower limit. Embodiments of the Invention In a first embodiment, a composition for preparing a wood plastic composite article comprises: 40 weight % to 70 weight % of (a) a lignocellulosic-based filler; 29 weight % to 59 weight % of (b) an ethylene-based polymer; 1 weight % to 4 weight % of (c) a polydiorganosiloxane of formula where each R is an independently selected alkyl group of 1 to 18 carbon atoms, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of 5,000 mPa·s to 50,000 mPa·s as measured at 25° C. at 0.1 RPM to 50 RPM on a Brookfield DV-III cone & plate viscometer with #CP-52 spindle; and 0 to 4 weight % of (d) a maleated ethylene-based polymer; each based on combined weights of starting materials (a), (b), (c), and (d) in said composition. In a second embodiment, in the composition of the first embodiment, starting material (a) the lignocellulosic-based filler comprises a lignocellulosic material derived from wood, plants, agricultural by-products, chaff, sisal, bagasse, wheat straw, kapok, ramie, henequen, corn fiber or coir, nut shells, flax, jute, hemp, kenaf, rice hulls, abaca, peanut hull, bamboo, straw, lignin, starch, or cellulose and cellulose-containing products, and combinations thereof, and starting material (a) is present in an amount of 45 weight % to 65 weight %. In a third embodiment, in the composition of the first embodiment or the second embodiment, (a) the lignocellulosic-based filler is a wood filler comprising lignin in an amount of 18 weight % to 35 weight % and carbohydrate in an amount of 65 weight % to 75 weight %, and optionally inorganic minerals in an amount up to 10 weight %. In a fourth embodiment, in the composition of any one of the preceding embodiments, (a) the lignocellulosic-based filler is a wood filler comprising 29 weight % to 57 weight % alpha-cellulose. In a fifth embodiment, in the composition of any one of the preceding embodiments, starting material (b) the ethylene-based polymer is selected from the group consisting of High Density Polyethylene (HDPE), Medium Density Polyethylene (MDPE), Low Density Polyethylene (LDPE), Linear Low Density Polyethylene (LLDPE), Low Density Low Molecular Weight Polyethylene (LDLMWPE), and a combination thereof, and starting material (b) is present in an amount of 31 weight % to 51 weight %. In a sixth embodiment, in the composition of any one of the preceding embodiments, (b) the ethylene-based polymer is selected from the group consisting of HDPE, LLDPE, and a combination thereof. In a seventh embodiment, in the composition of any one of the preceding embodiments, (b) the ethylene-based polymer comprises ≥50% recycled polyethylene. In an eighth embodiment, in the composition of any one of the preceding embodiments, in starting material (c) the polydiorganosiloxane, each R is an alkyl group of 1 to 12 carbon atoms, subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of 5,000 mPa·s to 20,000 mPa·s, and starting material (c) is present in an amount of 1 weight % to 2 weight %. In a ninth embodiment, in the composition of any one of the preceding embodiments, in starting material (c) the polydiorganosiloxane each R is an alkyl group of 1 to 6 carbon atoms, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of 5,000 mPa·s to 15,000 mPa·s. In a tenth embodiment, in the composition of any one of the preceding embodiments, starting material (c) is a tri methylsiloxy-terminated polydimethylsiloxane. In an eleventh embodiment, in the composition of any one of the preceding embodiments, starting material (d) the maleated ethylene-based polymer is present and starting material (d) has a melt index of 2 g/10 min to 25 g/10 min measured according to ASTM D1238-13 at 190° C. and 2.16 Kg and a maleic anhydride content of 0.25 weight % to 2.5 weight %. In a twelfth embodiment, in the composition of any one of the preceding embodiments, the composition further comprises an additional starting material selected from the group consisting of (e) an additional filler which is distinct from the lignocellulosic-based filler of starting material (a), (f) a colorant, (g) a blowing agent, (h) a UV stabilizer, (i) an antioxidant, (j) a process aid, (k) a preservative, (l) a biocide, (m) a flame retardant, (n) an impact modifier, and (o) a combination of two or more of (e) to (n). In a thirteenth embodiment, in the composition of any one of the preceding embodiments, starting material (e) the additional filler is present in an amount of 10 weight % to 15 weight %, and starting material (e) is a mineral filler. In a fourteenth embodiment, a method for preparing a wood plastic composite article comprises: (1) preparing the composition of any one of the preceding claims by combining the starting materials; and (2) forming the wood plastic composite article from the composition. In a fifteenth embodiment, the method of the fourteenth embodiment further comprises (i) mixing (a) the lignocellulosic based filler and (b) the ethylene-based polymer before adding (c) the polydiorganosiloxane; (ii) heating (b) the ethylene-based polymer to melt (b) the ethylene-based polymer before and/or during forming the composition; (iii) mixing a mixture of (a) the lignocellulosic-based filler and (c) the polydiorganosiloxane before adding (b) the ethylene-based polymer or (iv) any combination of (ii) and (i) or (iii). In a sixteenth embodiment, the method of the fourteenth embodiment further comprises: (i) (c) the polydiorganosiloxane is a liquid when combining (c) the polydiorganosiloxane with another starting material of the composition; or (ii) (c) the polydiorganosiloxane is present within a solid carrier component, and the method further comprises melting the solid carrier component when combining (c) the polydiorganosiloxane with another starting material of the composition. In a seventeenth embodiment, the method of any one of the fourteenth to sixteenth embodiments further comprises: (i) forming the wood plastic composite article from the composition further comprises forming the composition into a desired shape; (ii) forming the wood plastic composite article from the composition comprises extruding the composition; (iii) forming the wood plastic composite article from the composition comprises molding the composition; or (iv) any combinations of (i) to (iii). In an eighteenth embodiment, the method of any one of the fourteenth to seventeenth embodiments further comprises that the wood plastic composite article is useful as a building material selected from the group consisting of decking, railing, fencing, siding, trim, skirts, and window framing. In a nineteenth embodiment, the building material of the method of the eighteenth embodiment is decking and the method further comprises: 3) adding a cap stock layer to the decking after step 2). In a twentieth embodiment, a solid carrier component comprises: 10 weight % to 30 weight % of (i) a polydiorganosiloxane of formula where each R is an independently selected alkyl of 1 to 18 carbon atoms, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of 5,000 mPa·s to 50,000 mPa·s measured at 25° C. at 0.1 RPM to 50 RPM on a Brookfield cone & plate viscometer with #CP-52 spindle; and 70 weight % to 90 weight % of (ii) a polymer component selected from the group consisting of: an ethylene-based polymer, a maleated ethylene-based polymer, and a combination of both (b) and (d); and 0 to 10% of (iii) a filler. In a twenty-first embodiment, where in the polydiorganosiloxane in the solid carrier component of the twentieth embodiment each R is an alkyl group of 1 to 12 carbon atoms, subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of 5,000 mPa·s to 20,000 mPa·s, and the polydiorganosiloxane is present in an amount of 15 weight % to 25 weight % based on combined weights of all starting materials in the solid carrier component. In a twenty-second embodiment, the polydiorganosiloxane in the solid carrier component of the twentieth embodiment or the twenty-first embodiment has each R is an alkyl group of 1 to 6 carbon atoms, and subscript x has a value sufficient to give the polydiorganosiloxane a viscosity of 5,000 mPa·s to 15,000 mPa·s, and the polydiorganosiloxane is present in an amount of 18 weight % to 22 weight % based on combined weights of all starting materials in the solid carrier component. In a twenty-third embodiment, the polydiorganosiloxane in the solid carrier component of any one of the twentieth to twenty-second embodiments is a trimethylsiloxy-terminated polydimethylsiloxane. In a twenty-fourth embodiment, the polymer component in the solid carrier component of any one of the twentieth to twenty-third embodiments comprises the ethylene-based polymer. In a twenty-fifth embodiment, the polymer component in the solid carrier component of any one of the twentieth to twenty-fourth embodiments comprises high density polyethylene. In a twenty-sixth embodiment, the polymer component in the solid carrier component in any one of the twentieth to twenty-fifth embodiments comprises high density polyethylene with a melt index of 2.3 g/10 min to 20 g/10 min. In a twenty-seventh embodiment, the polymer component in any one of the twentieth to twenty-sixth embodiments further comprises the maleated ethylene-based polymer. In a twenty-eighth embodiment, the polymer component in any one of the twentieth to twenty-sixth embodiments does not include the maleated ethylene-based polymer. In a twenty-ninth embodiment, the polymer component in any one of the twentieth to twenty-third embodiments comprises the maleated ethylene-based polymer and does not include the ethylene-based polymer. In a thirtieth embodiment, the filler is present in the solid carrier component in any one of the twentieth to twenty-ninth embodiments, and the filler comprises talc. | 89,122 |
11859087 | DETAILED DESCRIPTION The technical solutions of the present application will be described clearly and completely with reference to the accompanying drawings. It is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without any creative efforts are within the scope of the present application. Further, the technical features involved in the different embodiments of the present application described below may be combined with each other as long as a conflict is constituted. The basic chemical raw materials such as reagents used in the embodiments of the present application can be purchased in the domestic chemical product market, or can be customized in the relevant intermediate preparation factory. Example 1 In example 1, a method of preparing an intermediate (I′) was provided. The preparation method was as follows: (1) Preparation of an Intermediate I-1 Phenylhydrazine was added to glacial acetic acid with stirring, and then 3-methyl-2-butanone was slowly added dropwise, heating to 60° C. for reaction for 3-4 hours to obtain a reaction product after completing such addition of 3-methyl-2-butanone. The reaction product was extracted, concentrated, and refined to obtain the intermediate I-1. Wherein, a molar ratio of phenylhydrazine to 3-methyl-2-butanone was 1:1.0. (2) Preparation of an Intermediate I-2 The intermediate I-1 and 1,2-dibromoethylene were added into toluene, and then heated to reflux in the presence of nitrogen for 16 hours to obtain a reaction product. The reaction product was cooled to precipitate a solid to obtain the intermediate I-2. Wherein, a molar ratio of the intermediate I-1 to 1,2-dibromoethylene was 1:1.5. (3) Preparation of an Intermediate I-4 Dry N,N-dimethylformamide was added to dry dichloromethane, and a solution of phosphorus oxychloride in dichloromethane was added with stirring in an ice bath, and cyclohexanone was added and then the ice bath was removed, followed by heating to reflux for 2 hours to obtain a reaction solution. Finally the reaction solution was poured into crushed ice, and left standing overnight to precipitate a solid to obtain the intermediate I-4. Wherein, the molar ratio of the cyclohexanone, N,N-dimethylformamide, and phosphorus oxychloride was 1:1.0:1.0. (4) Preparation of an Intermediate I′ The intermediate I-2 and the intermediate I-4 were added to a mixture of n-butanol and toluene, heating to reflux for 2 hours and precipitating a solid which was filtered to obtain the intermediate (I′). In this example, the nuclear magnetic spectrum of the intermediate I′ was shown inFIG.1, and the intermediate I′ was detected and characterized, the obtained data were as follows: Elemental Analysis Calculated Value: C34H36Br3N3 Mass Spectrum (MS+): 723.05 (M+) m/z: 725.04 (100.0%), 727.04 (97.7%), 726.05 (37.2%), 728.05 (37.0%), 723.05 (34.3%), 729.04 (31.9%), 724.05 (12.7%), 730.04 (12.0%), 727.05 (6.7%), 729.05 (6.5%), 725.05 (2.4%), 731.05 (2.1%), 726.04 (1.1%), 728.04 (1.1%). Elemental analysis: C, 56.22; H, 5.00; Br, 33.00; N, 5.78. Example 2 In example 2, a method of preparing an intermediate (I′) was provided. The preparation method was as follows: (1) Preparation of an Intermediate I-1 Phenylhydrazine was added to glacial acetic acid with stirring, and then 3-methyl-2-butanone was slowly added dropwise, heating to 62.5° C. for reaction for 3-4 hours to obtain a reaction product after completing such addition of 3-methyl-2-butanone. The reaction product was extracted, concentrated, and refined to obtain the intermediate I-1. Wherein, a molar ratio of phenylhydrazine to 3-methyl-2-butanone was 1:1.1. (2) Preparation of an Intermediate I-2 The intermediate I-1 and 1,2-dibromoethylene were added into toluene, and then heated to reflux in the presence of nitrogen for 17 hours to obtain a reaction product. The reaction product was cooled to precipitate a solid to obtain the intermediate I-2. Wherein, a molar ratio of the intermediate I-1 to 1,2-dibromoethylene was 1:1.75. (3) Preparation of an Intermediate I-4 Dry N,N-dimethylformamide was added to dry dichloromethane, and a solution of phosphorus oxychloride in dichloromethane was added with stirring in an ice bath, and cyclohexanone was added, and then the ice bath was removed, followed by heating to reflux for 2.5 hours to obtain a reaction solution. Finally the reaction solution was poured into crushed ice, and left standing overnight to precipitate a solid to obtain the intermediate I-4. Wherein, the molar ratio of the cyclohexanone, N,N-dimethylformamide, and phosphorus oxychloride was 1:1.05:1.025. (4) Preparation of an Intermediate I′ The intermediate I-2 and the intermediate I-4 were added to a mixture of n-butanol and toluene, heating to reflux for 2.5 hours and precipitating a solid, which was filtered to obtain the intermediate (I′). Example 3 In example 3, a method of preparing an intermediate (I′) was provided. The preparation method was as follows: (1) Preparation of an Intermediate I-1 Phenylhydrazine was added to glacial acetic acid with stirring, and then 3-methyl-2-butanone was slowly added dropwise, heating to 65° C. for reaction for 4 hours to obtain a reaction product after completing such addition of 3-methyl-2-butanone. The reaction product was extracted, concentrated, and refined to obtain the intermediate I-1. Wherein, a molar ratio of phenylhydrazine to 3-methyl-2-butanone was 1:1.2. (2) Preparation of an Intermediate I-2 The intermediate I-1 and 1,2-dibromoethylene were added into toluene, and then heated to reflux in the presence of nitrogen for 18 hours to obtain a reaction product. The reaction product was cooled to precipitate a solid to obtain the intermediate I-2. Wherein, a molar ratio of the intermediate I-1 to 1,2-dibromoethylene was 1:2.0. (3) Preparation of an Intermediate I-4 Dry N,N-dimethylformamide was added to dry dichloromethane, and a solution of phosphorus oxychloride in dichloromethane was added with stirring in an ice bath, and cyclohexanone was added, and then the ice bath was removed, followed by heating to reflux for 3 hours to obtain a reaction solution. Finally the reaction solution was poured into crushed ice, and left standing overnight to precipitate a solid to obtain the intermediate I-4. Wherein, the molar ratio of the cyclohexanone, N,N-dimethylformamide, and phosphorus oxychloride was 1:1.1:1.05. (4) Preparation of an Intermediate I′ The intermediate I-2 and the intermediate I-4 were added to a mixture of n-butanol and toluene, heating to reflux for 2-3 hours and precipitating a solid, which was filtered to obtain the intermediate (I′). Example 4 In example 4, the compound (A) was prepared by using the intermediate (I′) obtained in Example 1 as a raw material for carrying out a conventional amino substitution reaction. Bromoacetic acid was added to the intermediate (I′) for a reaction, and NaOH was added to obtain the desired compound (A). The nuclear magnetic spectrum of the compound (A) was shown inFIG.2, and the structure of the obtained compound (A) was confirmed to be correct after detection. Elemental Analysis Calculated Value: C36H38Br2N3O2+ Mass Spectrum (MS+): 702.13 (M+) m/z: 704.13 (100.0%), 702.13 (51.3%), 706.13 (49.0%), 705.13 (40.0%), 703.14 (20.2%), 707.13 (19.6%), 706.14 (8.0%), 704.14 (4.1%)), 708.14 (3.8%), 707.14 (1.3%), 705.14 (1.1%). Elemental analysis: C, 61.37; H, 5.44; Br, 22.68; N, 5.96; 0, 4.54. Example 5 In example 5, the compound (B) was prepared by using the intermediate (I′) obtained in Example 1 as a raw material for carrying out a conventional amino substitution reaction. Bromomethanol was added to the intermediate (I′) for a reaction, and NaOH was added to obtain the desired compound (B). The structure of the obtained compound (B) was confirmed to be correct after detection. Elemental Analysis Calculated Value: C35H38Br2N3O+ Mass Spectrum (MS+): 674.14 (M+) m/z: 676.14 (100.0%), 674.14 (49.5%), 678.13 (46.8%), 677.14 (36.9%), 675.14 (19.5%), 679.14 (18.1%), 678.14 (7.3%), 680.14 (3.5%), 677.13 (1.1%). Elemental analysis: C, 62.14; H, 5.66; Br, 23.62; N, 6.21; 0, 2.37. Example 6 In example 6, the compound (C) was prepared by using the intermediate (I′) obtained in Example 1 as a raw material for carrying out a conventional amino substitution reaction. 1-bromo-m-methylbenzoic acid was added to the intermediate (I′) for a reaction, and NaOH was added to obtain the desired compound (C). The structure of the obtained compound (C) was confirmed to be correct after detection. Elemental Analysis Calculated Value: C42H42Br2N3O2+ Mass Spectrum (MS+): 778.16 (M+) m/z: 780.16 (100.0%), 778.16 (51.3%), 782.16 (49.0%), 781.17 (46.8%), 779.17 (23.6%), 783.16 (22.6%), 782.17 (10.7%), 780.17 (5.5%), 784.17 (5.1%), 783.17 (2.0%), 781.16 (1.1%). Elemental analysis: C, 64.62; H, 5.42; Br, 20.47; N, 5.38; 0, 4.10. Example 7 In example 7, the compound (D) was prepared by using the intermediate (I′) obtained in Example 1 as a raw material for carrying out a conventional amino substitution reaction. Bromomethylphosphonic acid was added to the intermediate (I′) for a reaction, and NaOH was added to obtain the desired compound (D). The structure of the obtained compound (D) was confirmed to be correct after detection. Elemental Analysis Calculated Value: C35H39Br3N3O3P Mass Spectrum (MS+): 817.03 (M+) m/z: 819.03 (100.0%), 821.02 (94.7%), 820.03 (37.4%), 822.03 (36.7%), 817.03 (33.4%), 823.02 (31.1%), 824.03 (12.9%), 818.03 (12.8%), 821.03 (7.8%), 823.03 (7.4%), 825.03 (2.4%), 820.02 (1.1%), 822.02 (1.1%). Elemental analysis: C, 51.24; H, 4.79; Br, 29.22; N, 5.12; 0, 5.85; P, 3.78. Examples 8-15 In examples 8-15, compounds (E)-(L) were synthesized. The principle and method of preparing compounds (E)-(L) were the same as those in examples 4-7, i.e. a conventional substitution of the amino group on the intermediate (I′) was carried out with a suitable raw material. EXPERIMENTAL EXAMPLES In order to verify the fluorescence performance of fluorescent dyes of the present application, fluorescence spectrum, molar extinction coefficient, and fluorescence quantum yield were measured. Experimental Example 1 Determination of the Absorption Spectrum of Fluorescent Dyes The compounds prepared in Example 1 and Examples 4-6 were accurately weighed to prepare a solution having a concentration of 1.0×10−5mol/L using 50% by volume of ethanol. The absorption spectrum of the solution was measured, and the result was shown inFIG.2. InFIG.2,1represents the intermediate prepared in Example 1, and 2-4 represent the compound prepared in Examples 4-6, respectively. Experimental Example 2 Determination of the Fluorescence Spectrum of Fluorescent Dyes The fluorescence spectrum was measured using the maximum absorption wavelength in the measured near-infrared spectrum as the excitation wavelength of the fluorescence spectrum. The compounds prepared in Example 1 and Examples 4-6 were accurately weighed to prepare a solution of ethanol and water (50:50, v/v) at a concentration of 1.0×10−6mol/L. The emission spectrum of the solution was measured, and the result was shown inFIG.1. InFIG.1,1represents the intermediate prepared in Example 1, and 2-4 represent the compounds prepared in Examples 4-6, respectively. Experimental Example 3 Determination of Molar Extinction Coefficient of Fluorescent Dyes The molar extinction coefficient of the compound was determined by UV-visible absorption spectroscopy. The formula for calculating molar extinction coefficient is as shown in equation (1): A=εclEquation (1), wherein A represents ultraviolet absorption value, ε represents molar extinction coefficient, c represents concentration of the compound, and l represents thickness of the quartz cell for detection. Experimental Example 4 Determination of Fluorescence Quantum Yield of Fluorescent Dyes The fluorescence quantum yield of the fluorescent dyes was determined at 20° C., and quinine sulfate (Solvent: 0.1M H2SO4, Quantum yield: 0.56) was used as a reference compound, then the fluorescence quantum yield was calculated by measuring fluorescence integral intensities obtained from the dilute solutions of the fluorescent dyes and the reference compound under the same excitation conditions and the ultraviolet absorption value at this excitation wavelength. The product was dissolved in absolute ethanol. The formula for calculating fluorescence quantum yield is as shown in equation (2): Φ=ΦB×IIR×ARA×η2ηR2,Equation(2) wherein Φ represents quantum yield of the compound to be measured, the subscript R represents reference compound, I represents fluorescence integral intensity, A represents ultraviolet absorption value, and η represents solvent refractive index. Generally, the ultraviolet absorption values A and AR are less than 0.1. TABLE 1Spectroscopic properties of the fluorescent dyesλabsλemε × 104ΦStokes(max/nm)(max/nm)(M−1cm−1)(%)shift (nm)Example 17788098.080.3935Example 47858158.584.9938Example 57878208.686.0040Example 67908299.089.8634 As shown in Table 1, the intermediates and fluorescent dyes of the present application have the maximum absorption wavelength, corresponding to the maximum emission wavelength of 829 nm, the maximum molar absorption coefficient of 9.0, and the maximum fluorescence quantum yield of 89.86%, which indicates that the compounds have the advantage of being used for covalent fluorescent labeling of biological macromolecules such as nucleic acids or proteins. It is apparent that the above embodiments are merely examples for clarity of illustration, and are not intended to limit the embodiments. Other variations or modifications of the various forms may be made by those skilled in the art in view of the above description. There is no need and no way to present all of the embodiments herein. The obvious variations or modifications derived therefrom are still within the scope of protection of the present application. | 14,219 |
11859088 | DESCRIPTION OF EMBODIMENTS <Color Material> Hereafter, the present invention will be described on the basis of a suitable embodiment. A color material aqueous solution according to the present invention contains phycocyanin (A), a polyvalent carboxylic acid (B) containing one or more hydroxy groups, and at least one kind of nonionic emulsifier (C) selected from the group consisting of sucrose esters and polyglycerol esters, the at least one kind of nonionic emulsifier (C) having an HLB of 15 or more. (Phycocyanin) Phycocyanin (A) is one kind of phycobiliprotein and is a blue pigment containing phycocyanobilin and protein and being a solid at a normal temperature. The phycocyanin (A) may be C-phycocyanin, allophycocyanin, or R-phycocyanin. The phycocyanin (A) can be obtained, for example, through extraction thereof from algae using a freely selected technique. As the phycocyanin (A), any of publicly known and commonly used phycocyanin (A) can be used, and examples thereof include algal pigments derived from various algae such as blue-green algae and red algae. The phycocyanin (A) may be a natural product. However, the phycocyanin (A) is preferably a Spirulina pigment containing, as a main component, phycobiliprotein of blue-green algae belonging to the genus Arthrospira or the genus Spirulina (which hereafter may be referred to as “Spirulina”) due to its capability of being artificially cultivated through various culture methods and its ease of availability. The phycocyanin (A) per se mainly exhibits a blue color and thus is used as a blue color material. The method for obtaining phycocyanin from “Spirulina” is not particularly limited. However, phycocyanin can be extracted, for example, through a method of extraction thereof from Spirulina and into a buffer liquid. For example, phycocyanin can be obtained through a method disclosed in the literature (Japanese Unexamined Patent Application Publication No. 52-134058). (Polyvalent Carboxylic Acid Containing One or More Hydroxy Groups) The polyvalent carboxylic acid (B) containing one or more hydroxy groups is a carboxylic acid containing one or more hydroxy groups and two or more carboxy groups [and is hereafter abbreviated as “carboxylic acid (B)”]. In the present invention, the definition of the carboxylic acid (B) does not include a salt of the carboxylic acid. The carboxylic acid (B) is a compound containing free carboxyl groups not converted to salts. The carboxylic acid (B) may be a solid or a liquid at a normal temperature. Because of being used in combination with the phycocyanin (A), the carboxylic acid (B) per se preferably has the same color, taste, and smell as the phycocyanin (A) or is preferably colorless, tasteless, and odorless. When the (A) and (B) according to the present invention are used under acidic conditions, the carboxylic acid (B) may have a sour taste. Examples of the carboxylic acid (B) include citric acid, malic acid, and tartaric acid. Citric acid is particularly preferably used as the carboxylic acid (B) because the structural stability of the phycocyanin (A) under acidic conditions is increased when the phycocyanin (A) and the polyvalent carboxylic acid (B) containing one or more hydroxy groups are combined. The inventors have found that the emulsifier (C) has an effect of enhancing the thermal stability of the phycocyanin (A), particularly under acidic conditions, as illustrated in Examples described later. The effect is inherent to the emulsifier (C). In the present invention, the dispersion stability of the phycocyanin (A) in an aqueous solution can be enhanced when at least one kind of nonionic emulsifier (C) selected from the group consisting of sucrose esters and polyglycerol esters, the at least one kind of nonionic emulsifier (C) having an HLB of 15 or more [and hereafter being abbreviated as “emulsifier (C)”], is further contained. The term “HLB” is an acronym for “Hydrophilic-Lipophilic Balance” and is an indicator indicating the balance between the hydrophobicity and hydrophilicity of an emulsifier. The HLB value can be a value of 0 to 20, and a larger HLB value denotes higher hydrophilicity. It is important that this emulsifier (C) is neither an anionic emulsifier nor a cationic emulsifier but a nonionic emulsifier with which an object to be emulsified undergoes less changes in emulsion stability with respect to pH changes and that this emulsifier (C) is highly hydrophilic. Examples of the emulsifier (C) include sucrose esters and polyglycerol esters, and at least one kind selected from the group consisting of the foregoing can be used. A sucrose ester is an ester of sucrose and a fatty acid derived from vegetable oil, existing in various HLBs depending on the fatty acid composition. A polyglycerol ester is an ester of polyglycerol obtained by dehydration-condensing glycerol derived from vegetable oil and a fatty acid derived from vegetable oil, existing in various HLBs depending on the degree of polymerization of the polyglycerol and the kind and degree of esterification of the fatty acid. Examples of the sucrose ester include RYOTO (registered trade mark) Sugar Ester (Mitsubishi-Chemical Foods Corporation) and DK ESTER (registered trade mark) (Dai-ichi Kogyo Seiyaku Co., Ltd.) with various corresponding stock numbers. Examples of the polyglycerol ester include RYOTO (registered trade mark) Polyglyester and SY-Glyster (Sakamoto Yakuhin Kogyo, Co., Ltd.) with various corresponding stock numbers. Compared with emulsifiers having a different chemical structure, these have an excellent affinity with the phycocyanin (A), an excellent affinity with water (D), and excellent emulsifying properties regardless of fluctuations in pH, thereby contributing to a more excellent stabilizing effect with a smaller amount used. In the present invention, the amount of the emulsifier (C) used is not particularly limited. However, when the emulsifier (C) is contained as a non-volatile component in an amount of, for example, 1 g to 30 g, particularly 4 g to 14 g, and most preferably 4 g to 8 g with respect to 1 g of the phycocyanin (A), the dispersion stability of the phycocyanin (A) in an aqueous solution can be enhanced, and changes in the color value per se (color value retention rate) and changes in the color tone (color difference) before and after being subjected to a thermal history can be kept small. Thus, such an amount is preferable. With what is not the emulsifier (C), the above-described superiority when the emulsifier (C) as used in the present invention is selected for use is less likely to be obtained. For example, an anionic surfactant such as sodium dodecyl sulfate, when assumed to be used for food purposes, is largely restricted in the amount used due to various regulations of each country, and an anionic surfactant such as oleic acid is likely to be subjected to changes in stability depending on the pH when used. In preparation of the color material aqueous solution according to the present invention, the above-described emulsifier (C) may be used alone, but as needed, the emulsifier (C) may be used in combination with other emulsifiers. Examples of such emulsifiers that can be used in combination include sorbitan fatty acid esters, propylene glycol fatty acid esters, lecithins, lysolecithins, citric acid monoglyceride, saponins, and casein sodium. Examples other than the foregoing and limited in use include oxyethylene fatty acid alcohol, sodium oleate, morpholine fatty acid salts, polyoxyethylene higher fatty acid alcohol, calcium stearoyl lactylate, and monoglyceride ammonium phosphate. As the water (D), any of publicly known and commonly used water can be used, and, for example, distilled water, ion-exchanged water, or purified water can be used. In the present invention, because the above-described carboxylic acid (B) is used, it is preferable to use water having a pH of 7, the water containing no ionic component or containing an ionic component as little as possible. (Regarding Component Proportion) The color material aqueous solution according to the present invention contains, as essential components, the phycocyanin (A), the polyvalent carboxylic acid (B) containing one or more hydroxy groups, the emulsifier (C), and the water (D) and is prepared such that, with respect to 1 g of the phycocyanin (A), the polyvalent carboxylic acid (B) containing one or more hydroxy groups is contained in an amount of 200 mmol to 400 mmol in terms of carboxylic acid equivalent and the emulsifier (C) is contained in an amount of 1 g to 30 g. The content of the water (D) in the color material aqueous solution is not particularly limited, but the water (D) can be used such that, by mass, with respect to a total of 100 parts of the phycocyanin (A) and the polyvalent carboxylic acid (B) containing one or more hydroxy groups, 4000 parts to 25000 parts of the water (D) is contained. When the phycocyanin (A), the polyvalent carboxylic acid (B) containing one or more hydroxy groups and the emulsifier (C) are dissolved in water (D) to achieve the above-described component proportion, the pH of the resulting aqueous solution is 3 or less. Although depending on the combination proportion of the carboxylic acid (B) and the emulsifier (C) dissolved in the water (D), as the total amount of the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) is increased, a more concentrated color material aqueous solution having a darker blue color is obtained, in which case, for example, dilution is performed as needed. This is preferable because when coloring with the same color value is performed, such coloring can be performed on a larger quantity of objects to be colored. Reasonably, instead of once preparing a concentrated color material aqueous solution and thereafter diluting the color material aqueous solution, a dilute color material aqueous solution may be immediately obtained to achieve a concentration that matches the use and purpose without once preparing a concentrated color material aqueous solution. (Absorbance) Furthermore, the color material aqueous solution according to the present invention has an optical density of 0.05 or less with respect to 1 cm of optical path length at 800 nm at a hydrogen ion concentration index (pH) of 3 or less when all the (A), (B), and (C) are added such that the color value expressed as the absorbance at 620 nm is 0.4 to 1.0. Here, the expression “all the (A), (B), and (C) are added such that the color value expressed as the absorbance at 620 nm is 0.4 to 1.0” refers to a status in which all the (A), (B), and (C) are added such that the color value is a color value at any one point selected in the range of 0.4 to 1.0. Likewise, the expression “an optical density at a hydrogen ion concentration index (pH) of 3 or less” refers to an optical density at any one point selected in the range of a hydrogen ion concentration index (pH) of 3 or less. The excellent thermal stability of coloring with the color material aqueous solution according to the present invention under acidic conditions can be confirmed by performing a comparison of optical density between an existing publicly known color material aqueous solution and the color material aqueous solution according to the present invention under the conditions in which the color value and the pH are fixed in the above-described range. In the present invention, it is defined as “all the (A), (B), and (C) are added such that the color value expressed as the absorbance at 620 nm is 0.4 to 1.0″, but the color value of 0.4 to 1.0 is merely a measurement condition for determining the optical density with respect to 1 cm of optical path length at 800 nm using a color material aqueous solution having a hydrogen ion concentration index (pH) of 3 or less. As described above, not only a concentrated color material aqueous solution but also a dilute color material aqueous solution is acceptable, and a color material aqueous solution whose color value deviates from the range of 0.4 to 1.0 does not indicate the uniform exclusion thereof from the technical scope of the present invention. That is, in the present invention, it is assumed that the optical density is measured in the color value range of 0.4 to 1.0, and in the case of a color material aqueous solution having a color value of less than 0.4, for example, concentration is performed to measure the optical density at any point in the color value range of 0.4 to 1.0. On the other hand, in the case of a color material aqueous solution having a color value of more than 1.0, for example, dilution is performed to measure the optical density at any point in the color value range of 0.4 to 1.0. When the results are 0.05 or less, the color material aqueous solution can be determined as belonging to the technical scope of the present invention. When coloring is performed on an object to be colored using a color material aqueous solution, in the case where the color material aqueous solution contains a certain component that is insoluble, the color material aqueous solution per se looks turbid, providing a negative sensory impression. Even if coloring per se can be performed on an object to be colored to some extent, partial coloring unevenness may result. In the case where the object to be colored is, for example, a beverage, substantial inconvenience, such as poor texture when ingested, may result. The degree of presence of water insolubles represented by phycocyanin aggregates can be evaluated by measuring the optical density at an incident light wavelength of 800 nm. As the optical density measurement value (OD) is smaller, the content of water insolubles is smaller, and a color material aqueous solution having smaller or no defects such as those described above is obtained. (Method for Manufacturing Color Material Aqueous Solution) The color material aqueous solution according to the present invention can be prepared by mixing the phycocyanin (A), the polyvalent carboxylic acid (B) containing one or more hydroxy groups, the emulsifier (C), and the water (D) in a predetermined component proportion as described above by mass and by dissolving the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) in the water (D). In a method for manufacturing a color material aqueous solution according to the present invention, the phycocyanin (A) per se may be used as the raw material, but as needed, the phycocyanin (A) may be replaced with publicly known and commonly used phycobiliprotein containing the phycocyanin (A). When the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) are dissolved in the water (D), examples of the method include a method in which a solution of the phycocyanin (A) in the water (D) is prepared and thereafter the carboxylic acid (B) and the emulsifier (C) are further dissolved in the solution, a method in which a solution of the carboxylic acid (B) in the water (D) is prepared and thereafter the phycocyanin (A) and the emulsifier (C) are further dissolved in the solution, and a method in which the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) are dissolved in the water (D) at the same time in parallel. As the method of making the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) in contact with one another in the water (D), a publicly known mixing method can be employed. For example, various dynamic and static mixing methods using a juicer, a mixer, a mill, a micromixer, or the like in which stirring with stirring blades, collision of liquids with one another, and repeated liquid coalescence and separation, and the like are performed can be operated in a freely selected manner. The phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) may be mixed at one time or in portions or an aqueous solution of each of the foregoing may be prepared and thereafter mixed with the water (D). In preparation of an aqueous solution, the temperatures of the phycocyanin (A), the carboxylic acid (B), the emulsifier (C), and the water (D) are not particularly limited, and may be appropriately set according to the kinds of the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) used. However, for example, any of these temperatures is 0° C. or more and 50° C. or less. The difference in temperature between the foregoing four is preferably as small as possible, because in this case flaws such as aggregation are less likely to occur during mixing. In the method for manufacturing a color material aqueous solution according to the present invention, for example, a step of dissolving phycocyanin (A) the polyvalent carboxylic acid (B) containing one or more hydroxy groups and emulsifiers (C) in water (D) is performed as an essential step to thereby manufacture a color material aqueous solution having a hydrogen ion concentration index (pH) of 3 or less. Color material aqueous solutions having various concentrations obtained through this step of dissolution in water can be immediately used for coloring performed on an object to be colored, but an aging step, for example, aging for 5 to 48 hours and at a temperature of 15° C. to 30° C., may be included. In the method for manufacturing a color material aqueous solution, in the step of dissolving the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) in the water (D), when a tiny amount of water insolubles including phycocyanin aggregates is produced, it is more preferable to perform a step of removing such water insolubles following the step of dissolution. It is acceptable to once prepare a concentrated color material aqueous solution and thereafter dilute the color material aqueous solution to thereby prepare a dilute color material aqueous solution having a desired concentration. However, preparing a dilute color material aqueous solution having a desired concentration without passing through a step of preparing a concentrated color material aqueous solution is preferable. This is because this approach makes flaws such as aggregation as described above less likely to occur, makes the degree of aggregation minor if any aggregation occurs, and enables the step of removing water insolubles to be skipped or enables increased ease and increased reduction in the load involved in the removal step, thereby contributing to the enhancement of productivity. In the step of removing water insolubles, for example, techniques such as decantation, filtration, or centrifugation can be employed. In the method for manufacturing a color material aqueous solution according to the present invention, as needed, under conditions in which water insolubles such as those mentioned above are less likely to be produced, a concentration step may be further included as an essential step to concentrate a color material aqueous solution. In the present invention, when the carboxylic acid (B) and the emulsifier (C) are dissolved in the water (D) together with the phycocyanin (A), the solubility of the phycocyanin (A) in the resulting aqueous solution is enhanced by a certain interaction. The details are unknown, but conceivably, the carboxyl groups of the carboxylic acid (B) and the hydrophobic portions of the emulsifier (C) are selectively oriented toward the surface side of the phycocyanin (A), and the hydroxy groups (hydroxyl groups) are selectively oriented toward the water (D) side, thereby further enhancing the solubility of the phycocyanin (A) in the water (D) on the basis of the hydroxy groups. On the other hand, very surprisingly, the present inventors have found that even a color material aqueous solution from which water insolubles such as those mentioned above have been removed per se has excellent thermal stability. Furthermore, an aqueous solution thus obtained is, because of comprising the emulsifier (C), can be suitably used for food purposes. In the color material aqueous solution according to the present invention, due to the effect of the carboxylic acid (B) and the emulsifier (C), the production of water insolubles is effectively prevented or reduced, and, even after being subjected to a thermal history, a status in which the phycocyanin (A) is stably and uniformly dissolved in the liquid can be retained. In the method for manufacturing a color material aqueous solution according to the present invention, the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C) are contained in the water (D), and, as needed, water insolubles, such as those mentioned above, that can be produced at an initial stage of mixing the foregoing are removed to thereby be able to easily obtain a color material aqueous solution having excellent thermal stability, the color material aqueous solution undergoing less fading under acidic conditions. (Other Components) In the color material aqueous solution, in addition to the phycocyanin (A), the carboxylic acid (B), and the emulsifier (C), other components not corresponding to the foregoing may be contained as non-volatile components. Examples of such other components which are non-volatile components include salts of polyvalent carboxylic acids containing one or more hydroxy groups, excipients, preservatives, various vitamins, various minerals, various saccharides, substances derived from the above-described algae other than protein pigments, and substances derived from culture medium components of the above-described algae. Other than non-volatile components, the color material aqueous solution according to the present invention may further contain an organic solvent having a miscibility with water. As the organic solvent, for example, ethanol or isopropanol can be used. Examples of a salt (E) of a polyvalent carboxylic acid containing one or more hydroxy groups include metal salts and amine salts (hereafter referred to as conjugate bases) of the carboxylic acid (B) exemplified above. Examples of the metal salts include sodium salts, potassium salts, calcium salts, and aluminum salts. A polyvalent carboxylic acid containing one or more hydroxy groups, with at least some of the carboxyl groups contained therein having the above-described salt structure, corresponds to the above-mentioned salt, but one in which all the carboxyl groups have the above-described salt structure is preferably used in view of the ease of handling and raw material procurement. The technical effect of the present invention is on the basis of a function inherent and unique to the carboxylic acid (B), but a salt (E) of the carboxylic acid equivalent to the conjugate base of the carboxylic acid (B) may be further contained in a small amount within the scope that does not impair the technical effect. The number of moles used of the salt (E) of the carboxylic acid is in a range smaller than that of the carboxylic acid (B), and specifically, it is preferable that a salt (E) of the carboxylic acid/the carboxylic acid (B)=1/99 to 20/80 by number of moles is satisfied because the thermal stability under acidic conditions is excellent. When citric acid is used as the carboxylic acid (B) and a salt (E) thereof is used, specifically, it is preferable that a salt (e) of citric acid/citric acid (b)=10/90 to 20/80 by number of moles is satisfied because the thermal stability under acidic conditions is excellent. For example, color materials containing at least one kind of compound selected from the group consisting of saccharides, sugar alcohols, and polyvalent alcohols have been known. Existing color materials may have contained these compounds in a large amount. On the other hand, the color material according to one embodiment of the present invention can improve the thermal stability of the phycocyanin (A) only with the carboxylic acid (B) and the emulsifier (C) and without containing these compounds. Saccharides such as trehalose may be contained in the color material aqueous solution according to the present invention, but their contribution to the enhancement of thermal stability under acidic conditions cannot be expected. The proportion (% by mass) of the phycocyanin (A) in 100% by mass of non-volatile components of the color material can be obtained through a publicly known analysis method/measurement method. The proportion (% by mass) of the carboxylic acid (B) in 100% by mass of a solid content of the color material is obtained through a publicly known analysis method/measurement method. For example, the mass of the phycocyanin (A) can be measured on the basis of the absorbance of a color material aqueous solution in which the phycocyanin (A) is dissolved in a solvent such as water. In generally known phycocyanin (A), the relationship between the maximum absorption wavelength of phycocyanin (A) in a solution and the concentration % (w/v) of phycocyanin (A) is known, and the proportion (% by mass) of phycocyanin (A) can be calculated on the basis of the absorbance at the maximum absorption wavelength of the color material solution. For example, in accordance with a method described in the literature (Yoshikawa, N. and Belay, A. (2008) “Single-laboratory validation of a method for the determination of c-phycocyanin and allophycocyanin in Spirulina (Arthrospira) supplements and raw materials by spectrophotometry” Journal of AOAC International VOL. 91, 524-529), the concentration of C-phycocyanin (cPC) in a sample (g/L) and the concentration of allophycocyanin (aPC) in a sample (g/L) can be calculated from the value of the absorbance at the maximum absorption wavelength. Here, the maximum absorption wavelength of cPC in the color material solution is 620 nm and the maximum absorption wavelength of aPC in the color material solution is 650 nm. Phycocyanin in Spirulina can be determined as a sum of cPC and aPC. For example, the concentration of cPC in a sample can be calculated using the formula below. cPC(mg/mL)=0.162×Abs620−0.098×Abs650 For example, the concentration of aPC in a sample can be calculated using the formula below. aPC(mg/mL)=0.180×Abs650−0.042×Abs620 The measurement wavelength is appropriately determined according to the maximum absorption wavelength of a sample solution. For example, when the following phycocyanin (A) is contained as a main component among the pigments contained in the sample solution, the measurement wavelength is 610 nm to 630 nm. An optimal point is determined in this range to perform measurement. <Use> The color materials according to the embodiment is suitable to be added to: confections and breads such as ice cream, soft cream, cakes, Bavarian cream, yokan, jelly, gum, gummies, and chocolate; noodles such as soba noodles, udon noodles, and somen noodles; various foods such as tofu, fish cakes, and boiled flat fish cakes; beverages such as matcha green tea beverages, green tea beverages, milk beverages, soy milk beverages, vegetable beverages, fruit beverages, and soft beverages; and medicines, such as tablets, and cosmetics. With the color material aqueous solution according to the present invention, a beverage colored in blue (blue-colored beverage) having an intended color value can be easily prepared, for example, by diluting a concentrated color material aqueous solution with a solvent as needed to achieve a targeted and desired color value or by concentrating a dilute color material aqueous solution as needed to achieve a targeted and desired color value and thereafter further mixing the resulting color material aqueous solution with a beverage. Particularly in a soft beverage or a fruit beverage (juice) exhibiting its own acidity as a beverage, the color material aqueous solution according to the present invention has a remarkable effect in which excellent thermal resistance is exhibited and even after being subjected to a thermal history such as heat sterilization, no or less fading occurs. Because a soft beverage or a fruit beverage often serves as an acidic buffer solution, for the sake of convenience, such an acidic buffer solution is regarded as a simulated beverage which is an object to be colored. Thus, by diluting the color material aqueous solution according to the present invention and observing a change in the color value before and after heating, the degree of fading under acidic conditions can be confirmed. The color material aqueous solution according to the present invention alone is suitable for coloring, but the color material aqueous solution may be provided in the form of a composite with other color materials. Examples of such other color materials include not only safflower yellow, gardenia yellow, matcha green tea, and green tea, but also green-colored powders of, for example, young barley leaves, kale, mulberry, bamboo grass, Molokhia, chlorella, green shiso, broccoli, spinach, bell pepper, and ashitaba. EXAMPLES Hereafter, the present invention will be described on the basis of Examples. <Measurement of Absorbance of Solution> The absorbance of a solution was measured with a UV/Vis spectrophotometer (U-3900H, manufactured by Hitachi High-Tech Science Corporation) using a quartz cell having an optical path length of 1 cm. The number following the display of “Abs” referring to an absorbance denotes a measurement wavelength (nm). <Thermal Resistance and Acid Resistance Evaluation of Phycocyanin-Containing Blue Color Material Aqueous Solution> The color value retention rate Abs % before and after heating at Abs620is calculated using the formula below. The thermal resistance and acid resistance can be evaluated according to the value of this Abs %. Abs %=(Abs620after)/(Abs620before)×100 Here, the absorbances before and after heating are expressed as “Abs before” and “Abs after”, respectively. Example 1 A total of 0.12% by mass of LINA BLUE G1 (a vegetable blue pigment manufactured by DIC LIFETECH Corporation containing 30% by mass of phycocyanin equivalent, 5% by mass of trisodium citrate, 55% by mass of trehalose, and 10% by mass of other components), 0.125% by mass of RYOTO (registered trade mark) Polyglyester L-7D (HLB=17) (a mass ratio of phycocyanin/emulsifier=0.288), and citric acid and trisodium citrate were dispersed in pure water to achieve a final citric acid concentration of 50 mM and a pH of 2.5. The resulting mixture was centrifuged under conditions of 25° C.×9300 G×5 minutes to thereby remove a precipitate formed of water insolubles including phycocyan aggregates, and the resulting supernatant was collected to obtain a simulated blue-colored beverage. Next, 1 ml of the simulated blue-colored beverage was dispensed into a 1.5 ml-plastic tube to obtain a simulated blue-colored beverage before heating serving as a sample. The sample was heat treated under conditions of 70° C.×30 minutes in a tabletop incubator (WSL-2610, manufactured by ATTO Corporation), iced for 5 minutes, and thereafter centrifuged under conditions of 25° C.×9300 G×5 minutes to obtain a simulated blue-colored beverage after heating. The absorbance of the simulated blue-colored beverage before and after heating was measured under the following conditions, and thereafter the color value retention rate was calculated, the color value retention rate serving as an indicator of the thermal stability of pigments under acidic conditions. The color value retention rate calculated in accordance with the above was 85.5%. Example 2 The same procedure was performed as in Example 1 to obtain a simulated blue-colored beverage (a mass ratio of phycocyanin/emulsifier=0.036) except that the content of RYOTO (registered trade mark) Polyglyester L-7D was changed from 0.125% by mass to 1.00% by mass. The same procedure was performed as in Example 1 to heat treat the simulated blue-colored beverage, and the color value retention rate before and after heating was measured. The results indicated that the color value retention rate was 91.7%. Example 3 The same procedure was performed as in Example 1 to obtain a simulated blue-colored beverage (a mass ratio of phycocyanin/emulsifier=0.576) except that 0.125% by mass of RYOTO (registered trade mark) Polyglyester L-7D was replaced with 0.0625% by mass of RYOTO (registered trade mark) Sugar Ester L-1695 (HLB=16). The same procedure was performed as in Example 1 to heat treat the simulated blue-colored beverage, and the color value retention rate before and after heating was measured. The results indicated that the color value retention rate was 85.1%. Example 4 The same procedure was performed as in Example 1 to obtain a simulated blue-colored beverage (a mass ratio of phycocyanin/emulsifier=0.36) except that 0.125% by mass of RYOTO (registered trade mark) Polyglyester L-7D was replaced with 0.1% by mass of RYOTO (registered trade mark) Sugar Ester P-1670 (HLB=16). The same procedure was performed as in Example 1 to heat treat the simulated blue-colored beverage, and the color value retention rate before and after heating was measured. The results indicated that the color value retention rate was 81.7%. All the simulated blue-colored beverages of the above-described Examples exhibited excellent safety to the human body and were suited for food purposes (beverage purposes). Comparative Example 1 The same procedure was performed as in Example 1 to obtain a simulated blue-colored beverage (containing no emulsifier) except that no RYOTO (registered trade mark) Polyglyester L-7D was used. The same procedure was performed as in Example 1 to heat treat the simulated blue-colored beverage, and the color value retention rate before and after heating was measured. The results indicated that the color value retention rate was 61.6%. Comparative Example 2 The same procedure was performed as in Example 1 to obtain a simulated blue-colored beverage (a mass ratio of phycocyanin/emulsifier=0.36) except that 0.125% by mass of RYOTO (registered trade mark) Polyglyester L-7D was replaced with 0.1% by mass of RYOTO (registered trade mark) Sugar Ester S-570 (HLB=5). The same procedure was performed as in Example 1 to heat treat the simulated blue-colored beverage, and the color value retention rate before and after heating was measured. The results indicated that the color value retention rate was 58.2%. As revealed in a comparison of color value retention rates between Example 4 and Comparative Example 2, it is clear that even when the same amount of nonionic emulsifier formed of the same sucrose ester is used in these Examples, the thermal resistance under acidity is largely different within or outside a threshold of an HLB of 15 and that unless the HLB is 15 or more, no excellent technical effect according to the present invention is exhibited. INDUSTRIAL APPLICABILITY The color material aqueous solution according to the present invention contains phycocyanin (A), a polyvalent carboxylic acid (B) containing one or more hydroxy groups, and at least one kind of nonionic emulsifier (C) selected from the group consisting of sucrose esters and polyglycerol esters, the at least one kind of nonionic emulsifier (C) having an HLB of 15 or more. Thus, the color material aqueous solution exhibits excellent thermal stability under acidic conditions and can be suited for use in, for example, confections and breads, noodles, various foods, various beverages, and medicines and cosmetics. | 35,399 |
11859089 | EMBODIMENTS OF THE INVENTION 1. A process for the production of crystalline carbon structure networks in a reactor3which contains a reaction zone3band a termination zone3c, by injecting a water-in-oil or bicontinuous micro-emulsion c comprising metal catalyst nanoparticles, into the reaction zone3bwhich is at a temperature of above 600° C., preferably above 700° C., more preferably above 900° C., even more preferably above 1000° C., more preferably above 1100° C., preferably up to 3000° C., more preferably up to 2500° C., most preferably up to 2000° C., to produce crystalline carbon structure networks e, transferring these networks e to the termination zone3c, and quenching or stopping the formation of crystalline carbon structure networks in the termination zone by spraying in water d.2. The process according to embodiment 1, said reactor being a furnace carbon black reactor3which contains, along the axis of the reactor3, a combustion zone3a, a reaction zone3band a termination zone3c, by producing a stream of hot waste gas a1in the combustion zone by burning a fuel a in an oxygen-containing gas b and passing the waste gas a1from the combustion zone3ainto the reaction zone3b, spraying a water-in-oil or bicontinuous micro-emulsion c comprising metal catalyst nanoparticles, in the reaction zone3bcontaining the hot waste gas, carbonizing said emulsion at a temperature of above 600° C., preferably above 700° C., more preferably above 900° C., even more preferably above 1000° C., more preferably above 1100° C., preferably up to 3000° C., more preferably up to 2500° C., most preferably up to 2000° C., and quenching or stopping the reaction in the termination zone3cby spraying in water d, to yield crystalline carbon structure networks e.3. The process according to any one of the preceding embodiments, wherein the oil phase in the emulsion is aromatic and/or aliphatic, preferably comprising at least 50 wt % C14 or higher, based on the total weight of the oil phase.4. The process according to any one of the preceding embodiments, said emulsion comprising at least 1 mM metal catalyst nanoparticles, preferably having an average particle size between 1 and 100 nm.5. A crystalline carbon structure network obtainable by the process according to any one of the preceding embodiments, wherein said carbon structures are chemically interconnected through a multitude of junctions, including Y- and H-junctions.6. The network according to embodiment 5, having at least one, preferably at least two, more preferably at least three, most preferably all of the following properties:(i) Iodine Adsorption Number (IAN) of at least 250 mg/g according to ASTM D1510;(ii) Nitrogen Surface Area (N2SA) of at least 250 m2/g according to ASTM D6556;(iii) Statistical Thickness Surface Area (STSA) of at least 120 m2/g according to ASTM D6556;(iv) Oil Absorption Number (OAN) of at least 150 cc/100 g according to ASTM D2414.7. The network according to embodiment 5 or 6, wherein said structures have an average thickness of 1-400 nm, preferably between 5 and 350 nm, more preferably up to 100 nm, in one embodiment between 50 and 100 nm, and/or an average length in the range of 100-10000 nm, preferably 200-5000 nm, more preferably 500-5000 nm; and/or wherein the structures have an average aspect ratio of length to thickness of at least 2.8. A composite comprising carbon structure networks according to any one of embodiments 5-7, further comprising one or more polymers, for instance for adding mechanical strength, electrical conductivity or thermal conductivity to said polymer-based composite, and wherein said networks are in any amount of 1-70 wt %, preferably 10-50 wt %, more preferably between 20-40 wt %, based on the total polymer weight in the composite.9. The composite according to embodiment 8, showing an E modulus increasing with network concentration as measured according to ISO 527.10. Use of an emulsified carbon black feedstock in a carbon black manufacture process, preferably a furnace carbon black manufacture process, for producing crystalline carbon structure networks.11. A process for the semi-batch production of the crystalline carbon structure networks in a reactor3where a water-in-oil or bicontinuous micro-emulsion c comprising metal catalyst nanoparticles is injected from the top of the reactor3, preferably through spraying using an aerosol inlet4, to obtain an aerosol, and wherein said networks e are formed at an increased temperature of at least 600° C., preferably 700-1200° C. and deposited at the bottom of the reactor, and wherein the increased temperature is obtained using pyrolysis (e.g. heat source outside reactor, using N2, depleted of oxygen) or by combustion (heat source inside reactor, using air or oxygen).12. A process for the continuous production of the crystalline carbon structure networks in a reactor3where a water-in-oil or bicontinuous micro-emulsion c comprising metal catalyst nanoparticles is injected from the top of the reactor3, said reactor preferably being a thermal black reactor, preferably through spraying using an aerosol inlet4, to obtain an aerosol, and wherein said networks e are formed at an increased temperature of at least 600° C., preferably 700-1200° C. and deposited at the bottom of the reactor, and wherein the increased temperature is obtained using combustion (heat source inside reactor, using air or oxygen), but wherein the emulsion is injected only under pyrolysis conditions. DETAILED DESCRIPTION The invention can be described best as a modified carbon black manufacturing process, wherein ‘modified’ is understood that a suitable oil, preferably an oil comprising at least 14 C atoms (>C14) such as carbon black feedstock oil (CBFS), is provided to the reaction zone of a carbon black reactor as part of a single-phase emulsion, being a thermodynamically stable micro-emulsion, comprising metal catalyst nanoparticles. The emulsion is preferably provided to the reaction zone by spraying, thus atomizing the emulsion to droplets. While the process can be carried out batch or semi-batch wise, the modified carbon black manufacturing process is advantageously carried out as a continuous process. The single-phase emulsion is a micro-emulsion comprising metal catalyst nanoparticles. The preferred single-phase emulsion comprises CBFS oil, and may be referred to as ‘emulsified CBFS’ in the context of the invention. In one embodiment, the invention pertains to a process for the production of the crystalline carbon structure networks according to the invention in a reactor3which contains a reaction zone3band a termination zone3c, by injecting a single-phase emulsion c, being a micro-emulsion comprising metal catalyst nanoparticles, preferably a CBFS-comprising emulsion, according to the invention into the reaction zone3bwhich is at a temperature of above 600° C., preferably above 700° C., more preferably above 900° C., even more preferably above 1000° C., more preferably above 1100° C., preferably up to 3000° C., more preferably up to 2500° C., most preferably up to 2000° C., to produce crystalline carbon structure networks e, transferring these networks e to the termination zone3c, and quenching or stopping the formation of crystalline carbon structure networks in the termination zone by spraying in water d. The single-phase emulsion is preferably sprayed into the reaction zone. Reference is made toFIG.1A. In a preferred embodiment, the invention pertains to a process for the production of the crystalline carbon structure networks according to the invention in a furnace carbon black reactor3which contains, along the axis of the reactor3, a combustion zone3a, a reaction zone3band a termination zone3c, by producing a stream of hot waste gas a1in the combustion zone by burning a fuel a in an oxygen-containing gas b and passing the waste gas a1from the combustion zone3ainto the reaction zone3b, spraying (atomizing) a single-phase emulsion c according to the invention, preferably a micro-emulsion comprising metal catalyst nanoparticles, preferably a CBFS-comprising emulsion, in the reaction zone3bcontaining the hot waste gas, carbonizing said emulsion at increased temperatures (at a temperature of above 600° C., preferably above 700° C., more preferably above 900° C., even more preferably above 1000° C., more preferably above 1100° C., preferably up to 3000° C., more preferably up to 2500° C., most preferably up to 2000° C.), and quenching or stopping the reaction (i.e. the formation of crystalline carbon structure networks e) in the termination zone3cby spraying in water d. The reaction zone3bcomprises at least one inlet (preferably a nozzle) for introducing the emulsion, preferably by atomization. Reference is made toFIG.1A. Residence times for the emulsion in the reaction zone of the furnace carbon black reactor can be relatively short, preferably ranging from 1-1000 ms, more preferably 10-100 ms. In accordance with conventional carbon black manufacturing processes, the oil phase can be aromatic and/or aliphatic, preferably comprising at least 50 wt % C14 or higher, more preferably at least 70 wt % C14 or higher (based on the total weight of the oil). List of typical oils which can be used, but not limited to obtain stable emulsions are carbon black feedstock oils (CBFS), phenolic oil, anthracene oils, (short-medium-long chain) fatty acids, fatty acids esters and paraffins. The oil is preferably a C14 or higher. In one embodiment, the oil preferably has high aromaticity. Within the field, the aromaticity is preferably characterized in terms of the Bureau of Mines Correlation Index (BMCI). The oil preferably has a BMCI>50. In one embodiment, the oil is low in aromaticity, preferably having a BMCI<15. CBFS is an economically attractive oil source in the context of the invention, and is preferably a heavy hydrocarbon mix comprising predominantly C14 to C50, the sum of C14-C50 preferably amounting to at least 50 wt %, more preferably at least 70 wt % of the feedstock. Some of the most important feedstocks used for producing carbon black include clarified slurry oil (CSO) obtained from fluid catalytic cracking of gas oils, ethylene cracker residue from naphtha steam cracking and coal tar oils. The presence of paraffins (<C15) substantially reduces their suitability, and a higher aromaticity is preferred. The concentration of aromatics determines the rate at which carbon nuclei are formed. The carbon black feedstock preferably has a high BMCI to be able to offer a high yield with minimum heat input hence reducing the cost of manufacturing. In a preferred embodiment, and in accordance with current CBFS specifications, the oil, including mixtures of oil, has a BMCI value of more than 120. While the skilled person has no difficulties understanding which are suitable CBFS, merely as a guide it is noted that—from a yield perspective—a BMCI value for CBFS is preferably more than 120, even more preferably more than 132. The amount of asphaltene in the oil is preferably lower than 10 wt %, preferably lower than 5.0 wt % of the CBFS weight. The CBFS preferably has low sulfur content, as sulfur adversely affects the product quality, leads to lower yield and corrodes the equipment. It is preferred that the sulfur content of the oil according to ASTM D1619 is less than 8.0 wt %, preferably below 4.0 wt % more preferably less than 2.0 wt %. The emulsion, preferably a CBFS-comprising emulsion, is a “single-phase emulsion” which is understood to mean that the oil phase and the water phase optically appear as one miscible mixture showing no physical separation of oil, water or surfactant to the naked eye. The single-phase emulsion can be a macro-emulsion or a micro-emulsion, and can be either kinetically or thermodynamically stable. The process by which an emulsion completely breaks (coalescence), i.e. the system separates into bulk oil and water phases, is generally considered to be controlled by four different droplet loss mechanisms, i.e., Brownian flocculation, creaming, sedimentation flocculation and disproportionation. A ‘stable single-phase emulsion’ within the context of the invention is understood to mean that the emulsion shows no physical separation visible to the eye, preferably reflected in terms of the emulsion not showing any change in pH by more than 1.0 pH unit and/or the emulsion not showing any change in viscosity by more than 20%, over a period of time that exceeds the carbon structure network production time. The term ‘stable’ can mean ‘thermodynamically stable’ or ‘kinetically stable’ (by adding energy, i.e. through mixing). In practice, the single-phase emulsion is regarded stable if no de-mixing optically arises, i.e. a single-phase is retained, for a period of at least 1 minute after preparation of the emulsion. It is thus preferred that the emulsion maintains its pH within 1.0 pH unit and/or its viscosity with less than 20% variation over a period of time of at least 1 minute, preferably at least 5 minutes after preparation. While for handling purposes an extended stability is preferred, it is noted that the manufacturing process can still benefit from using emulsions stable over relatively short time spans of 1 minute, preferably 5 minutes: By adding energy (mixing) the stability of the emulsion can be extended, and short-term stability can be extended using in-line mixing. While macro-emulsions are not thermodynamically stable, and will always revert to their original, immiscible separate oil and water phases, the break down rate can be sufficiently slow to render it kinetically stable for the length of the manufacturing process. Provided that a stable, single-phase emulsion is obtained, the amounts of water and oil are not regarded limiting, but it is noted that reduced amounts of water (and increased amounts of oil) improve yields. The water content is typically between 5 and 50 wt % of the emulsion, preferably 10-40 wt %, even more preferably up to 30 wt %, more preferably 10-20 wt % of the emulsion. While higher amounts of water can be considered, it will be at the cost of yield. Without wishing to be bound by any theory, the inventors believe that the water phase attributes to the shape and morphology of the networks thus obtained. The choice of surfactant(s) is not regarded a limiting factor, provided that the combination of the oil, water and surfactant(s) results in a stable micro-emulsion as defined here above. As further guidance to the skilled person, it is noted that the surfactant can be selected on the basis of the hydrophobicity or hydrophilicity of the system, i.e. the hydrophilic-lipophilic balance (HLB). The HLB of a surfactant is a measure of the degree to which it is hydrophilic or lipophilic, determined by calculating values for the different regions of the molecule, according to the Griffin or Davies method. The appropriate HLB value depends on the type of oil and the amount of oil and water in the emulsion, and can be readily determined by the skilled person on the basis of the requirements of retaining a thermodynamically stable, single phase emulsion as defined above. It is found that an emulsion comprising more than 50 wt % oil, preferably having less than 30 wt % water phase, would be stabilized best with a surfactant having an HLB value above 7, preferably above 8, more preferably above 9, most preferably above 10. On the other hand, an emulsion with at most 50 wt % oil would be stabilized best with a surfactant having an HLB value below 12, preferably below 11, more preferably below 10, most preferably below 9, particularly below 8. The surfactant is preferably selected to be compatible with the oil phase. In case the oil is a CBFS-comprising emulsion with a CBFS, a surfactant with high aromaticity is preferred, while an oil with low BMCI, such as characterized by BMCI<15, would be stabilized best using aliphatic surfactants. The surfactant(s) can be cationic, anionic or non-ionic, or a mixture thereof. One or more non-ionic surfactants are preferred, in order to increase the yields since no residual ions will be left in the final product. In order to obtain a clean tail gas stream, the surfactant structure is preferably low in sulfur and nitrogen, preferably free from sulfur and nitrogen. Non-limiting examples of typical non-ionic surfactants which can be used to obtain stables emulsions are commercially available series of tween, span, Hypermer, Pluronic, Emulan, Neodol, Triton X and Tergitol. In the context of the invention, a micro-emulsion is a dispersion made of water, oil (preferably CBFS), and surfactant(s) that is a single optically isotropic and thermodynamically stable liquid with dispersed domain diameter varying approximately from 1 to 500 nm, preferably 1 to 100 nm, usually 10 to 50 nm. In a micro-emulsion the domains of the dispersed phase are either globular (i.e. droplets) or interconnected (to give a bicontinuous micro-emulsion). In a preferred embodiment, the surfactant tails form a continuous network in the oil-phase of a water-in-oil (w/o) emulsion or bicontinuous emulsion. The water domains should contain a metal catalyst, preferably having an average particle size between 1 nm and 100 nm. The single-phase emulsion, i.e. a w/o or bicontinuous micro-emulsion, preferably a bicontinuous micro-emulsion, further comprises metal catalyst nanoparticles preferably having an average particle size between 1 and 100 nm. The skilled person will find ample guidance in the field of carbon nanotubes (CNTs) to produce and use these kinds of nanoparticles. These metal nanoparticles are found to improve network formation in terms of both rates and yields, and reproducibility. Methods for manufacturing suitable metal nanoparticles are found in Vinciguerra et al. “Growth mechanisms in chemical vapour deposited carbon nanotubes” Nanotechnology (2003) 14, 655; Perez-Cabero et al. “Growing mechanism of CNTs: a kinetic approach” J. Catal. (2004) 224, 197-205; Gavillet et al. “Microscopic mechanisms for the catalyst assisted growth of single-wall carbon nanotubes” Carbon. (2002) 40, 1649-1663 and Amelinckx et al. “A formation mechanism for catalytically grown helix-shaped graphite nanotubes” Science (1994) 265, 635-639, their contents about manufacturing metal nanoparticles herein incorporated by reference. The metal catalyst nanoparticles are used in a bicontinuous or w/o microemulsion, preferably a CBFS-comprising bicontinuous or w/o micro-emulsion. In one embodiment, a bicontinous micro-emulsion is most preferred. Advantageously, the uniformity of the metal particles is controlled in said (bicontinuous) micro-emulsion by mixing a first (bicontinuous) micro-emulsion in which the aqueous phase contains a metal complex salt capable of being reduced to the ultimate metal particles, and a second (bicontinuous) micro-emulsion in which the aqueous phase contains a reductor capable of reducing said metal complex salt; upon mixing the metal complex is reduced, thus forming metal particles. The controlled (bicontinuous) emulsion environment stabilizes the particles against sintering or Ostwald ripening. Size, concentrations and durability of the catalyst particles are readily controlled. It is considered routine experimentation to tune the average metal particle size within the above range, for instance by amending the molar ratio of metal precursor vs. the reducing agent. An increase in the relative amount of reducing agent yields smaller particles. The metal particles thus obtained are monodisperse, deviations from the average particle size are preferably within 10%, more preferably within 5%. Also, the present technology provides no restraint on the actual metal precursor, provided it can be reduced. Non-limiting examples of effective catalyst species are the noble metals (Pt, Pd, Au, Ag), iron-family elements (Fe, Co and Ni), Ru, and Cu. Suitable metal complexes are but are not limited to (i) platinum precursors such as H2PtCl6; H2PtCl6·xH2O; K2PtCl4; K2PtCl4·xH2O; Pt(NH3)4(NO3)2; Pt(C5H7O2)2, (ii) ruthenium precursors such as Ru(NO)(NO3)3; Ru(dip)3Cl2 [dip=4,7-diphenyl-1,10-fenanthroline]; RuCl3, or (iii) palladium precursors such as Pd(NO3)2, or (iv) nickel precursors such as NiCl2 or NiCl2·xH2O; Ni(NO3)2; Ni(NO3)2·xH2O; Ni(CH3COO)2; Ni(CH3COO)2·xH2O; Ni(AOT)2 [AOT=bis(2-ethylhexyl)sulphosuccinate]. Non-limiting suitable reducing agents are hydrogen gas, sodium boron hydride, sodium bisulphate, hydrazine or hydrazine hydrate, ethylene glycol, methanol and ethanol. Also suited are citric acid and dodecylamine. The type of metal precursor is not an essential part of the invention. The metal of the particles of the (bicontinuous) micro-emulsion are preferably selected from the group consisting of Pt, Pd, Au, Ag, Fe, Co, Ni, Ru and Cu, and mixtures thereof, in order to control morphology of the carbon structures networks ultimately formed. The metal nanoparticles end up embedded inside these structures where the metal particles are physically attached to the structures. While there is no minimum concentration of metal particles at which these networks are formed—in fact networks are formed using the modified carbon black manufacturing process according to the invention—it was found that the yields increase with the metal particle concentrations. In a preferred embodiment, the active metal concentration is at least 1 mM, preferably at least 5 mM, preferably at least 10 mM, more preferably at least 15 mM, more preferably at least 20 mM, particularly at least 25 mM, most preferably up to 3500 mM, preferably up to 3000 mM. In one embodiment, the metal nanoparticles comprise up to 250 mM. These are concentrations of the catalyst relative to the amount of the aqueous phase of the (bicontinuous) micro-emulsion. Atomization of the single-phase emulsion, preferably a CBFS-comprising emulsion, is preferably realized by spraying, using a nozzle-system4, which allows the emulsion droplets to come in contact with the hot waste gas a1in the reaction zone3b, resulting in traditional carbonization, network formation and subsequent agglomeration, to produce crystalline carbon structure networks e according to the invention. The injection step preferably involves increased temperatures above 600° C., preferably between 700 and 3000° C., more preferably between 900 and 2500° C., more preferably between 1100 and 2000° C. In a different but related aspect of the invention, the invention pertains to a process for the semi-batch production of the crystalline carbon structure networks according to the invention in a reactor3where a single-phase emulsion c according to the invention is injected from the top of the reactor3, preferably through spraying using an aerosol inlet4, to obtain an aerosol, and wherein said networks e are formed at a temperature of at least 600° C., preferably 700-1200° C. and deposited at the bottom of the reactor furnace. The elevated temperature and reaction conditions may be achieved using pyrolysis (e.g. heat source outside reactor, using N2, depleted of oxygen) or by combustion (heat source inside reactor, using air or oxygen). In a further embodiment, the semi-batch process is conveniently operated with a carbon feed gas above its cracking temperature such as methane, ethane, propane, butane, ethylene, acetylene and propylene, carbon monoxide, oxygenated hydrocarbons such as methanol; aromatic hydrocarbons such as toluene, benzene and naphthalene, and mixtures of the above, for example carbon monoxide and methane. Reference is made toFIG.1B. Typical residence times are extended compared to the preferred furnace black process, with residence times of the emulsion in the reactor typically in the order of 1 hours to 7 days, more preferably 8 hours to 3 days. The single-phase emulsion is as defined before, i.e. a water-in-oil (w/o) micro-emulsion or a bicontinuous micro-emulsion comprising metal catalyst nanoparticles. Related therewith, the invention also pertains to a process for the continuous production of the crystalline carbon structure networks in a reactor3where a single-phase emulsion c according to the invention is injected from the top of the reactor3, said reactor preferably being a thermal black reactor, preferably through spraying using an aerosol inlet4, to obtain an aerosol, and wherein said networks e are formed at an increased temperature of at least 600° C., preferably 700-1200° C. and deposited at the bottom of the reactor, and wherein the increased temperature is obtained using combustion (heat source inside reactor, using air or oxygen), but wherein the emulsion is injected only under pyrolysis conditions. In a further embodiment, the continuous ‘pyrolysis’ process encompassing an initial combustion step is conveniently operated with a carbon feed gas above its cracking temperature such as methane, ethane, propane, butane, ethylene, acetylene and propylene, carbon monoxide, oxygenated hydrocarbons such as methanol; aromatic hydrocarbons such as toluene, benzene and naphthalene, and mixtures of the above, for example carbon monoxide and methane. Reference is made toFIG.1B. The residence time for the emulsion in the reactor is preferably in the range of 1 to 600 seconds, more preferably 5 to 60 seconds. The single-phase emulsion is as defined before, i.e. a water-in-oil (w/o) micro-emulsion or a bicontinuous micro-emulsion comprising metal catalyst nanoparticles. According to the above semi-batch and continuous processes of the invention, crystalline carbon structure networks (i.e. networks of crystalline carbon structures) can be produced. In a related aspect, the invention thus pertains to crystalline carbon structure networks obtained by or obtainable by the process of the invention. The term “carbon structures” are understood to comprise crystalline sp2-based carbon allotropes, i.e. substances in which a carbon atom is bonded to neighbouring three carbon atoms in a hexagonal pattern, including graphene, fullerene, carbon nanofibers and carbon nanotubes. The method of the invention allows for the growth of crystalline carbon structure networks formed from carbon structures that are chemically interconnected through a multitude of junctions, including Y- and H-junctions. In the context of the invention, a ‘network’ is preferably understood to comprise at least 3, preferably at least 5, more preferably at least 10, more preferably at least 100, more preferably at least 500 chemically connected nodes. The networks preferably have at least one, preferably at least two, more preferably at least three, most preferably all of the following properties:(i) Iodine Adsorption Number (IAN) of at least 250 mg/g, more preferably at least 300 mg/g, preferably 300-1000 mg/g, according to ASTM D1510;(ii) Nitrogen Surface Area (N2SA) of at least 250 m2/g, more preferably at least 300 m2/g, preferably 300-1000 m2/g, according to ASTM D6556;(iii) Statistical Thickness Surface Area (STSA) of at least 120 m2/g, more preferably at least 150 m2/g, preferably 150-1000 m2/g, according to ASTM D6556;(iv) Oil Absorption Number (OAN) of at least 150 cc/100 g, preferably 150-500 cc/100 g according to ASTM D2414, wherein: IAN=Iodine Adsorption Number: the number of grams of iodine adsorbed per kilogram of carbon black under specified conditions as defined in ASTM D1510; N2SA=nitrogen surface area: the total surface area of carbon black that is calculated from nitrogen adsorption data using the B.E.T. theory, according to ASTM D6556; STSA=statistical thickness surface area: the external surface area of carbon black that is calculated from nitrogen adsorption data using the de Boer theory and a carbon black model, according to ASTM D6556; and OAN=Oil Absorption Number: the number of cubic centimeters of dibutyl phthalate (DBP) or paraffin oil absorbed by 100 g of carbon black under specified conditions. The OAN value is proportional to the degree of aggregation of structure level of the carbon black, determined according to ASTM D2414. For each of IAN, N2SA (or NSA), STSA and OAN—all typical parameters for characterizing carbon black materials—the networks exhibit superior properties compared to traditional carbon black. The networks of the invention are preferably characterized by at least one, preferably at least two, more preferably all of (i), (ii) and (iii) since these are typical ways of characterized the surface area properties of the materials. In one embodiment, the networks exhibit at least one of (i), (ii) and (iii), and further comply with (iv). These structures forming the network may be described as nanofibers, which are solid (i.e. non-hollow), preferably having an average diameter or thickness of 1-400 nm, more preferably between 5 and 350 nm, more preferably up to 100 nm, in one embodiment 50-100 nm, compared to the average particle size of 8-500 nm for spherical carbon black particles. In one embodiment, the average fiber length (i.e. the average distance between two junctions) is preferably in the range of 100-10,000 nm, more preferably 200-5000 nm, more preferably 500-5000 nm, as for instance can be determined using SEM. Alternatively, the nanofibers or structures may preferably be described in terms of an average aspect ratio of fiber length-to-thickness of at least 2, preferably at least 3, more preferably at least 4, most preferably at least 5; in sharp contrast with the amorphous (physically associated) aggregates formed from spherical particles obtained through conventional carbon black manufacturing. The aggregates of carbon structure networks according to the invention are typically of the order of 0.1-100 microns, preferably 1-50 microns, which is observed with Laser Diffraction and Dynamic Light Scattering analysis. The invention also pertains to a composite comprising carbon structure networks according to the invention, further comprising one or more polymers, for instance for adding mechanical strength, electrical conductivity or thermal conductivity to said polymer-based composite. The networks may be added in any amount adapted to the desired performance, e.g. 1-70 wt %, more preferably 10-50 wt %, even more preferably between 20-40 wt %, based on the total polymer weight in the composite. In one aspect, the composite shows a network concentration-dependent elasticity modulus (E-modulus, i.e. an increase with increasing concentration of networks) for instance as measured according to ISO 527. EXAMPLES Example 1A. Preparation of Crystalline Carbon Structure Network 100 gallon of feedstock were prepared comprising of:a) Carbon Black slurry oil (CBO or CBFS oil)b) Water phase containing 3500 mM metal precursor salt (FeCl3)c) Water phase containing reducing agent (3650 mM citric acid)d) Surfactant (TritonX; HLB 13.4). The exact composition of the micro-emulsions (a+b+d) and (a+c+d) was detailed below: EmulsionCEOWater/FeCl3Water/CATritonXa + b + d70%10%0%20%a + c + d70%0%10%20% Both micro-emulsions (a+b+d) and (a+c+d) were added together and a single-phase micro-emulsion was obtained by stirring, and said micro-emulsion was stable for more than one hour, which was longer than the entire length of the experiment. The networks thus obtained had the following characteristics: IAN=382.5 mg/g, according to ASTM D1510 N2SA=350 m2/g (ASTM D6556) STSA=160.6 m2/g (ASTM D6556) OAN=170 cc/100 g (ASTM D2414). Example 2. Carbon Black vs Network The carbon networks according to example 1 were compared to conventional carbon black produced using (a). Standard grade carbon black typically has a nitrogen surface area (NSA or N2SA) varying up to 150 m2/g (N100-grade rubber carbon black). The morphology of the carbon networks was assessed by Scanning Electron Microscopy (SEM). It was found that the carbon network building blocks were chemically covalently linked solid carbon (nano)fibers with average fibre diameters below 100 nm. On the other hand, the carbon black building blocks were nodules in which graphitic layers are organized in a spherical shape (8-300 nm diameter). SEM pictures of carbon black and carbon networks building blocks are shown inFIGS.2A and2B, respectively. It was found that the carbon networks were organized in aggregate size 1-100 μm, while carbon blacks aggregates ranged typically from 85-500 nm. Example 3: Effect of Metal Nanoparticles The metal catalyst concentration had an effect on the final yields of the reactions: Three 20 g bicontinuous micro-emulsions were made from isopropylpalmitate (35% wt), butanol (11.25% wt), Tween 80 (33.75% wt), water (20% wt). While the first batch was prepared without any metal nanoparticles, two batches involved 50 and 200 mM FeCl3 metal nanoparticles (based on citric acid and FeCl3 with ratio 10:1). Each of the emulsions were stable over the full length of the experiments. The experiment without metal nanoparticles was carried out at least 10 times. In each case, the emulsions were introduced in the middle of a quartz-tube of a thermal horizontal tube reactor. The reactor was heated up to 750° C. (3 K/min) under 130 sccm of nitrogen flow and kept for 90 min at the same temperature. In the first 60 min the nitrogen gas flow was reduced to 100 sccm and ethylene gas was added at 100 sccm flow. During the last 30 minutes at 750° C. the ethylene was purged out from nitrogen at 130 sccm for the last 30 min and the reactor was then cooled down. It is only with metal nanoparticles that carbon structure networks were obtained. In none of the ten experiments without metal nanoparticles networks were found. The test done in the presence of 200 mM FeCl3 showed a yield increase of carbon structure networks, compared to the results reported with 50 mM FeCl3. A SEM picture of the networks obtained with a bicontinuous micro-emulsion based on isopropylpalmitate (35% wt), butanol (11.25% wt), Tween 80 (33.75% wt) and water (20% wt), with 100 mM Fe nanoparticles is shown inFIG.3. Example 4: Graph E-Modulus in PA6 Carbon Network powder such as prepared according to the recipe of example 1 was compounded in different loadings (10, 20, 30, 40% wt) in Polyamide 6 (Akulon F223D), by means of a twin screw extruder (LID=38, D=25 mm) and compared to glass fiber (Chopvantage 3540) compounded at 10, 20, 30% wt loadings under the same conditions. The E modulus was measured according to ISO 527, dried as molded tensile bars. The results are plotted inFIG.4, and indicate a performance of carbon networks, which is comparable to that of glass fibers. Carbon black was found not to provide significant reinforcement in thermoplastic, at whatever concentration. Example 5: Graph Electrical Conductivity PA6 and PET Volume resistivity was measured on different compounds prepared with carbon network prepared using the recipe according to example 1, in different loadings in Polyamide 6 (Akulon F223D) and PET (Ramapet N1), by means of a twin-screw extruder (LID=38, D=25 mm). The results are plotted inFIG.5. The percolation curves show good dosage control in the static dissipative range and that high conductive performances are achieved at high loadings. At the opposite, carbon black percolation threshold for conductive applications was found at lower dosages, i.e. <20% wt, and dosage control in the static dissipative range was unsatisfactory. Moreover, carbon networks compounds did not slough up to 30% wt loading, whereas carbon black compounds are known to be sloughing also at low filling degree. Example 6: Mechanical Strength Carbon nanofiber networks (low IAN, high crystallinity) obtained through the modified carbon black manufacturing process according to the invention were found capable of enhancing the mechanical properties of thermoplastic (and thermoset) polymer resins. Adding 10% by weight of carbon nanofiber networks to a polypropylene co-polymer resulted in an increase in tensile strength (at break) of 15% and an elasticity modulus increase of 16% compared to the neat polymer reference. A Brabender©Plasticorder© was used for mixing sufficient amount of carbon nanofiber networks and polypropylene at 210° C. and 80 rpm. Samples were compression moulded and tested with an Instron 3366 10 kN tensile tester at 23° C., 50% RH. Modulus(Young'sTensileTensileTensileTensileTensilestress atstrain atstress atstrain atstressYieldYieldBreakBreak10%0.05% -(Zero(Zero(Automatic(AutomaticCarbonX/0.25%)Slope)Slope)Load Drop)Load Drop)PP(MPa)(MPa)(%)(MPa)(%)Average1459.9920.057.6819.769.80Std dev149.721.130.201.140.9110.3%5.6%2.6%5.8%9.3% Modulus(Young'sTensileTensileTensileTensileTensilestress atstrain atstress atstrain atstressYieldYieldBreakBreak0.05% -(Zero(Zero(Automatic(AutomaticPP0.25%)Slope)Slope)Load Drop)Load Drop)reference(MPa)(MPa)(%)(MPa)(%)Average1258.3518.958.7617.1413.54Std dev141.141.170.891.374.3011.2%6.2%10.2%8.0%31.7% Example 7: Production by Means of Plasma Reactor Carbon nanofiber networks produced by means of using a plasma instead of combustion of a carbon gas. The plasma gas used was nitrogen (N2) at 60 kW with an initial plasma flow rate of 12 Nm3/h. Argon flow rate was set at 0.6 Nm3/h. Feedstock (emulsion) flow rate was set at 2.5 kg/h. GC-measurements were done to monitor H2 and progress of the carbon conversion. Temperature at injection was set at 1400° C., approximated residence time was 4 seconds. The collected material has a density of 0.13 g/cc and showed presence of carbon nanofiber networks observed via SEM and TEM, see figures. The average fiber diameter was determined to be 70 nm, while the length in-between was 5 to 10 times fiber diameter. | 37,699 |
11859090 | DETAILED DESCRIPTION The present invention provides protective inorganic coating compositions comprising: a liquid composition portion comprising by weight percent of the liquid composition portion: from 48 to 65 weight percent water, from 20 to 28 weight percent of an alkali metal oxide component comprising potassium oxide, and from 18 to 28 weight percent of a silicate-containing component; and a powder composition portion comprising by weight percent of the powder composition portion; from 20 to 80 weight percent microspheres, from 1 to 60 weight percent of at least one metal oxide powder comprising a Group II metal, Group IV metal, Group VI metal, Group X metal, Group XII metal or a combination thereof, and up to 50 weight percent microfibers. In certain embodiments, the liquid composition portion comprises from 50 to 62 weight percent water, from 22 to 25 weigh percent alkali metal oxide component, and from 21 to 27 weight percent silicate-containing component, and the powder composition portion comprises from 25 to 50 weight percent microspheres, from 1 to 50 weight percent metal oxide powder, and from 1 to 20 weight percent microfibers. For example, the powder composition portion may comprise from 30 to 40 weight percent microspheres, from 2 to 30 weight percent metal oxide powder, and from 2 to 10 weight percent microfibers. The liquid composition portion may comprise from 10 to 55 weight percent of the total weight of the coating composition, and the powder composition portion may comprise from 45 to 90 weight percent of the total weight of the coating composition. In certain embodiments, the liquid composition portion may comprise from 15 to 50 weight percent of the total weight of the coating composition, and the powder composition portion may comprise from 50 to 85 weight percent of the total weight of the coating composition. For example, the liquid composition portion may comprise from 20 to 45 weight percent of the total weight of the coating composition, and the powder composition portion may comprise from 55 to 80 weight percent of the total weight of the coating composition. The silicate-containing component may comprise potassium silicate, and may, be provided in the form of a water-based solution containing the potassium silicate. The microspheres may have a particle size from about 0.05 to about 10 μm, and may comprise at least one material selected from cenospheres, glass, pozzolan, ceramic, and composite. The Group II metal oxide may be selected from calcium, beryllium, and magnesium oxides. The Group IV metal oxide ma be selected from titanium, zirconium, and hafnium oxides. The Groups II and IV metal oxides may have a maximum particle size of 25 μm, for example from 0.05 to 5 μm. The coating compositions may also include microfibers such as silica, alumina, carbon, wollastonite, silicon carbide or a combination thereof. The microfibers may have an average aspect ratio of greater than 2:1, a maximum length of 500 μm, and a maximum diameter of 50 μm. For example, the microfibers may have an average aspect ratio of from 2:1 to 5:1 or 10:1, an average length of from 10 to 200 microns, and an average diameter of from 0.1 to 10 microns. The microfibers may comprise from 1 to 30 weight percent of the powder composition portion, for example, from 2 to 20 or from 5 to 10 weight percent of the powder composition portion. The coating composition may further comprise alumina powder having an average particle size of from 50 nanometers to 5 μm in an amount up to 5 or 10 weight percent of the powder composition portion. The coating composition may further comprise a sugar in an amount of from about 0.1 to about 1.5 or 2 weight percent of the total composition. The compositions may further comprise a densifier such as silicic acid in an amount up to 10 weight percent of the total composition, ford example, from 0.5 to 5 weight percent, or from 1 to 3 weight percent. The coating composition may further comprise standard colored pigments, for example, in an amount of from 0.1 to 10 weight percent of the coating composition. In accordance with an embodiment of the present invention, a method for coating a substrate is provided, comprising applying the coating compositions described above onto a galvanized steel substrate and allowing the composition to cure or dry. The coating compositions may be applied by any suitable method such as spraying, painting, or dip coating the substrate with this composition. The substrate may comprise iron, steel, or alloys thereof, with zinc deposited on its surface by hot-dip galvanizing, continuous sheet galvanizing, zinc rich painting, met lining, mechanical plating, electro-galvanizing, or zinc plating. As used herein, the term “galvanized steel” includes any type of iron, steel or alloys thereof having a zinc-containing layer applied to at least a portion of the surface thereof. The present coatings chemically stabilize the deposited zinc, and may provide protection to the deposited zinc by isolating it from moisture, oxygen, carbon dioxide, carbonic acids, chlorides, and sulfur compounds. An embodiment of the present invention provides a colorized ceramic coating that seals the galvanized zinc layer away from corrosive elements. Different colors, textures, and finishes may be provided to the galvanized steel surfaces. For example, the coating system may provide a natural satin finish and a nature-based color pallet. The coatings may also blend power towers and poles into their surroundings, while also preserving them from abrasion damage and graffiti. Ceramic fibers and barrier particles added to the coating compositions may increase physical properties such as surface hardness, density and friction resistance to protect higher value assets in industrial settings. The present coatings provide highly durable barriers against physical abuse that can be easily repaired without the need for stripping back down to the metal. The ability of this system to bond into itself means that some assets that experience high abrasion can be recoated while preserving the steel substrate indefinitely. Current repair methods rely on sacrificial, dimensionally unstable zinc that lacks resistance to abrasion, corrosive chlorides, and weathering. The present coatings may be applied over clean, e.g., degreased, zinc-containing surfaces. Smooth surfaces should be lightly abraded and loose rust should be removed to provide a proper surface for attachment. The extreme durability of the present coatings provide extreme long life for structures. Unlike leachable zing in galvanizing, the present coatings do not leach or deteriorate heavy metals to the environment, making them safe around critical streams, estuaries, ocean environments and the like. The following examples are intended to illustrate various aspects of the present invention, and are not intended to limit the scope of the invention. Example 1 A coating composition comprising three components is prepared: liquid, powder, and densifier. The liquid is made by blending a silicate-containing solution and potassium hydroxide flakes in water to create a binder solution. The powder is made by mixing the various raw components listed above. Microspheres represent 45-65% of the powder, by weight. Titanium, zirconium, hafnium, and aluminum oxides represent 20-35% of the powder, by weight. Calcium oxide represents 1% to about 3% of the powder, by weight. Sugar represents 1% to about 3% of the powder, by weight. Microfiber composed of wollastonite represents 5-10% of the powder, by weight. Discrete carbon fibers represent 5-10% of the powder, by weight. Densifier consisting of silicic acid is added to the liquid and powder at 1-5%, by weight. Example 2 A coating consisting of the components of Example 1 is prepared, with 0.1-10% pigment(s) added, by weight, to create a colored coating. Example 3 A coating consisting of the components of Example 1 is prepared by mixing the liquid, powder, and densifier components in either a high or low shear mixer (such as a paint mixing drill bit) to form a low-viscosity solution that can be applied to the surface of galvanized steel. The coating composition is applied to galvanized steel surfaces either by brush, roller, sprayer, or any other method typically used to apply coatings. Example 4 A coating consisting of the components of Example 1 is applied to galvanized steel and allowed to dry, harden, and cure at room temperature on the galvanized steel surface. Example 5 A coating consisting of the components of Example 1 is applied to a hot dipped galvanized surface. This stabilizes the surface chemistry, prevents the ingress of salts and other chemicals that negatively react with zinc, creates a surface resistance to abrasion, and creates an aesthetically pleasing surface. Example 6 A coating consisting of the components of Example 1 is applied to a cold-galvanized surface. This stabilizes the surface chemistry, prevents the ingress of salts and other chemicals that negatively react with zinc, creates a surface resistance to abrasion, and creates an aesthetically pleasing surface. Example 7 Physical testing, results for coated steel substrates are listed in Table 1 below. The coating composition included three components: liquid, powder, and densifier. The liquid portion is blend of a silicate-containing water-based solution 70% by weight, potassium hydroxide flakes 15% by weight, and water 15% by weight. The silicate-containing solution comprises about 61 weight percent water and about 39 weight percent potassium silicate and a combination of silicic acid and potassium salt, and is commercially available under the designation KASIL 6 Potassium Silicate Solution from PQ Corporation. The water contained in the silicate-containing solution and the separately added water provide a total water content of about 54 weight percent of the liquid portion. The powder portion is made by mixing pozzolan microspheres 60% by weight, zirconium oxide 12% by weight, titanium dioxide 15% by weight, microfiber wollastonite 9% by weight, discrete carbon fibers 2% by weight, calcium oxide 1% by weight, sugar 1% by weight. The densifier is silicic acid 100% by weight. The final composition comprises 38% by weight of the liquid portion, 61% by weight of the powder portion, and 1% by weight of the silicic acid densifier. TABLE 1PHYSICAL TESTINGDry Film Thickness4-8 Mils (100-200 Microns)Adhesion by Knife (ASTM D6677)Rating: 10/10Thermal ExpansionGalvanized Steel CompatibleImmersion in Water (ASTM D870)1000 Hours (No Corrosion)Immersion in Saltwater (ASTM D870)1000 Hours (No Corrosion)Hardness, Shore D (ASTM D2240)85.0 ± 5.0UVA/B 370 nmλNo Chalking OccurredNo Cracking OccurredNo Delamination OccurredFire Rating (ASTM E84-15b)Zero (0) Flame SpreadSmoke Generation (ASTM E84-15b)Zero (0) Smoke For purposes, of the detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers such as those expressing values, amounts, percentages, ranges, subranges and fractions may be read as if prefaced by the word “about,” even if the term does not expressly appear. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where a closed or open-ended numerical range is described herein, all numbers, values, amounts, percentages, subranges and fractions within or encompassed by the numerical range are to be considered as being specifically included in and belonging to the original disclosure of this application as if these numbers, values, amounts, percentages, subranges and fractions had been explicitly written out in their entirety. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements. As used herein, “including,” “containing” and like terms are understood in the context of this application to be synonymous with “comprising” and are therefore open-ended and do not exclude the presence of additional undescribed or unrecited elements, materials, ingredients or method steps. As used herein, “consisting of” is understood in the context of this application to exclude the presence of any unspecified element, ingredient or method step. As used herein, “consisting essentially of” is understood in the context of this application to include the specified elements, materials, ingredients or method steps “and those that do not materially affect the basic and novel characteristic(s)” of what is being described. As used herein, the terms “on,” “onto,” “applied on,” “applied onto,” “formed on,” “deposited on,” “deposited onto,” mean formed, overlaid, deposited, or provided on but not necessarily in contact with the suffice. For example, a coating composition “deposited onto” a galvanized steel substrate does not preclude the presence of one or, more other intervening coating layers of the same or different composition located between the coating composition and the substrate. Whereas particular embodiments of this invention have been described above for purposes of illustration, it will be evident to those skilled in the art that numerous variations of the details of the present invention may be made without departing from the invention as defined in the appended claims. | 14,209 |
11859091 | DETAILED DESCRIPTION The present invention provides a protective coating composition for coating ferrous substrates comprising a liquid composition portion comprising by weight percent of the liquid composition portion: from 50 to 70 weight percent water, from 17 to 27 weight percent of an alkali metal oxide component comprising potassium oxide, and from 18 to 28 weight percent of a silicate-containing component; and a powder composition portion comprising by weight percent of the powder composition portion: from 15 to 80 weight percent of microspheres, from 2 to 70 weight percent of at least one metal oxide powder comprising a Group II metal, Group IV metal, Group VI metal, Group X metal, Group XII metal or a combination thereof, and up to 50 weight percent microfibers. In certain embodiments, the liquid composition portion comprises from 52 to 65 weight percent water, from 20 to 24 weight percent alkali metal oxide component, and from 21 to 25 weight percent silicate-containing component, and the powder composition portion comprises from 20 to 60 weight percent microspheres, from 3 to 50 weight percent metal oxide powder, and from 1 to 40 weight percent microfibers. For example, the powder composition portion comprises from 30 to 50 weight percent microspheres, and from 4 to 20 weight percent metal oxide powder, and from 2 or 5 to 20 or 30 weight percent microfibers. The liquid composition portion may comprise from 10 to 55 weight percent of the total weight of the coating composition, and the powder composition portion may comprise from 45 to 90 weight percent of the total weight of the coating composition. In certain embodiments, the liquid composition portion may comprise from 15 to 50 weight percent of the total weight of the coating composition, and the powder composition portion may comprise from 50 to 85 weight percent of the total weight of the coating composition. For example, the liquid composition portion may comprise from 20 to 45 weight percent of the total weight of the coating composition, and the powder composition portion may comprise from 55 to 80 weight percent of the total weight of the coating composition. The silicate-containing component may comprise potassium silicate, and may be provided in the form of a water-based solution containing the potassium silicate. The microspheres may have a particle size from about 0.05 to about 10 μm, and may comprise at least one material selected from cenospheres, glass, pozzolan, ceramic, and composite. The Group II metal oxide may be selected from the group consisting of calcium, beryllium, and magnesium. The Group IV metal oxides may be selected from the group consisting of titanium, zirconium, and hafnium. The Group VI metal oxides may be selected from the group consisting of chromium, molybdenum, and tungsten. The Group XI metal oxide may be zinc and cadmium. Each of the Groups II, IV, VI, XII metal oxides may have a maximum particle size of about 10 μm. The coating compositions may also include microfibers such as silica, alumina, carbon, wollastonite, silicon carbide or a combination thereof. The microfibers may have an average aspect ratio of greater than 2:1, a maximum length of 500 μm, and a maximum diameter of 50 μm. For example, the microfibers may have an average aspect ratio of from 2:1 to 5:1 or 10:1, an average length of from 10 to 200 microns, and an average diameter of from 0.1 to 10 microns. The microfibers may comprise from 1 to 30 weight percent of the powder composition portion, for example, from 2 to 20 or from 5 to 10 weight percent of the powder composition portion. The coating composition may further comprise alumina powder in the 5 nanometers to 5 μm size in an amount up to 5 or 10 weight percent of the powder composition portion. The coating composition may further comprise a sugar in an amount of from about 0.1 to about 1.5 or 2 weight percent of the total composition. The compositions may further comprise a densifier such as silicic acid in an amount up to 10 weight percent of the total composition, for example, from 0.5 to 5 weight percent, or from 1 to 3 weight percent. The coating composition may further comprise standard colored pigments, for example, in an amount of from 0.1 to 10 weight percent of the coating composition. The coating composition may further comprise nano-diameter tubes composed of carbon, graphene, alumina, silica, or nitrides. Such nanotubes may be functionalized and may have a maximum length of 50 μm and a maximum diameter of 500 nanometers. The coating compositions may further comprise at least one additional component selected from carbides, nitrides, borides, silicides, zeolites, or a combination thereof. Such additional components may have a maximum particle size of 25 μm and may be provided in the form of elongated single crystal whiskers. The compositions may further comprise metakaolin in an amount of from 0.1 to 10 weight percent, or from 1 to 5 weight percent, based on the weight of the powder composition portion. In accordance with an embodiment of the present invention, a method for coating a substrate is provided, comprising applying the coating compositions described above onto a steel substrate and allowing the composition to cure or dry. The coating compositions may be applied by any suitable method such as spraying, painting, or dip coating the substrate with this composition. As used herein the terms “steel” and “steel substrate” include iron, steel and alloys thereof. The present coatings reduce corrosion, can provide abrasion resistance, and can electrically insulate such ferrous substrates. The present coatings may passivate steel in several ways: form a glass barrier layer that stops any humidity, salts, or contaminants from ever reaching the surface; attach iron atoms to molecules of silica glass, passivating them by bonding iron atoms chemically, e.g., covalently, so that the iron atoms do not form iron oxide; form a permanent alkali protective layer that passivates steel; penetrate and neutralize corrosion cells and passivate iron oxide; insulate against stray currents; provide a biologically impervious surface that inhibits bacteria which causes microbially induced corrosion (MIC). The present coatings reduce or eliminate the main cause of coating failure, rust creep, giving the top coating an extended lifespan. The present coatings can be considered to be a non-sacrificial primer. The coatings may form an integral composite with steel surfaces that cannot be separated. The coatings thermally expand and contract with the steel, and will not delaminate, crack or peel. The addition of fibers and nano-ceramic barrier particles adds flexibility and abrasion resistance. The present coatings may be applied over clean, degreased steel surfaces. Smooth surfaces may be lightly abraded and loose rust may be removed to provide a proper surface for attachment. The present coatings can be applied by any suitable method, such as spraying, and room temperature cured. The coatings may include ceramics particles, such as silica carbide for increased abrasion resistance, to imbue the coating with certain properties tailored for its end use. This makes the coatings extremely useful for industry and infrastructure. The present coatings may lower toxicity to workers and the environment. The following examples are intended to illustrate various aspects of the present invention, and are not intended to limit the scope of the invention. Example 1 A coating composition comprising three components is prepared: liquid, powder, and densifier. The liquid is made by blending a silicate-containing solution and potassium hydroxide flakes in water to create a binder solution. The powder is made by mixing the various raw components listed above. Pozzolan microspheres represent 45-50% of the powder, by weight. Metakaolin represents 1-5% of the powder, by weight. Titanium, zirconium, hafnium, iron, and aluminum oxides represent 25-40% of the powder, by weight. Microfiber composed of wollastonite represents 5-10% of the powder, by weight. Zinc oxide represents 5-10% of the powder, by weight. Discrete carbon fibers represent 5-10% of the powder, by weight. Densifier consisting of silicic acid is added to the liquid and powder at 1-5%, by weight. Example 2 A coating consisting of the components of Example 1 is prepared, 0.1-10% silicon carbide in the form of whiskers added, by weight. Example 3 A coating consisting of the components of Example 1 is prepared by mixing the liquid, powder, and densifier components in either a high or low shear mixer (such as a paint mixing drill bit) to form a low-viscosity solution that can be applied to the surface of concrete. The coating composition is applied to steel surfaces either by brush, roller, sprayer, or any other method typically used to apply coatings. Example 4 A coating consisting of the components of Example 1 is applied to steel and allowed to dry, harden, and cure at room temperature on the steel surface. Example 5 A coating consisting of the components of Example 1 is applied to low-carbon steel. This prevents oxidation from occurring, chemically stabilizes and neutralizes any surface contaminants, prevents abrasion of the steel surface, and creates a primer surface for a topcoat. Example 6 A coating consisting of the components of Example 1 is applied to high-carbon steel. This prevents oxidation from occurring, chemically stabilizes and neutralizes any surface contaminants, prevents abrasion of the steel surface, and creates a primer surface for a topcoat. Example 7 A coating consisting of the components of Example 1 is submerged in water. The coating prevents the surface from oxidizing/rusting. Example 8 A coating consisting of the components of Example 1 is submerged in saltwater. The coating prevents the surface from oxidizing/rusting. Example 9 Physical testing results for coated steel substrates are listed in Table I below. The coating composition included three components: liquid, powder, and densifier. The liquid portion is blend of a silicate-containing water-based solution 70% by weight, potassium hydroxide flakes 10% by weight, and water 20% by weight. The silicate-containing solution comprises about 61 weight percent water and about 39 weight percent potassium silicate and a combination of silicic acid and potassium salt, and is commercially available under the designation KASIL 6 Potassium Silicate Solution from PQ Corporation. The water contained in the silicate-containing solution and the separately added water provide a total water content of about 58 weight percent of the liquid portion. The powder portion is made by mixing pozzolan microspheres 48% by weight, metakaolin 3% by weight, zirconium oxide 24% by weight, zinc oxide 7% by weight, aluminum oxide 2% by weight, microfiber wollastonite 7% by weight, discrete carbon fibers 9% by weight. The densifier is silicic acid 100% by weight. The final composition comprises 33% by weight of the liquid portion, 65% by weight of the powder portion, and 2% by weight of the silicic acid densifier. TABLE 1PHYSICAL TESTINGDry Film Thickness4-8 Mils (100-200 Microns)Direct Pull-Off Adhesion>1500 PSI on Steel & Iron(ASTM D4541)Adhesion by KnifeRating: 10/10(ASTM D6677)Thermal ExpansionSteel & Iron CompatibleImmersion in Water1000 Hours (No Corrosion)(ASTM D870)Immersion in Saltwater1000 Hours (No Corrosion)(ASTM D870)Saltwater “Corrosion Creep”0 millimeters(ASTM D1654)**performed by PacificNorthwest National LaboratoryHardness, Shore D85.0 ± 5.0(ASTM D2240)UVA/B 370 nmλNo Chalking OccurredNo Cracking OccurredNo Delamination OccurredFire RatingZero (0) Flame Spread(ASTM E84 - 15b)Smoke GenerationZero (0) Smoke(ASTM E84 - 15b) For purposes of the detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers such as those expressing values, amounts, percentages, ranges, subranges and fractions may be read as if prefaced by the word “about,” even if the term does not expressly appear. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Where a closed or open-ended numerical range is described herein, all numbers, values, amounts, percentages, subranges and fractions within or encompassed by the numerical range are to be considered as being specifically included in and belonging to the original disclosure of this application as if these numbers, values, amounts, percentages, subranges and fractions had been explicitly written out in their entirety. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements. As used herein, “including,” “containing” and like terms are understood in the context of this application to be synonymous with “comprising” and are therefore open-ended and do not exclude the presence of additional undescribed or unrecited elements, materials, ingredients or method steps. As used herein, “consisting of” is understood in the context of this application to exclude the presence of any unspecified element, ingredient or method step. As used herein, “consisting essentially of” is understood in the context of this application to include the specified elements, materials, ingredients or method steps “and those that do not materially affect the basic and novel characteristic(s)” of what is being described. As used herein, the terms “on,” “onto,” “applied on,” “applied onto,” “formed on,” “deposited on,” “deposited onto,” mean formed, overlaid, deposited, or provided on but not necessarily in contact with the surface. For example, a coating composition “deposited onto” a substrate does not preclude the presence of one or more other intervening coating layers of the same or different composition located between the coating composition and the substrate. Whereas particular embodiments of this invention have been described above for purposes of illustration, it will be evident to those skilled in the art that numerous variations of the details of the present invention may be made without departing from the invention as defined in the appended claims. | 15,073 |
11859092 | DETAILED DESCRIPTION OF THE INVENTION In the description of embodiments of the invention disclosed herein, any reference to direction or orientation is merely intended for convenience of description and is not intended in any way to limit the scope of the present invention. Relative terms such as “lower,” “upper,” “horizontal,” “vertical,” “above,” “below,” “up,” “down,” “top” and “bottom” as well as derivative thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description only and do not require that the apparatus be constructed or operated in a particular orientation unless explicitly indicated as such. Terms such as “attached,” “affixed,” “connected,” “coupled,” “interconnected,” and similar refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. Moreover, the features and benefits of the invention are illustrated by reference to the preferred embodiments. Accordingly, the invention expressly should not be limited to such embodiments illustrating some possible non-limiting combination of features that may exist alone or in other combinations of features, the scope of the invention being defined by the claims appended hereto. The invention is directed to a non-curable, thixotropic hot melt composition formed by combining a hot melt wax with a thickener. The wax is converted into a thixotropic composition which can be used as a resist. The non-curable, thixotropic hot melt composition is preferably printable. In this specification, the term “non-curable” includes any composition that cannot be cured under conventional conditions, including but not limited to exposure to UV light, heat or moisture. Illustrative embodiments of the invention are directed to converting non-Newtonian hot melt waxes into printable non-curable thixotropic hot melt waxes which are used as resists by adding the thickeners to such waxes. By converting these non-curable waxes into non-curable thixotropic hot melt waxes, they can still be printable under high shear rate and printing temperatures. However, the droplets of these printable non-curable thixotropic hot melt waxes will not flow and will not form thin edges after application to the parts due to their high viscosity under low shear rate. Examples of suitable non-Newtonian non-curable hot melt waxes which can be used in the instant invention, include, but are not limited to, LS2535 wax or LS2538 wax, both manufactured by Koster Keunen, Inc. Preferably, these waxes are blends of polyolefin waves with acid functional groups. Many other non-curable hot melt waxes which are printable can also be used in the hot melt non-curable thixotropic composition of the instant invention. For example, for acidic gold and palladium plating waxes, the waxes must have an acid functional groups or anhydride functional groups so they can withstand the acidic plating solution but be stripped off in a basic (or alkaline) solution after plating. Examples of suitable non-curable hot melt waxes to be used as gold and palladium plating waxes include but are not limited to Licowax S flakes acid wax from Clariant AG and stearic acid. For alkaline plating solutions such as cyanide silver plating waxes, the waxes must have amine functional groups so they can withstand the alkaline plating solution and be stripped off in acid solution after plating. Examples of suitable non-curable hot melt waxes that can be used in silver plating include but are not limited to octadecylamine and Fentamine DA102 from Solvay S.A. Mixtures of the various waxes can be used provided the desired properties of the thixotropic hot melt composition are obtained. Thickeners such as fumed silica, polymeric particles or metal complexes can be added to the wax to form the printable non-curable thixotropic hot melt composition of this invention. For a composition which is used in inkjet printing, the preferred size of the thickener particles is less than one tenth of the nozzle size of the inkjet printer. In illustrative embodiments, the size of the thickener particles is ten microns or less. Any thickener that exhibits thickening efficiency and has the small size required may be used. The amount by weight of fillers can be between approximately 0.50% to approximately 30% in weight of the total composition. In one example, a non-Newtonian non-curable hot melt wax (LS2535) was converted into a non-curable thixotropic hot melt wax by adding fumed silica to the wax. Examples of suitable fumed silicas include but are not limited to, Aerosil 200 and Aerosil R974, both manufactured by Evonik Industries. The sizes of fumed silica particles are generally sub-micron. Such thickeners can be between approximately 0.50% to approximately 5% in weight of the total composition. The resultant non-curable thixotropic wax exhibited strong shear thinning character. The resultant non-curable thixotropic hot melt waxes have high viscosities under low shear rate and slightly higher viscosities under high shear rate when compared with a control sample which is the formulation without the thickener. The viscosities of the resultant non-curable thixotropic hot melt wax can be further lowered under higher shear rate present in the printer. The viscosity of the resultant non-curable thixotropic hot melt wax can also be adjusted by the addition of low viscosity acid waxes such as stearic acid. The amount of the low viscosity acid wax to be added is well within the scope of one of ordinary skill in the art. Fumed silica can form aggregations if processed in an inkjet printer during the dispersion process due to its high surface area and surface property compatibility to the plating wax. Additionally, the fumed silica can precipitate from the composition. Therefore, it is desirable to use a fumed silica which is highly compatible with the wax used. Polymeric particles or micron size powder products may also be used as thickeners in the composition of the instant invention. The polymeric particles must have high enough melting points to be above the mixing and printing temperature of the composition so as to survive the mixing and printing process. Generally, small particle sizes for the polymeric particles or powder products give better thickening and thixotropic performance. For polymeric particles, precipitation and low shear thinning efficiency may need to be considered. An example of a polymeric particle is MPP-635XF from Micro Powders Inc. Preferably, the mean particle size is from about 4 to about 6 microns. In one embodiment, the polymeric micron powder product used as a thickener is between approximately 5% to approximately 20% by weight of the composition. In addition to the above thickeners, metallic complexes can also be used to transform the waxes to the printable non-curable hot melt compositions of the instant invention. Examples of such include but are not limited to lithium complexes and aluminum complexes. Other thickeners can be incorporated into the non-curable thixotropic hot melt composition of the instant invention. Examples of such other thickeners include, but are not limited to, aerogel, carbon nanotubes, graphene and other inorganic or organic micron or nano particles. These thickeners may also be used so long as the hot melt composition retains its desired properties and they can be dispersed in the wax to form a thixotropic composition. Other additives may be included in the composition in conventional amounts provided that the additives do not detract from the thixotropic properties of the composition. Examples of such additives include dyes, opacifiers, antistatic agents, foaming agents, plasticizers, binders, antioxidants, surfactants, antistatic agents, metal adhesion agents and stabilizers. Such additives may be included in conventional amounts. Another embodiment of the instant invention uses the in-situ formation of thickeners in a hot melt wax, to form the non-curable thixotropic hot melt composition. In this specific embodiment, a thickener component is used to form the thickener and therefore provide the thixotropic properties to the wax. In this particular embodiment, the thickener component must have a different solubility than the rest of the composition under different temperatures using the process of the instant invention. The thickener component must be miscible or partially miscible with the other components of the composition. The thickener component, the wax and other components of the composition are mixed at a temperature to achieve complete mixing. The temperature can be easily chosen by one of ordinary skill in the art in this embodiment dealing with the in-situ formation of thickeners. When the composition is cooled to a lower temperature with vigorous stirring, the thickener component precipitates to form fine particles and thereby renders the composition thixotropic. The amount by weight of thickener component of the total composition is between approximately 0.5% to approximately 20% by weight, preferably between approximately 1% to approximately 5% by weight. Examples of thickener components that can be used in the instant invention include but are not limited to Licocene PP 6102 granules from Clariant A.G. (low viscous metallocene catalyzed polypropylene wax), Licocene PP MA 6252 granules from Clariant A.G. (maleic anhydride grafted polypropylene wax), A-C 540 ethylene acrylic acid copolymer from Honeywell International, Inc. and A-C 325 high density oxidized polyethylene homopolymer from Honeywell International, Inc. To obtain the in-situ formation of the thickener, the thickener component and the hot melt wax are heated to a temperature that is sufficiently high so as to achieve mixing of the thickener component and the hot melt wax. In an embodiment, the mixing temperature is approximately 10° C. to approximately 30° C. above the melting temperature of thickener component. The heating time depends upon how long it takes the thickener component and plating wax to be completely mixed. With the thickener component fully mixed with the hot melt wax, the composition is cooled down. As the mixture is cooled down, it is subjected to high speed rotation, stirring, shaking or vibration, for example, by the use of a speed mixer or other equipment. In an illustrative embodiment, the mixture is cooled down to a temperature which is approximately 10° C. to approximately 30° C. below the melting temperature of the wax. Under these conditions, the thickener component will precipitate from the wax to form fine particles due to its reduced solubility. As a result, small solid particles down to micron size are formed due to vigorous shear or shaking before the composition solidifies. The solid particles function as thickeners for the wax forming the non-curable thixotropic hot melt composition of the instant invention. In yet another embodiment of the invention, a component is added to the wax to form a non-curable hot melt composition of the instant invention. The component is not miscible with the wax. This component is then dispersed to form fine particles under a sufficiently vigorous mixing process. The printable non-curable thixotropic hot melt composition of the instant invention can be used as a resist. The resist can be used as an etch resist or a plating resist. In general, the resist is selectively deposited on a substrate. Hot melt compositions are typically applied by inkjet printers. In addition, the hot melt resist compositions of the instant invention can be applied using screen printing or by spray apparatus having nano- to macro-deposition capability. All of these are known methods in the art and can be used to apply the printable non-curable thixotropic hot melt composition of the instant invention to a substrate. If used as an etch resist, the uncovered section of the substrate may be etched to a desired depth. The etchant does not remove the resist from the substrate during etching, therefore, the composition functions as an etch resist. The etch resist is then stripped from the substrate leaving a patterned substrate which can be further processed using conventional methods known in the art. In the alternative, the substrate may be plated with a metal to form a pattern on the substrate, thus the resist functions as a plating resist. The plating resist is then stripped from the substrate leaving a substrate with a metal pattern for further processing by conventional methods known in the art. Etching can be done by methods known in the art appropriate to the material of which the substrate is composed. Typically, etching is done with acids, such as hydrofluoric acid, nitric acid, phosphoric acid, hydrochloric acid, organic acids such as carboxylic acids and mixtures thereof, or with industrial etches such as cupric chloride and ferric chloride. Such etches are well known in the art and may be obtained from the literature. Etching is typically done at temperatures of 20° C. to 100° C., more typically from 25° C. to 60° C. Etching includes spraying or dipping the resist coated substrate with the etchant in either a vertical or horizontal position. Typically, spraying is done when the substrate is in the horizontal position. This allows for quicker removal of the etchant. The speed of etching may be accelerated by agitating the etchant, for example using sonic agitation or oscillating sprays. After the substrate has been treated with the etchant it is typically rinsed with water to remove traces of the etchant. One or more metal layers may be deposited in the pattern formed on the substrate. Metals may be deposited electroless, electrolytically, by immersion or light induced plating. Conventional electroless, electrolytic and immersion baths and methods may be used to deposit metal or metal alloy layers. Many such baths are commercially available or described in the literature. Metals include but are not limited to noble and non-noble methods and their alloys. Examples of such suitable noble metals are gold, silver, platinum, palladium and their alloys. Examples of suitable non-noble metals are copper, nickel, cobalt, bismuth, zinc, indium, tin, and their alloys. The hot melt composition wax can be used on various substrates, including but not limited to semiconductors, photovoltaic or solar cells, components for electronic devices such as lead frames and printed circuit boards, in metal finishing of parts and precision tooling. After being deposited on the desired substrate as part of the plating process, the non-curable hot melt composition can be removed from the substrate using a stripping bath. For a resist that is stable in an acid plating bath, the resist would be strippable in an alkali stripping bath. For a resist that is stable in an alkaline plating bath, the resist would be strippable in an acidic stripping bath. While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention as defined in the accompanying claims. One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, proportions, sizes, materials and components and otherwise used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being defined by the appended claims, and not limited to the foregoing description or embodiments. | 16,123 |
11859093 | DETAILED DESCRIPTION OF THE INVENTION In the description of embodiments of the invention disclosed herein, any reference to direction or orientation is merely intended for convenience of description and is not intended in any way to limit the scope of the present invention. Relative terms such as “lower,” “upper,” “horizontal,” “vertical,” “above,” “below,” “up,” “down,” “top” and “bottom” as well as derivative thereof (e.g., “horizontally,” “downwardly,” “upwardly,” etc.) should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description only and do not require that the apparatus be constructed or operated in a particular orientation unless explicitly indicated as such. Terms such as “attached,” “affixed,” “connected,” “coupled,” “interconnected,” and similar refer to a relationship wherein structures are secured or attached to one another either directly or indirectly through intervening structures, as well as both movable or rigid attachments or relationships, unless expressly described otherwise. Moreover, the features and benefits of the invention are illustrated by reference to the preferred embodiments. Accordingly, the invention expressly should not be limited to such embodiments illustrating some possible non-limiting combination of features that may exist alone or in other combinations of features, the scope of the invention being defined by the claims appended hereto. The invention is directed to a non-curable, thixotropic hot melt composition formed by combining a hot melt wax with a thickener. The wax is converted into a thixotropic composition which can be used as a resist. The non-curable, thixotropic hot melt composition is preferably printable. In this specification, the term “non-curable” includes any composition that cannot be cured under conventional conditions, including but not limited to exposure to UV light, heat or moisture. Illustrative embodiments of the invention are directed to converting non-Newtonian hot melt waxes into printable non-curable thixotropic hot melt waxes which are used as resists by adding the thickeners to such waxes. By converting these non-curable waxes into non-curable thixotropic hot melt waxes, they can still be printable under high shear rate and printing temperatures. However, the droplets of these printable non-curable thixotropic hot melt waxes will not flow and will not form thin edges after application to the parts due to their high viscosity under low shear rate. Examples of suitable non-Newtonian non-curable hot melt waxes which can be used in the instant invention, include, but are not limited to, LS2535 wax or LS2538 wax, both manufactured by Koster Keunen, Inc. Preferably, these waxes are blends of polyolefin waxes having one or more types of acid functional group. Many other non-curable hot melt waxes which are printable can also be used in the hot melt non-curable thixotropic composition of the instant invention. For example, for acidic gold and palladium plating waxes, the waxes must have an acid functional group or anhydride functional group so they can withstand the acidic plating solution but can be stripped off in a basic (or alkaline) solution after plating. Examples of suitable non-curable hot melt waxes to be used as gold and palladium plating waxes include but are not limited to Licowax S flakes acid wax from Clariant AG and stearic acid. For alkaline plating solutions such as cyanide silver plating waxes, the waxes must have an amine functional group so they can withstand the alkaline plating solution and be stripped off in acid solution after plating. Examples of suitable non-curable hot melt waxes that can be used in silver plating include but are not limited to octadecylamine and Fentamine DA102 from Solvay S.A. Mixtures of the various waxes can be used provided the desired properties of the thixotropic hot melt composition are obtained. Thickeners such as fumed silica, polymeric particles, or metal complexes can be added to the wax to form the printable non-curable thixotropic hot melt composition of this invention. For a composition which is used in inkjet printing, the preferred size of the thickener particles is less than one tenth of the nozzle size of the inkjet printer. In illustrative embodiments, the size of the thickener particles is ten microns or less. Any thickener that exhibits thickening efficiency and has the small size required may be used. The amount by weight of fillers can be between approximately 0.50% to approximately 30% by weight of the total composition. In one example, a non-Newtonian non-curable hot melt wax (LS2535) was converted into a non-curable thixotropic hot melt wax by adding fumed silica to the wax. Examples of suitable fumed silicas include but are not limited to, Aerosil 200 and Aerosil R974, both manufactured by Evonik Industries. The sizes of fumed silica particles are generally sub-micron. Such thickeners can be between approximately 0.50% to approximately 5% by weight of the total composition. The resultant non-curable thixotropic wax exhibited strong shear thinning character. The resultant non-curable thixotropic hot melt waxes have high viscosities under low shear rate and slightly higher viscosities under high shear rate when compared with a control sample which is the formulation without the thickener. The viscosities of the resultant non-curable thixotropic hot melt wax can be further lowered under higher shear rate present in the printer. The viscosity of the resultant non-curable thixotropic hot melt wax can also be adjusted by the addition of low viscosity acid waxes such as stearic acid. The amount of the low viscosity acid wax to be added is well within the scope of one of ordinary skill in the art. Fumed silica can form aggregations if processed in an inkjet printer during the dispersion process due to its high surface area and surface property compatibility to the plating wax. Additionally, the fumed silica can precipitate from the composition. Therefore, it is desirable to use a fumed silica which is highly compatible with the wax used. Polymeric particles or micron size powder products may also be used as thickeners in the composition of the instant invention. One or more types of polymeric particles may be used. Preferably the polymeric particles are polyolefin particles. An example of polyolefin particles are polyethylene particles. The polymeric particles must have high enough melting points to be above the mixing and printing temperature of the composition so as to survive the mixing and printing process. Generally, small particle sizes for the polymeric particles or powder products give better thickening and thixotropic performance. For polymeric particles, precipitation and low shear thinning efficiency may need to be considered. An example of a polymeric particle is MPP-635XF high molecular weight polyethylene wax from Micro Powders Inc. Preferably, the mean particle size is from about 4 to about 6 microns. In one embodiment, the polymeric micron powder product used as a thickener is between approximately 5% to approximately 20% by weight of the composition. In addition to the above thickeners, metallic complexes can also be used to transform the waxes to the printable non-curable hot melt compositions of the instant invention. Examples of such include but are not limited to lithium complexes and aluminum complexes. Other thickeners can be incorporated into the non-curable thixotropic hot melt composition of the instant invention. Examples of such other thickeners include, but are not limited to, aerogel, carbon nanotubes, graphene and other inorganic or organic micron or nano particles. These thickeners may also be used so long as the hot melt composition retains its desired properties and they can be dispersed in the wax to form a thixotropic composition. Other additives may be included in the composition in conventional amounts provided that the additives do not detract from the thixotropic properties of the composition. Examples of such additives include dyes, opacifiers, antistatic agents, foaming agents, plasticizers, binders, antioxidants, surfactants, antistatic agents, metal adhesion agents and stabilizers. Such additives may be included in conventional amounts. Another embodiment of the instant invention uses the in-situ formation of thickeners in a hot melt wax, to form the non-curable thixotropic hot melt composition. In this specific embodiment, a thickener component is used to form the thickener and therefore provide the thixotropic properties to the wax. In this particular embodiment, the thickener component must have a different solubility than the rest of the composition under different temperatures using the process of the instant invention. The thickener component must be miscible or partially miscible with the other components of the composition. The thickener component, the wax and other components of the composition are mixed at a temperature to achieve complete mixing. The temperature can be easily chosen by one of ordinary skill in the art in this embodiment dealing with the in-situ formation of thickeners. When the composition is cooled to a lower temperature with vigorous stirring or other agitation, the thickener component precipitates to form fine particles and thereby renders the composition thixotropic. The amount by weight of thickener component of the total composition is between approximately 0.5% to approximately 20% by weight, preferably between approximately 1% to approximately 5% by weight. Examples of thickener components that can be used in the instant invention include but are not limited to Licocene PP 6102 granules from Clariant A.G. (low viscous metallocene catalyzed polypropylene wax), Licocene PP MA 6252 granules from Clariant A.G. (maleic anhydride grafted polypropylene wax), A-C 540 ethylene acrylic acid copolymer from Honeywell International, Inc. and A-C 325 high density oxidized polyethylene homopolymer from Honeywell International, Inc. To obtain the in-situ formation of the thickener, the thickener component and the hot melt wax are heated to a temperature that is sufficiently high so as to achieve mixing of the thickener component and the hot melt wax. In an embodiment, the mixing temperature is approximately 10° C. to approximately 30° C. above the melting temperature of thickener component. The heating time depends upon how long it takes the thickener component and plating wax to be completely mixed. With the thickener component fully mixed with the hot melt wax, the composition is cooled down. As the mixture is cooled down, it is subjected to high speed rotation, stirring, shaking or vibration, for example, by the use of a speed mixer or other equipment. In an illustrative embodiment, the mixture is cooled down to a temperature which is approximately 10° C. to approximately 30° C. below the melting temperature of the wax. Under these conditions, the thickener component will precipitate from the wax to form fine particles due to its reduced solubility. As a result, small solid particles down to micron size are formed due to vigorous shear or shaking before the composition solidifies. The solid particles function as thickeners for the wax forming the non-curable thixotropic hot melt composition of the instant invention. In another embodiment of the invention, thickener particles are uniformly dispersed in a mixture of hot melt waxes. Preferably in this embodiment, the thickener particles are organic. The thickener, the hot melt wax and any other additives are mixed at a temperature to achieve complete mixing. Generally, the temperature of mixing is determined by the component in the mixture with highest melting point. Preferably, the composition is mixed at about 10 to about 50° C. above the melting temperature of the thickener. After being mixed, the composition is then deposited on the substrate. Preferably the composition is deposited on the substrate by an inkjet printer. After the composition is deposited, the thickener starts to precipitate and form small particles and converts the mixture into a thixotropic composition with reduced flowability, preventing formation of thin resist edges. In this embodiment, the amount by weight of the thickener component of the total composition is between approximately 0.1% to about 20% by weight, preferably between approximately 1% to approximately 10% by weight. Examples of suitable thickeners to be used in this embodiment include polyethylene waxes. Preferably, the polyethylene waxes are lower molecular weight polyethylenes and have a molecular weight in the range of about 400 to about 1000. In addition, the melting point of the polyethylene wax used in this embodiment is between 70° C. to 110° C., preferably between 80° C. and 100° C. An example of a suitable polyethylene wax to be used in the instant invention is Polywax® wax, available from Baker Hughes, Inc. Once the mixed composition is applied to the substrate, the thickener starts to precipitate as the composition cools and forms small particles due to its high crystallization temperature and limited solubility in the hot melt wax. These precipitated thickener particles convert the mixture containing the hot melt wax into a thixotropic hot melt composition with reduced flowability. This embodiment provides for improved printability of the printable non-curable thixotropic hot melt composition. In yet another embodiment of the invention, a component is added to the wax to form a non-curable hot melt composition of the instant invention. The component is not miscible with the wax. This component is then dispersed to form fine particles under a sufficiently vigorous mixing process. The printable non-curable thixotropic hot melt composition of the instant invention can be used as a resist. The resist can be used as an etch resist or a plating resist. In general, the resist is selectively deposited on a substrate. Hot melt compositions are typically applied by inkjet printers. In addition, the hot melt resist compositions of the instant invention can be applied using screen printing or by spray apparatus having nano- to macro-deposition capability. All of these are known methods in the art and can be used to apply the printable non-curable thixotropic hot melt composition of the instant invention to a substrate. If used as an etch resist, the uncovered section of the substrate may be etched to a desired depth. The etchant does not remove the resist from the substrate during etching, therefore, the composition functions as an etch resist. The etch resist is then stripped from the substrate leaving a patterned substrate which can be further processed using conventional methods known in the art. In the alternative, the substrate may be plated with a metal to form a pattern on the substrate, thus the resist functions as a plating resist. The plating resist is then stripped from the substrate leaving a substrate with a metal pattern for further processing by conventional methods known in the art. Etching can be done by methods known in the art appropriate to the material of which the substrate is composed. Typically, etching is done with acids, such as hydrofluoric acid, nitric acid, phosphoric acid, hydrochloric acid, organic acids such as carboxylic acids and mixtures thereof, or with industrial etches such as cupric chloride and ferric chloride. Such etches are well known in the art and may be obtained from the literature. Etching is typically done at temperatures of 20° C. to 100° C., more typically from 25° C. to 60° C. Etching includes spraying or dipping the resist coated substrate with the etchant in either a vertical or horizontal position. Typically, spraying is done when the substrate is in the horizontal position. This allows for quicker removal of the etchant. The speed of etching may be accelerated by agitating the etchant, for example using sonic agitation or oscillating sprays. After the substrate has been treated with the etchant it is typically rinsed with water to remove traces of the etchant. One or more metal layers may be deposited in the pattern formed on the substrate. Metals may be deposited electroless, electrolytically, by immersion or light induced plating. Conventional electroless, electrolytic and immersion baths and methods may be used to deposit metal or metal alloy layers. Many such baths are commercially available or described in the literature. Metals include but are not limited to noble and non-noble methods and their alloys. Examples of such suitable noble metals are gold, silver, platinum, palladium and their alloys. Examples of suitable non-noble metals are copper, nickel, cobalt, bismuth, zinc, indium, tin, and their alloys. The hot melt composition wax can be used on various substrates, including but not limited to semiconductors, photovoltaic or solar cells, components for electronic devices such as lead frames and printed circuit boards, in metal finishing of parts and precision tooling. After being deposited on the desired substrate as part of the plating process, the non-curable hot melt composition can be removed from the substrate using a stripping bath. For a resist that is stable in an acid plating bath, the resist would be strippable in an alkali stripping bath. For a resist that is stable in an alkaline plating bath, the resist would be strippable in an acidic stripping bath. While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention as defined in the accompanying claims. One skilled in the art will appreciate that the invention may be used with many modifications of structure, arrangement, proportions, sizes, materials and components and otherwise used in the practice of the invention, which are particularly adapted to specific environments and operative requirements without departing from the principles of the present invention. The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being defined by the appended claims, and not limited to the foregoing description or embodiments. | 18,504 |
11859094 | While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments or implementations have been shown by way of example in the drawings and will be described in detail herein. It should be understood, however, that the disclosure is not intended to be limited to the particular forms disclosed. Rather, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of an invention as defined by the appended claims. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS FIG.1shows the relationship of the PTC ratio RT/R25versus temperature T of a typical PTC ink composition. When the temperature T is in excess of 70° C., the PTC ratio RT/R25(also known as a “resistance magnification” or “resistivity magnification”) begins to decline. The PTC ratio RT/R25is defined as the ratio between a resistance RTat temperature T and the resistance R25at temperature 25° C. The lower resistance at the onset of NTC leads to excessive current flow and the heating element is overpowered. Therefore, the NTC temperature region is a potential safety-risk temperature region. FIG.2shows the relative resistance magnification of a high-resistance magnification (HRM) PTC ink suitable for applications in the present disclosure. The HRM PTC ink exhibits the following properties: its switching temperature is at about 40° C.; its resistance magnification (or PTC ratio RT/R25) rises to about 200 within 30° C. of the switching temperature; and it exhibits only PTC behavior 30° C. beyond the switching temperature. That is, within a span of 30° C. above the switching temperature, the current flowing through the HRM PTC ink is reduced by a factor of 200, thereby providing a self-regulating mechanism by which the power consumption is reduced by 200. In fact, further investigation reveals that the HRM PTC ink has an upper resistance magnification greater than 200 above a temperature of 70° C. Furthermore, no NTC effect is observable 30° C. above the switching temperature, implying use of the HRM PTC ink is safe within this temperature range. For example, common commercially available PTC inks typically manifest a resistance multiplication of 10-15, as shown inFIG.1. On the other hand, the resistance magnification of an HRM PTC ink reaches 200 as shown inFIG.2. Moreover, the NTC effect is well above the switching temperature as compared to the NTC effect depicted inFIG.1. In addition, hysteresis effects are absent, transition regions are short and resistance recovery times are short. Finally, the slope of the curve for the HRM PTC ink inFIG.2is steeper than that depicted inFIG.1, which allows more tightly controlled temperature regulation. These properties imply that the HRM PTC ink ofFIG.2may be used in applications where a PTC effect, along with a high resistance magnification (i.e. power consumption reduction) are required between about 40° C. and 70° C. The high resistance magnification can be greater than 50, or greater than 100, or greater than 150, in the temperature range of interest. It follows that applications that require a PTC effect and high magnification resistance in a different temperature range can use an HRM PTC ink with a) a switching temperature; b) PTC effect; and c) high resistance magnification in the required temperature range. An example of an HRM PTC ink is disclosed in US Pat. Pub. No. 20170327707, incorporated herein by reference. However, as discussed above, any HRM PTC ink that exhibits substantially similar characteristics may be used. The requisite HRM PTC ink exhibits a switching temperature, high resistance magnification and a PTC effect over the temperature range required for the application. For example, the HRM PTC ink may comprise a first resin that provides a first PTC effect in a first temperature range and a second resin that provides a second PTC effect in a second temperature range, wherein the second temperature range is higher than the first temperature range. As an example, the double-resin HRM PTC ink may comprise about 10-30 wt % conductive particles; about 5-15 wt % of a first polymer resin; about 5-15 wt % of a second polymer resin; about 40-80 wt % of an organic solvent; and about 0-5 wt % other additives. The conductive particles can be one of, or a mixture of: a metallic powder, a metal oxide, carbon black and graphite. The first polymer resin may be a kind of crystalline or semi-crystalline polymer, such as polyurethane, nylon, and polyester. The second polymer resin may be a kind of non-crystalline polymer, such as acrylic resin. The selection of the solvent is based on its proper boiling point and the solubility of polymer resins since the polymer resins are completely dissolved in the organic solvent prior to blending with other components. Any organic, inert liquid may be used as the solvent for the so long as the polymer is fully solubilized. As examples, the solvent may be selected from MEK, N-methyl pyrolidone (NMP), toluene, xylene, and the like. The other additives include a dispersing/wetting additive and a rheology additive. As an example of the double resin composition, the HRM PTC ink may comprise about 5-15 wt % of a thermally active polymer resin-1 having a melting point of 30-70° C. and providing a first temperature coefficient characteristic in the first temperature range below 70° C.; about 5-15 wt % thermally active polymer resin-2 having a melting point of 70-140° C. and providing a second positive temperature coefficient characteristic in the second temperature range above 70° C.; about 10-30 wt % conductive particles; about 40-80 wt % organic solvent having a boing point higher than 100° C., the organic solvent being capable of dissolve both the polymer resin-1 and polymer resin-2, and about 0-5 wt % additives. The additives may comprise dispersing additives, wetting additives, and rheological additives, with the additives having enhanced dispersing/wetting and rheology properties. The first polymer resin may be a kind of crystalline or semi-crystalline polymer, such as polyurethane, nylon, and polyester. The second polymer resin may be a kind of non-crystalline polymer, such as acrylic resin. Thermal Substrate In a thermal substrate comprising a substrate and an HRM PTC ink, the HRM PTC ink can provide the elimination of NTC and therefore avoid catastrophic failure. Moreover, the high resistivity of the HRM PTC ink in a temperature window of 20° C. to 40° C. above the switch temperature can provide precise temperature self-regulation with rapid time-to-temperature. Non-limiting examples of a substrate include a fabric, a mesh, and a film. The present disclosure describes applications of the HRM PTC ink described above that extends or eliminates the onset of the NTC effect, offers magnification factors greater than 15, 25, 50, 100, 150 or 200, and switches in the range of 0°-160° C. Such applications are therefore safer, more reliable and dissipate minimal power at the switch temperature. Moreover, the wide switch temperature range of HRM PTC inks offers greater design flexibility and the steep temperature-resistance transition enables tighter temperature control. Thermal substrates that use HRM PTC ink may be created using various materials—depending on the specific application. For example, for outdoor wear, a nylon fabric may be used. For an industrial application that requires a higher operating temperature, a woven glass fiber mesh may be used. Similarly, various substrates may be used depending on the parameters of the application. For example, for clothing where low-weight and flexibility are required, thermoplastic polyurethane (TPU), polyester or a natural fabric such as cotton or a cotton blend is appropriate. In general, all-natural fibers, many polymer films and, in the case of meshes, metal wires are amenable to heating with a HRM PTC ink. First Embodiment of a Thermal Substrate In some embodiments, a thermal substrate may be made by deposition of a HRM PTC ink onto a fabric or mesh. The HRM PTC ink may be deposited on the substrate by various techniques. For example, screen printing onto a substrate may be used successfully because of the favorable dispersion of the HRM PTC ink. Other suitable techniques include gravure or rotogravure (e.g. “doctor blade”) methods. The HRM PTC ink may also be dispensed over simple or complex surfaces using nozzles mounted on programmable robots or embedded in components by 3-D printing. Other methods of depositing a HRM PTC ink with substantial accuracy are known in the art. Once the HRM PTC ink has been deposited on a fabric or mesh, silver or other conductive paste may be deposited on the substrate to create electrical interconnects (e.g. contacts and bus lines) for use in heater applications. In some embodiments, other metals (e.g. metal foils or wires), metal alloys or electrically conductive materials such as, but not limited to, aluminum, copper, nickel and alloys thereof, or highly conductivity electronic polymers may be deposited on the substrate as a paste or ink to create interconnects In all cases, interconnects can be designed for minimal length to lessen the possibility of cracking. An HRM PTC ink is methodically deposited on a substrate such that the resulting circuit pattern provides for optimum power delivered by the resulting thermal substrate. For any given heating application, whereas the switch temperature is determined by the composition of the HRM PTC ink, the power delivered by the thermal substrate is determined by the HRM PTC ink's circuit pattern. FIG.3depicts a printed circuit that comprises multiple deposits of printed HRM PTC ink. Each deposit of ink380acts as a resistor that regulates temperatures independently over a small area covered by the individual resistor380. Each resistor380in a column is powered by line voltage busses390and interconnects391. The printed circuit arises from deposition of the HRM PTC ink and conductive interconnects onto the substrate, as described above. Since the HRM PTC resistor380material typically has high sheet resistance, the power, P, is determined by arranging the printed resistors in parallel in a column on the substrate, as shown inFIG.3. The power dissipated by a column, Pcol, is: Pcol=V2/Rcol, where V is the applied voltage and Rcolis the total resistance of the column. If the number of resistors in parallel within a column is ncoland the resistance of individual resistor380is R, the resistance of the column, Rcol, is: Rcol=R/ncol. For a total of N columns (as depicted inFIG.3), the total power dissipated by the sheet, P, is P=ncolNV2/R. The resistors may have a length of from about 0.2 cm to about 10 cm. The temperature at each resistor is independently regulated. This circuit pattern allows independent temperature control of small areas, controlled power delivery and temperature uniformity, or non-uniformity if desired, over the surface of the substrate regardless of the local thermal load. The gap between discrete resistors may also be reduced to zero to form a contiguous line of resistors with identical behavior of the heater, i.e. local self-regulation in response to local thermal load conditions. In all cases, the resistivity of the HRM PTC ink may be adjusted appropriately. Second Embodiment of a Thermal Substrate In other embodiments, the HRM PTC ink may be deposited on a thread, yarn, or mesh element for weaving into a fabric or mesh to create a thermal fabric or mesh. FIG.4Aillustrates a cross-section of an HRM PTC coating420on an insulating thread, yarn, or filament410. The HRM PTC ink is deposited as a coating420on the insulating thread, yarn, or filament410and preferably sealed for electrical isolation on the outer surface with an insulating polymer430. Coating and sealing may be done by dipping, extrusion, or vapor deposition. Conduction of current is therefore along the length of the thread, yarn, or filament. In this configuration, the HRM PTC ink is formulated for low resistivity, while multiple coated threads, yarns or filaments may be connected in parallel. In another embodiment shown inFIG.4B, the HRM PTC ink472can be deposited on a conductive thread, yarn, or filament471(e.g. such as a copper wire) and coated with another electrically conductive layer473such as copper or silver. As above, there can be an optional insulating layer474around the conductive layer473. In this case, the HRM PTC ink may be formulated for high resistivity and electrical current flows radially inward from the outer conductive layer473to the conductive thread471. FIG.4Cillustrates an example of how the coated threads460may be woven into the weft of a fabric or mesh to form a thermal substrate using an insulator as a thread, yarn, or filament. Wires450and451carry supply voltage and are woven into the warp. Contact with the heater threads can be made by coating with pressure-sensitive adhesive and subsequently simultaneously applying heat and pressure, pulse welding, swaging, sealing with an overcoat or other means known in the art. The remaining threads470in the warp are standard fabric or mesh materials, e.g. polyester. FIG.4Dis a schematic of a mesh or fabric that incorporates a conductive yarn or filament constructed as shown inFIG.4C. One wire carrying supply voltage480makes contact with the outer conductor layer of the heater threads490, which constitute the weft; the other wire481makes contact with the inner conductor of the coated threads. The remaining threads495are the customary fabric material. The inner conductors are exposed by stripping the HRM PTC ink from the threads with solvent or mechanical means. Third Embodiment of a Thermal Substrate In yet another embodiment a thermal substrate505may be made by depositing the HRM PTC ink510and conductive interconnects511onto a polymer film520, as shown inFIG.5A. The printed elements may be subsequently encapsulated by optionally laminating a second film530of the same composition (as that of film520), resulting in a laminated thermal substrate535, as shown inFIG.5B. Lamination may be achieved by using a pressure and temperature adhesive. Suitable substrate and encapsulation materials include, but are not limited to: polyester, polyimide, polypropylene, rubber, silicone, thermoplastic polyurethane, laminates, ethylene-vinyl acetate (EVA) adhesive film, acrylate adhesive film and silicon adhesive film, fabric, silicone, and polyethylene terephthalate (PET). Additionally, the fabric or mesh heated by the thermal substrate505may have other layers of materials bonded to it such as, but not limited to: adhesive films, thermal barriers, reflective films, high or low emissivity films, absorptive films, alkaline resistant films, ground planes or EMI/RFI protective layers. If the primary heat transfer mechanism is conduction, the laminated thermal substrate535can be positioned in thermal communication with a fabric or mesh560in order to heat the fabric or mesh560, as shown inFIG.5C. In the embodiment shown, the laminated thermal substrate535may be merely positioned close to the fabric or mesh560to be in contact therewith. While laminated thermal substrate535(ofFIG.5B) is shown, thermal substrate505(ofFIG.5A) may be used in place of the laminated thermal substrate535. Alternatively, the laminated thermal substrate535may be attached to the fabric or mesh560by a fastener570(such as, but not limited to: a rivet, snap, clasp or stud), as shown in FIG.5D, or by various other means such as adhesives, sewing or removable clips. While laminated thermal substrate535(ofFIG.5B) is shown in inFIG.5C, the thermal substrate505(ofFIG.5A) may be used in place of the laminated thermal substrate535. If the primary mode of heat transfer is infrared radiation or convection, the laminated thermal substrate535may not need to be proximate to the fabric or mesh560. Then, an air gap580may be configured, as shown inFIG.5E, between the laminated thermal substrate535and the fabric or mesh560. While laminated thermal substrate535(ofFIG.5B) is shown, the thermal substrate505(ofFIG.5A) may be used in place of the laminated thermal substrate535. Whether the laminated thermal substrate535(or thermal substrate505) is adjacent to the mesh or fabric560(as inFIG.5C), fastened to the mesh or fabric560(as inFIG.5D), or separated from the mesh or fabric560by an air gap580(as inFIG.5E), an optional layer of material may be used for thermal insulation, water proofing, etc.FIG.5Fillustrated a configuration where a laminated thermal substrate535is fastened to a mesh or fabric560, and placed near a layer of material590, with an air gap580. For example, in an embodiment, a heated jacket can have a fabric lining that has an HRM PTC film attached thereto, along with a waterproof fabric that forms an outer layer of the jacket. In various applications, the fabric or mesh heated by the thermal film may have a sensor positioned proximate to it or laminated in it, Furthermore, it may use a feedback loop to adjust its temperature based on the sensor. In other applications, the HRM PTC ink itself may be used as its own temperature sensor since it manifests such a strong and repeatable relationship between resistance and temperature. In such an application, an auxiliary circuit may be configured to measure real-time heater resistance for an accurate temperature integrated over the entire thermal film. Application: Body Warming/Pain Relief A flexible heater comprising a HRM PTC deposited on a flexible substrate may be used as an article for body warming and/or pain relief applications such as, but not limited to, heating pads, heat wraps, heated blankets, heated throws, heated body pillows and heated mattress pads. The HRM PTC ink has a positive temperature coefficient (PTC) and a resistance magnification of at least 15 in a temperature range of at least 20 degrees Celsius above a switching temperature of the HRM PTC ink. The resistance magnification of the HRM PTC ink may be at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200. In addition, the temperature range may be 25 degrees Celsius, 30 degrees Celsius, 35 degrees Celsius or 40 degrees Celsius above the switching temperature. Furthermore, the switching temperature of the HRM PTC ink may be between 0 and 160 degrees Celsius. As an example, the HRM PTC ink may be a double resin ink that comprises: a first resin that provides a first PTC effect in a first temperature range; and a second resin that provides a second PTC effect in a second temperature range, wherein the second temperature range is higher than the first temperature range. In some embodiments, the first temperature range is between 30 C-70 C. Suitable substrates for this application are low-weight and flexible, and can include for example, thermoplastic polyurethane (TPU) or fabric itself such as polyester blend or nylon. The advantage of using HRM PTC heaters is the elimination of a temperature sensor, elimination of controller or power supply, elimination of physical connectors (e.g. wires), excellent temperature uniformity and efficiency. The latter advantage arises from the HRM PTC ink being formulated to switch at a desired level, as well as the layout/design of the article. In addition, the article can achieve heating temperatures with lower voltage. In addition, the article can be laundered. In some embodiments, the HRM PTC ink600is deposited onto a TPU substrate610using a method of screen printing, as shown inFIG.6. In some embodiments, the HRM PTC ink/TPU substrate assembly (i.e. the flexible heater) is cured using heat, and then encapsulated in an encapsulation material700, as shown inFIG.7. Suitable encapsulation materials for use in this application include a dielectric ink or other thin film. In some embodiments, the encapsulated heater is inserted, sewn, or otherwise attached to a fabric or shell (that is placed adjacent to a user) to form the article for body warming and/or pain relief. FIG.8is a plan view of a heater used in a body warming/pain relief application according to an embodiment of the present invention.FIG.8illustrates a thin film panel800comprising a HRM PTC ink810screen printed onto a TPU substrate815, cured with heat, and encapsulated with a dielectric ink (not shown). Panel800also comprises a series of printed conductive traces820that are screen printed onto the TPU, cured, and laminated with a dielectric ink. The conductive traces820are designed to carry the electrical current. InFIG.8, the conductive traces820run perpendicular through the printed HRM PTC ink, which creates a plurality of individual resistors810that generate heat. Each individual resistor is also self-regulating due to the properties of the HRM PTC ink properties. Furthermore, the HRM effect of the ink relate to the ability of each individual resistor resisting the electrical current enough to prevent at least one of overheating, fires, and failure. In some embodiments, printed silver traces are used. In one example of body warming/pain relief application, a HRM PTC ink was deposited onto a TPU substrate using a method of screen printing. A voltage of 120 VAC was applied to the article, resulting in a watt density of between 5-10 watts/ft2, or around 7 watts/ft2. While the article can be any shape, in this example, it was square or rectangular. The square measured from 6 in×6 in to 24 in×24 in, or about 12 in×12 in. The rectangle measured from 6 in×12 in to 18 in×30 in, or about 12 in×24 in. The self-regulating temperature of the double-switching ink was about 60° C. C, while the temperature of the article where applied to a user was about 50° C. to about 55° C. C or about 52° C. In general, an article can be designed to obtain a range of self-regulating temperatures and watt densities, in order to achieve specific metrics required by a customer. In a second example of body warming/pain relief application, a HRM PTC ink is deposited onto a TPU substrate using a method of screen printing. A voltage of about 11.1 VDC was applied to the article, resulting in a watt density of between 60-70 watts/ft2, or about 65 watts/ft2. While the article can be any shape, in this example, it was square, measuring from 6 in×6 in to 24 in×24 in, or about 12 in×12 in. The self-regulating temperature of the double-switching PTC ink was about 73° C., while the temperature of the article where applied to a user was about 60° C. to about 70° C. or about 65 C ° C. In general, an article can be designed to obtain a range of self-regulating temperatures and watt densities, in order to achieve specific metrics required by a customer. While particular implementations and applications of the present disclosure have been illustrated and described, it is to be understood that the present disclosure is not limited to the precise construction disclosed herein and that various modifications, changes, and variations can be apparent from the foregoing descriptions without departing from the spirit and scope of an invention as defined in the appended claims. | 23,169 |
11859095 | DETAILED DESCRIPTION The following describes embodiments of the present disclosure. Note that in the following, measurement values of volume median diameter (D50) are values measured using a dynamic light scattering type particle size distribution analyzer (“Zetasizer Nano ZS”, product of Malvern Instruments Ltd.) unless otherwise specified. In the following, measurement values of acid value are values measured according to “Japanese Industrial Standards (JIS) K0070-1992” unless otherwise specified. Furthermore, measurement values of mass average molecular weight (Mw) are values measured using gel permeation chromatography unless otherwise specified. In the present specification, acrylic and methacrylic may be collectively referred to as “(meth)acrylic”. First Embodiment: Ink Set The following describes an ink set according to a first embodiment of the present disclosure. The ink set of the present disclosure includes an inkjet ink (may be referred to below simply as an “ink”) and a recording head filling liquid (may be referred to below simply as a “filling liquid”). The ink contains a pigment, a pigment coating resin, a first alkali metal ion, and water. The filling liquid contains polyethylene glycol, a second alkali metal ion, a surfactant, and water. The polyethylene glycol has a mass average molecular weight of at least 180 and no greater than 800. The content ratio of the surfactant in the filling liquid is greater than 0.00% by mass and no greater than 0.10% by mass. The first and second alkali metal ions each contain at least one of Li+, Na+, and K+. The first and second alkali metal ions are identical to each other. The filling liquid has a pH of at least 7.0 and no higher than 11.5. In the ink set of the present disclosure, the filling liquid is used to fill a recording head in which ink remains. For example, when the recording head is temporarily unused for some reason after the recording head has discharged the ink, the filling liquid is used to fill the recording head. Specifically, the filling liquid is used to fill the recording head during shipment of the recording head, long-term storage of the recording head, or transportation of the recording head. The ink set of the present disclosure is suitable as an ink set used in a recording head testing method according to a second embodiment. By including the configuration described above, the ink set of the present disclosure facilitates introduction of the filling liquid into the ink flow channel in the recording head and can effectively inhibit the pigment component (pigment and pigment coating resin) in the ink from agglomerating in the recording head. The reasons for this are surmised to be as follows. The ink contains a first alkali metal ion including at least one of Li+, Na+, and K+. The first alkali metal ion included in the ink is a monovalent cation and has high ionic strength. Therefore, the first alkali metal ion functions as an excellent counter ion for the pigment coating resin and contributes to improved dispersibility of the pigment component. In this way, the first alkali metal ion is an essential component to stably disperse the pigment component in the ink. Here, when the filling liquid does not contain an alkali metal ion, or when an alkali metal ion contained in the filling liquid differs from an alkali metal ion contained in the ink, the dispersibility of the pigment component becomes unstable when the ink and filling liquid are mixed together. By contrast, the filling liquid contains the second alkali metal ion. The first and second alkali metal ions are identical to each other. As such, the ink set of the present disclosure can inhibit the dispersibility of the pigment component from becoming unstable when the ink and the filling liquid are mixed together. Additionally, the pigment component tends to be stably dispersed in neutral to slightly alkaline solutions. Because the filling liquid is neutral to slightly alkaline, the filling liquid can maintain the dispersibility of the pigment component when mixed with the ink. Additionally, the filling liquid contains a surfactant. The surfactant improves the dispersibility of the pigment component and facilitates the introduction of the filling liquid into the ink flow channel in the recording head. However, a large amount of surfactant can reduce the dispersibility of the pigment component. As such, the content ratio of the surfactant in the filling liquid is very small. The ink flow channel in the recording head is connected to outside air through an opening (e.g., a nozzle orifice). As such, in the filling liquid in the recording head, water gradually evaporates and other components are concentrated. From the above, the filling liquid must maintain the dissolved state of the surfactant even when water has evaporated. By contrast, the filling liquid contains polyethylene glycol. Polyethylene glycol is a component that easily dissolves surfactants (particularly highly hydrophobic surfactants) and does not volatilize easily. By containing polyethylene glycol, the filling liquid can maintain the dissolved state of the surfactant even when water evaporates after the filling liquid has been filled into the recording head. From the above, the ink set of the present disclosure can effectively inhibit the pigment component in the ink from agglomerating in the recording head. Additionally, the polyethylene glycol contained in the filling liquid has a relatively low molecular weight with a mass average molecular weight of at least 180 and no greater than 800, and has a low viscosity. From the above, the ink set of the present disclosure facilitates the introduction of the filling liquid into the ink flow channel in the recording head. The following describes the ink set of the present disclosure in further detail. Note that each component in the following description may be used as one type thereof independently, or may be used as a combination of two or more types thereof. [Ink] The ink contains a pigment, a pigment coating resin, a first alkali metal ion, and water. The pigment in the ink forms pigment particles together with a pigment coating resin, for example. The pigment particles are dispersed in a solvent. From the viewpoint of improving the color density, hue, or stability of the ink, the D50of the pigment particles is preferably at least 30 nm and no greater than 200 nm, and more preferably at least 70 nm and no greater than 130 nm. (Pigment) Examples of the pigment included in the ink include a yellow pigment, an orange pigment, a red pigment, a blue pigment, a violet pigment, and a black pigment. Examples of the yellow pigment include C.I. Pigment Yellow (74, 93, 95, 109, 110, 120, 128, 138, 139, 151, 154, 155, 173, 180, 185, or 193). Examples of the orange pigment include C.I. Pigment Orange (34, 36, 43, 61, 63, or 71). Examples of the red pigment include C.I. Pigment Red (122 or 202). Examples of the blue pigment include C.I. Pigment Blue (15, more specifically 15:3). Examples of the violet pigment include C.I. Pigment Violet (19, 23, or 33). Examples of the black pigment include C.I. Pigment Black (7). The content ratio of the pigment in the ink is preferably at least 1.0% by mass and no greater than 12.0% by mass, and more preferably at least 4.0% by mass and no greater than 8.0% by mass. By setting the content ratio of the pigment to at least 1.0% by mass, the image density of an image formed by the ink can be set to a desired value. Furthermore, by setting the content ratio of the pigment to no greater than 12.0% by mass, the fluidity of the ink can be improved. (Pigment Coating Resin) The pigment coating resin is a resin that is soluble in the ink. A portion of the pigment coating resin, for example, is present on the surfaces of the pigment particles to improve the dispersibility of the pigment particles. A portion of the pigment coating resin, for example, is present as dissolved in the ink. Examples of the pigment coating resin include a copolymer of at least one monomer among (meth)acrylic acid alkyl ester, styrene, and vinylnaphthalene and at least one monomer among (meth)acrylic acid and maleic acid. The pigment coating resin is preferably a resin with a repeating unit derived from (meth)acrylic acid (a (meth)acrylic acid unit), a repeating unit derived from (meth)acrylic acid alkyl ester (a (meth)acrylic acid alkyl ester unit), and a styrene unit. In this case, the ratio of the (meth)acrylic acid unit to all repeating units in the pigment coating resin is preferably at least 20% by mass and no greater than 60% by mass. The ratio of the (meth)acrylic acid alkyl ester unit to all repeating units in the pigment coating resin is preferably at least 30% by mass and no greater than 65% by mass. The ratio of the styrene unit to all repeating units included in the pigment coating resin is preferably at least 5% by mass and no greater than 25% by mass. The pigment coating resin is more preferably a resin with a repeating unit derived from methacrylic acid, a repeating unit derived from methyl methacrylate, a repeating unit derived from butyl acrylate, and a styrene unit. The content ratio of the pigment coating resin in the ink is preferably at least 0.5% by mass and no greater than 8.0% by mass, and more preferably at least 1.5% by mass and no greater than 4.0% by mass. By setting the content ratio of the pigment coating resin to at least 0.5% by mass, the dispersibility of the pigment component can be improved. By setting the content ratio of the pigment coating resin to no greater than 8.0% by mass, the ink can be inhibited from causing nozzle clogging. The pigment coating resin has an acid value of at least 50 mgKOH/g and no greater than 150 mgKOH/g, for example. By setting the acid value of the pigment coating resin to at least 50 mgKOH/g and no greater than 150 mgKOH/g, the preservation stability of the ink can be improved while further improving the dispersibility of the pigment component. The acid value of the pigment coating resin can be adjusted by changing the amount of monomer used in synthesizing the pigment coating resin. For example, by using a monomer (more specifically acrylic acid, methacrylic acid, or the like) with an acidic functional group (e.g., a carboxy group) in synthesizing the pigment coating resin, the acid value of the pigment coating resin can be increased. The pigment coating resin has an Mw of at least 10,000 and no greater than 50,000, for example. By setting the Mw of the pigment coating resin to at least 10,000 and no greater than 50,000, the image density of an image formed by the ink can be set to a desired value while inhibiting an increase in the viscosity of the ink. The Mw of the pigment coating resin can be adjusted by changing the polymerization conditions (more specifically the amount of polymerization initiator used, the polymerization temperature, the polymerization time, and the like) of the pigment coating resin. In the polymerization of the pigment coating resin, the amount of polymerization initiator used is preferably at least 0.001 mole and no greater than 5 moles to 1 mole of the monomer mixture, and more preferably at least 0.01 mole and no greater than 2 moles. In the polymerization of the pigment coating resin, for example, the polymerization temperature can be set to at least 50° C. and no higher than 70° C., and the polymerization time can be set to at least 10 hours and no longer than 24 hours. Note that the polymerized pigment coating resin may be used as a raw material of the ink, or may be used as a raw material of the ink after being neutralized with an equal amount of a basic compound. The basic compound is preferably a hydroxide of the first alkali metal ion. (First Alkali Metal Ion) The first alkali metal ion functions as a counter ion of the pigment coating resin. The first alkali metal ion includes at least one of Li+, Na+, and K+. The first alkali metal ion preferably includes only one of Li+, Na+, and K+. The first alkali metal ion is added to the ink as a salt (particularly a hydroxide), for example. That is, the ink contains lithium hydroxide, sodium hydroxide, or potassium hydroxide, for example. The amount of the first alkali metal ion in the ink is an amount capable of neutralizing the pigment coating resin in equal amounts (more specifically at least 90% by mass and no greater than 120% by mass to an amount capable of neutralizing the pigment coating resin in equal amounts), for example. (Water) Water is the main solvent of the ink. The content ratio of water in the ink is at least 30.0% by mass and no greater than 60.0% by mass, for example. (Water-Soluble Organic Solvent) The ink preferably further contains a water-soluble organic solvent. Examples of the water-soluble organic solvent in the ink include a glycol compound, a glycol ether compound, a lactam compound, a nitrogen-containing compound, an acetate compound, thiodiglycol, glycerin, and dimethyl sulfoxide. Examples of the glycol compound include ethylene glycol, 1,3-propanediol, propylene glycol, 1,5-pentanediol, 1,2-octanediol, 1,8-octanediol, 3-methyl-1,5-pentanediol, diethylene glycol, triethylene glycol, and tetraethylene glycol. Examples of the glycol ether compound include diethylene glycol diethyl ether, diethylene glycol monobutyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, diethylene glycol diethyl ether, triethylene glycol monomethyl ether, triethylene glycol monoethyl ether, triethylene glycol monobutyl ether, and propylene glycol monomethyl ether. Examples of the lactam compound include 2-pyrrolidone and N-methyl-2-pyrrolidone. Examples of the nitrogen-containing compound include 1,3-dimethylimidazolidinone, formamide, and dimethyl formamide. Examples of the acetate compound include diethylene glycol monoethyl ether acetate. The water-soluble organic solvent in the ink is preferably triethylene glycol monobutyl ether, 2-pyrrolidone, or glycerin. The content ratio of the water-soluble organic solvent in the ink is preferably at least 10.0% by mass and no greater than 45.0% by mass, and more preferably at least 25.0% by mass and no greater than 35.0% by mass. By setting the content ratio of the water-soluble organic solvent to at least 10.0% by mass and no greater than 45.0% by mass, the discharge stability of the ink can be improved. (Surfactant) The ink preferably further contains a surfactant. The surfactant improves the compatibility and dispersion stability of each component included in the ink. The surfactant also improves the permeability (wettability) of the ink to a recording medium. The surfactant in the ink is preferably a non-ionic surfactant. Examples of the non-ionic surfactant in the ink include an acetylene glycol surfactant (surfactant including an acetylene glycol compound), a silicone surfactant (surfactant including a silicone compound), and a fluorine surfactant (surfactant including a fluororesin or a fluorine-containing compound). Examples of the acetylene glycol surfactant include an ethylene oxide adduct of acetylene glycol and a propylene oxide adduct of acetylene glycol. When the ink contains a non-ionic surfactant, the content ratio of the non-ionic surfactant in the ink is preferably at least 0.1% by mass and no greater than 2.0% by mass, and more preferably at least 0.2% by mass and no greater than 0.6% by mass. (Additives) The ink may further contain known additives (e.g., a solution stabilizer, an anti-drying agent, an antioxidant, a viscosity modifier, a pH adjuster, and an antifungal agent) as necessary. (Ink Production Method) The ink can be produced, for example, by mixing water, a pigment dispersion, and additional components (e.g., water-soluble organic solvent and non-ionic surfactant) as necessary. The pigment dispersion contains the pigment, the pigment coating resin, and the first alkali metal ion. The pigment coating resin is prepared by neutralizing an equal amount of an alkali-soluble resin with a salt (e.g., hydroxide) including the first alkali metal ion, for example. The pigment dispersion can be prepared by adding the pigment to an aqueous solution containing the pigment coating resin and then performing dispersion processing. Examples of an apparatus used in the dispersion processing include a bead mill. After the dispersion processing in the production of the ink, foreign objects and coarse particles may be removed by a filter (e.g., a filter with a pore size of no larger than 5 μm). [Filling Liquid] The filling liquid contains polyethylene glycol, a second alkali metal ion, a surfactant, and water. The pH of the filling liquid is at least 7.0 and no higher than 11.5, and preferably at least 7.5 and no higher than 9.0. By setting the pH of the filling liquid to at least 7.0 and no higher than 11.5, the ink set of the present disclosure can effectively inhibit the pigment component in the ink from agglomerating in the recording head. The contact angle of the filling liquid to a stainless-steel plate is preferably no larger than 60°. Here, the ink flow channels of recording heads are often composed of stainless-steel material. By setting the contact angle of the filling liquid to the stainless-steel plate to no larger than 60°, the filling liquid is more easily introduced into the ink flow channel in the recording head. The static surface tension of the filling liquid at 20° C. is preferably at least 20 mN/m and no greater than 30 mN/m. By setting the static surface tension of the filling liquid to at least 20 mN/m and no greater than 30 mN/m, the filling liquid is more easily introduced into the ink flow channel in the recording head. (Polyethylene Glycol) The polyethylene glycol has a mass average molecular weight (Mw) of at least 180 and no greater than 800, preferably at least 250 and no greater than 500. By setting the mass average molecular weight of the polyethylene glycol to at least 180, the polyethylene glycol can be inhibited from volatilizing in the recording head after the filling liquid is filled into the recording head. By setting the mass average molecular weight of the polyethylene glycol to no greater than 800, the viscosity of the filling liquid can be suitably reduced. As a result, the filling liquid is easily introduced into the ink flow channel in the recording head. The content ratio of the polyethylene glycol in the filling liquid is preferably at least 3.0% by mass and no greater than 60.0% by mass, and more preferably at least 15.0% by mass and no greater than 60.0% by mass. By setting the content ratio of the polyethylene glycol in the filling liquid to at least 3.0% by mass, the pigment component in the ink can be more effectively inhibited from agglomerating in the recording head. By setting the content ratio of the polyethylene glycol in the filling liquid to no greater than 60.0% by mass, the viscosity of the filling liquid can be suitably reduced. As a result, the filling liquid is more easily introduced into the ink flow channel in the recording head. (Second Alkali Metal Ion) The second alkali metal ion includes at least one of Li+, Na+, and K+. The second alkali metal ion is the same as the first alkali metal ion contained in the ink. The second alkali metal ion preferably includes only one of Li+, Na+, and K+. The second alkali metal ion is added to the filling liquid as a salt (e.g., a hydroxide), for example. That is, the filling liquid contains lithium hydroxide, sodium hydroxide, or potassium hydroxide, for example. The second alkali metal ion imparts a weak alkalinity to the filling liquid. (Surfactant) The surfactant contained in the filling liquid is preferably a non-ionic surfactant. Examples of the non-ionic surfactant contained in the filling liquid include the same surfactants given as examples of the non-ionic surfactant in the ink. The content ratio of the surfactant in the filling liquid is more than 0.00% by mass and no greater than 0.10% by mass, preferably at least 0.01% by mass and no greater than 0.06% by mass. By setting the content ratio of the surfactant in the filling liquid to more than 0.00% by mass, the filling liquid is easily introduced into the ink flow channel in the recording head and can effectively inhibit the pigment component in the ink from agglomerating in the recording head. By setting the content ratio of the surfactant in the filling liquid to no greater than 0.10% by mass, the filling liquid can effectively inhibit the pigment component in the ink from agglomerating in the recording head. (Water) Water is the main solvent of the filling liquid. The content ratio of water in the filling liquid is at least 30.0% by mass and no greater than 60.0% by mass, for example. (Water-Soluble Organic Solvent) The filling liquid preferably further contains a water-soluble organic solvent. Due to the filling liquid containing a water-soluble organic solvent, the filling liquid is easily discharged from the recording head. Through the above, when it becomes necessary to eject the filling liquid from the recording head filled with the filling liquid, the filling liquid can be easily ejected by discharging from the recording head. Examples of the water-soluble organic solvent in the filling liquid include the same water-soluble organic solvents given as examples of the water-soluble organic solvent in the ink. The water-soluble organic solvent in the filling liquid is preferably glycerin or propylene glycol. The content ratio of the water-soluble organic solvent in the filling liquid is preferably at least 1.0% by mass and no greater than 30.0% by mass, and more preferably at least 10.0% by mass and no greater than 20.0% by mass. By setting the content ratio of the water-soluble organic solvent in the filling liquid to at least 1.0% by mass and no greater than 30.0% by mass, the filling liquid is more easily discharged from the recording head. (Additives) The filling liquid may further contain known additives (e.g., a solution stabilizer, an anti-drying agent, an antioxidant, a viscosity modifier, a pH adjuster, and an antifungal agent) as necessary. (Filling Liquid Production Method) The filling liquid can be produced by, for example, mixing the polyethylene glycol, the surfactant, and water and then adjusting the pH by adding a salt including the second alkali metal ion. Second Embodiment: Recording Head Testing Method The following describes a recording head testing method according to a second embodiment of the present disclosure. The recording head testing method of the present disclosure is a method by which a recording head is tested using the ink set according to the first embodiment, and includes a testing process of testing the discharge performance of the recording head and a filling process of filling the recording head with the filling liquid after the testing process. In the testing process, the discharge performance of the recording head is tested by the recording head discharging the ink. The recording head testing method of the present disclosure can inhibit discharge failure from occurring in the recording head after testing because the ink set according to the first embodiment is used. The recording head testing method of the present disclosure is performed by the manufacturer of the recording head before shipping the recording head, for example. The recording head to be tested by the recording head testing method of the present disclosure is not particularly limited, and examples thereof include a piezoelectric inkjet recording head and a thermal recording head. [Testing Process] In the present process, the discharge performance of the recording head is tested. Specifically, in the present process, the discharge performance of the recording head is tested by the recording head discharging ink. In the recording head tested in the present process, ink remains in the ink flow channel. In the present process, the recording head may be washed after the testing process. The method of washing the recording head is not particularly limited, and examples thereof include a method of discharging a wash fluid from the recording head after the recording head is filled with the wash fluid. Examples of the wash fluid include a wash fluid including water or a water-soluble organic solvent. In the present process, it is difficult to completely remove the ink in the ink flow channel even when the recording head is washed. [Filling Process] In the present process, the recording head is filled with the filling liquid. After the present process, the recording head is for example stored in preparation for shipment or transported for shipment. After the recording head has been delivered to a user, the filling liquid can be ejected from the recording head by discharging the filling liquid from the recording head. EXAMPLE The following describes Example of the present disclosure. However, the present disclosure is not limited to the following Example. <Study A: Mass Average Molecular Weight of Polyethylene Glycol> In Example, the mass average molecular weight of the polyethylene glycol used in the filling liquid was first studied. The following illustrates the preparation method of each raw material used in the production of the ink. [Preparation of Pigment Coating Resin (R-1)] An alkali-soluble resin with a repeating unit derived from methacrylic acid (an MAA unit), a repeating unit derived from methyl methacrylate (an MMA unit), a repeating unit derived from butyl acrylate (a BA unit), and a repeating unit derived from styrene (an ST unit) was prepared. This alkali-soluble resin had a mass average molecular weight (Mw) of 20,000 and an acid value of 100 mgKOH/g. The mass ratio of each repeating unit in this alkali-soluble resin was “MAA unit:MMA unit:BA unit:ST unit=40:15:30:15”. This alkali-soluble resin was mixed with an aqueous sodium hydroxide solution containing sodium hydroxide (neutralization processing). Through the neutralization processing, the alkali-soluble resin was neutralized with an equal amount (105%, strictly speaking) of NaOH. Through the above, a pigment coating resin solution containing a pigment coating resin (R-1) and water was obtained. The Mw of the alkali-soluble resin described above was measured using gel permeation chromatography (“HLC-8020GPC”, product of Tosoh Corporation) under the following conditions. Calibration curves were prepared using F-40, F-20, F-4, F-1, A-5000, A-2500, and A-1000, which are TSKgel standard polystyrene produced by Tosoh Corporation, and n-propylbenzene. (Measurement Conditions for Mass Average Molecular Weight) Columns: “TSKgel SuperMultipore HZ-H”, product of Tosoh Corporation (4.6 mm I.D.×15 cm semi-microcolumns)Number of columns: 3Eluent: TetrahydrofuranFlow rate: 0.35 mL/minSample injection amount: 10 μLMeasurement temperature: 40° C.Detector: IR detector [Preparation of Pigment Dispersion (D-1)] To achieve the composition shown in Table 1 below, a pigment (“RIONOL (registered Japanese trademark) Blue FG-7330”, product of TOYOCOLOR CO., LTD., component: copper phthalocyanine, color index: Pigment Blue 15:3), the pigment coating resin solution described above containing the pigment coating resin (R-1), an acetylene glycol surfactant “OLFINE (registered Japanese trademark) E1010” (ethylene oxide adduct of acetylene glycol), product of Nissin Chemical Industry Co., Ltd, and ion exchange water were charged into a vessel of 0.6 L capacity. Next, the contents of the vessel were wet dispersed using a media type wet disperser (“DYNO (registered Japanese trademark)-MILL”, product of Willy A. Bachofen (WAB) AG). Note that the content ratio of “water” in Table 1 below indicates the total content ratio of ion exchange water charged to the vessel described above and water included in the pigment coating resin solution (specifically, water included in the aqueous sodium hydroxide solution used to neutralize the alkali-soluble resin and water occurring due to the neutralization reaction of the alkali-soluble resin and the sodium hydroxide). TABLE 1Content ratioPigment dispersion[% by mass]Water78.5Pigment coating resin (R-1)6.0(NaOH neutralized)Pigment15.0Acetylene glycol surfactant0.5Total100.0 Continuing, the contents of the vessel described above underwent dispersion processing using a wet disperser (“Nano Grain Mill”, product of Asada Iron Works Co., Ltd.) and zirconia beads (0.5 mm in particle diameter) as a media. Dispersion conditions were set at a temperature of 10° C. and a peripheral speed of 8 m/sec. Through the above, a pigment dispersion (D-1) was obtained. The volume median diameter (D50) of the pigment particles included in the obtained pigment dispersion (D-1) were measured. In detail, the obtained pigment dispersion (D-1) was diluted 300 times with ion exchange water, and this was used as a measurement sample. The D50of the pigment particles in the measurement sample were measured using a dynamic light scattering type particle size distribution analyzer (“Zetasizer Nano ZS”, product of Malvern Instruments Ltd.). The D50of the pigment particles in the measurement sample was set as the D50of the pigment particles included in the pigment dispersion (D-1). The pigment particles included in the pigment dispersion (D-1) had a D50of 100 nm. [Preparation of Ink (I-1)] Ion exchange water was charged into a flask equipped with a stirrer (“THREE-ONE MOTOR (registered Japanese trademark) BL-600”, product of Shinto Scientific Co., Ltd.). While stirring the contents with the stirrer described above (stirring speed: 400 rpm), the pigment dispersion liquid (D-1) described above, an acetylene glycol surfactant (“SURFYNOL (registered Japanese trademark) 420”, product of Nissin Chemical Industry Co., Ltd.), triethylene glycol monobutyl ether, 2-pyrrolidone, glycerin, and water were added in the stated order. The proportion of the charged amount of each raw material was as shown in Table 2 below. TABLE 2ChargedamountInk[% by mass]Pigment dispersion (D-1)40.0SURFYNOL (R) 4200.3Triethylene glycol monobutyl ether4.02-pyrrolidone5.0Glycerin20.0Ion exchange waterRemainderTotal100.0 In order to remove foreign objects and coarse particles from the obtained mixture, the mixture was filtered using a filter with a pore size of 5 μm. Through the above, an ink (I-1) was obtained. [Preparation of Filling Liquids (F-1) to (F-7)] Filling liquids (F-1) to (F-7) were prepared by the following method. First, the polyethylene glycol (PEG), triethylene glycol (TEG), and polypropylene glycol (PPG) used to prepare the filling liquids (F-1) to (F-7) are illustrated below.TEG: Triethylene glycol (product of Maruzen Petrochemical Co., Ltd.), molecular weight 150PEG-200: Polyethylene glycol (“PEG-200”, product of Sanyo Chemical Industries, Ltd.), mass average molecular weight 200PEG-300: Polyethylene glycol (“PEG-300”, product of Sanyo Chemical Industries, Ltd.), mass average molecular weight 300PEG-400: Polyethylene glycol (“PEG-400”, product of Sanyo Chemical Industries, Ltd.), mass average molecular weight 400PEG-600: Polyethylene glycol (“PEG-600”, product of Sanyo Chemical Industries, Ltd.), mass average molecular weight 600PEG-1000: Polyethylene glycol (“PEG-1000”, product of Sanyo Chemical Industries, Ltd.), mass average molecular weight 1000PPG-400: Polypropylene glycol (“Polypropylene glycol 400”, product of FUJIFILM Wako Pure Chemical Corporation), mass average molecular weight 400 (Preparation of Filling Liquid (F-1)) A mixed liquid was obtained by mixing 30.0 parts by mass of triethylene glycol, 0.04 parts by mass of the surfactant (S-1) (“OLFINE (registered Japanese trademark) Exp 4300”, product of Nissin Chemical Industry Co., Ltd., acetylene glycol surfactant), 15.0 parts by mass of glycerin, 1N aqueous sodium hydroxide solution, and water. The additive amount of 1N aqueous sodium hydroxide solution was set to an amount bringing the pH of the mixed liquid to 8.0 (approximately 0.03 to 0.04 parts by mass). The additive amount of water was an amount bringing the total amount of the mixed liquid to 100 parts by mass. This mixed liquid was used as the filling liquid (F-1). (Preparation of Filling Liquids (F-2) to (F-7)) The filling liquids (F-2) to (F-7) were prepared by the same method as for the preparation of the filling liquid (F-1) aside from that the additive amounts of each component were changed as shown in Table 3 below. TABLE 3Filling liquidF-1F-2F-3F-4F-5F-6F-7ChargedTEG30.0——————amountPEG-200—30.0—————[parts byPEG-300——30.0————mass]PEG-400———30.0———PEG-600————30.0——PEG-1000—————30.0—PPG-400——————30.0OLFINE (R)0.040.040.040.040.040.040.04Exp 4300Glycerin15.015.015.015.015.015.015.0Aqueous NaAdjusted to pH 8.0hydroxide(approx. 0.03~0.04 parts by mass)solutionWaterRemainderRemainderRemainderRemainderRemainderRemainderRemainderTotal100.0100.0100.0100.0100.0100.0100.0 [Preparation of Ink Sets (IS-1) to (IS-7)] The ink (I-1) was combined with any one of the filling liquids (F-1) to (F-7) as shown in Table 4 below. Through the above, ink sets (IS-1) to (IS-7) were prepared. [Evaluation] With respect to the ink sets (IS-1) to (IS-7), the following methods were used to measure whether or not ink agglomeration could be inhibited, the introductivity of the filling liquid (ease of introducing the filling liquid into the ink flow channel in the recording head), and the contact angle of the filling liquid. The measurement results are shown in Table 4 below. (Agglomeration Inhibition) One part by mass of the ink (ink (I-1) in Study A) and 50 parts by mass of the filling liquid (any one of the filling liquids (F-1) to (F-7) in Study A) included in the ink set to be evaluated were mixed in a beaker. Next, the beaker containing the mixed liquid was stored in a thermostatic chamber at 40° C. for one month (storage processing). After processing, the volume of the mixed liquid was reduced by approximately 50% by evaporation. After processing, the mixed liquid was analyzed for the presence or absence of aggregates with a particle diameter of 3 μm or greater using a particle shape image analyzer (“FPIA (registered Japanese trademark)-3000”, product of Malvern Panalytical Ltd.). For the inhibition of ink agglomeration, “A (pass)” was defined as no occurrence of aggregates with a particle diameter of 3 μm or greater after the storage processing, and “B (fail)” was defined as occurrence of aggregates with a particle diameter of 3 μm or greater after the storage processing. Note that when the recording head was filled with the filling liquid after the recording head was tested, the residual ink and the filling liquid were mixed inside the recording head. The mixing ratio of the residual ink and the filling liquid (amount of ink/amount of filling liquid) varied depending on the part of the recording head but was assumed to be approximately 1/50 at maximum. As such, the mixing ratio of the ink and the filling liquid was set to one part by mass of the ink to 50 parts by mass of the filling liquid. Furthermore, aggregates with a particle diameter of 3 μm or greater occurring inside the recording head may clog a filter arranged inside the recording head and cause discharge failure of the ink. As such, whether or not aggregates with a particle diameter of 3 μm or greater occurred after storage processing was used as a determination reference of whether or not ink agglomeration could be inhibited. (Introductivity) An unused recording head (“KJ4B-QA”, product of Kyocera Corporation, total nozzle count: 2,656) was washed with pure water and thoroughly dried. Next, the recording head was filled with 25 mL of the filling liquid (any one of the filling liquids (F-1) to (F-7) in Study A) included in the ink set to be evaluated. Then, the filling liquid was ejected from the recording head by discharging the filling liquid from the recording head. This operation was performed a total of 10 times (total filling of 250 mL). Then, the recording head was refilled with the filling liquid. Nozzle check pattern printing was then performed on a glass plate using the recording head filled with the filling liquid. Through the above, a nozzle check pattern including the filling liquid was formed on the glass plate. Next, the number of discharge nozzles that could discharge the filling liquid (discharge nozzle count) was counted by reading the glass plate described above with a scanner. The ratio (%) of the discharge nozzle count to the total nozzle count (2,656) of the recording head (introduction rate) was determined by the following formula. The introductivity of the filling liquid was determined according to the following reference. Introduction rate=100×discharge nozzle count/total nozzle count (Introductivity Determination Reference)A (pass): introductivity 90% or higherB (fail): introductivity less than 90% (Contact Angle) The filling liquid (any one of the filling liquids (F-1) to (F-7) in Study A) included in the ink set to be evaluated was dropped onto a stainless-steel plate (SUS plate 304), and the static contact angle thereof was measured. The contact angle was measured at a temperature of 23° C. using a contact angle measuring apparatus (“OCA 40”, product of EKO Instruments B.V.). TABLE 4Ink setIS-1IS-2IS-3IS-4IS-5IS-6IS-7InkI-1I-1I-1I-1I-1I-1I-1FillingTypeF-1F-2F-3F-4F-5F-6F-7liquidCompoundTEGPEGPEGPEGPEGPEGPPGMw1502003004006001000400EvaluationAgglomerationBAAAAABresultinhibitionIntroductivityAAAAABAContact angle [°]60606060606460 As shown in Tables 1 to 4, the filling liquids (F-2) to (F-5) included in the respective ink sets (IS-2) to (IS-5) each contained polyethylene glycol with a mass average molecular weight of at least 180 and no greater than 800. The ink sets (IS-2) to (IS-5) facilitated introduction of the filling liquids into the ink flow channel in the recording head and effectively inhibited the pigment component in the ink from agglomerating in the recording head. By contrast, the filling liquid (F-1) included in the ink set (IS-1) contained tetraethylene glycol instead of polyethylene glycol. Due to having a small mass average molecular weight, tetraetylene glycol was determined not to be capable of sufficiently maintaining the solubility of the surfactant. As a result, the ink set (IS-1) could not inhibit the pigment component in the ink from agglomerating in the recording head. The filling liquid (F-6) included in the ink set (IS-6) contained polyethylene glycol with a mass average molecular weight of greater than 800. The filling liquid (F-6) was determined to have high viscosity due to containing polyethylene glycol with an excessively large mass average molecular weight. As a result, the filling liquid (F-6) included in the ink set (IS-6) was difficult to introduce into the ink flow channel in the recording head. The filling liquid (F-7) included in the ink set (IS-7) contained polypropylene glycol. Polypropylene glycol was determined to reduce the dispersibility of the pigment component due to having high hydrophobicity. As a result, the ink set (IS-7) could not inhibit the pigment component in the ink from agglomerating in the recording head. <Study B: Type and Amount of Surfactant> Next, the type and amount of the surfactant used in the filling liquid was studied. [Preparation of Filling Liquids (F-8) to (F-15)] Filling liquids (F-8) to (F-15) were prepared by the following method. First, the surfactants used in the preparation of the filling liquids (F-8) to (F-15) are shown below.S-1: Acetylene glycol surfactant (“OLFINE (registered Japanese trademark) Exp 4300”, product of Nissin Chemical Industry Co., Ltd)S-2: Acetylene glycol surfactant (“SURFYNOL (registered Japanese trademark) 104”, product of Nissin Chemical Industry Co., Ltd)S-3: Silicone surfactant (“BYK (registered Japanese trademark) 345”, product of BYK Japan K.K.)S-4: Fluorine surfactant (“SURFLON (registered Japanese trademark) S242”, product of AGC Seimi Chemical Co., Ltd.) The filling liquids (F-8) to (F-15) were prepared by the same method as for the preparation of the filling liquid (F-1) aside from that the additive amounts of each component were changed as shown in Tables 5 and 6 below. TABLE 5Filling liquidF-8F-9F-10F-11ChargedPEG-30030.030.030.030.0amountSurfactant (S-1)0.05———[parts bySurfactant (S-2)—0.02——mass]Surfactant (S-3)——0.07—Surfactant (S-4)———0.02Propylene17.017.017.017.0glycol1N aqueousAdjusted to pH 8.0 (approx. 0.03 tosodium0.04 parts by mass)hydroxidesolutionWaterRemainderRemainderRemainderRemainderTotal100.0100.0100.0100.0 TABLE 6Filling liquidF-12F-13F-14F-15ChargedPEG-30030.030.030.030.0amountSurfactant (S-1)0.080.12——[parts bySurfactant (S-2)————mass]Surfactant (S-3)——0.090.11Surfactant (S-4)————Propylene17.017.017.017.0glycol1N aqueousAdjusted to pH 8.0 (approx. 0.03 tosodium0.04 parts by mass)hydroxidesolutionWaterRemainderRemainderRemainderRemainderTotal100.0100.0100.0100.0 [Preparation of Ink Sets (IS-8) to (IS-15)] The ink (I-1) was combined with any one of the filling liquids (F-8) to (F-15) as shown in Table 7 below. Through the above, ink sets (IS-8) to (IS-15) were prepared. [Evaluation] With respect to the ink sets (IS-8) to (IS-15), whether or not ink agglomeration could be inhibited, the introductivity of the filling liquid, and the contact angle of the filling liquid were measured by the same method as for the measurement of the ink sets (IS-1) to (IS-7). The measurement results are shown in Table 7 below. TABLE 7Ink setIS-8IS-9IS-10IS-11IS-12IS-13IS-14IS-15InkI-1I-1I-1I-1I-1I-1I-1I-1FillingTypeF-8F-9F-10F-11F-12F-13F-14F-15liquidSurfactantTypeS-1S-2S-3S-4S-1S-1S-3S-3Parts0.050.020.070.020.080.120.090.11by massEvaluationAgglomerationAAAAABABresultinhibitionIntroductivityAAAAAAAAContact angle [°]5858575658575856 As shown in Tables 5 to 7, the filling liquids (F-8) to (F-12) and (F-14) included respectively in the ink sets (IS-8) to (IS-12) and (IS-14) contained acetylene glycol surfactant, silicone surfactant, or fluorine surfactant as surfactants. The content ratio of the surfactant in the filling liquids (F-8) to (F-12) and (F-14) was greater than 0.00% by mass and no greater than 0.10% by mass. The ink sets (IS-8) to (IS-12) and (IS-14) facilitated introduction of the respective filling liquids into the ink flow channel in the recording head and effectively inhibited the pigment component in the ink from agglomerating in the recording head. By contrast, the filling liquids (F-13) and (F-15) included in the respective ink sets (IS-13) and (IS-15) had a surfactant content ratio of more than 0.10% by mass. Due to the filling liquids of the ink sets (IS-13) and (IS-15) containing large amounts of surfactant, it was determined that the surfactant interacted with the pigment coating resin to cause agglomeration of the pigment component. As a result, the ink sets (IS-13) and (IS-15) could not inhibit the pigment component in the ink from agglomerating in the recording head. <Study C: Type of Alkali Metal Ion> Next, the types of the alkali metal ion included in the ink and the filling liquid were studied. [Preparation of Pigment Coating Resins (R-2) to (R-5)] Pigment coating resins (R-2) to (R-5) were prepared by the same method as for the preparation of the pigment coating resin (R-1) aside from the following changes. In the preparation of pigment coating resins (R-2) to (R-5), an aqueous lithium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous cesium hydroxide solution, or an aqueous magnesium hydroxide solution was used instead of an aqueous sodium hydroxide solution in the neutralization processing of the alkali-soluble resin. That is, the alkali-soluble resin was neutralized with an equal amount (105%, strictly speaking) of LiOH, KOH, CsOH, or Mg(OH)2. Through the above, a pigment coating resin solution containing any one of the pigment coating resins (R-2) to (R-5) and water was obtained. [Preparation of Pigment Dispersions (D-2) to (D-5)] Aside from the following changes, the pigment dispersions (D-2) to (D-5) were prepared by the same method as for the preparation of the pigment dispersion (D-1). In the preparation of the pigment dispersions (D-2) to (D-5), a pigment coating resin solution containing any one of the pigment coating resins (R-2) to (R-5) described above was used to achieve the compositions shown in Table 8 below. TABLE 8Pigment dispersionD-1D-2D-3D-4D-5Content ratioWater78.578.578.578.578.5[% by mass]Pigment coating resin (R-1) (NaOH neutralized)6.0————Pigment coating resin (R-2) (KOH neutralized)—6.0———Pigment coating resin (R-3) (LiOH neutralized)——6.0——Pigment coating resin (R-4) (CsOH neutralized)———6.0—Pigment coating resin (R-5) (Mg(OH)2neutralized)————6.0Pigment15.015.015.015.015.0Acetylene glycol surfactant0.50.50.50.50.5Total100.0100.0100.0100.0100.0 [Preparation of Inks (I-2) to (I-5)] As shown in Table 9 below, the inks (I-2) to (I-5) were prepared by the same method as for the preparation of the ink (I-1) aside from the pigment dispersions (D-2) to (D-5) being used instead of the pigment dispersion (D-1). TABLE 9InkI-1I-2I-3I-4I-5Charged amountPigment dispersion (D-1)40.0————[% by mass]Pigment dispersion (D-2)—40.0———Pigment dispersion (D-3)——40.0——Pigment dispersion (D-4)———40.0—Pigment dispersion (D-5)————40.0SURFYNOL (R) 4200.30.30.30.30.3Triethylene glycol monobutyl ether4.04.04.04.04.02-pyrrolidone5.05.05.05.05.0Glycerin20.020.020.020.020.0Ion exchange waterRemainderRemainderRemainderRemainderRemainderTotal100.0100.0100.0100.0100.0 [Preparation of Filling Liquids (F-16) to (F-20)] A mixed liquid was obtained by mixing 35.0 parts by mass of “PEG-400” described above, 0.04 parts by mass of the surfactant (S-1) (“OLFINE (registered Japanese trademark) Exp 4300”, product of Nissin Chemical Industry Co., Ltd., acetylene glycol surfactant), 10.0 parts by mass of glycerin, the alkali component shown in Table 10 below, and water. The additive amount of the alkali component was set to an amount (trace amount) bringing the pH of the mixed liquid to 8.0 to 9.0. The additive amount of water was an amount bringing the total amount of the mixed liquid to 100 parts by mass. The obtained mixed liquids were set as filling liquids (F-16) to (F-20). Note that the concentrations of the aqueous sodium hydroxide (NaOH) solution, the aqueous potassium hydroxide (KOH) solution, and the aqueous lithium hydroxide (LiOH) solution shown in Table 10 below were each 1N. TABLE 10Filling liquidF-16F-17F-18F-19F-20ChargedPEG-40035.035.035.035.035.0amountSurfactant (S-1)0.040.040.040.040.04[parts byGlycerin10.010.010.010.010.0mass]AlkaliTypeNaOHKOHLiOHCsOHMg(OH)2componentaqueousaqueousaqueousaqueoussolutionsolutionsolutionsolutionAmountTrace amount (adjusted to pH 8.0 to 9.0)WaterRemainderRemainderRemainderRemainderRemainderTotal100.0100.0100.0100.0100.0 [Preparation of Ink Sets (IS-16) to (IS-23)] Any one of the inks (I-1) to (I-5) was combined with any one of the filling liquids (F-16) to (F-20) as shown in Table 11 below. Through the above, ink sets (IS-16) to (IS-23) were prepared. In Table 11 below, the ions (alkali metal ions) included in each ink or filling liquid are also shown. [Evaluation] With respect to the ink sets (IS-16) to (IS-23), whether or not ink agglomeration could be inhibited, the introductivity of the filling liquid, and the contact angle of the filling liquid were measured by the same method as for the measurement of the ink sets (IS-1) to (IS-7). The measurement results are shown in Table 11 below. TABLE 11Ink setIS-16IS-17IS-18IS-19IS-20IS-21IS-22IS-23InkTypeI-1I-2I-3I-4I-5I-1I-2I-3IonNa+K+Li+Cs+Mg2+Na+K+Li+FillingTypeF-16F-17F-18F-19F-20F-17F-18F-16liquidIonNa+K+Li+Cs+Mg2+K+Li+Na+EvaluationAgglomerationAAABBBBBresultinhibitionIntroductivityAAAAAAAAContact angle [°]5857585859575858 As shown in Tables 8 to 11, the ink sets (IS-16) to (IS-18) contained the same respective alkali metal ions in the inks and filling liquids thereof. The alkali metal ion included at least one of Li+, Na+, and K+. The ink sets (IS-16) to (IS-18) facilitated introduction of the filling liquids into the ink flow channel in the recording head and effectively inhibited the pigment component in the ink from agglomerating in the recording head. By contrast, the ink sets (IS-19) and (IS-20) contained Cs+or Mg2+in the inks and filling liquids thereof. Due to having low ionic strength, Cs+was determined not to be capable of stably dispersing the pigment components. Due to being a divalent cation, Mg2+was determined to have agglomerated the pigment component. As a result, the ink sets (IS-19) and (IS-20) could not inhibit the pigment component in the ink from agglomerating in the recording head. In the ink sets (IS-21) to (IS-23), the ink and the filling liquid contained different alkali metal ions (Li+, Na+or K+) from each other. When the type of alkali metal ion contained in the filling liquid differed from the type of alkali metal ion contained in the ink, it was determined that the dispersibility of the pigment component was reduced when the filling liquid and ink were mixed. As a result, the ink sets (IS-21) to (IS-23) could not inhibit the pigment component in the ink from agglomerating in the recording head. <Study D: pH of Filling Liquid> [Preparation of Filling Liquids (F-21) to (F-24)] A mixed liquid was obtained by mixing 35.0 parts by mass of “PEG-400” described above, 0.04 parts by mass of the surfactant (S-1) (“OLFINE (registered Japanese trademark) Exp 4300”, product of Nissin Chemical Industry Co., Ltd., acetylene glycol surfactant), 10.0 parts by mass of glycerin, 1N aqueous sodium hydroxide solution, and water. The additive amount of 1N aqueous sodium hydroxide solution was set to an amount (trace amount) bringing the pH of the mixed liquid to between 8.0 and 12.0 as shown in Table 12 below. The additive amount of water was an amount bringing the total amount of the mixed liquid to 100 parts by mass. The obtained mixed liquids were set as filling liquids (F-21) to (F-24). TABLE 12Filling liquidF-21F-22F-23F-24ChargedPEG-40035.035.035.035.0amountSurfactant (S-1)0.040.040.040.04[parts byGlycerin10.010.010.010.0mass]1N NaOHAmountTraceTraceTraceTraceaqueousamountamountamountamountsolutionpH8.010.011.012.0WaterRemainderRemainderRemainderRemainderTotal100.0100.0100.0100.0 [Preparation of Ink Sets (IS-24) to (IS-27)] The ink (I-1) was combined with any one of the filling liquids (F-21) to (F-24) as shown in Table 13 below. Through the above, ink sets (IS-24) to (IS-27) were prepared. [Evaluation] With respect to the ink sets (IS-24) to (IS-27), whether or not ink agglomeration could be inhibited, the introductivity of the filling liquid, and the contact angle of the filling liquid were measured by the same method as for the measurement of the ink sets (IS-1) to (IS-7). The measurement results are shown in Table 13 below. TABLE 13Ink setIS-24IS-25IS-26IS-27InkI-1I-1I-1I-1FillingTypeF-21F-22F-23F-24liquidpH8.010.011.012.0EvaluationAgglomerationAAABresultinhibitionIntroductivityAAAAContact angle [°]58575858 As shown in Tables 12 and 13, the filling liquids (F-21) to (F-23) included in the respective ink sets (IS-24) to (IS-26) had a pH of at least 7.0 and no higher than 11.5. The ink sets (IS-24) to (IS-26) facilitated introduction of the filling liquids into the ink flow channel in the recording head and could effectively inhibit the pigment component in the ink from agglomerating in the recording head. By contrast, the filling liquid (F-24) included in the ink set (IS-27) had a pH higher than 11.5. When the pH of the filling liquid was too high, it was determined that the dispersibility of the pigment component was reduced. As a result, the ink set (IS-27) could not inhibit the pigment component in the ink from agglomerating in the recording head. <Study E: Amount of Polyethylene Glycol> [Preparation of Filling Liquids (F-25) and (F-26)] A mixed liquid was obtained by mixing “PEG-300” described above, 0.04 parts by mass of the surfactant (S-1) (“OLFINE (registered Japanese trademark) Exp 4300”, product of Nissin Chemical Industry Co., Ltd., acetylene glycol surfactant), 10.0 parts by mass of glycerin, 1N aqueous sodium hydroxide solution, and water. The additive amount of “PEG-300” was set as shown in Table 14 below. The additive amount of 1N aqueous sodium hydroxide solution was set to an amount (trace amount) bringing the pH of the mixed liquid to 8.0. The additive amount of water was an amount bringing the total amount of the mixed liquid to 100 parts by mass. The obtained mixed liquids were set as filling liquids (F-25) and (F-26). TABLE 14Filling liquidF-25F-26ChargedPEG-3005.055.0amountSurfactant (S-1)0.040.04[parts byGlycerin10.010.0mass]1N aqueous sodiumAdjusted to pH 8.0 (approx. 0.03hydroxide solutionto 0.04 parts by mass)WaterRemainderRemainderTotal100.0100.0 [Preparation of Ink Sets (IS-28) and (IS-29)] The ink (I-1) was combined with any one of the filling liquids (F-25) and (F-26) as shown in Table 15 below. Through the above, ink sets (IS-28) and (IS-29) were prepared. [Evaluation] With respect to the ink sets (IS-28) and (IS-29), whether or not ink agglomeration could be inhibited, the introductivity of the filling liquid, and the contact angle of the filling liquid were measured by the same method as for the measurement of the ink sets (IS-1) to (IS-7). The measurement results are shown in Table 15 below. TABLE 15Ink setIS-28IS-29InkI-1I-1FillingTypeF-25F-26liquidPEG-300 content ratio5.055.0[% by mass]EvaluationAgglomeration inhibitionAAresultIntroductivityAAContact angle [°]5859 As shown in Tables 14 and 15, the content ratio of polyethylene glycol in the filling liquids (F-25) and (F-26) included in the respective ink sets (IS-28) and (IS-29) was at least 3.0% by mass and no greater than 60.0% by mass. The ink sets (IS-28) and (IS-29) facilitated introduction of the filling liquids into the ink flow channel in the recording head and could effectively inhibit the pigment component in the ink from agglomerating in the recording head. | 54,417 |
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